CN115927215A - A targeted attenuated vaccinia virus vaccine - Google Patents

Abstract

The invention relates to a recombinant vaccinia virus and application thereof. Compared with the Tiantan strain of vaccinia virus, the recombinant vaccinia virus lacks at least one of TK gene, F4L gene and B2R gene. The recombinant vaccinia virus of the invention has the characteristics of low toxicity, complete replication and higher immunogenicity. Therefore, the recombinant vaccinia virus provided by the present invention has higher safety and enhanced immunogenicity. The recombinant vaccinia virus provided by the invention has higher application value as a vaccine, can be used to prevent infection of poxviridae viruses, and can be used as a virus vector to construct other infectious disease vaccines and tumor vaccines.

Description

A targeted attenuated vaccinia virus vaccine

technical field

The invention belongs to the field of vaccines, in particular to a recombinant vaccinia virus and its application.

Background technique

Poxvirus is a type of DNA virus with the largest virions, including various types: Vaccinia virus, Variola virus, Cowpox virus and Monkey poxvirus, etc. Before the last century, smallpox virus had spread around the world, with a fatality rate of 30%. Later, vaccinia virus was used as a vaccine strain for the prevention and immunity of smallpox, and played an important role in the process of eliminating smallpox. Later studies found that using After inoculation against smallpox vaccinia virus strains, it also has a protective effect against monkeypox and other orthopoxvirus infections. In 1978, the WHO announced that smallpox virus was eliminated, and the vaccination of vaccinia virus was also stopped. However, because vaccinia virus has the characteristics of wide host range, many non-essential genes, high conservation, large capacity of foreign genes, and cytoplasmic replication, As a vaccine carrier, it still has further research value for the development of new infectious disease vaccine carriers and oncolytic viruses.

The vaccinia virus used to prevent smallpox, the original vaccinia virus (VACV), has full replication competence in mammalian cells. The production of the first-generation vaccine strains is mostly carried out in animals. After the vaccine strains are inoculated in the skin lymph nodes of animals to replicate and amplify, they are made into vaccine strains for human immunization, such as the New York City Board of Health (NYCBH) stored and produced in the United States. ) strain, the Lister strain stored and used in many European countries, the Tiantan strain (VACV Tiantan, VTT) in China, the Copenhagen (Cop) strain in Denmark, etc. Although the first-generation vaccine strain has a high protection rate, it has certain side effects in the population, 40-47% of the injection site pain, 5-9% of fever, and some individuals with abnormal immunity developed eczema, encephalitis, etc. symptom.

With the development of vaccine production technology, the production of vaccine strains has shifted to tissue or chicken embryo cell culture, thereby generating the second generation of vaccinia virus strains. For example, the RIVM strain produced by the lisiter strain cultured in rabbit kidney cells, the Lister/CEP strain cultured in the chicken embryo allantoic membrane, the CCSV strain formed by the NYCBH strain cultured in MRC-5 cells and ACAM1000, ACAM2000 strains, etc. The second-generation vaccine strain has a similar immune effect to the first-generation vaccine strain, and the neurotoxicity of the ACAM2000 vaccine strain is relatively lower than that of the first-generation vaccine strain, but it still produces certain side effects after being immunized as a vaccine strain.

In order to solve the relative safety problems of the first two generations of vaccine strains, an attenuated third-generation vaccine strain was further developed. The first- and second-generation vaccine strains with replication ability are continuously passaged in cultured cells, gene mutations accumulate between generations, host range factors, virus virulence genes, etc. change, and then vaccine strains with low toxicity are screened out. The third-generation vaccine strains mainly include MVA strain, LC16m8 strain and DI strain. Japanese researchers used the Lister strain and passaged it in rabbit kidney epithelial cells (PRK) under low temperature conditions to obtain an attenuated strain LC16m8, which has relatively few gene mutation sites and can still replicate in human cells. After the Japanese first-generation Dairen vaccine strain was subcultured in 1-day-old chicken embryos, the attenuated strain DI was obtained, with more gene deletion sites than the LC16m8 strain, 19 open reading frames were deleted, and it could not replicate in most mammalian cells. The third-generation vaccine strain with the weakest toxicity is the MVA strain, which was isolated from the COP strain after more than 500 passages in chicken embryo fibroblasts. Compared with the parent CVA strain, its genome is missing about 15%. Although it can infect human cells and encode early, middle and late proteins, it cannot be assembled into infectious virus particles, so it cannot replicate in various mammalian cells including humans, and the toxicity is significantly reduced, and the effect of virus passage Better than the DI strain, as a new generation of vaccine vector, it is widely used in the prevention and treatment of infectious diseases and tumors. However, comparing the immune effects of different vaccine strains, it is found that at the same dose, the replication-deficient strain needs to have a virus titer 1-2 logarithmically higher than the original vaccine strain or adopt a booster immunization plan to achieve the immune effect of the original vaccine. , and the antibody protection time, neutralization effect, specific cellular immunity, etc. are still not as effective as replicating the complete first-generation vaccine.

Therefore, it is necessary to develop a new generation of candidate vaccinia virus with complete replication ability, reduced toxicity and enhanced immunogenicity, which can be used to prevent the spread of zoonotic and human-to-human infections caused by poxviridae viruses, and as a new type of vaccine vector ,necessary.

Contents of the invention

In view of this, the object of the present invention is to propose a recombinant vaccinia virus and its application. The recombinant vaccinia virus has the characteristics of low toxicity, complete replication and higher immunogenicity, and can be used to prevent poxviridae virus infection. And as a viral vector for constructing other infectious disease vaccines and tumor vaccines.

Based on the above purpose, the first aspect of the present invention provides a recombinant vaccinia virus, which has deleted at least one of the TK gene, F4L gene and B2R gene relative to the vaccinia virus Tiantan strain.

In a preferred embodiment of the present invention, the recombinant vaccinia virus is one of the following recombinant vaccinia viruses:

(1) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dTK that has deleted the TK gene;

(2) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dF4L that has deleted the F4L gene;

(3) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dB2R that has deleted the B2R gene;

(4) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dT/F that has simultaneously deleted the TK gene and the F4L gene;

(5) Compared with the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dB/T/F that simultaneously deleted the TK gene, the F4L gene and the B2R gene.

In a preferred embodiment of the present invention, the recombinant vaccinia virus is a recombinant vaccinia virus VTT-dB/T/F that has simultaneously deleted the TK gene, the F4L gene and the B2R gene relative to the vaccinia virus Tiantan strain.

In a preferred embodiment of the present invention, the sequences of the TK gene, the F4L gene and the B2R gene are respectively shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

In a preferred embodiment of the present invention, the recombinant vaccinia virus is:

(1) The genome sequence of the recombinant vaccinia virus VTT-dTK is relative to the genome sequence of the vaccinia virus Tiantan strain, the partial sequence of the TK region is deleted and the p11 promoter sequence and the p7.5 promoter sequence are inserted; wherein the partial sequence of the TK region is GenBank: 80439-80465 in JX489139.1, p11 promoter sequence and p7.5 promoter sequence are GenBank: 43077-43168 in JX489139.1 and GenBank: 189237-189494 in JX489139.1;

(2) The genome sequence of the recombinant vaccinia virus VTT-dF4L is relative to the genome sequence of the vaccinia virus Tiantan strain, the partial sequence of the F4L region is deleted and the p11 promoter sequence and the p7.5 promoter sequence are inserted; wherein the partial sequence of the F4L region is GenBank: 32451-33293 in JX489139.1, p11 promoter sequence and p7.5 promoter sequence are GenBank: 43077-43168 in JX489139.1, GenBank: 189237-189494 in JX489139.1;

(3) The genome sequence of the recombinant vaccinia virus VTT-dB2R is relative to the genome sequence of the vaccinia virus Tiantan strain, the B2R region sequence is deleted and the p11 promoter sequence and p7.5 promoter sequence are inserted; wherein the B2R region sequence is GenBank: 164449-165126 in JX489139.1, p11 promoter sequence and p7.5 promoter sequence are GenBank: 43077-43168 in JX489139.1, GenBank: 189237-189494 in JX489139.1;

(4) The genome sequence of the recombinant vaccinia virus VTT-dT/F is relative to the genome sequence of the vaccinia virus Tiantan strain, and part of the sequence of the TK region and part of the F4L region are deleted at the same time; the part of the sequence of the TK region is in GenBank: JX489139.1 80439-80465, the partial sequence of F4L region is 32451-33293 in GenBank: JX489139.1;

(5) The genome sequence of the recombinant vaccinia virus VTT-dB/T/F is relative to the genome sequence of the vaccinia virus Tiantan strain, and the partial sequence of the TK region, the partial sequence of the F4L region and the sequence of the B2R region are deleted at the same time; wherein the partial sequence of the TK region is GenBank: 80439-80465 in JX489139.1, the partial sequence of F4L region is GenBank: 32451-33293 in JX489139.1, and the sequence of B2R region is 164449-165126 in GenBank: JX489139.1;

Wherein, the genome sequence of the vaccinia virus Tiantan strain is the sequence represented by the gene accession number Gene Bank No.AF095689.

The second aspect of the present invention provides a method for constructing a recombinant vaccinia virus, which uses homologous recombination to construct a recombinant vaccinia virus.

