CN114805500B - Application of African swine fever virus I73R protein as immunosuppressant and construction of immunosuppression site mutant strain - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of African swine fever virus I73R protein as an immunosuppressant and construction of an immunosuppression site mutant strain. The invention firstly discovers that the African swine fever virus I73R protein can inhibit the expression of IFN-beta induced by SeV, has stronger immunosuppression effect, and can be used for preparing immunosuppression; secondly, the invention discovers an immunosuppression site of an African swine fever virus I73R protein, and constructs an African swine fever recombinant virus with mutation of the immunosuppression site of the I73R protein, the natural immunity of the African swine fever recombinant virus is obviously enhanced, the toxicity is obviously weakened, the survival rate of an infected recombinant virus experimental animal is obviously improved compared with that of an infected wild virus experimental animal, the biological safety and the immunoprotection efficacy are improved, and the African swine fever recombinant virus can be used as a recombinant vaccine strain and has good application prospect.

Description

Application of African swine fever virus I73R protein as immunosuppressant and construction of immunosuppression site mutant strain

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of African swine fever virus I73R protein as an immunosuppressant and construction of an immunosuppression site mutant strain.

Background

African swine fever (African Swine Fever, ASF) is an acute virulent infectious disease characterized by fever and systemic visceral hemorrhage in pigs caused by African Swine Fever Virus (ASFV) infection, and the mortality rate of domestic pigs can be as high as 100%. ASFV is a large intracytoplasmic replicating virus with icosahedral symmetry and 200nm diameter virus particles in concentric structure. The genome of African swine fever virus is single-molecule linear double-stranded DNA with covalently closed ends, the whole length of the genome is 170-190 kb, and the genome lengths of different strains are inconsistent. The whole genome of ASFV contains more than 160 open reading frames and can code for 150-200 proteins. The central conserved region (C region) in the genome is 125kb in length, and the variable regions on both sides of the C region are 13-22kb in length and contain 5 polygenic families (MGF). This region is involved in the mechanism of viral antigen variation, host defense system evasion, and the like.

Vaccines are the most effective and economical means of preventing and controlling infectious diseases, and the development of effective vaccines for ASF has been highly valued by many countries, especially european research institutions, after the transfer from ASF to europe, and has made significant progress. However, as the pathogenesis of ASFV is complex, the viral infection and the immune mechanism are unclear, so far, no effective vaccine has been used for preventing and controlling the disease. Although research into ASF vaccines began at the end of the 60 s of the 20 th century, many reports have been made in recent years, but have not been successfully applied to practice.

I73R belongs to an African swine fever virus early transcription gene, and the I73R can be integrated on an asymptomatic host tissue cell genome. The invention firstly discovers that the African swine fever virus I73R protein can inhibit the activation of IFN-beta induced by SeV, has stronger immunosuppression effect, and can be applied as immunosuppression.

Based on the findings, the invention constructs I73R protein mutant plasmid by mutation PCR, gene synthesis and other methods, wherein the mutation site is a key site for playing an immunosuppression function; the I73R protein immunosuppression site is mutated in an African swine fever CN/GS/2018 isolate, so that the African swine fever recombinant virus with the I73R protein immunosuppression site mutation is constructed, the natural immunity of the African swine fever recombinant virus is obviously weakened, the African swine fever recombinant virus can be used as a recombinant vaccine strain, the early immune response can be promoted, the high-level antibody production can be induced, the biosafety and the immune protection efficacy can be improved, and the African swine fever recombinant virus has a good application prospect.

Disclosure of Invention

Firstly, the invention discovers that the African swine fever virus I73R protein can inhibit the activation of IFN-beta induced by SeV, has stronger immunosuppression effect, and can be applied as immunosuppression. Accordingly, a first object of the present invention is to:

provides an application of African swine fever virus I73R protein in preparing immunosuppressants.

Provides an application of African swine fever virus I73R protein in preparation of SeV inhibitors.

Provides an application of African swine fever virus I73R protein in preparing a medicine for inhibiting IFN-beta activity.

