CN115197949A - Recombinant Newcastle disease virus rNDV-OX40L, genome thereof, preparation method and application thereof - Google Patents

Abstract

The invention relates to a recombinant newcastle disease virus genome, a recombinant newcastle disease virus rNDV-OX40L containing the genome, a preparation method thereof, a DNA molecule for coding the recombinant newcastle disease virus genome and application of the DNA molecule in preparing a medicament for treating or improving cancer. The recombinant newcastle disease virus provided by the invention has the advantages that the coding gene of OX40L is inserted into the genome of the recombinant newcastle disease virus, so that the obtained recombinant newcastle disease virus improves the oncolytic efficiency and shows a good tumor treatment effect.

Description

A kind of recombinant Newcastle disease virus rNDV-OX40L, its genome, preparation method and the same use

technical field

The invention belongs to the field of cancer treatment, and specifically relates to a recombinant Newcastle disease virus genome, a recombinant Newcastle disease virus comprising the genome and a preparation method thereof, a DNA molecule encoding the above-mentioned recombinant Newcastle disease virus genome, and uses thereof .

Background technique

Cancer has become the number one killer of health. my country is a high incidence area of cancer, especially lung cancer, stomach cancer, liver cancer and rectal cancer. According to statistics, in 2016 alone, there were 4.8 million new cases of various types of cancer in the country, and 2.3 million patients died of various types of cancer. With the advancement of technology, various new treatment methods, especially biological drug treatment, have been continuously put into clinical use. However, the needs of drug safety, efficacy and quality of life of patients are far from being met. It is imperative to develop new drugs or treatments.

In 1991, Martuza et al. published an article in the journal Science, proving that transgenic herpes simplex virus has a certain effect in the treatment of malignant glioma. Since then, the development of oncolytic viruses to treat cancer has received increasing attention. The principle of oncolytic virus treatment of cancer is to genetically modify some weakly pathogenic viruses that exist in nature, so that they can selectively infect tumor cells, replicate in large numbers in cells, and finally destroy tumor cells. At the same time, it can also stimulate an immune response, attracting immune cells to continue to kill residual cancer cells or to kill migrated cancer cells through an immune response. In recent decades, great progress has been made in the research on oncolytic viruses. People have successively used Newcastle disease virus (NDV), herpes simplex virus type 1 (HSV-1), reovirus (reovirus), oncolytic adenovirus (oncolyticadenovirus) to develop oncolytic virus, and animal experiments show that it is safe efficient. However, the clinical manifestations were far below expectations. The inventors note that these poor clinical manifestations are related to current oncolytic virus design strategies.

NDV, an avian paramyxovirus with a negative-sense single-stranded RNA genome, has been a promising approach for cancer therapy. However, the effect of NDV as a single drug treatment is limited. On the one hand, the body's antiviral immune response can clear the virus, and on the other hand, the body can produce neutralizing antibodies to resist the virus. To improve the therapeutic efficiency of NDV in cancer, these viruses are subsequently used to deliver genes with antitumor activity, which can be expected to further enhance activity. Such genes include genes encoding immune checkpoint molecules, tumor suppressor proteins or immune stimulating proteins.

However, in tumor animal models, the therapeutic effect of NDV combined with some immune checkpoint inhibitors is not ideal, and the clinical response rate of NDV combined with PD1 antibody is also limited, which makes some patients unable to receive effective treatment However, oncolytic viruses combined with certain immune checkpoint inhibitors and immune stimulating molecules are still under study, such as immune checkpoint LAG-3 (Lymphocyte-activation gene 3), SIRPα (Signal regulatory proteinα), CTLA-4 (cytotoxic T lymphocyte-associated protein 4), TIGIT (T cell immunoglobulin and ITIM domain), immune costimulatory molecules ICOS (Inducible costimulatory), OX40 (TNFRSF4, CD134), 4-1BB (TNFRSF9, CD137), etc.

LAG-3 belongs to the immunoglobulin superfamily and consists of three parts: the extracellular domain, the transmembrane domain and the cytoplasmic domain. The gene of LAG-3 is located on chromosome 12 (12P13), which is similar to the location and structure of CD4 molecule on chromosome. Inhibition of LAG-3 can allow T cells to regain cytotoxicity, thereby enhancing the killing effect of tumors, while inhibiting LAG-3 can also reduce the function of regulatory T cells to suppress immune responses. Therefore, LAG-3 is considered a more attractive target than other immune checkpoint proteins.

SIRPα is a transmembrane protein whose extracellular domain consists of three Ig-like domains and a cytoplasmic domain (containing an immunoreceptor tyrosine inhibitory motif that mediates the binding of protein tyrosine phosphatases SHP1 and SHP2). SIRPα is especially abundant in myeloid cells such as macrophages and dendritic cells (DC), while its expression levels are extremely low in T, B, NK and NKT cells. SIRPα inhibits macrophage phagocytosis after interaction with its ligand CD47, which is normally upregulated on the surface of malignant tumor cells. Therefore, antibodies that block CD47-SIRPα interaction should enhance phagocytosis of macrophages in the tumor microenvironment and inhibit tumor growth, making blocking CD47-SIRPα a promising tool for cancer immunotherapy.

The immune co-stimulatory factor OX40 ligand (OX40L) is a member of the tumor necrosis factor ligand superfamily. Tumor necrosis factor ligand superfamily member 4 (TNFSF4) is expressed on the surface of antigen-presenting cells such as dendritic cells and B cells, and the receptor is OX40 expressed on the surface of activated T cells, which can provide T cell proliferation and stimulation signals, induce Th2 cells produce cytokines, promote the differentiation of B cells into plasma cells, and promote antibody production.

SUMMARY OF THE INVENTION

In view of the technical problems that the limited therapeutic effect of NDV as a single drug, the low response rate and tumor inhibition rate limit the clinical development and utilization of oncolytic viruses, the inventors of the present invention provide a recombinant oncolytic virus through research. The above-mentioned more typical immune checkpoints and immune costimulatory factor ligands are used to construct recombinant Newcastle disease virus, which aims to improve the anti-tumor immunity of the virus and enhance its oncolytic effect. After pharmacodynamic tests, the inventors unexpectedly found that, compared with other immune checkpoints, NDV combined with OX40L can overcome the problem of poor anti-tumor immune effect of viruses, and can significantly inhibit tumors: OX40L acts as an immune costimulatory molecule, It can activate T cells, and the biological effects produced by T cells will effectively kill tumor cells; in addition, because NDV is tumor-specific, it can selectively infect tumor cells, making the effect of OX40L more targeted, and it will be more targeted at the tumor site. Stimulating the production of more tumor-infiltrating lymphocytes, resulting in a stronger immune response, can show a more pronounced therapeutic effect. The oncolytic virus is a genetically engineered recombinant Newcastle disease virus, which can replicate in cancer cells with strong replication ability to kill host cancer cells, and has reliable safety for non-cancer cells.

In one aspect, the present invention provides a recombinant Newcastle disease virus genome, wherein the genome includes an encoding gene of OX40L, and the encoding gene of OX40L is located between the P gene and the M gene of the Newcastle disease virus genome.

In another aspect, the present application provides a recombinant Newcastle disease virus, wherein the virus comprises the above-mentioned recombinant Newcastle disease virus genome.

In another aspect, the present application provides a DNA molecule encoding the above-mentioned recombinant Newcastle disease virus genome.

In yet another aspect, the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus and/or DNA molecules.

In another aspect, the application provides a method for preparing the above-mentioned recombinant Newcastle disease virus, comprising:

(1) The cloning vector containing the DNA sequence of the coding gene of OX40L and the NDV viral vector are respectively digested with enzymes, and the DNA sequence of the coding gene of the OX40L obtained by the enzyme cutting is connected with the NDV viral vector to obtain recombination Newcastle disease virus plasmid;

(2) Transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain the recombinant Newcastle disease virus.

In yet another aspect, the present application provides the use of the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition in the preparation of a medicament for treating or ameliorating cancer.

The recombinant Newcastle disease virus prepared by the invention significantly improves the antitumor effect and oncolytic efficiency of the oncolytic virus.

Description of drawings

In order to illustrate the exemplary technical solutions of the present invention more clearly, the accompanying drawings will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as protection Scope limitation.

Figure 1 shows the genomic framework of recombinant Newcastle disease virus rNDV-OX40L.

Figure 2 shows the growth curves of the parental strain rNDV and the recombinant Newcastle disease viruses rNDV-hOX40L (a) and rNDV-mOX40L (b).

Figure 3 shows the expression of OX40L protein in cells treated with parental strain rNDV and recombinant Newcastle disease virus rNDV-mOX40L (a) and rNDV-hOX40L (b).

Figure 4 shows the tumor inhibition of the parental strain rNDV and recombinant Newcastle disease virus rNDV-mOX40L, rNDV-Lag3 and rNDV-SIRPα on mouse colon cancer model in vivo.

Figure 5 shows that rNDV-mOX40L induces tumor necrosis and tumor-infiltrating T lymphocyte proliferation.

Figure 6 shows that rNDV-mOX40L stimulates splenic T lymphocytosis.

Figure 7 shows that rNDV-mOX40L induces an increase in OX40+ T lymphocytes at the tumor site.

Figure 8 shows that rNDV-mOX40L induces increased expression of interferon-gamma, perforin and granzyme B at the tumor site.

Figure 9 shows the rClone30-Anh-(F) treatment group and the recombinant Newcastle disease virus rClone30-Anh-(F)-SIRPα treatment group, rClone30-Anh-(F)-Lag3 treatment group and rClone30-Anh-(F) - Inhibition of tumor growth in mouse liver cancer model by mOX40L treatment group.

Figure 10 shows the genome sequence of the recombinant Newcastle disease virus rNDV-hOX40L prepared in Example 1.

Figure 11 shows the genome sequence of the recombinant Newcastle disease virus rNDV-mOX40L prepared in Example 1.

Figure 12 shows the genome sequence of the recombinant Newcastle disease virus rClone30-Anh-(F)-hOX40L prepared in Example 9.

13 shows the genome sequence of the recombinant Newcastle disease virus rClone30-Anh-(F)-mOX40L prepared in Example 9.

Detailed ways

Next, exemplary embodiments of the present invention will be described, but the scope of the present invention is not limited thereto.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, eg, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989).

As used herein, unless otherwise specified, the term "treating" means curing, alleviating, alleviating, alleviating, alleviating, or ameliorating a disease or a symptom associated with a disease, or preventing, delaying, arresting, suspending, or cessation of a disease or disease in a statistically significant manner. Onset or further development of associated symptoms.

The present invention utilizes reverse genetic manipulation technology to construct a recombinant Newcastle disease virus (rNDV-OX40L) expressing OX40L. The gene encoding OX40L was inserted between the P and M genes of the Newcastle disease virus vector, and then the recombinant Newcastle disease virus plasmid (prNDV-OX40L) and the helper plasmid were co-transfected into BHK-21 cells to rescue the recombinant Newcastle disease virus. The proliferation curve of recombinant Newcastle disease virus was determined by DF-1 cells, and the ability of recombinant Newcastle disease virus to express OX40L was detected at the cellular level. At the same time, it was confirmed by mouse colon cancer and liver cancer models that the recombinant Newcastle disease virus rNDV-OX40L of the present invention is more targeted, can more significantly reduce tumor volume, induce tumor necrosis and increase tumor-infiltrating T lymphocytes, stimulate Splenic T lymphocytes increased, induced increased OX40+ T lymphocytes at tumor sites, induced increased expression of interferon-γ, perforin, and granzyme B at tumor sites, resulting in stronger immune responses and higher oncolytic efficiency .

It is known that Newcastle disease virus (NDV) belongs to the Paramyxoviridae family of single-molecule negative-stranded RNA viruses, and has an envelope; the nucleocapsid is located in the envelope and contains the RNA genome and nucleocapsid protein. The full-length genome of the classic Newcastle disease virus is about 15-16kb, and it contains NP gene, P gene, M gene, F gene, HN gene and L gene from the 3' end to the 5' end, which are respectively used to encode the following 6 species Main proteins: Nucleocapsid Protein (NP), Phosphate Protein (P), Matrix Protein (M), Fusion Protein (F), Hemagglutinin-Neuraminidase Protein (Haemagglutinin Neuraminidase Protein, HN) and RNA-dependent RNA polymerase (LargeProtein, L). In this paper, the open reading frame of the immune costimulatory molecule is inserted into the sequence between the P gene and the M gene of the NDV genome through GE and GS, and SacII and PmeI restriction endonuclease sites are introduced at both ends, and the NDV The restriction site of the vector is matched.

In one embodiment, the present invention provides a recombinant Newcastle disease virus genome, wherein the genome includes an OX40L encoding gene, and the OX40L encoding gene is located between the P gene and the M gene of the Newcastle disease virus genome.

In some preferred embodiments, the gene encoding OX40L may be in the form of DNA or RNA.

In some preferred embodiments, the gene encoding OX40L is the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or has at least 80% (eg 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical sequences. Herein, the sequence shown in SEQ ID NO.1 is as follows:

CAGGTATCACATCGGTATCCTCGAATTCAAAGTATCAAAGTACAATTTACCGAATATAAGAAGGAGAAAGGTTTCATCCTCACTTCCCAAAAGGAGGATGAAATCATGAAGGTGCAGAACAACTCAGTCATCATCAACTGTGATGGGTTTTATCTCATCTCCCTGAAGGGCTACTTCTCCCAGGAAGTCAACATTAGCCTTCATTACCAGAAGGATGAGGAGCCCCTCTTCCAACTGAAGAAGGTCAGGTCTGTCAACTCCTTGATGGTGGCCTCTCTGACTTACAAAGACAAAGTCTACTTGAATGTGACCACTGACAATACCTCCCTGGATGACTTCCATGTGAATGGCGGAGAACTGATTCTTATCCATCAAAATCCTGGTGAATTCTGTGTCCTTTGA。

The sequence shown in SEQ ID NO.2 is as follows:

CAACTCTCTTCCTCTCCGGCAAAGGACCCTCCAATCCAAAGACTCAGAGGAGCAGTTACCAGATGTGAGGATGGGCAACTATTCATCAGCTCATACAAGAATGAGTATCAAACTATGGAGGTGCAGAACAATTCGGTTGTCATCAAGTGTGATGGGCTTTATATCATCTACCTGAAGGGCTCCTTTTTCCAGGAGGTCAAGATTGACCTTCATTTCCGGGAGGATCATAATCCCATCTCTATTCCAATGCTGAACGATGGTCGAAGGATTGTCTTCACTGTGGTGGCCTCTTTGGCTTTCAAAGATAAAGTTTACCTGACTGTAAATGCTCCTGATACTCTCTGCGAACACCTCCAGATAAATGATGGGGAGCTGATTGTTGTCCAGCTAACGCCTGGATACTGTGCTCCTGAAGGATCTTACCACAGCACTGTGAACCAAGTACCACTGTGA。

In some preferred embodiments, the sequence of the recombinant Newcastle disease virus genome is shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO.8 (see Figure 10-Figure 13).

In one embodiment, the present application provides a recombinant Newcastle disease virus, wherein the virus comprises the above-mentioned recombinant Newcastle disease virus genome.

In some preferred embodiments, the starting strains of the Newcastle disease virus can be: attenuated strains LaSota, Hitchner B1, V4, virulent strains Mukteswar, Anhinga, virulent strains F48E9, JS/7/05/Ch, Italien, Herts/33, NDV-BJ; and any chimeric strain constructed by genetic engineering based on the originating strain, but not limited thereto.

In one embodiment, the present application provides a DNA molecule (eg, recombinant Newcastle disease virus plasmid) encoding the above-mentioned recombinant Newcastle disease virus genome.

In one embodiment, the present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus and/or DNA molecules.

In some preferred embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. The pharmaceutically acceptable pharmaceutical excipients can be selected from, for example, but not limited to, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure Regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants, diluents, etc. For other available pharmaceutically acceptable excipients, see, for example, "Handbook of Pharmaceutical Excipients" (4th Edition), R.C. Luo et al., translated by Zheng Zemin, 2005, Chemical Industry Press.

In one embodiment, the application provides a method for preparing the above-mentioned recombinant Newcastle disease virus, comprising:

(1) The cloning vector containing the DNA sequence of the coding gene of OX40L and the NDV viral vector are respectively digested with enzymes, and the DNA sequence of the coding gene of the OX40L obtained by the enzyme cutting is connected with the NDV viral vector to obtain recombination Newcastle disease virus plasmid;

(2) Transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain the recombinant Newcastle disease virus.

In some preferred embodiments, the cloning vector can be constructed using a vector selected from the group consisting of: PUC57 vector, pMD18-T vector, pMD19-T vector, pBlueScript SK (+/-) vector, pBluescript II KS (+/-) ).

In some preferred embodiments, the NDV viral vector may be a full-length cDNA sequence of the genome of the NDV virus selected from the group consisting of: attenuated strains LaSota, Hitchner B1, V4, strains Mukteswar, Anhinga, virulent strains F48E9, JS /7/05/Ch, Italien, Herts/33, NDV-BJ, but not limited to.

Herein, the recombinant Newcastle disease virus plasmid was combined with helper plasmids NP, P and L capable of expressing nucleocapsid protein NP, phosphorylated protein P and RNA-dependent RNA polymerase L (they can be constructed to Any NP, P, L recombinant plasmids on any known eukaryotic expression vector) were co-transfected into the cells. Herein, the genes of the helper plasmids NP, P and L can be derived from any strain of NDV, such as LaSota, Anhinga, F48E9 and the like. In some preferred embodiments, the recombinant Newcastle disease virus plasmid is co-transfected into the cell with a helper plasmid selected from the group consisting of: pTM-NP, pTM-P and pTM-L; pCI-neo-NP, pCI-neo-P and pCI-neo-L; or pBluescript II KS(+/-)-NP, pBluescript IIKS(+/-)-P and pBluescript II KS(+/-)-L, but not limited thereto.

In this context, transfection is a technique for introducing exogenous nucleic acid substances (including DNA and RNA) into cells, mainly including physical mediation (electroporation, microinjection and gene gun), chemical mediation (calcium phosphate co- Precipitation method, liposome transfection, cationic substance-mediated) and biologically-mediated (protoplast transfection, virus-mediated transfection) three types of pathways. Specific operations can be selected by those skilled in the art based on common knowledge in the field (for example, please refer to "Molecular Cloning Experiment Guide" (4th Edition), edited by J. Sambrook et al., translated by He Fuchu, Science Press, 2017) Appropriate experimental conditions and procedures, or according to the instructions in commercially available kits.

In some preferred embodiments, the cells may be selected from, but not limited to, BHK-21 cells, BSR-T7/5 cells, VERO cells, DF-1 cells, 293 cells, or MDCK cells.

In this paper, the culture of the transfected cells can be carried out by those skilled in the art by selecting conventional culture medium and culture conditions according to the type of cells (Liu Bin, editor-in-chief, "Cell Culture (3rd Edition)", World Book Publishing Company, January 2018; Editor-in-chief of Lan Rong and Zhou Zhenhui, "Cell Culture Technology", Chemical Industry Press, August 2007; Editor-in-chief of Zhang Jingbo, "Tissue and Cell Culture Technology (3rd Edition)", People's Medical Publishing House, 2014 June, etc.).

In one embodiment, the present application provides the use of the above-mentioned recombinant Newcastle disease virus genome, recombinant Newcastle disease virus, DNA molecule and/or pharmaceutical composition in the preparation of a medicament for treating or ameliorating cancer.

In some preferred embodiments, the cancer may be selected from, but not limited to, colon cancer, liver cancer (eg, hepatocellular carcinoma), lung cancer (eg, non-small cell lung cancer, small cell lung cancer), gastric cancer, rectal cancer, leukemia, lymphatic Tumor, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urothelial cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophageal cancer, renal cell cancer, thyroid cancer, head and neck cancer cancer, testicular cancer, endocrine adenocarcinoma, adrenal cancer, pituitary gland cancer, skin cancer, soft tissue cancer, vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, Sarcoma, Uterine Cancer, Glioblastoma, Medulloblastoma, Neuroblastoma, Kidney Cancer, Astrocytoma, Glioma, Meningioma, Gastrinoma, Neuroblastoma, Melanoma , acute myeloid leukemia, myelodysplastic syndrome, or sarcoma.

The exemplary technical solution of the present invention can be described by the content of the following numbered paragraphs:

What is claimed is: 1. A recombinant Newcastle disease virus genome, wherein the genome comprises an encoding gene of OX40L, and the encoding gene of OX40L is located between the P gene and the M gene of the Newcastle disease virus genome.

2. The recombinant Newcastle disease virus genome of paragraph 1, wherein the gene encoding OX40L is in the form of DNA or RNA.

3. The Newcastle disease virus genome of the recombination as described in paragraph 1 or 2, wherein, the encoding gene of described OX40L is the sequence shown with SEQ ID NO.1 or SEQ ID NO.2 or has at least 80 with the sequence % sequence of identity.

4. The Newcastle disease virus genome of the reorganization as described in any one of paragraphs 1-3, wherein the sequence of the recombination Newcastle disease virus genome is with SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO. 8.

5. A recombinant Newcastle disease virus, wherein the virus comprises the recombinant Newcastle disease virus genome of any of paragraphs 1-4.

6. recombinant Newcastle disease virus as described in paragraph 5, wherein, the starting strain of described Newcastle disease virus is selected from: attenuated strain LaSota, Hitchner B1, V4, poisonous strain Mukteswar, Anhinga, virulent strain F48E9, JS/7 /05/Ch, Italien, Herts/33, NDV-BJ; and any chimeric strain constructed by genetic engineering based on the originating strain.

