CN110551695A - African swine fever virus four-gene deletion low virulent strain and application thereof - Google Patents

Abstract

The invention discloses a four-gene-deleted attenuated strain of African swine fever virus. The attenuated strain is a four-gene-deleted attenuated strain of African swine fever virus SY18 isolate, which lacks the functional proteins of the following genes: CD2v gene coding product and three polynucleotides Gene family genes (MGF360‑12L, MGF360‑13L, MGF360‑14L) encode products. The invention also discloses the application of the attenuated African swine fever virus strain in preparing a vaccine for preventing or treating the African swine fever. The attenuated African swine fever virus strain of the invention can provide complete immune protection against the attack of ASFV parental strains, has high safety, and is suitable as a vaccine candidate strain for preventing African swine fever.

Description

African swine fever virus four-gene deletion attenuated strain and its application

technical field

The invention relates to the technical field of bioengineering, in particular to a four-gene-deleted attenuated strain of African swine fever virus and its application.

Background technique

African swine fever (ASF) is an acute and severe infectious disease of pigs caused by African swine fever virus (ASFV) infection, characterized by fever and systemic organ hemorrhage, and the fatality rate of domestic pigs can be as high as 100%. The disease first broke out in Kenya in 1921 and then became widespread in domestic and wild pigs throughout Africa, gradually evolving into 24 genotypes. It was introduced into Europe in the 1950s, and it took 40 years to eliminate the disease throughout Europe. However, the disease was introduced to Georgia from East Africa again in 2007, and then spread widely in Eastern Europe, and in 2017, it was introduced to Irkutsk in the Russian Far East. In early August 2018, researcher Hu Rongliang took the lead in reporting the first African swine fever epidemic in my country. In just one year, the disease spread to 30 provinces and autonomous regions across the country, causing more than 20% of the country's production reduction rate and a loss of more than 10 billion yuan. , the current situation of the prevention and control of the disease is extremely severe and complicated.

ASFV is a large virus that replicates in the cytoplasm. Virus particles are icosahedrally symmetrical, with a diameter of 200 nm and a concentric circular structure. The African swine fever virus genome is a single-molecule linear double-stranded DNA whose ends are covalently closed. The total length of the genome is 170-190kb, and the genome length of different strains is inconsistent. The entire genome of ASFV contains more than 160 open reading frames, which can encode 150-200 proteins. The central part of the genome is the central conserved region (C region), which is about 125kb in length. There is a variable region on each side of the C region, and the right variable region (VR) is 13-22kb in length and contains 5 multigene families (MGF). This region is related to mechanisms such as virus antigen mutation and evasion of host defense system.

Vaccines are the most effective and economical means of preventing and controlling infectious diseases. Since ASF was introduced to Europe, the research and development of effective ASF vaccines has been highly valued by many countries, especially European research institutions, and important progress has been made, such as the discovery of ASFV Live vaccines cannot induce effective neutralizing antibodies, and both humoral and cellular immunity must be considered; subunit vaccines are safer than live attenuated vaccines. From the current progress, the combined expression of different antigen combinations by viral vectors can improve ASFV genetic engineering. The immune efficacy of subunit vaccines is a feasible idea; live attenuated vaccines through knockout of virulence genes have the best protective effect and are the most promising for development. Side effects, field poisoning, and excessive attenuation caused by multiple gene deletions will cause the attenuated strains to lose their protective effects. It is necessary to take into account safety (biological safety risks such as side effects, persistent infection, and field poisoning) and protection in research. sexual balance.

Due to the complex pathogenic mechanism of ASFV and the unclear mechanism of virus infection and immunity, there is still no effective vaccine to prevent and control the disease so far. Although the research on ASF vaccine started in the late 1960s, and there have been many reports in recent years, it has not been successfully applied in practice. So far, a number of ASFV gene-deleted vaccine candidate strains have been obtained internationally by means of genetic engineering, and attenuated vaccine candidate strains have been obtained through cell passage attenuation and isolation of attenuated strains from nature, some of which are targeted at Homologous virus strains have a 50% to 100% protection rate, but the protection effect against heterologous virus is not ideal. In addition, whether it is an artificial gene deletion attenuated strain or a natural deletion attenuated strain, the pigs have dose-dependent side effects after immunization, such as depression, loss of appetite, fever, viremia, hypergammaglobulinemia, pneumonia, joint swelling, Lameness, chronic disease and dead pigs, etc., reduce the actual use value of the vaccine. At present, people are generally concerned about the safety of African swine fever gene deletion strains and natural attenuated strains as vaccines. Due to various reasons, the research and storage of African swine fever vaccine in my country is seriously lagging behind. After the outbreak of African swine fever, the research of African swine fever vaccine has been put on the agenda again.

At present, the situation of prevention and control of African swine fever (ASF) in my country is extremely severe, and it is urgent to develop a safe and efficient ASF vaccine.

Contents of the invention

The present invention aims to solve the technical problem of the lack of safe and effective African swine fever vaccine in China at present, and provides a gene-deleted attenuated strain of African swine fever virus, which can provide complete immune protection against the attack of ASFV parental strains, And the safety is high, and it is suitable as a vaccine candidate strain for preventing African swine fever.

In order to solve the problems of the technologies described above, the present invention is realized through the following technical solutions:

In one aspect of the present invention, a kind of attenuated strain of African swine fever virus is provided, which is a gene deletion attenuated strain of African swine fever virus SY18 isolate, which lacks the functional protein of the following genes:

CD2v gene and three multigene family genes MGF360-12L, MGF360-13L, MGF360-14L.

Preferably, the deleted gene sequence includes: a nucleotide sequence encoding the amino acid sequence of the CD2v protein shown in SEQ ID NO.1, a nucleotide sequence encoding the amino acid sequence of the MGF360-12L protein shown in SEQ ID NO.2, encoding The nucleotide sequence of the amino acid sequence of the MGF360-13L protein shown in SEQ ID NO.3 encodes the nucleotide sequence of the amino acid sequence of the MGF360-14L protein shown in SEQ ID NO.4.

More preferably, the deleted gene sequence includes: the CD2v gene nucleotide sequence shown in SEQ ID NO.5, the MGF360-12L gene nucleotide sequence shown in SEQ ID NO.6, the MGF360-12L gene sequence shown in SEQ ID NO.7. 13L gene nucleotide sequence, MGF360-14L gene nucleotide sequence shown in SEQ ID NO.8.

In another aspect of the present invention, a recombinant virus is provided, which is a recombinant virus in which the CD2v gene in the African swine fever virus SY18 isolate is deleted.

Preferably, the recombinant virus also lacks the following three multigene family genes: MGF360-12L, MGF360-13L, and MGF360-14L.

In another aspect of the present invention, application of the above-mentioned attenuated African swine fever virus strain in preparing a vaccine for preventing or treating African swine fever is also provided.