Preferably, the amino acid sequence of the African swine fever virus I73R protein is shown as SEQ ID NO. 1.

Preferably, the nucleotide sequence of the encoding African swine fever virus I73R protein is shown as SEQ ID NO. 2.

Secondly, the invention constructs I73R protein mutant plasmid by mutation PCR, gene synthesis and other methods, wherein the mutation site is a key site for playing the function of immunosuppression; the I73R protein immunosuppression site is mutated in an African swine fever CN/GS/2018 isolate, so that the African swine fever recombinant virus with the I73R protein immunosuppression site mutation is constructed, the natural immunity of the African swine fever recombinant virus is obviously weakened, and the African swine fever recombinant virus can be used as a recombinant vaccine strain. Accordingly, another object of the present invention is to:

provides an application of preparing an African swine fever recombinant virus by inhibiting an immunosuppression function of an African swine fever virus I73R protein.

Preferably, the immunosuppressive function of the african swine fever virus I73R protein is: amino acids 59-60 of the African swine fever virus I73R protein are mutated into asparagine and alanine.

Preferably, the African swine fever virus is a CN/GS/2018 isolate, and the gene for encoding 59-60 amino acids of the I73R protein of the African swine fever virus is positioned at 172292-172297 of the complete gene sequence of the CN/GS/2018 isolate of the African swine fever virus.

Preferably, the amino acid sequence of the African swine fever virus I73R protein is shown as SEQ ID NO. 1; the nucleotide sequence of the encoding African swine fever virus I73R protein is shown as SEQ ID NO. 2; the amino acid sequence of the mutated I73R protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coded mutated I73R protein is shown as SEQ ID NO. 4.

An african swine fever recombinant virus having a loss of immunosuppressive function of an I73R protein is provided, which comprises mutating amino acids 59-60 of the I73R protein of the african swine fever virus to asparagine and alanine.

Preferably, the African swine fever virus is a CN/GS/2018 isolate, and the gene for encoding 59-60 amino acids of the I73R protein of the African swine fever virus is positioned at 172292-172297 of the complete gene sequence of the CN/GS/2018 isolate of the African swine fever virus.

Preferably, the amino acid sequence of the African swine fever virus I73R protein is shown as SEQ ID NO. 1; the nucleotide sequence of the encoding African swine fever virus I73R protein is shown as SEQ ID NO. 2; the amino acid sequence of the mutated I73R protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coded mutated I73R protein is shown as SEQ ID NO. 4.

An african swine fever vaccine is provided, comprising an african swine fever recombinant virus having a loss of immunosuppressive function of the I73R protein.

The preparation method of the African swine fever recombinant virus with the I73R protein immune suppression function lost is provided, and comprises the following steps: the 59-60 amino acids of the African swine fever virus I73R protein are mutated into asparagine and alanine by genetic engineering means.

Preferably, the African swine fever virus is a CN/GS/2018 isolate, and the gene for encoding 59-60 amino acids of the I73R protein of the African swine fever virus is positioned at 172292-172297 of the complete gene sequence of the CN/GS/2018 isolate of the African swine fever virus.

Preferably, the amino acid sequence of the African swine fever virus I73R protein is shown as SEQ ID NO. 1; the nucleotide sequence of the encoding African swine fever virus I73R protein is shown as SEQ ID NO. 2; the amino acid sequence of the mutated I73R protein is shown as SEQ ID NO. 3; the nucleotide sequence of the coded mutated I73R protein is shown as SEQ ID NO. 4.

Preferably, the method comprises the steps of:

(1) Constructing an I73R protein gene mutation sequence, wherein the gene mutation sequence is a gene sequence for encoding 59-72 amino acids of the I73R protein and mutating 59-60 amino acids into asparagine and alanine, and the gene mutation sequence is shown as SEQ ID NO. 5;

(2) Taking about 1.0kb of an upstream gene of 172292 th site of a full genome of a CN/GS/2018 isolate as a left arm of homologous recombination, taking about 1.0kb of a downstream gene sequence of 172510 th site of the full genome of the CN/GS/2018 isolate as a right arm of homologous recombination, and cloning the right arm into pUC19 vectors respectively to obtain a recombinant transfer vector;

(3) Inserting the gene mutation sequences in the step (1) into the left and right arm gene sequences of the recombinant transfer vector in the step (2), and screening the gene fragments of the expression cassette to obtain a homologous recombinant transfer vector;

(4) And (3) transfecting the homologous recombination transfer vector in the step (3) into BMDM cells infected with the parent African swine fever strain, and purifying and screening to obtain the African swine fever recombinant virus with the I73R protein having a lost immune suppression function.