7. A DNA molecule encoding the recombinant Newcastle disease virus genome of any of paragraphs 1-4.

8. a pharmaceutical composition, wherein, described pharmaceutical composition comprises the Newcastle disease virus genome of the reorganization described in any one paragraph in paragraph 1-4, the recombination Newcastle disease virus described in paragraph 5 or 6 and/or the recombination Newcastle disease virus described in paragraph 7. the DNA molecule described.

9. The recombinant Newcastle disease virus of paragraph 8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

10. The recombinant Newcastle disease virus as described in paragraph 8 or 9, wherein the pharmaceutically acceptable pharmaceutical adjuvant is selected from the group consisting of solvent, propellant, solubilizer, cosolvent, emulsifier, colorant, disintegrant, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH regulators, surfactants or thinner.

11. A method of preparing the recombinant Newcastle disease virus of paragraph 5 or 6, comprising:

(1) The cloning vector containing the DNA sequence of the coding gene of OX40L and the NDV viral vector are respectively digested with enzymes, and the DNA sequence of the coding gene of the OX40L obtained by the enzyme cutting is connected with the NDV viral vector to obtain recombination Newcastle disease virus plasmid;

(2) Transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain the recombinant Newcastle disease virus.

12. The method of paragraph 11, wherein the cloning vector is constructed using a vector selected from the group consisting of: PUC57 vector, pMD18-T vector, pMD19-T vector, pBlueScript SK (+/-) vector, pBluescript II KS ( +/-).

13. The method of paragraph 11 or 12, wherein the NDV viral vector is a full-length cDNA sequence selected from the genome of the following NDV viruses: attenuated strains LaSota, Hitchner B1, V4, strains Mukteswar, Anhinga, strong strains Strain F48E9, JS/7/05/Ch, Italien, Herts/33, NDV-BJ.

14. The method of any of paragraphs 11-13, wherein the recombinant Newcastle disease virus plasmid is co-transfected into the cell with a helper plasmid selected from the group consisting of: pTM-NP, pTM-P and pTM -L; pCI-neo-NP, pCI-neo-P and pCI-neo-L; or pBluescript II KS(+/-)-NP, pBluescript II KS(+/-)-P and pBluescriptII KS(+/- )-L.

15. The method of any of paragraphs 11-14, wherein the cells are selected from BHK-21 cells, BSR-T7/5 cells, VERO cells, DF-1 cells, 293 cells, or MDCK cells.

16. The recombinant Newcastle disease virus genome of any one of paragraphs 1-4, the recombinant Newcastle disease virus of paragraphs 5 or 6, the DNA molecule of paragraph 7 and/or the one of paragraphs 8-10 Use of the pharmaceutical composition in the preparation of a medicament for treating or improving cancer.

17. The use of paragraph 16, wherein the cancer is selected from colon cancer, liver cancer, lung cancer, stomach cancer, rectal cancer, leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urinary Epithelial cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophagus cancer, renal cell cancer, thyroid cancer, head and neck cancer, testicular cancer, endocrine adenocarcinoma, adrenal cancer, pituitary gland cancer, Skin cancer, soft tissue cancer, vascular cancer, brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, sarcoma, uterine cancer, glioblastoma, medulloblastoma , neuroblastoma, kidney cancer, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, acute myeloid leukemia, myelodysplastic syndrome, or sarcoma.

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. Unless otherwise stated, the reagents, materials or instruments used without the manufacturer's indication are conventional products that can be purchased from the market. The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example

Unless otherwise specified, the design, synthesis and cloning of genes involved in this application, as well as the construction and transfection of vectors and operations such as electrophoresis, can be performed according to techniques known in the art (for example, see the description of CURRENT PROTOCOLS INMOLECULAR BIOLOGY). Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

The oncolytic virus rClone30-F48E9 (F) as the parental strain provided in the following example starts from Newcastle disease virus LaSota (purchased from Harbin Veterinary Epidemic Prevention Station), according to Wang Yong et al. The effect of force, Acta Microbiol, 2008, 48(5): 638～643), the engineering method of "gene replacement" was described, using the F gene of the virulent strain of Newcastle disease virus F48E9 (GenBank accession number: AY508514.1 ) was obtained by replacing the F gene of the LaSota strain, and the parent strain was named rNDV hereinafter. The genome of the recombinant Newcastle disease virus expressing OX40L in this example is shown in SEQ ID NO.3 and SEQ ID NO.4; the nucleic acid sequence of OX40L is shown in SEQ ID NO.1 and SEQ ID NO.2. Next, the recombinant Newcastle disease virus plasmid and recombinant Newcastle disease virus NDV-OX40L were constructed by the following exemplary methods.

The human OX40L gene (NM_003326.5) and the mouse OX40L gene (U12763) were searched through Genebank, and exogenous gene fragments were designed according to the 6-base principle. After adding SacII and PmeI restriction sites, they were synthesized by Shanghai Sangon Bioengineering Company The respective DNA sequences of the above exogenous genes (the vector is pUC57 vector) were obtained to obtain plasmids pUC57-hOX40L (containing human OX40L gene) and pUC57-mOX40L (containing mouse OX40L gene). The plasmids pUC57-hOX40L, pUC57-mOX40L and NDV vector (the full-length cDNA sequence of the genome of the above rNDV strain) were digested with restriction endonucleases SacII and PmeI (purchased from NEB Company), respectively. Agarose gel electrophoresis was carried out for identification by enzyme digestion. After the identification of enzyme digestion was correct, agarose gel DNA recovery kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: DP219) was used to recover the gel according to the instructions, and the gel was recovered. The recovered product was subjected to gene sequence determination. After the gel recovery product of each exogenous gene OX40L and NDV vector with correct sequencing was purified with TaKaRa MiniBEST DNAFragmentPurification Kit (purchased from Takara, item number: 9761), the purified product of human OX40L gene and mouse OX40L gene were purified. The products were respectively ligated with purified NDV vector using T4 DNA ligase (purchased from NEB Company) according to the instructions at 4°C overnight.

Each ligation product was transformed into stbl2 Escherichia coli (E.coli) competent cells, and the transformed competent cells were spread on LB plates containing 100 μg/mL ampicillin, and cultured in a bacterial incubator at 20°C for 24 hours. hours, pick a single colony, inoculate it in the ampicillin-resistant LB medium containing 100 μg/mL ampicillin, and cultivate at 30 °C for 16-18 h for amplification. Plasmid extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: DP103) was used for plasmid extraction, and the obtained plasmid was digested with SacII and PmeI enzymes (purchased from NEB Company) and passed through nucleic acid agarose Gel electrophoresis was used for restriction enzyme digestion identification, and the plasmid identified by restriction enzyme digestion was used for gene sequence determination to determine whether the position and sequence of the exogenous gene OX40L were correct.

The results of enzyme digestion identification showed that two bands were obtained, one was about 18000bp in size, which was consistent with the size of the NDV vector; The results of gene sequence determination showed that the exogenous gene OX40L was located between the P gene and the M gene of the NDV vector, and there was no mutation, and the sequence alignment consistency was 100%. The successfully constructed recombinant Newcastle disease virus plasmids were named prNDV-hOX40L (containing human OX40L gene) and prNDV-mOX40L (containing murine OX40L gene).

Next, rescue and identification of recombinant Newcastle disease virus was performed.

After purifying the correctly identified recombinant Newcastle disease virus plasmids prNDV-hOX40L and prNDV-mOX40L, 2μg of prNDV-hOX40L and 2μg of prNDV-mOX40L were mixed with the helper plasmids pTM-NP (1μg) and pTM-P (0.5μg), respectively. BHK-21 cells (purchased from ATCC) in logarithmic growth phase were co-transfected with pTM-L (0.25 μg) by liposome 3000. After 72 h, the cells were freeze-thawed at -80°C for 3 times to lyse the cells, and then Cell supernatants were obtained by centrifugation at 800 rpm. Take 100 μL of cell supernatant and inoculate 9-day-old SPF chicken embryos. After inoculation, the chicken embryos were cultured in an incubator at 37°C and 5% CO ₂ for 72 hours, and the allantoic fluid was collected and tested for hemagglutination titer (for example, see the following). The method described in: Zhou Ling, Li Yanfang, Ma Xiali, Isolation and identification of a chicken-derived Newcastle disease virus [J]. Zhejiang Animal Husbandry and Veterinary Medicine, 2015, 40(03):8-10) to determine whether the rescue was successful. The rescued recombinant Newcastle disease viruses were named rNDV-hOX40L and rNDV-mOX40L, respectively.

The viral RNA in the above-mentioned allantoic fluid was extracted by a viral RNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., item number: DP315-R), and synthesized according to the instructions by RT-PCR (Thermo Fisher RT-PCR kit). cDNA first strand and PCR; PCR primers are: upstream primer 5'TCAAGCGCCTTGCTCTAAATGGC 3' (SEQ ID NO.5); downstream primer 5'GGGCAGAATCAAAGTACAGCCCAAT 3' (SEQ ID NO.6)), nucleic acid agarose gel electrophoresis and gene The purity and gene sequence of rNDV-hOX40L and rNDV-mOX40L were identified by sequencing.

The results of nucleic acid agarose gel electrophoresis showed that the purity of recombinant Newcastle disease virus rNDV-hOX40L and rNDV-mOX40L were high, and there was no contamination by other Newcastle disease viruses; PCR product sequencing results showed that the rescued recombinant Newcastle disease virus rNDV-hOX40L There is no mutation in the exogenous gene OX40L in rNDV-mOX40L and rNDV-mOX40L. After sequence alignment, the sequence identity is 100%.

The effects of the insertion of foreign genes on the replication ability of the virus and the growth ability of the recombinant viruses rNDV-hOX40L and rNDV-mOX40L obtained above were identified by the following methods.

rNDV-hOX40L and rNDV-mOX40L were respectively infected with DF-1 cells (purchased from Wuhan Proceeds) at 1MOI (multiplicity of infection) for 12h, 24h, 36h, 48h, 60h and 72h, and rNDV was used as a control, The TCID50 of the virus contained in the cell supernatant at different time points was detected, and the growth curves of the recombinant viruses rNDV-hOX40L (Fig. 2(a)) and rNDV-mOX40L (Fig. 2(b)) relative to rNDV were drawn respectively. The results showed that the growth characteristics of the recombinant viruses rNDV-hOX40L and rNDV-mOX40L were consistent with rNDV, indicating that the insertion of foreign genes did not affect the replication ability of the virus, and the recombinant viruses rNDV-hOX40L and rNDV-mOX40L both showed good performance at the cellular level. growth ability (results are shown in Figure 2).

Example 2

The titer, virulence and pathogenicity of recombinant Newcastle disease virus rNDV-hOX40L and rNDV-mOX40L were determined by the following methods.

In order to compare the titer, virulence, and pathogenicity of rNDV and recombinant Newcastle disease virus rNDV-hOX40L and rNDV-mOX40L, the recombinant Newcastle disease virus rNDV-hOX40L and rNDV-mOX40L and rNDV were subjected to large scale analysis according to the method disclosed in the OIE standard. Bulk propagation, then the allantoic fluid of parental strain rNDV and recombinant Newcastle disease virus rNDV-hOX40L and rNDV-mOX40L were collected, and the titer was determined by the hemagglutination titer (HA) detection described in Example 1, according to OIE standards The method disclosed in TCID50, EID50, MDT was used for virulence determination, and the virulence determination was performed by ICPI according to the method disclosed in the OIE standard.

The results of hemagglutination titer test showed that in the hemagglutination titer test of recombinant Newcastle disease virus rNDV-mOX40L, the hemagglutination titer (HA) of rNDV was 2 ⁹ , and the hemagglutination titer (HA) of rNDV-mOX40L was 2 9 . In the hemagglutination titer detection of recombinant Newcastle disease virus rNDV-hOX40L, the hemagglutination titer (HA) of rNDV was 2 ¹⁰ , and the hemagglutination titer (HA) of rNDV-hOX40L was ^{2 10 .} The virulence assay results showed that there was no significant difference in virulence between rNDV and recombinant Newcastle disease virus rNDV-hOX40L and rNDV-mOX40L. The virulence assay results showed that there was no significant difference in virulence between rNDV and recombinant Newcastle disease viruses rNDV-hOX40L and rNDV-mOX40L (see Table 1 and Table 2). These results indicated that the insertion of hOX40L and mOX40L genes did not affect the titer, virulence and pathogenicity of recombinant Newcastle disease virus.

Table 1 Titer, virulence and pathogenicity of recombinant Newcastle disease virus rNDV-hOX40L

Table 2 Titer, virulence and pathogenicity of recombinant Newcastle disease virus rNDV-mOX40L

Example 3

The expressions of exogenous genes hOX40L and mOX40L contained in the recombinant Newcastle disease viruses rNDV-hOX40L and rNDV-mOX40L prepared in Example 1 in tumor cells were detected by Western blot.

Each successfully rescued rNDV-OX40L and parental NDV (rNDV) were infected with CT26 cells at 10 MOI, cultured at 37° C., 5% CO ₂ for 24 h, and then centrifuged at 800 rpm to harvest the cell supernatant, and detected by Western blot (for example, according to the method disclosed below). Carry out: An Y, et al. Recombinant Newcastle disease virus expressing P53demonstrates promising antitumor efficiency in hepatoma model [J]. Journal of Biomedical Science, 2016 23(1): 55) The expression levels of mOX40L and hOX40L proteins in the cell supernatant. As shown in Fig. 3, the rNDV-mOX40L and rNDV-hOX40L treatment groups expressed more mOX40L protein (Fig. 3(a)) and hOX40L protein (Fig. 3(b)), respectively, while the rNDV-treated group (NDV group) No OX40L protein was detected.

Example 4

In this example, the effects of recombinant Newcastle disease virus rNDV-mOX40L, rNDV-Lag3, and rNDV-SIRPα on tumor inhibition in mice were tested.

According to the method described in Example 1, the Lag3 gene (NCBI accession number: NM_002286.6) and the SIRPα gene (Genbank accession number: CAA71403) were used to construct recombinant Newcastle disease viruses rNDV-Lag3 and rNDV-SIRPα, respectively.

A mouse colon cancer model was established by subcutaneously injecting mouse colon cancer CT26 cells (purchased from ATCC). When the tumor grew to about 100 mm, 100 μL of the above oncolytic viruses (rNDV ^- mOX40L, rNDV-Lag3 and rNDV-SIRPα) and the parental NDV strain (rNDV) in PBS suspension (prepared with 1×PBS buffer). In each experimental group (rNDV-mOX40L treatment group, rNDV-Lag3 treatment group and rNDV-SIRPα treatment group and rNDV treatment group), each oncolytic virus (rNDV-mOX40L, rNDV-Lag3, rNDV-SIRPα and rNDV) were It was injected into the tumor once a day for a total of 14 days, with 1×10 ⁷ PFU per injection; 1×PBS buffer (without oncolytic virus) was injected into the tumor of the mouse colon cancer model as a negative control group (also called "PBS-treated group"); 6 animals per group. The antitumor effects of the above-mentioned oncolytic viruses on the mouse colon cancer model were evaluated by plotting the change curve of mouse tumor volume. Meanwhile, 6 non-tumor-bearing mice were set as normal controls to compare the physiological state of mice in each treatment group. At the end of the experiment, the tumor size of each group of mice was tested and compared with the negative control group of mice, and the relative inhibition rate was calculated.

Relative inhibition rate (%)=(tumor volume of negative control group-tumor volume of experimental group)/tumor volume of negative control group×100%.

The results showed that the average tumor volume of the negative control group was 1408.15 mm ³ , the average tumor volume of the parental NDV (rNDV) treatment group was 355.33 mm ³ , and the average tumor volume of the rNDV-mOX40L treatment group was 163.36 mm ³ ; rNDV-SIPRα treatment The mean tumor volume of the group was 633.72 mm ³ and the mean tumor volume of the rNDV-Lag3 treated group was 644.43 mm ³ . The tumor volume in the rNDV-mOX40L-treated group was significantly suppressed relative to the PBS-treated and rNDV-treated groups (as shown in Figure 4).

The relative tumor inhibition rates of rNDV treatment group and rNDV-mOX40L treatment group were 74.77% and 88.40%, respectively. The tumor inhibition rate of rNDV-mOX40L treatment group was 13.63% higher than that of rNDV treatment group. At the same time, compared with the negative control group, the mean tumor volume of the rNDV-SIPRα treatment group, rNDV-Lag3 treatment group and rNDV-mOX40L treatment group was smaller, especially, the mean tumor volume of the rNDV-mOX40L treatment group was the smallest.

Example 5

In this example, it was found that rNDV-mOX40L induced tumor necrosis and increased tumor-infiltrating T lymphocytes by HE section and immunohistochemical detection.

In order to detect the anti-tumor immune effect of rNDV-mOX40L, tumor tissue was excised after the 14th day of treatment described in Example 4, and the tumor necrosis and the expression of tumor-infiltrating T lymphocytes were detected by HE section and immunohistochemistry.

The results of HE sections showed that the tumor tissue of the negative control group (PBS-treated group) xenografts were tightly arranged, with larger nuclei and prominent nucleoli. Tumor tissue in parental rNDV-treated xenografts exhibited nuclear pyknosis, and tumor cells in rNDV-treated xenografts decreased in size relative to PBS-treated xenografts. The xenografts in the rNDV-mOX40L-treated group were loosely arranged and had large necrotic areas. In addition, tumor cells in the rNDV-mOX40L-treated xenografts exhibited nuclear deformation, nuclear pyknosis, disappearance of nucleoli, unclear nuclear structure, and nuclear disorganization (Figure 5). Immunohistochemical staining results showed that in a mouse model of colon cancer, the xenografts in the rNDV-mOX40L-treated group presented more tumors compared to the parental rNDV-treated xenografts and the PBS-treated xenografts Infiltrating CD4+ and CD8+ T lymphocytes are expressed, which can generate a stronger immune response and show a more significant therapeutic effect.

Example 6

In this example, rNDV-mOX40L was found to stimulate splenic T lymphocytosis by FACS analysis.

To further investigate the immune response induced by rNDV-mOX40L, the spleens of mice were excised after the end of treatment on day 14 as described in Example 4, and the percentages of CD3+, CD4+ and CD8+ T cells in the spleens of mice were analyzed by FACS (results shown in in Figure 6).

As shown in Figure 6, the percentages of CD3+ T cells in the spleen of the normal control, PBS-treated, rNDV-treated and rNDV-mOX40L-treated mice were 34.93%, 17.43%, 22.27%, and 33.9%, respectively. The percentages of CD4+ T cells in the spleen of the normal control, PBS-treated, rNDV-treated and rNDV-mOX40L-treated mice were 25.9%, 11.16%, 13.16% and 24.16%, respectively. The percentages of CD8+ T cells in the spleen of the normal control, PBS-treated, rNDV-treated and rNDV-mOX40L-treated mice were 17.47%, 8.3%, 8.73%, and 16.7%, respectively. It can be seen that there is no significant difference in the percentages of CD3+ T cells, CD4+ T cells and CD8+ T cells between the normal control group and the rNDV-mOX40L treatment group. However, the percentages of CD3+ T cells, CD4+ T cells, and CD8+ T cells were significantly increased after rNDV-mOX40L treatment compared with rNDV-treated and PBS-treated groups, suggesting that rNDV-mOX40L promotes anti-tumor by increasing splenic T cells reaction.

Example 7

In this example, it was found by immunohistochemical staining that rNDV-mOX40L induced an increase in OX40+ T lymphocytes at the tumor site.

To examine the activation effect of mOX40L on T cells, the T cell activation marker OX40 in tumor tissues was examined by immunohistochemical staining. Tumor tissue was excised after the 14th day of treatment described in Example 4, and immunohistochemical staining was performed with anti-CD4 and anti-CD8 antibodies (the corresponding secondary antibodies were goat anti-rabbit secondary antibodies, purchased from Abcam) as follows , and then use anti-OX40 antibody (the corresponding secondary antibody is goat anti-rabbit secondary antibody, purchased from Abcam) in the region rich in CD4+ and CD8+ T cells to carry out immunohistochemical staining on tumor tissue. The specific operations are as follows:

1. Dewaxing and hydration: Immerse the prepared paraffin sections in xylene for dewaxing, immerse twice for 5 minutes each time, and then put in 100vol%, 95vol%, 90vol%, 80vol% and 70vol% of alcohol at all levels In the solution for 5min each, then rinsed twice in distilled water, 3min each time.

2. Antigen retrieval: After heating the 0.01M sodium citrate buffer (PH=6.0) to 95°C in a water bath, place the slices into the slices and heat for 10 minutes, and then rinse with 1×PBS buffer for 3 times, 5 minutes each time.

3. Inactivate endogenous peroxidase: Add endogenous peroxidase blocking solution (purchased from Beyotime; Item No. P0100A) dropwise to completely cover the sample, incubate at room temperature for 10 min, and then wash with 1×PBS buffer 3 times, 3 minutes each time.

4. Blocking: A blocking solution (purchased from Beyotime; Product No. P0260 QuickBlock) was added dropwise to block the tissue section for 10 min.

5. Incubate the primary antibody: use the blocking solution to prepare the primary antibody working solution according to the dilution ratio of the antibody instruction, add the primary antibody working solution dropwise to the tissue section, and incubate at 4°C overnight; after the primary antibody incubation, rinse the tissue with 1×PBST buffer Slice 3 times, 5 min each time.

6. Incubate the secondary antibody: prepare the working solution of the secondary antibody with the blocking solution according to the dilution ratio of the antibody specification, drop the working solution of the secondary antibody to the tissue section, and incubate at room temperature for 1 h; after the secondary antibody incubation, rinse with 1×PBST buffer The tissue was sectioned 3 times, 5 min each time.

