CN1405312A - A recombinant virus capable of specifically killing Epstein-Barr virus-related tumors and its construction method - Google Patents

Abstract

The present invention provides a type of proliferative recombinant virus that can specifically kill Epstein-Barr virus-related tumor cells and a method for constructing the same. By inserting a certain cis-acting element into the upstream region of the virus early gene, the recombinant virus can and only proliferate in cells latently infected or infected by Epstein-Barr virus, and at the same time, the target gene is inserted into a non-proliferation essential region of the viral genome that proliferates in the tumor cell, so that the target gene is efficiently expressed in the tumor cell, thereby specifically killing the tumor cell latently infected or infected by Epstein-Barr virus. The recombinant virus can be used to treat Epstein-Barr virus-related tumors, such as nasopharyngeal carcinoma, Hodgkin's lymphoma, gastric cancer, etc.

Description

A recombinant virus capable of specifically killing Epstein-Barr virus-related tumors and its construction method

The invention relates to the field of life sciences, in particular to a recombination capable of proliferating and expressing late genes in Epstein-Barr virus-infected or latently infected tumor cells, but not proliferating and not expressing late genes in Epstein-Barr virus-negative normal cells Cytolytic viruses, and methods of construction and propagation thereof.

Malignant tumors seriously threaten human life and health. At present, the conventional treatment for malignant tumors is still surgery, radiotherapy, and chemotherapy. The curative effect of this conventional treatment on most tumors is still not very satisfactory. For most chemotherapy drugs, its therapeutic index is still low, that is, its treatment The dose is close to the toxic dose. Therefore, this treatment is usually accompanied by significant toxic effects, including life-threatening bone marrow suppression. Therefore, it is very important to study the method of selectively killing tumor cells without affecting normal cells. This method of selectively killing tumor cells mainly depends on the specific markers of tumor cells, and its curative effect is also determined by the tumor markers. Whether it is strictly restricted to tumor cells.

About 15% of human tumors are related to viruses. Several viruses associated with human tumors have latent infection in tumor cells, while in normal cells, the latent infection rate is very low and the viral load is very small. For example, Epstein-Barr virus (according to conventional abbreviation EB virus, denoted as EBV, the same below) can be latently infected in all cells of Barkitt's lymphoma and undifferentiated nasopharyngeal carcinoma, and its viral gene EBNA-1 is expressed in the above tumor cells , but it is very low in normal cells, the positive rate in mononuclear cells in peripheral blood is 10 ^-5 -10 ^-6 , and it is almost negative in other normal cells in adults. Another example is that HPV virus can be latently infected in 90% of cervical cancer cells, and its cervical cancer cells usually express the E6 and E7 genes of HPV virus, and the expression rate of this gene in normal cells is extremely low or negative. Therefore, in patients with tumors associated with this virus, its viral protein can serve as a very good tumor marker. Viral proteins on these tumor cells have been used as specific targets for immunotherapy. However, during the occurrence and development of tumors, in order to avoid immune surveillance, many changes occur in tumor cells, such as the expression of MHC1 genes and antigens in many tumor cells. The ability to present is down-regulated and lacks the second signal of immune cell stimulation, so it cannot effectively activate cytotoxic T cells to kill tumor cells, so the clinical efficacy of immunotherapy is not ideal.

Gene therapy is a new method for treating malignant tumors that has emerged in recent years. There are two methods of gene transfection: viral method and non-viral method. Viral methods usually use retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and vaccinia virus. Retrovirus has a high transfection rate in vitro, but the virus titer is low, and the transfection rate in vivo is low, and it can only infect cells in the division phase, and it has the disadvantage of integrating into chromosomes and the possibility of cancer . Adeno-associated virus has the ability to transfect dividing and quiescent cells, and can express persistently. Non-viral methods include methods such as liposomes and gene guns, whose transfection gene expression time is shorter and the transfection rate is lower. Adenovirus is currently the most common viral vector in tumor gene therapy. It has been widely used in human gene therapy programs. It is easy to produce and purify. It can effectively transfect dividing and resting cells in vivo and in vitro without carcinogenicity. .

The main obstacle of current tumor gene therapy is that the target gene cannot be effectively transfected into all tumor cells, and the curative effect of gene therapy is closely related to the number of tumor cells transfected with the gene. Therefore, the research and development of specific transfection of all tumor cells The viral vector system of tumor cells is crucial in tumor gene therapy.

In recent years, research on tumor-specific proliferating viruses has emerged internationally. According to the differences in certain biological characteristics between tumor cells and normal cells, the modified virus can only specifically proliferate in tumor cells, lyse tumor cells, and then Release virus particles, reinfect other tumor cells, proliferate and lyse again, and thus produce an amplification effect. Since the virus can diffuse to various tissues and organs throughout the body, it can infect all tumor cells, thereby killing local and metastatic tumors. Normal cells are not affected.

There are three typical examples, the first one is E1B 55Kda protein inactivated adenovirus ONYX-015, its main mechanism is: Many researchers have found that P53 plays an important role in the process of tumor carcinogenesis, and the mutation or inactivation of P53 can make DNA Damaged cells continue to divide and replicate, and can become cancerous when other genes are also mutated. P53 is also the main anti-virus protein of host cells. P53 can be activated after virus infection in normal cells. For example, the E1A region of the gene necessary for the proliferation of adenovirus can lead to abnormal proliferation of cells and activate P53 at the same time, eventually leading to cell apoptosis. Virus replication terminated. However, in most cases, normal cells do not undergo immediate apoptosis after being infected with adenovirus. The main reason is that adenovirus still has two proteins, E1B 19Kda and 55Kda, that shut down and activate the function of P53. The former acts on the downstream of P53 to prevent Apoptosis, the latter combined with P53 to prevent the activation of P53, so the wild-type adenovirus can survive and replicate in normal cells. When the adenovirus lacks the 55Kda protein in the E1B region, in normal cells, due to the normal function of P53, it is quickly activated, thereby causing cell apoptosis, preventing the adenovirus from effectively replicating and multiplying, and thus terminating the infection. In tumor cells with P53 mutation or inactivation, P53 cannot be activated after virus infection, so adenovirus lacking E1B can still replicate and proliferate, causing the virus to replicate in large numbers in tumor cells, eventually leading to tumor cell lysis and death, and New viruses are released to infect other tumor cells, which makes all tumor cells infected and die. ONYX-015 has obvious curative effect on tumors with P53 mutation and abnormal function in vivo and in vitro, and it is found that ONYX-015 and chemotherapy drug 5-fluorouracil (5-Fu) cisplatin have obvious synergistic effect. In March 1999, the phase II clinical trial was completed. For 30 patients with head and neck tumors who were ineffective after conventional treatment, the virus was combined with conventional chemotherapy. The results showed that the tumor size of 19 cases shrunk by more than half (accounting for 63%), and 8 of them The tumor regressed completely (accounting for 27%), and only 17% of patients were ineffective. So far, they have been followed up for 1-11 months, and there is no recurrence and no obvious toxic effect (1.Bischoff JR, Kirn DH, Williams A, et al. Anadenovirus mutant that replicaties selective in p53-deficientes human cells. Science 1996, 274, 373-376.2. Heise C, Sampson-Johannes A, Williams A, et al. Onyx-015, an E1b gene-attenuated adenovirus, cause to be specific antitumoral efficacy that can be augmentedby standard chemotherapeutic agents.Nature Medicine，1997，3，639-645.3.McCormic k.Cytopathic for therapy and prophylaxis of neoplasia.1998，United States Patent 5,801,029.4.McCormick.Cytopathic for therapyand prophylaxis of neoplasia.1997， United States Patent 5,677,178.5.McCormick.Cytopathic for therapy and prophylaxis of neoplasia.1998, United States Patent 5,846,945.6.McCormick.Cytopathic for therapy and prophylaxis of neoplasia.1999, United States 5, 18, Patent 5)