Provides the African swine fever recombinant virus with the I73R protein immune suppression function lost, which is prepared by the method.

The beneficial effects of the invention are as follows:

(1) firstly, the invention discovers that the African swine fever virus I73R protein can inhibit the activation of IFN-beta induced by SeV, which indicates that the I73R protein has stronger immunosuppression effect and can be applied as immunosuppression agent; the amino acid at the 59-60 locus of the I73R protein is mutated into asparagine and alanine, so that the activation of IFN-beta by SeV is recovered,

(2) the invention surprisingly discovers that after amino acid mutation of the 59-60 locus of the I73R protein is changed into asparagine and alanine, the activation of the IFN-beta by SeV is recovered, the immunosuppression locus of the I73R protein of the African swine fever virus is discovered, and the immunosuppression locus mutated African swine fever recombinant virus of the I73R protein is constructed by a genetic engineering means;

(3) the natural immunity of the African swine fever recombinant virus is obviously weakened, the African swine fever recombinant virus can be used as a recombinant vaccine strain, can promote early immune response, obviously weakens the toxicity of the recombinant strain, obviously increases the survival rate of an infected recombinant virus experimental animal compared with an infected wild virus experimental animal, improves the biological safety, improves the immune protection efficacy, and has good application prospect.

Drawings

FIG. 1 is a graph showing the results of inhibition of SeV-induced IFN- β activation by the African swine fever virus I73R protein;

FIG. 2 is a graph showing the results of IFN- β activation by SeV after mutation at the 59-60 locus of the I73R protein of African swine fever virus;

FIG. 3 is a schematic diagram of a construction strategy of an African swine fever virus I73R protein mutant strain with amino acids 59-60;

FIG. 4 fluorescence imaging of single GPF positive cells after 72h inoculation of PAM cells in 96-well plates;

FIG. 5 is a graph showing the temperature change results of swine infected with African swine fever virus I73R KT59/60NA mutant strain;

FIG. 6 is a graph showing the mortality rate of pigs infected with African swine fever virus I73R KT59/60NA mutant strain;

FIG. 7 is a graph showing the results of infection of swine blood viruses with African swine fever virus I73R KT59/60NA mutant strain;

FIG. 8 purity detection of African swine fever virus I73R KT59/60NA mutant strain.

Detailed Description

The invention is further described in connection with the following detailed description, in order to make the technical means, the creation characteristics, the achievement of the purpose and the effect of the invention easy to understand. The scope of the invention is not limited to the examples described below.

The experiments described in the examples below obtained biosafety permissions and african swine fever laboratory activity permissions:

the national institute of agricultural science, lanzhou, according to the biological safety 3-level laboratory (BSL-3) and the related requirements of African swine fever related biosafety, was reported by the biological safety committee of the national institute of veterinary science, the ethical committee of laboratory animals, the biosafety committee of the national academy of agricultural science, the ethical committee of laboratory animals of the national veterinary institute, the biosafety committee of the national institute of veterinary science, and the permissions of the department of agriculture for developing highly pathogenic ASFV pathogens and animal research, and has been filed in the rural department of agriculture, conforming to the requirements of the national biosafety level.