7. Color development: Add 100 μl of DAB color development working solution (purchased from Beyotime) dropwise, fully cover the sample, and incubate in the dark at room temperature for 15 minutes. After color development, remove the DAB color development working solution and wash with distilled water 1-2 times to stop the development. color reaction.

As can be seen from the results in Figure 7, in the region enriched in CD4+ and CD8+ T cells, the xenografts in the rNDV-mOX40L treated group compared to the xenografts in the rNDV treated group and the xenografts in the PBS treated group Immunohistochemical staining of the samples showed more OX40+ T cell infiltration, indicating that rNDV-mOX40L promotes antitumor responses by increasing the infiltration of OX40+ T cells in tumor tissue in a mouse colon cancer model.

Example 8

In this example, it was found by Western blot detection that rNDV-mOX40L induced increased expression of interferon-γ, perforin and granzyme B in the tumor site.

In humans and mice, the OX40/OX40L signaling pathway can activate and induce T cell activity. To examine the biological effects of T cells in tumor tissue induced by rNDV-mOX40L, tumor tissue was excised after the 14th day of treatment as described in Example 4, according to methods known in the art (see, e.g., Shuai Li et al. .Fibroblast growthfactor 21ameliorates high glucose-induced fibrogenesis in mesangial cells through inhibiting STAT5 signaling pathway[J]. Biomedicine & Pharmacotherapy, 2017, 93: 695-704) Detection of IFN-γ, granzyme B and perforin by Western blot and immunohistochemical staining Express.

As a result, a band of about 20 kDa corresponding to the molecular weight of mouse IFN-γ protein was detected from the tumor tissues of the rNDV-mOX40L-treated group, the rNDV-treated group and the PBS-treated group of the mouse colon cancer model, and the IFN-γ of the rNDV-mOX40L-treated group was detected. The level of -γ protein was significantly higher than that in the rNDV-treated group and the PBS-treated group. As shown in Figure 8, immunohistochemical staining of xenografts in the rNDV-mOX40L-treated group showed more IFN-γ, granzyme B, and perforin compared to the xenografts in the rNDV-treated and PBS-treated groups expression. These results indicate that rNDV-mOX40L can induce the activity of T cells in tumor tissue, resulting in biological effects of cytotoxic T lymphocytes.

Example 9

The oncolytic virus rClone30-Anh-(F) as the parental strain provided in the following example is based on the LaSota strain of Newcastle disease virus (purchased from Harbin Veterinary Epidemic Prevention Station), according to Wang Yong et al. (Acta Microbiology, 2008, 48 (5). ): 638-643, the same as above) The engineering method for "gene replacement" is obtained by replacing the F gene of the LaSota strain with the F gene of the Newcastle disease virus strain Anhinga (GenBank accession number: EF065682.1). The genome of the recombinant Newcastle disease virus expressing OX40L of this example is shown in SEQ ID NO. 7 and SEQ ID NO. 8 (see Figures 12 and 13); the nucleic acid sequence of OX40L is shown in SEQ ID NO. 1 and SEQ ID NO. 2 is shown.

First, human OX40L gene (NM_003326.5), mouse OX40L gene (U12763), Lag3 gene (NCBI accession number: NM_002286.6) and SIRPα gene (Genbank accession number: CAA71403) were used according to the method described in Example 1. ) were respectively constructed to obtain the corresponding recombinant Newcastle disease virus plasmid and recombinant Newcastle disease virus, and the recombinant virus was successfully rescued. The successfully rescued and correctly identified recombinant Newcastle disease viruses were named rClone30-Anh-(F)-hOX40L, rClone30-Anh-(F)-mOX40L, rClone30-Anh-(F)-SIRPα and rClone30-Anh-(F )-Lag3.

Then, according to the method described in Example 4, H22 cells (purchased from Nanjing Kebai) were used to establish an H22 subcutaneous tumor-bearing model (ie, a mouse liver cancer model). When the tumor grows to about 100 mm, start to inject 100 μL ^of each of the above oncolytic viruses (rClone30-Anh-(F)-mOX40L, rClone30-Anh-(F)-Lag3 and rClone30-Anh-(F)-SIRPα) into the tumor. ) and the parental strain rClone30-Anh-(F) in PBS (prepared with 1×PBS buffer). In each treatment group, each oncolytic virus (rClone30-Anh-(F)-mOX40L, rClone30-Anh-(F)-Lag3, rClone30-Anh-(F)-SIRPα and rClone30-Anh-(F)) They were injected into the tumor once a day for a total of 14 days, with 1×10 ⁷ PFU per injection; 1×PBS buffer (without oncolytic virus) was injected into the tumor of the mouse liver cancer model as a negative control group (also known as "PBS treatment group"); there were 6 animals in each group, and the therapeutic effect of each recombinant virus was observed by dissecting tumor tissue.

As shown in Figure 9, after the treatment, the average tumor volume of the negative control group was 1421.77 mm ³ , the average tumor volume of the parental rClone30-Anh-(F) treatment group was 807.30 mm ³ , and the rClone30-Anh-(F)-mOX40L The mean tumor volume in the treatment group was 306.42 mm ³ ; the mean tumor volume in the rClone30-Anh-(F)-SIRPα treated group was 608.05 mm ³ and the mean tumor volume in the rClone30-Anh-(F)-Lag3 treated group was 824.49 mm ³ . The results showed that, compared with the negative control group, the parental strains rClone30-Anh-(F) treatment group, rClone30-Anh-(F)-SIRPα treatment group, rClone30-Anh-(F)-Lag3 treatment group and rClone30-Anh- (F)-mOX40L treatment group could inhibit tumor growth, especially the rClone30-Anh-(F)-mOX40L treatment group had the smallest mean tumor volume.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

sequence listing

<110> Jiangsu Kangyuan Ruiao Biomedical Technology Co., Ltd.

<120> A kind of recombinant Newcastle disease virus rNDV-OX40L, its genome, preparation method and use thereof

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<170> PatentIn version 3.5

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<223> Nucleic acid sequence of human OX40L

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caggtatcac atcggtatcc tcgaattcaa agtatcaaag tacaatttac cgaatataag 60

aaggagaaag gtttcatcct cacttcccaa aaggaggatg aaatcatgaa ggtgcagaac 120

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caactctctt cctctccggc aaaggaccct ccaatccaaa gactcagagg agcagttacc 60

agatgtgagg atgggcaact attcatcagc tcatacaaga atgagtatca aactatggag 120

gtgcagaaca attcggttgt catcaagtgt gatgggcttt atatcatcta cctgaagggc 180

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aaagataaag tttacctgac tgtaaatgct cctgatactc tctgcgaaca cctccagata 360

aatgatgggg agctgattgt tgtccagcta acgcctggat actgtgctcc tgaaggatct 420

taccacagca ctgtgaacca agtaccactg tga 453

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ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gtagcctgaa 60

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 120

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 180

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 240

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 300

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 360

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 420

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 480

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 540

tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt caaatatgta 600

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 660

gagtattcaa catttccgtg tcgcccttat tcccttttttt gcggcatttt gccttcctgt 720

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 780

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 840

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 900

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 960

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 1020

cagtgctgcc ataagcatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1080

aggaccgaag gagctaaccg ctttttttca caacatgggg gatcatgtaa ctcgccttga 1140

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1200

tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1260

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1320

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1380

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1440

gacgggcagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1500

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1560

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1620

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1680

aggatcttct tgagatcctt ttttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1740

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1800

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1860

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1920

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1980

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 2040

gcgaacgacc tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct 2100

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2160

cacgagggag cttccagggg ggaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2220