Another successful example comes from research on the herpes simplex virus. Herpes simplex virus has obvious neurotropic characteristics. In quiescent cells, its ribonucleotide reductase and thymidine kinase (HSV-TK) are genes necessary for viral DNA replication, which are essential for rapid growth cells. For example, the above two genes are not needed for virus DNA replication in tumor cells. This is because there are sufficient nucleotides in rapidly growing cells. Therefore, the virus can also be replicated in the absence of HSV-TK, due to normal The central nervous cells are in the quiescent phase, while the intracranial tumor cells are in the growth phase. Therefore, Martuza RL et al. applied herpes simplex virus lacking HSV-TK to treat glioma, which was also effective in lysing growing glioma cells in vitro. Directly injecting the virus into glioma cells or other central nervous system tumors in nude mice can make the virus multiply obviously and inhibit tumor growth. This experiment was subsequently confirmed by other groups in the immunocompetent rat 9L glioma model. Phase I clinical trials began in 1998 (Mineta T, Rabkin SD, Yazaki T, ea al. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nature Med, 1995, 1, 938-943). The third example comes from the report of Rodriguez R et al. The application of the prostate specific antigen (PSA) promoter was inserted into the upstream region of the gene (E1A) necessary for the proliferation of adenovirus type 5, named CN706, and the expression of the E1A gene of the virus was strictly regulated by the PSA promoter. Control, in the prostate cancer cell line LNCaP that secretes PSA, the expression of E1A gene decreased by 99%, and the proliferation ability of its adenovirus was significantly reduced, and the virus titer produced was 150 times lower than that of the prostate cancer cell line LNCaP that secreted PSA. It shows that the adenovirus CN706 can replicate and proliferate quite specifically in the cell line that secretes PSA. One-time injection of this virus into nude mice can effectively kill 1×10 ⁹ LNCaP tumor cells that secrete PSA and make the secretion of PSA disappear, but it is ineffective for DU145 that does not secrete PSA. Currently, the research has been carried out in Phase I in the United States. Clinical Trials. In this study, certain tissues of eukaryotic cells were used to specifically activate cis-acting elements to control the proliferation of adenovirus early genes of the virus (1. Rodriguez R, Schuur ER, Lim HY, et al. Prostate attenuated replication competent adenovirus (ARCA) CN706: Aselective cytotoxic for prostate-specific antigen-positive prostate cancer cells. Cancer Res, 1997, 57, 2559-2563.2. Henderson, et al. Tissue specific and tumor growth supperssion by adenovirus comprising prostate specific antigen. 1999, United States Patent 5,871. Tissue specific viral vectors. 1997, United States Patent 5,698,443).

But so far, there has been no report on the use of cis-acting elements specifically activated by Epstein-Barr virus infection or latently infected cells to control the proliferation of early genes of the virus

The object of the present invention is to provide a kind of proliferative recombinant virus capable of specifically killing Epstein-Barr virus-related tumors and its construction method, that is, the virus can selectively replicate and proliferate in Epstein-Barr virus-infected or latently infected tumor cells, and In Epstein-Barr virus-negative normal cells, there is basically no replication and basically no proliferation, so as to specifically inhibit or kill Epstein-Barr virus-infected or latently infected tumor cells.

The proliferative recombinant virus capable of specifically killing Epstein-Barr virus-related tumors provided by the present invention modifies the non-proliferation-essential region of the virus genome and inserts a purpose gene. The target gene is one of (1) tumor suppressor gene, (2) blood vessel suppressor gene, (3) cytokine gene, (4) prodrug conversion enzyme gene and (5) cell apoptosis gene.

The target gene carried by the above-mentioned recombinant virus provided by the present invention is (1) tumor suppressor gene, which can inhibit the growth of tumor cells, and the tumor suppressor gene includes one of the following genes: P53, Rb, NF1, VHL and APC.

The target gene carried by the above-mentioned recombinant virus provided by the present invention is (2) blood vessel inhibitory gene, which inhibits tumor neovascularization and can block the nutrient supply of tumor cells, thereby causing tumor cells to die due to insufficient nutrition, and the tumor shrinks significantly even fade away completely. At the same time, it inhibits the formation of tumor neovascularization, and also achieves the purpose of blocking the pathway of tumor metastasis. Vascular inhibitory genes include one of the following genes: endostatin gene, angiostatin gene, Kringle1-4 structure, Kringle1-5 structure, Kringle1-3 structure and Kringle1-3 plus Kringle5 structure in plasma plasminogen, Interferon-α gene, interferon-β gene, interferon-γ gene, thrombospondin gene, platelet factor 4 gene, plasminogen activator inhibitor (PAI) gene, interleukin-12 and fibronectin Gene. The vasopressor gene contains a coded secreted signal peptide. The secretory signal peptide can be any one of the following: signal peptide of angiogenesis inhibitor itself, signal peptide of M-oncogenesis protein, signal peptide of immunoglobulin K chain.

The target gene carried by the above-mentioned recombinant virus provided by the present invention is (3) cytokine gene, which can activate immune cells, increase hematopoietic function, etc., and the cytokine gene includes the following genes: interleukin 2, interleukin 12, Granules - single colony stimulating factor, tumor necrosis factor, interferon-alpha, interferon-beta, interferon-gamma, Light and Flt3 ligand.

The target gene carried by the recombinant virus provided by the present invention is (4) prodrug converting enzyme gene, which can convert non-toxic drugs into toxic drugs, thereby enhancing the killing of tumor cells. The prodrug converting enzyme gene comprises one of the following genes: herpes simplex virus thymidine kinase, varicella-zoster virus thymidine kinase, and Escherichia coli cytosine deaminase.

The target gene carried by the above-mentioned recombinant virus provided by the present invention is (5) apoptosis gene, which can lead to apoptosis of eukaryotic cells, and the apoptosis gene includes one of the following genes: ICE, capase-3, capase-8 , capase-9.

As the virus replicates in tumor cells, the copy number of the target gene increases, allowing the tumor cells to express the target gene efficiently.

Regulation of gene transcriptional activation is influenced by the interaction of trans-acting factors (such as transcription factors) with cis-acting elements. When certain transcription factors are lacking or appearing, the transcription level of genes will be affected. When the host cell is infected or latently infected by the virus, the virus can change the transcriptional regulatory factors in the cell, which is beneficial to the expression of the virus gene, the replication and proliferation of the virus. In the present invention, certain cis-acting elements of viral genes are used to control the target gene, so that the target gene is specifically expressed in the virus-infected or latently infected cells, but not expressed or expressed at a low level in virus-negative cells. Some cis-acting elements of the virus are used to control the essential genes for virus proliferation and replication, so that the genes necessary for virus proliferation can only be expressed in the cells infected by the above viruses, and the virus can only proliferate in the cells infected by the above viruses or latently infected , and basically no proliferation in the above-mentioned virus-negative cells. This modified viral vector can be used in certain cell mixtures to kill special target cells infected or latently infected by a certain virus, and can selectively proliferate in this target cell by administering the modified virus, So that the target cells are selectively killed by the proliferating virus; in vitro culture or in animals by mixing the modified virus and cell complexes, the virus can only proliferate in the target cells, that is to say, except for the target cells, Other cells cannot be killed by this virus. Since the virus proliferates and amplifies in the target cell, the target cell in the mixed cells is killed. Once the target cell is destroyed, the virus can no longer proliferate.

The present invention adopts the cell-specific response element to be composed of Epstein-Barr virus-infected or latently infected cells to specifically activate the cis-acting element, and the cis-acting element EB virus Orip is combined with the reporter gene controlled by the Bam HI C-promoter. The expression level of latently infected cells (EBNA-1 positive) cells is about 1000 times higher than that of Epstein-Barr virus negative (EBNA-1 negative) cells, and the cis-acting element EB virus Orip combined with herpes simplex virus thymidine kinase is basically used The reporter gene controlled by the promoter (mini-HSV-TK) or SV40 basic promoter (mini-SV40 promoter) has a higher expression level in Epstein-Barr virus latently infected cells (EBNA-1 positive) cells than in EB virus negative (EBNA-1 positive) cells. 1 negative) the expression level is 100-1000 times higher.

The proliferative recombinant virus that can specifically kill Epstein-Barr virus-related tumor cells proposed by the present invention has at least one expression of a gene necessary for virus propagation controlled by a cis-acting element specifically activated by Epstein-Barr virus infection or latently infected cells. It consists of inserting a certain cis-acting element between the transcription start site and the coding start site of the gene necessary for virus proliferation. The cis-acting element is specifically activated in Epstein-Barr virus-infected or latently infected cells to produce transcriptional activity, but not activated in Epstein-Barr virus-negative cells and cannot produce transcriptional activity. The cis element contains at least one of the following sequences: Orip in Epstein-Barr virus, family of 30bp repeats (referred to as FR) in Epstein-Barr virus Orip, Bam HI C-promoter of Epstein-Barr virus, Orip in Epstein-Barr virus combined with Epstein-Barr virus Bam HI C-promoter, FR in Epstein-Barr virus Orip combined with herpes simplex virus thymidine kinase basic promoter (mini-HSV-TK) or SV40 basic promoter (mini-SV40 promoter) and in Epstein-Barr virus infection or A cis-acting element that is specifically activated in latently infected cells. What is necessary for the proliferation of the above viruses is the early expression gene of the virus, such as the early early expression gene ICE4 of herpes simplex virus.

In the present invention, adenovirus can be used as the above-mentioned virus. The essential gene for virus proliferation contains at least one of the following early expression genes of adenovirus: E1A, E1B, E2, E4.

Proliferating recombinant virus and chemotherapeutic drugs (such as cisplatin, 5-fluorouracil, mitomycin C, carboplatin, cyclophosphamide, etc.) (such as snake venom), and monoclonal antibodies (such as anti-nasopharyngeal carcinoma cell antibodies, etc.) can form a complex together, which has a better effect as an anti-tumor drug.