In a preferred embodiment of the present invention, it comprises the following steps:

(1) Construct respectively the first recombination plasmid comprising the left arm of the TK gene homology arm and the right arm of the TK gene homology arm, the second recombination plasmid comprising the left arm of the F4L gene homology arm and the right arm of the F4L gene homology arm, and the second recombination plasmid comprising The third recombinant plasmid of the left arm of the B2R gene homology arm and the right arm of the B2R gene homology arm;

(2) Infect the cells with the vaccinia virus Tiantan strain, and then transfect the first recombinant plasmid, the second recombinant plasmid and the third recombinant plasmid, respectively, to obtain recombinant vaccinia virus VTT-dTK with respect to the vaccinia virus Tiantan strain, which has deleted the TK gene , a recombinant vaccinia virus VTT-dF4L with the deletion of the F4L gene, and a recombinant vaccinia virus VTT-dB2R with the deletion of the B2R gene.

In a preferred embodiment of the present invention, the cells are infected with the recombinant vaccinia virus VTT-dTK and transfected with the second recombinant plasmid again to obtain the recombinant vaccinia virus VTT- dT/F.

In a preferred embodiment of the present invention, the cells are infected with the recombinant vaccinia virus VTT-dT/F and transfected with the third recombinant plasmid again to obtain the TK gene, F4L gene and B2R gene that have been deleted at the same time relative to the vaccinia virus Tiantan strain. Recombinant vaccinia virus VTT-dB/T/F.

The third aspect of the present invention provides the use of the above-mentioned recombinant vaccinia virus in the preparation of poxvirus vaccine or as a vaccine carrier.

The fourth aspect of the present invention provides a poxvirus vaccine comprising the above-mentioned recombinant vaccinia virus.

The beneficial effects of the present invention are:

Compared with the vaccinia virus Tiantan strain, the recombinant vaccinia virus provided by the present invention has the characteristics of low toxicity, is an attenuated vaccinia virus, and has higher safety; moreover, the recombinant vaccinia virus provided by the present invention also has the ability to fully replicate , It has the characteristics of higher immunogenicity and can better activate the natural immune response. In other words, the recombinant vaccinia virus provided by the present invention has higher safety and enhanced immunogenicity.

The recombinant vaccinia virus provided by the invention can be used to develop a new generation of vaccinia virus vaccines, to prevent infection of poxviridae viruses, and to construct other infectious disease vaccines and tumor vaccines as virus vectors.

Description of drawings

Figure 1 Schematic diagram of homologous recombination for deletion of TK;

Fig. 2 is the PCR identification result to recombinant virus VTT-dTK, and wherein WT is wild-type virus, and dTK is VTT-dTK recombinant virus;

Figure 3 Schematic diagram of homologous recombination for deletion of F4L;

Fig. 4 is the PCR identification result to recombinant virus VTT-dF4L, and wherein WT is wild-type virus, and dF4L is VTT-dF4L recombinant virus;

Figure 5 is a schematic diagram of homologous recombination for deletion of B2R;

Figure 6 is a schematic diagram of PCR primers required for identification of recombinant virus VTT-dB2R;

Fig. 7 is the identification result of the recombinant virus VTT-dB2R. Wherein, N is a negative control, WT is a wild virus, and delB2R is a recombinant virus VTT-dB2R;

Figure 8 is the level of innate immune response induced by the recombinant virus VTT-dB2R, wherein mock is a negative control, WT is wild virus, and delB2R is the recombinant virus VTT-dB2R;

Fig. 9 is the identification result of the recombinant virus VTT-dT/F. Wherein, WT is the wild virus, and dT/F is the recombinant virus VTT-dT/F;

Figure 10 is the identification result of the recombinant virus VTT-dB/T/F. Among them, 1-6 is the recombinant virus VTT-dB/T/F, and WT is the wild type virus;

Figure 11 is a comparison of the replication levels of different recombinant viruses in mammalian cells;

Figure 12 shows the results of body weight changes in mice infected with different viruses;

Figure 13 is the survival situation of mice infected with different viruses;

Figure 14 is a comparison of the size of erythema at the injection site after rabbits were immunized with different viruses;

Figure 15 is the comparative statistics of the size of erythema at the injection site after rabbits were immunized with different viruses;

Figure 16 is the antibody titer level induced after different virus immunization mice;

Figure 17 shows the levels of serum neutralizing antibodies after mice were immunized with different viruses.

Detailed ways

It should be noted that, unless otherwise defined, the technical terms or scientific terms used in this application shall have the usual meanings understood by those skilled in the art.

The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

As used herein and in the appended claims, the singular forms "a," "an," "another," and "the" include plural referents unless the context clearly dictates otherwise.

In this application, the term "comprises/comprises" generally means including specifically specified features, but not excluding other elements.

In the present application, "targeted attenuation" refers to the use of genetic engineering (such as homologous recombination) to make vaccinia virus Tiantan strain produce targeted mutations, and at least one of the TK gene, F4L gene and B2R gene is deleted, so that Greatly reduce its toxicity, the vaccine made of this weakened recombinant vaccinia virus is called attenuated vaccine.

Vaccinia virus is a DNA virus, and a variety of enzymes related to deoxyribonucleic acid synthesis play an important role in its replication process. These enzymes include thymidylate kinase (TK), ribonucleotide reductase (ribonucleotide reductyase), deoxyuridine triphosphatase (deoxyuridine triphosphatase) and so on. When vaccinia virus replicates, a high-concentration nucleotide pool can be formed under the action of these enzymes to ensure the smooth progress of progeny DNA replication. In normal cells, the concentration of nucleotides is low, so TK is necessary for virus proliferation in normal cells; tumor cells have a higher concentration of nucleotides, so TK is not necessary for virus proliferation in tumor cells. It can be known from the above that the poxvirus with deleted TK gene can preferentially replicate in tumor cells that provide enough nucleotides for it, but cannot replicate in normal cells. Ribonucleic acid reductase F4L is produced shortly after the virus infects cells, and its structure is 70% to 80% similar to that of eukaryotic ribonucleic acid reductase. It contains a catalytic small subunit and a regulatory large subunit, which can catalyze Nucleoside diphosphates form deoxynucleoside diphosphates. Mutations in the large subunit would inhibit reductase activity, and the virus mutants in mice would be less virulent. There is a sensory molecule of viral DNA in the host cell, that is, cyclic GMP-AMP synthase (cGAS). After recognizing foreign DNA molecules such as viruses, its enzymatic activity is activated, and the downstream is activated by synthesizing 2', 3'-cGAMP STING signaling pathway, and then induce the expression of interferon, in order to achieve the host's anti-viral natural immune response. The B2R gene of vaccinia virus encodes poxin protein, which can specifically degrade intracellular 2',3'-cGAMP, thereby blocking the natural immune activation signal induced by intracellular cGAS. After deleting B2R, vaccinia virus infection can be activated more strongly Innate immunity, which in turn induces a higher level of specific immune response.

The present invention attempts to delete at least one of the above genes on the basis of the vaccinia virus Tiantan strain, in order to obtain a recombinant vaccinia virus with low toxicity, complete replication and higher immunogenicity.

In a preferred embodiment of the present invention, the recombinant vaccinia virus is one of the following recombinant viruses:

(1) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dTK that has deleted the TK gene;

(2) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dF4L that has deleted the F4L gene;

(3) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dB2R that has deleted the B2R gene;

(4) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dT/F that has simultaneously deleted the TK gene and the F4L gene;

(5) Compared with the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dB/T/F that simultaneously deleted the TK gene, the F4L gene and the B2R gene.

In a preferred embodiment of the present invention, the recombinant vaccinia virus is a recombinant vaccinia virus VTT-dB/T/F that has simultaneously deleted the TK gene, the F4L gene and the B2R gene relative to the vaccinia virus Tiantan strain. Recombinant vaccinia virus VTT-dB/T/F has the following characteristics:

1. Compared with the vaccinia virus Tiantan strain, the recombinant virus VTT-dB/T/F that simultaneously deletes the TK gene, the F4L gene and the B2R gene has a lower replication level in mammalian cells.

2. Compared with the vaccinia virus Tiantan strain, the recombinant virus VTT-dB/T/F lacking TK gene, F4L gene and B2R gene at the same time has significantly lower virulence.

3. The recombinant virus VTT-dB/T/F, which simultaneously lacks TK gene, F4L gene and B2R gene, can still induce higher serum antibody levels after immunizing mice.

In a word, the recombinant vaccinia virus VTT-dB/T/F obtained by simultaneously deleting the TK gene, the F4L gene and the B2R gene on the basis of the vaccinia virus Tiantan strain (VTT) in the present invention, compared with VTT, has significantly lower toxicity and more It is safe and will not cause serious pathological reactions in the body, and the recombinant vaccinia virus VTT-dB/T/F can better activate the natural immune response and has higher immunogenicity. In other words, the recombinant vaccinia virus VTT-dB/T/F provided by the present invention, which simultaneously deletes the TK gene, the F4L gene and the B2R gene, has higher safety and enhanced immunogenicity.

The above-mentioned recombinant vaccinia virus is constructed by homologous recombination. The basic principle is: construct a recombinant plasmid containing a homologous arm, and transfect the recombinant plasmid containing a homologous arm into the cells after inoculation, so that the virus undergoes homologous recombination in the cell. Source recombination to delete or insert a gene of interest.

The present invention also provides a poxvirus vaccine, which comprises the above-mentioned recombinant vaccinia virus, and the poxvirus vaccine can be used to prevent the infection of poxviridae virus, and can be used as a virus vector to construct other infectious disease vaccines and tumor vaccines.

The technical solutions provided by the present invention will be further described below in conjunction with specific embodiments. The following examples are only used to illustrate the present invention, and do not limit the protection scope of the present invention.