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggccgagcc tatggaaaaa 2280

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2340

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2400

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2460

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 2520

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttacct 2580

cactcattag gcaccccagg ctttacactt tatgcttccg gctcctatgt tgtgtggaat 2640

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcgg 2700

aagcggccgc taatacgact cactataggg accaaacaga gaatccgtaa gttacgataa 2760

aaggcgaagg agcaattgaa gtcgcacggg tagaaggtgt gaatctcgag tgcgagcccg 2820

aagcacaaac tcgagaaagc cttctgccaa catgtcttcc gtatttgatg agtacgaaca 2880

gctcctcgcg gctcagactc gccccaatgg agctcatgga gggggagaaa aagggagtac 2940

cttaaaagta gacgtcccgg tattcactct taacagtgat gacccagaag atagatggag 3000

ctttgtggta ttctgcctcc ggattgctgt tagcgaagat gccaacaaac cactcaggca 3060

aggtgctctc atatctcttt tatgctccca ctcacaggta atgaggaacc atgttgccct 3120

tgcagggaaa cagaatgaag ccacattggc cgtgcttgag attgatggct ttgccaacgg 3180

cacgccccag ttcaacaata ggagtggagt gtctgaagag agagcacaga gatttgcgat 3240

gatagcagga tctctccctc gggcatgcag caacggaacc ccgttcgtca cagccggggc 3300

cgaagatgat gcaccagaag acatcaccga taccctggag aggatcctct ctatccaggc 3360

tcaagtatgg gtcacagtag caaaagccat gactgcgtat gagactgcag atgagtcgga 3420

aacaaggcga atcaataagt atatgcagca aggcagggtc caaaagaaat acatcctcta 3480

ccccgtatgc aggagcacaa tccaactcac gatcagacag tctcttgcag tccgcatctt 3540

tttggttagc gagctcaaga gaggccgcaa cacggcaggt ggtacctcta cttattataa 3600

cctggtaggg gacgtagact catacatcag gaataccggg cttactgcat tcttcttgac 3660

actcaagtac ggaatcaaca ccaagacatc agcccttgca cttagtagcc tctcaggcga 3720

catccagaag atgaagcagc tcatgcgttt gtatcggatg aaaggagata atgcgccgta 3780

catgacatta cttggtgata gtgaccagat gagctttgcg cctgccgagt atgcacaact 3840

ttactccttt gccatgggta tggcatcagt cctagataaa ggtactggga aataccaatt 3900

tgccagggac tttatgagca catcattctg gagacttgga gtagagtacg ctcaggctca 3960

gggaagtagc attaacgagg atatggctgc cgagctaaag ctaaccccag cagcaaggag 4020

gggcctggca gctgctgccc aacgggtctc cgaggagacc agcagcatag acatgcctac 4080

tcaacaagtc ggagtcctca ctgggcttag cgaggggggg tcccaagctc tacaaggcgg 4140

atcgaataga tcgcaagggc aaccagaagc cggggatggg gagacccaat tcctggatct 4200

gatgagagcg gtagcaaata gcatgaggga ggcgccaaac tctgcacagg gcactcccca 4260

atcggggcct cccccaactc ctgggccatc ccaagataac gacaccgact gggggtattg 4320

atggacaaaa cccagcctgc ttccacaaaa acatcccaat gccctcaccc gtagtcgacc 4380

cctcgatttg cggctctata tgaccacacc ctcaaacaaa catccccctc tttcctccct 4440

ccccctgctg tacaactccg cacgccctag ataccacagg cacaatgcgg ctcactaaca 4500

atcaaaacag agccgaggga attagaaaaa agtacgggta gaagagggat attcagagat 4560

cagggcaagt ctcccgagtc tctgctctct cctctacctg atagaccagg acaaacatgg 4620

ccacctttac agatgcagag atcgacgagc tatttgagac aagtggaact gtcattgaca 4680

acataattac agcccagggt aaaccagcag agactgttgg aaggagtgca atcccacaag 4740

gcaagaccaa ggtgctgagc gcagcatggg agaagcatgg gagcatccag ccaccggcca 4800

gtcaagacaa ccccgatcga caggacagat ctgacaaaca accatccaca cccgagcaaa 4860

cgaccccgca tgacagcccg ccggccacat ccgccgacca gccccccacc caggccacag 4920

acgaagccgt cgacacacag ctcaggaccg gagcaagcaa ctctctgctg ttgatgcttg 4980

acaagctcag caataaatcg tccaatgcta aaaagggccc atggtcgagc ccccaagagg 5040

ggaatcacca acgtccgact caacagcagg ggagtcaacc cagtcgcgga aacagtcagg 5100

aaagaccgca gaaccaagtc aaggccgccc ctggaaacca gggcacagac gtgaacacag 5160

catatcatgg acaatgggag gagtcacaac tatcagctgg tgcaacccct catgctctcc 5220

gatcaaggca gagccaagac aatacccttg tatctgcgga tcatgtccag ccacctgtag 5280

actttgtgca agcgatgatg tctatgatgg aggcgatatc acagagagta agtaaggttg 5340

actatcagct agatcttgtc ttgaaacaga catcctccat ccctatgatg cggtccgaaa 5400

tccaacagct gaaaacatct gttgcagtca tggaagccaa cttgggaatg atgaagattc 5460

tggatcccgg ttgtgccaac atttcatctc tgagtgatct acgggcagtt gcccgatctc 5520

acccggtttt agtttcaggc cctggagacc cctctcccta tgtgacacaa ggaggcgaaa 5580

tggcacttaa taaactttcg caaccagtgc cacatccatc tgaattgatt aaacccgcca 5640

ctgcatgcgg gcctgatata ggagtggaaa aggacactgt ccgtgcattg atcatgtcac 5700

gcccaatgca cccgagttct tcagccaagc tcctaagcaa gttagatgca gccgggtcga 5760

tcgaggaaat caggaaaatc aagcgccttg ctctaaatgg ctaattacta ctgccacacg 5820

tagcgggtcc ctgtccactc ggcatcacac ggaatctgca ccgagttccc ccccgcagac 5880

ccaaggtcca actctccaag cggcaatcct ctctcgcttc ctcagcccca ctgaatgatc 5940

gcgtaaccgt aattaatcta gctacattta agattaagaa aaaatacggg tagaatcccc 6000

gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 6060

ggatccactg gtcaggtatc acatcggtat cctcgaattc aaagtatcaa agtacaattt 6120

accgaatata agaaggagaa aggtttcatc ctcacttccc aaaaggagga tgaaatcatg 6180

aaggtgcaga acaactcagt catcatcaac tgtgatgggt tttatctcat ctccctgaag 6240

ggctacttct cccaggaagt caacattagc cttcattacc agaaggatga ggagcccctc 6300

ttccaactga agaaggtcag gtctgtcaac tccttgatgg tggcctctct gacttacaaa 6360

gacaaagtct acttgaatgt gaccactgac aatacctccc tggatgactt ccatgtgaat 6420

ggcggagaac tgattcttat ccatcaaaat cctggtgaat tctgtgtcct ttgataagaa 6480

aaaatacggg tagaagttta aacccttgga gtgccccaat tgtgccaaga tggactcatc 6540

taggacaatt gggctgtact ttgattctgc ccattcttct agcaacctgt tagcatttcc 6600

gatcgtccta caagacacag gagatgggaa gaagcaaatc gccccgcaat ataggatcca 6660

gcgccttgac ttgtggactg atagtaagga ggactcagta ttcatcacca cctatggatt 6720

catctttcaa gttgggaatg aagaagccac tgtcggcatg atcgatgata aacccaagcg 6780

cgagttactt tccgctgcga tgctctgcct aggaagcgtc ccaaataccg gagaccttat 6840

tgagctggca agggcctgtc tcactatgat agtcacatgc aagaagagtg caactaatac 6900

tgagagaatg gttttctcag tagtgcaggc accccaagtg ctgcaaagct gtagggttgt 6960

ggcaaacaaa tactcatcag tgaatgcagt caagcacgtg aaagcgccag agaagattcc 7020

cgggagtgga accctagaat acaaggtgaa ctttgtctcc ttgactgtgg taccgaagaa 7080

ggatgtctac aagatcccag ctgcagtatt gaaggtttct ggctcgagtc tgtacaatct 7140

tgcgctcaat gtcactatta atgtggaggt agacccgagg agtcctttgg ttaaatctct 7200

gtctaagtct gacagcggat actatgctaa cctcttcttg catattggac ttatgaccac 7260

cgtagatagg aaggggaaga aagtgacatt tgacaagctg gaaaagaaaa taaggagcct 7320

tgatctatct gtcgggctca gtgatgtgct cgggccttcc gtgttggtaa aagcaagagg 7380

tgcacggact aagcttttgg cacctttctt ctctagcagt gggacagcct gctatcccat 7440

agcaaatgct tctcctcagg tggccaagat actctggagt caaaccgcgt gcctgcggag 7500

cgttaaaatc attatccaag caggtaccca acgcgctgtc gcagtgaccg ccgaccacga 7560

ggttacctct actaagctgg agaaggggca cacccttgcc aaatacaatc cttttaagaa 7620

ataagctgcg tctctgagat tgcgctccgc ccactcaccc agatcatcat gacacaaaaa 7680

actaatctgt cttgattatt tacagttagt ttacctgtct atcaagttag aaaaaacacg 7740

ggtagaagat tctggatccc ggttggcgcc ctccaggtgc aagatgggcc ccaaatcttc 7800

taccaatgtc ccagcacctc tgatgctgac cgtcaggatt gcgctggcac tgagctgtgt 7860

ccgtctgaca aattctctcg atggaaggcc tcttgcagct gcagggattg tagtaacggg 7920

agacaaagca gtcaacatat acacctcatc tcagacagggg tcaataatag tcaagttact 7980

cccaaatatg cctaaggata aagaggcgtg tgcaaaagcc ccgttggagg catacaacag 8040

gacactgact actttgctca ccccccttgg tgattctatc cgcaggatac aagagtctgc 8100

gactacgtcc ggaggaagga ggcagagacg ctttataggt gccattatcg gcagtgtagc 8160

tcttggggtt gccacagatg cccagataac agcagcctca gctctgatac aagccaacca 8220

gaatgctgcc aacatcctcc ggcttaaaga gagcattgct gcaactaatg aagctgtaca 8280

tgaagtcact gacggattat cgcaactagc agtggcagtt gggaagatgc agcagtttgt 8340

taatgaccag tttaataaca cagctcagga attggactgt ataaaaatta cacagcaggt 8400

tggtgtagaa ctcaacctgt acctaactga attgactaca gtattcgggc cacaaatcac 8460

ttcccctgcc ttaactcagc tgactatcca ggcgctttac aatctagctg gtggtaatat 8520

ggattatttg ttgactaagt taggtgttgg gaacaaccaa ctcagctcat taatcggtag 8580

cggcttgatc accggtaacc ctattctgta cgattcacag actcaactct taggtataca 8640

ggtaacttta ccctcagtcg gtaacctaaa taatatgcgt gctacctact tggagacctt 8700

gtctgtaagc acaaccaagg gatttgcctc agcacttgtc ccaaaagtgg tgacacaggt 8760

cgggtctgtg atagaggaac ttgacacctc atactgtgta gagaccgatt tggatttata 8820

ttgtacaaga atagtgacat tccctatgtc tcctggtatt tattcctgtc tgagcggtaa 8880

tacatcagct tgcatgtatt caaagactga aggtgcactt actacgccat atatgactat 8940

caagggctca gttattgcca attgcaagat gacaacatgc agatgtgcag accctccggg 9000

tatcatatcg caaaattatg gagaagctgt gtctctaata gataggcact catgcaatgt 9060

cttatcctta gacgggataa ctttgaggct cagtggagaa tttgacgtaa cttatcaaaa 9120

gaatatctca atattagatt ctcaggtaat agtgacaggc aatctcgata tctcaactga 9180

acttgggaat gtcaacaact cgataagtaa tgctttggat aagttagagg aaagcaatag 9240

caaacttgac aaagtcaatg tcaagctgac cggcacgtct gctctcatta cctatatagt 9300

tttaactatc atatctcttg tttgtggtat acttagcctg gttctagcat gctatctgat 9360

gtataagcaa aaggcgcaac aaaagacctt attatggctt gggaataata ccctaaatca 9420

gatgagggcc actacaagaa tctgaacaca gatgaggaac gaaggtttcc ctaatagtaa 9480

tttgtgtgaa agttctggta gtctgtcagt taagaaaaaa tacgggtaga aggttaaccg 9540

gcccgcggcg acgcgtggcc tgagaggcct tcagagagtt aagaaaaaac taccggttgt 9600

agatgaccaa aggacgatat acgggtagaa cggtaagaga ggccgcccct caattgcgag 9660

ccaggcttca caacctccgt tctaccgctt caccgacaac agtcctcaat catggaccgc 9720

gccgttagcc aagttgcgtt agagaatgat gaaagagagg caaaaaatac atggcgcttg 9780

atattccgga ttgcaatctt attcttaaca gtagtgacct tggctatatc tgtagcctcc 9840

cttttatata gcatgggggc tagcacacct agcgatcttg taggcatacc gactaggatt 9900

tccagggcag aagaaaagat tacatctaca cttggttcca atcaagatgt agtagatagg 9960

atatataagc aagtggccct tgagtctccg ttggcattgt taaatactga gaccacaatt 10020

atgaacgcaa taacatctct ctcttatcag attaatggag ctgcaaacaa cagtgggtgg 10080

ggggcaccta tccatgaccc agattatata ggggggatag gcaaagaact cattgtagat 10140

gatgctagtg atgtcacatc attctatccc tctgcatttc aagaacatct gaattttatc 10200

ccggcgccta ctacaggatc aggttgcact cgaataccct catttgacat gagtgctacc 10260

cattactgct acacccataa tgtaatattg tctggatgca gagatcactc acattcatat 10320

cagtatttag cacttggtgt gctccggaca tctgcaacag ggagggtatt cttttctact 10380

ctgcgttcca tcaacctgga cgacacccaa aatcggaagt cttgcagtgt gagtgcaact 10440

cccctgggtt gtgatatgct gtgctcgaaa gtcacggaga cagaggaaga agattataac 10500

tcagctgtcc ctacgcggat ggtacatggg aggttagggt tcgacggcca gtaccacgaa 10560

aaggacctag atgtcacaac attattcggg gactgggtgg ccaactaccc aggagtaggg 10620

ggtggatctt ttattgacag ccgcgtatgg ttctcagtct acggagggtt aaaacccaat 10680

tcacccagtg acactgtaca ggaagggaaa tatgtgatat acaagcgata caatgacaca 10740

tgcccagatg agcaagacta ccagattcga atggccaagt cttcgtataa gcctggacgg 10800

tttggtggga aacgcataca gcaggctatc ttatctatca aggtgtcaac atccttaggc 10860

gaagacccgg tactgactgt accgcccaac acagtcacac tcatgggggc cgaaggcaga 10920

attctcacag tagggacatc tcatttcttg tatcaacgag ggtcatcata cttctctccc 10980

gcgttattat atcctatgac agtcagcaac aaaacagcca ctcttcatag tccttataca 11040

ttcaatgcct tcactcggcc aggtagtatc ccttgccagg cttcagcaag atgccccaac 11100

tcgtgtgtta ctggagtcta tacagatcca tatcccctaa tcttctatag aaaccacacc 11160

ttgcgagggg tattcgggac aatgcttgat ggtgtacaag caagacttaa ccctgcgtct 11220

gcagtattcg atagcacatc ccgcagtcgc attactcgag tgagttcaag cagtaccaaa 11280

gcagcataca caacatcaac ttgttttaaa gtggtcaaga ctaataagac ctattgtctc 11340

agcattgctg aaatatctaa tactctcttc ggagaattca gaatcgtccc gttactagtt 11400

gagatcctca aagatgacgg ggttagagaa gccaggtctg gctagttgag tcaattataa 11460

aggagttgga aagatggcat tgtatcacct atcttctgcg acatcaagaa tcaaaccgaa 11520

tgccggcgcg tgctcgaatt ccatgttgcc agttgaccac aatcagccag tgctcatgcg 11580

atcagattaa gccttgtcaa tagtctcttg attaagaaaa aatgtaagtg gcaatgagat 11640

acaaggcaaa acagctcatg gtaaataata cgggtaggac atggcgagct ccggtcctga 11700

aagggcagag catcagatta tcctaccaga gtcacacctg tcttcaccat tggtcaagca 11760

caaactactc tattactgga aattaactgg gctaccgctt cctgatgaat gtgacttcga 11820

ccacctcatt ctcagccgac aatggaaaaa aatacttgaa tcggcctctc ctgatactga 11880

gagaatgata aaactcggaa gggcagtaca ccaaactctt aaccacaatt ccagaataac 11940

cggagtgctc caccccaggt gtttagaaga actggctaat attgaggtcc cagattcaac 12000

caacaaattt cggaagattg agaagaagat ccaaattcac aacacgagat atggagaact 12060

gttcacaagg ctgtgtacgc atatagagaa gaaactgctg gggtcatctt ggtctaacaa 12120

tgtcccccgg tcagaggagt tcagcagcat tcgtacggat ccggcattct ggtttcactc 12180

aaaatggtcc acagccaagt ttgcatggct ccatataaaa cagatccaga ggcatctgat 12240

ggtggcagct aggacaaggt ctgcggccaa caaattggtg atgctaaccc ataaggtagg 12300

ccaagtcttt gtcactcctg aacttgtcgt tgtgacgcat acgaatgaga acaagttcac 12360

atgtcttacc caggaacttg tattgatgta tgcagatatg atggagggca gagatatggt 12420

caacataata tcaaccacgg cggtgcatct cagaagctta tcagagaaaa ttgatgacat 12480

tttgcggtta atagacgctc tggcaaaaga cttgggtaat caagtctacg atgttgtatc 12540

actaatggag ggatttgcat acggagctgt ccagctactc gagccgtcag gtacatttgc 12600

aggagatttc ttcgcattca acctgcagga gcttaaagac attctaattg gcctcctccc 12660

caatgatata gcagaatccg tgactcatgc aatcgctact gtattctctg gtttagaaca 12720

gaatcaagca gctgagatgt tgtgtctgtt gcgtctgtgg ggtcacccac tgcttgagtc 12780

ccgtattgca gcaaaggcag tcaggagcca aatgtgcgca ccgaaaatgg tagactttga 12840

tatgatcctt caggtactgt ctttcttcaa gggaacaatc atcaacgggt acagaaagaa 12900

gaatgcaggt gtgtggccgc gagtcaaagt ggatacaata tatgggaagg tcattgggca 12960

actacatgca gattcagcag agatttcaca cgatatcatg ttgagagagt ataagagttt 13020

atctgcactt gaatttgagc catgtataga atatgaccct gtcaccaacc tgagcatgtt 13080

cctaaaagac aaggcaatcg cacaccccaa cgataattgg cttgcctcgt ttaggcggaa 13140

ccttctctcc gaagaccaga agaaacatgt aaaagaagca acttcgacta atcgcctctt 13200

gatagagtttt ttagagtcaa atgattttga tccatataaa gagatggaat atctgacgac 13260

ccttgagtac cttagagatg acaatgtggc agtatcatac tcgctcaagg agaaggaagt 13320

gaaagttaat ggacggatct tcgctaagct gacaaagaag ttaaggaact gtcaggtgat 13380

ggcggaaggg atcctagccg atcagattgc acctttcttt cagggaaatg gagtcattca 13440

ggatagcata tccttgacca agagtatgct agcgatgagt caactgtctt ttaacagcaa 13500

taagaaacgt atcactgact gtaaagaaag agtatcttca aaccgcaatc atgatccgaa 13560

aagcaagaac cgtcggagag ttgcaacctt cataacaact gacctgcaaa agtactgtct 13620

taattggaga tatcagacaa tcaaattgtt cgctcatgcc atcaatcagt tgatgggcct 13680

acctcacttc ttcgaatgga ttcacctaag actgatggac actacgatgt tcgtaggaga 13740

ccctttcaat cctccaagtg accctactga ctgtgacctc tcaagagtcc ctaatgatga 13800

catatatatt gtcagtgcca gagggggtat cgaaggatta tgccagaagc tatggacaat 13860

gatctcaatt gctgcaatcc aacttgctgc agctagatcg cattgtcgtg ttgcctgtat 13920

ggtacagggt gataatcaag taatagcagt aacgagagag gtaagatcag acgactctcc 13980

ggagatggtg ttgacacagt tgcatcaagc cagtgataat ttcttcaagg aattaattca 14040

tgtcaatcat ttgattggcc ataatttgaa ggatcgtgaa accatcaggt cagacacatt 14100

cttcatatac agcaaacgaa tcttcaaaga tggagcaatc ctcagtcaag tcctcaaaaa 14160

ttcatctaaa ttagtgctag tgtcaggtga tctcagtgaa aacaccgtaa tgtcctgtgc 14220

caacattgcc tctactgtag cacggctatg cgagaacggg cttcccaaag acttctgtta 14280

ctatttaaac tatataatga gttgtgtgca gacatacttt gactctgagt tctccatcac 14340

caacaattcg caccccgatc ttaatcagtc gtggattgag gacatctctt ttgtgcactc 14400

atatgttctg actcctgccc aattaggggg actgagtaac cttcaatact caaggctcta 14460

cactagaaat atcggtgacc cggggactac tgcttttgca gagatcaagc gactagaagc 14520

agtgggatta ctgagtccta acattatgac taatatctta actaggccgc ctgggaatgg 14580

agattgggcc agtctgtgca acgacccata ctctttcaat tttgagactg ttgcaagccc 14640

aaatattgtt cttaagaaac atacgcaaag agtcctattt gaaacttgtt caaatccctt 14700

attgtctgga gtgcacacag aggataatga ggcagaagag aaggcattgg ctgaattctt 14760

gcttaatcaa gaggtgattc atccccgcgt tgcgcatgcc atcatggagg caagctctgt 14820

aggtaggaga aagcaaattc aagggcttgt tgacacaaca aacaccgtaa ttaagattgc 14880

gcttactagg aggccattag gcatcaagag gctgatgcgg atagtcaatt attctagcat 14940

gcatgcaatg ctgtttagag acgatgtttt ttcctccagt agatccaacc accccttagt 15000

ctcttctaat atgtgttctc tgacactggc agactatgca cggaatagaa gctggtcacc 15060

tttgacggga ggcaggaaaa tactgggtgt atctaatcct gatacgatag aactcgtaga 15120

gggtgagatt cttagtgtaa gcggagggtg tacaagatgt gacagcggag atgaacaatt 15180

tacttggttc catcttccaa gcaatataga attgaccgat gacaccagca agaatcctcc 15240

gatgagggta ccatatctcg ggtcaaagac acaggagagg agagctgcct cacttgcaaa 15300

aatagctcat atgtcgccac atgtaaaggc tgccctaagg gcatcatccg tgttgatctg 15360

ggcttatggg gataatgaag taaattggac tgctgctctt acgattgcaa aatctcggtg 15420

taatgtaaac ttagagtatc ttcggttact gtccccttta cccacggctg ggaatcttca 15480

acatagacta gatgatggta taactcagat gacattcacc cctgcatctc tctacagggt 15540

gtcaccttac attcacatat ccaatgattc tcaaaggctg ttcactgaag aaggagtcaa 15600

agaggggaat gtggtttacc aacagatcat gctcttgggt ttatctctaa tcgaatcgat 15660

ctttccaata acaacaacca ggacatatga tgagatcaca ctgcacctac atagtaaatt 15720

tagttgctgt atcagagaag cacctgttgc ggttcctttc gagctacttg gggtggtacc 15780

ggaactgagg acagtgacct caaataagtt tatgtatgat cctagccctg tatcggaggg 15840

agactttgcg agacttgact tagctatctt caagagttat gagcttaatc tggagtcata 15900

tcccacgata gagctaatga acattctttc aatatccagc gggaagttga ttggccagtc 15960

tgtggtttct tatgatgaag atacctccat aaagaatgac gccataatag tgtatgacaa 16020

tacccgaaat tggatcagtg aagctcagaa ttcagatgtg gtccgcctat ttgaatatgc 16080

agcacttgaa gtgctcctcg actgttctta ccaactctat tacctgagag taagaggcct 16140

agacaatatt gtcttatata tgggtgattt atacaagaat atgccaggaa ttctactttc 16200

caacattgca gctacaatat ctcatcccgt cattcattca aggttacatg cagtgggcct 16260

ggtcaaccat gacggatcac accaacttgc agatacggat tttatcgaaa tgtctgcaaa 16320

actattagta tcttgcaccc gacgtgtgat ctccggctta tattcaggaa ataagtatga 16380

tctgctgttc ccatctgtct tagatgataa cctgaatgag aagatgcttc agctgatatc 16440

ccggttatgc tgtctgtaca cggtactctt tgctacaaca agagaaatcc cgaaaataag 16500

aggcttaact gcagaagaga aatgttcaat actcactgag tatttactgt cggatgctgt 16560

gaaaccatta cttagtcccg atcaagtgag ctctatcatg tctcctaaca taattacatt 16620

cccagctaat ctgtactaca tgtctcggaa gagcctcaat ttgatcaggg aaagggagga 16680

cagggatact atcctggcgt tgttgttccc ccaagagcca ttattagagt tcccttctgt 16740

gcaagatatt ggtgctcgag tgaaagatcc attcacccga caacctgcgg catttttgca 16800

agagttagat ttgagtgctc cagcaaggta tgacgcattc acacttagtc agattcatcc 16860

tgaactcaca tctccaaatc cggaggaaga ctacttagta cgatacttgt tcagagggat 16920

agggactgca tcttcctctt ggtataaggc atctcatctc ctttctgtac ccgaggtaag 16980

atgtgcaaga cacgggaact ccttatactt agctgaaggg agcggagcca tcatgagtct 17040

tctcgaactg catgtaccac atgaaactat ctattacaat acgctctttt caaatgagat 17100

gaaccccccg caacgacatt tcgggccgac cccaactcag tttttgaatt cggttgttta 17160

taggaatcta caggcggagg taacatgcaa agatggattt gtccaagagt tccgtccatt 17220

atggagagaa aatacagagg aaagtgacct gacctcagat aaagtagtgg ggtatattac 17280

atctgcagtg ccctacagat ctgtatcatt gctgcattgt gacattgaaa ttcctccagg 17340

gtccaatcaa agcttactag atcaactagc tatcaattta tctctgattg ccatgcattc 17400

tgtaagggag ggcggggtag taatcatcaa agtgttgtat gcaatgggat actactttca 17460

tctactcatg aacttgtttg ctccgtgttc cacaaaagga tatattctct ctaatggtta 17520

tgcatgtcga ggagatatgg agtgttacct ggtatttgtc atgggttacc tgggcgggcc 17580

tacatttgta catgaggtgg tgaggatggc gaaaactctg gtgcagcggc acggtacgct 17640

tttgtctaaa tcagatgaga tcacactgac caggttattc acctcacagc ggcagcgtgt 17700

gacagacatc ctatccagtc ctttaccaag attaataaag tacttgagga agaatattga 17760

cactgcgctg attgaagccg ggggacagcc cgtccgtcca ttctgtgcgg agagtctggt 17820

gagcacgcta gcgaacataa ctcagataac ccagatcatc gctagtcaca ttgacacagt 17880

tatccggtct gtgatatata tggaagctga gggtgatctc gctgacacag tatttctatt 17940

taccccttac aatctctcta ctgacgggaa aaagaggaca tcacttaaac agtgcacgag 18000

acagatccta gaggttacaa tactaggtct tagagtcgaa aatctcaata aaataggcga 18060

tataatcagc ctagtgctta aaggcatgat ctccatggag gaccttatcc cactaaggac 18120

atacttgaag catagtacct gccctaaata tttgaaggct gtcctaggta ttaccaaact 18180

caaagaaatg tttacagaca cttctgtact gtacttgact cgtgctcaac aaaaattcta 18240

catgaaaact ataggcaatg cagtcaaagg atattacagt aactgtgact cttaacgaaa 18300

atcacatatt aataggctcc ttttttggcc aattgtattc ttgttgattt aatcatatta 18360

tgttagaaaa aagttgaacc ctgactcctt aggactcgaa ttcgaactca aataaatgtc 18420

ttaaaaaaag gttgcgcaca attattcttg agtgtagtct cgtcattcac caaatctttg 18480

tttggtttgg tggccggcat ggtcccagcc tcctcgctgg cgccggctgg gcaacattcc 18540

gaggggaccg tcccctcggt aatggcgaat gggacgcggc cgatccggct gctaacaaag 18600

cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg 18660

gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatcgg 18720

ccgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 18780

aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 18840

gaggaactat atccggatgg ccgccaccgg tgggccttgc agcacatccc cccttcgcca 18900

g 18901

<210> 4

<211> 18955

<212> DNA

<213> Artificial Sequence

<220>

<223> Genome sequence of recombinant Newcastle disease virus rNDV-mOX40L

<400> 4

ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gtagcctgaa 60

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 120

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 180

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 240

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 300

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 360

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 420

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 480

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 540

tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt caaatatgta 600

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 660

gagtattcaa catttccgtg tcgcccttat tcccttttttt gcggcatttt gccttcctgt 720

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 780

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 840

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 900

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 960

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 1020

cagtgctgcc ataagcatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1080

aggaccgaag gagctaaccg ctttttttca caacatgggg gatcatgtaa ctcgccttga 1140

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1200

tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1260

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1320

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1380

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1440

gacgggcagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1500

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1560

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1620

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1680

aggatcttct tgagatcctt ttttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1740

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1800

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1860

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1920

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1980

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 2040

gcgaacgacc tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct 2100

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2160

cacgagggag cttccagggg ggaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2220