The method for constructing a proliferative recombinant adenovirus that specifically kills Epstein-Barr virus-related tumor cells proposed by the present invention is as follows. Two vectors containing the left and right arms of adenovirus are co-transfected into cells that can produce recombinant adenovirus. At least one of the two adenoviral vectors has a non-essential region for adenoviral proliferation inserted into the target gene, and at least one of the two adenoviral vectors has adenoviral early expression genes E1A, E1B, E2, and E4 region transcription initiation A certain cis-acting element is inserted between the site and the coding start site, and the cis-acting element is specifically activated in Epstein-Barr virus-infected or latently infected cells to produce transcriptional activity, while in Epstein-Barr virus-negative cells Inactive, no transcriptional activity can be produced. The cis-operating element contains at least one of the following sequences: Orip in Epstein-Barr virus, FR in Epstein-Barr virus Orip, Bam HI C-promoter in Epstein-Barr virus, Orip in Epstein-Barr virus combined with Bam HI C-promoter in Epstein-Barr virus, EB virus Bam HI C-promoter, EB virus The FR in the virus Orip is combined with the basic promoter of herpes simplex virus thymidine kinase (denoted as mini-HSV-TK) or the basic promoter of SV40 (denoted as mini-SV40 promoter), and in Epstein-Barr virus-infected or latently infected cells A cis-acting element specifically activated in

The cis-acting elements that are specifically activated in Epstein-Barr virus infection or latently infected cells can be inserted into the region between the transcription initiation site of the adenovirus early gene and the coding initiation site by the following methods: by polymerase chain reaction (PCR ) technology in the region between the adenovirus early gene transcription start site and the coding start site to make a restriction site, and insert the above-mentioned cis-acting element into this site through restriction digestion, so that the adenovirus early gene is regulated A cis-acting element that controls the specific activation of Epstein-Barr virus infection or latently infected cells.

There are six different subgenera of human adenoviruses, A, B, C, D, E, and F. Their affinity for host cells, tumorigenicity and disease history are not the same. The present invention takes Adenovirus type 5 (Ad5) in subgenus C as an example to further illustrate the present invention, but is not limited to this example. Adenovirus genes are divided into two categories, early genes and late genes, and early genes include E1A, E1B, E2, and E4. The E1a gene is expressed immediately after virus infection (0-2 hours). It is the earliest expression of adenovirus protein. It acts as a transactivation transcriptional regulator and is necessary for the expression of early gene proteins of other viruses. At the same time, it transactivates the initiation of other viruses. son. Therefore, lacking the expression of the E1A gene, the gene products necessary for adenoviral DNA replication cannot be produced, and the adenovirus cannot replicate and proliferate efficiently.

The transcription of E1b gene is activated by E1A protein, and its protein function is necessary for the transfer of late viral gene mRNA from the nucleus to the cytoplasm. The loss of E1B gene expression can lead to poor gene expression in the late stage of the virus and the inability to shut down the synthesis of host cell proteins.

The E4 gene is located at the right end of the viral genome, and its open reading frame 3 (ORF3) and ORF6 can increase the mRNA level of the main late gene of adenovirus. Lack of ORF3 and ORF6 protein functions, the virus titer is 10 ⁶ times lower than the wild titer.

The adenovirus system is composed of two vectors, one vector provides the left arm part of the adenovirus, and the other vector provides the right arm. These two vectors have at least a 500nt homologous recombination region, but the transfected cells produce recombinant adenovirus, The vector system plasmid pXC1 (Mckinnon, Gene 1982, 19:39-42), contains the wild-type Ad5 left arm. pBHG10 provides the Ad5 right arm lacking the E3 region or pBHGE3 provides the Ad5 right arm (containing the E3 region).

The transcription start site and coding start site of Ad5 E1A are respectively located at about 560nt and 610nt of the viral genome, and a certain viral cis-acting element is inserted in this region, such as the Orip joint BamH I C promoter of Epstein-Barr virus. First, use polymerase chain reaction (PCR) technology to construct a restriction endonuclease site in this region, and PCR primers will be limited to the Ad5 genome or a plasmid carrying part of the Ad5 genome, such as primers with pBR322 as the background. Using the pBR322 sequence containing the EcoR I site and the Ad5 sequence containing the Xba I site, a new unique restriction restriction site is created in this region through the double stack PCR method, and the exact site will be used to insert the virus in cis element.

A similar protocol was used to insert a viral cis-acting element to regulate the E1B promoter of E1b Ad5, consisting of a single high-affinity recognition for Sp1 and a TATAbox located at 1636 to Sub-1701 nt. By inserting a viral cis-acting element in this region, cell-specific transcription of E1B will be provided.

The present invention also proposes the above-mentioned proliferative recombinant virus propagation method, that is, to infect mammalian cells infected with Epstein-Barr virus (such as recombinant adenovirus) or latently infected with the recombinant virus, so that the recombinant virus can specifically infect the mammalian cells infected with Epstein-Barr virus or latently infected. proliferate in cells.

Using an adenovirus that can only proliferate in specific target cells infected with Epstein-Barr virus or latently infected, the adenovirus is allowed to proliferate and cause cell death in the target cells. Cultured in vitro or in animals by mixing the modified adenovirus with cell complexes, the adenovirus can only proliferate in EB virus-infected or latently infected cells, that is to say, except for EB virus-infected or latently infected cells, Other cells cannot be killed by this adenovirus, because the adenovirus proliferates and amplifies in the Epstein-Barr virus-infected or latently infected cells, so that the Epstein-Barr virus-infected or latently infected cells in the mixed cells are killed. Epstein-Barr virus-infected or latently infected cells are destroyed and the adenovirus is no longer able to reproduce.

The present invention proposes the practical application of the above-mentioned recombinant virus. For example, the recombinant virus (such as recombinant adenovirus) is used to infect Epstein-Barr virus-infected or latently infected tumor cells in vitro, resulting in cytotoxicity. Recombinant viruses, such as recombinant adenoviruses, are used to inhibit the growth of Epstein-Barr virus-infected or latently infected tumor cells. Recombinant viruses (such as recombinant adenoviruses) are used to selectively kill Epstein-Barr virus-infected or latently infected tumor cells in vivo.

The present invention has the following beneficial effects

1. The present invention provides a class of proliferative recombinant viruses for the treatment of Epstein-Barr virus-related tumors,

Animal experiments have proved that this recombinant virus can be used to treat Epstein-Barr virus-related tumors,

Including nasopharyngeal carcinoma, Hodgkin's lymphoma, Burkitt's lymphoma, gastric cancer, etc.

2. The present invention provides a class of proliferative recombinant virus for the treatment of Epstein-Barr virus-related tumors

build method. The method is simple and easy and can be used to construct a variety of therapeutic Epstein-Barr virus phases

Tumor-related proliferative recombinant virus.

3. The present invention provides a method for killing EB virus latent infection or infection in vivo and in vitro

Tumor cells without affecting Epstein-Barr virus-negative normal cells.

Example 1: Construction of a vector containing Epstein-Barr virus cis-acting elements

Using pCAT-Basic as the basic vector, insert the family of 30bp repeats (FR, located at 7337-8190bp of the Epstein-Barr virus) in the multiple cloning site of the Epstein-Barr virus combined with the SV40 basic promoter (mini-SV40), and purchase pCAT-Basic from Promega. PCR technology was used to amplify the family of 30bp repeats and mini-SV40 promoter in Epstein-Barr virus Orip. The former template Epstein-Barr virus DNA was extracted from the lymphoma cell line B95-8. For the method of extracting viral DNA, refer to the operating instructions of the QIAamp Blood kit from QIAGEN Company. The latter template is pCAT-Control (purchased from Promega). The primers of FR in Epstein-Barr virus Orip are:

prime1: 5'-primer (containing Hind III and AgeI restriction sites) GGG AAG CTTACC GGT GCA TGC AGG AAA AGG ACA AGC

primer2: 3'-primer (containing part of the mini-SV40 promoter sequence) GAG ATGCAG ATC AAT GGC ACC CCG GGG AAT ACC

The primers for the mini-SV40 promoter are

primer3: 5'-primer (containing part of the FR sequence in Orip) GTG CCA TTG ATCTGC ATC TCA ATT AGT CAG

primer4: 3'-primer (containing Sal I and Age I restriction sites) GCT AAA GTCGAC ACC GGT AAG CTT TTT GCA AAA GCC TAG

Apply two stacking PCR techniques (see PCR Protools Current Methods and Applications for the method, edited by White BA, published by Humana Press Inc. in 1993—document 1) to produce a fusion promoter from the FR sequence in the Epstein-Barr virus Orip and the mini-SV40 promoter. The fusion promoter PCR fragment was inserted into the Hind III and Sal I sites of the pCAT-Basic vector using Hind III and Sal I double enzyme digestion (for the method, refer to the Molecular Cloning Experiment Guide, Science Press 1992 publication-document 2). The fragment was subjected to DNA sequencing, and its fusion promoter sequence was completely correct, which was recorded as CHEB-FRSV40.