In the present invention, the whole genome sequence of the vaccinia virus Tiantan strain has been deposited in the International Gene Bank, numbered Gene Bank, No.AF095689. In the present invention, the source of the vaccinia virus Tiantan strain is not particularly limited, and conventional methods can be used. HEK293T, Vero, and HeLa cells used in the present invention use DMEM culture fluid containing 10% fetal bovine serum, 100U/mL penicillin, and 100 μg/mL streptomycin; THP1 cells use 10% fetal bovine serum, 100 U/mL penicillin, 100 μg /mL streptomycin 1640 culture medium, cultured in a 37°C, 5% CO ₂ incubator, and passaged once every 2-3 days.

The technical solutions provided by the present invention will be described in detail below in conjunction with the examples, but they should not be interpreted as limiting the protection scope of the present invention.

Those skilled in the art can query the corresponding nucleotide sequence in the GenBank: JX489139.1 mentioned in the following examples in the NICBI database (https://www.ncbi.nlm.nih.gov/) The nucleotide sequence of the position, for example, the nucleotide sequence of 79716-80438 in GenBank: JX489139.1 found in NCBI data is (shown as the sequence with underline):

79681 gtataaagat tgtgcaaagc ttttgcgatc aataa atgga tcacaaccag tatctcttaa

79741 cgatgttctt cgcagatgat gattcatttt ttaagtattt ggctagtcaa gatgatgaat

79801 cttcattatc tgatatattg caaatcactc aatatctaga ctttctgtta ttattattga

79861 tccaatcaaa aaataaatta gaagccgtgg gtcattgtta tgaatctctt tcagaggaat

79921 acagacaatt gacaaaattc acagactctc aagattttaa aaaactgttt aacaaggtcc

79981 ctattgttac agatggaagg gtcaaactta ataaaggata tttgttcgac tttgtgatta

80041 gtttgatgcg attcaaaaaa gaatcctctc tagctaccac cgcaaatagat cctattagat

80101 acatagatcc tcgtcgcgat atcgcatttt ctaacgtgat ggatatatta aagtcgaata

80161 aagtgaacaa taattaattc tttatgtca tcatgaacgg cggacatatt cagttgataa

80221 tcggccccat gttttcaggt aaaagtacag aattaattag acgagttaga cgttatcaaa

80281 tagctcaata taaatgcgtg actataaaat attctaacga taatagatac ggaacgggac

80341 tatggacgca tgataagaat aattttgaag cattggaagc aactaaacta tgtgatgtct

80401 tggaatcaat tacagatttc tccgtgatag gtatcgat ga aggacagttctttccagaca

The primer sequences referred to in the following examples are summarized in the table below.

Primer Sequence List

Embodiment 1 constructs the recombinant virus VTT-dTK of TK gene deletion

1. Method

1.1 Reorganization

The recombinant plasmid pdelJ2R is synthesized by the company, mainly including p7.5 promoter (early promoter), p11 promoter (late promoter), TK region homology arm.

The pdelJ2R plasmid preferably contains p7.5 promoter, p11 promoter and TK region homology arm sequence. The pdelJ2R plasmid sequence is shown in SEQ ID NO:4. The key sequences are the left arm of the homology arm of the TK region (GenBank: 79716-80438 in JX489139.1), p11 and p7.5 promoter sequences (GenBank: 43077-43168 in JX489139.1, GenBank: 189237 in JX489139.1) -189494), the right arm of the homology arm of the TK region (GenBank: 80466-81460 in JX489139.1).

293T cells (5×10 ⁵ ) were inoculated in a six-well plate in DMEM complete medium without antibiotics, and cultured overnight. When the cell growth was 60% to 70%, the wild-type virus (that is, vaccinia virus Tiantan strain) was diluted with DMEM, and infected Cells; after 2 hours, transfect the recombinant plasmid pdelJ2R; after 4 hours, replace the medium with 2% FBS; after 48 hours, discard 1.2mL supernatant, blow out the cells, and freeze-thaw three times.

1.2 Screening and purification of recombinant virus

Vero (1×10 ⁶ ) was inoculated in a six-well plate in complete antibiotic-free DMEM medium and cultured overnight. When the cell growth was 80% to 90% flat, the virus supernatant collected in the above 1.2.1 freeze-thawed three times and centrifuged was suspended. A 10-fold serial dilution of the solution was used to re-infect Vero cells. After 2 hours, wash with PBS and replace with methylcellulose medium with a final concentration of 1.2%. Three days later, a single plaque was selected by means of viral plaque purification and identified by PCR. The primer sequences used are shown in Table 1 below, which are respectively located in the left and right homology arms of the TK gene. According to the amplification of wild-type virus and recombinant virus, According to the difference of the fragments, the recombinant poxvirus was identified, and the purification steps were repeated until pure recombinant virus VTT-dTK was obtained.

Table 1 Primer Sequence

The specific steps of PCR are as follows:

Table 2 Operation steps of PCR amplification

(1) Add the dosage according to the above table into a 0.2mL nucleic acid-free thin-walled PCR tube (see the above primer table for the primer sequence).

(3) Mix gently and centrifuge briefly to ensure that all reaction components are at the bottom of the amplification tube.

(4) Put the reaction system into the PCR instrument that has been set up according to the above method for reaction. The PCR reaction products were collected and stored at -20°C.

PCR product electrophoresis:

Detection of viral PCR products by 1% agarose gel electrophoresis:

(1) Add 0.3 g of accurately weighed agarose powder to the conical flask that has been added with 30 mL of 1×TAE electrophoresis buffer.

(2) Place the Erlenmeyer flask into a microwave oven and heat it over medium heat until the agarose is completely dissolved, then cool it to about 60-70°C at room temperature, add 2 μL of ethidium bromide (EB), and shake it gently to mix well.

(3) Put the rubber mold into the glue tank and fix it, and place a comb at a distance of 0.5-1.0 mm from the bottom plate, so that a complete sample hole can be formed after adding agarose.

(4) Pour warm agarose solution into the rubber mold, the thickness of the gel is between 3-5mm.

(5) After the gel is completely solidified (place it at room temperature for 10-25 minutes or in a refrigerator at 4°C for 5 minutes), carefully remove the comb and put the gel into the electrophoresis tank.

(6) Add a sufficient amount of electrophoresis buffer just below the surface of the gel to a depth of about 1 mm.

(7) After mixing 10 μL of DNA sample with 2 μL of 6×Loading Buffer, slowly add the mixture into the sample well with a micropipette.

(8) Cover the electrophoresis tank and turn on the electricity to move the DNA to the anode. After a few minutes, bromophenol blue migrates from the wells into the gel. Continue electrophoresis until the bromophenol blue has migrated an appropriate distance in the gel.

(9) Cut off the current, unplug the wire from the electrophoresis tank, and open the tank cover. Remove the gel.

(10) Observe the movement of the sample under ultraviolet light and take pictures.

1.3 Identification of recombinant virus

DNAMini Kit说明书提取DNA，然后将DNA溶于无菌水，用Nanodrop测定DNA含量。将提取的野生型、重组型病毒基因组进行PCR扩增，鉴定引物如表1所示。The obtained recombinant virus and wild-type virus samples were

The DNAMini Kit manual extracts DNA, then dissolves the DNA in sterile water, and uses Nanodrop to measure the DNA content. The extracted wild-type and recombinant virus genomes were amplified by PCR, and the primers for identification are shown in Table 1.

2. Results

2.1 Construction of recombinant plasmid pdelJ2R

The constructed recombinant plasmid pdelJ2R mainly includes a p7.5 promoter, a p11 promoter, and a homology arm of the TK region (as shown in FIG. 1 ).

2.2 Screening and identification of recombinant virus VTT-dTK

DNAMini Kit说明书提取DNA，然后利用PCR扩增验证(PCR验证所用引物如表1所示)，重组病毒中缺失了TK区(如图2所示)，再将PCR产物测序，重组病毒中缺失了TK区部分序列(GenBank:JX489139.1中80439-80465)，插入了p11启动子序列和p7.5启动子序列(GenBank:JX489139.1中43077-43168，GenBank:JX489139.1中189237-189494)，确证重组病毒VTT的TK区不表达。Press the recombinant virus

The DNAMini Kit manual extracts DNA, and then uses PCR amplification verification (primers used in PCR verification are shown in Table 1), the TK region is missing in the recombinant virus (as shown in Figure 2), and then the PCR product is sequenced, and the TK region is missing in the recombinant virus Partial sequence of TK region (GenBank: 80439-80465 in JX489139.1), inserting p11 promoter sequence and p7.5 promoter sequence (GenBank: 43077-43168 in JX489139.1, GenBank: 189237-189494 in JX489139.1) , confirmed that the TK region of the recombinant virus VTT was not expressed.

Example 2 Construction of recombinant virus VTT-dF4L with F4L gene deletion

1. Method

1.1 Construction of recombinant virus VTT-dF4L

The recombinant plasmid pdelF4L was synthesized by the company, mainly including the constructed recombinant plasmid including p7.5 promoter, p11 promoter and homology arm of F4L region.

The pdelF4L plasmid preferably contains p7.5 promoter, p11 promoter and F4L region homology arm sequence. The pdelF4L plasmid is shown in SEQ ID NO:5. The key sequences are the left arm of the F4L region homology arm (GenBank: 31818-32450 in JX489139.1), p11 promoter and p7.5 promoter (GenBank: 43077-43168, 189237-189494 in JX489139.1), F4L Region homology arm right arm (GenBank: 33281-33900 in JX489139.1).

The method is the same as in Example 1. 293T cells were infected with wild vaccinia virus (ie vaccinia virus Tiantan strain) and transfected with recombinant plasmid pdelF4L to obtain a suspension containing recombinant virus VTT-dF4L. The primers for virus identification are shown in Table 2 below.

Table 2 Primer Sequence

Virus purification and identification methods are the same as in Example 1.

