Recombinant African swine fever attenuated vaccine as well as preparation method and application thereof

Abstract

The invention discloses a recombinant African swine fever attenuated vaccine as well as a preparation method and application thereof, and relates to the technical field of biology, the vaccine comprises p54 protein deleted by an apoptosis-promoting motif, the p54 protein is encoded by an E183L gene, and the nucleotide sequence is shown as SEQ ID NO.1; the deleted pro-apoptosis motif is the nucleotide sequence of the whole genome sequence 162311 to 162355 and the nucleotide sequence 162251 to 162355 of the ASFV GZ isolated strain; the preparation method comprises the following steps: constructing a homologous recombinant vector and recombinant viruses ASFV-p54 DBM and ASFV-p54 CTM; the vaccine is applied to prevention of African swine fever. According to the invention, a key motif for p54 protein to exert a p54 apoptosis promoting function is screened out, and the recombinant ASFV obtained by knocking out the p54 protein apoptosis promoting motif is constructed, so that the pathogenicity of the recombinant ASFV to domestic pigs is obviously reduced, the host immune response can be promoted, and a high-level antibody can be induced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a recombinant African swine fever attenuated vaccine and a preparation method and application thereof. Background Art

[0002] African swine fever (ASF) is a highly contagious and fatal infectious disease caused by the African swine fever virus (ASFV), with a mortality rate of up to 100% for domestic pigs. To date, ASF continues to occur worldwide, posing a threat not only to the global pig industry, but also to human public health and safety. There is currently no effective vaccine to prevent and treat the disease. For the prevention and control of ASF, the main measures currently adopted are culling and tooth extraction.

[0003] ASFV has a complex structure and is the only DNA arbovirus. It is a member of the genus ASFV of the family ASFVidae and is an enveloped double-stranded DNA virus. ASFV has an icosahedral symmetric structure with a diameter of 175-215nm. It can encode more than 54 structural proteins and has a complex immune escape mechanism. The genome structure is similar to that of poxviruses. It is a single-molecule linear double-stranded DNA with covalently closed ends, 170-190kb in size, and the terminal bases are connected to each other. ASFV mainly infects reticuloendothelial cells and mononuclear macrophages in various tissues. After animals are infected with highly virulent strains, their tissues show extensive damage; after being infected with low-virulence strains, the scope of infected tissues and damage to tissues are relatively mild. Studies have shown that genes that affect the virulence of ASFV are also the main factors causing cytopathic effects. Several genes of ASFV are related to the pathogenicity of domestic pigs, but do not affect the replication of the virus in macrophages, such as DP96R and DP71L. ASFV infection also affects and regulates the host immune response. The virus manipulates the cell death pathway to provide powerful conditions for its own replication, including apoptosis, pyroptosis, and necroptosis. Apoptosis, as a programmed cell death mechanism, plays a vital role in clearing virus-infected cells and maintaining tissue homeostasis. A large number of studies have shown that ASFV infection significantly induces apoptosis of immune cells, which plays a pivotal role in the pathogenesis of ASF. Previous studies have shown that in the early stages of viral infection, the virus tends to inhibit apoptosis to establish a microenvironment that is conducive to its replication. As the virus infection enters the late stage, virus-induced apoptosis produces a large number of apoptotic bodies carrying non-enveloped virus particles; once engulfed by neighboring cells, these ASFVs can cleverly evade host immune surveillance in a "Trojan horse" manner, triggering a new round of infection.

[0004] The p54 protein is present in the inner envelope of the virus particle and is an important structural protein of ASFV. It is encoded by the E183L gene, has a molecular mass of approximately 25 kDa, and contains a transmembrane region, which is mainly concentrated in the derived endoplasmic reticulum. The transmembrane structure of the p54 protein plays an important role in the conversion of viral proteins into viral membrane precursors through the endoplasmic reticulum. Previous studies have shown that the carboxyl terminal region of the p54 protein contains a binding motif for the LC8 chain of the dynein protein, which is involved in the induction of cell apoptosis. However, there is currently no direct evidence to prove the role of the p54 protein in apoptosis during ASFV infection.

[0005] Therefore, technicians in this field are committed to developing a recombinant African swine fever attenuated vaccine lacking the pro-apoptotic motif of the p54 protein, as well as its preparation method and application. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to develop a recombinant African swine fever attenuated vaccine lacking the pro-apoptotic motif of the p54 protein, and a preparation method and application thereof.