Experimental cells, viruses and plasmid sources as described in the examples below:

primary Pig Alveolar Macrophages (PAM) and primary bone marrow macrophages (BMDM) were obtained from healthy white pigs of 2-4 months of age, after aseptic harvesting of the cells, red blood cells were removed with a red blood cell lysate (from Biosharp Corp.), the supernatant was discarded after low-speed centrifugation, and the cell pellet was resuspended in RPMI 1640 complete medium (from Gibco Corp.) containing 10% FBS (from PAN Corp.) at 37℃in 5% CO ₂ Culturing in an incubator. BMDM cell culture requires an additional addition of GM-CSF (available from R) at a final concentration of 10ng/mL in RPMI 1640 complete medium&D SystemsCompany) at 37℃with 5% CO ₂ Inducing in incubator, washing once every 2-3 days, centrifuging non-adherent cells, adding into new cell dish again, changing liquid, inducing, and freezing or using after 3-7 days. ASFV was expanded using PAM cells and virus content titrations were performed, BMDM cells were used for plasmid transfection and virus recombination experiments.

Type II African swine fever virus strain ASFV CN/GS 2018 is from the national African swine fever area laboratory (Lanzhou), belongs to type II genes, and has a virus titer of 5×10 ⁷ TCID50/mL is the 4 th generation seed virus after PAM cell proliferation; the rice is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of CCTCC NO: v202096; preservation address: chinese university of armed chinese; contact phone: 027-68752319.

The pepGFP-N1 vector and pUC19 vector were purchased from Lanzhou Rableacher Biotech Co., ltd; endotoxin-free plasmid extraction kit, purchased from OMEGA company.

HEK-293T cells purchased from ATCC; IFN-beta promoter plasmid, TK plasmid, FLAG-I73R and mutant plasmid are constructed by Lanzhou Rableacher biotechnology Co., ltd; lipofectamine TM 3000, available from Invitrogen corporation.

The experimental methods in the following examples, unless otherwise specified, are all procedures known in the art; the test materials used in the examples described below, unless otherwise specified, were purchased from conventional Biochemical reagent companies.

EXAMPLE 1I 73R protein inhibits SeV-induced IFN- β activation

Effect of the I73R protein on SeV-induced IFN- β expression

HEK-293T cells were plated into individual wells of 24-well plates. When the cells were grown to 70% -80% confluence, IFN- β promoter plasmid (100 ng/well) and TK plasmid (10 ng/well) and FLAG-I73R plasmid (100 ng) were transfected with liposome reagents, respectively, for 24 hours, and then SeV (MOI=1) was inoculated for 16 hours, and the activity of IFN- β was detected with luciferase kit.

The results are shown in FIG. 1, where EV is a non-transfected FLAG-I73R plasmid. The results showed that IFN- β expression levels were higher after 16h infection with SeV (MOI=1) and transfection of FLAG-tagged empty plasmid alone (100 ng/well); and IFN- β expression was significantly reduced 16h after transfection of FLAG-I73R plasmid (100 ng/well) and infection with SeV (MOI=1). The African swine fever virus I73R protein can inhibit the activation of IFN-beta induced by SeV, and has an immunosuppressive effect.

Effect of the KT59/60NA mutation of the I73R protein on SeV-induced IFN- β expression

HEK-293T cells were plated into individual wells of 24-well plates. When the cells were grown to 70% -80% confluence, IFN- β promoter plasmid (100 ng/well), TK plasmid (10 ng/well), FLAG-I73R plasmid (100 ng), and I73R 59/60 site amino acid mutant plasmid FLAG-I73R KT59/60NA plasmid (100 ng/well) were transfected with liposome reagents, respectively, for 24 hours, and then SeV (MOI=1) was inoculated for 16 hours, and the activity of IFN- β was detected with luciferase kit.

The results are shown in FIG. 2, where EV was not transfected with FLAG-I73R and FLAG-I73R KT59/60NA plasmids (100 ng/well). The results showed that IFN- β expression levels were higher after 16h infection with SeV (MOI=1) and transfection of FLAG-tagged empty plasmid alone (100 ng/well); after 16h transfection of FLAG-I73R plasmid (100 ng/well) and infection with SeV (MOI=1), IFN- β expression was significantly reduced; and after 16h of transfection of the I73R 59/60 site amino acid mutant plasmid FLAG-I73R KT59/60NA plasmid (100 ng/well) and infection of SeV (MOI=1), the IFN- β expression level was significantly increased compared to the transfected FLAG-I73R plasmid, comparable to the EV group level. It is shown that the mutation of the I73R protein KT59/60NA of African swine fever virus results in the loss of the function of inhibiting the activation of IFN-beta induced by SeV by the I73R, namely the I73R protein 59/60 site is a key site for the I73R to play a role in natural immunosuppression.

