African swine fever virus attenuated live vaccine strain and application thereof

Abstract

The invention discloses a genetically engineered virus strain of an African swine fever virus, and in a genome of the genetically engineered virus strain, a D250R gene is an inactivated gene. The microbial preservation number of a parent strain for preparing the genetic engineering virus strain is CCTCC (China Center For Type Culture Collection) NO: V 202318, and the GenBank accession number of a genomic sequence of the strain is OQ504956.1. The invention also discloses a preparation method of the genetically engineered virus strain, a vaccine composition containing the genetically engineered virus strain, a preparation method of the vaccine composition, and application of the genetically engineered virus strain in preparation of a preparation for preventing African swine fever. The genetically engineered virus strain is good in safety and immune effectiveness, and has the potential of preparing clinically applied vaccines.

Description

Technical Field

[0001] The invention belongs to the field of veterinary virology and relates to an attenuated live vaccine strain of African swine fever virus and an application thereof. Background Art

[0002] African swine fever (ASF) is an acute, severe and highly contagious disease caused by African swine fever virus (ASFV), with a mortality rate of nearly 100%. In view of the severity of the harm caused by African swine fever, the World Organization for Animal Health (WOAH) has listed ASF as a notifiable animal disease, and my country has listed it as a Class I animal disease. According to the WOAH animal epidemic report, ASF has broken out in Africa, Asia, America, Europe and Oceania, seriously endangering the development of the global pig industry. As ASFV continues to spread in China, ASFV is also constantly evolving, posing huge challenges to disease prevention and control and vaccine development.

[0003] Since the first report of ASF in 1921, many countries have conducted a large number of vaccine studies, and the results show that gene-deleted attenuated live vaccines and cell-passaged attenuated live vaccines can provide effective protection against homologous strains. By specifically deleting ASFV virulence-related genes, gene-deleted candidate vaccines that can effectively protect pigs from ASFV infection are produced. Although some experimental ASF vaccines are promising, safety still needs to be further improved. The combined deletion of multiple genes may be used to improve safety, but it often affects the replication of the virus in pigs, resulting in reduced protective efficacy. In addition, more importantly, the effect of gene deletion on viral virulence varies with different virus strains, which highlights the importance of identifying and identifying new virulence genes. It is especially important for the development of attenuated live vaccine candidates against new epidemic ASFV strains. The ideal gene-deleted ASFV vaccine strain is one that loses virulence by only deleting one gene, with basically unchanged reproductive capacity and immunogenicity.

[0004] The D250R gene of ASFV (encoding the g5Rp protein) is an early protein of ASFV and the only one known to have decapping enzyme activity. Summary of the invention

[0005] In order to study whether the D250R gene affects the virulence or immunogenicity of the virus, the present invention uses homologous recombination technology to construct the D250R gene deletion strains (HLJ / 18ΔD250R strain and JS / LG / 21ΔD250R strain) of the genotype II African swine fever virulent HLJ / 18 strain and the genotype I / II naturally recombinant virulent JS / LG / 21 strain. The pigs inoculated with the HLJ / 18ΔD250R strain and the JS / LG / 21ΔD250R strain all had normal body temperature and no adverse clinical symptoms, indicating that the virulence was significantly reduced. At the same time, the pigs inoculated with the HLJ / 18ΔD250R strain and the JS / LG / 21ΔD250R strain can produce a high level of ASFV-specific antibody response, and can provide effective protection against the attack of homologous virulent strains.

[0006] The first aspect of the present invention provides a genetically engineered virus strain of African swine fever virus, in the genome of the genetically engineered virus strain, the D250R gene is an inactivated gene;

[0007] The parent used to prepare the genetically engineered virus strain is selected from the following strains:

[0008] The GenBank accession number of the genome sequence is OQ504956.1, and the microbial deposit number is CCTCCNO: V 202318;

[0009] The GenBank accession number of the genome sequence is OQ504954.1;

[0010] The GenBank accession number of the genome sequence is OQ504955.1.