[0007] To achieve the above-mentioned purpose, the present invention provides a recombinant African swine fever attenuated vaccine, comprising a p54 protein lacking a pro-apoptotic motif, encoded by the E183L gene, and the nucleotide sequence is shown in SEQ ID NO.1; the lacking pro-apoptotic motif is the nucleotide sequence 162311-162355 and 162251-162355 of the whole genome sequence of the ASFV GZ isolate, and the nucleotide sequence of the p54 protein lacking a pro-apoptotic motif is shown in SEQ ID NO.4.

[0008] The present invention also provides a method for preparing a recombinant African swine fever attenuated vaccine, comprising the following steps:

[0009] Step 1. Construct homologous recombination vector: Use pUC19 vector as the backbone vector to construct homologous recombination transfer vector for knocking out amino acids 150-164 and 150-184 of p54 protein, and obtain homologous recombination vector plasmids p72GFP-p54△DBM (p54△150-164) and p72GFP-p54△CTM (p54△150-184).

[0010] Step 2, construct recombinant ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses: transfect the homologous recombinant vector plasmid p72GFP-p54△DBM or p72GFP-p54△CTM obtained in step 1 into PAM cells, and then infect ASFV GZ virus; then obtain ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses by continuous EGFP-positive single cell purification.

[0011] Furthermore, step 1 also includes: the p54 protein is encoded by the E183L gene, the nucleotide sequence of the E183L gene is shown in SEQ ID NO.1, and the knocked-out E183L gene sequence is the nucleotide sequence 162311-162355 and 162251-162355 of the whole genome sequence of the ASFV GZ isolate.

[0012] Furthermore, step 1 also includes: cloning 1200 bp of the upstream and downstream sequences of the gene fragment of amino acids 150-164 or 150-184 of the p54 protein as homologous recombination homology arms, ASFVp72 promoter and PCR fragments of the GFP gene into the pUC19 vector through a fusion PCR kit, wherein the GFP gene is expressed under the control of the ASFVp72 late promoter.

[0013] Furthermore, the homologous recombination homology arm includes a left arm and a right arm, the nucleotide sequence of the left arm is shown in SEQ ID NO.2; the nucleotide sequence of the right arm is shown in SEQ ID NO.3.

[0014] Furthermore, step 2 also includes:

[0015] 2 μg of p72GFP-p54△DBM or p72GFP-p54△CTM was transfected into PAM cells, and then 6 h after transfection, ASFV GZ virus was infected at a dose of 1 MOI; 24 h after infection, cells expressing EGFP were selected from the monolayer of PAM cells and rinsed with sterile PBS, and then inoculated into new PAM cells, and then ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses were obtained by continuous EGFP-positive single cell purification.

[0016] Furthermore, the transfection kit used was -macrophage.

[0017] Furthermore, step 2 also includes evaluating and identifying the purity of the recombinant virus by conventional PCR procedures, using primers p54-check-F and p54-check-R; the p54-check-F sequence is: 5'-GTTTTGATGCATTTAGATACCA-3'; the p54-check-R sequence is: 5'-CAACATACTGTTGTATATTATAAG-3'.

[0018] Furthermore, the full-length sequence of ASFV GZ virus is shown in GenBank accession number: MT496893.1.

[0019] The present invention also provides the use of the recombinant African swine fever attenuated vaccine in preventing African swine fever.

[0020] In the preferred embodiment 1 of the present invention, the preparation process of the recombinant African swine fever attenuated vaccine is described in detail;

[0021] In another preferred embodiment 2 of the present invention, the identification process of the interaction between the ASFV p54 protein and the host factor is described in detail;

[0022] In another preferred embodiment 3 of the present invention, the effect of the interaction between ASFV p54 and SCAMP3 on cell apoptosis is described in detail;

[0023] In another preferred embodiment 4 of the present invention, the key motif identification process of ASFVp54 inducing apoptosis is described in detail;

[0024] In another preferred embodiment 5 of the present invention, it is described in detail that the deletion of the pro-apoptotic motif of ASFV p54 significantly reduces the intercellular spread of ASFV through the apoptosome pathway;

[0025] In another preferred embodiment 6 of the present invention, the biological activity analysis process of ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses is described in detail.