Using pbluescript II KS(+) as the base vector (purchased from ATCC, USA), insert the family of 30bp repeats (FR, located at 7337-8190bp of Epstein-Barr virus) in the multiple cloning site of the Epstein-Barr virus Orip combined with the HSV-TK basic vector Promoter (mini-TK), pbluescript II was purchased from Promega Company. PCR technology was used to amplify the family of 30bp repeats and mini-SV40 promoter in Epstein-Barr virus Orip. The former template Epstein-Barr virus DNA was extracted from the lymphoma cell line B95-8. For the method of extracting viral DNA, refer to the operating instructions of the QIAamp Blood kit from QIAGEN Company. The latter template is pTAL-Luc (purchased from Clontech).

The primers of FR in Epstein-Barr virus Orip are:

Primer 5: 5'-primer (contains XhoI restriction site) GGG CAT CTC GAG GCATGC AGG AAA AGG ACA AGC

Primer 6: 3'-primer (containing part of the mini-HSV-TK promoter sequence) AGT CGGGGC GGC AAT GGC ACC CCG GGG AAT ACC

The primers for the mini-HSV-TK promoter are

Primer 7: 5'-primer (containing part of the FR sequence in Orip) GGT GCC ATT GCCGCC CCG ACT GCA TCT GC

Primer8: 3'-primer (containing Xba I restriction site) TTT TCT AGA CTT CTG CTTCAT CCC CGT G

Apply two stacked PCR techniques (the method is the same as before) to generate a fusion promoter from the FR sequence in Epstein-Barr virus Orip and the mini-TK promoter, and insert the PCR fragment of the fusion promoter into pbluescript II KS using Xho I and Xba I double enzyme digestion (+) In the Xho I and Xba I sites of the vector (purchased from ATCC, USA) (see reference 2 for the method). The fragment was subjected to DNA sequencing, and its fusion promoter sequence was completely correct, which was denoted as CHEB-FRTK.

Example 2: Construction of an attenuated proliferating adenovirus vector carrying endostatin or angiostatin in cis-acting elements of Epstein-Barr virus to control the expression of E1A and E1B.

The pXC.1 vector was purchased from Microbix Biosystem Inc. (Toronto), Canada, and pXC.1 contains the sequence bp22-5790 of type 5 adenovirus. A new and unique Age I restriction site was created at 552bp in the vector, which was located at 12bp before the E1A start codon, and the method used double-PCR technology for positional mutation (see the aforementioned document 1 for the method). Its primers are

primer9: 5'-primer (contains EcoR I restriction site) TTC AAG AAT TCT CATGTT TG

primer10: 3'-primer (insert an A to generate an Age I restriction site) CAG TCA CCG GTG TCG GAG C

primer11: 5'-primer (insert a T to generate an Age I restriction site) GCT CCG ACA CCG GTG ACT GA

primer12: 3'-primer (contains Xba I restriction site) TTC TCT AGA CAC AGGTGA TG Using positional mutation double PCR technology, the PCR product fragment is inserted into the pGEM-T-easy vector (for the method, refer to the operation manual of Promega Company), named is pGEM-T-E1a. The fragment was subjected to DNA sequencing, and the sequencing results showed that a T was inserted into the bp552 site of the pXC.1 plasmid, thereby generating a new Age I restriction site, and the other sequences were the same as those of pXC.1. Use EcoR I and Xba I to double digest the pGEM-T-E1A and pXC.1 plasmids, and insert the excised fragment from pGEM-T-E1A into the EcoR I and BamH I sites in the pXC.1 plasmid to make pXC.1 552 to insert a T, thereby generating an Age I restriction site, which is located in the E1A start codon 12bp before, and the plasmid was named pXC.1-Age I.

CHEB was digested with Age I respectively, and the digested fragments were inserted into the Age I restriction sites in the pXC-Age I plasmid, respectively, and primers 24 and 19 were used for PCR amplification, and 2050bp was amplified, which indicated that the Epstein-Barr virus Orip The family of 30bp repeats combined with the mini-SV40 promoter has been inserted forward into the Age I site of the pXC-Age I plasmid, which is the 12bp upstream region of the E1A initiation codon of adenovirus type 5, named pXC-FRSVE1A. In order to further confirm that the family of 30bp repeats combined with the mini-SV40 promoter in the Epstein-Barr virus Orip has been inserted forward into the Age I site of the pXC-Age I plasmid, the present invention uses EcoR I plus Xba I for double digestion of the pXC-FRSVE1A vector, The 2823bp fragment was recovered and inserted into the pBluescript II SK vector EcoR I and Xba I restriction sites for sequencing. The results confirmed that the family of 30bp repeats combined with the mini-SV40 promoter in the Epstein-Barr virus Orip had been inserted forward into the Age I site of the pXC-Age I plasmid point.

The pXC.1 vector was purchased from Microbix Biosystem Inc. (Toronto), Canada, and pXC.1 contains the sequence bp22-5790 of type 5 adenovirus. Multiple restriction sites were established at 1686bp in the vector, including Bgl II, BamH I, Xho I, and Xba I. The restriction sites are located between the E1B transcription site and the start codon. Secondary PCR technology (see the above-mentioned literature 1 for the method). Its primers are

primer13: 5'-primer (contains Hind III and Hpa I restriction sites) TTT TGCAAG CTT GTT AAC GCC TTT GTT TGC TGA

primer14: 3'-primer (generates four enzyme cutting sites) CTC GAG GGA TCC AGATCT GCG CAT TAT ATA CCC TTT AAG

primer15: 5'-primer (generates four restriction sites) AGA TCT GGA TCC CTC GAGTGA TCT AGA GGG CTA ATC TTG GTT ACA TC

primer16: 3'-primer (contains Kpa I restriction site) CCA GAA AAT CCA GCAGGT AAC uses positional mutation double PCR technology, the PCR product fragment is digested with Hind III and Kpn I, and inserted into the pUC19 vector Hind III and Kpn I The enzyme cutting point, pUC19 was purchased from ATCC Company in the United States and named pUC-E1B. The fragment was subjected to DNA sequencing, and the sequencing results showed that Bgl II, BamH I, Xho I, and Xba I restriction sites were inserted into the E1b transcription start site and coding start site, and the other sequences were the same as pXC.1 .

Digest CHEB-FRTK with Xho I and Xba I, and insert its fragment into the XhoI and Xba I restriction sites in pUC-E1B, that is, insert the EB virus Orip in the E1B transcription start site and coding start site forward The family of 30bp repeats combined with the mini-TK promoter, named pUC-E1B-FRTK, was amplified by PCR with primers 28 and 23, and 1159 bp was amplified, which indicated that the FR combined with the mini-TK promoter in Epstein-Barr virus Orip had been positively Insert the pUC plasmid Xho I and Xba I sites.

Example 3. Construction of an adenocarrier carrying a deletion in the E1 region of the human endostatin (endostatin) gene

The pCA13 vector was purchased from Microbix Biosystem Inc. (Toronto), Canada. pCA13 contains the adenovirus type 5 sequence bp22-5790 and deletes the 342 to 3523bp fragment of the E1 region, and inserts the human cytomegalovirus (HCMV) IE promoter (- 299-+72) and SV40 poly A tailed signal. The human endostatin (endostatin) gene was extracted by polymerase chain reaction (PCR) technology, and total RNA was extracted from fresh normal human liver tissue, and random primers were used for reverse transcription reaction to amplify human endostatin (endostatin) gene, Two polymerase chain reaction (PCR) techniques were used (see reference 1 for the method) to add M-oncostatin-M signal peptide before the gene and two restriction sites of EcoRI and XbaI were introduced before the signal peptide and at the end of the gene.

Primer17: GGG GAA TTC ACC ATG GGG GTA CTG CTC ACA CAG AGGACG CTG CTC AGT CTG GTC CTT GCA CTC

Primer18: CTG CTC AGT CTG GTC CTT GCA CTC CTG TTT CCA AGCATG GCG AGC CAC CGC GAC TTC CAG

Primer19: GCT CTA GAC TAT TAC TTG GAG GCA GTC ATG AAG CTGTTC TCA ATG CAT AGC ACG ATG TAG GCG TG

Primer18 and primer19 were used for the first PCR amplification, and the 615bp fragment was recovered, and primer17 and primer19 were used for the second PCR amplification of the 615bp fragment, and the 647bp fragment was recovered, and EcoR I+Xba I was used for digestion, and the gene was inserted into pbluescriptIIKS (+) carrier (purchased from ATCC, USA) for sequencing, the sequencing results are as follows:

GAATTCACCATGGGGGTACTGCTCACACAGAGGACGCTGCTCAGTCTGGTCCTTGCACTCCTGTTTCCAAGCATGGCGAGCCACAGCCACCGCGACTTCCAGCCGGTGCTCCACCTGGTTGCGCTCAACAGCCCCCTGTCAGGCGGCATGCGGGGCATCCGCGGGGCCGACTTCCAGTGCTTCCAGCAGGCGCGGGCCGTGGGGCTGGCGGGCACCTTCCGCGCCTTCCTGTCCTCGCGCCTGCAGGACCTGTACAGCATCGTGCGCCGTGCCGACCGCGCAGCCGTGCCCATCGTCAACCTCAAGGACGAGCTGCTGTTTCCCAGCTGGGAGGCTCTGTTCTCAGGCTCTGAGGGTCCGCTGAAGCCCGGGGCACGCATCTTCTCCTTTAACGGCAAGGACGTCCTGAGGCACCCCACCTGGCCCCAGAAGAGCGTGTGGCATGGCTCGGACCCCAACGGGCGCAGGCTGACCGAGAGCTACTGTGAGACGTGGCGGACGGAGGCTCCCTCGGCCACGGGCCAGGCCTCCTCGCTGCTGGGGGGCAGGCTCCTGGGGCAGAGTGCCGCGAGCTGCCATCACGCCTACATCGTGCTATGCATTGAGAACAGCTTCATGACTGCCTCCAAGTAATAGTCTAG

Cut out its EcoR I+Xba I, insert it into pCA13 vector EcoR I+Xba I, and name it pCA13-human endostatin.