[0011] In some embodiments, the genetically engineered virus strain is a genetically inactivated attenuated strain of African swine fever virus; and / or

[0012] The D250R gene is obtained by point mutation, deletion mutation, insertion mutation or replacement mutation to obtain the genetically engineered virus strain.

[0013] The second aspect of the present invention provides a method for preparing the genetically engineered virus strain described in the first aspect of the present invention, wherein the preparation method comprises: inactivating the D250R gene of the parent strain of the genetically engineered virus strain by genetic manipulation.

[0014] In some embodiments, the D250R gene of the parent strain of the genetically engineered virus strain is inactivated by gene coding sequence fragment or base deletion mutation, gene coding sequence fragment or base insertion mutation, gene coding sequence point mutation, gene coding sequence fragment replacement mutation, gene regulatory region mutation or gene transcriptional regulation.

[0015] In some embodiments, the preparation method is: knocking out the D250R gene of the parent strain of the genetically engineered virus strain by homologous recombination method.

[0016] In some embodiments, the steps of the preparation method are:

[0017] P1: constructing a homologous recombination vector, wherein the homologous recombination vector contains a marker gene expression cassette, wherein the upstream sequence of the marker gene expression cassette is homologous to a sequence on one side of the D250R gene coding sequence, and the downstream sequence of the marker gene expression cassette is homologous to a sequence on the other side of the D250R gene coding sequence;

[0018] P2: Infecting African swine fever virus susceptible cells with the parent strain of the genetically engineered virus strain, and then transfecting African swine fever virus susceptible cells infected with the parent strain of the genetically engineered virus strain with the homologous recombination vector to obtain double genetically manipulated susceptible cells;

[0019] P3: performing monoclonal plaque culture on the susceptible cells subjected to the dual genetic manipulation;

[0020] P4: Selecting monoclonal plaques capable of detecting marker gene products to obtain the genetically engineered virus strain.

[0021] In some embodiments, the marker gene is a fluorescent protein gene; and / or

[0022] The African swine fever virus susceptible cells are primary porcine alveolar macrophages, Vero cells, primary porcine peripheral blood mononuclear cells or primary porcine bone marrow cells.

[0023] The third aspect of the present invention provides a method for culturing the genetically engineered virus strain described in the first aspect of the present invention or the genetically engineered virus strain prepared by the preparation method described in the second aspect of the present invention, wherein the culturing method comprises: inoculating the genetically engineered virus strain into cultured cells or susceptible animals, and performing proliferation culture of the genetically engineered virus strain to obtain a propagated genetically engineered virus strain.

[0024] In some embodiments, the cultured cells are primary porcine alveolar macrophages, Vero cells, primary porcine peripheral blood mononuclear cells or primary porcine bone marrow cells.

[0025] The fourth aspect of the present invention provides a biomaterial, wherein the biomaterial is selected from any one or a combination of the following P1, P2, P3 and P4:

[0026] P1: homologous recombination vector

[0027] The homologous recombination vector contains a marker gene expression cassette, the upstream sequence of the marker gene expression cassette is homologous to a sequence on one side of the D250R gene coding sequence of the African swine fever virus JS / LG / 21 strain, and the downstream sequence of the marker gene expression cassette is homologous to a sequence on the other side of the D250R gene coding sequence of the African swine fever virus JS / LG / 21 strain;

[0028] The GenBank accession number of the African swine fever virus JS / LG / 21 strain is OQ504956.1; the microbial preservation number of the African swine fever virus JS / LG / 21 strain is CCTCC NO: V 202318;

[0029] The backbone of the genetic engineering vector can be transfected into cells susceptible to African swine fever virus and express the gene in the marker gene expression cassette;

[0030] P2: Recombinant cells

[0031] The recombinant cell contains the homologous recombination vector described in P1;

[0032] P3: Composition

[0033] The composition contains the homologous recombination vector described in P1 or the recombinant cell described in P2;

[0034] P4: Test kit

[0035] The kit contains the homologous recombination vector described in P1 or the recombinant cell described in P2.