[0026] The beneficial technical effects of the present invention are as follows:

[0027] The present invention screened out the host factor SCAMP3 that interacts with the p54 protein during the ASFV infection process through immunoprecipitation-mass spectrometry analysis; and found that the key motif of the p54 protein that plays a pro-apoptotic function is involved in the production of ASFV-induced budding apoptotic bodies, thereby promoting the intercellular transmission of ASFV; by deleting the pro-apoptotic motif of the p54 protein in the ASFV GZ isolate, a recombinant ASFV with a p54 protein pro-apoptotic motif knocked out was constructed, and the pro-apoptotic ability of the ASFV recombinant virus was significantly weakened; and the recombinant ASFV lacking the p54 protein pro-apoptotic motif weakened the interaction with SCAMP3, resulting in the obstruction of the virus transmission through the apoptotic body; animal experimental results show that the recombinant ASFV with a p54 protein pro-apoptotic motif knocked out constructed by the present invention has significantly reduced pathogenicity to domestic pigs, can be used as a recombinant vaccine strain, can promote host immune response, induce high-level antibodies, and effectively resist lethal dose attacks of wild-type strong toxins, and has good application prospects.

[0028] The present invention screens and identifies the virulence targets encoded by ASFV and related to pathogenicity, and constructs a recombinant virus with a deletion of the related virulence gene by genetic engineering, so that it loses its pathogenicity and retains its immunogenicity. A recombinant African swine fever attenuated vaccine based on the deletion of the pro-apoptotic motif of the p54 protein is prepared.

[0029] The present invention identified the pro-apoptotic motif of the p54 protein, and successfully obtained a recombinant ASFV lacking the pro-apoptotic motif of the p54 protein through gene editing technology. By partially deleting the key motif of the p54 protein that plays a pro-apoptotic role, it loses its pro-apoptotic function, but does not affect its participation in the assembly of virus particles. The problem that the ASFV membrane protein p54 cannot be artificially deleted was solved, and a recombinant ASFV lacking the pro-apoptotic motif of the p54 protein was obtained.

[0030] The present invention screened out the p54 protein through immunoprecipitation-mass spectrometry analysis to play a pro-apoptotic function by interacting with SCAMP3. It proved that the host factor SCAMP3 plays an important role in the release of apoptotic bodies. The molecular mechanism of the p54 protein playing a pro-apoptotic function was analyzed, enriching our theoretical understanding of ASFV infection and pathogenicity.

[0031] By deleting the key amino acid segment where the p54 protein interacts with the host factor SCAMP3, the intercellular transmission of the apoptosome pathway is weakened. The recombinant ASFV with weakened intercellular transmission ability of the apoptosome pathway has significantly reduced pathogenicity to domestic pigs and can be used as a recombinant vaccine strain to promote host immune response, induce high levels of antibodies, and effectively resist lethal doses of wild-type virulent attacks. It was found that the p54 protein interacts with the SCAMP3 protein in the host cell through the 150-184aa motif of the extracellular domain, participates in the production of ASFV-induced budding apoptosomes, and thus promotes the intercellular transmission of ASFV.

[0032] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of the construction of a recombinant virus with two amino acid segments at the carboxyl terminus of the ASFV p54 protein deleted according to a preferred embodiment 1 of the present invention;

[0034] Figure 2 This is a diagram showing the results of the immunoprecipitation verification of the ASFV p54 protein and the host SCAMP3 protein according to a preferred embodiment 2 of the present invention;

[0035] Figure 3 This is a diagram showing the co-localization verification result of the ASFV p54 protein and the host SCAMP3 protein according to a preferred embodiment 2 of the present invention;

[0036] Figure 4 A preferred embodiment 3 of the present invention is a diagram showing the interaction between ASFV p54 protein and host SCAMP3 protein to promote cell apoptosis;

[0037] Figure 5This is a diagram showing that knocking down SCAMP3 in WSL cells significantly reduces the apoptosis-promoting ability of ASFV p54 protein, according to a preferred embodiment 3 of the present invention;

[0038] Figure 6 This is an amino acid segment diagram of the interaction between ASFV p54 protein and host SCAMP3 protein predicted by Alphafold of a preferred embodiment 4 of the present invention (white: SCAMP3 protein structure prediction; black: p54 protein structure prediction);

[0039] Figure 7 A diagram showing the interaction between the ASFV p54 protein and the host SCAMP3 protein through the amino acid segment 150-184 at the carboxyl terminal of a preferred embodiment 4 of the present invention;

[0040] Figure 8 This is a diagram showing that the apoptosis-promoting ability of p54 protein lacking the 150-184 amino acid segment is significantly reduced according to a preferred embodiment 4 of the present invention;