In another aspect of the present invention, a vaccine comprising the above-mentioned attenuated strain of African swine fever virus is also provided.

The vaccine is preferably suitable for oral or intramuscular injection.

In another aspect of the present invention, there is also provided a detection kit for distinguishing the above-mentioned African swine fever virus attenuated strain from wild strain infection, comprising: CD2v, MGF360-12L, MGF360-13L for African swine fever virus SY18 isolate A pair of primers designed for at least one of the four genes MGF360-14L. The attenuated African swine fever virus strain and the wild strain of the present invention can be distinguished and identified by using the designed and synthesized primer pair through PCR amplification reaction.

In another aspect of the present invention, a detection kit for distinguishing the above-mentioned African swine fever virus attenuated strain from wild strain infection is also provided, comprising: CD2v, MGF360-12L, MGF360-13L, Antibodies to proteins expressed by at least one of the four genes in MGF360-14L. By detecting CD2v, MGF360-12L, MGF360-13L, and/or MGF360-14L protein antibodies in infected pigs, the attenuated African swine fever virus strain of the present invention can be distinguished from the infection of wild strains.

The African swine fever virus gene deletion attenuated strain of the present invention, 28 days after the piglets were inoculated and immunized, the ASFV parental virus was used to challenge the virus. Any abnormal clinical manifestations, safe and reliable, can be used as a good vaccine candidate strain for preventing African swine fever.

Description of drawings

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Fig. 1 is the schematic diagram of the ASFV recombination deletion gene position and insertion marker gene expression cassette of embodiment 1 of the present invention;

Fig. 2 is the observation picture under the fluorescence microscope of the ASFV SY18ΔC recombinant virus construction first generation (*10) of the embodiment 1 of the present invention;

Fig. 3 is the observation diagram of the growth situation (*10) of the ASFV SY18ΔC recombinant virus P2 generation recombinant virus of the embodiment 1 of the present invention;

Fig. 4 is the ASFV SY18ΔC recombinant virus P7 generation PCR detection result (1～23 represent different clones) figure of embodiment 1 of the present invention;

Fig. 5 is the ASFV SY18ΔC recombinant virus P8 generation PCR detection result (1～23 represent different clones) figure of embodiment 1 of the present invention;

Fig. 6 is the ASFV SY18ΔC recombinant virus P9-P11 generation PCR detection result figure of embodiment 1 of the present invention;

Fig. 7 is the ASFV SY18ΔMC4 recombinant virus P2 generation fluorescence detection result (*10) figure of embodiment 1 of the present invention;

Fig. 8 is the result of PCR detection (1～23 represent different clones) of ASFV SY18ΔMC4 recombinant virus P3 generation of embodiment 1 of the present invention;

Fig. 9 is the result of PCR detection (1～23 represent different clones) of ASFV SY18ΔMC4 recombinant virus P5 generation of embodiment 1 of the present invention;

Fig. 10 is the result figure of PCR detection of the P6-P8 generation of ASFV SY18ΔMC4 recombinant virus of embodiment 1 of the present invention;

Fig. 11 is the ASFV SY18ΔMC4 recombinant virus P8 generation recombinant virus fluorescence detection result (24hpi) (*20) figure of embodiment 1 of the present invention;

Fig. 12 is the virus growth curve figure of embodiment 2 of the present invention;

Fig. 13 is a graph showing the death curve of the recombinant virus in Example 3 of the present invention.

Detailed ways

The results of the examples of the present invention show that the single deletion of the CD2v gene has a limited impact on the virulence of the Chinese strain, and the preliminary results also show that another kind of MGF multigene family 6 genes (containing 6 MGF multigene families, including MGF360- After immunizing pigs with 12L, 13L and 14L) deletion strains, although the body temperature of the pigs would rise for a long time (not exceeding 41.3°C), it did not affect the mental state and feed intake.

The present invention further clones and optimizes the expression of all possible MGF multi-gene family member genes in the whole genome of the Chinese strain SY-18, and compares the inhibitory activity of the transcription level of the interferon reporter gene to screen and determine MGF360-12L, MGF360-12L, MGF360-13L and MGF360-14L are particularly critical for viral virulence and immunosuppression. Based on the above preliminary results, the present invention further constructs a vaccine candidate strain that lacks the key virulence genes of ASFV CD2v and MGF360 by using genetic engineering methods, and further deletes CD2v After genetic modification, the long-term fever caused by virus inoculation was basically eliminated, which indicated that although CD2v single gene deletion strains were highly virulent, CD2v gene deletion could further reduce the virulence of MGF deletion strains. From the preliminary results of the immune efficacy and safety experiments, it can be evaluated that the four-gene deletion strain has a complete protective effect against the challenge of the ASFV parent strain, which shows that it can be used as a good African swine fever vaccine candidate strain.

Example 1 Construction and purification identification of recombinant viruses SY18ΔC and SY18ΔMC4

1. Materials and Methods

1.1 Biosafety license and African swine fever laboratory activity license

The Military Veterinary Research Institute has approved the Biosafety Committee of the Military Veterinary Research Institute, the Experimental Animal Ethics Committee, the Biosafety Committee of the Military Medical Research Institute, the National The Army Experimental Animal Ethics Committee and the Biosafety Committee of the Academy of Military Sciences reported it step by step, and obtained the permission from the Health Bureau of the Logistics Support Department of the Military Commission to carry out research on highly pathogenic ASFV pathogens and animals, and it has been filed with the Ministry of Agriculture and Rural Affairs, which meets the requirements of the national biosafety level. .

1.2 Cells and viruses

Primary porcine alveolar macrophages (PAM) and primary bone marrow macrophages (BMDM) were obtained from 2-4 month-old healthy SPF Bama miniature pigs (purchased from Jiangsu Academy of Agricultural Sciences). The lysate (purchased from Biosharp Company) was used to remove red blood cells, and after low-speed centrifugation, the supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 complete medium (purchased from Gibco Company) containing 10% FBS (purchased from PAN Company), Place them in a 37°C, 5% _CO2 incubator. BMDM cell culture needs to add 10 ng/mL final concentration of recombinant porcine GM-CSF (purchased from R&D Systems) to RPMI 1640 complete medium, and place it in a 37°C, 5% _CO2 incubator for induction. Wash once every day, centrifuge the non-adherent cells and put them back into a new cell dish, change the medium to continue induction, freeze or use after 3-7 days. PAM cells were used to amplify ASFV and titrate the virus content, and BMDM cells were used for plasmid transfection and virus recombination experiments. The ASFV SY18 isolate is the first domestic virus isolate reported by our team. It belongs to genotype II, and the virus titer is 5×10 ⁷ TCID ₅₀ /mL. Store at -80°C for later use. The Genbank accession number of the ASFV SY18 isolate is MH766894.1, and its complete genome sequence is provided by Genbank accession number MH766894.1.