[0036] In some embodiments, any one or a combination thereof is selected from the following B1, B2, B3, B4, B5, B6 and B7;

[0037] B1: The backbone of the homologous recombination vector is a pUC57 vector;

[0038] B2: The marker gene is a fluorescent protein gene;

[0039] B3: The promoter in the marker gene expression cassette is the promoter of the African swine fever virus P72 gene;

[0040] B4: The African swine fever virus susceptible cells are primary porcine alveolar macrophages, Vero cells, primary porcine peripheral blood mononuclear cells or primary porcine bone marrow cells;

[0041] B5: The upstream sequence of the marker gene expression cassette is the sequence of positions 132420-133419 of the full-length genome sequence shown in Genbank sequence number OQ504956.1; the downstream sequence of the marker gene expression cassette is the sequence of positions 134173-135172 of the full-length genome sequence shown in Genbank sequence number OQ504956.1;

[0042] B6: The host cell of the recombinant cell is an Escherichia coli cell;

[0043] B7: The coding sequence encoded by the D250R gene is shown in the sequence of positions 133420-134172 of the full-length genome sequence shown in Genbank sequence number OQ504956.1.

[0044] The fifth aspect of the present invention provides the use of the biological material described in the fourth aspect of the present invention in preparing a genetically engineered virus strain of African swine fever virus. In the genome of the genetically engineered virus strain, the D250R gene is an inactivated gene.

[0045] The sixth aspect of the present invention provides a composition, wherein the active biological material in the composition contains the genetically engineered virus strain described in the first aspect of the present invention, the genetically engineered virus strain prepared by the preparation method described in the second aspect of the present invention, or the genetically engineered virus strain cultured by the culture method described in the third aspect of the present invention.

[0046] In some embodiments, the composition uses the genetically engineered virus strain described in the first aspect of the present invention, the genetically engineered virus strain prepared by the preparation method described in the second aspect of the present invention, or the genetically engineered virus strain cultured by the culture method described in the third aspect of the present invention as the sole immunogen, an immunogen in a multivalent vaccine, or an immunogen in a combined vaccine.

[0047] In some embodiments, the composition is a live African swine fever virus vaccine composition; and / or

[0048] The composition also includes auxiliary materials.

[0049] In some embodiments, the excipient is a lyoprotectant; in the raw materials of the vaccine composition, the lyoprotectant is an aqueous solution containing 4-12 w / w% gelatin and 30-50 w / w% sucrose; and / or

[0050] In the raw materials of the vaccine composition, the dry weight ratio of the African swine fever virus strain to the lyophilized protective agent is 1-20×10 6.0 TCID 50 :1g;

[0051] TCID of the African swine fever virus strain 50 It is calculated based on primary porcine alveolar macrophage culture using immunofluorescence analysis according to the Reed-Muench method.

[0052] The seventh aspect of the present invention provides a method for preparing the composition described in the sixth aspect of the present invention, wherein the preparation method comprises: using the genetically engineered virus strain as an immunogen to prepare a vaccine composition.

[0053] In some embodiments, the preparation method is: mixing the genetically engineered virus strain with the lyophilization protectant to obtain a mixture, and freeze-drying the mixture to obtain the vaccine composition.

[0054] The eighth aspect of the present invention provides the genetically engineered virus strain described in the first aspect of the present invention, the genetically engineered virus strain prepared by the preparation method described in the second aspect of the present invention, or the genetically engineered virus strain cultured by the culture method described in the third aspect of the present invention, in the preparation of a preparation for single use, for use in combination with other immune preparations and / or drugs, as a component of a compound preparation composed of other immune preparations and / or drugs, or as a parent virus of a genetically engineered virus to prevent, mitigate and / or control African swine fever, or in the preparation of a preparation for the identification and detection of wild African swine fever virus strains and African swine fever vaccine virus strains.