[0041] Fig. 9 This is a morphological comparison diagram of three ASFV recombinant viruses after infecting PAM cells according to a preferred embodiment 4 of the present invention;

[0042] Fig.10 This is a statistical diagram of the number of apoptotic bodies produced by cells induced by three ASFV recombinant viruses in a preferred embodiment 4 of the present invention;

[0043] Fig.11 This is a diagram showing that the level of Caspase 3 cleavage induced by ASFV-p54△CTM infection of cells is significantly reduced according to a preferred embodiment 4 of the present invention;

[0044] Fig.12 This is a graph of ASFV titer in apoptotic bodies, cell supernatant and cells according to a preferred embodiment 5 of the present invention;

[0045] Fig.13 This is a temperature monitoring chart of domestic pigs inoculated with three ASFV recombinant viruses according to a preferred embodiment 6 of the present invention;

[0046] Fig.14 This is a survival curve diagram of domestic pigs inoculated with three ASFV recombinant viruses according to a preferred embodiment 6 of the present invention;

[0047] Fig.15 This is a diagram showing the results of virus DNA testing in blood, oral, nasal and anal swabs of domestic pigs after inoculation of three ASFV recombinant viruses in a preferred embodiment 6 of the present invention;

[0048] Fig.16This is a diagram showing the results of viral DNA detection in organs of domestic pigs after inoculation of three ASFV recombinant viruses according to a preferred embodiment 6 of the present invention;

[0049] Fig.17 This is a temperature monitoring chart after ASFV-p54△CTM immune challenge according to a preferred embodiment 6 of the present invention;

[0050] Fig.18 This is a survival curve of ASFV-p54△CTM after immune challenge according to a preferred embodiment 6 of the present invention;

[0051] Fig.19 This is a graph of p72 antibody levels after ASFV-p54△CTM immunization according to a preferred embodiment 6 of the present invention. DETAILED DESCRIPTION

[0052] The following describes several preferred embodiments of the present invention with reference to the drawings in the specification, so that the technical content is clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned in the text.

[0053] Example 1 Preparation of recombinant African swine fever attenuated vaccine

[0054] Biosafety Statement: All ASFV infections in this invention were conducted under animal biosafety level 3 (ABSL-3) conditions of Jinyu Baoling Biological Pharmaceutical Co., Ltd. The experiments in the following implementation cases have been accredited and approved by the China National Accreditation Service for Conformity Assessment (CNAS) (CNAS-BL0101) and approved by the Ministry of Agriculture and Rural Affairs.

[0055] Virus: ASFV Chinese epidemic strain (ASFV GZ201801 strain, or ASFV GZ strain), isolated and preserved by South China Agricultural University (for its full-length sequence, see GenBank accession number: MT496893.1).

[0056] Cells: Primary porcine alveolar macrophages (PAM) were isolated from the lungs of 40-day-old piglets, and the specific operation method was as described above. ASFV, classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus type 1 and type 2 (PCV-1 / 2), porcine reproductive and respiratory syndrome (PRRSV) and porcine parvovirus (PPV) were detected according to national standard methods to ensure that the isolated PAM cells were not contaminated with the above viruses. PAM cells were maintained in a medium supplemented with 10% fetal bovine serum (FBS), 1% Pen-Strep and 2mM L-glutamine (Gibco TM, Thermo Fisher Scientific, Waltham, MA, USA) and 10 ng / mL rpGM-CSF (R&D System, Minneapolis, MN, USA) in RPMI 1640 basal medium. HEK-293T cells and WSL cells were purchased from ATCC.

[0057] Plasmids: pCAGGS vector and pUC19 vector were purchased from Shanghai Sangon Biotechnology Co., Ltd.; p54-EGFP, p54-EGFP△150-164 (or p54-EGFP△DBM), p54-EGFP△150-184 (or p54-EGFP△CTM), and pSCAMP3-Flag plasmids were constructed by Shanghai Sangon Biotechnology Co., Ltd.

[0058] Homologous recombination vector construction: pUC19 vector was used as a backbone vector to construct a homologous recombination transfer vector for knocking out amino acids 150-164 and 150-184 of the p54 protein (encoded by the E183L gene). The nucleotide sequence of the E183L gene is shown in SEQ ID NO.1. The knocked-out E183L gene sequence is the nucleotide sequence of 162311-162355 (amino acids 150-164) and 162251-162355 (amino acids 150-184) of the whole genome sequence of the ASFV GZ isolate. The construction strategy is as follows: Figure 1 As shown. 1200 bp of the upstream and downstream sequences of the gene fragment of amino acids 150-164 or 150-184 of the p54 protein were used as homologous recombination homologous arms (the left arm is shown in SEQ ID NO.2; the right arm is shown in SEQ ID NO.3), PCR fragments of four genes including the ASFVp72 promoter and GFP were cloned into the pUC19 vector by a fusion PCR kit, wherein the GFP gene was expressed under the control of the ASFVp72 late promoter, thereby obtaining homologous recombination vector plasmids p72GFP-p54△DBM (p54△150-164) and p72GFP-p54△CTM (p54△150-184).