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggccgagcc tatggaaaaa 2280

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2340

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2400

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2460

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 2520

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttacct 2580

cactcattag gcaccccagg ctttacactt tatgcttccg gctcctatgt tgtgtggaat 2640

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcgg 2700

aagcggccgc taatacgact cactataggg accaaacaga gaatccgtaa gttacgataa 2760

aaggcgaagg agcaattgaa gtcgcacggg tagaaggtgt gaatctcgag tgcgagcccg 2820

aagcacaaac tcgagaaagc cttctgccaa catgtcttcc gtatttgatg agtacgaaca 2880

gctcctcgcg gctcagactc gccccaatgg agctcatgga gggggagaaa aagggagtac 2940

cttaaaagta gacgtcccgg tattcactct taacagtgat gacccagaag atagatggag 3000

ctttgtggta ttctgcctcc ggattgctgt tagcgaagat gccaacaaac cactcaggca 3060

aggtgctctc atatctcttt tatgctccca ctcacaggta atgaggaacc atgttgccct 3120

tgcagggaaa cagaatgaag ccacattggc cgtgcttgag attgatggct ttgccaacgg 3180

cacgccccag ttcaacaata ggagtggagt gtctgaagag agagcacaga gatttgcgat 3240

gatagcagga tctctccctc gggcatgcag caacggaacc ccgttcgtca cagccggggc 3300

cgaagatgat gcaccagaag acatcaccga taccctggag aggatcctct ctatccaggc 3360

tcaagtatgg gtcacagtag caaaagccat gactgcgtat gagactgcag atgagtcgga 3420

aacaaggcga atcaataagt atatgcagca aggcagggtc caaaagaaat acatcctcta 3480

ccccgtatgc aggagcacaa tccaactcac gatcagacag tctcttgcag tccgcatctt 3540

tttggttagc gagctcaaga gaggccgcaa cacggcaggt ggtacctcta cttattataa 3600

cctggtaggg gacgtagact catacatcag gaataccggg cttactgcat tcttcttgac 3660

actcaagtac ggaatcaaca ccaagacatc agcccttgca cttagtagcc tctcaggcga 3720

catccagaag atgaagcagc tcatgcgttt gtatcggatg aaaggagata atgcgccgta 3780

catgacatta cttggtgata gtgaccagat gagctttgcg cctgccgagt atgcacaact 3840

ttactccttt gccatgggta tggcatcagt cctagataaa ggtactggga aataccaatt 3900

tgccagggac tttatgagca catcattctg gagacttgga gtagagtacg ctcaggctca 3960

gggaagtagc attaacgagg atatggctgc cgagctaaag ctaaccccag cagcaaggag 4020

gggcctggca gctgctgccc aacgggtctc cgaggagacc agcagcatag acatgcctac 4080

tcaacaagtc ggagtcctca ctgggcttag cgaggggggg tcccaagctc tacaaggcgg 4140

atcgaataga tcgcaagggc aaccagaagc cggggatggg gagacccaat tcctggatct 4200

gatgagagcg gtagcaaata gcatgaggga ggcgccaaac tctgcacagg gcactcccca 4260

atcggggcct cccccaactc ctgggccatc ccaagataac gacaccgact gggggtattg 4320

atggacaaaa cccagcctgc ttccacaaaa acatcccaat gccctcaccc gtagtcgacc 4380

cctcgatttg cggctctata tgaccacacc ctcaaacaaa catccccctc tttcctccct 4440

ccccctgctg tacaactccg cacgccctag ataccacagg cacaatgcgg ctcactaaca 4500

atcaaaacag agccgaggga attagaaaaa agtacgggta gaagagggat attcagagat 4560

cagggcaagt ctcccgagtc tctgctctct cctctacctg atagaccagg acaaacatgg 4620

ccacctttac agatgcagag atcgacgagc tatttgagac aagtggaact gtcattgaca 4680

acataattac agcccagggt aaaccagcag agactgttgg aaggagtgca atcccacaag 4740

gcaagaccaa ggtgctgagc gcagcatggg agaagcatgg gagcatccag ccaccggcca 4800

gtcaagacaa ccccgatcga caggacagat ctgacaaaca accatccaca cccgagcaaa 4860

cgaccccgca tgacagcccg ccggccacat ccgccgacca gccccccacc caggccacag 4920

acgaagccgt cgacacacag ctcaggaccg gagcaagcaa ctctctgctg ttgatgcttg 4980

acaagctcag caataaatcg tccaatgcta aaaagggccc atggtcgagc ccccaagagg 5040

ggaatcacca acgtccgact caacagcagg ggagtcaacc cagtcgcgga aacagtcagg 5100

aaagaccgca gaaccaagtc aaggccgccc ctggaaacca gggcacagac gtgaacacag 5160

catatcatgg acaatgggag gagtcacaac tatcagctgg tgcaacccct catgctctcc 5220

gatcaaggca gagccaagac aatacccttg tatctgcgga tcatgtccag ccacctgtag 5280

actttgtgca agcgatgatg tctatgatgg aggcgatatc acagagagta agtaaggttg 5340

actatcagct agatcttgtc ttgaaacaga catcctccat ccctatgatg cggtccgaaa 5400

tccaacagct gaaaacatct gttgcagtca tggaagccaa cttgggaatg atgaagattc 5460

tggatcccgg ttgtgccaac atttcatctc tgagtgatct acgggcagtt gcccgatctc 5520

acccggtttt agtttcaggc cctggagacc cctctcccta tgtgacacaa ggaggcgaaa 5580

tggcacttaa taaactttcg caaccagtgc cacatccatc tgaattgatt aaacccgcca 5640

ctgcatgcgg gcctgatata ggagtggaaa aggacactgt ccgtgcattg atcatgtcac 5700

gcccaatgca cccgagttct tcagccaagc tcctaagcaa gttagatgca gccgggtcga 5760

tcgaggaaat caggaaaatc aagcgccttg ctctaaatgg ctaattacta ctgccacacg 5820

tagcgggtcc ctgtccactc ggcatcacac ggaatctgca ccgagttccc ccccgcagac 5880

ccaaggtcca actctccaag cggcaatcct ctctcgcttc ctcagcccca ctgaatgatc 5940

gcgtaaccgt aattaatcta gctacattta agattaagaa aaaatacggg tagaatcccc 6000

gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 6060

ggatccactg gtcaactctc ttcctctccg gcaaaggacc ctccaatcca aagactcaga 6120

ggagcagtta ccagatgtga ggatgggcaa ctattcatca gctcatacaa gaatgagtat 6180

caaactatgg aggtgcagaa caattcggtt gtcatcaagt gtgatgggct ttatatcatc 6240

tacctgaagg gctcctttttt ccaggaggtc aagattgacc ttcatttccg ggaggatcat 6300

aatcccatct ctattccaat gctgaacgat ggtcgaagga ttgtcttcac tgtggtggcc 6360

tctttggctt tcaaagataa agtttacctg actgtaaatg ctcctgatac tctctgcgaa 6420

cacctccaga taaatgatgg ggagctgatt gttgtccagc taacgcctgg atactgtgct 6480

cctgaaggat cttaccacag cactgtgaac caagtaccac tgtgattaag aaaaaatacg 6540

ggtagaaggg tttaaaccct tggagtgccc caattgtgcc aagatggact catctaggac 6600

aattgggctg tactttgatt ctgcccattc ttctagcaac ctgttagcat ttccgatcgt 6660

cctacaagac acaggagatg ggaagaagca aatcgccccg caatatagga tccagcgcct 6720

tgacttgtgg actgatagta aggaggactc agtattcatc accacctatg gattcatctt 6780

tcaagttggg aatgaagaag ccactgtcgg catgatcgat gataaaccca agcgcgagtt 6840

actttccgct gcgatgctct gcctaggaag cgtcccaaat accggagacc ttattgagct 6900

ggcaagggcc tgtctcacta tgatagtcac atgcaagaag agtgcaacta atactgagag 6960

aatggttttc tcagtagtgc aggcacccca agtgctgcaa agctgtaggg ttgtggcaaa 7020

caaatactca tcagtgaatg cagtcaagca cgtgaaagcg ccagagaaga ttcccgggag 7080

tggaacccta gaatacaagg tgaactttgt ctccttgact gtggtaccga agaaggatgt 7140

ctacaagatc ccagctgcag tattgaaggt ttctggctcg agtctgtaca atcttgcgct 7200

caatgtcact attaatgtgg aggtagaccc gaggagtcct ttggttaaat ctctgtctaa 7260

gtctgacagc ggatactatg ctaacctctt cttgcatatt ggacttatga ccaccgtaga 7320

taggaagggg aagaaagtga catttgacaa gctggaaaag aaaataagga gccttgatct 7380

atctgtcggg ctcagtgatg tgctcgggcc ttccgtgttg gtaaaagcaa gaggtgcacg 7440

gactaagctt ttggcacctt tcttctctag cagtgggaca gcctgctatc ccatagcaaa 7500

tgcttctcct caggtggcca agatactctg gagtcaaacc gcgtgcctgc ggagcgttaa 7560

aatcattatc caagcaggta cccaacgcgc tgtcgcagtg accgccgacc acgaggttac 7620

ctctactaag ctggagaagg ggcacaccct tgccaaatac aatcctttta agaaataagc 7680

tgcgtctctg agattgcgct ccgcccactc acccagatca tcatgacaca aaaaactaat 7740

ctgtcttgat tatttacagt tagtttacct gtctatcaag ttagaaaaaa cacgggtaga 7800

agattctgga tcccggttgg cgccctccag gtgcaagatg ggccccaaat cttctaccaa 7860

tgtcccagca cctctgatgc tgaccgtcag gattgcgctg gcactgagct gtgtccgtct 7920

gacaaattct ctcgatggaa ggcctcttgc agctgcaggg attgtagtaa cgggagacaa 7980

agcagtcaac atatacacct catctcagac agggtcaata atagtcaagt tactcccaaa 8040

tatgcctaag gataaagagg cgtgtgcaaa agccccgttg gaggcataca acaggacact 8100

gactactttg ctcacccccc ttggtgattc tatccgcagg atacaagagt ctgcgactac 8160

gtccggagga aggaggcaga gacgctttat aggtgccatt atcggcagtg tagctcttgg 8220

ggttgccaca gatgcccaga taacagcagc ctcagctctg atacaagcca accagaatgc 8280

tgccaacatc ctccggctta aagagagcat tgctgcaact aatgaagctg tacatgaagt 8340

cactgacgga ttatcgcaac tagcagtggc agttgggaag atgcagcagt ttgttaatga 8400

ccagtttaat aacacagctc aggaattgga ctgtataaaa attacacagc aggttggtgt 8460

agaactcaac ctgtacctaa ctgaattgac tacagtattc gggccacaaa tcacttcccc 8520

tgccttaact cagctgacta tccaggcgct ttacaatcta gctggtggta atatggatta 8580

tttgttgact aagttaggtg ttgggaacaa ccaactcagc tcattaatcg gtagcggctt 8640

gatcaccggt aaccctattc tgtacgattc acagactcaa ctcttaggta tacaggtaac 8700

tttaccctca gtcggtaacc taaataatat gcgtgctacc tacttggaga ccttgtctgt 8760

aagcacaacc aagggatttg cctcagcact tgtcccaaaa gtggtgacac aggtcgggtc 8820

tgtgatagag gaacttgaca cctcatactg tgtagagacc gatttggatt tatattgtac 8880

aagaatagtg acattcccta tgtctcctgg tatttattcc tgtctgagcg gtaatacatc 8940

agcttgcatg tattcaaaga ctgaaggtgc acttactacg ccatatatga ctatcaaggg 9000

ctcagttatt gccaattgca agatgacaac atgcagatgt gcagaccctc cgggtatcat 9060

atcgcaaaat tatggagaag ctgtgtctct aatagatagg cactcatgca atgtcttatc 9120

cttagacggg ataactttga ggctcagtgg agaatttgac gtaacttatc aaaagaatat 9180

ctcaatatta gattctcagg taatagtgac aggcaatctc gatatctcaa ctgaacttgg 9240

gaatgtcaac aactcgataa gtaatgcttt ggataagtta gaggaaagca atagcaaact 9300

tgacaaagtc aatgtcaagc tgaccggcac gtctgctctc attacctata tagttttaac 9360

tatcatatct cttgtttgtg gtatacttag cctggttcta gcatgctatc tgatgtataa 9420

gcaaaaggcg caacaaaaga ccttattatg gcttgggaat aataccctaa atcagatgag 9480

ggccactaca agaatctgaa cacagatgag gaacgaaggt ttccctaata gtaatttgtg 9540

tgaaagttct ggtagtctgt cagttaagaa aaaatacggg tagaaggtta accggcccgc 9600

ggcgacgcgt ggcctgagag gccttcagag agttaagaaa aaactaccgg ttgtagatga 9660

ccaaaggacg atatacgggt agaacggtaa gagaggccgc ccctcaattg cgagccaggc 9720

ttcacaacct ccgttctacc gcttcaccga caacagtcct caatcatgga ccgcgccgtt 9780

agccaagttg cgttagagaa tgatgaaaga gaggcaaaaa atacatggcg cttgatattc 9840

cggattgcaa tcttattctt aacagtagtg accttggcta tatctgtagc ctccctttta 9900

tatagcatgg gggctagcac acctagcgat cttgtaggca taccgactag gatttccagg 9960

gcagaagaaa agattacatc tacacttggt tccaatcaag atgtagtaga taggatat 10020

aagcaagtgg cccttgagtc tccgttggca ttgttaaata ctgagaccac aattatgaac 10080

gcaataacat ctctctctta tcagattaat ggagctgcaa acaacagtgg gtggggggca 10140

cctatccatg acccagatta tatagggggg ataggcaaag aactcattgt agatgatgct 10200

agtgatgtca catcattcta tccctctgca tttcaagaac atctgaattt tatcccggcg 10260

cctactacag gatcaggttg cactcgaata ccctcatttg acatgagtgc tacccattac 10320

tgctacaccc ataatgtaat attgtctgga tgcagagatc actcacattc atatcagtat 10380

ttagcacttg gtgtgctccg gacatctgca acagggaggg tattcttttc tactctgcgt 10440

tccatcaacc tggacgacac ccaaaatcgg aagtcttgca gtgtgagtgc aactcccctg 10500

ggttgtgata tgctgtgctc gaaagtcacg gagacagagg aagaagatta taactcagct 10560

gtccctacgc ggatggtaca tgggaggtta gggttcgacg gccagtacca cgaaaaggac 10620

ctagatgtca caacattatt cggggactgg gtggccaact acccaggagt agggggtgga 10680

tcttttattg acagccgcgt atggttctca gtctacggag ggttaaaacc caattcaccc 10740

agtgacactg tacaggaagg gaaatatgtg atatacaagc gatacaatga cacatgccca 10800

gatgagcaag actaccagat tcgaatggcc aagtcttcgt ataagcctgg acggtttggt 10860

gggaaacgca tacagcaggc tatcttatct atcaaggtgt caacatcctt aggcgaagac 10920

ccggtactga ctgtaccgcc caacacagtc acactcatgg gggccgaagg cagaattctc 10980

acagtaggga catctcattt cttgtatcaa cgagggtcat catacttctc tcccgcgtta 11040

ttatatccta tgacagtcag caacaaaaca gccactcttc atagtcctta tacattcaat 11100

gccttcactc ggccaggtag tatcccttgc caggcttcag caagatgccc caactcgtgt 11160

gttactggag tctatacaga tccatatccc ctaatcttct atagaaacca caccttgcga 11220

ggggtattcg ggacaatgct tgatggtgta caagcaagac ttaaccctgc gtctgcagta 11280

ttcgatagca catcccgcag tcgcattact cgagtgagtt caagcagtac caaagcagca 11340

tacacaacat caacttgttt taaagtggtc aagactaata agacctattg tctcagcatt 11400

gctgaaatat ctaatactct cttcggagaa ttcagaatcg tcccgttact agttgagatc 11460

ctcaaagatg acggggttag agaagccagg tctggctagt tgagtcaatt ataaaggagt 11520

tggaaagatg gcattgtatc acctatcttc tgcgacatca agaatcaaac cgaatgccgg 11580

cgcgtgctcg aattccatgt tgccagttga ccacaatcag ccagtgctca tgcgatcaga 11640

ttaagccttg tcaatagtct cttgattaag aaaaaatgta agtggcaatg agatacaagg 11700

caaaacagct catggtaaat aatacgggta ggacatggcg agctccggtc ctgaaagggc 11760

agagcatcag attatcctac cagagtcaca cctgtcttca ccattggtca agcacaaact 11820

actctattac tggaaattaa ctgggctacc gcttcctgat gaatgtgact tcgaccacct 11880

cattctcagc cgacaatgga aaaaaatact tgaatcggcc tctcctgata ctgagagaat 11940

gataaaactc ggaagggcag tacaccaaac tcttaaccac aattccagaa taaccggagt 12000

gctccacccc aggtgtttag aagaactggc taatattgag gtcccagatt caaccaacaa 12060

atttcggaag attgagaaga agatccaaat tcacaacacg agatatggag aactgttcac 12120

aaggctgtgt acgcatatag agaagaaact gctggggtca tcttggtcta acaatgtccc 12180

ccggtcagag gagttcagca gcattcgtac ggatccggca ttctggtttc actcaaaatg 12240

gtccacagcc aagtttgcat ggctccatat aaaacagatc cagaggcatc tgatggtggc 12300

agctaggaca aggtctgcgg ccaacaaatt ggtgatgcta acccataagg taggccaagt 12360

ctttgtcact cctgaacttg tcgttgtgac gcatacgaat gagaacaagt tcacatgtct 12420

tacccaggaa cttgtattga tgtatgcaga tatgatggag ggcagagata tggtcaacat 12480

aatatcaacc acggcggtgc atctcagaag cttatcagag aaaattgatg acattttgcg 12540

gttaatagac gctctggcaa aagacttggg taatcaagtc tacgatgttg tatcactaat 12600

ggagggattt gcatacggag ctgtccagct actcgagccg tcaggtacat ttgcaggaga 12660

tttcttcgca ttcaacctgc aggagcttaa agacattcta attggcctcc tccccaatga 12720

tatagcagaa tccgtgactc atgcaatcgc tactgtattc tctggtttag aacagaatca 12780

agcagctgag atgttgtgtc tgttgcgtct gtggggtcac ccactgcttg agtcccgtat 12840

tgcagcaaag gcagtcagga gccaaatgtg cgcaccgaaa atggtagact ttgatatgat 12900

ccttcaggta ctgtctttct tcaagggaac aatcatcaac gggtacagaa agaagaatgc 12960

aggtgtgtgg ccgcgagtca aagtggatac aatatatggg aaggtcattg ggcaactaca 13020

tgcagattca gcagagattt cacacgatat catgttgaga gagtataaga gtttatctgc 13080

acttgaattt gagccatgta tagaatatga ccctgtcacc aacctgagca tgttcctaaa 13140

agacaaggca atcgcacacc ccaacgataa ttggcttgcc tcgtttaggc ggaaccttct 13200

ctccgaagac cagaagaaac atgtaaaaga agcaacttcg actaatcgcc tcttgataga 13260

gtttttagag tcaaatgatt ttgatccata taaagagatg gaatatctga cgacccttga 13320

gtaccttaga gatgacaatg tggcagtatc atactcgctc aaggagaagg aagtgaaagt 13380

taatggacgg atcttcgcta agctgacaaa gaagttaagg aactgtcagg tgatggcgga 13440

agggatccta gccgatcaga ttgcaccttt ctttcaggga aatggagtca ttcaggatag 13500

catatccttg accaagagta tgctagcgat gagtcaactg tcttttaaca gcaataagaa 13560

acgtatcact gactgtaaag aaagagtatc ttcaaaccgc aatcatgatc cgaaaagcaa 13620

gaaccgtcgg agagttgcaa ccttcataac aactgacctg caaaagtact gtcttaattg 13680

gagatatcag acaatcaaat tgttcgctca tgccatcaat cagttgatgg gcctacctca 13740

cttcttcgaa tggattcacc taagactgat ggacactacg atgttcgtag gagacccttt 13800

caatcctcca agtgacccta ctgactgtga cctctcaaga gtccctaatg atgacatata 13860

tattgtcagt gccagagggg gtatcgaagg attatgccag aagctatgga caatgatctc 13920

aattgctgca atccaacttg ctgcagctag atcgcattgt cgtgttgcct gtatggtaca 13980

gggtgataat caagtaatag cagtaacgag agaggtaaga tcagacgact ctccggagat 14040

ggtgttgaca cagttgcatc aagccagtga taatttcttc aaggaattaa ttcatgtcaa 14100

tcatttgatt ggccataatt tgaaggatcg tgaaaccatc aggtcagaca cattcttcat 14160

atacagcaaa cgaatcttca aagatggagc aatcctcagt caagtcctca aaaattcatc 14220

taaattagtg ctagtgtcag gtgatctcag tgaaaacacc gtaatgtcct gtgccaacat 14280

tgcctctact gtagcacggc tatgcgagaa cgggcttccc aaagacttct gttactattt 14340

aaactatata atgagttgtg tgcagacata ctttgactct gagttctcca tcaccaacaa 14400

ttcgcacccc gatcttaatc agtcgtggat tgaggacatc tcttttgtgc actcatatgt 14460

tctgactcct gcccaattag ggggactgag taaccttcaa tactcaaggc tctacactag 14520

aaatatcggt gacccgggga ctactgcttt tgcagagatc aagcgactag aagcagtggg 14580

attactgagt cctaacatta tgactaatat cttaactagg ccgcctggga atggagattg 14640

ggccagtctg tgcaacgacc catactcttt caattttgag actgttgcaa gcccaaatat 14700

tgttcttaag aaacatacgc aaagagtcct atttgaaact tgttcaaatc ccttattgtc 14760

tggagtgcac acagaggata atgaggcaga agagaaggca ttggctgaat tcttgcttaa 14820

tcaagaggtg attcatcccc gcgttgcgca tgccatcatg gaggcaagct ctgtaggtag 14880

gagaaagcaa attcaagggc ttgttgacac aacaaacacc gtaattaaga ttgcgcttac 14940

taggaggcca ttaggcatca agaggctgat gcggatagtc aattattcta gcatgcatgc 15000

aatgctgttt agagacgatg ttttttcctc cagtagatcc aaccacccct tagtctcttc 15060

taatatgtgt tctctgacac tggcagacta tgcacggaat agaagctggt cacctttgac 15120

gggaggcagg aaaatactgg gtgtatctaa tcctgatacg atagaactcg tagagggtga 15180

gattcttagt gtaagcggag ggtgtacaag atgtgacagc ggagatgaac aatttacttg 15240

gttccatctt ccaagcaata tagaattgac cgatgacacc agcaagaatc ctccgatgag 15300

ggtaccatat ctcgggtcaa agacacagga gaggagagct gcctcacttg caaaaatagc 15360

tcatatgtcg ccacatgtaa aggctgccct aagggcatca tccgtgttga tctgggctta 15420

tggggataat gaagtaaatt ggactgctgc tcttacgatt gcaaaatctc ggtgtaatgt 15480

aaacttagag tatcttcggt tactgtcccc tttacccacg gctgggaatc ttcaacatag 15540

actagatgat ggtataactc agatgacatt cacccctgca tctctctaca gggtgtcacc 15600

ttacattcac atatccaatg attctcaaag gctgttcact gaagaaggag tcaaagaggg 15660

gaatgtggtt taccaacaga tcatgctctt gggtttatct ctaatcgaat cgatctttcc 15720

aataacaaca accaggacat atgatgagat cacactgcac ctacatagta aatttagttg 15780

ctgtatcaga gaagcacctg ttgcggttcc tttcgagcta cttggggtgg taccggaact 15840

gaggacagtg acctcaaata agtttatgta tgatcctagc cctgtatcgg agggagactt 15900

tgcgagactt gacttagcta tcttcaagag ttatgagctt aatctggagt catatcccac 15960

gatagagcta atgaacattc tttcaatatc cagcgggaag ttgattggcc agtctgtggt 16020

ttcttatgat gaagatacct ccataaagaa tgacgccata atagtgtatg acaatacccg 16080

aaattggatc agtgaagctc agaattcaga tgtggtccgc ctatttgaat atgcagcact 16140

tgaagtgctc ctcgactgtt cttaccaact ctattacctg agagtaagag gcctagacaa 16200

tattgtctta tatatgggtg atttatacaa gaatatgcca ggaattctac tttccaacat 16260

tgcagctaca atatctcatc ccgtcattca ttcaaggtta catgcagtgg gcctggtcaa 16320

ccatgacgga tcacaccaac ttgcagatac ggattttatc gaaatgtctg caaaactatt 16380

agtatcttgc acccgacgtg tgatctccgg cttatattca ggaaataagt atgatctgct 16440

gttcccatct gtcttagatg ataacctgaa tgagaagatg cttcagctga tatcccggtt 16500

atgctgtctg tacacggtac tctttgctac aacaagagaa atcccgaaaa taagaggctt 16560

aactgcagaa gagaaatgtt caatactcac tgagtattta ctgtcggatg ctgtgaaacc 16620

attacttagt cccgatcaag tgagctctat catgtctcct aacataatta cattcccagc 16680

taatctgtac tacatgtctc ggaagagcct caatttgatc agggaaaggg aggacaggga 16740

tactatcctg gcgttgttgt tcccccaaga gccattatta gagttccctt ctgtgcaaga 16800

tattggtgct cgagtgaaag atccattcac ccgacaacct gcggcatttt tgcaagagtt 16860

agatttgagt gctccagcaa ggtatgacgc attcacactt agtcagattc atcctgaact 16920

cacatctcca aatccggagg aagactactt agtacgatac ttgttcagag ggatagggac 16980

tgcatcttcc tcttggtata aggcatctca tctcctttct gtacccgagg taagatgtgc 17040

aagacacggg aactccttat acttagctga agggagcgga gccatcatga gtcttctcga 17100

actgcatgta ccacatgaaa ctatctatta caatacgctc ttttcaaatg agatgaaccc 17160

cccgcaacga catttcgggc cgaccccaac tcagtttttg aattcggttg tttataggaa 17220

tctacaggcg gaggtaacat gcaaagatgg atttgtccaa gagttccgtc cattatggag 17280

agaaaataca gaggaaagtg acctgacctc agataaagta gtggggtata ttacatctgc 17340

agtgccctac agatctgtat cattgctgca ttgtgacatt gaaattcctc cagggtccaa 17400

tcaaagctta ctagatcaac tagctatcaa tttatctctg attgccatgc attctgtaag 17460

ggagggcggg gtagtaatca tcaaagtgtt gtatgcaatg ggatactact ttcatctact 17520

catgaacttg tttgctccgt gttccacaaa aggatatatt ctctctaatg gttatgcatg 17580

tcgaggagat atggagtgtt acctggtatt tgtcatgggt tacctgggcg ggcctacatt 17640

tgtacatgag gtggtgagga tggcgaaaac tctggtgcag cggcacggta cgcttttgtc 17700

taaatcagat gagatcacac tgaccaggtt attcacctca cagcggcagc gtgtgacaga 17760

catcctatcc agtcctttac caagattaat aaagtacttg aggaagaata ttgacactgc 17820

gctgattgaa gccggggggac agcccgtccg tccattctgt gcggagagtc tggtgagcac 17880

gctagcgaac ataactcaga taacccagat catcgctagt cacattgaca cagttatccg 17940

gtctgtgata tatatggaag ctgagggtga tctcgctgac acagtatttc tatttacccc 18000

ttacaatctc tctactgacg ggaaaaagag gacatcactt aaacagtgca cgagacagat 18060

cctagaggtt acaatactag gtcttagagt cgaaaatctc aataaaatag gcgatataat 18120

cagcctagtg cttaaaggca tgatctccat ggaggacctt atcccactaa ggacatactt 18180

gaagcatagt acctgcccta aatatttgaa ggctgtccta ggtattacca aactcaaaga 18240

aatgtttaca gacacttctg tactgtactt gactcgtgct caacaaaaat tctacatgaa 18300

aactataggc aatgcagtca aaggatatta cagtaactgt gactcttaac gaaaatcaca 18360

tattaatagg ctcctttttt ggccaattgt attcttgttg atttaatcat attatgttag 18420

aaaaaagttg aaccctgact ccttaggact cgaattcgaa ctcaaataaa tgtcttaaaa 18480

aaaggttgcg cacaattatt cttgagtgta gtctcgtcat tcaccaaatc tttgtttggt 18540

ttggtggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca ttccgagggg 18600

accgtcccct cggtaatggc gaatgggacg cggccgatcc ggctgctaac aaagcccgaa 18660

aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct 18720

ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tcggccgatc 18780

cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac 18840

tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa 18900

ctatatccgg atggccgcca ccggtgggcc ttgcagcaca tccccccttc gccag 18955

<210> 5

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Upstream primer

<400> 5

tcaagcgcct tgctctaaat ggc 23

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Downstream Primers

<400> 6

gggcagaatc aaagtacagc ccaat 25

<210> 7

<211> 18901

<212> DNA

<213> Artificial Sequence

<220>

<223> Genome sequence of recombinant Newcastle disease virus rClone30-Anh-(F)-hOX40L