[0055] The genetically engineered virus strain of the present invention has good safety and immune effectiveness and has the potential to be used in the preparation of vaccines for clinical application.

[0056] The present invention deletes the D250R gene in the HLJ / 18 and JS / LG / 21 viruses respectively, weakens the virulence of the parental strains, and obtains a highly safe attenuated African swine fever candidate vaccine strain; after pigs are inoculated with the genetically engineered virus strain as an immunogen, the pigs are completely attenuated and the immune pigs are healthy, and can provide complete protection against strong strains of African swine fever, and can be used as a recombinant live African swine fever vaccine for preventing and controlling the epidemic. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the plasmid construction principle for homologous recombination replacement of the D250R gene.

[0058] Figure 2 Schematic diagram of PCR identification of recombinant virus HLJ / 18ΔD250R.

[0059] Figure 3 Schematic diagram and fluorescent photograph of the corresponding genomic location of the recombinant virus HLJ / 18ΔD250R.

[0060] Figure 4 This is a photo of the PCR identification results of the recombinant virus HLJ / 18ΔD250R.

[0061] Figure 5 Shown are the body temperature monitoring and survival results of piglets after immunization with the HLJ / 18ΔD250R recombinant virus.

[0062] Figure 6 The induction of antibodies by the HLJ / 18ΔD250R strain is shown.

[0063] Figure 7 Shown are the body temperature monitoring and survival results of piglets immunized with the HLJ / 18ΔD250R recombinant virus after being challenged with the homologous virulent HLJ / 18 strain.

[0064] Figure 8 This is a photo of the PCR identification results of the recombinant virus JS / LG / 21ΔD250R.

[0065] Fig. 9 This is a fluorescent photograph of the infection of the recombinant virus JS / LG / 21ΔD250R.

[0066] Fig.10 Shown are the body temperature monitoring and survival results of piglets immunized with JS / LG / 21ΔD250R recombinant virus.

[0067] Fig.11 The induction of antibodies by the JS / LG / 21ΔD250R strain is shown.

[0068] Fig.12 Shown are the body temperature monitoring and survival results of piglets immunized with JS / LG / 21ΔD250R recombinant virus after being challenged with the homologous virulent JS / LG / 21 strain.

[0069] Fig.13 Shown are the body temperature monitoring and survival results of piglets immunized with JS / LG / 21ΔD250R recombinant virus after being challenged with the heterologous virulent HLJ / 18 strain. DETAILED DESCRIPTION

[0070] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0071] Materials and instruments not described in the present invention are conventional materials and instruments in the art, operation details not described in the present invention are conventional operations in the art, the software used in the present invention is operated by conventional methods with reference to the instructions of the software provider, and the kit used in the present invention is operated by conventional methods with reference to the kit instruction manual.

[0072] The nucleic acid sequences shown in the present invention are all written from left to right in the 5' to 3' direction.

[0073] Statement: The experiments described in the following examples of this application have obtained laboratory accreditation from the China National Accreditation Service for Conformity Assessment, and have obtained biosafety license and African swine fever experimental activity license from the Ministry of Agriculture and Rural Affairs of the People's Republic of China.

[0074] Strains and cells

[0075] (1) African swine fever virus HLJ / 18 strain

[0076] Pig / Heilongjiang / 2018 (abbreviated as HLJ / 18) strain is a virulent strain of genotype II African swine fever virus identified and preserved by the National African Swine Fever Professional Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The Genbank sequence number is MK333180.1, and its full name is Pig / HLJ / 2018.

[0077] The isolated African swine fever virus HLJ / 18 strain was submitted to a patent procedure approved depository for preservation. The depository is China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microbial deposit number is CCTCC NO: V202403; the Chinese classification name is: African swine fever virus; the English classification name is: African Swine Fever Virus; the preservation time is January 5, 2024; the identification survival time is January 11, 2024. The culture name is: African swine fever virus ASFV HLJ / 18African swine fever virus ASFV HLJ / 18.