[0059] Construction of recombinant ASFV-p54△DBM and ASFV-p54△CTM viruses: -macrophage (Polyplus-transfection Inc., Illkirch, France) was used to transfect 2 μg of p72GFP-p54△DBM or p72GFP-p54△CTM into PAM cells, and then 6 h after transfection, ASFV GZ virus was infected at a dose of 1 MOI. 24 h after infection, cells expressing EGFP were selected from the monolayer of PAM cells and rinsed 3 times with sterile PBS, then inoculated into new PAM cells, and then ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses were obtained by continuous EGFP-positive single cell purification. The purity of ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses was evaluated and identified by conventional PCR procedures, and the primer pairs were as follows: p54-check-F: 5'-GTTTTGATGCATTTAGATACCA-3' and p54-check-R: 5'-CAACATACTGTTGTATATTATAAG-3'.

[0060] Example 2 Identification of the interaction between ASFVp54 protein and host factors

[0061] PAM cells were inoculated in a 10 cm cell culture dish, and cell lysate samples were collected for protein immunoprecipitation after infection with 1 MOI of ASFV GZ virus for 72 hours. The detailed steps are as follows: cells were collected and lysed in RIPA cell lysis buffer (Beyotime). After ultrasonic disruption, the cell lysate samples were flipped and incubated overnight at 4 ° C with magnetic beads coupled to anti-p54 monoclonal antibodies. The next day, the magnetic beads were washed 3 times with PBS, and the captured proteins were eluted. 15% SDS-PAGE was used for separation and Coomassie Brilliant Blue (Beyotime) staining. The gel was then excised and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The LC-MS / MS signals were placed in the NCBI protein database for comparison analysis using Mascot Server (Matrix Science) software.

[0062] Mass spectrometry results showed that SCAMP3 protein in host cells was significantly enriched by ASFV p54 protein. To further confirm the interaction between SCAMP3 and p54, we performed co-immunoprecipitation (Co-IP) and indirect immunofluorescence (IFA) experiments. Figure 2 and Figure 3 As shown, it was shown that SCAMP3 interacts with p54.

[0063] Example 3 Effect of the interaction between ASFVp54 and SCAMP3 on cell apoptosis

[0064] WSL cells were plated into a single well of a 6-well plate and transfected with pCAGGS vector, pSCAMP3-Flag plasmid, p54-EGFP plasmid or co-transfected with pSCAMP3-Flag / p54-EGFP plasmid using Lipofectamine 3000 liposome reagent. 48 hours after transfection, the level of cell apoptosis was detected by flow cytometry.

[0065] The results are as follows Figure 4 As shown in Figure 2, cells transfected with pCAGGS vector or pSCAMP3-Flag plasmid did not show obvious apoptosis; however, the apoptosis level of cells co-transfected with pSCAMP3-Flag / p54-EGFP plasmid was significantly higher than that of cells expressing p54 alone. Subsequently, we knocked down SCAMP3 in WSL cells using siRNA and transfected with p54-EGFP plasmid. The results are shown in Figure 2. Figure 5 As shown in the figure, the apoptosis level of cells in the SCAMP3 knockdown group was significantly lower than that in the control group, indicating that the ability of p54 to induce apoptosis depends on the expression level of SCAMP3. The increase in SCAMP3 expression level promotes p54-induced apoptosis, while when the SCAMP3 expression level is reduced, p54-induced apoptosis is reduced. In summary, the interaction between p54 and SCAMP3 is crucial for the pro-apoptotic function of p54 protein.

[0066] Example 4 Identification of key motifs for ASFVp54 inducing cell apoptosis

[0067] Alphafold was used to predict the tertiary structures of p54 and SCAMP3, and through molecular docking, a potential interaction region was found at amino acids 150-184 of the carboxyl terminal amino acids of the p54 protein, such as Figure 6 As shown in the figure, it includes the pro-apoptotic motif (amino acids 150-164) that DLC8 binds to as previously reported in the literature. Therefore, we constructed mutant plasmids with two amino acid segments missing at the carboxyl terminus of p54 protein: p54-EGFP△150-164aa (or p54-EGFP△DBM) and p54-EGFP△150-184aa (or p54-EGFP△CTM). Subsequently, a series of biochemical verifications were performed on cells.