In conclusion, the African swine fever virus I73R protein can inhibit the activation of related interferon such as IFN-beta induced by SeV, has an immunosuppressive effect, can be used as an immunosuppressant to inhibit natural immunity, and can be used for preparing medicines or medicine compositions for inhibiting SeV infection.

EXAMPLE 2 construction, purification and identification of ASFV I73R-KT59/60NA recombinant strain

1. Screening expression cassette construction

To facilitate the screening, a set of expression cassettes for screening marker genes, namely, the construction of the enhanced green fluorescent protein (Enhance Green fluorescent protein, eGFP) gene screening expression cassettes, are constructed together

Referring to the literature, the CMV pro promoter is amplified by PCR for use; the amplification primers are as follows: forward primer 5'-CAAGGCTTGACCGACAATTGCAT-3' (shown as SEQ ID NO. 6) and reverse primer 5'-TAGTGAGTCGTATTAGCGGCC-3' (shown as SEQ ID NO. 7); using peGFP-N1 carrier as template to amplify eGFP gene, the amplification primer is: forward primer 5'-ATGGTGAGCAAGGGCGAGGAG-3' (shown as SEQ ID NO. 8) and reverse primer 5'-ACCACAACTAGAATGCAGTG-3' (shown as SEQ ID NO. 9);

reference (Borca MV, holinka LG, berggren KA, glade DP. CRISPR-Cas9, a tool toefficiently increase the development of recombinant African swine fever viruses. Sci Rep.2018;8 (1): 3154.) the CMV pro promoter amplified in the above procedure and the eGFP gene obtained by amplification in the above procedure were joined by fusion PCR to obtain a fragment of the eGFP selection expression cassette gene designated CMV pro-eGFP-SV40PA (SEQ ID NO. 10) containing the SV40PA termination sequence.

2. Construction of homologous recombinant transfer vector

The pUC19 vector is used as a skeleton vector to construct a homologous recombination transfer vector for mutation of 59-60 amino acids of the I73R protein, the nucleotide sequence of the I73R gene is shown as SEQ ID NO.2, the sequence of the mutated I73R gene is shown as SEQ ID NO.4, and the construction strategy is shown in FIG. 3.

The method comprises the following specific steps:

(1) Constructing elements for expressing a gene mutation sequence encoding amino acids 59-72 of the I73R protein and mutating amino acids 59-60 of the I73R protein into asparagine and alanine (shown as I73R mut. Pat in the figure); the gene mutation sequence is shown as SEQ ID NO. 5.

(2) Designing a gene upstream sequence of 59 th amino acid (172292 th of the complete genome of CN/GS/2018 isolate) of the I73R protein with about 1.0kb as a homologous recombination Left arm (shown in SEQ ID NO. 11); taking a gene fragment of about 1.0kb downstream of the I73R protein (CN/GS/2018 isolate genome 172510) as a homologous recombination Right arm (shown in SEQ ID NO. 12), and cloning the gene fragment into pUC19 vectors respectively to obtain recombinant transfer vectors of 59-60 amino acid mutations of the I73R gene;

(3) An element of a gene mutation sequence (shown as I73R mut. Pat in the figure, SEQ ID NO. 5), a DNA sequence of CMV pro. Shown as SEQ ID NO. 13) and an eGFP screening expression cassette gene fragment eGFP-SV40PA (shown as eGFP-SV40 PA) are inserted in sequence between the left and right arm gene sequences of the recombinant transfer vector of the I73R gene; after sequencing correctly, the homologous recombination transfer vector is named I73R-mut-eGFP; extracting DNA with endotoxin-free plasmid extraction kit, measuring concentration, and preserving at-20deg.C.