[0078] (2) African swine fever virus strain JS / LG / 21

[0079] ASFV JS / LG / 21 strain (referred to as JS / LG / 21 strain) is a naturally isolated strong strain of African swine fever virus. According to the analysis of genome sequence characteristics, JS / LG / 21 strain is a natural recombinant virus of genotype I / II African swine fever virus, isolated, identified and preserved by the National African Swine Fever Professional Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The literature (Highly lethalgenotype I and II recombinant African swine fever viruses detected in pigs. Dongming Zhao et al, Nature Communications. Volume 14, Article number: 3096 (2023). https: / / doi.org / 10.1038 / s41467-023-38868-w) records the isolation and characterization of Pig / Jiangsu / LG / 2021 (JS / LG / 21 strain). The document also records two other strains of natural recombinant viruses of genotype I / II African swine fever virus with GenBank accession numbers OQ504954.1 and OQ504955.1. The genome sequence of the JS / LG / 21 strain can be found in GenBank No. OQ504956.1, in which the full name of the strain is Pig / Jiangsu / LG / 2021. The acquisition process of the JS / LG / 21 strain is recorded in the Chinese patent application document with application number 202310363647.X. The isolated African swine fever virus JS / LG / 21 strain has been submitted to a patent procedure recognized depository for preservation. The preservation unit is China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the microorganism preservation number is CCTCC NO: V 202318; the culture name is African swine fever virus ASFVJS / LG / 21ASFV Pig / Jiangsu / LG / 2021; the Chinese classification name is: African swine fever virus; the English classification name is: African Swine Fever Virus; the preservation time is March 29, 2023; the identification survival time is March 29, 2023.

[0080] (3) Primary porcine alveolar macrophages

[0081] Primary porcine alveolar macrophages (PAMs) were obtained from 30-50 day old healthy SPF pigs and routinely cultured in 1640 medium containing 10% FBS in a 37°C, 5% CO2 incubator. They were prepared and used by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0082] Example 1: Pathogenicity analysis of ASFV HLJ / 18 strain

[0083] The African swine fever (ASF) virus Pig / HLJ / 18 belongs to genotype II ASF virus and replicates in primary porcine alveolar macrophages (PAMs) with a titer of up to 10 7.2 HAD 50 / ml. Intramuscular injection 10 3.5 -10 6.5 HAD 50 SPF pigs with different virus doses showed acute disease with signs of fever and bleeding. The incubation period was 3-5 days for pigs inoculated with the virus and 9 days for cohabiting pigs. All virus-inoculated pigs died between 6-9 days post-inoculation (pi), and cohabiting pigs died between 13-14 days (pc). These results indicate that Pig / HLJ / 18 is highly virulent and transmissible in domestic pigs.

[0084] Example 2: Construction of genetically engineered virus strains

[0085] As can be seen from Example 1, the ASFV HLJ / 18 strain has high virulence and high lethality. In order to study the effect of inactivation of the D250R gene on the virulence of the ASFV HLJ / 18 strain, the present invention prepared an inactivated strain of the D250R gene of the ASFV HLJ / 18 strain.

[0086] 1. Construction of homologous recombination vector

[0087] See the construction strategy Figure 1 In order to replace the D250R gene of the HLJ / 18 strain with the EGFP gene by homologous recombination, a homologous recombination transfer vector for D250R gene knockout was constructed using the pUC57 vector as a backbone vector, and the replaced gene sequence corresponds to positions 137523-138275 of the full-length genome sequence of the HLJ / 18 strain. The full-length genome sequence of the HLJ / 18 strain in the present invention is based on the genome sequence recorded by Genbank sequence number MK333180.1.