[0068] The results are as follows Figure 7As shown in the figure, there was no significant difference in the ability of p54-EGFP△DBM to interact with SCAMP3 compared with p54-EGFP; however, the interaction between p54-EGFP△CTM and SCAMP3 was significantly weakened. Subsequently, we evaluated the ability of the three p54 plasmids to induce cell apoptosis after transfection of cells. Flow cytometry results showed that the level of cell apoptosis induced by p54-EGFP△CTM transfection was significantly reduced compared with the p54-EGFP and p54-EGFP△DBM transfection groups, as shown in the figure. Figure 8 These results indicate that p54 protein promotes cell apoptosis by interacting with SCAMP3 through the 150-184 amino acid motif at the carboxyl terminal.

[0069] In addition, we constructed mutant recombinant viruses with two amino acids missing at the carboxyl terminus of ASFV p54 protein based on ASFV GZ strain by homologous recombination technology: ASFV-p54△DBM and ASFV-p54△CTM. ASFV GZ isolate and two recombinant ASFV were inoculated into PAM cells at an infection dose of 0.1 MOI, and 24 hours after infection, the cell morphology after virus infection was observed by DIC imaging technology, and the Caspase 3 cleavage level was analyzed by western blot (WB) technology.

[0070] The results are as follows Fig. 9 As shown in the results, all three virus-infected PAM cells showed an apoptotic phenotype, characterized by membrane blebbing and the presence of apoptotic bodies, which is consistent with previous reports (Gao P et al.). However, by statistical analysis of the number of apoptotic cells after virus infection, it was found that the number of apoptotic cells induced by ASFV-p54△CTM was significantly lower than that in the wild-type ASFV GZ infection group and the ASFV-p54△DBM infection group, as shown in Figure 2. Fig.10 As shown in Figure 2, similarly, WB also observed that the level of Caspase 3 cleavage induced by ASFV-p54△CTM infection in cells was significantly lower than that in the other two virus infection groups, such as Fig.11 In summary, the amino acid sequence from position 150 to 184 at the carboxyl terminus of p54 protein is the key segment for p54 to exert its pro-apoptotic function.

[0071] Example 5 Loss of the pro-apoptotic motif in ASFVp54 significantly reduces the intercellular spread of ASFV via the apoptosome pathway

[0072] PAM cells were inoculated with wild-type ASFV, ASFV-p54△DBM or ASFV-p54△CTM at an infection dose of 0.1 MOI. 24 h after infection, the cell supernatant was harvested, the apoptotic bodies were separated and purified by differential centrifugation, and the virus titer in the apoptotic bodies was determined by the Reed-Munch method.

[0073] The results are as follows Fig.12 As shown, the apoptotic bodies produced by PAM cells infected with ASFV-p54△CTM and the virus content in the cell supernatant were significantly lower than those in the wild-type ASFV GZ infection group and the ASFV-p54△DBM infection group; however, the virus content inside the cells was significantly higher than that in the wild-type ASFV GZ infection group and the ASFV-p54△DBM infection group. The above results indicate that ASFV lacking the pro-apoptotic motif of the p54 protein is blocked through the apoptotic body secretion pathway, thereby reducing intercellular transmission.

[0074] Example 6 Biological Activity Analysis of ASFV-p54ΔDBM and ASFV-p54ΔCTM Recombinant Viruses

[0075] 1. Virus Titer Determination

[0076] ASFV titer was determined using 50% tissue culture infectious dose (TCID 50 ) method. The steps are as follows: the virus stock solution was serially diluted 10 times, and 8 wells were inoculated at each dilution, with 0.1 mL per well; five days after inoculation, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 (purchased from Shanghai Bioengineering Co., Ltd.), blocked with 2% BSA for 1 hour, and then incubated with ASFVp30 mAb (stored in this laboratory) at 37°C for 1 hour, and then incubated with goat anti-mouse IgG (H+L) secondary antibody Alexa Fluor 488 (purchased from ThermoFisher, USA) at a dilution of 1:600, and then the infection of each cell well was observed under a Leica fluorescence microscope (Leica Microsystems, Wetzlar, Germany), and the ASFV titer was calculated by the Reed-Muench method.