2. Results

2.1 Construction of recombinant plasmid pdelF4L

The constructed recombinant plasmid pdelF4L mainly includes a p7.5 promoter, a p11 promoter, and a homology arm of the F4L region (as shown in FIG. 3 ).

2.2 Screening and identification of recombinant virus VTT-dF4L

DNAMini Kit说明书提取DNA，然后利用PCR扩增验证(PCR验证所用引物如表2所示)，重组病毒中缺失了F4L区(如图4所示)，再将PCR产物测序，重组病毒中缺失了F4L区部分序列(GenBank:JX489139.1中32451-33293)，插入了p11启动子序列和p7.5启动子序列(GenBank:JX489139.1中43077-43168，GenBank:JX489139.1中189237-189494)，确证重组病毒VTT敲除了F4L区。Press the recombinant virus

DNAMini Kit manual extracts DNA, and then uses PCR amplification verification (primers used for PCR verification are shown in Table 2), the F4L region is missing in the recombinant virus (as shown in Figure 4), and then the PCR product is sequenced, and the recombinant virus is missing Partial sequence of F4L region (GenBank: 32451-33293 in JX489139.1), inserting p11 promoter sequence and p7.5 promoter sequence (GenBank: 43077-43168 in JX489139.1, GenBank: 189237-189494 in JX489139.1) , confirmed that the recombinant virus VTT knocked out the F4L region.

Example 3 Construction of recombinant virus VTT-dB2R with B2R gene deletion

1. Method

1.1 Acquisition of recombinant virus VTT-dB2R

The recombinant plasmid pdelB2R was synthesized by the company, mainly including p7.5 promoter, p11 promoter and homologous arm sequence of B2R region.

The pdelB2R plasmid preferably contains a p7.5 promoter, a p11 promoter and a B2R region homology arm sequence. The pdelB2R plasmid is shown in SEQ ID NO:6. The key sequences are the left arm of the B2R region homology arm (GenBank: 163789-164448 in JX489139.1), p11 promoter and p7.5 promoter (GenBank: 43077-43168, 189237-189494 in JX489139.1), B2R Region homology arm right arm (GenBank: 165109-165768 in JX489139.1).

The recombination, purification and identification process methods are the same as in Example 1. 293T cells were infected with wild vaccinia virus and transfected with recombinant plasmid pdelB2R to obtain a suspension containing recombinant virus VTT-dB2R. The primers for virus identification are shown in Table 3 below. Virus purification and identification methods are the same as in Example 1.

Table 3 Primer Sequence

1.2. Evaluation of the effect of recombinant virus VTT-dB2R on activating innate immunity

1.2.1 Infection of THP1 cells with recombinant virus

18-24 hours before infection, seed THP1 cells in a six-well plate, 1×10 ⁶ per well. The same amount of recombinant virus VTT-dB2R and wild vaccinia virus (MOI: 1) were used to infect THP1 cells, and placed in a cell culture incubator at 37°C, 5% CO ₂ for 6 hours, and then the cells were harvested.

1.2.2. Protein extraction

Centrifuge at 3000rpm for 3min, wash once with PBS, add 100μL cell lysate RIPA (20mM Tris-HCl pH7.5, 150mM NaCl, 0.25% sodium deoxycholate, 1mM EDTA, 1% NP40), add loading buffer to supernatant after centrifugation , for Western Blot.

1.3.3. Western Blot to detect the expression of p-IRF3 and p-TBK1 and other proteins

(l) First prepare 12% separating gel (4mL 30% acrylamide solution, 2.5mL Tris/HCI pH8.8, 100μL 10% SDS, 100μL 10% ammonium persulfate, 5μL TEMED), fill the gel, and add water, polymerized at room temperature for 60min.

(2) Pour off the upper layer of water, and then prepare 5% stacking gel (0.5mL 30% acrylamide solution, 0.5mL Tris/HCI pH6.8, 40μL 10% SDS, 50μL 10% ammonium persulfate, 5μL TEMED), in Insert the comb teeth on the laminating glue, and polymerize at room temperature for 50 minutes. After the glue is completely coagulated, carefully pull out the comb teeth.

(3) The harvested lysate was centrifuged at 12000 rpm for 10 min at 4°C. Take 60 μL, add 20 μL of 4× sample buffer, cook at 98°C for 10 min.

(4) Put the prepared electrophoresis gel into the electrophoresis tank, fill the inner tank with freshly prepared electrophoresis buffer (without the gel plate), take 20 μL of the sample processed in the previous step and slowly add it to the comb hole of the stacking gel , and then add an appropriate amount of electrophoresis buffer in the outer tank of the electrophoresis tank, and perform electrophoresis: firstly, 120V, electrophoresis for 20 minutes, observe that the sample is well compressed into a line, and the protein pre-stained Marker begins to separate, indicating that the sample enters the separation gel, which can be Adjust the current to 160V, continue the electrophoresis for 40 minutes, observe that the protein pre-stained markers are well separated, and end the electrophoresis.

(5) Cut off the excess glue block, and stack them in the transfer film splint in the order of filter paper, glue, nitrocellulose membrane (NC film), and filter paper from the negative electrode to the positive electrode, and put it into the film transfer device. Transmembrane solution, on ice or at 4°C, insert the electrode to transfer the membrane, 80V for 120min, transfer the protein on the gel to the NC membrane.

(6) The transferred membrane was sealed with 5% skimmed milk powder, incubated at room temperature for 1 h at 25 rpm on a horizontal shaker.

(7) Soak the membrane in 5% skimmed milk powder containing appropriately diluted antibodies (p-IRF3, p-TBK1 and other antibodies diluted 1:1000) on a horizontal shaker at 25 rpm at 4°C overnight.

(8) On a 50rpm horizontal shaker, wash the membrane 4 times with TBST, 5min each time.

488goat anti-Rabbit二抗，水平摇床上25rpm，室温避光孵育lh。(9) Discard the lotion and add 1:10000 diluted Alexa

488goat anti-Rabbit secondary antibody, 25rpm on a horizontal shaker, incubated at room temperature in the dark for 1h.

(10) The secondary antibody was discarded, and the membrane was washed 4 times with TBST on a horizontal shaker at 25 rpm, 5 min each time.

(11) Take out the membrane and scan the membrane with LI-COR Odyssey instrument

2. Results

2.1 Construction and identification of recombinant plasmid pdelB2R

The constructed recombinant plasmid pdelB2R includes homology arms (Left arm, Right arm) in P7.5, P11, and B2R regions, as shown in FIG. 5 .

2.2 Screening of recombinant virus VTT-dB2R

The recombination and screening process is the same as in Example 1. Screening primers (B2Rup-F+B2Rdown-R) were used to perform PCR to screen viral plaques, and the genetic differences between the wild-type virus and VTT-dB2R are shown in FIG. 6 . The results of PCR identification of the purified recombinant virus VTT-dB2R are shown in Figure 7, which confirmed that the gene B2R was deleted in the recombinant virus. And the PCR product was sequenced, the B2R region sequence was deleted in the recombinant virus (164449-165126 in GenBank: JX489139.1), and the p11 promoter sequence and p7.5 promoter sequence were inserted (43077-43168 in GenBank: JX489139.1, GenBank: 189237-189494 in JX489139.1), confirmed that the recombinant virus VTT knocked out the B2R region.

2.3 Evaluation of the effect of recombinant virus VTT-dB2R on activating innate immunity

After the recombinant virus VTT-dB2R and wild vaccinia virus WT infected THP1 cells at the same time, compared with the wild type, the B2R deletion strain can activate innate immunity and induce host cells to produce higher levels of p-IRF3 (human phosphorylated interferon regulated Factor 3) and p-TBK1 (human phosphorylated TANK-binding kinase) (as shown in Figure 8).

Embodiment 4 constructs the recombinant virus VTT-dT/F that deletes F4L gene and TK gene simultaneously

In this embodiment, the TK gene and the F4L gene are simultaneously deleted in the vaccinia virus Tiantan strain to obtain a recombinant virus VTT-dT/F that lacks the TK gene and the F4L gene, and the method for constructing the recombinant virus that lacks the TK gene and the F4L gene Same as embodiment 2, specifically:

293T cells were infected with the recombinant virus VTT-dTK and transfected with the recombinant plasmid pdelF4L to obtain a suspension containing the recombinant virus VTT-dT/F. The further screening and purification methods of the recombinant virus were the same as in Example 2. The obtained purified virus was identified using the F4L and TK region identification primers at the same time. The size of the PCR fragment was different from that of the wild type (ie, vaccinia virus Tiantan strain) (as shown in Figure 9), and it was verified by sequencing that the recombinant virus lacked The partial sequence of the TK region (GenBank: 80439-80465 in JX489139.1) and the partial sequence of the F4L region (GenBank: 32451-33293 in JX489139.1) confirmed that a recombinant virus VTT-dT/F with simultaneous deletion of the TK gene and the F4L gene was obtained.