[0088] Taking the direction of the positive strand of the D250R gene as a reference, the DNA sequence at positions 136523-137522 corresponding to the full-length genome sequence of the HLJ / 18 strain upstream of the D250R gene was used as the left homologous arm (LR) of homologous recombination, and the DNA sequence at positions 138276-139275 corresponding to the full-length genome sequence of the HLJ / 18 strain downstream of the D250R gene was used as the right homologous arm (RR) of homologous recombination, and an EGFP expression cassette (the promoter was the P72 promoter of the African swine fever virus P72 gene, and the coding sequence was the EGFP gene) was inserted between the left homologous arm sequence (LR) and the right homologous arm sequence (RR) of the recombinant transfer vector.

[0089] The African swine fever virus P72 promoter sequence used in the present invention is as follows (SEQ ID NO.1):

[0090] TTGTTATTATCAAGATCCTTCGCATAAACCGCCATATTTAATAAAAACAATAAATTATTTTTATAACATTATATA

[0091] The coding sequence of EGFP refers to Yang, TT et al. "Optimized codon usage and chromophore mutations provide enhanced sensitivity with the green fluorescent protein." Nucleic acids research vol. 24, 22 (1996): 4592-3. doi: 10.1093 / nar / 24.22.4592.

[0092] The EGFP gene expression cassette sequence containing the ASFVP72 promoter sequence was commissioned to be synthesized by Jilin Kumei Biotechnology Co., Ltd. The three fragments of the left homology arm, the P72EGFP expression cassette, and the right homology arm were cloned into the pUC57 vector. After DNA sequencing verified that the recombination was correct, the homologous recombination transfer vector was named pUC57-P72EGFP. pUC57-P72EGFP uses the pUC57 vector as a skeleton, and the regions outside the pUC57 vector used to support homologous recombination are the aforementioned RR, P72 promoter, EGFP coding sequence, LR in order. The purpose is that after homologous recombination occurs between the LR and RR of pUC57-P72EGFP and the homologous sequence of the HLJ / 18 strain, the EGFP carried by the recombinant HLJ / 18 strain can express green fluorescent protein under the action of the P72 promoter, and then distinguish the original strain from the recombinant strain by whether it can emit green fluorescence.

[0093] 2. Construction and identification of the recombinant African swine fever virus HLJ / 18ΔD250R strain

[0094] The plasmid pUC57-P72EGFP was transfected into PAMs cells infected with HLJ / 18 (MOI=1) using Trans IT-LT1 transfection reagent (purchased from MirusBio, USA, catalog number MIR 2300) according to the instructions of the kit, and the recombinant African swine fever virus plaques expressing EGFP fluorescent protein were picked and purified using the plaque cloning method. The recombinant African swine fever virus is called HLJ / 18ΔD250R. In theory, the EGFP expression cassette replaces the coding sequence of the D250R gene, which will be experimentally verified below.

[0095] The genome templates of the recombinant virus strain HLJ / 18ΔD250R and the parental strain HLJ / 18 were extracted using a viral genome extraction kit (purchased from Beijing Tiangen Biotechnology Co., Ltd., catalog number DP304-03). Upstream primer F was designed upstream of the D250R coding sequence, downstream primer R1 was designed downstream of the D250R coding sequence, and primer R2 was designed inside the D250R coding sequence. Primer pair F / R1 can amplify HLJ / 18 and HLJ / 18ΔD250R, and primer pair F / R2 can only amplify HLJ / 18. After PCR identification with two pairs of primers (F / R1, F / R2), it was confirmed whether the deletion was successful. The principle is shown in Figure 2 .

[0096] F (SEQ ID NO. 2): 5'-GAGTCAGTACTATTACGTGATAGTG-3'.

[0097] R1 (SEQ ID NO. 3): 5'-GAATCACATGATGTTCTCGATGATC-3'.

[0098] R2 (SEQ ID NO. 4): 5'-ctcccgtatggcacaggtaaggtc-3'.