[0077] 2. Evaluation of the pathogenicity of ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses to domestic pigs

[0078] To evaluate the pathogenicity of ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses to domestic pigs, we introduced 13 Du Da Chang hybrid piglets weighing 10-15 kg from a pig farm that met the biosafety and hygiene standards in Hohhot, Inner Mongolia. Common swine infectious diseases such as ASFV, classical swine fever (CSFV), pseudorabies virus (PRV), porcine circovirus type 1 and type 2 (PCV-1 / 2), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine parvovirus (PPV) were detected using national standard methods. The 13 piglets that were negative for the above pathogens were randomly divided into 3 groups. The first two groups (n = 5) were inoculated with 10 3 TCID 50 The ASFV-p54△DBM or ASFV-p54△CTM recombinant virus was used, and the third group (n=3) was inoculated with 10^3TCID50 of wild-type ASFV GZ strain in the same way. After inoculation, the animal's body temperature was monitored daily and the animal's mental state (eating, drinking, defecation, etc.) was observed. Whole blood, oral swabs, nasal swabs, and anal swabs were collected 1, 3, 5, 7, 9, 11, 13, 15, 21, and 28 days after inoculation to detect viremia and detoxification levels. 28 days after inoculation, all surviving piglets were euthanized by injection of sodium pentobarbital, and an autopsy was performed to collect spleen, liver, lungs, kidneys, tonsils, mesenteric lymph nodes, and inguinal lymph nodes for organ viral load testing. Post-vaccination body temperature monitoring data showed that ( Fig.13 ), piglets inoculated with ASFV GZ strain showed elevated body temperature (>40°C) 2-4 days after infection, followed by ASF-related clinical symptoms, including depression, loss of appetite, cyanosis of the skin, and blood in the stool. These clinical symptoms gradually worsened with prolonged infection time, and all piglets died within 8 days. Fig.14 As shown in the figure, ASFV-p54△DBM piglets began to show elevated body temperature on days 4-6. Fig.15 As shown in the figure, 4 piglets showed varying degrees of depression and decreased food intake. The fever lasted for 5-7 days before gradually returning to normal body temperature. Only 1 piglet died at 13 days old. The survival rate was 80%. Fig.16 However, all piglets inoculated with ASFV-p54ΔCTM had normal body temperatures during the 28-day observation period, as shown in Fig.13 As shown, they did not show any clinical symptoms related to ASF and had a 100% survival rate. Fig.14 As shown. Fig.15As shown in the figure, the results of viremia detection showed that the viremia level of piglets inoculated with ASFV-p54△CTM recombinant virus was significantly lower than that of ASFV GZ and ASFV-p54△DBM inoculation groups, and no viremia was detected 11-28 days after inoculation. The results of oral, nasal and anal shedding tests showed that high levels of shedding were detected in all piglets inoculated with ASFV GZ and ASFV-p54△DBM, while low levels of shedding were detected in piglets inoculated with ASFV-p54△CTM recombinant virus only from 5 to 15 days, and no shedding was detected in all piglets after 15 days. Fig.15 The results of organ virus level detection showed that high viral loads were detected in the organs of all piglets inoculated with ASFV GZ and ASFV-p54△DBM, regardless of whether they died from ASFV infection or were euthanized (10 3.54 Up to 10 5.97 In the piglets inoculated with ASFV-p54△CTM recombinant virus, only one piglet was detected with low levels of viral load in the liver and kidney, and no organ infection was detected in the other four piglets. Fig.16 In summary, the pathogenicity of ASFV-p54△CTM recombinant virus to domestic pigs was significantly reduced.

[0079] 3. Immune challenge experiment

[0080] According to the results of the above pathogenicity evaluation test, we selected the ASFV-p54△CTM recombinant virus as a vaccine to evaluate whether the ASFV-p54△CTM that has lost pathogenicity is immunogenic. Eight Durham-Land hybrid piglets weighing 10-15 kg were introduced and randomly divided into two groups. One group (n=5) was inoculated with 10^3TCID50 of ASFV-p54△CTM recombinant virus by intramuscular injection (IM), and the other group (n=3) was used as a placebo group and injected with 1mL of normal saline in the same way; 21 days after immunization, the same dose was used for booster immunization; 14 days after the second immunization, the two groups of experimental pigs were challenged with ASFV GZ strong toxin, with an attack dose of 10^3TCID50. 3 TCID 50 , 21 days after challenge, all surviving piglets were euthanized by injection of sodium pentobarbital.