<400> 7

ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gtagcctgaa 60

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 120

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 180

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 240

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 300

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 360

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 420

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 480

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 540

tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt caaatatgta 600

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 660

gagtattcaa catttccgtg tcgcccttat tcccttttttt gcggcatttt gccttcctgt 720

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 780

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 840

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 900

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 960

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 1020

cagtgctgcc ataagcatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1080

aggaccgaag gagctaaccg ctttttttca caacatgggg gatcatgtaa ctcgccttga 1140

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1200

tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1260

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1320

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1380

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1440

gacgggcagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1500

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1560

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1620

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1680

aggatcttct tgagatcctt ttttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1740

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1800

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1860

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1920

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1980

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 2040

gcgaacgacc tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct 2100

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2160

cacgagggag cttccagggg ggaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2220

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggccgagcc tatggaaaaa 2280

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2340

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2400

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2460

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 2520

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttacct 2580

cactcattag gcaccccagg ctttacactt tatgcttccg gctcctatgt tgtgtggaat 2640

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcgg 2700

aagcggccgc taatacgact cactataggg accaaacaga gaatccgtaa gttacgataa 2760

aaggcgaagg agcaattgaa gtcgcacggg tagaaggtgt gaatctcgag tgcgagcccg 2820

aagcacaaac tcgagaaagc cttctgccaa catgtcttcc gtatttgatg agtacgaaca 2880

gctcctcgcg gctcagactc gccccaatgg agctcatgga gggggagaaa aagggagtac 2940

cttaaaagta gacgtcccgg tattcactct taacagtgat gacccagaag atagatggag 3000

ctttgtggta ttctgcctcc ggattgctgt tagcgaagat gccaacaaac cactcaggca 3060

aggtgctctc atatctcttt tatgctccca ctcacaggta atgaggaacc atgttgccct 3120

tgcagggaaa cagaatgaag ccacattggc cgtgcttgag attgatggct ttgccaacgg 3180

cacgccccag ttcaacaata ggagtggagt gtctgaagag agagcacaga gatttgcgat 3240

gatagcagga tctctccctc gggcatgcag caacggaacc ccgttcgtca cagccggggc 3300

cgaagatgat gcaccagaag acatcaccga taccctggag aggatcctct ctatccaggc 3360

tcaagtatgg gtcacagtag caaaagccat gactgcgtat gagactgcag atgagtcgga 3420

aacaaggcga atcaataagt atatgcagca aggcagggtc caaaagaaat acatcctcta 3480

ccccgtatgc aggagcacaa tccaactcac gatcagacag tctcttgcag tccgcatctt 3540

tttggttagc gagctcaaga gaggccgcaa cacggcaggt ggtacctcta cttattataa 3600

cctggtaggg gacgtagact catacatcag gaataccggg cttactgcat tcttcttgac 3660

actcaagtac ggaatcaaca ccaagacatc agcccttgca cttagtagcc tctcaggcga 3720

catccagaag atgaagcagc tcatgcgttt gtatcggatg aaaggagata atgcgccgta 3780

catgacatta cttggtgata gtgaccagat gagctttgcg cctgccgagt atgcacaact 3840

ttactccttt gccatgggta tggcatcagt cctagataaa ggtactggga aataccaatt 3900

tgccagggac tttatgagca catcattctg gagacttgga gtagagtacg ctcaggctca 3960

gggaagtagc attaacgagg atatggctgc cgagctaaag ctaaccccag cagcaaggag 4020

gggcctggca gctgctgccc aacgggtctc cgaggagacc agcagcatag acatgcctac 4080

tcaacaagtc ggagtcctca ctgggcttag cgaggggggg tcccaagctc tacaaggcgg 4140

atcgaataga tcgcaagggc aaccagaagc cggggatggg gagacccaat tcctggatct 4200

gatgagagcg gtagcaaata gcatgaggga ggcgccaaac tctgcacagg gcactcccca 4260

atcggggcct cccccaactc ctgggccatc ccaagataac gacaccgact gggggtattg 4320

atggacaaaa cccagcctgc ttccacaaaa acatcccaat gccctcaccc gtagtcgacc 4380

cctcgatttg cggctctata tgaccacacc ctcaaacaaa catccccctc tttcctccct 4440

ccccctgctg tacaactccg cacgccctag ataccacagg cacaatgcgg ctcactaaca 4500

atcaaaacag agccgaggga attagaaaaa agtacgggta gaagagggat attcagagat 4560

cagggcaagt ctcccgagtc tctgctctct cctctacctg atagaccagg acaaacatgg 4620

ccacctttac agatgcagag atcgacgagc tatttgagac aagtggaact gtcattgaca 4680

acataattac agcccagggt aaaccagcag agactgttgg aaggagtgca atcccacaag 4740

gcaagaccaa ggtgctgagc gcagcatggg agaagcatgg gagcatccag ccaccggcca 4800

gtcaagacaa ccccgatcga caggacagat ctgacaaaca accatccaca cccgagcaaa 4860

cgaccccgca tgacagcccg ccggccacat ccgccgacca gccccccacc caggccacag 4920

acgaagccgt cgacacacag ctcaggaccg gagcaagcaa ctctctgctg ttgatgcttg 4980

acaagctcag caataaatcg tccaatgcta aaaagggccc atggtcgagc ccccaagagg 5040

ggaatcacca acgtccgact caacagcagg ggagtcaacc cagtcgcgga aacagtcagg 5100

aaagaccgca gaaccaagtc aaggccgccc ctggaaacca gggcacagac gtgaacacag 5160

catatcatgg acaatgggag gagtcacaac tatcagctgg tgcaacccct catgctctcc 5220

gatcaaggca gagccaagac aatacccttg tatctgcgga tcatgtccag ccacctgtag 5280

actttgtgca agcgatgatg tctatgatgg aggcgatatc acagagagta agtaaggttg 5340

actatcagct agatcttgtc ttgaaacaga catcctccat ccctatgatg cggtccgaaa 5400

tccaacagct gaaaacatct gttgcagtca tggaagccaa cttgggaatg atgaagattc 5460

tggatcccgg ttgtgccaac atttcatctc tgagtgatct acgggcagtt gcccgatctc 5520

acccggtttt agtttcaggc cctggagacc cctctcccta tgtgacacaa ggaggcgaaa 5580

tggcacttaa taaactttcg caaccagtgc cacatccatc tgaattgatt aaacccgcca 5640

ctgcatgcgg gcctgatata ggagtggaaa aggacactgt ccgtgcattg atcatgtcac 5700

gcccaatgca cccgagttct tcagccaagc tcctaagcaa gttagatgca gccgggtcga 5760

tcgaggaaat caggaaaatc aagcgccttg ctctaaatgg ctaattacta ctgccacacg 5820

tagcgggtcc ctgtccactc ggcatcacac ggaatctgca ccgagttccc ccccgcagac 5880

ccaaggtcca actctccaag cggcaatcct ctctcgcttc ctcagcccca ctgaatgatc 5940

gcgtaaccgt aattaatcta gctacattta agattaagaa aaaatacggg tagaatcccc 6000

gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 6060

ggatccactg gtcaggtatc acatcggtat cctcgaattc aaagtatcaa agtacaattt 6120

accgaatata agaaggagaa aggtttcatc ctcacttccc aaaaggagga tgaaatcatg 6180

aaggtgcaga acaactcagt catcatcaac tgtgatgggt tttatctcat ctccctgaag 6240

ggctacttct cccaggaagt caacattagc cttcattacc agaaggatga ggagcccctc 6300

ttccaactga agaaggtcag gtctgtcaac tccttgatgg tggcctctct gacttacaaa 6360

gacaaagtct acttgaatgt gaccactgac aatacctccc tggatgactt ccatgtgaat 6420

ggcggagaac tgattcttat ccatcaaaat cctggtgaat tctgtgtcct ttgataagaa 6480

aaaatacggg tagaagttta aacccttgga gtgccccaat tgtgccaaga tggactcatc 6540

taggacaatt gggctgtact ttgattctgc ccattcttct agcaacctgt tagcatttcc 6600

gatcgtccta caagacacag gagatgggaa gaagcaaatc gccccgcaat ataggatcca 6660

gcgccttgac ttgtggactg atagtaagga ggactcagta ttcatcacca cctatggatt 6720

catctttcaa gttgggaatg aagaagccac tgtcggcatg atcgatgata aacccaagcg 6780

cgagttactt tccgctgcga tgctctgcct aggaagcgtc ccaaataccg gagaccttat 6840

tgagctggca agggcctgtc tcactatgat agtcacatgc aagaagagtg caactaatac 6900

tgagagaatg gttttctcag tagtgcaggc accccaagtg ctgcaaagct gtagggttgt 6960

ggcaaacaaa tactcatcag tgaatgcagt caagcacgtg aaagcgccag agaagattcc 7020

cgggagtgga accctagaat acaaggtgaa ctttgtctcc ttgactgtgg taccgaagaa 7080

ggatgtctac aagatcccag ctgcagtatt gaaggtttct ggctcgagtc tgtacaatct 7140

tgcgctcaat gtcactatta atgtggaggt agacccgagg agtcctttgg ttaaatctct 7200

gtctaagtct gacagcggat actatgctaa cctcttcttg catattggac ttatgaccac 7260

cgtagatagg aaggggaaga aagtgacatt tgacaagctg gaaaagaaaa taaggagcct 7320

tgatctatct gtcgggctca gtgatgtgct cgggccttcc gtgttggtaa aagcaagagg 7380

tgcacggact aagcttttgg cacctttctt ctctagcagt gggacagcct gctatcccat 7440

agcaaatgct tctcctcagg tggccaagat actctggagt caaaccgcgt gcctgcggag 7500

cgttaaaatc attatccaag caggtaccca acgcgctgtc gcagtgaccg ccgaccacga 7560

ggttacctct actaagctgg agaaggggca cacccttgcc aaatacaatc cttttaagaa 7620

ataagctgcg tctctgagat tgcgctccgc ccactcaccc agatcatcat gacacaaaaa 7680

actaatctgt cttgattatt tacagttagt ttacctgtct atcaagttag aaaaaacacg 7740

ggtagaagat tctggatccc ggttggcgcc ctccaggtgc aagatgggcc ccaaaccccc 7800

caccggaacc ccagcgcctc tggtgctgat cgcccggacc gcgctggcgt tgggctgtgt 7860

ctgtccggcg ggctctcttg acggcagacc tcttgcagct gcagggattg tggtaacgag 7920

agataaagca gtcaatatat acacttcatc tcaaacgggg tcaatcatag tcaagttact 7980

cccaaatatg cccaaagaca aggaggcgtg cgcaaaagcc ccattagagg cgtacaatag 8040

aacactgacc actttactca ctcctcttgg cgactccatc cgcaggatac aagggtctgc 8100

aactacatct agaggaagga gacagaaacg ttttgtaggt gctatcattg gcagtatagc 8160

tcttggggtt gcgacagctg cacaagtaac agcagctgca gctctgatac aagccaacca 8220

gaacgctgcc aacatcctcc ggcttaagga gagcattgct gcaaccaatg aagctgtgca 8280

cgaggtcact gacggattat cacaactagc gatggcgatt gggaagatgc agcagtttgt 8340

taatgaccag tttaataata cggcgcgaga attggactgc atcaaaatta cacaacaggt 8400

tggtgtcgaa ctcaatttgt atctaactga actgactaca gtattcgggc cacaaatcac 8460

ttcccctgct ttaactcagc taactatcca ggcactttat aatttagctg gtggcaatat 8520

gaattactta ttgactaagt taggtgtagg gaacaatcaa cttagctcat taattagtag 8580

tggcctgatc actggcaacc ccattttata tgactcacag acccaactct taggcataca 8640

gataaatgta ccctcagtcg ggagcctaaa taatatgcgt gccacctact tggagacctt 8700

atccgtaagc acaaccaggg ggttcgcctc agcacttgtc ccgaaagttg tgacgcaagt 8760

tggttctgtg atagaagaac ttgacacctc atattgtata gaatctaatc tggatttata 8820

ttgtacaagg atagtgacat tccccatgtc tcccggcatt tattcctgtc tgagcggtaa 8880

tacgtcagct tgtatgtatt caaagactga gggtgcactc actacaccat acatagctct 8940

caagggctca gttattgcta attgcaagat gattacatgt agatgtgcag accccccagg 9000

tatcatatcg caaaattacg gagaagctgt gtccctaata gataaacatt catgtaatgt 9060

cttatcccta gacggaataa ccctgaggct cagtggggaa tttgatgcga cctatcaaaa 9120

gaacatctta atactagatt cccaggtcat cgtgacaggc aatctcgata tatcaactga 9180

acttgggaat gtcaacaact cgataagcag tgctctggac aaattagcgg aaagtaacag 9240

caagttaaac aaagtcaatg tcaacctaac tagcacatct gctctcatta cttatattgt 9300

tctagctgtc atatctcttg ttttcggcgt aattagcctg attctagcgt gctgcttgat 9360

gtataaacaa aaagcacaac aaaagacctt actatggctt gggaacaata ccctcgatca 9420

gatgagagcc accacaaaaa catgaacaca gatgaggaac gaaggtttcc ctaatagtaa 9480

tttgtgtgaa agttctggta gtctgtcagt taagaaaaaa tacgggtaga aggttaaccg 9540

gcccgcggcg acgcgtggcc tgagaggcct tcagagagtt aagaaaaaac taccggttgt 9600

agatgaccaa aggacgatat acgggtagaa cggtaagaga ggccgcccct caattgcgag 9660

ccaggcttca caacctccgt tctaccgctt caccgacaac agtcctcaat catggaccgc 9720

gccgttagcc aagttgcgtt agagaatgat gaaagagagg caaaaaatac atggcgcttg 9780

atattccgga ttgcaatctt attcttaaca gtagtgacct tggctatatc tgtagcctcc 9840

cttttatata gcatgggggc tagcacacct agcgatcttg taggcatacc gactaggatt 9900

tccagggcag aagaaaagat tacatctaca cttggttcca atcaagatgt agtagatagg 9960

atatataagc aagtggccct tgagtctccg ttggcattgt taaatactga gaccacaatt 10020

atgaacgcaa taacatctct ctcttatcag attaatggag ctgcaaacaa cagtgggtgg 10080

ggggcaccta tccatgaccc agattatata ggggggatag gcaaagaact cattgtagat 10140

gatgctagtg atgtcacatc attctatccc tctgcatttc aagaacatct gaattttatc 10200

ccggcgccta ctacaggatc aggttgcact cgaataccct catttgacat gagtgctacc 10260

cattactgct acacccataa tgtaatattg tctggatgca gagatcactc acattcatat 10320

cagtatttag cacttggtgt gctccggaca tctgcaacag ggagggtatt cttttctact 10380

ctgcgttcca tcaacctgga cgacacccaa aatcggaagt cttgcagtgt gagtgcaact 10440

cccctgggtt gtgatatgct gtgctcgaaa gtcacggaga cagaggaaga agattataac 10500

tcagctgtcc ctacgcggat ggtacatggg aggttagggt tcgacggcca gtaccacgaa 10560

aaggacctag atgtcacaac attattcggg gactgggtgg ccaactaccc aggagtaggg 10620

ggtggatctt ttattgacag ccgcgtatgg ttctcagtct acggagggtt aaaacccaat 10680

tcacccagtg acactgtaca ggaagggaaa tatgtgatat acaagcgata caatgacaca 10740

tgcccagatg agcaagacta ccagattcga atggccaagt cttcgtataa gcctggacgg 10800

tttggtggga aacgcataca gcaggctatc ttatctatca aggtgtcaac atccttaggc 10860

gaagacccgg tactgactgt accgcccaac acagtcacac tcatgggggc cgaaggcaga 10920

attctcacag tagggacatc tcatttcttg tatcaacgag ggtcatcata cttctctccc 10980

gcgttattat atcctatgac agtcagcaac aaaacagcca ctcttcatag tccttataca 11040

ttcaatgcct tcactcggcc aggtagtatc ccttgccagg cttcagcaag atgccccaac 11100

tcgtgtgtta ctggagtcta tacagatcca tatcccctaa tcttctatag aaaccacacc 11160

ttgcgagggg tattcgggac aatgcttgat ggtgtacaag caagacttaa ccctgcgtct 11220

gcagtattcg atagcacatc ccgcagtcgc attactcgag tgagttcaag cagtaccaaa 11280

gcagcataca caacatcaac ttgttttaaa gtggtcaaga ctaataagac ctattgtctc 11340

agcattgctg aaatatctaa tactctcttc ggagaattca gaatcgtccc gttactagtt 11400

gagatcctca aagatgacgg ggttagagaa gccaggtctg gctagttgag tcaattataa 11460

aggagttgga aagatggcat tgtatcacct atcttctgcg acatcaagaa tcaaaccgaa 11520

tgccggcgcg tgctcgaatt ccatgttgcc agttgaccac aatcagccag tgctcatgcg 11580

atcagattaa gccttgtcaa tagtctcttg attaagaaaa aatgtaagtg gcaatgagat 11640

acaaggcaaa acagctcatg gtaaataata cgggtaggac atggcgagct ccggtcctga 11700

aagggcagag catcagatta tcctaccaga gtcacacctg tcttcaccat tggtcaagca 11760

caaactactc tattactgga aattaactgg gctaccgctt cctgatgaat gtgacttcga 11820

ccacctcatt ctcagccgac aatggaaaaa aatacttgaa tcggcctctc ctgatactga 11880

gagaatgata aaactcggaa gggcagtaca ccaaactctt aaccacaatt ccagaataac 11940

cggagtgctc caccccaggt gtttagaaga actggctaat attgaggtcc cagattcaac 12000

caacaaattt cggaagattg agaagaagat ccaaattcac aacacgagat atggagaact 12060

gttcacaagg ctgtgtacgc atatagagaa gaaactgctg gggtcatctt ggtctaacaa 12120

tgtcccccgg tcagaggagt tcagcagcat tcgtacggat ccggcattct ggtttcactc 12180

aaaatggtcc acagccaagt ttgcatggct ccatataaaa cagatccaga ggcatctgat 12240

ggtggcagct aggacaaggt ctgcggccaa caaattggtg atgctaaccc ataaggtagg 12300

ccaagtcttt gtcactcctg aacttgtcgt tgtgacgcat acgaatgaga acaagttcac 12360

atgtcttacc caggaacttg tattgatgta tgcagatatg atggagggca gagatatggt 12420

caacataata tcaaccacgg cggtgcatct cagaagctta tcagagaaaa ttgatgacat 12480

tttgcggtta atagacgctc tggcaaaaga cttgggtaat caagtctacg atgttgtatc 12540

actaatggag ggatttgcat acggagctgt ccagctactc gagccgtcag gtacatttgc 12600

aggagatttc ttcgcattca acctgcagga gcttaaagac attctaattg gcctcctccc 12660

caatgatata gcagaatccg tgactcatgc aatcgctact gtattctctg gtttagaaca 12720

gaatcaagca gctgagatgt tgtgtctgtt gcgtctgtgg ggtcacccac tgcttgagtc 12780

ccgtattgca gcaaaggcag tcaggagcca aatgtgcgca ccgaaaatgg tagactttga 12840

tatgatcctt caggtactgt ctttcttcaa gggaacaatc atcaacgggt acagaaagaa 12900

gaatgcaggt gtgtggccgc gagtcaaagt ggatacaata tatgggaagg tcattgggca 12960

actacatgca gattcagcag agatttcaca cgatatcatg ttgagagagt ataagagttt 13020

atctgcactt gaatttgagc catgtataga atatgaccct gtcaccaacc tgagcatgtt 13080

cctaaaagac aaggcaatcg cacaccccaa cgataattgg cttgcctcgt ttaggcggaa 13140

ccttctctcc gaagaccaga agaaacatgt aaaagaagca acttcgacta atcgcctctt 13200

gatagagtttt ttagagtcaa atgattttga tccatataaa gagatggaat atctgacgac 13260

ccttgagtac cttagagatg acaatgtggc agtatcatac tcgctcaagg agaaggaagt 13320

gaaagttaat ggacggatct tcgctaagct gacaaagaag ttaaggaact gtcaggtgat 13380

ggcggaaggg atcctagccg atcagattgc acctttcttt cagggaaatg gagtcattca 13440

ggatagcata tccttgacca agagtatgct agcgatgagt caactgtctt ttaacagcaa 13500

taagaaacgt atcactgact gtaaagaaag agtatcttca aaccgcaatc atgatccgaa 13560

aagcaagaac cgtcggagag ttgcaacctt cataacaact gacctgcaaa agtactgtct 13620

taattggaga tatcagacaa tcaaattgtt cgctcatgcc atcaatcagt tgatgggcct 13680

acctcacttc ttcgaatgga ttcacctaag actgatggac actacgatgt tcgtaggaga 13740

ccctttcaat cctccaagtg accctactga ctgtgacctc tcaagagtcc ctaatgatga 13800

catatatatt gtcagtgcca gagggggtat cgaaggatta tgccagaagc tatggacaat 13860

gatctcaatt gctgcaatcc aacttgctgc agctagatcg cattgtcgtg ttgcctgtat 13920

ggtacagggt gataatcaag taatagcagt aacgagagag gtaagatcag acgactctcc 13980

ggagatggtg ttgacacagt tgcatcaagc cagtgataat ttcttcaagg aattaattca 14040

tgtcaatcat ttgattggcc ataatttgaa ggatcgtgaa accatcaggt cagacacatt 14100

cttcatatac agcaaacgaa tcttcaaaga tggagcaatc ctcagtcaag tcctcaaaaa 14160

ttcatctaaa ttagtgctag tgtcaggtga tctcagtgaa aacaccgtaa tgtcctgtgc 14220

caacattgcc tctactgtag cacggctatg cgagaacggg cttcccaaag acttctgtta 14280

ctatttaaac tatataatga gttgtgtgca gacatacttt gactctgagt tctccatcac 14340

caacaattcg caccccgatc ttaatcagtc gtggattgag gacatctctt ttgtgcactc 14400

atatgttctg actcctgccc aattaggggg actgagtaac cttcaatact caaggctcta 14460

cactagaaat atcggtgacc cggggactac tgcttttgca gagatcaagc gactagaagc 14520

agtgggatta ctgagtccta acattatgac taatatctta actaggccgc ctgggaatgg 14580

agattgggcc agtctgtgca acgacccata ctctttcaat tttgagactg ttgcaagccc 14640

aaatattgtt cttaagaaac atacgcaaag agtcctattt gaaacttgtt caaatccctt 14700

attgtctgga gtgcacacag aggataatga ggcagaagag aaggcattgg ctgaattctt 14760

gcttaatcaa gaggtgattc atccccgcgt tgcgcatgcc atcatggagg caagctctgt 14820

aggtaggaga aagcaaattc aagggcttgt tgacacaaca aacaccgtaa ttaagattgc 14880

gcttactagg aggccattag gcatcaagag gctgatgcgg atagtcaatt attctagcat 14940

gcatgcaatg ctgtttagag acgatgtttt ttcctccagt agatccaacc accccttagt 15000

ctcttctaat atgtgttctc tgacactggc agactatgca cggaatagaa gctggtcacc 15060

tttgacggga ggcaggaaaa tactgggtgt atctaatcct gatacgatag aactcgtaga 15120

gggtgagatt cttagtgtaa gcggagggtg tacaagatgt gacagcggag atgaacaatt 15180

tacttggttc catcttccaa gcaatataga attgaccgat gacaccagca agaatcctcc 15240

gatgagggta ccatatctcg ggtcaaagac acaggagagg agagctgcct cacttgcaaa 15300

aatagctcat atgtcgccac atgtaaaggc tgccctaagg gcatcatccg tgttgatctg 15360

ggcttatggg gataatgaag taaattggac tgctgctctt acgattgcaa aatctcggtg 15420

taatgtaaac ttagagtatc ttcggttact gtccccttta cccacggctg ggaatcttca 15480

acatagacta gatgatggta taactcagat gacattcacc cctgcatctc tctacagggt 15540

gtcaccttac attcacatat ccaatgattc tcaaaggctg ttcactgaag aaggagtcaa 15600

agaggggaat gtggtttacc aacagatcat gctcttgggt ttatctctaa tcgaatcgat 15660

ctttccaata acaacaacca ggacatatga tgagatcaca ctgcacctac atagtaaatt 15720

tagttgctgt atcagagaag cacctgttgc ggttcctttc gagctacttg gggtggtacc 15780

ggaactgagg acagtgacct caaataagtt tatgtatgat cctagccctg tatcggaggg 15840

agactttgcg agacttgact tagctatctt caagagttat gagcttaatc tggagtcata 15900

tcccacgata gagctaatga acattctttc aatatccagc gggaagttga ttggccagtc 15960

tgtggtttct tatgatgaag atacctccat aaagaatgac gccataatag tgtatgacaa 16020

tacccgaaat tggatcagtg aagctcagaa ttcagatgtg gtccgcctat ttgaatatgc 16080

agcacttgaa gtgctcctcg actgttctta ccaactctat tacctgagag taagaggcct 16140

agacaatatt gtcttatata tgggtgattt atacaagaat atgccaggaa ttctactttc 16200

caacattgca gctacaatat ctcatcccgt cattcattca aggttacatg cagtgggcct 16260

ggtcaaccat gacggatcac accaacttgc agatacggat tttatcgaaa tgtctgcaaa 16320

actattagta tcttgcaccc gacgtgtgat ctccggctta tattcaggaa ataagtatga 16380

tctgctgttc ccatctgtct tagatgataa cctgaatgag aagatgcttc agctgatatc 16440

ccggttatgc tgtctgtaca cggtactctt tgctacaaca agagaaatcc cgaaaataag 16500

aggcttaact gcagaagaga aatgttcaat actcactgag tatttactgt cggatgctgt 16560

gaaaccatta cttagtcccg atcaagtgag ctctatcatg tctcctaaca taattacatt 16620

cccagctaat ctgtactaca tgtctcggaa gagcctcaat ttgatcaggg aaagggagga 16680

cagggatact atcctggcgt tgttgttccc ccaagagcca ttattagagt tcccttctgt 16740

gcaagatatt ggtgctcgag tgaaagatcc attcacccga caacctgcgg catttttgca 16800

agagttagat ttgagtgctc cagcaaggta tgacgcattc acacttagtc agattcatcc 16860

tgaactcaca tctccaaatc cggaggaaga ctacttagta cgatacttgt tcagagggat 16920

agggactgca tcttcctctt ggtataaggc atctcatctc ctttctgtac ccgaggtaag 16980

atgtgcaaga cacgggaact ccttatactt agctgaaggg agcggagcca tcatgagtct 17040

tctcgaactg catgtaccac atgaaactat ctattacaat acgctctttt caaatgagat 17100

gaaccccccg caacgacatt tcgggccgac cccaactcag tttttgaatt cggttgttta 17160

taggaatcta caggcggagg taacatgcaa agatggattt gtccaagagt tccgtccatt 17220

atggagagaa aatacagagg aaagtgacct gacctcagat aaagtagtgg ggtatattac 17280

atctgcagtg ccctacagat ctgtatcatt gctgcattgt gacattgaaa ttcctccagg 17340

gtccaatcaa agcttactag atcaactagc tatcaattta tctctgattg ccatgcattc 17400

tgtaagggag ggcggggtag taatcatcaa agtgttgtat gcaatgggat actactttca 17460

tctactcatg aacttgtttg ctccgtgttc cacaaaagga tatattctct ctaatggtta 17520

tgcatgtcga ggagatatgg agtgttacct ggtatttgtc atgggttacc tgggcgggcc 17580

tacatttgta catgaggtgg tgaggatggc gaaaactctg gtgcagcggc acggtacgct 17640

tttgtctaaa tcagatgaga tcacactgac caggttattc acctcacagc ggcagcgtgt 17700

gacagacatc ctatccagtc ctttaccaag attaataaag tacttgagga agaatattga 17760

cactgcgctg attgaagccg ggggacagcc cgtccgtcca ttctgtgcgg agagtctggt 17820

gagcacgcta gcgaacataa ctcagataac ccagatcatc gctagtcaca ttgacacagt 17880

tatccggtct gtgatatata tggaagctga gggtgatctc gctgacacag tatttctatt 17940

taccccttac aatctctcta ctgacgggaa aaagaggaca tcacttaaac agtgcacgag 18000

acagatccta gaggttacaa tactaggtct tagagtcgaa aatctcaata aaataggcga 18060

tataatcagc ctagtgctta aaggcatgat ctccatggag gaccttatcc cactaaggac 18120

atacttgaag catagtacct gccctaaata tttgaaggct gtcctaggta ttaccaaact 18180

caaagaaatg tttacagaca cttctgtact gtacttgact cgtgctcaac aaaaattcta 18240

catgaaaact ataggcaatg cagtcaaagg atattacagt aactgtgact cttaacgaaa 18300

atcacatatt aataggctcc ttttttggcc aattgtattc ttgttgattt aatcatatta 18360

tgttagaaaa aagttgaacc ctgactcctt aggactcgaa ttcgaactca aataaatgtc 18420

ttaaaaaaag gttgcgcaca attattcttg agtgtagtct cgtcattcac caaatctttg 18480

tttggtttgg tggccggcat ggtcccagcc tcctcgctgg cgccggctgg gcaacattcc 18540

gaggggaccg tcccctcggt aatggcgaat gggacgcggc cgatccggct gctaacaaag 18600

cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca taaccccttg 18660

gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata tccggatcgg 18720

ccgatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc 18780

aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag 18840

gaggaactat atccggatgg ccgccaccgg tgggccttgc agcacatccc cccttcgcca 18900

g 18901

<210> 8

<211> 18955

<212> DNA

<213> Artificial Sequence

<220>

<223> Genome sequence of recombinant Newcastle disease virus rClone30-Anh-(F)-mOX40L

<400> 8

ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gtagcctgaa 60

tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 120

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 180

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 240

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 300

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 360

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 420

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 480

acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 540

tcggggaaat gtgcgcggaa cccctatttg ttattttttc taaatacatt caaatatgta 600

tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 660

gagtattcaa catttccgtg tcgcccttat tcccttttttt gcggcatttt gccttcctgt 720

ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 780

agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 840

agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 900

tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 960

tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 1020

cagtgctgcc ataagcatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1080

aggaccgaag gagctaaccg ctttttttca caacatgggg gatcatgtaa ctcgccttga 1140

tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1200

tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1260

ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1320

ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1380

cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1440

gacgggcagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1500

actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1560

aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1620

caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1680

aggatcttct tgagatcctt ttttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1740

accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1800

aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1860

ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1920

agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1980

accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 2040

gcgaacgacc tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct 2100

tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2160

cacgagggag cttccagggg ggaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2220

cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggccgagcc tatggaaaaa 2280

cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2340

ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2400

taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2460

gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 2520

cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttacct 2580

cactcattag gcaccccagg ctttacactt tatgcttccg gctcctatgt tgtgtggaat 2640

tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagctcgg 2700

aagcggccgc taatacgact cactataggg accaaacaga gaatccgtaa gttacgataa 2760

aaggcgaagg agcaattgaa gtcgcacggg tagaaggtgt gaatctcgag tgcgagcccg 2820

aagcacaaac tcgagaaagc cttctgccaa catgtcttcc gtatttgatg agtacgaaca 2880

gctcctcgcg gctcagactc gccccaatgg agctcatgga gggggagaaa aagggagtac 2940

cttaaaagta gacgtcccgg tattcactct taacagtgat gacccagaag atagatggag 3000

ctttgtggta ttctgcctcc ggattgctgt tagcgaagat gccaacaaac cactcaggca 3060

aggtgctctc atatctcttt tatgctccca ctcacaggta atgaggaacc atgttgccct 3120

tgcagggaaa cagaatgaag ccacattggc cgtgcttgag attgatggct ttgccaacgg 3180

cacgccccag ttcaacaata ggagtggagt gtctgaagag agagcacaga gatttgcgat 3240

gatagcagga tctctccctc gggcatgcag caacggaacc ccgttcgtca cagccggggc 3300

cgaagatgat gcaccagaag acatcaccga taccctggag aggatcctct ctatccaggc 3360

tcaagtatgg gtcacagtag caaaagccat gactgcgtat gagactgcag atgagtcgga 3420

aacaaggcga atcaataagt atatgcagca aggcagggtc caaaagaaat acatcctcta 3480

ccccgtatgc aggagcacaa tccaactcac gatcagacag tctcttgcag tccgcatctt 3540

tttggttagc gagctcaaga gaggccgcaa cacggcaggt ggtacctcta cttattataa 3600

cctggtaggg gacgtagact catacatcag gaataccggg cttactgcat tcttcttgac 3660

actcaagtac ggaatcaaca ccaagacatc agcccttgca cttagtagcc tctcaggcga 3720

catccagaag atgaagcagc tcatgcgttt gtatcggatg aaaggagata atgcgccgta 3780

catgacatta cttggtgata gtgaccagat gagctttgcg cctgccgagt atgcacaact 3840

ttactccttt gccatgggta tggcatcagt cctagataaa ggtactggga aataccaatt 3900

tgccagggac tttatgagca catcattctg gagacttgga gtagagtacg ctcaggctca 3960

gggaagtagc attaacgagg atatggctgc cgagctaaag ctaaccccag cagcaaggag 4020

gggcctggca gctgctgccc aacgggtctc cgaggagacc agcagcatag acatgcctac 4080

tcaacaagtc ggagtcctca ctgggcttag cgaggggggg tcccaagctc tacaaggcgg 4140

atcgaataga tcgcaagggc aaccagaagc cggggatggg gagacccaat tcctggatct 4200

gatgagagcg gtagcaaata gcatgaggga ggcgccaaac tctgcacagg gcactcccca 4260

atcggggcct cccccaactc ctgggccatc ccaagataac gacaccgact gggggtattg 4320

atggacaaaa cccagcctgc ttccacaaaa acatcccaat gccctcaccc gtagtcgacc 4380

cctcgatttg cggctctata tgaccacacc ctcaaacaaa catccccctc tttcctccct 4440

ccccctgctg tacaactccg cacgccctag ataccacagg cacaatgcgg ctcactaaca 4500

atcaaaacag agccgaggga attagaaaaa agtacgggta gaagagggat attcagagat 4560

cagggcaagt ctcccgagtc tctgctctct cctctacctg atagaccagg acaaacatgg 4620

ccacctttac agatgcagag atcgacgagc tatttgagac aagtggaact gtcattgaca 4680

acataattac agcccagggt aaaccagcag agactgttgg aaggagtgca atcccacaag 4740

gcaagaccaa ggtgctgagc gcagcatggg agaagcatgg gagcatccag ccaccggcca 4800

gtcaagacaa ccccgatcga caggacagat ctgacaaaca accatccaca cccgagcaaa 4860

cgaccccgca tgacagcccg ccggccacat ccgccgacca gccccccacc caggccacag 4920

acgaagccgt cgacacacag ctcaggaccg gagcaagcaa ctctctgctg ttgatgcttg 4980

acaagctcag caataaatcg tccaatgcta aaaagggccc atggtcgagc ccccaagagg 5040

ggaatcacca acgtccgact caacagcagg ggagtcaacc cagtcgcgga aacagtcagg 5100

aaagaccgca gaaccaagtc aaggccgccc ctggaaacca gggcacagac gtgaacacag 5160

catatcatgg acaatgggag gagtcacaac tatcagctgg tgcaacccct catgctctcc 5220

gatcaaggca gagccaagac aatacccttg tatctgcgga tcatgtccag ccacctgtag 5280

actttgtgca agcgatgatg tctatgatgg aggcgatatc acagagagta agtaaggttg 5340

actatcagct agatcttgtc ttgaaacaga catcctccat ccctatgatg cggtccgaaa 5400

tccaacagct gaaaacatct gttgcagtca tggaagccaa cttgggaatg atgaagattc 5460

tggatcccgg ttgtgccaac atttcatctc tgagtgatct acgggcagtt gcccgatctc 5520

acccggtttt agtttcaggc cctggagacc cctctcccta tgtgacacaa ggaggcgaaa 5580

tggcacttaa taaactttcg caaccagtgc cacatccatc tgaattgatt aaacccgcca 5640

ctgcatgcgg gcctgatata ggagtggaaa aggacactgt ccgtgcattg atcatgtcac 5700

gcccaatgca cccgagttct tcagccaagc tcctaagcaa gttagatgca gccgggtcga 5760

tcgaggaaat caggaaaatc aagcgccttg ctctaaatgg ctaattacta ctgccacacg 5820

tagcgggtcc ctgtccactc ggcatcacac ggaatctgca ccgagttccc ccccgcagac 5880

ccaaggtcca actctccaag cggcaatcct ctctcgcttc ctcagcccca ctgaatgatc 5940

gcgtaaccgt aattaatcta gctacattta agattaagaa aaaatacggg tagaatcccc 6000

gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 6060

ggatccactg gtcaactctc ttcctctccg gcaaaggacc ctccaatcca aagactcaga 6120

ggagcagtta ccagatgtga ggatgggcaa ctattcatca gctcatacaa gaatgagtat 6180

caaactatgg aggtgcagaa caattcggtt gtcatcaagt gtgatgggct ttatatcatc 6240

tacctgaagg gctcctttttt ccaggaggtc aagattgacc ttcatttccg ggaggatcat 6300

aatcccatct ctattccaat gctgaacgat ggtcgaagga ttgtcttcac tgtggtggcc 6360

tctttggctt tcaaagataa agtttacctg actgtaaatg ctcctgatac tctctgcgaa 6420

cacctccaga taaatgatgg ggagctgatt gttgtccagc taacgcctgg atactgtgct 6480

cctgaaggat cttaccacag cactgtgaac caagtaccac tgtgattaag aaaaaatacg 6540

ggtagaaggg tttaaaccct tggagtgccc caattgtgcc aagatggact catctaggac 6600

aattgggctg tactttgatt ctgcccattc ttctagcaac ctgttagcat ttccgatcgt 6660

cctacaagac acaggagatg ggaagaagca aatcgccccg caatatagga tccagcgcct 6720

tgacttgtgg actgatagta aggaggactc agtattcatc accacctatg gattcatctt 6780

tcaagttggg aatgaagaag ccactgtcgg catgatcgat gataaaccca agcgcgagtt 6840

actttccgct gcgatgctct gcctaggaag cgtcccaaat accggagacc ttattgagct 6900

ggcaagggcc tgtctcacta tgatagtcac atgcaagaag agtgcaacta atactgagag 6960

aatggttttc tcagtagtgc aggcacccca agtgctgcaa agctgtaggg ttgtggcaaa 7020

caaatactca tcagtgaatg cagtcaagca cgtgaaagcg ccagagaaga ttcccgggag 7080

tggaacccta gaatacaagg tgaactttgt ctccttgact gtggtaccga agaaggatgt 7140

ctacaagatc ccagctgcag tattgaaggt ttctggctcg agtctgtaca atcttgcgct 7200

caatgtcact attaatgtgg aggtagaccc gaggagtcct ttggttaaat ctctgtctaa 7260

gtctgacagc ggatactatg ctaacctctt cttgcatatt ggacttatga ccaccgtaga 7320

taggaagggg aagaaagtga catttgacaa gctggaaaag aaaataagga gccttgatct 7380

atctgtcggg ctcagtgatg tgctcgggcc ttccgtgttg gtaaaagcaa gaggtgcacg 7440

gactaagctt ttggcacctt tcttctctag cagtgggaca gcctgctatc ccatagcaaa 7500

tgcttctcct caggtggcca agatactctg gagtcaaacc gcgtgcctgc ggagcgttaa 7560

aatcattatc caagcaggta cccaacgcgc tgtcgcagtg accgccgacc acgaggttac 7620

ctctactaag ctggagaagg ggcacaccct tgccaaatac aatcctttta agaaataagc 7680

tgcgtctctg agattgcgct ccgcccactc acccagatca tcatgacaca aaaaactaat 7740

ctgtcttgat tatttacagt tagtttacct gtctatcaag ttagaaaaaa cacgggtaga 7800

agattctgga tcccggttgg cgccctccag gtgcaagatg ggccccaaac cccccaccgg 7860

aaccccagcg cctctggtgc tgatcgcccg gaccgcgctg gcgttgggct gtgtctgtcc 7920

ggcgggctct cttgacggca gacctcttgc agctgcaggg attgtggtaa cgagagataa 7980

agcagtcaat atatacactt catctcaaac ggggtcaatc atagtcaagt tactcccaaa 8040

tatgcccaaa gacaaggagg cgtgcgcaaa agccccatta gaggcgtaca atagaacact 8100

gaccacttta ctcactcctc ttggcgactc catccgcagg atacaagggt ctgcaactac 8160

atctagagga aggagacaga aacgttttgt aggtgctatc attggcagta tagctcttgg 8220

ggttgcgaca gctgcacaag taacagcagc tgcagctctg atacaagcca accagaacgc 8280

tgccaacatc ctccggctta aggagagcat tgctgcaacc aatgaagctg tgcacgaggt 8340

cactgacgga ttatcacaac tagcgatggc gattgggaag atgcagcagt ttgttaatga 8400

ccagtttaat aatacggcgc gagaattgga ctgcatcaaa attacacaac aggttggtgt 8460

cgaactcaat ttgtatctaa ctgaactgac tacagtattc gggccacaaa tcacttcccc 8520

tgctttaact cagctaacta tccaggcact ttataattta gctggtggca atatgaatta 8580

cttattgact aagttaggtg tagggaacaa tcaacttagc tcattaatta gtagtggcct 8640

gatcactggc aaccccattt tatatgactc acagacccaa ctcttaggca tacagataaa 8700

tgtaccctca gtcgggagcc taaataatat gcgtgccacc tacttggaga ccttatccgt 8760

aagcacaacc agggggttcg cctcagcact tgtcccgaaa gttgtgacgc aagttggttc 8820

tgtgatagaa gaacttgaca cctcatattg tatagaatct aatctggatt tatattgtac 8880

aaggatagtg acattcccca tgtctcccgg catttattcc tgtctgagcg gtaatacgtc 8940

agcttgtatg tattcaaaga ctgagggtgc actcactaca ccatacatag ctctcaaggg 9000

ctcagttatt gctaattgca agatgattac atgtagatgt gcagaccccc caggtatcat 9060

atcgcaaaat tacggagaag ctgtgtccct aatagataaa cattcatgta atgtcttatc 9120

cctagacgga ataaccctga ggctcagtgg ggaatttgat gcgacctatc aaaagaacat 9180

cttaatacta gattcccagg tcatcgtgac aggcaatctc gatatatcaa ctgaacttgg 9240

gaatgtcaac aactcgataa gcagtgctct ggacaaatta gcggaaagta acagcaagtt 9300

aaacaaagtc aatgtcaacc taactagcac atctgctctc attacttata ttgttctagc 9360

tgtcatatct cttgttttcg gcgtaattag cctgattcta gcgtgctgct tgatgtataa 9420

acaaaaagca caacaaaaga ccttactatg gcttgggaac aataccctcg atcagatgag 9480

agccaccaca aaaacatgaa cacagatgag gaacgaaggt ttccctaata gtaatttgtg 9540

tgaaagttct ggtagtctgt cagttaagaa aaaatacggg tagaaggtta accggcccgc 9600

ggcgacgcgt ggcctgagag gccttcagag agttaagaaa aaactaccgg ttgtagatga 9660

ccaaaggacg atatacgggt agaacggtaa gagaggccgc ccctcaattg cgagccaggc 9720

ttcacaacct ccgttctacc gcttcaccga caacagtcct caatcatgga ccgcgccgtt 9780

agccaagttg cgttagagaa tgatgaaaga gaggcaaaaa atacatggcg cttgatattc 9840

cggattgcaa tcttattctt aacagtagtg accttggcta tatctgtagc ctccctttta 9900

tatagcatgg gggctagcac acctagcgat cttgtaggca taccgactag gatttccagg 9960

gcagaagaaa agattacatc tacacttggt tccaatcaag atgtagtaga taggatat 10020

aagcaagtgg cccttgagtc tccgttggca ttgttaaata ctgagaccac aattatgaac 10080

gcaataacat ctctctctta tcagattaat ggagctgcaa acaacagtgg gtggggggca 10140

cctatccatg acccagatta tatagggggg ataggcaaag aactcattgt agatgatgct 10200

agtgatgtca catcattcta tccctctgca tttcaagaac atctgaattt tatcccggcg 10260

cctactacag gatcaggttg cactcgaata ccctcatttg acatgagtgc tacccattac 10320

tgctacaccc ataatgtaat attgtctgga tgcagagatc actcacattc atatcagtat 10380

ttagcacttg gtgtgctccg gacatctgca acagggaggg tattcttttc tactctgcgt 10440

tccatcaacc tggacgacac ccaaaatcgg aagtcttgca gtgtgagtgc aactcccctg 10500

ggttgtgata tgctgtgctc gaaagtcacg gagacagagg aagaagatta taactcagct 10560

gtccctacgc ggatggtaca tgggaggtta gggttcgacg gccagtacca cgaaaaggac 10620

ctagatgtca caacattatt cggggactgg gtggccaact acccaggagt agggggtgga 10680

tcttttattg acagccgcgt atggttctca gtctacggag ggttaaaacc caattcaccc 10740

agtgacactg tacaggaagg gaaatatgtg atatacaagc gatacaatga cacatgccca 10800

gatgagcaag actaccagat tcgaatggcc aagtcttcgt ataagcctgg acggtttggt 10860

gggaaacgca tacagcaggc tatcttatct atcaaggtgt caacatcctt aggcgaagac 10920

ccggtactga ctgtaccgcc caacacagtc acactcatgg gggccgaagg cagaattctc 10980

acagtaggga catctcattt cttgtatcaa cgagggtcat catacttctc tcccgcgtta 11040

ttatatccta tgacagtcag caacaaaaca gccactcttc atagtcctta tacattcaat 11100

gccttcactc ggccaggtag tatcccttgc caggcttcag caagatgccc caactcgtgt 11160

gttactggag tctatacaga tccatatccc ctaatcttct atagaaacca caccttgcga 11220

ggggtattcg ggacaatgct tgatggtgta caagcaagac ttaaccctgc gtctgcagta 11280

ttcgatagca catcccgcag tcgcattact cgagtgagtt caagcagtac caaagcagca 11340

tacacaacat caacttgttt taaagtggtc aagactaata agacctattg tctcagcatt 11400

gctgaaatat ctaatactct cttcggagaa ttcagaatcg tcccgttact agttgagatc 11460

ctcaaagatg acggggttag agaagccagg tctggctagt tgagtcaatt ataaaggagt 11520

tggaaagatg gcattgtatc acctatcttc tgcgacatca agaatcaaac cgaatgccgg 11580

cgcgtgctcg aattccatgt tgccagttga ccacaatcag ccagtgctca tgcgatcaga 11640

ttaagccttg tcaatagtct cttgattaag aaaaaatgta agtggcaatg agatacaagg 11700

caaaacagct catggtaaat aatacgggta ggacatggcg agctccggtc ctgaaagggc 11760

agagcatcag attatcctac cagagtcaca cctgtcttca ccattggtca agcacaaact 11820

actctattac tggaaattaa ctgggctacc gcttcctgat gaatgtgact tcgaccacct 11880

cattctcagc cgacaatgga aaaaaatact tgaatcggcc tctcctgata ctgagagaat 11940

gataaaactc ggaagggcag tacaccaaac tcttaaccac aattccagaa taaccggagt 12000

gctccacccc aggtgtttag aagaactggc taatattgag gtcccagatt caaccaacaa 12060

atttcggaag attgagaaga agatccaaat tcacaacacg agatatggag aactgttcac 12120

aaggctgtgt acgcatatag agaagaaact gctggggtca tcttggtcta acaatgtccc 12180

ccggtcagag gagttcagca gcattcgtac ggatccggca ttctggtttc actcaaaatg 12240

gtccacagcc aagtttgcat ggctccatat aaaacagatc cagaggcatc tgatggtggc 12300

agctaggaca aggtctgcgg ccaacaaatt ggtgatgcta acccataagg taggccaagt 12360

ctttgtcact cctgaacttg tcgttgtgac gcatacgaat gagaacaagt tcacatgtct 12420

tacccaggaa cttgtattga tgtatgcaga tatgatggag ggcagagata tggtcaacat 12480

aatatcaacc acggcggtgc atctcagaag cttatcagag aaaattgatg acattttgcg 12540

gttaatagac gctctggcaa aagacttggg taatcaagtc tacgatgttg tatcactaat 12600