1.3 Detection of exogenous pathogens in primary cells and virus seeds

A small sample of the above cells was taken to detect exogenous virus DNA, bacteria, mold and Mycoplasma hyopneumoniae. Routine virus detection pathogens include: ASFV, porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), swine fever virus (CSFV), Japanese encephalitis virus (JEV), porcine epidemic diarrhea virus (PEDV) ), H3N2 subtype swine influenza virus, porcine pseudorabies virus (PRV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2), Seneca virus (SVV), porcine serotype 4 adenovirus (PAdV-4), porcine cytomegalovirus (PCMV), etc. The detection of Seneca virus (SVV) uses RT-PCR to amplify the VP2 gene, the upstream primer VP2F: 5'-CGGGAT CCATGGATCACAATACCGAAGAAA-3' (SEQ ID NO.9); the downstream primer VP2R: 5'-CGGAATC TGCTCCTCGTCCGTCCCGGTC-3 '(SEQ ID NO.10); detection of porcine serum adenovirus type 4 (PAdV-4) using self-designed primers for PCR, the upstream primer PAdV4-F is: CCTCATGCCGGCTTTCTAT (SEQ ID NO.11), the downstream primer PAdV4-R is: AGGGTATCTTGGTCTTTCATT (SEQ ID NO.12); porcine cytomegalovirus (PCMV) detection using self-designed primers for PCR, the upstream primer PCMVF1 is: CCTATGTTGGCACTGATACTTGAC (SEQ ID NO.13), the downstream primer PCMVR1 is: CCCTGAAAATCACCGTCTGAGAGA (SEQ ID NO.14 ).

1.4 CRISPR/Cas9 vector construction

Because African swine fever virus mainly replicates in the cytoplasmic virus factory, pX335 was first optimized when constructing the pCRISPR/Cas9 vector. That is, the nuclear localization signal (NLS) at both ends of the Cas9 enzyme was removed by the ClonExpress II one-step cloning method, and it was named pX335ΔN. Subsequently, three gene-targeting gRNAs targeting ASFV EP402R (CD2v), MGF360-12L, and MGF360-14L were designed, and a total of 4 pairs of oligonucleotides were designed. The names and sequences were: CD2v-gRNA-LF: CACCGCTAGCTACATGTGGAAAAGC (SEQ ID NO .15); CD2v-gRNA-LR: AAACGCTTTT CCACATGTAGCTAGC (SEQ ID NO.16); CD2v-gRNA-RF: CACCGTTGGGTAGTAGCGGGATACT (SEQ ID NO.17); CD2v-gRNA-RR: AAACAG TATCCCGCTACTACCCAAC (SEQ ID NO.18 ); MGF36012L-gRNA-LF: CACCGCTAATACCAAGGTCACATC A (SEQ ID NO.19); MGF36012L-gRNA-LR: AAACTGATGTGACCTTGGTATTAGC (SEQ ID NO.20); MGF36014L-gRNA-RF: CACCGGGATGATCTTTGTCTCGAAG (SEQ ID NO.21); 4MGF360 - RR: AAACCTTCGAGACA AAG ATCATCCC (SEQ ID NO. 22). Through the cloning method recommended by ZhangF et al., insert the oligonucleotide pair into the pX335-ΔN vector, extract the plasmid DNA, and name the positive cloned plasmids after correct sequencing: pX335ΔN-CD2vL, pX335ΔN-CD2vR, pX335ΔN-12LL, pX335ΔN-14LR. DNA was extracted with an endotoxin-free plasmid extraction kit, the concentration was measured, and stored at -20°C for later use.

1.5 Construction of Screening Expression Cassette

In order to facilitate the screening, two sets of expression cassettes for screening marker genes were constructed: 1) EGFP screening expression cassette construction: amplify the p72 promoter by PCR, that is, from -199nt to the sequence before the ATG start codon; at the same time, use pEGFP-N1 The carrier is used as a template to amplify the green fluorescent protein (Enhanced green fluorescent protein, EGFP) gene. The two genes were connected by the method of fusion PCR to obtain the EGFP screening expression cassette gene fragment, which was named p72-EGFP-SV40polyA, and the expression cassette sequence contained the SV40polyA termination sequence; 2) mCherry screening expression cassette construction: using pcDNA3-mCherry vector as Template, amplify the red fluorescent protein (mCherry) gene, and at the same time the artificially synthesized nopaline synthase (Nopaline synthase, NOS) termination sequence (Genbank accession number is AB697057.1), that is, 1870-2192nt, as the p72 promoter The transcription termination sequence is named NOSpolyA sequence, and the p72 promoter, mCherry gene sequence, and NOSpolyA sequence are connected by fusion PCR to obtain the mCherry screening expression cassette gene fragment, which is named p72-mCherry-NOSpolyA.

1.6 Construction of homologous recombination transfer vector

Using the pUC19 vector as the backbone vector, the homologous recombination transfer vectors for gene cluster knockout of EP402R (CD2v) and MGF360-12L~MGF360-14L were respectively constructed. The specific steps are as follows: design 1.2 kb of the upstream and downstream sequences of the CD2v gene and 1.5 kb on both sides of the MGF360-12L ~ MGF360-14L gene cluster as homologous recombination arms, and clone them into the pUC19 vector respectively. The p72-EGFP-SV40polyA screening marker gene expression cassette gene fragment was inserted in the middle of the right arm gene sequence. After the sequencing was correct, the homologous recombination transfer vector was named pCD2vLR-EGFP; the p72-mCherry-NOSpolyA gene fragment was inserted between the left and right arm sequences of the recombination transfer vector of the MGF360-12L~MGF360-14L gene cluster as a screening marker gene expression cassette. After the sequencing was correct, the homologous recombination transfer vector was named pMGF3LR-mCherry. DNA was extracted with an endotoxin-free plasmid extraction kit, the concentration was measured, and stored at -20°C for later use. The deletion positions selected by the recombination strategy are shown in Figure 1. As shown in Figure 1, the deleted CD2v gene (i.e. EP402R gene) is the nucleic acid sequence (SEQ ID NO.5) located at 73369-74451 in the whole gene sequence, its amino acid sequence is shown in SEQ ID NO.1, the three missing A multi-gene family gene MGF360-12L, MGF360-13L, MGF360-14L, wherein the MGF360-12L gene is a nucleic acid sequence (SEQ ID NO.6) located at 29382-30434 in the whole gene sequence, and its amino acid sequence is as SEQ ID NO. 2. The MGF360-13L gene is a nucleic acid sequence (SEQ ID NO.7) located at 30595-31656, its amino acid sequence is as shown in SEQ ID NO.3, and the MGF360-14L gene is a nucleic acid sequence located at 31840-32913 (SEQ ID NO.8 ), its amino acid sequence is as SEQ ID NO.4.

1.7 Cell transfection and recombinant virus screening

Whole cell transfection and recombinant virus selection are performed in two steps.