Example 4. Construction of an adenocarrier carrying angiostatin (angiostatin) gene deletion in the E1 region

Using polymerase chain reaction (PCR) technology to angiostatin (angiostatin) gene, total RNA was extracted from fresh normal human liver tissue, the first round of PCR was performed after reverse transcription reaction with random primers, and two polymerase chain reactions were used to (PCR) technology (method see document 1) introduces two restriction sites of EcoR I and Xho I at the end of the gene,

primer20: 5'-AGCGAATTCCAAAATGGAACATAAGG-3'

    EcoR I plasma plasminogen: 49-67

Primer21: 5'-ACACTTTTTCCTTGACCTGATTTCAG-3'

    Plasma plasminogen: 348-343+111-94

primer22: 5'-CTGAAATCAGGTCAAGGAAAAGTGTATCTCTCA

GAGTGC-3'

    Plasma plasminogen: 94-111+343-363

primer23: 5'-AGCCTCGAGCTATTACGCTTCTGTTCCTGAG-3'

  Xho I (2 Stop) plasma plasminogen: 1431-1416

Primer20 and primer21, primer22 and primer23 were subjected to the first PCR reaction respectively, and the reaction products were mixed, and primer20 and primer23 were used to perform PCR reaction again, and a 1168bp fragment was recovered. Use EcoR I+Xho I to digest, insert the gene into the pbluescript IIKS (+) vector (purchased from ATCC, USA) for sequencing, and the sequencing results are as follows:

GAATTCCAAAATGGAACATAAGGAAGTGGTTCTTCTACTTCTTTTATTTCTGAAATCAGGTCAAGGAAAAGTGTATCTCTCAGAGTGCAAGACTGGGAATGGAAAGAACTACAGAGGGACGATGTCCAAAACAAAAAATGGCATCACCTGTCAAAAATGGAGTTCCACTTCTCCCCACAGACCTAGATTCTCACCTGCTACACACCCCTCAGAGGGACTGGAGGAGAACTACTGCAGGAATCCAGACAACGATCCGCAGGGGCCCTGGTGCTATACTACTGATCCAGAAAAGAGATATGACTACTGCGACATTCTTGAGTGTGAAGAGGAATGTATGCATTGCAGTGGAGAAAACTATGACGGCAAAATTTCCAAGACCATGTCTGGACTGGAATGCCAGGCCTGGGACTCTCAGAGCCCACACGCTCATGGATACATTCCTTCCAAATTTCCAAACAAGAACCTGAAGAAGAATTACTGTCGTAACCCCGATAGGGAGCTGCGGCCTTGGTGTTTCACCACCGACCCCAACAAGCGCTGGGAACTTTGCGACATCCCCCGCTGCACAACACCTCCACCATCTTCTGGTCCCACCTACCAGTGTCTGAAGGGAACAGGTGAAAACTATCGCGGGAATGTGGCTGTTACCGTTTCCGGGCACACCTGTCAGCACTGGAGTGCACAGACCCCTCACACACATAACAGGACACCAGAAAACTTCCCCTGCAAAAATTTGGATGAAAACTACTGCCGCAATCCTGACGGAAAAAGGGCCCCATGGTGCCATACAACCAACAGCCAAGTGCGGTGGGAGTACTGTAAGATACCGTCCTGTGACTCCTCCCCAGTATCCACGGAACAATTGGCTCCCACAGCACCACCTGAGCTAACCCCTGTGGTCCAGGACTGCTACCATGGTGATGGACAGAGCTACCGAGGCACATCCTCCACCACCACCACAGGAAAGAAGTGTCAGTCTTGGTCATCTATGACACCACACCGGCACCAGAAGACCCCAGAAAACTACCCAAATGCTGGCCTGACAATGAACTACTGCAGGAATCCAGATGCCGATAAAGGCCCCTGGTGTTTTACCACAGACCCCAGCGTCAGGTGGGAGTACTGCAACCTGAAAAAATGCTCAGGAACAGAAGCGTAATAGCTCGAG

Cut out its EcoR I+Xho I, insert it into the pCA13 vector EcoR I+Xho I, and name it pCA13-human angiostatin.

Example 5: Construction of an adenoviral vector that controls the expression of E1A and E1B by EB virus cis-acting elements carrying endostatin or angiostatin:

Use Bgl II to digest pCA13-human endostatin and pCA13-humanangiostatin respectively, and recover 1237bp (human endothelial cells containing human cytomegalovirus (HCMV) IE promoter (-299--+72) and M-oncogene signal peptide) respectively. Inhibitin gene and SV40 poly A tail signal) and 1758bp (human cytomegalovirus (HCMV) IE promoter (-299--+72), human angiogenesis inhibition and SV40 poly A tail signal), insert it into pUC- For the Bgl II restriction site in E1B-FRTK, PCR technology was used to verify the positive and negative directions of its insertion: the upstream primer of Bgl II

primer24: GTT AAC GCC TTT GTT TGC TGA

Amplified with human endostatin 5' and 3'-primer respectively

primer25 human endostatin 5'-primer (located in M-oncogene signal peptide and human endostatin gene 110-131bp) TCC ACC TGG TTG CGC TCA ACA G

primer26 human endostatin 3'-primer (located in M-oncogene signal peptide and human endostatin gene 577-600bp) AGC ACG ATG TAG GCG TGA TGG C

Amplified with human angiostatin 5' and 3'-primer respectively

primer27 human angiostatin 5'-primer (located at 11-32bp of human angiostatin) ATG GAA CAT AAG GAA GTG GTT C

primer28 human angiostatin 3'-primer (located at 823-842bp of human angiostatin) AGG AGT CAC AGG ACG GTA TC

Its primer24 and primer26 can be amplified to 1122bp, indicating that it contains human cytomegalovirus (HCMV) IE promoter (-299--+72), human endostatin gene containing M-oncogene signal peptide and SV40 poly A plus The tail signal was inserted forward into the pUC-E1B-FRTK Bgl II restriction site, named pUC-E1B-FRTK-endostatin1. Its primer24 and primer25 can be amplified to 818bp, indicating that it contains human cytomegalovirus (HCMV) IE promoter (-299--+72), human endostatin gene containing M-oncogene signal peptide and SV40poly A tail The signal is reversely inserted into the pUC-E1B-FRTK Bgl II restriction site. Named pUC-E1B-FRTK-endostatin2.

Its primer24 and primer28 can be amplified to 1364bp, indicating that it contains human cytomegalovirus (HCMV) IE promoter (-299--+72), human angiostatin gene and SV40 poly A tailing signal inserted forward into pUC-E1B- FRTK Bgl II restriction site, named pUC-E1B-FRTK-angiostatin1. Its primer24 and primer27 can be amplified to 1440bp, indicating that it contains human cytomegalovirus (HCMV) IE promoter (-299--+72), human angiostatin gene and SV40 poly A tailing signal reversely inserted into pUC-E1B -FRTKBgl II restriction site, named pUC-E1B-FRTK-angiostatin2.

Use Hpa I and Kpn I to digest pUC-E1B-FRTK-endostatin1, pUC-E1B-FRTK-endostatin2, pUC-E1B-FRTK-angiostatin1 and pUC-E1B-FRTK-angiostatin2, recover the fragments, and insert them into pXC-FRSVE1A respectively The restriction sites of Hpa I and Kpn I were named pXC-FRSVE1A-FRTKE1B-endostatin1, pXC-FRSVE1A-FRTKE1B-endostatin2, pXC-FRSVE1A-FRTKE1B-angio-statin1 and pXC-FRSVE1A-FRTKE1B-angiostatin2, respectively.

Example 6: Recombination of Epstein-Barr virus cis-acting elements carrying endostatin or angiostatin to control the expression of E1A and E1B.