3. Cell transfection and recombinant virus screening

The homologous recombinant transfer vector pEI R-mut-eGFP (2. Mu.g) and 6. Mu.L of the-Macrophage DNA transfection reagent were thoroughly mixed and co-transfected into porcine BMDM cells (cell number: about 106 cells/well), and after 6h of transfection, african swine fever virus CN/GS/2018 isolate was directly infected (at 1MOI dose), without changing the solution, until 48h of transfection, and the fluorescent cell number was observed under a fluorescent microscope. As a result, as shown in FIG. 4, there was a sporadic green fluorescence under the fluorescence microscope, i.e., cells infected with suspected recombinant viruses were seen. Fluorescent cells are selected, carefully blown off in a new culture dish, settled for 1h, single fluorescent cells are selected, repeatedly frozen and thawed for 3 times after collection, inoculated into pre-paved 96-well plate PAM cells, observed once every 12h, observed and marked in fluorescent cell holes, and continuously observed for 72h. The proportion of fluorescent cells up to 100% was a full positive well, indicating that recombinant toxin construction was essentially successful.

And (3) carrying out 6 times of limiting dilution expansion culture on the full positive hole, picking the 8 th generation recombinant virus hole, digesting into single cells, carefully sucking 10 fluorescent cells, respectively inoculating the 10 fluorescent cells into the pre-paved 96-well plate PAM cells, and continuing to grow for 72 hours. And (3) selecting cells with more GFP fluorescence, extracting genome DNA, carrying out PCR identification on the purity of the cells by using an I73R-F/R primer, and carrying out PCR identification on the mutation condition of the cells by using the I73R-check-F/R primer. The I73R-F/R primer pair is as follows: I73R-F: CCCCGCTTTGGATACGGAAA (SEQ ID NO. 14) and I73R-R: GCGGGAATAAGCCAGGACATGA (SEQ ID NO. 15). The I73R-check-F/R primer pair is: I73R-check-F: CCCGACAACCACTACCTGAGCA (SEQ ID NO. 16) and I73R-check-R: CTGCGGGAATAAGCCAGGACAT (SEQ ID NO. 17). The PCR amplification result shows that when the gene encoding wild type ASFV I73R protein is used as a template, the I73R-F/R primer can amplify a distinct band (FIG. 8, endogenous band wt), and when the gene encoding mutant ASFV I73R protein 59-60 is used as a template, the I73R-F/R primer cannot amplify a band (FIG. 8, exogenous band wt), indicating that the ASFV I73R protein 59-60 has been mutated and purified. When the gene encoding amino acids 59-60 of the mutated ASFV I73R protein was used as a template, the I73R-check-F/R primer was able to amplify the band (FIG. 8, exogenous bands 1, 2, 3), indicating that amino acids 59-60 of the ASFV I73R protein were deleted and that the nucleotide sequence encoding amino acids 59-60 of the ASFV I73R protein had been successfully mutated as a result of sequencing the product and was designated as I73R-KT59/60NA recombinant strain.

EXAMPLE 3 evaluation of virulence of mutant Virus I73R-KT59/60NA

In order to detect virulence of the I73R 59/60 gene mutant attenuated African swine fever virus strain I73R-KT59/60NA, the experiment was evaluated for virulence by intramuscular injection of piglets at a 10HAD50 dose.

The experiment was performed with 12 healthy and white pigs negative for African swine fever antigen antibodies, which were divided into 2 groups of six African swine fever virus CN/GS/2018 isolates and I73R 59/60 gene mutation attenuated African swine fever virus strains I73R-KT59/60NA, after the virus was tapped, the temperature change condition was measured every day, peripheral blood was collected, the survival condition was observed, and the experimental results were shown in FIG. 7, which were obtained by reference to King DP, reid SM, hutchings GH, grierson SS, wilkinson PJ, dixon LK, bastos AD, drew TW 2003.Development of a TaqMan PCR assay with internal amplification control for the detection of African swine fever virus J Virol Methods 107:53-61.