ggagggattt gcatacggag ctgtccagct actcgagccg tcaggtacat ttgcaggaga 12660

tttcttcgca ttcaacctgc aggagcttaa agacattcta attggcctcc tccccaatga 12720

tatagcagaa tccgtgactc atgcaatcgc tactgtattc tctggtttag aacagaatca 12780

agcagctgag atgttgtgtc tgttgcgtct gtggggtcac ccactgcttg agtcccgtat 12840

tgcagcaaag gcagtcagga gccaaatgtg cgcaccgaaa atggtagact ttgatatgat 12900

ccttcaggta ctgtctttct tcaagggaac aatcatcaac gggtacagaa agaagaatgc 12960

aggtgtgtgg ccgcgagtca aagtggatac aatatatggg aaggtcattg ggcaactaca 13020

tgcagattca gcagagattt cacacgatat catgttgaga gagtataaga gtttatctgc 13080

acttgaattt gagccatgta tagaatatga ccctgtcacc aacctgagca tgttcctaaa 13140

agacaaggca atcgcacacc ccaacgataa ttggcttgcc tcgtttaggc ggaaccttct 13200

ctccgaagac cagaagaaac atgtaaaaga agcaacttcg actaatcgcc tcttgataga 13260

gtttttagag tcaaatgatt ttgatccata taaagagatg gaatatctga cgacccttga 13320

gtaccttaga gatgacaatg tggcagtatc atactcgctc aaggagaagg aagtgaaagt 13380

taatggacgg atcttcgcta agctgacaaa gaagttaagg aactgtcagg tgatggcgga 13440

agggatccta gccgatcaga ttgcaccttt ctttcaggga aatggagtca ttcaggatag 13500

catatccttg accaagagta tgctagcgat gagtcaactg tcttttaaca gcaataagaa 13560

acgtatcact gactgtaaag aaagagtatc ttcaaaccgc aatcatgatc cgaaaagcaa 13620

gaaccgtcgg agagttgcaa ccttcataac aactgacctg caaaagtact gtcttaattg 13680

gagatatcag acaatcaaat tgttcgctca tgccatcaat cagttgatgg gcctacctca 13740

cttcttcgaa tggattcacc taagactgat ggacactacg atgttcgtag gagacccttt 13800

caatcctcca agtgacccta ctgactgtga cctctcaaga gtccctaatg atgacatata 13860

tattgtcagt gccagagggg gtatcgaagg attatgccag aagctatgga caatgatctc 13920

aattgctgca atccaacttg ctgcagctag atcgcattgt cgtgttgcct gtatggtaca 13980

gggtgataat caagtaatag cagtaacgag agaggtaaga tcagacgact ctccggagat 14040

ggtgttgaca cagttgcatc aagccagtga taatttcttc aaggaattaa ttcatgtcaa 14100

tcatttgatt ggccataatt tgaaggatcg tgaaaccatc aggtcagaca cattcttcat 14160

atacagcaaa cgaatcttca aagatggagc aatcctcagt caagtcctca aaaattcatc 14220

taaattagtg ctagtgtcag gtgatctcag tgaaaacacc gtaatgtcct gtgccaacat 14280

tgcctctact gtagcacggc tatgcgagaa cgggcttccc aaagacttct gttactattt 14340

aaactatata atgagttgtg tgcagacata ctttgactct gagttctcca tcaccaacaa 14400

ttcgcacccc gatcttaatc agtcgtggat tgaggacatc tcttttgtgc actcatatgt 14460

tctgactcct gcccaattag ggggactgag taaccttcaa tactcaaggc tctacactag 14520

aaatatcggt gacccgggga ctactgcttt tgcagagatc aagcgactag aagcagtggg 14580

attactgagt cctaacatta tgactaatat cttaactagg ccgcctggga atggagattg 14640

ggccagtctg tgcaacgacc catactcttt caattttgag actgttgcaa gcccaaatat 14700

tgttcttaag aaacatacgc aaagagtcct atttgaaact tgttcaaatc ccttattgtc 14760

tggagtgcac acagaggata atgaggcaga agagaaggca ttggctgaat tcttgcttaa 14820

tcaagaggtg attcatcccc gcgttgcgca tgccatcatg gaggcaagct ctgtaggtag 14880

gagaaagcaa attcaagggc ttgttgacac aacaaacacc gtaattaaga ttgcgcttac 14940

taggaggcca ttaggcatca agaggctgat gcggatagtc aattattcta gcatgcatgc 15000

aatgctgttt agagacgatg ttttttcctc cagtagatcc aaccacccct tagtctcttc 15060

taatatgtgt tctctgacac tggcagacta tgcacggaat agaagctggt cacctttgac 15120

gggaggcagg aaaatactgg gtgtatctaa tcctgatacg atagaactcg tagagggtga 15180

gattcttagt gtaagcggag ggtgtacaag atgtgacagc ggagatgaac aatttacttg 15240

gttccatctt ccaagcaata tagaattgac cgatgacacc agcaagaatc ctccgatgag 15300

ggtaccatat ctcgggtcaa agacacagga gaggagagct gcctcacttg caaaaatagc 15360

tcatatgtcg ccacatgtaa aggctgccct aagggcatca tccgtgttga tctgggctta 15420

tggggataat gaagtaaatt ggactgctgc tcttacgatt gcaaaatctc ggtgtaatgt 15480

aaacttagag tatcttcggt tactgtcccc tttacccacg gctgggaatc ttcaacatag 15540

actagatgat ggtataactc agatgacatt cacccctgca tctctctaca gggtgtcacc 15600

ttacattcac atatccaatg attctcaaag gctgttcact gaagaaggag tcaaagaggg 15660

gaatgtggtt taccaacaga tcatgctctt gggtttatct ctaatcgaat cgatctttcc 15720

aataacaaca accaggacat atgatgagat cacactgcac ctacatagta aatttagttg 15780

ctgtatcaga gaagcacctg ttgcggttcc tttcgagcta cttggggtgg taccggaact 15840

gaggacagtg acctcaaata agtttatgta tgatcctagc cctgtatcgg agggagactt 15900

tgcgagactt gacttagcta tcttcaagag ttatgagctt aatctggagt catatcccac 15960

gatagagcta atgaacattc tttcaatatc cagcgggaag ttgattggcc agtctgtggt 16020

ttcttatgat gaagatacct ccataaagaa tgacgccata atagtgtatg acaatacccg 16080

aaattggatc agtgaagctc agaattcaga tgtggtccgc ctatttgaat atgcagcact 16140

tgaagtgctc ctcgactgtt cttaccaact ctattacctg agagtaagag gcctagacaa 16200

tattgtctta tatatgggtg atttatacaa gaatatgcca ggaattctac tttccaacat 16260

tgcagctaca atatctcatc ccgtcattca ttcaaggtta catgcagtgg gcctggtcaa 16320

ccatgacgga tcacaccaac ttgcagatac ggattttatc gaaatgtctg caaaactatt 16380

agtatcttgc acccgacgtg tgatctccgg cttatattca ggaaataagt atgatctgct 16440

gttcccatct gtcttagatg ataacctgaa tgagaagatg cttcagctga tatcccggtt 16500

atgctgtctg tacacggtac tctttgctac aacaagagaa atcccgaaaa taagaggctt 16560

aactgcagaa gagaaatgtt caatactcac tgagtattta ctgtcggatg ctgtgaaacc 16620

attacttagt cccgatcaag tgagctctat catgtctcct aacataatta cattcccagc 16680

taatctgtac tacatgtctc ggaagagcct caatttgatc agggaaaggg aggacaggga 16740

tactatcctg gcgttgttgt tcccccaaga gccattatta gagttccctt ctgtgcaaga 16800

tattggtgct cgagtgaaag atccattcac ccgacaacct gcggcatttt tgcaagagtt 16860

agatttgagt gctccagcaa ggtatgacgc attcacactt agtcagattc atcctgaact 16920

cacatctcca aatccggagg aagactactt agtacgatac ttgttcagag ggatagggac 16980

tgcatcttcc tcttggtata aggcatctca tctcctttct gtacccgagg taagatgtgc 17040

aagacacggg aactccttat acttagctga agggagcgga gccatcatga gtcttctcga 17100

actgcatgta ccacatgaaa ctatctatta caatacgctc ttttcaaatg agatgaaccc 17160

cccgcaacga catttcgggc cgaccccaac tcagtttttg aattcggttg tttataggaa 17220

tctacaggcg gaggtaacat gcaaagatgg atttgtccaa gagttccgtc cattatggag 17280

agaaaataca gaggaaagtg acctgacctc agataaagta gtggggtata ttacatctgc 17340

agtgccctac agatctgtat cattgctgca ttgtgacatt gaaattcctc cagggtccaa 17400

tcaaagctta ctagatcaac tagctatcaa tttatctctg attgccatgc attctgtaag 17460

ggagggcggg gtagtaatca tcaaagtgtt gtatgcaatg ggatactact ttcatctact 17520

catgaacttg tttgctccgt gttccacaaa aggatatatt ctctctaatg gttatgcatg 17580

tcgaggagat atggagtgtt acctggtatt tgtcatgggt tacctgggcg ggcctacatt 17640

tgtacatgag gtggtgagga tggcgaaaac tctggtgcag cggcacggta cgcttttgtc 17700

taaatcagat gagatcacac tgaccaggtt attcacctca cagcggcagc gtgtgacaga 17760

catcctatcc agtcctttac caagattaat aaagtacttg aggaagaata ttgacactgc 17820

gctgattgaa gccggggggac agcccgtccg tccattctgt gcggagagtc tggtgagcac 17880

gctagcgaac ataactcaga taacccagat catcgctagt cacattgaca cagttatccg 17940

gtctgtgata tatatggaag ctgagggtga tctcgctgac acagtatttc tatttacccc 18000

ttacaatctc tctactgacg ggaaaaagag gacatcactt aaacagtgca cgagacagat 18060

cctagaggtt acaatactag gtcttagagt cgaaaatctc aataaaatag gcgatataat 18120

cagcctagtg cttaaaggca tgatctccat ggaggacctt atcccactaa ggacatactt 18180

gaagcatagt acctgcccta aatatttgaa ggctgtccta ggtattacca aactcaaaga 18240

aatgtttaca gacacttctg tactgtactt gactcgtgct caacaaaaat tctacatgaa 18300

aactataggc aatgcagtca aaggatatta cagtaactgt gactcttaac gaaaatcaca 18360

tattaatagg ctcctttttt ggccaattgt attcttgttg atttaatcat attatgttag 18420

aaaaaagttg aaccctgact ccttaggact cgaattcgaa ctcaaataaa tgtcttaaaa 18480

aaaggttgcg cacaattatt cttgagtgta gtctcgtcat tcaccaaatc tttgtttggt 18540

ttggtggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca ttccgagggg 18600

accgtcccct cggtaatggc gaatgggacg cggccgatcc ggctgctaac aaagcccgaa 18660

aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct 18720

ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga tcggccgatc 18780

cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac 18840

tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa 18900

ctatatccgg atggccgcca ccggtgggcc ttgcagcaca tccccccttc gccag 18955

Claims (10)

What is claimed is: 1. A recombinant Newcastle disease virus genome, wherein the genome comprises an encoding gene of OX40L, and the encoding gene of OX40L is located between the P gene and the M gene of the Newcastle disease virus genome. 2. The recombinant Newcastle disease virus genome of claim 1, wherein the encoding gene of the OX40L is in the form of DNA or RNA; Preferably, the gene encoding OX40L is the sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 or a sequence with at least 80% identity to the sequence. 3. the Newcastle disease virus genome of the reorganization as claimed in claim 1 or 2, wherein, the sequence of the Newcastle disease virus genome of the reorganization is with SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.7 or SEQ ID NO. ID No. 8 is shown. 4. A recombinant Newcastle disease virus, wherein the virus comprises the recombinant Newcastle disease virus genome of any one of claims 1-3. 5. recombinant Newcastle disease virus as claimed in claim 4, wherein, the starting strain of described Newcastle disease virus is selected from: attenuated strain LaSota, Hitchner B1, V4, poisonous strain Mukteswar, Anhinga, virulent strain F48E9, JS/ 7/05/Ch, Italien, Herts/33, NDV-BJ; and any chimeric strain constructed by genetic engineering based on the originating strain. 6. A DNA molecule encoding the recombinant Newcastle disease virus genome of any one of claims 1-3. 7. A pharmaceutical composition, wherein, the pharmaceutical composition comprises the recombinant Newcastle disease virus genome of any one of claims 1-3, the recombinant Newcastle disease virus of claim 4 or 5 and/or The DNA molecule of claim 6; Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable pharmaceutical excipients are selected from solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators , stabilizers, glidants, flavoring agents, preservatives, suspending agents, antioxidants, penetration enhancers, pH adjusters, surfactants or diluents. 8. a method for preparing the described recombinant Newcastle disease virus of claim 4 or 5, comprising: (1) The cloning vector containing the DNA sequence of the coding gene of OX40L and the NDV viral vector are respectively digested with enzymes, and the DNA sequence of the coding gene of the OX40L obtained by the enzyme cutting is connected with the NDV viral vector to obtain recombination Newcastle disease virus plasmid; (2) Transfecting the recombinant Newcastle disease virus plasmid into cells and culturing the transfected cells to obtain the recombinant Newcastle disease virus. 9. The method of claim 8, wherein the cloning vector is constructed using a vector selected from the group consisting of: PUC57 vector, pMD18-T vector, pMD19-T vector, pBlueScript SK (+/-) vector, pBluescript II KS (+/-); Preferably, the NDV viral vector is a full-length cDNA sequence selected from the genome of the following NDV viruses: attenuated strains LaSota, Hitchner B1, V4, strains Mukteswar, Anhinga, virulent strains F48E9, JS/7/05/Ch , Italien, Herts/33, NDV-BJ; Preferably, the recombinant Newcastle disease virus plasmid is co-transfected into the cell with a helper plasmid selected from the group consisting of: pTM-NP, pTM-P and pTM-L; pCI-neo-NP, pCI-neo-P and pCI-neo-L; or pBluescript II KS(+/-)-NP, pBluescript II KS(+/-)-P and pBluescript II KS(+/-)-L; Preferably, the cells are selected from BHK-21 cells, BSR-T7/5 cells, VERO cells, DF-1 cells, 293 cells or MDCK cells. 10. the described recombinant Newcastle disease virus genome of any one of claim 1-3, the described recombinant Newcastle disease virus of claim 4 or 5, the described DNA molecule of claim 6 and/or described in claim 7 Use of the pharmaceutical composition in the preparation of a medicament for treating or improving cancer; Preferably, the cancer is selected from colon cancer, liver cancer, lung cancer, stomach cancer, rectal cancer, leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, bladder cancer, urothelial cancer, bronchial cancer, bone cancer , prostate cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, esophagus cancer, renal cell cancer, thyroid cancer, head and neck cancer, testicular cancer, endocrine adenocarcinoma, adrenal cancer, pituitary gland cancer, skin cancer, soft tissue cancer, vascular cancer, Brain cancer, nerve cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharyngeal cancer, cervical cancer, sarcoma, uterine cancer, glioblastoma, medulloblastoma, neuroblastoma, kidney cancer, Astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, acute myeloid leukemia, myelodysplastic syndrome, or sarcoma.

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