Embodiment 5 constructs the recombinant virus VTT-dB/T/F that simultaneously deletes F4L gene, TK gene and B2R gene

In the present invention, when the TK gene, the F4L gene and the B2R gene are simultaneously deleted in the vaccinia virus Tiantan strain to obtain a recombinant virus that simultaneously deletes the TK gene, the F4L gene and the B2R gene, the simultaneous deletion of the TK gene, the F4L gene and the B2R gene The construction method of the recombinant virus VTT-dB/T/F is the same as Example 3, specifically:

293T cells were infected with the recombinant virus VTT-dT/F and transfected with the recombinant plasmid pdelB2R to obtain a suspension containing the recombinant virus VTT-dB/T/F. The further screening and purification methods of the recombinant virus were the same as in Example 3. The obtained purified virus was identified using B2R, F4L and TK region identification primers at the same time. The size of the PCR fragment was different from that of the wild type (i.e. vaccinia virus Tiantan strain) (as shown in Figure 10), and it was verified by sequencing. The partial sequence of TK region (GenBank: 80439-80465 in JX489139.1), the partial sequence of F4L region (GenBank: 32451-33293 in JX489139.1) and the sequence of B2R region (GenBank: 164449-165126 in JX489139.1) were deleted, confirming The recombinant virus VTT-dB/T/F which simultaneously deleted the B2R gene, the TK gene and the F4L gene was obtained.

Example 6 compares the replication level of recombinant viruses lacking TK, F4L and B2 in mammalian cells

1. Method

1.1 Recombinant virus infection of mammalian cells

Human hepatocytes LO2 (2*10 ⁵ /well) were inoculated in a 12-well plate in complete antibiotic-free DMEM medium, cultured overnight, and when the cell growth was 70%-80%, the recombinant virus was added for infection (MOI=0.005). There are 6 kinds of recombinant viruses, the first group is the prototype strain of vaccinia virus VACV tiantan strain (VTT); the second group is VACV tiantan strain with deletion of TK region (VTT-dTK); the third group is VACV tiantan strain with deletion of F4L region (VTT -dF4L); the fourth group is VACVtiantan strain with double deletion of TK region and F4L region (VTT-dT/F); the fifth group is VACV tiantan strain with deletion of B2R region (VTT-dB2); the sixth group is simultaneous deletion of TK region, VACV tiantan strain (VTT-dB/T/F) in F4L and B2 regions. After 2 hours of infection, wash with 1mL PBS, replace with DMEM medium with 2% serum and incubate at 37°C; collect culture plates at 12 hours, 24 hours, 36 hours, and 48 hours after infection, and freeze and thaw between -80°C and room temperature Three times, the frozen-thawed cell solution was centrifuged at 1500 g for 10 minutes, the supernatant was taken, and the obtained virus suspension was tested for virus titer by plaque.

1.2 Detection of replication titer of recombinant virus by plaque formation

Vero cells (2*10 ⁵ ) were inoculated in a 12-well plate in DMEM complete medium without antibiotics and cultured overnight. When the cell growth was 80%-90% flat, the collected virus suspension was serially diluted 10 times to reinfect the Vero cells. After 2 hours, wash with 1mL PBS and replace with DMEM medium with a final concentration of 1% low melting point agar. Three days later, the plaques formed were counted, and the titer after virus replication was calculated.

2. Results

2.1 Replication of recombinant viruses lacking TK, F4L and B2 in mammalian cells

Virus titration tests were performed on cells harvested at different infection times (12 hours, 24 hours, 36 hours, 48 hours) with recombinant viruses that deleted TK, F4L and B2 infecting human hepatocytes LO2. The results showed that wild virus VTT and VTT-dB2R with single deletion B2 had the strongest replication ability, reaching 10 ⁶ PFU/mL at 48 hours after infection; The replication ability of recombinant vaccinia virus was slightly worse than that of VTT, reaching 2*10 ⁵ /mL 48 hours after infection; double deletion of TK and F4L (VTT-dT/F) and triple deletion of TK, F4L and B2 (VTT-dB/T/ F) The recombinant vaccinia virus has the lowest replication ability, reaching 5*10 ⁴ PFU/mL and 1.5*10 ⁴ PFU/mL 48 hours after infection, but several recombinant viruses can replicate effectively in mammalian cells (such as Figure 11).

Example 7 Recombinant virus VTT-dB/T/F virulence verification

When the recombinant virus VTT-dB/T/F that vaccinia virus simultaneously deletes the TK gene, the F4L gene and the B2R gene infects animals, when the virulence is significantly reduced, the recombinant virus virulence experiment includes the following steps:

1. Method

1.1 Dosing regimen of mouse recombinant virus

Six-week-old BALB/c female mice were divided into 13 groups, with 6 mice in each group. The first group was a negative control, given PBS; the remaining 12 groups were immunized with two virus concentrations of 2.5*10 ⁵ PFU/mouse and 2.5*10 ⁶ PFU/mouse by intranasal drops, respectively. The second group is the prototype strain of vaccinia virus VACV tiantan (VTT, 2.5*10 ⁵ PFU/monkey); the third group is the VACV tiantan strain with deletion of TK region (VTT-dTK, 2.5*10 ⁵ PFU/bird); the fourth group is VACV tiantan strain with deletion of F4L region (VTT-dF4L, 2.5*10 ⁵ PFU/bird); the fifth group was VACV tiantan strain with double deletion of TK region and F4L region (VTT-dT/F, 2.5*10 ⁵ PFU/bird); The sixth group was VACV tiantan strains with deletion of B2 region (VTT-dB2, 2.5*10 ⁵ PFU/bird); the seventh group was VACV tiantan strains with deletion of TK region, F4L region and B2 region at the same time (VTT-dB/T/F , 2.5*10 ⁵ PFU/bird); the eighth group was the prototype vaccinia virus VACV tiantan strain (VTT, 2.5*10 ⁶ PFU/bird); the ninth group was the VACV tiantan strain with deletion of TK region (VTT-dTK, 2.5*10 ⁶ PFU/bird); the tenth group was VACV tiantan strains with deletion of F4L region (VTT-dF4L, 2.5*10 ⁶ PFU/rause); the eleventh group was VACV tiantan strains with double deletion of TK region and F4L region (VTT-dT/ F, 2.5*10 ⁶ PFU/bird); the twelfth group was the VACV tiantan strain with deletion of the B2 region (VTT-dB2, 2.5*10 ⁶ PFU/bird); the thirteenth group was the simultaneous deletion of the TK region, F4L region and B2 VACV tiantan strain in the area (VTT-dB/T/F, 2.5*10 ⁶ PFU/bird). On day 0, the above-mentioned different recombinant viruses and nasal drops of different concentrations were immunized with 50 μl/mouse, and then the survival and body weight of the mice were observed at the same time every day for a total of 21 days.

1.2 Dosing regimen of recombinant virus in rabbits

Six New Zealand white rabbits weighing 2-2.5 kg were taken and divided into three groups, two in each group, and immunized with different concentrations of the recombinant virus. The administration concentration of the first group was 1*10 ⁶ PFU/pin, the second group was 1*10 ⁵ /pin, and the third group was 1*10 ⁴ /pin. First, prepare the skin on the back of the rabbit, and then inject six recombinant VACV tiantan strains subcutaneously on the back of the hairless rabbit in turn, inject 2 injections of each virus laterally, 100 μl/needle; the six recombinant viruses start from the direction close to the head of the rabbit to Subcutaneous injection was performed in the direction of the tail, followed by prototype strain VACV tiantan (VTT), VACV tiantan strain with deletion of TK region (VTT-dTK), VACV tiantan strain with deletion of F4L region (VTT-dF4L), VACV tiantan strain with double deletion of TK region and F4L region (VTT-dT/F), VACV tiantan strain with deletion of B2 region (VTT-dB2) and triple deletion VACV tiantan strain with deletion of TK region, F4L region and B2 region (VTT-dBTF). The subcutaneous injection was the 0th day, and the diameter of the erythema at the injection site on the back of the rabbit was observed and photographed every day thereafter.

2. Results

2.1 Changes in body weight of mice after immunization

The body weight of the mice immunized by intranasal drops was measured every day, and the obtained data were used to plot the change curve of the body weight of the mice. The results showed similar trends in body weight for both immune concentrations. The wild strain (VTT) and the knockout B2R (VTT-dB2) group had a significant weight loss compared with the control group injected with PBS, and the other knockout strains included VTT-dTK, VTT-dF4L, VTT-dT/F and Compared with the control group (injected with PBS), the body weight of the mice in the VTT-B/T/F group did not change significantly (as shown in Figure 12). It indicated that compared with the wild type, VTT-dT/F and VTT-B/T/F were successfully attenuated.

2.2 Survival of mice after immunization

The survival of the mice immunized with nasal drops was observed every day, and the survival curve was drawn according to the change of time. The results showed that in the 2.5*10 ⁶ PFU/dosage group, the mice in the VTT and VTT-dB2 groups all died on the fifth day and the sixth day, and the rest of the injection groups (VTT-dTK, VTT-dF4L, VTT-dT/ F and VTT-B/T/F) were in good condition during the observation period; in the 2.5*10 ⁵ PFU/dose group, all the mice in the VTT group died after 7 days, and two mice in the VTT-dB2 group died after 5 days. died; the remaining mice survived well during the observation period (as shown in Figure 13). It indicated that compared with the wild type, VTT-dT/F and VTT-B/T/F were successfully attenuated.

2.3 Erythema on the back of rabbits after administration

After skin preparation on the rabbit's back, the erythema at the injection site of subcutaneous injection of different recombinant viruses was observed, measured and photographed. The size of erythema at three concentrations (1*10 ⁶ PFU/needle, 1*10 ⁵ PFU/needle, 1*10 ⁴ PFU/needle) showed that the erythema caused by the subcutaneous injection of prototype strain VTT and VTT-dB2 lacking B2 was obvious In the low-dose group, only the prototype strain VTT and VTT-dB2 lacking B2 produced obvious erythema, and the rest of the groups (VTT-dTK, VTT-dF4L, VTT-dT/F and VTT -B/T/F) produced only slight skin erythema and fully recovered in about a week after injection (results are shown in Figure 14 and Figure 15). It indicated that compared with the wild type, VTT-dT/F and VTT-B/T/F were successfully attenuated.