[0081] As expected, all experimental piglets survived the 28-day immunization period without any ASFV-related clinical symptoms. After ASFV virulent challenge, piglets in the placebo group showed elevated body temperature (>40°C) 1-3 days after challenge. Fig.17As shown in the figure, clinical symptoms associated with classic ASF subsequently appeared, and all died within 7 days after the virus challenge; however, piglets vaccinated with the ASFV-p54△CTM vaccine only had a short low-grade fever (<41°C) after the virus challenge, which then returned to physiological levels. All piglets were alive and well until 21 days after the virus challenge, as shown in the figure. Fig.18 As shown in Figure 2, the vaccinated piglets were able to resist the attack of the parent strain. Fig.19 As shown, 2 / 5 of the piglets vaccinated with ASFV-p54△CTM had turned positive 14 days after immunization, and high levels of antibodies were detected in all piglets 21-28 days after immunization. The above data show that ASFV-p54△CTM can provide effective protection against strong virus attacks in piglets.

[0082] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A recombinant African swine fever attenuated vaccine, It is characterized in that The vaccine comprises a p54 protein lacking a pro-apoptotic motif, and the nucleotide sequence of the p54 protein lacking a pro-apoptotic motif is shown as SEQ ID NO.

4.

2. A method for preparing a recombinant African swine fever attenuated vaccine, It is characterized in that The method comprises the following steps: Step 1, constructing homologous recombination vector: using pUC19 vector as a backbone vector, constructing homologous recombination transfer vectors for knocking out amino acids 150-164 and 150-184 of p54 protein, and obtaining homologous recombination vector plasmids p72GFP-p54△DBM and p72GFP-p54△CTM; Step 2, construct recombinant ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses: transfect the homologous recombinant vector plasmid p72GFP-p54△DBM or p72GFP-p54△CTM obtained in step 1 into PAM cells, and then infect ASFV GZ virus; then obtain ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses by continuous EGFP-positive single cell purification.

3. The method according to claim 2, It is characterized in that The step 1 also includes: the p54 protein is encoded by the E183L gene, the nucleotide sequence of the E183L gene is shown in SEQ ID NO.1, and the knocked-out E183L gene sequence is the nucleotide sequence 162311-162355 and 162251-162355 of the whole genome sequence of the ASFVGZ isolate.

4. The method according to claim 3, It is characterized in that The knockout also includes: cloning 1200 bp of the upstream and downstream sequences of the gene fragment of amino acids 150-164 or 150-184 of the p54 protein as homologous recombination homologous arms, ASFVp72 promoter and PCR fragments of the GFP gene into the pUC19 vector through a fusion PCR kit, wherein the GFP gene is expressed under the control of the ASFVp72 late promoter.

5. The method according to claim 4, It is characterized in that The homologous recombination homology arm includes a left arm and a right arm, the nucleotide sequence of the left arm is shown as SEQ ID NO.2; the nucleotide sequence of the right arm is shown as SEQ ID NO.

3.

6. The method according to claim 2, It is characterized in that The step 2 also includes: The p72GFP-p54△DBM or p72GFP-p54△CTM is transfected into the PAM cells, and then after transfection, the ASFV GZ virus is infected; after infection, cells expressing EGFP are selected from the monolayer of PAM cells and rinsed with sterile PBS, and then inoculated into new PAM cells, and then the ASFV-p54△DBM and ASFV-p54△CTM recombinant viruses are obtained by continuous purification of the EGFP-positive single cells.

7. The method according to claim 6, It is characterized in that The transfection further comprises transfecting 2 μg of the p72GFP-p54△DBM or p72GFP-p54△CTM into the PAM cells; the transfection time is 6 hours, the infection dose is 1 MOI, and the time after infection is 24 hours.

8. The method according to claim 2, It is characterized in that The step 2 also includes evaluating and identifying the purity of the recombinant virus by conventional PCR procedures, using primers p54-check-F and p54-check-R; the p54-check-F sequence is: 5'-GTTTTGATGCATTTAGATACCA-3'; the p54-check-R sequence is: 5'-CAACATACTGTTGTATATTATAAG-3'.

9. The method according to claim 2, It is characterized in that The full-length sequence of the ASFV GZ virus is shown in GenBank accession number: MT496893.

1.

10. Use of the recombinant African swine fever attenuated vaccine as claimed in claim 1 in preventing African swine fever.

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