The 293 cell line was purchased from Microbix Biosystem Inc. (Toronto) in Canada. It was transformed from cut type 5 adenovirus DNA into human embryonic kidney cells. It contains and expresses type 5 adenovirus E1 region, and adenovirus DNA has a high Transfection rate. The plasmid containing the left arm of type 5 adenovirus combined with the plasmid containing the right arm of type 5 adenovirus was co-transfected into 293 cells, and an infectious adenovirus could be produced through homologous recombination. We co-transfected pXC-FRSVE1A-FRTKE1B-endostatin1, pXC-FRSVE1A-FRTKE1B-endostatin2, pXC-FRSVE1A-FRTKE1B-angiostatin1 and pXC-FRSVE1A-FRTKE1B-angiostatin2 with plasmid pBHG10 containing the right arm of adenovirus type 5 into 293 thin strains, its specific method is referring to the operating instruction of GIBCO BRL company. PBHG10 was purchased from Microbix Biosystems Inc. (Ontario), Canada, and contains the right arm of adenovirus type 5, but lacks the E3 region. Virus plaques appeared 9-14 days after co-transfection. After three times of virus plaque purification, E1A and E1B were activated by EB virus cis-acting elements Orip FR combined with mini-SV40 promoter and Orip FR combined with mini-HSV-TK The adenoviruses controlled by the progeny, and carry human endostatin or angiostatin, named as EBV FRSVEIA-FRTKE1B-endostatin1, EBVFRSVEIA-FRTKE1B-endostatin2, EBV FRSVEIA-FRTKE1B-angiostatin1 and FRSVEIA-FRTKE1B-angiostatin2, respectively, denoted as CNHKEBV-endostatin1, CNHKEBV-endostatin2, CNHKEBV-angiostatin1 and CNHKEBV-angiostatin2.

The list of recombinant viruses constructed by the above method is as follows:

Virus Name Containing Ad5 Left Arm Plasmid Containing Ad5 Right

Armed plasmid EBV FRSVEIA-FRTK CNHKEBV- pXC-FRSVE1A-FRTKE1B-

PBHG10E1B-endostatin1 endostatin1 endostatin1EBV FRSVEIA-FRTK CNHKEBV-B-FRT pXC-FRSE1A

PBHG10E1B-endostatin2 endostatin2 endostatin2EBV FRSVEIA-FRTK CNHKEBV-B-FRT PXC-FRSE1A

PBHG10E1B-angiostatin1 angiostatin1 angiostatin1EBV FRSVEIA-FRTK -CNHKEBV- FXC-FRSKVEE1B-

PBHG10E1B-angiostatin2 angiostatin2 angiostatin2

The adenoviruses reproduced in large quantities in 293 cells, and the cesium chloride gradient centrifugation method was used to purify a large amount of adenoviruses (for specific methods, see the publication of the aforementioned document 2). EBV FRSVEIA-FRTKE1B-endostatin1 (CNHKEBV-endostatin1) is a type 5 adenovirus, and the EB virus cis-acting element Orip FR combined with the mini-SV40 promoter is inserted between the E1A and E1B transcription start sites and the coding start sites, respectively and Orip FR joint mini-HSV-TK promoter, and create a new Bgl II restriction site between the upstream region of the Orip FR joint mini-HSV-TK promoter after the E1A stop codon, and in this restriction site Insert human cytomegalovirus (HCMV) IE promoter (-299--+72), human endostatin gene containing M-oncogene signal peptide and SV40 poly A tailed signal gene sequence, accompanied by 28133 -30818bp (partial sequence of E3 region) was deleted, and other DNA sequences of the virus were the same as type 5 adenovirus. EBV FRSVEIA-FRTK E1B-endostatin2 (CNHKEBV-endostatin2) is a type 5 adenovirus, and the EB virus cis-acting element Orip FR combined with mini-SV40 is inserted between the E1A and E1B transcription start sites and the coding start sites, respectively. and the Orip FR joint mini-HSV-TK promoter, and create a new Bgl II restriction site between the E1A stop codon and the upstream region of the Orip FR joint mini-HSV-TK promoter. Reverse insertion of human cytomegalovirus (HCMV) IE promoter (-299--+72), human endostatin gene containing M-oncogene signal peptide and SV40 poly A tailed signal gene sequence, accompanied by There is 28133-30818bp (partial sequence of E3 region) missing, and other DNA sequences of the virus are the same as type 5 adenovirus.

EBV FRSVEIA-FRTK E1B-angiostatin1 (CNHKEBV-angiostatin1) is a type 5 adenovirus, and the EB virus cis-acting element Orip FR combined with mini-SV40 is inserted between the E1A and E1B transcription start sites and the coding start sites, respectively. and the OripFR joint mini-HSV-TK promoter, and create a new Bgl II restriction site between the E1A stop codon and the upstream region of the Orip FR joint mini-HSV-TK promoter. The insertion contains human cytomegalovirus (HCMV) IE promoter (-299--+72), human angiostatin gene and SV40 poly A tailed signal gene sequence, accompanied by 28133-30818bp (partial sequence of E3 region ) is missing, and the other DNA sequences of the virus are the same as those of type 5 adenovirus. EBV FRSVEIA-FRTK E1B-angiostatin2 (CNHKEBV-angiostatin2) is a type 5 adenovirus, and the EB virus cis-acting element Orip FR combined with mini-SV40 is inserted between the E1A and E1B transcription start sites and the coding start sites to start and the Orip FR joint mini-HSV-TK promoter, and create a new Bgl II restriction site between the E1A stop codon and the upstream region of the Orip FR joint mini-HSV-TK promoter. The reverse insertion contains the human cytomegalovirus (HCMV) IE promoter (-299--+72), contains the human angiostatin gene and the SV40 poly A tailed signal gene sequence, and is accompanied by 28133-30818bp (part of the E3 region sequence) is missing, and the other DNA sequences of the virus are the same as those of type 5 adenovirus.

Example 7: EB virus cis-acting elements carrying human endostatin or human angiostatin control E1A and E1B expression of attenuated proliferating adenovirus in vitro in EB-infected or latently infected tumor cells to proliferate, replicate and highly express human Endostatin or human angiostatin factor and specifically kill tumor cells.

CNHKEBV-endostatin1, CNHKEBV-endostatin1, CNHKEBV-angiostatin1 and CNHKEBV-angiostatin2 were respectively infected with Epstein-Barr virus-infected lymphoma cell lines Jijoye, 293 and normal human fibroblasts, and the cells were seeded in 6-well plates at 2×10 ⁵ /well, respectively Infect recombinant adenoviruses CNHKEBV-endostatin1, CNHKEBV-endostatin1, CNHKEBV-angiostatin1, CNHKEBV-angiostatin2 and wild type 5 adenovirus 4×10 ⁵ pfu, and use 293 cell line to measure the virus titer after 48 hours. For specific methods, refer to the above literature 2, the result is:

                                    

293 Jijoye

                                                                             , 

Virus 1×10 ⁵ 7×10 ⁴ 5×10 ⁴

CNHKEBV-

1×10 ⁵ 4×10 ⁵ 3×10endostatin1

CNHKEBV-

1×10 ⁵ 4×10 ⁵ 4×10endostatin2

CNHKEBV-

1×10 ⁵ 2.5×10 ⁵ 2×10angiostatin1

CNHKEBV-

1×10 ⁵ 2.5×10 ⁵ 2×10 ² angiostatin1

Lymphoma cell line Jijoye infected with EB virus and normal human fibroblasts were respectively infected with recombinant adenoviruses CNHKEBV-endostatin1, CNHKEBV-endostatin2, CNHKEBV-angiostatin1 and CNHKEBV-angiostatin2, MOI was 1, incubated at 37°C for 1 hour, after infection After 7 days, the cells were collected, and the viral DNA was extracted using the QIAamp DNA Bloodmini kit (QIAGEN, Germany). For the method, refer to the operating instructions of QIAGEN. Apply Nhe I and Xho I double digestion, use 0.8% agarose electrophoresis, transfer it to a nylon membrane, use ³² P to label human type 5 adenovirus 1178bp BstXI plus Xho I fragment (located at adenovirus nt4611-5789), and carry out Southern blot hybridization, with pXC.1 as the virus copy number control (see literature 2 for the method), the virus copy number per cell of CNHKEBV-endostatin1, CNHKEBV-endostatin2, CNHKEBV-angiostatin1 and CNHKEBV-angiostatin2 in Jijoye and normal human fibroblasts They are 5×10 ⁴ , 5×10 ⁴ , 4×10 ⁴ , 4×10 ⁴ and 0, 0, 0, 0, respectively. The DNA extracted from the above-mentioned CNHKEBV-endostatin1 and CNHKEBV-endostatin2 was respectively digested with Bgl ^II , electrophoresed with 1% agarose, transferred to a nylon membrane, and the cDNA fragments of human endostatin (EcoR I and Xba I double digested pCA13-humanendostatin, recovered 637bp) as a probe, carried out souther blot hybridization, and used pCA13-human endostatin as a virus copy number control (see literature 2 for the method). The virus copy numbers per cell of fibroblasts were 5×10 ⁴ , 5×10 ⁴ and <10, <10, respectively.