The viral loads of six pigs surviving the challenge mutant are all at a low level, and ASFV I73R 59/60 gene mutant attenuated African swine fever virus strain I73R-KT59/60NA group is significantly different from African swine fever virus CN/GS/2018 isolate group, and the results are shown in FIG. 7. Experimental results prove that after the mutation of the African swine fever virus CN/GS/2018 isolate I73R 59/60 amino acid mutant gene, the toxicity of the obtained ASFV I73R 59/60 amino acid mutant gene deletion attenuated African swine fever virus strain I73R-KT59/60NA is obviously weaker than that of the African swine fever virus CN/GS/2018 isolate.

Typical symptoms of ASFV appear after the African swine fever virus CN/GS/2018 isolate is subjected to intramuscular injection of 10HAD50, the ASFV is heated at high temperature and dies in the later period, and the result of ASFV I73R 59/60 gene mutation attenuated African swine fever virus strain I73R-KT59/60NA group shows that most animals are reduced to normal body temperature after being heated at high temperature, the survival rate is 100 percent, and the result is shown in figures 5 and 6.

The foregoing examples merely illustrate embodiments of the invention, which are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that other modifications can be made by those skilled in the art without departing from the spirit of the invention, which falls within the scope of the invention.

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ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 1140

ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 1200

cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 1260

gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aattaacttg 1320

tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa 1380

gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaag 1438

<210> 11

<211> 998

<212> DNA

<213> African swine fever virus (African swine fever virus)

<400> 11

tcaggaatgt aaagaaacta tttttgattt aaaggtggta ggaaatgttt agccaataaa 60

ctcatgcccg cattttttac aggtacaaaa tatcgtggat ggctcatcga gggcgcgtgt 120

ttgtacttct ctgtaggtac acatacgctg cttgcagttg ggacacttat aaagttgtga 180

cgtcttttcg gcgacctttt gctgcgaacg tagagtaatt tctgtcttct cctttaaggc 240

ggcagagggg caaagctcgg cgaacgtcat gctaccaatt gcctccggtt ttagctcgcc 300

agaaattagc ttattaaggg catcgttatc ctgttgttgg tgactttttt tttcgcagtt 360

aataatatga ttgatcgtcc cacaacgggt tgaatattct tctaaaaagg ttttttcttg 420

ttgctggtac gtataatgat aacacgaggc ctcgattttt tgcgcgtatt cggtgcataa 480

atcagtatgt tccttaaaaa acatatgttt ttgaagcgtt ctaaaaaaca tcatttggat 540

gatatcacgc atttccaaaa taatataggg ttctagtctt ttggaatctt tcataactag 600

atcggtggta atattcttag tcatacaatt tattaaaaat ggtttaatat attgtaaata 660

ttttttaggc gtgtcagcct gtaaaaaaca ttcttgttca atcttatttg taaggatagt 720

attttgcaaa tacttattta gcaaaaatac gatagaatcg cgggctatat gcattttcat 780

ataatttttt ttttaaaatt taatacaaaa aaaagaagta tagactcttc ttctagtccg 840

gttagttcgt tggttgcctc aacatggaga ctcagaagtt gatttccatg gttaaggaag 900

ccttagaaaa atatcaatac cctcttactg ctaaaaatat taaagtagtg atacaaaaag 960

agcacaatgt cgtcttacct acaggatcta taaatagc 998

<210> 12

<211> 998

<212> DNA

<213> African swine fever virus (African swine fever virus)