The first step is the construction of CD2v gene knockout virus. Homologous recombination transfer plasmids pCD2vLR-EGFP (2 μg), pX335ΔN-CD2vL (1 μg) and pX335ΔN-CD2vR (1 μg) with 12 μL -Macrophage DNA transfection reagent was mixed well, and co-transfected into porcine BMDM cells (the number of cells was about 10 ⁶ cells/well). After 6 hours of transfection, directly infect the SY18 purified virus strain (according to the infection amount of 1moi), without changing the medium , to 48 hours after infection, observe the number of fluorescent cells under a fluorescent microscope, digest the cells and pick all the fluorescent cells in a single well, blow them carefully in a new culture dish, settle for 1 hour, pick a single fluorescent cell, and collect Freezing and thawing was repeated 3 times, followed by 10 rounds of limited dilution on the PAM cell monolayer, expanded culture, purity test, target gene PCR, marker gene expression and insertion sequence determination to confirm that the purified ASFV CD2v gene knockout virus was obtained, and detected The primers are CD2v-check-F: TGATATAAATGGA GTATCATGGA (SEQ ID NO. 23), CD2v-check-R: AGGACATGGTTTAGGTGGAGGAC (SEQ ID NO. 24). The recombinant virus obtained in the first step was named SY18ΔC.

The second step is the construction of MGF360 family 3 gene knockout virus. Homologous recombination transfer plasmids pMGF3LR-mCherry (2 μg), pX335ΔN-12LL (1 μg), pX335ΔN-14LR (1 μg) with 12 μL -Macrophage DNA transfection reagent was well mixed, and co-transfected into porcine BMDM cells (the number of cells was about 10 ⁶ /well), and 6 hours after transfection, SY18ΔC virus (1moi) was inoculated. 48h after infection, the specific red fluorescent expression of the recombinant virus in the corresponding green fluorescent cells was observed. 2mM EDTA solution digested cells and picked all fluorescent cells in a single well, repeated freezing and thawing 3 times, 10 rounds of limited dilution on PAM cell monolayer, expanded culture, purity test, target gene PCR, marker gene expression and insertion Sequence determination, to determine the purified 4-gene knockout seed virus, detection primers are: MGF360-12L-check-F:TAACCGCCACGTATTCAGGTGTG (SEQ ID NO.25); MGF360-14L-check-R:CAGCTCAGATGCTTCCTTGCCAC (SEQ ID NO .26). The recombinant virus obtained in the second step was named SY18ΔMC4.

2. Experimental results

2.1 CD2v single gene knockout process and purification identification results

Mix pX335ΔN-CD2vL, pX335ΔN-CD2vR and pCD2vLR-EGFP plasmids, and co-transfect BMDM cells. After 6 hours of transfection, change the complete culture medium, inoculate BMDM cells with SY-18 strain at 1moi, and do not change the medium after infection , cultivated to 48h, and photographed under a fluorescent microscope, it can be seen that there are sporadic fluorescence, which is regarded as cells infected by the suspected recombinant virus (Figure 2A). Control the visible light (Figure 2B), pick all the single fluorescent cells in the transfection wells, freeze and thaw repeatedly, inoculate the PAM cells in the pre-plated 96-well plate, observe once every 12 hours, observe the fluorescent cell wells, after marking , continue to observe until 72h, the results show that the proportion of fluorescent cells in some wells can reach 100%. This shows that the construction of the recombinant virus is basically successful (Fig. 3).

Then carry out 4 limited dilution and expansion cultures on the all positive wells, pick the 6th generation recombinant virus wells and digest them into single cells, carefully absorb 23 fluorescent cells, inoculate them in the pre-laid 96-well plate PAM cells, and continue to grow At 72 hours, pick some cells from the fluorescent cell wells, extract genomic DNA, and use the CD2v-check-F/R primer pair for PCR identification. The results show that 11 of all 23 single-cell seeded cell wells still contain wild virus , while no CD2v gene could be detected in 12 wells (Fig. 4). Digest the cells in the No. 7 well, pick 23 single cells, and inoculate them into 96-well PAM cells respectively. After 72 hours of infection, observe the fluorescence-positive cell wells. The PCR identification shows that all the wells are CD2v gene-deleted GFP recombinant virus (Fig. 5). This indicates that the recombinant virus has been purified. Pick the 7th well of the virus wells of the P8 generation, and pass it in the PAM cells of the 6-well plate for three generations, that is, the P9, P10, and P11 generations, respectively, and identify them by PCR. ). In Fig. 6, 1: P9 generation virus; 2: P10 generation virus; 3: P11 generation virus; 4: negative control; 5: SY18 genomic DNA.

2.2 MGF360-12L～14L triple gene knockout process and purification identification results

Use pure ASFV SY18ΔC as the parental strain virus, and continue to delete three consecutive multi-gene family members, MGF360-12L, MGF360-13L, and MGF360-14L, and the steps are the same as CD2v deletion, that is, transfect pX335ΔN in BMDM cells -12LL, pX335ΔN-14LR and pMGF3LR-mCherry three plasmids were used to infect SY18ΔC P10 generation virus purifiers, single cells with both red and green fluorescence in P1 generation were picked and inoculated in PAM cells in 96-well plates, and infected 72h later detection. The results show that some wells can reach almost 100% positive ( FIG. 7 ). In FIG. 7 , A: EGFP fluorescence detection result; B: red fluorescence detection result. Pick 23 single cells from one of the wells in the P2 generation, inoculate new pre-plated PAM cells in a 96-well plate, and use MGF360-12L-check-F, MGF360-14L-check-R primers for PCR detection. The results proved that 16 of them were PCR-negative, and cells in 7 wells were PCR-positive ( FIG. 8 ). Subsequently, 23 single cells were picked in the fifth well infected by the P3 virus, digested and inoculated in a new 96-well plate PAM cells, and the PCR detection results of the primers for detection of CD2v and MGF proved that the P4 generation recombination deleted the virus. , There are still 2 holes with wild poison. Immediately, 23 single cells were picked again from the seventh well of the P4 generation and inoculated into PAM cells in a new 96-well plate. After PCR detection, it was found that only one well of infected cells was positive in PCR detection ( FIG. 9 ). The infected cells of the P5 passage were expanded and cultured in PAM cells in a 6-well plate, and continuously expanded for 3 passages, respectively marked as P6, P7, and P8 passages, and detected by PCR, and the results were all negative (Figure 10). This proves that the SY18ΔMC4 gene deletion virus has been purified. In Fig. 10, 1: P6 generation virus; 2: P7 generation virus; 3: P8 generation virus; 4: negative control; 5: SY18 genomic DNA control. The fluorescence detection results (24hpi) (*20) of ASFVSY18ΔMC4P8 generation recombinant virus are shown in Figure 11. In Figure 11, A. EGFP fluorescence detection results; B. mCherry fluorescence detection results; C. EGFP and mCherry superposition results.