The mouse immunization experiment of the recombinant virus of embodiment 8 deletion TK, F4L and B2

1. Method

1.1 Mouse immunization scheme

The 6-week-old BALB/c mice were divided into 6 groups, 5 mice in each group. The first group is the prototype strain VACV tiantan (VTT), the second group is dTK with single deletion of TK, the third group is dF4L with single deletion of F4L region, the fourth group is dT/F with double deletion of TK and F4L region, and the fifth group is dT/F with double deletion of TK and F4L region. The first group is dB2 with single deletion of B2 region, and the sixth group is dB/T/F with triple deletion of TK, F4L and B2 region. Mice were immunized with nasal drops of each recombinant virus at a titer of 2.5*10 ⁶ PFU/mouse. After 21 days of immunization, the serum of the mice was harvested, inactivated at 56°C for 30 minutes, and then stored in a -80°C refrigerator.

1.2 Anti-VACV antibody detection in mouse serum

Anti-VACV antibody titers in mouse serum were detected by ELISA method. The specific method is as follows. First, the purified VACV tiantan (VTT) virus was inactivated at 56°C for 30 minutes, and then the inactivated VTT was diluted to 1*10 ⁵ PFU/100 μL/well with the coating solution, and added to the high Coating was carried out in a 96-well plate dedicated to binding ELISA at 37°C for 2 hours. After coating, wash the plate with 1× washing buffer, soak in 300 μL/well for 1 min, pat dry the microplate plate, and perform the next wash, wash the plate 5 times in total; then add 200 μL/well of blocking solution, and carry out at 37°C for 2 hours closed. After blocking, wash the plate with 1× washing buffer, soak in 300 μL/well for 1 min, pat dry the microplate plate, and perform the next wash, washing the plate 5 times in total. The mouse serum was diluted 4-fold with 1× dilution buffer, and 100 μL/well of the diluted mouse serum was added to the microtiter plate, and incubated at room temperature for 2 hours. Discard the liquid in the wells, pat dry the microplate, wash the plate with 1× washing buffer, soak in 300 μL/well for 1 min, pat dry the microplate, and perform the next wash, washing the plate 5 times in total. Dilute the anti-mouse IgG antibody-HRP 1:100 with 1× dilution buffer for use, add 100 μL/well to the microtiter plate, mix well, and incubate at room temperature for 1 hour. Discard the liquid in the well, pat dry the microplate, wash the plate with 1× washing buffer, soak in 300 μL/well for 1 min, pat dry the microplate, and perform the next wash, washing the plate 5 times in total. Add the pre-prepared substrate solution (mix substrate A and substrate B in equal volumes 1:1, prepare 10 minutes before use) into the microtiter plate, 200 μL/well, mix well, and incubate at room temperature in the dark for 20 minute. Add 50 μL/well stop solution to the microtiter plate, shake the microtiter plate gently until the color develops uniformly. Read the light absorption value at OD450nm within 20 minutes.

1.3 Detection of neutralizing antibodies in mouse serum

The mouse serum was diluted 4-fold with DMEM, and the diluted mouse serum was incubated with the VACVtiantan strain (VTT-GFP) expressing GFP in equal amounts at 37°C for 1 hour, and then added to a 48-well plate covered with Vero cells for incubation. After 24 hours, the GFP signal expressed by VACV was detected by flow cytometry to indicate the level of neutralizing antibody in mouse serum.

2. Results

2.1 Detection of VACV antibody in mouse serum

After the mouse serum was serially diluted 4 times with 1× dilution buffer, the S antibody in the serum was detected by ELISA. The results are shown in Figure 16, the prototype strain VTT and VTT-dB2 with single deletion of B2 induced higher antibody titers, single deletion of TK (VTT-dTK) and F4L (VTT-dF4L) and double deletion of VTT-dT/ Compared with the prototype strain and VTT-dB2, the antibody titer of F and triple-deleted VTT-dB/T/F decreased, but the serum antibody titer induced by triple-deleted VTT-dB/T/F was higher than that of double-deleted VTT-dB/T/F VTT-dT/F rebounded. It indicated that deletion of TK and F4L attenuation followed by deletion of B2R improved the immunogenicity of triple-deficient recombinant virus VTT-dB/T/F.

2.2 Detection of neutralizing antibodies in mouse serum

After the mouse serum was diluted 4-fold with DMEM, it was incubated with the same amount of VACV-GFP at 37°C for 1 hour, and then added to a 48-well plate with Vero cells. Infection of GFP. The results are shown in Figure 17, the prototype strain VTT and VTT-dB2 with single deletion of B2 induced higher neutralizing antibody activity, single deletion of TK (VTT-dTK) and F4L (VTT-dF4L) and double deletion of VTT-dT /F and triple-deleted VTT-dB/T/F compared with the prototype strain and VTT-dB2, the neutralizing antibody activity decreased, but the neutralizing antibody activity induced by triple-deleted VTT-dB/T/F Higher than double-deleted VTT-dT/F.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence description

SEQ ID NO: 1 (TK gene)

ATGAACGGCGGACATATTCAGTTGATAATCGGCCCCATGTTTTTCAGGTAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCAAAATAGCTCAATAAATGCGTGACTATAAAATATTCTAACGATAATAGATACGGAACGGGACTATGGACGCATGATAAGAATAATTTTGAAGCATTGGAAGCAACTAAACTATGTGATGTCTTGGAATCAATTACA GATTTCTCCGTGATAGGTATCGATGAAGGACAGTTCCTTTCCAGACATTGTTGAATTCTGTGAGCGTATGGCAAACGAAGGAAAAATAGTTATAGTAGCCGCACTCGATGGGACATTTCAACGTAAACCGTTTAATAATATTTTGAATCTTATTCCATTATCTGAAATGGTGGTAAAACTAACTGCTGTGTGTATGAAATGCTTTAAGGAGGCTTCCTTTCTAAAC GATTGGGTGAGGAAACCGAGATAGAAATAATAGGAGGTAATGATATGTATCAATCGGTGTGTAGAAAGTGTTACATCGACTCATAA

SEQ ID NO:2 (F4L gene)

ATGGAACCCATCCTTGCACCAAATCCAAATAGATTTGTTATT

TTCCCAAATCCAATATTATGACATCTGGAACATGTATAAAAAAGGCA

GAGGCATCATTTTGGACAGTGGAAGAAGTAGATATATCTAAAGA

TATCAATGATTGGAATAAACTAACACCAGACGAAAAATATTTTAT

AAAACATGTATTGGCGTTTTTTGCAGCCAGTGACGGAATAGTGA

ATGAAAATTTGGCGGAACGATTTTGTACAGAAGTACAGATTACC

GAGGCTAGATGTTTCTACGGATTTCAGATGGCCATTGAAAACAT

TCATTCGGAAATGTATAGTCTTTTGATCGATACTTATGTTAAAGAT

AGTAATGAAAAAAACTATCTCTTTAATGCCATAGAAACGATGCCT

TGTGTAAAAAAAGAAGGCCGATTGGGCTCAAAAGTGGATACATGA

CAGCGCCGGTTATGGAGAGAGACTTATTGCCTTTGCTGCAGTAG

AAGGAATCTTCTTTTCCGGATCATTCGCTTCCATATTTTGGCTTA

AAAAGCGTGGCCTAATGCCCGGACTCACGTTTTTCCAACGAATTG

ATTAGTAGAGACGAGGGTCTGCACTGCGATTTCGCATGTTTGAT

GTTTAAACATTTATTGCATCCACCGAGTGAAGAAACCGTTAGAT

CTATTATAACAGATGCGGTATCCATTGAACAAGAATTTCTTACTG

CGGCTCTTCCAGTTAAACTTATAGGAATGAATTGTGAAATGATG

AAAACATATATAGAATTCGTCGCGGATAGATTGATTTCTGAATTG

GGATTTAAAAAATTTATAATGTTACCAATCCGTTTGATTTCATG

GAAAATATATCATTGGAAGGAAAAACTAATTTTTTCGAAAAACGT

GTGGGTGAATACCAAAAAATGGGAGTTATGTCTCAAGAAGACAA

TCATTTTTCTTTAGATGTTGACTTTTAA

SEQ ID NO:3 (B2R gene)

ATGGCGATGTTTTACGCACACGCTCTCGGTGGGTACGACGA

GAATCTTCATGCCTTTCCTGGAATATCATCGACTGTTGCCAATGA

TGTCAGAAAATATTCTGTTGTGTCAGTTTATAATAACAAGTATGA

CATTGTAAAAGACAAATATATGTGGTGTTACAGTCAGGTGAACA

AGAGATATATTGGAGCACTGCTGCCTATGTTTGAGTGCAATGAAT

ATCTACAAATTGGAGATCCGATCCATGATCAAGAAGGAAATCAA

ATCTCTATCATCACATATCGCCACAAAAACTACTATGCTCTAAGC

GGAATCGGGTACGAGAGTCTAGACTTGTGTTTGGAAGGAGTAG

GGATTCATCATCACACACTTGAAGCAGGAAACGCTGTATATGGA

AAAGTTCAACATGATTATTCTACTATCAAAGAGAAGGCCAAAGA

AATGAGTACACTTAGTCCAGGACCTATCATTGATTACCACGTCTG

GATAGGAGATTGTATCTGTCAAGTTACTGCTGTGGACGTACATG

GAAAGGAAATTATGAGAATGAGATTCAAAAAGGGTGCGGTGCTT

CCGATCCCAAATCTGGTAAAAGTTAAACTTGGGGAGAATGATAC

AGAAAATCTTTCTTCTACTATATCGGCGGCACCATCGAGGTAA

SEQ ID NO:4 (pdelJ2R plasmid)

GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA

CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCT

GTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTAT

GCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAAT

ACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAA

ACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGA

GATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCA

GCATCTTTTACTTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG

AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA

TGTTGAATACTCATACTCTTCCTTTTTCAATATTTATTGAAGCATTT

ATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTT

AGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAA

GTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACC

TATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAAGC

TTTTGCGATCAATAAATGGATCACAACCAGTATCTCTAACGATG

TTCTTCGCAGATGATGATTCATTTTTTAAGTATTTGGCTAGTCAA

GATGATGAATCTTCATTTATCTGATATATTGCAAATCACTCAATATC

TAGACTTTCTGTTATTTATTATTGATCCAATCAAAAAATAAATTAGA

AGCCGTGGGTCATTGTTATGAATCTCTTTCAGAGGAATACAGAC

AATTGACAAAATTCACAGACTTCTCAAGATTTTAAAAACTGTTTA

ACAAGGTCCCTATTGTTACAGATGGAAGGGTCAAACTTAATAAA

GGATATTTGTTCGACTTTGTGATTAGTTTGATGCGATTCAAAAAA

GAATCCTCTCTAGCTACCACCGCAATAGATCCTATTAGATACATA

GATCCTCGTCGCGATATCGCATTTTCTAACGTGATGGATATATTA

AAGTCGAATAAAGTGAACAATAATTAATTCTTTATTGTCATCATG

AACGGCGGACATATTCAGTTGATAATCGGCCCCATGTTTTTCAGG

TAAAAGTACAGAATTAATTAGACGAGTTAGACGTTATCAAATAGC

TCAATATAAATGCGTGACTATAAAATATTTCTAACGATAATAGATAC

GGAACGGGACTATGGACGCATGATAAGAATAATTTTGAAGCATT

GGAAGCAACTAAACTATGTGATGTCTTGGAATCAATTACAGATTT

CTCCGTGATAGGTATCGATAGATCTGCTAGCCACGTGTTAATTAA

GCCCGGGCCTCGAGGAATTCATTTATAGCATAGAAAAAAACAAA

ATGAAATTCTACTATATTTTTACATACATATATTTCTAAATATGAAAG

TGGTGATTGTGACTAGCGTAGCATCGCTTCTAGACATCTATAC

TATATAGTAATACCAATACTCAAGACTACGAAACTGATACAATCT

CTTATCATGTGGGTAATGTTCTCGATGTCGATAGCCATATGCCCG

GTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCCACCC

GCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAAT

TAATTTCTCGTAAAAGTAGAAAATATATTTCTAATTTATTGCACGGT

AAGGAAGTAGAATCATAAAGAACAGTGACGGATCCCCGGGCCAT

GGGCGGCCGCGTCGACGTAGAAAGTGTTACATCGACTCATAATA

TTATATTTTTTTATCTAAAAAACTAAAAAATAAACATTGATTAAATTT

TAATATAATACTTAAAAATGGATGTTGTGTCGTTAGATAAACCGT

TTATGTATTTTGAGGAAATTGATAATGAGTTAGATTACGAACCAG

AAAGTGCAAATGAGGTCGCAAAAAAACTGCCGTATCAAGGACA

GTTAAAACTATTACTAGGAGAATTATTTTTTCTTAGTAAGTTACA

GCGACACGGTATATTAGATGGTGCCACCGTAGTGTATATAGGATC

TGCTCCTGGTACACATATACGTTATTTGAGAGATCATTTCTATAAT

TTAGGAGTGATCATCAAATGGATGCTAATTGACGGCCGCCATCA

TGATCCTATTTTAAATGGATTGCGTGATGTGACTCTAGTGACTCG

GTTCGTTGATGAGGAATATCTACGATCCATCAAAAAACAACTGC

ATCCTTCTAAGATTTATTTTAATTTCTGATGTGAGATCCAAACGAG

GAGGAAATGAACCTAGTACGGCGGATTTACTAAGTAATTACGCT

CTACAAAATGTCATGATTAGTATTTTAAACCCCGTGGCATCTAGT

CTTAAATGGAGATGCCCGTTTCCAGATCAATGGATCAAGGACTT

TTATATCCCACACGGTAATAAAATGTTACAACCTTTTGCTCCTTC

ATATTCAGCTGAAATGAGATTATTAAGTATTTACCGGTGAGAA

CATGAGACTGACTCGACCGATGCCCTTGAGAGCCTTCAACCCA

GTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCG

CACTTATGACTGTCTTTCTTTATCATGCAACTCGTAGGACAGGTG

CCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCT

GGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAAT

CTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACC

AAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGG

CCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGG

CTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCA

TCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGA

TGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTT

ACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGAT

TTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATT

GTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCG

CGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGC

GGCACCTCGCTAACGGATTCACCACTCCAAAGAATTGGAGCCAAT

CAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGC

AGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGG

CGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGA

TCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTG

CCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGT

GAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAAC

ATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGC

GGAAGTCAGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGC

GCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA

GGCGGTAATACGGTTATTCCACAGAATCAGGGGATAACGCAGGAA

AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA

AAAGGCCGCGTTGCTGGCGTTTTTTCCATAGGCTCCGCCCCCCT

GACGAGCATCACAAAAATCGACGCTCCAAGTCAGAGGTGGCGAA

ACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCCTGGAAGC

TCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATA

CCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATA

GCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC

AAGCTGGGCTGTGTGCACGAACCCCCGTTCAGCCCGACCGCT

GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGA

CACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAG

CAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGG

TGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTG

CGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC

TCTTGATCCGGCAAAACAAACCACCCGCTGGTAGCGGTGGTTTTT

TTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA

GAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAA

CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAA

GGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT

CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAAT

GCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT

CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC

GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG

AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGC

CAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATC

CGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAA

GTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCT

GCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATT

CAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCA

TGTTGTGTCAAAAAAGCGGTTAGTCCTTCGGTCCTCCGATCGTT

GTCAGAA

SEQ ID NO:5 (pdelF4L plasmid)

GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTTCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTTGAAGCATTTATCAGGGT TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAAGCTTTGACAAAATCAACTACATAATCCTCATCTGGAACATTTAGTTCGTCGCTTTCTAGAAT AAGTTTCATAGATAGATAATCAAAATTGTCTATGATGTCATCTTCCAGTTCCAAAAAGTGTTTGGCAATAAAGTTTTTAGTATGACATAAGAGATTGGATAGTCCGTATTCTATACCCATCATGTAACACTCGACACAATATTTCCTTTCTAAAATCTCGTAAGATAAAGTTTACAAGTGTAGATGATAAATTCTACAGAGGTTAATAGAAGCACGTAATAAATTGACGACGT TATGACTATCTATATATACCTTTCCAGTATATGAGTAAATAACTATAGAAGTTAAACTGTGAATGTCAAGGTCTAGACAAACCCTtGTAACTGGATCTTTATTTTTCGTGTATTTTTGACGTAAATGTGTGCGAAAGTAAGGAGATAACTTTTTCAATATCGACTATTATATTGCCTCCTATGGCATCAATAATTGTTTTGAATTTCTTAGTCATAGACAATGCTAAT ATATTTCTTACAGTACACAGTATTaACAAATATCGGCATTTATGTTTCTTAAAAGTCAACATCTAgAGAAAAATGATTGTCTTTCTTGAGACATAACTCCCATTGCTAGCCACGTGTTAATTAAGCCCGGGCCTCGAGGAATTCAgggcctcgaggaattcaTTTATAGCATAGAAAAAAACAAAATGAAATTCTACTATATTTTTACATACATATATTTCTAAATATGAAAGT GGTGATTGTGACTAGCGTAGCATCGCTTCTAGACATCTATATACTATATAGTAATACCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGATGTCGATAGCCATATGCCCGGTAGTTGCGATATACATAAACTGATCACTAATTCCAAACCACCCGCTTTTTATAGTAAGTTTTTCACCCATAAATAATAAATACAATAATTAATTTCTC GTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACAGTGACGGATCCCCGGGCCATGGGCGGCCGCCATGTTCCAGATGTCATgATATTGGATTGGGAAAATAACAAATCTATTTGGATTTGGTGCAAGGATGGGTTCCATAACTAAATTAACAATAtCgATAAATTTTTTTTCAGTTATCTATATGC CTGTACTTGGATtTTTTGTACATCGATATCGCCGCAATCACTACAATAATTACAAGTATTGATAGCATTGTTATTAGTACTATCATAATTAAATTATCgACATTCATGGGTGCTGAATAATCGTTATTATCATTATCATTTTGTAATTGTGACATCATACTAGATAAATCGTTTGCGAGATTGTTGTGGGAAGCGGGCATGGAGGATGAATTATCGTTA TTATTATTTAAAGCCTCCCATTCGGATTCACAAATATGGCGCGCGTTCAACATTTATGGAAACTATAATTTTGTGAAAACAGATAACAAGAAAACTCGTCATCGTTCAAAATTTTTAACGATAGTAAACCGATTAAACGTCGAGCTAATTTCTAACGCTAGCGACTCTGTTGGATATGGGTTTCCAGATATATCTTTTCAGTTCCCCCTACGTATCTATAATCATCTGT AGGAAATGGAAGATATTTCCATTTATCTACTGTTCCTAATATCGACTATCGTCGGTACCGCCGCACTTATGACTGTCTTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCTC GTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACA GCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGA TTCACCACTCCAAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCA GAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATC CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGA CCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGC CACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCCGCTGGTAGCGGTGGTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAA AAAAAGGATCTCCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAG AGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGTCAAAAAAGCGGTTAGTCCTTCGGTCCTCCGATCGTTGTCAGAA