The above-mentioned CNHKEBV-angiostatin1 and CNHKEBV-angiostatin2 extracted DNAs were respectively digested with ^Bgl II, electrophoresed with 1% agarose, transferred to nylon membranes, and human angiostatin cDNA fragments (EcoR I and Xba I double digested pCA13-human angiostatin, recovered 1168bp) as a probe, performed souther blot hybridization, and used pCA13-human angiostatin as a virus copy number control (see literature 2 for the method). The virus copy numbers per cell of human fibroblasts were 4×10 ⁴ , 4×10 ⁴ and <10, <10, respectively.

Example 8: Epstein-Barr virus cis-acting elements carrying human endostatin or human angiostatin to control the expression of E1A and E1B attenuated proliferating adenovirus in SCID mice to treat Epstein-Barr virus-infected or latently infected tumor cell xenografts

SCID mice aged 4-5 weeks were subcutaneously inoculated with 5×10 ⁷ lymphoma cell line Jijoye cells infected with Epstein-Barr virus, and treated with 5×10 ⁸ pfu proliferative recombinant adenovirus CNHK101 two weeks later or treated with the same dose of control adenovirus For the virus Ad5-LacZ, the tumor size of the untreated group and the control group treated with adenovirus increased by more than 3 times after 4 weeks, while the tumors in the treated group shrunk significantly, and some tumors completely disappeared.

Example 9: Construction of an adenocarrier with deletion of the E1 region of the interleukin-12 fusion gene

The pCA14 vector was purchased from Microbix Biosystem Inc. (Toronto), Canada. pCA14 contains the adenovirus type 5 sequence bp22-5790 and deletes the 342 to 3523 bp fragment in the E1 region, and inserts the human cytomegalovirus (HCMV) IE promoter in the E1 deletion region (- 299--+72) and SV40 poly A tailed signal.

Polymerase chain reaction (PCR) technique was used to amplify the interleukin-12 fusion gene, total RNA was extracted from fresh normal human T cells, the first round of PCR was performed after reverse transcription reaction with random primers, and polymerase chain reaction was performed twice Reaction (PCR) technology (method see 1) introduces the Sal I restriction site at the end of the gene,

primer29: TTT GTC GAC CAT GGG TCA CCA GCA GTT GGT CAT

Primer30: AGA TCC GCC GCC ACC GCC ACC ACT GCA GGG CAC AGA

TGC CCA

Primer31: GGT GGC GGT GGC GGC GGA TCT AGA AAC CTC CCC GTG

GCC ACT

Primer32: TAT AAA GTC GAC TCA TTA GGA AGC ATT CAG ATA GCT

CG

Primer29 and primer30, primer31 and primer32 were subjected to the first PCR reaction respectively, and the reaction products were mixed, and primer29 and primer32 were used to perform PCR reaction again, and a 1610bp fragment was recovered. The gene was inserted into the pbluescriptIIKS (+) vector (purchased from ATCC, USA) for sequencing with Sal I digestion, and the sequencing result was correct.

Cut off its Sal I, insert it into the pCA14 vector Sal I, and use BamH I enzyme digestion to identify the forward and reverse directions of gene insertion. The BamH I enzyme digestion shows 1422bp and 6919bp, which indicates that the hIL-12 fusion gene is inserted into the pCA14 vector in the forward direction , named pCA14-humanIL-12.

Example 10: Construction of an adenoviral vector carrying human interleukin-12 cis-acting elements to control the expression of E1A and E1B:

Use Bgl II to partially digest pCA14-human IL-12, and recover 2191bp (containing human cytomegalovirus (HCMV) IE promoter (-299--+72), human IL-12 fusion gene and SV40 poly A tailing signal respectively) ), insert it into the Bgl II restriction site in pUC-E1B-FRTK, and use PCR technology to verify the forward and reverse directions of its insertion: its Bgl II upstream primer

primer24: GTT AAC GCC TTT GTT TGC TGA

Amplified with primer29 and primer32 of human interleukin-12, respectively

Its primer24 and primer32 can be amplified to 2122bp, indicating that it contains human cytomegalovirus (HCMV) IE promoter (-299--+72), contains human IL12 fusion gene and SV40 poly A tailing signal is inserted forward into pUC-E1B-FRTK Bgl II restriction site, named pUC-E1B-FRTK1-IL12. Its primer24 and primer29 can be amplified to 2122bp, indicating that it contains human cytomegalovirus (HCMV) IE promoter (-299--+72), contains human IL12 fusion gene and SV40 poly A tailing signal is reversely inserted into pUC-E1B- FRTK Bgl II restriction site. Designated as pUC-E1B-FRTK2-IL12.

pUC-E1B-FRTK1-IL12 and pUC-E1B-FRTK2-IL12 were digested with HpaI and KpnI, and the fragments were recovered, inserted into the HpaI and KpnI restriction sites in pXC-FRSVE1A, respectively, and named pXC-FRSVE1A- FRTKE1B1-IL12, pXC-FRSVE1A-FRTKE1B2-IL12.

Example 11: Recombination of Epstein-Barr virus cis-acting elements carrying interleukin-12 fusion gene to control the expression of E1A and E1B.

The 293 cell line was purchased from Microbix Biosystem Inc. (Toronto) in Canada. It was transformed from cut type 5 adenovirus DNA into human embryonic kidney cells. It contains and expresses type 5 adenovirus E1 region, and adenovirus DNA has a high Transfection rate. The plasmid containing the left arm of type 5 adenovirus combined with the plasmid containing the right arm of type 5 adenovirus was co-transfected into 293 cells, and an infectious adenovirus could be produced through homologous recombination. We co-transfected pXC-FRSVE1A-FRTKE1B1-IL12 and pXC-FRSVE1A-FRTKE1B2-IL12 and plasmid pBHG10 containing the right arm of type 5 adenovirus into 293 strains by Lipofectamine. For specific methods, please refer to the operation instructions of GIBCO BRL Company. PBHG10 was purchased from Microbix Biosystems Inc. (Ontario), Canada, and contains the right arm of adenovirus type 5, but lacks the E3 region. Virus plaques appeared 9-14 days after co-transfection, and after three times of virus plaque purification, E1A and E1B cis-acting elements Orip FR combined with mini-SV40 promoter and OripFR combined with mini-HSV-TK promoter were obtained. The controlled adenovirus, carrying human interleukin 12 fusion gene, was named EBV FRSVEIA-FRTKE1B1-IL12 and FRSVEIA-FRTKE1B2-IL12, respectively, and recorded as CNHKEBV1-IL12 and CNHKEBV2-IL12, respectively.

The list of recombinant viruses constructed by the above method is as follows:

Contains Ad5 right

Virus Name Containing Ad5 Left Arm Plasmid

                                                                                                                                                                                                                                                                                                       |

CNHKEBV1-IL12 PBHG10

E1B1-IL12 FRTKE1B1-IL12EBV FRSVEIA-FRTK pXC-FRSVE1A-

CNHKEBV2-IL12 PBHG10

E1B2-IL12 FRTKE1B2-IL12

The adenoviruses reproduced in large quantities in 293 cells, and the cesium chloride gradient centrifugation method was used to purify a large number of adenoviruses (see reference 2 for the method). EBV FRSVEIA-FRTK E1B1-IL12 (CNHKEBV1-IL12) is a type 5 adenovirus, and the EB virus cis-acting element Orip FR combined with mini-SV40 is inserted between the E1A and E1B transcription start sites and the coding start sites, respectively. and the Orip FR joint mini-HSV-TK promoter, and create a new BglII restriction site between the E1A stop codon and the upstream region of the Orip FR joint mini-HSV-TK promoter. The insertion contains human cytomegalovirus (HCMV) IE promoter (-299--+72), human interleukin 12 fusion gene and SV40 poly A tailed signal gene sequence, accompanied by 28133-30818bp (E3 region part sequence) is missing, and the other DNA sequences of the virus are the same as those of type 5 adenovirus. EBV FRSVEIA-FRTK E1B2-IL12 (CNHKEBV2-IL12) is a type 5 adenovirus, and the EB virus cis-acting element Orip FR combined with mini-SV40 is inserted between the E1A and E1B transcription start sites and the coding start sites, respectively. and the Orip FR joint mini-HSV-TK promoter, and create a new BglII restriction site between the E1A stop codon and the upstream region of the Orip FR joint mini-HSV-TK promoter. Reverse insertion contains human cytomegalovirus (HCMV) IE promoter (-299--+72), human interleukin 12 fusion gene and SV40 poly A tailed signal gene sequence, accompanied by 28133-30818bp (E3 region Part of the sequence) is missing, and the other DNA sequences of the virus are the same as those of type 5 adenovirus.