Example 2 Determination of Growth Characteristics of Recombinant Viruses SY18ΔC and SY18ΔMC4 in PAM Cells

1. Determination of virus growth curve

Spread the primary porcine PAM cells into a 6-well plate, grow into a monolayer, inoculate with 0.1moi virus solution, incubate at 37°C for 2h, and harvest at 0h, 6h, 12h, 18h, 24h, 30h, 36h, and 48h after infection The virus was amplified, frozen and thawed three times, and then subpackaged by high-speed centrifugation. The titers of the virus were measured respectively, and the growth curves of the wild virus and the gene knockout virus were drawn.

2. Virus titer titration

Dilute the above-mentioned recombinant virus solution according to a 10-fold gradient, infect the porcine primary PAM cells that have been spread in a 96-well plate to form a monolayer, fix the infected cells with 4% paraformaldehyde solution 72 hours after infection, and add P30 diluted at an appropriate concentration. Monoclonal antibody 1E10 was stained with a fluorescently labeled secondary antibody; the two virus species with gene deletions could be directly observed with a fluorescence microscope, and the titer of the virus was calculated according to the Reed-Muench method, with TCID ₅₀ /mL as the unit.

result:

The three viruses SY18, SY18ΔC, and SY18ΔMC4 were inoculated into PAM cell monolayer with the same amount of virus, and the culture supernatant and cell mixture were harvested at 0h, 6h, 12h, 24h, and 48h after infection, and were repeatedly frozen and thawed for 3 times, and then assayed in PAM cells Viral titer. The results showed that the average titers of the two knockout viruses were slightly lower than those of the SY18 wild virus. At 48 hours after infection, the average titer of SY18ΔC gene deletion virus was 1.679×10 ⁷ TCID ₅₀ /mL, the average titer of SY18ΔMC4 gene deletion virus was 8.472×10 ⁶ TCID ₅₀ /mL, and the average titer of SY18 parent virus was 4.295×10 ⁷ TCID ₅₀ /mL ( FIG. 12 ).

Example 3 Safety Evaluation Experiment of Recombinant Viruses SY18ΔC and SY18ΔMC4

In order to detect whether the above two gene knockout viruses have no pathogenicity at all, this experiment firstly investigated the safety of the recombinant virus on piglets by intramuscular injection at different infection doses (10 ³ , 10 ⁴ , 10 ⁵ TCID ₅₀ ). Evaluation. In this experiment, 30 African swine fever antigen antibody-negative healthy Sanyuan piglets were divided into 6 groups. After immunization, the daily feed intake, body weight and body temperature were measured, and peripheral blood and saliva were collected every 5 days. The blood content of ASFV virus was measured by fluorescent quantitative PCR method, and the observation was terminated after 60 days. The experimental scheme is shown in Table 1.

Table 1 Safety Evaluation

result:

Through the safety evaluation, it was found that when the SY18ΔC strain was inoculated intramuscularly with 10 ³ TCID ₅₀ of the virus, all piglets developed high fever within 3 days, as high as above 42°C, depressed, and began to die in about 4-6 days, and all piglets died within 10 days. die. After oral inoculation with 10 ³ TCID ₅₀ of the virus, all piglets developed high fever and depression 10 days later, and died in about 20-30 days. Necropsy showed severe bleeding in lymph nodes, spleen, kidney and other parts, and the viremia continued until the pig died, and the saliva was positive for detoxification. This shows that the strain has no significant pathogenicity difference from the SY18 parent wild virus.

However, after inoculation with SY18ΔMC4, whether it was low dose (10 ³ TCID ₅₀ ), medium dose (10 ⁴ TCID ₅₀ ) or high dose (10 ⁵ TCID ₅₀ ), there was only a transient rise in body temperature on the 2nd to 3rd day after inoculation. It did not exceed 41.3°C, and then turned to normal body temperature. No abnormal body temperature was found in the later period. During the observation period, the mental state was normal, no abnormality in feed intake was found, and the body weight increased significantly. Autopsy showed no pathological findings. This indicated that the four-gene deletion strain showed good safety to piglets (Fig. 13).

Example 4 Piglet immune efficacy evaluation experiment

In order to clarify whether the deletion virus strain can be used as a vaccine candidate strain, this experiment carried out a preliminary study on the immune efficacy of the recombinant virus. A total of 30 healthy piglets with negative ASFV, PRRSV, PRV, CSFV, PPV, and PCV-2 antigen antibodies were divided into 3 groups: 1) The immunization group was inoculated intramuscularly with the ASFV four-gene knockout virus SY18ΔMC4, and the immune group was inoculated with After 28 days, 10 ³ TCID ₅₀ doses of SY18 virus were inoculated orally, and observation was continued for 35 days. The daily feed intake, body weight and body temperature changes were measured. Finally, all pigs were euthanized under anesthesia, and the lesions of various organs were observed and scored. Fluorescent quantitative PCR method was used to detect the virus copy number in each organ. 2) The challenge group was inoculated with PBS. After 28 days, 10 ³ TCID ₅₀ doses of SY18 virus were orally inoculated, and the observation was continued for 35 days. 3) The normal inoculated PBS control group.

result:

According to the results of the safety experiment, the SY18ΔMC4 attenuated strain was immunized by intramuscular injection, and the dose was 10 ³ TCID ₅₀ / head (group 1). After 28 days of immunization, the SY18 parental virus was used for the challenge test, and the way of challenge was oral administration , the dose was 10 ³ TCID ₅₀ / head, and the observation continued for 35 days. The results showed that all the immunized pigs were fully protected. During the observation period, the body temperature was within the normal range. There were no abnormal clinical manifestations, no viremia, and no detoxification in feces, saliva, lacrimal glands and other parts. Necropsy showed that no obvious pathological changes were found in all immune-protected pigs. The 5 pigs in the challenge control group were all sick, and their body temperature began to rise in about 10 days. After 9 to 11 days, they began to die. After 25 days, they all died; the 5 pigs in the normal control group (group 3) were normal. The above results indicated that intramuscular injection of SY18ΔMC4 had a good challenge protection effect and could be used as a good candidate strain of ASF vaccine.