<400> 12

tagaaatagc taagcttaat actaattcag cttttttttt aactaaaacc tgaatagatg 60

cgaagtagcg gacatataca tactaaaata agccatacat ttactttctt cttgaacatg 120

aaaccttttt ttcttctgtt gttggtatat aaacaatagg actgtttgct gaggttgtat 180

gatcttctac aactgctgtc tcaggatgac gatgtttttt taaactaaaa gtgtaggatg 240

gaatgagtgg aatatagtta tggctcgact tatcctgttt cgtacaggaa tattttttac 300

aaatagaacg caacaagcat atgaataaaa acagaaatga tatacaggag cataaaatag 360

atatgaacac taaggggtag cagcttttat aacgttccgt atttttctta gctatcaatt 420

gatttaccgt aatatttatc tcgggaaact ttgttctaca atattttgtt tggtattcca 480

gaaactcatg tcctggctta ttcccgcagc ttaaaaaatg atacaaaaat gtgttattgt 540

tactaaaatt aattcttctt aagaaaaact gcggaagacg ctttaggtac gtctgttcct 600

gttttagtag gaagtagtat aagggacaat ttctttttcc acacattaga ttattgtaat 660

ataggtaggt tggggtgttg gagcgaataa gttttctgag tatgttataa tctatgactt 720

gtaaatcgtt ataccttagg tccaaaaact tgagttcttt accaaagcca cctgcaattt 780

cagaaatatt tttcatcccg cagcggataa tacggatgtc ctgaaacgtc tttaaaatac 840

ttgtattgta gtgaatactt atgttatttt tttgtaaata atctatgtca tgacaagtgc 900

atgaaatgcc agcagcattg cttggtatag tattatatgc aggaagaact atactactat 960

tgagaatagt cacattgtac ttataccatg tattattt 998

<210> 13

<211> 587

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 60

gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 120

ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 180

ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 240

atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 300

cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 360

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 420

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 480

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 540

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagct 587

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ccccgctttg gatacggaaa 20

<210> 15

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gcgggaataa gccaggacat ga 22

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

cccgacaacc actacctgag ca 22

<210> 17

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

ctgcgggaat aagccaggac at 22

Claims (8)

1. The application of the African swine fever recombinant virus prepared by inhibiting the immunosuppression function of the African swine fever virus I73R protein is characterized in that the immunosuppression function of the African swine fever virus I73R protein is as follows: amino acids 59 and 60 of the African swine fever virus I73R protein were mutated to asparagine and alanine, respectively.

2. An african swine fever recombinant virus having a loss of immunosuppressive function of an I73R protein, characterized in that amino acids 59 and 60 of the I73R protein in the african swine fever virus are mutated into asparagine and alanine, respectively.

3. The african swine fever recombinant virus of claim 2, wherein the african swine fever virus is a CN/GS/2018 isolate.

4. An african swine fever vaccine comprising the african swine fever recombinant virus having a loss of immunosuppressive function of the I73R protein of claim 2 or 3.

5. A preparation method of an african swine fever recombinant virus with a loss of an immune suppression function of an I73R protein, which is characterized by comprising the following steps: the 59 th amino acid and the 60 th amino acid of the African swine fever virus I73R protein are mutated into asparagine and alanine respectively through genetic engineering means.

6. The method of claim 5, wherein the african swine fever virus is a CN/GS/2018 isolate.

7. The method according to claim 6, characterized in that the method comprises the steps of:

(1) Constructing an I73R protein gene mutation sequence, wherein the gene mutation sequence is a gene sequence for encoding 59-72 amino acids of the I73R protein and respectively mutating 59 and 60 amino acids into asparagine and alanine, and the gene mutation sequence is shown as SEQ ID NO. 5;

(2) Taking about 1.0kb of an upstream gene of 172292 th site of a full genome of a CN/GS/2018 isolate as a homologous recombination left arm, taking about 1.0kb of a downstream gene sequence of 172510 th site of the full genome of the CN/GS/2018 isolate as a homologous recombination right arm, and cloning the homologous recombination right arms into pUC19 vectors respectively to obtain a recombinant transfer vector;

(3) Inserting the gene mutation sequences in the step (1) into the left and right arm gene sequences of the recombinant transfer vector in the step (2), and screening the gene fragments of the expression cassette to obtain a homologous recombinant transfer vector;

(4) And (3) transfecting the homologous recombination transfer vector in the step (3) into BMDM cells infected with the parent African swine fever strain, and purifying and screening to obtain the African swine fever recombinant virus with the I73R protein having a lost immune suppression function.

8. The recombinant virus of african swine fever with loss of immunosuppressive function of the I73R protein prepared by the method of any one of claims 5 to 7.

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