Example 12: EB virus cis-acting element carrying human interleukin 12 fusion gene controls the expression of E1A and E1B in vitro Proliferation, replication and high-level expression of human interleukin in EB infected or latently infected tumor cells 12 and specifically kill tumor cells.

CNHKEBV1-IL12 and CNHKEBV2-IL12 were respectively infected with Epstein-Barr virus-infected lymphoma cell lines Jijoye, 293 and normal human fibroblasts. The cells were inoculated into 6-well plates at 2×10 ⁵ /well, and respectively infected with recombinant adenoviruses CNHKEBV1-IL12, CNHKEBV2-IL12 and wild-type 5 adenovirus 4×10 ⁵ pfu, 48 hours later, the 293 cell line was used to measure the virus titer. For the specific method, refer to the above-mentioned literature 2, and the results are as follows:

Normal normal fibroblast

293 Jijoye

Cellular wild-type adenovirus type 5 1×10 ⁵ 7×10 ⁴ 5×10 ⁴ CNHKEBV1-IL12 1×10 ⁵ 3×10 ⁵ 2×10CNHKEBV2-IL-12 1×10 ⁵ 2×10 ⁵ 3×10

Lymphoma cell line Jijoye and normal human fibroblasts infected with Epstein-Barr virus were infected with recombinant adenoviruses CNHKEBV1-IL12 and CNHKEBV2-IL12 respectively, MOI was 1, incubated at 37°C for 1 hour, cells were collected 7 days after infection, and QIAamp DNA was applied to Blood minikit (QIAGEN, Germany) was used to extract viral DNA. For the method, refer to the operating instructions of QIAGEN. Apply Nhe I and Xho I double digestion, use 0.8% agarose electrophoresis, transfer it to a nylon membrane, use ³² P to label human type 5 adenovirus 1178bp BstXI plus Xho I fragment (located at adenovirus nt4611-5789), and carry out Southern blot hybridization, using pXC.1 as the virus copy number control (for the method, refer to the Molecular Cloning Experiment Guide, published in Science Publishing 1992), the virus copy numbers of each cell of CNHKEBV1-IL12 and CNHKEBV2-IL12 in Jijoye and normal human fibroblasts were respectively 5×10 ⁴ , 5×10 ⁴ , and <10, <10. The DNA extracted from the above-mentioned CNHKEBV1-IL12 and CNHKEBV2-IL12 was respectively digested with Bgl II, electrophoresed with 1% agarose, transferred to a nylon membrane, and labeled with ³² P. Human endostatin cDNA fragments (EcoR I and Xba I double digested pCA14-human IL12, recovered 637bp) as a probe, carried out souther blot hybridization, and used pCA13-human endostatin as a virus copy number control (for the method, refer to the Molecular Cloning Experiment Guide, published by Science Press 1992), CNHKEBV1-IL12 and CNHKEBV2 - The virus copy numbers per cell of IL12 in Jijoye and normal human fibroblasts were 5×10 ⁴ , 5×10 ⁴ and <10, <10, respectively.

Claims (19)

1. A proliferative recombinant virus capable of specifically killing Epstein-Barr virus-related tumor cells is characterized in that the non-proliferation essential region in its modified virus genome is inserted with a purpose gene, and the purpose gene is a tumor suppressor gene, an angiosuppressor gene, a cell One of factor gene, prodrug converting enzyme gene and apoptosis gene. 2. The recombinant virus according to claim 1, characterized in that said tumor suppressor gene comprises one of the following genes: P53, P21, Rb, NF1, VHL and APC. 3. recombinant virus according to claim 1, it is characterized in that described blood vessel suppressing gene comprises following one gene: endostatin gene, angiostatin gene, are Kringle1-4 structure in plasma plasminogen, Kringle1-5 structure, Kringle1-3 structure and Kringle1-3 plus Kringle5 structure, interferon-α gene, interferon-β gene, interferon-γ gene, thrombospondin gene, platelet factor 4 gene, plasminogen Activator inhibitor (PAI) gene, interleukin-12 and fibronectin genes. 4. The recombinant virus according to claim 3, characterized in that said vascular inhibitory gene vascular inhibitory gene contains a coded secretory signal peptide. The secretory signal peptide can be any one of the following: signal peptide of angiogenesis inhibitor itself, signal peptide of M-oncogenesis protein, signal peptide of immunoglobulin K chain. 5. The recombinant virus according to claim 1, wherein said cytokine gene comprises one of the following genes: interleukin 2, interleukin 12, granulocyte-single colony stimulating factor, tumor necrosis factor, interferon- α, Interferon-β, Interferon-γ, Light and Flt3 Ligands. 6. recombinant virus according to claim 1, it is characterized in that described prodrug conversion enzyme gene comprises following one gene: herpes simplex virus thymidine kinase, varicella-zoster virus thymidine kinase and Escherichia coli cytosine desine ammonia enzyme. 7. The recombinant virus according to claim 1, characterized in that said apoptosis gene comprises one of the following genes: ICE, capase-3, capase-8, capase-9. 8. The recombinant virus according to claim 1, characterized in that a certain cis-acting element is inserted between the transcription initiation point and the coding initiation site of the essential gene for virus propagation, and the cis-acting element contains at least the following Sequence one: Orip in EB virus, FR in EB virus Orip, EB virus BamHI C-promoter, Orip in EB virus combined with EB virus BamHI C-promoter, FR in EB virus Orip combined with herpes simplex virus thymus Pyrimidine nucleoside kinase basic promoter or SV40 basic promoter, a cis-acting element specifically activated in Epstein-Barr virus-infected or latently infected cells, and an essential gene for virus proliferation is a viral early gene. 9. The recombinant virus according to claim 8, characterized in that the proliferative recombinant virus is an adenovirus, and the adenovirus proliferation essential gene contains at least one of the following early expression genes of the adenovirus: E1A, E1B, E2, E4 . 10. A method for constructing a proliferative recombinant virus capable of specifically killing Epstein-Barr virus-related tumor cells, characterized in that two vectors containing the left and right arms of the adenovirus are co-transfected into the recombinant adenovirus-producing Cells, at least one of the two adenoviral vectors has an adenoviral non-proliferation-essential region inserted into a target gene, and at least one of the two adenoviral vectors contains adenoviral early genes E1A, E1B, E2, and E4 A certain cis-acting element is inserted in the expression region between the transcription start site and the coding start site, and the cis-acting element contains at least one of the following sequences: Orip in Epstein-Barr virus, FR in Epstein-Barr virus Orip, EB Virus Bam HIC-promoter, Orip in Epstein-Barr virus combined with Epstein-Barr virus Bam HI C-promoter, FR in Epstein-Barr virus Orip combined with herpes simplex virus thymidine kinase basic promoter or SV40 basic promoter, and in Epstein-Barr virus A cis-acting element that is specifically activated in infected or latently infected cells. 11. The construction method of a kind of recombinant virus according to claim 10, it is characterized in that by polymerase chain reaction technology, an enzyme cut is made in the region between the adenovirus early gene transcription start site and the coding start site site, insert the above-mentioned cis-acting elements into this site by enzyme cutting, so that the adenovirus early gene is controlled by the cis-acting elements specifically activated by Epstein-Barr virus infection or latently infected cells. 12. A method for multiplying a proliferative recombinant virus that can specifically kill Epstein-Barr virus-related tumor cells, characterized in that the recombinant virus is infected with Epstein-Barr virus infection or latently infected mammalian cells, so that the recombinant virus can specifically infect Epstein-Barr virus Proliferates in infected or latently infected mammalian cells. 13. An application of the proliferative recombinant virus capable of specifically killing Epstein-Barr virus-related tumor cells as claimed in claim 1, characterized in that the recombinant virus is used to infect Epstein-Barr virus-infected or latently infected tumor cells in vitro, cause cytotoxicity. 14. The application of the proliferative recombinant virus capable of specifically killing Epstein-Barr virus-associated tumor cells as claimed in claim 1, characterized in that the recombinant virus is used to inhibit the growth of Epstein-Barr virus-infected or latently infected tumor cells . 15. An application of the proliferative recombinant virus capable of specifically killing Epstein-Barr virus-associated tumor cells as claimed in claim 1, characterized in that the recombinant virus is used to selectively kill Epstein-Barr virus infection or latent infection in vivo of tumor cells. 16. An antineoplastic drug, characterized in that the drug is a complex composed of the proliferative recombinant virus capable of specifically killing Epstein-Barr virus-related tumors as claimed in claim 1, chemotherapeutic drugs, biotoxins, and monoclonal antibodies. 17. The antineoplastic drug according to claim 16, characterized in that said chemotherapeutic drug is one of cisplatin, 5-fluorouracil, mitomycin C, carboplatin, and cyclophosphamide. 18. The antitumor drug according to claim 16, characterized in that said biological toxin is one of snake toxin and diphtheria toxin. 19. The antitumor drug according to claim 16, characterized in that said monoclonal antibody is nasopharyngeal carcinoma cell antibody.