The above-mentioned embodiments only express the implementation manner of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Sequence List

<110> Shanghai Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences (Shanghai Branch of China Center for Animal Health and Epidemiology)

<120> African swine fever virus four-gene deletion attenuated strain and its application

<160> 26

<170> PatentIn version 3.3

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Met Ile Ile Leu Ile Phe Leu Ile Phe Ser Asn Ile Val Leu Ser Ile

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Asp Tyr Trp Val Ser Phe Asn Lys Thr Ile Ile Leu Asp Ser Asn Ile

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Thr Asn Asp Asn Asn Asp Ile Asn Gly Val Ser Trp Asn Phe Phe Asn

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Asn Ser Phe Asn Thr Leu Ala Thr Cys Gly Lys Ala Gly Asn Phe Cys

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Val Val Trp Asn Gln Ile Ile Asn Tyr Thr Ile Lys Leu Leu Thr Pro

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Ala Thr Pro Pro Asn Ile Thr Tyr Asn Cys Thr Asn Phe Leu Ile Thr

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Cys Lys Lys Asn Asn Gly Thr Asn Thr Asn Ile Tyr Leu Asn Ile Asn

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Asp Thr Phe Val Lys Tyr Thr Asn Glu Ser Ile Leu Glu Tyr Asn Trp

145 150 155 160

Asn Asn Ser Asn Ile Asn Asn Phe Thr Ala Thr Cys Ile Ile Asn Asn

165 170 175

Thr Ile Ser Thr Ser Asn Glu Thr Thr Leu Ile Asn Cys Thr Tyr Leu

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Thr Leu Ser Ser Asn Tyr Phe Tyr Thr Phe Phe Lys Leu Tyr Tyr Ile

195 200 205

Pro Leu Ser Ile Ile Ile Ile Gly Ile Thr Ile Ser Ile Leu Leu Ile Ser

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Ile Ile Thr Phe Leu Ser Leu Arg Lys Arg Lys Lys His Val Glu Glu

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Ile Glu Ser Pro Pro Pro Glu Ser Asn Glu Glu Glu Gln Cys Gln His

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Asp Asp Thr Thr Ser Ile His Glu Pro Ser Pro Arg Glu Pro Leu Leu

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Pro Lys Pro Tyr Ser Arg Tyr Gln Tyr Asn Thr Pro Ile Tyr Tyr Met

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Arg Pro Ser Thr Gln Pro Leu Asn Pro Phe Pro Leu Pro Lys Pro Cys

290 295 300

Pro Pro Pro Lys Pro Cys Pro Pro Pro Lys Pro Cys Pro Pro Pro Lys

305 310 315 320

Pro Cys Pro Ser Ala Glu Ser Tyr Ser Pro Pro Lys Pro Leu Pro Ser

325 330 335

Ile Pro Leu Leu Pro Asn Ile Pro Pro Leu Ser Thr Gln Asn Ile Ser

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Leu Ile His Val Asp Arg Ile Ile

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Trp His Asp Ala Pro Ile Thr Phe Asp His Asn Leu Arg Leu Ile Lys

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Ser Ala Gly Ile Lys Glu Gly Leu Asn Leu Asn Thr Ala Leu Val Lys

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Ala Val Arg Glu Asn Asn Tyr Asn Leu Ile Lys Leu Phe Ala Glu Trp

65 70 75 80

Gly Ala Asp Ile Asn Tyr Gly Leu Val Ser Val Asn Thr Glu His Thr

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Trp Asp Leu Cys Arg Glu Leu Gly Ala Lys Glu Thr Leu Asn Glu Glu Glu

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Glu Ile Leu Gln Ile Phe Ile Asp Leu Lys Phe His Lys Thr Ser Ser

115 120 125

Asn Ile Ile Leu Cys His Glu Val Phe Ser Asn Asn Asn Pro Ile Leu Gln

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Lys Val Asn Asn Ile Lys Met Arg Ile Glu Ile Phe Trp Glu Leu Arg

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Glu Leu Ile Val Lys Thr Asp Leu Leu Asn Asn Glu Phe Ser Leu Ser

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Thr Leu Leu Leu Lys Tyr Trp Tyr Ala Ile Ala Ile Arg Tyr Asn Leu

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Lys Glu Ala Ile Gln Tyr Phe Tyr Gln Lys Tyr Thr His Leu Asn Thr

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Trp Arg Leu Thr Cys Ala Leu Cys Phe Asn Asn Val Phe Asp Leu His

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Tyr Tyr Asn Ile Glu Asn Met Phe Phe Cys Ile Asp Leu Gly Ala Asp

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Val Phe Glu Glu Gly Thr Thr Ala Leu Gly Glu Gly Tyr Glu Leu Ile

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Ser Gln Cys Leu Ser Ile Asp Glu His Cys Ile Leu Lys Tyr Cys Gly

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Leu Trp Trp His Asp Ala Pro Leu Lys Leu Cys Met Asp Arg Gly Arg

35 40 45

Ile Gln Ile Lys Ser Gly Phe Leu Gly Glu Asp Ile Asp Leu Arg Val

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Ala Leu Ile Ile Ala Val Lys Glu Asn Asn Tyr Ser Leu Ile Lys Leu

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Phe Thr Glu Trp Gly Ala Asn Ile Asn Tyr Gly Leu Leu Ser Ile Asn

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Thr Lys His Ile Arg Glu Leu Cys Arg Gln Leu Gly Ala Lys Glu Thr

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Leu Glu Asp Asn Asp Ile Phe Arg Ile Phe Thr Arg Ile Met His Asn

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Lys Thr Ser Gly Ser Ile Ile Leu Cys His Glu Ile Phe Met Asn Asn

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Pro Ile Leu Glu Asn Lys Phe Val Ile Gln Leu Arg Gly Leu Ile Tyr

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Lys Asp Ala Ile Cys Phe Leu Asp Glu Lys Tyr Thr Asp Leu Asn Glu

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Trp Arg Leu Lys Cys Leu Leu Tyr Tyr Asn Lys Ile Tyr Glu Leu His

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Glu Met Tyr His Lys Glu Asn Ile Gln Ile Asp Val His Asp Met Ile

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Cys Leu Ala Ser Thr Lys Asp Asn Asn Pro Leu Thr Ile Tyr Tyr Cys

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Tyr Ala Leu Gly Gly Asn Ile Asn Gln Ala Met Leu Thr Ser Val Gln

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Tyr Tyr Asn Ile Gly Asn Ile Phe Phe Cys Ile Asp Leu Gly Gly Asn

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Ala Phe Glu Glu Gly Arg Ala Ile Ala Glu Gln Lys Gly Tyr Asn Phe

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Leu Ser His Ser Leu Ala Leu Asp Ile Tyr Ser Ser Asp Ala Ser Leu

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Leu Ser Ser Asp Tyr Asp Tyr Thr Leu Gln Arg Phe Gly Leu Trp Trp

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Asp Leu Gly Pro Ile His Leu Cys Asn Asn Cys Lys Gln Val Phe Ser

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Tyr Lys His Leu Gln Cys Phe Ser Glu Asp Asp Leu Cys Leu Glu Ala

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Ala Leu Val Lys Ala Val Lys Ser Asp Asn Leu Glu Leu Ile Arg Leu

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Phe Val Asp Trp Gly Ala Asn Pro Glu Tyr Gly Leu Ile Arg Val Pro

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Ala Val Tyr Leu Lys Arg Leu Cys Ala Glu Leu Gly Gly Leu Thr Pro

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Val Ser Glu Pro Arg Leu Leu Glu Ile Leu Lys Glu Val Ala Arg Leu

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Lys Ser Cys Ala Gly Val Leu Leu Gly Tyr Asp Met Phe Cys His Asn