SEQ ID NO:6 (pdelB2R plasmid)

GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTTCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTTGAAGCATTTATCAGGGT TATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTTCAAGAAGCTTGATCAGAGCCAATAATAATAGATTACCAAAAGGTCGGTGATGTTGATCGGAAT CGAAATCTTAAATACCATACAATTTATGCACGAGCAAGGATATTCTCACGGAGATATTAAAGCGAGTAATAGTCTTGGATCAAATAGATAAGAATAAATTATATCTAGTGGATTACGGATTGGTTTCTAAATTCATGTCTAATGGCGAACATGTTCCATTTATAAGAAATCCAAATAAAATGGATAACGGTACTCTAGAATTTACACCTATAGATTCGCATAAAGGAT ACGTTGTATCTAGACGTGGAGATCTAGAAACACTTGGATATTGTATGATTAGATGGTTGGGAGGTATCTTGCCATGGACTAAGATATCTGAAACAAAGAATTGTGCATTAGTAAGTGCCACAAAACAGAAATATGTTAACAATACTGCGACTTTGTTAATGACCAGTTTGCAATATGCACCTAGAGAATTGCTGCAATATTACCATGGTAAACTCTTTGACATA TTTTGAGGAACCCAATTACGACGAGTTTCGGCACATATTAATGCAGGGTGTATATTATTAAGTGTGGTGTTTGGTCGATGTAAAATTTTTGTCGATAAAAATTAAAAAATAACTTAATTTATTATTGATCTCGTGTGTACAACCGAAATCTTAATTAAATAACTTCGTATAGCATAGAAAAAAACAAAATGAAATTCTACTATATTTTTACATACATATTTCTAAATAT GAAAGTGGTGATTGTGACTAGCGTAGCATCGCTTCTAGACATCTATATACTATATAGTAATACCAATACTCAAGACTACGAAACTGATACAATCTCTTATCATGTGGGTAATGTTCTCGATGTCGATAGCCATATGCCCGGTAGTTGCGATATACATAAACTGATCACTAATCCACCCGCTTTTTATAGTAAGTTTCACCCATAAATAATAAATACAATAATTAAT TTCTCGTAAAAGTAGAAAATATATTCTAATTTATTGCACGGTAAGGAAGTAGAATCATAAAGAACAGTGACGGATCCCCACCTCCTGGAAGACAGCGTGAATAATGTACTCATGAAACGTTTGGAAACTATACGCCATATGTGGTCTGTTGTATATGATCATTTTGATATTGTGAATGGTAAAGAATGCTGTTATGTGCATACGCATTTGTCTAATCAAAATCT TATACCGAGTACTGTAAAAACAAATTTGTACATGAAGACTATGGGATCATGCATTCAAATGGATTCCATGGAAGCTCTAGAGTATCTTAGCGAACTGAAGGAATCAGGTGGATGGAGTCCCAGACCAGAAATGCAGGAATTTGAATATCCAGATGGAGTGGAAGACACTGAATCAATTGAGAGATTGGTAGAGGAGTTTCTTCAATAGATCAGAACTTCAGGCTGGT AAATTAGTCAAATTTGGTAATTCTAATTAATTGTTAAACATACATCTGTTTCAGCTAAGCAACTAAGAACACGTATACGGCAGCAGCTTCCTTTATACTCTCATCTTTTACCAACACAAAGGGTGGATATTTGTTCATTGGAGTTGATAATAATACACACAAAGTATTTGGATTCACGGTGGGTTACGACTACCTCAGACTGGTAGAGAATGATATAGAAAAGCA TATCAAAAGACTTCGTGTTGTGCATTTCTGTGAGAAGAAAGAGGACATCAAGTACGCGTGTGGTACCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGC CTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGG GACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAAC GGATTCACCACTCCAAAGAATTGGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGCTAGGCTGGCGGGGGTTGCCTTACTGGTTA GCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGT TATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTC CGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAACCACCCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGC AGAAAAAAAGGATCCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCT GTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGTCAAAAAAGCGGTTAGTCCTTCGGTCCTCCGATCGTTGTCAGAA

Claims (10)

1. A recombinant vaccinia virus, which has deleted at least one of the TK gene, the F4L gene and the B2R gene relative to the vaccinia virus Tiantan strain. 2. The recombinant vaccinia virus according to claim 1, wherein the recombinant vaccinia virus is selected from one of the following recombinant vaccinia viruses: (1) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dTK that has deleted the TK gene; (2) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dF4L that has deleted the F4L gene; (3) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dB2R that has deleted the B2R gene; (4) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dT/F that has simultaneously deleted the TK gene and the F4L gene; (5) Relative to the vaccinia virus Tiantan strain, the recombinant vaccinia virus VTT-dB/T/F that simultaneously deleted the TK gene, the F4L gene and the B2R gene; Preferably, the recombinant vaccinia virus is a recombinant vaccinia virus VTT-dB/T/F that has simultaneously deleted the TK gene, the F4L gene and the B2R gene relative to the vaccinia virus Tiantan strain. 3. The recombinant vaccinia virus according to claim 1 or 2, wherein the sequences of the TK gene, the F4L gene and the B2R gene are respectively shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 . 4. The recombinant vaccinia virus according to any one of claims 1-3, wherein: (1) The genome sequence of the recombinant vaccinia virus VTT-dTK is relative to the genome sequence of the vaccinia virus Tiantan strain, the partial sequence of the TK region is deleted and the p11 promoter sequence and the p7.5 promoter sequence are inserted; wherein the partial sequence of the TK region is GenBank: 80439-80465 in JX489139.1, p11 promoter sequence and p7.5 promoter sequence are GenBank: 43077-43168 in JX489139.1 and GenBank: 189237-189494 in JX489139.1; (2) The genome sequence of the recombinant vaccinia virus VTT-dF4L is relative to the genome sequence of the vaccinia virus Tiantan strain, the partial sequence of the F4L region is deleted and the p11 promoter sequence and the p7.5 promoter sequence are inserted; wherein the partial sequence of the F4L region is GenBank: 32451-33293 in JX489139.1, p11 promoter sequence and p7.5 promoter sequence are GenBank: 43077-43168 in JX489139.1, GenBank: 189237-189494 in JX489139.1; (3) The genome sequence of the recombinant vaccinia virus VTT-dB2R is relative to the genome sequence of the vaccinia virus Tiantan strain, the B2R region sequence is deleted and the p11 promoter sequence and p7.5 promoter sequence are inserted; wherein the B2R region sequence is GenBank: 164449-165126 in JX489139.1, p11 promoter sequence and p7.5 promoter sequence are GenBank: 43077-43168 in JX489139.1, GenBank: 189237-189494 in JX489139.1; (4) The genome sequence of the recombinant vaccinia virus VTT-dT/F is relative to the genome sequence of the vaccinia virus Tiantan strain, and part of the sequence of the TK region and part of the F4L region are deleted at the same time; the part of the sequence of the TK region is in GenBank: JX489139.1 80439-80465, the partial sequence of F4L region is 32451-33293 in GenBank: JX489139.1; (5) The genome sequence of the recombinant vaccinia virus VTT-dB/T/F is relative to the genome sequence of the vaccinia virus Tiantan strain, and the partial sequence of the TK region, the partial sequence of the F4L region and the sequence of the B2R region are deleted at the same time; wherein the partial sequence of the TK region is GenBank: 80439-80465 in JX489139.1, the partial sequence of F4L region is GenBank: 32451-33293 in JX489139.1, and the sequence of B2R region is 164449-165126 in GenBank: JX489139.1; Wherein, the genome sequence of the vaccinia virus Tiantan strain is the sequence represented by the gene accession number Gene Bank No.AF095689. 5. A method for constructing recombinant vaccinia virus, which adopts the method of homologous recombination to construct recombinant vaccinia virus. 6. The method of claim 5, comprising the steps of: (1) Construct respectively the first recombination plasmid comprising the left arm of the TK gene homology arm and the right arm of the TK gene homology arm, the second recombination plasmid comprising the left arm of the F4L gene homology arm and the right arm of the F4L gene homology arm, and the second recombination plasmid comprising The third recombinant plasmid of the left arm of the B2R gene homology arm and the right arm of the B2R gene homology arm; (2) Infect the cells with the vaccinia virus Tiantan strain, and then transfect the first recombinant plasmid, the second recombinant plasmid and the third recombinant plasmid, respectively, to obtain recombinant vaccinia virus VTT-dTK with respect to the vaccinia virus Tiantan strain, which has deleted the TK gene , a recombinant vaccinia virus VTT-dF4L with the deletion of the F4L gene, and a recombinant vaccinia virus VTT-dB2R with the deletion of the B2R gene. 7. The method according to claim 6, wherein, the recombinant vaccinia virus VTT-dTK is used to infect the cells and the second recombinant plasmid is transfected again to obtain the recombinant vaccinia that has simultaneously deleted the TK gene and the F4L gene relative to the vaccinia virus Tiantan strain Virus VTT-dT/F. 8. The method according to claim 7, wherein, the cells are infected with recombinant vaccinia virus VTT-dT/F and the third recombinant plasmid is transfected again to obtain the simultaneous deletion of TK gene, F4L gene and Recombinant vaccinia virus VTT-dB/T/F with B2R gene. 9. Use of the recombinant vaccinia virus according to any one of claims 1-4 in the preparation of a poxvirus vaccine or as a vaccine carrier. 10. A poxvirus vaccine comprising the recombinant vaccinia virus according to any one of claims 1-4.

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