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Pro Leu Leu Glu Thr Val Thr Arg Thr Thr Leu Asp Thr Val Thr Tyr

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Thr Cys Ser Asn Ile Pro Leu Thr Gly Asp Thr Ala His His Leu Leu

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Thr Lys Phe Trp Phe Ala Leu Ala Leu Arg His Asn Phe Thr Lys Ala

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Ile His Tyr Phe Tyr Lys Arg His Lys Asn His Leu Tyr Trp Arg Val

195 200 205

Ala Cys Ser Leu Tyr Phe Asn Asn Ile Phe Asp Ile His Glu Leu Cys

210 215 220

Arg Glu Lys Glu Ile Cys Ile Ser Pro Asn Leu Met Met Lys Phe Ala

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Cys Leu Arg Glu Lys Asn Tyr Ala Ala Ile Tyr Tyr Cys His Arg Leu

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Gly Ala Ser Leu Asp Tyr Gly Met Asn Leu Ser Ile Tyr Asn Asn Asn

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Thr Leu Asn Met Phe Phe Cys Ile Asp Leu Gly Ala Ala Asp Phe Asp

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Arg Ala Gln Leu Ile Ala His Lys Ala Tyr Met Tyr Asn Leu Ser Asn

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Ile Phe Leu Val Lys Gln Leu Phe Ser Arg Asp Val Thr Leu Val Leu

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Ser Lys Asn Leu Lys Arg Ala Glu Glu Tyr Leu Thr Ala His Pro Glu

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Ile Ile Val Ile Asp

355

<210> 5

<211> 1083

<212>DNA

<213> African swine fever virus

<400> 5

atgataatac ttattttttt aatattttct aacatagttt taagtattga ttatgggtt 60

agttttaata aaacaataat tttagatagt aatattacta atgataataa tgatataaat 120

ggagtatcat ggaatttttt taataattct tttaatacac tagctacatg tggaaaagca 180

ggtaactttt gtgaatgttc taattatagt acatcaatat ataataac aaataattgt 240

agcttaacta tttttcctca taatgatgta tttgatacaa catatcaagt agtatggaat 300

caaataatta attatacaat aaaattatta acacctgcta ctcccccaaa tatcacatat 360

aattgtacta attttttaat aacatgtaaa aaaaataatg gaacaaacac taatatatat 420

ttaaatataa atgatacttt tgttaaatat actaatgaaa gtatacttga atataactgg 480

aataatagta acattaacaa ttttacagct acatgtataa ttaataatac aattagtaca 540

tctaatgaaa caacacttat aaattgtact tattaacat tgtcatctaa ctatttttat 600

acttttttta aattatatta tattccatta agcatcataa ttgggataac aataagtatt 660

cttcttatat ccatcataac ttttttatct ttacgaaaaa gaaaaaaaca tgttgaagaa 720

atagaaagtc caccacctga atctaatgaa gaagaacaat gtcagcatga tgacaccact 780

tccatacatg aaccatctcc cagagaacca ttacttccta agccttacag tcgttatcag 840

tataatacac ctatttacta catgcgtccc tcaacacaac cactcaaccc atttccctta 900

cctaaaccgt gtcctccacc caaaccatgt ccgccaccca aaccatgtcc tccacctaaa 960

ccatgtcctt cagctgaatc ctattctcca cccaaaccac tacctagtat cccgctacta 1020

cccaatatcc cgccattatc tacccaaaat atttcgctta ttcacgtaga tagaattatt 1080

taa 1083

<210> 6

<211> 1053

<212>DNA

<213> African swine fever virus

<400> 6

atgttgcctt ccctgcaatc tctgaccaaa aaggtgttgg ctggacaatg cgtgcccacg 60

aaccaacatt atcttttaaa gtgttatgac ctatggtggc atgatgctcc gatcacgttt 120

gatcataacc taaggctcat aaaatcagca ggtattaaag agggcttaaa cctaaatacg 180

gcgttagtga aggctgtaag ggaaaacaac tacaacctta taaaactgtt tgcagagtgg 240

ggggcggaca tcaactacgg gctggtatct gtcaacacgg agcacacctg ggacctctgt 300

cgagagctag gtgccaagga aaccttgaat gaagaagaaa ttttacaaat ttttatagat 360

ctaaagtttc ataaaactag tagtaacatt attttatgcc atgaggtgtt ttccaacaat 420

ccgatattac aaaaagtaaa taatataaaa atgaggatag aaattttctg ggagttaagg 480

gagttaatag taaaaaccga tctgctaaat aatgagtttt cgctcagtac attackactc 540

aaatactggt acgctatagc catacgctat aacctgaaag aggccataca gtatttttac 600

caaaaatata cacacctgaa tacgtggcgg ttaacatgtg ctctttgttt taataatgtg 660

tttgaccttc atgaggcgta tgaaaaggac aagatccata tggacataga agagatgatg 720

cggatcgcct gcatcaaaga ccacaacctt tcaaccatgt actactgcta tgtcctgggc 780

gccaacatca atcaagccat gcttagctca atacagtact ataatataga aaacatgttc 840

ttttgtatag atctgggggc tgatgttttc gaagaggta ctacagcttt agggggaaggg 900

tatgagctta taaagaacat tttatcccta aagattata gtccggccac caccccgttg 960

cctaaaagca cggaccctga aatcatagat catgcgttaa aaaattacgt ttcaaaaaat 1020

atgatgatct tccttaccta tgatttaaga tga 1053

<210> 7

<211> 1062

<212>DNA

<213> African swine fever virus

<400> 7

atgtcgttgc cgctctctct gcagaccctc gtcaaaaaga cgatagccag ccagtgtttg 60

tcaatagatg aacactgcat tttgaaatat tgtggcctat ggtggcatga tgctcctctc 120

aagctttgta tggatcgtgg ccgaatacaa ataaaatcag gatttttagg agaagatata 180

gaccttcgtg tggcattaat aatagctgtt aaggaaaaca actatagtct gataaagctc 240

tttacagagt ggggcgcaaa tatcaactat ggtttgcttt ctatcaatac gaagcacatc 300

cgagagttgt gtagacagct aggcgccaaa gaaactctag aggacaacga tattttccgt 360

atttttacca ggataatgca caataaaacc agcggcagta ttattttgtg ccatgaaatt 420

tttatgaata atcctatttt agaaaacaaa tttgttatac aattaagggg cttaatttt 480

aaaagactat gggggctcat agaaataaaa gaaacggacg agttaaatgg tttactagtg 540

aagtattggt acgccaaagc agtacaatac gattgtaagg acgccatttg ttttctagat 600

gagaaatata cggatcttaa tgaatggcga ttaaaatgtc tcctgtatta taacaaaata 660

tatgagcttc atgagatgta ccacaaggaa aacatccaaa tagacgtcca tgacatgata 720

tgtctggctt ctaccaagga taacaatcca ttaacaatat attackgtta cgcgctgggg 780

ggcaacatca accaagctat gcttacttca gtacaatatt ataacatcgg taatatattt 840

ttctgtatag atttgggtgg taatgccttt gaagagggtc gtgccatagc ggaacaaaaa 900

ggttataatt ttctgagcca tagtttggct ttggatattt acagctcaga tgcttccttg 960

ccactaaact taaaaggaccc cgaagaaata agcagtttat taaaagatta taaatcaaaa 1020

aacttatcca tcatttggga atattctcat aatatactat ag 1062

<210> 8

<211> 1074

<212>DNA

<213> African swine fever virus

<400> 8

atgttgtctt tacaaacgtt ggccaaaaaa gttgtggcat gcaattatct ttcaagtgac 60

tatgattata cgttgcagcg ttttggtttg tggtgggatt taggtcctat tcacctatgt 120

aacaattgta agcaagtttt ttcgtataaa catttacagt gtttttctga ggatgatctt 180

tgtctcgaag cggcgctagt aaaggccgtg aagagcgata atcttgaact tatacgttta 240

tttgtggatt ggggcgcaaa tcctgaatat gggcttatac gtgttcctgc cgtgtatcta 300

aagcggctgt gtgcggaact gggaggctta acgcctgtat ccgaaccccg tcttctggaa 360

attttaaaag aagtggccag gctaaaatcc tgtgcaggag ttctgctggg ttatgacatg 420

ttttgtcata atccactctt ggaaaccgta actagaacca ctttagacac agttacgtac 480

acctgttcaa acattccgtt gacgggggat acggcgcacc acctattaac aaagttttgg 540

tttgccctgg cattacgaca taattttaca aaggctattc actatttcta taaaaggcat 600

aaaaatcacc tctattggcg ggtagcttgt agcctttatt ttaataacat ttttgacata 660

cacgagttgt gtcgtgaaaa agagatttgc atcagcccta atctgatgat gaaatttgct 720

tgcttgcggg aaaaaaatta cgcggccatt tattactgtc ataggttggg ggctagtctc 780

gattatggca tgaatctttc tatctataac aataatactt taaacatgtt tttctgtatt 840

gatttggggg ctgccgattt tgatagggca caactcattg cgcacaaagc ttatatgtat 900

aacttgagca aattttctt agtaaagcag cttttcagcc gtgatgtgac cttggtatta 960

gatgtaaccg aaccccagga aatatatgat atgctaaaga catatacttc aaaaaatctg 1020

aaacgagccg aagagtatct tacagcccat ccagaaatta tagttataga ctaa 1074

<210> 9

<211> 30

<212>DNA

<213> Artificial sequence (Artificial)

<400> 9

cgggatccat ggatcacaat accgaagaaa 30

<210> 10

<211> 28

<212>DNA

<213> Artificial sequence (Artificial)

<400> 10

cggaatctgc tcctcgtccg tcccggtc 28

<210> 11

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 11

caccgctagc tacatgtgga aaagc 25

<210> 12

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 12

aaacgctttt ccacatgtag ctagc 25

<210> 13

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 13

caccgttggg tagtagcggg atact 25

<210> 14

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 14

aaacagtatc ccgctactac ccaac 25

<210> 15

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 15

caccgctaat accaaggtca catca 25

<210> 16

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 16

aaactgatgt gaccttggta ttagc 25

<210> 17

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 17

caccgggatg atctttgtct cgaag 25

<210> 18

<211> 25

<212>DNA

<213> Artificial sequence (Artificial)

<400> 18

aaaccttcga gacaaagatc atccc 25

<210> 19

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 19

tgatataaat ggagtatcat gga 23

<210> 20

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 20

aggacatggt ttaggtggag gac 23

<210> 21

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 21

taaccgccac gtattcaggt gtg 23

<210> 22

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 22

cagctcagat gcttccttgc cac 23

<210> 23

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 23

tgatataaat ggagtatcat gga 23

<210> 24

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 24

aggacatggt ttaggtggag gac 23

<210> 25

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 25

taaccgccac gtattcaggt gtg 23

<210> 26

<211> 23

<212>DNA

<213> Artificial sequence (Artificial)

<400> 26

cagctcagat gcttccttgc cac 23

Claims (10)

1. A four-gene-deleted low virulent strain of African swine fever virus is a gene-deleted low virulent strain of a SY18 isolate of the African swine fever virus, and the four-gene-deleted low virulent strain is deleted with the following functional genes:

CD2v gene and three polygene family genes MGF360-12L, MGF360-13L, MGF 360-14L.

2. The African swine fever virus four-gene-deleted attenuated strain of claim 1, wherein the deleted gene sequence comprises: a nucleotide sequence for coding the amino acid sequence of the CD2v protein shown in SEQ ID NO.1, a nucleotide sequence for coding the amino acid sequence of the MGF360-12L protein shown in SEQ ID NO.2, a nucleotide sequence for coding the amino acid sequence of the MGF360-13L protein shown in SEQ ID NO.3 and a nucleotide sequence for coding the amino acid sequence of the MGF360-14L protein shown in SEQ ID NO. 4.

3. The African swine fever virus four-gene-deleted attenuated strain of claim 2, wherein the deleted gene sequence comprises: the nucleotide sequence of the CD2v gene shown in SEQ ID NO.5, the nucleotide sequence of the MGF360-12L gene shown in SEQ ID NO.6, the nucleotide sequence of the MGF360-13L gene shown in SEQ ID NO.7 and the nucleotide sequence of the MGF360-14L gene shown in SEQ ID NO. 8.

4. A recombinant virus is characterized in that the recombinant virus is a recombinant virus with a CD2v gene deleted in a SY18 isolate of African swine fever virus.

5. The recombinant virus of claim 4, wherein the recombinant virus further lacks the following three polygene family genes: MGF360-12L, MGF360-13L, MGF 360-14L.

6. Use of the African swine fever virus four-gene deletion attenuated strain of any one of claims 1 to 3 in the preparation of a vaccine for preventing or treating African swine fever.

7. A vaccine comprising the african swine fever virus four-gene-deleted low virulent strain of any one of claims 1 to 3.

8. the vaccine of claim 7, wherein the vaccine is suitable for oral administration, or intramuscular injection.

9. a test kit for differentiating infection by the african swine fever virus attenuated strain with the four-gene deletion attenuated strain of claim 1, comprising: the primer pair is designed aiming at least one gene of four genes of CD2v and MGF360-12L, MGF360-13L, MGF360-14L of an African swine fever virus SY18 isolate.

10. A test kit for differentiating infection by the african swine fever virus attenuated strain with the four-gene deletion attenuated strain of claim 1, comprising: antibody of protein expressed by at least one gene of CD2v, MGF360-12L, MGF360-13L, MGF360-14L of SY18 isolate of African swine fever virus.