[0099] Theoretically, the PCR fragment size of the parental strain HLJ / 18 amplified by primer pair F / R1 is 1161bp, and the PCR product size amplified by primer pair F / R2 is 556bp, while the PCR fragment size of the recombinant virus strain HLJ / 18ΔD250R amplified by primer pair F / R1 is 1202bp, and no PCR product can be amplified by primer pair F / R2. Figure 3 The left figure shows a schematic diagram of the genomic difference segments between HLJ / 18 and HLJ / 18ΔD250R. Figure 4The gel electrophoresis diagram of the actual PCR results is shown, where lane 1 is the HLJ / 18ΔD250R genome template amplification result; lane 2 is the HLJ / 18 genome template amplification result; lane 3 is the negative control (distilled water) amplification result, the left picture is the F / R2 amplification result, and the right picture is the F / R1 amplification result. The above results show that the HLJ / 18ΔD250R recombinant virus has been successfully constructed.

[0100] HLJ / 18 and the recombinant virus strain HLJ / 18ΔD250R infected PAMs cells for 48 hours and then took photos using an inverted fluorescence microscope. For the photo results, see Figure 3 In the right picture, the upper part is a fluorescent photo of HLJ / 18 infection, and the lower part is a fluorescent photo of HLJ / 18ΔD250R infection.

[0101] It can be seen that the construction of HLJ / 18ΔD250R was in line with expectations, and a genetically recombinant strain in which the D250R gene was replaced by the EGFP gene was obtained.

[0102] Example 3: Virus content determination method

[0103] (I) Titration of virus titer

[0104] The titration of African swine fever virus was performed using the 50% tissue culture infectious dose (TCID 50 ) for characterization.

[0105] TCID 50 The titration was performed according to the following steps: the ASFV cell culture supernatant was diluted 10 times in RPMI Medium 1640 culture medium, and the cells were inoculated into 96-well culture plates containing PAMs cells with a cell density of 90-100%. Eight wells were inoculated in parallel for each dilution, and the inoculation volume per well was 0.02 mL. The cells were cultured at 37°C and 5% CO2 for 3-7 days. The cells were judged to be infected based on the green fluorescence, and the half cell infection dose (TCID) was calculated by the Reed-Muench method. 50 ).

[0106] TCID of the embodiment of the present invention 50 All were measured using this method.

[0107] (II) Determination of hematocrit (HAD) titer

[0108] The ASFV culture supernatant was diluted 10-fold in series with RPMI 1640 complete medium and inoculated into 96-well plates in RPMI 1640 complete medium suspension containing 0.1% porcine erythrocytes of PBMCs (porcine peripheral blood mononuclear cells). HAD was observed under a microscope every day for 7 consecutive days. HAD titer was calculated using the Reed-Muench method.

[0109] Example 4: Animal Immunization Experiment

[0110] The experiment was conducted in the ABSL-3 animal room of the National Animal Disease Prevention and Control High-Level Biosafety Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. 14 7-week-old Large White and Landrace inbred SPF pigs (purchased from the Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were grouped and inoculated as shown in Table 1. The experimental group was inoculated with the recombinant virus HLJ / 18ΔD250R by intramuscular injection in the neck, and the control group was not inoculated. After immunization, the animals' mental state and feeding were continuously observed, and the animals' body temperature was monitored.

[0111] Table 1. Design of pig immune challenge experiment

[0112]

[0113] All HLJ / 18ΔD250R immunized pigs showed no abnormal phenomena such as elevated body temperature and reduced food intake within 28 days after immunization, and all immunized pigs survived normally ( Figure 5 ). Figure 5 In the figure, A and C are the statistical information of HLJ / 18ΔD250R group one, B and D are the statistical information of HLJ / 18ΔD250R group two, A and B are the survival rates of pigs on each day after vaccination, and C and D are the body temperature statistics of pigs on each day after vaccination.

[0114] It can be seen that HLJ / 18ΔD250R has low toxicity and high safety. No clinical symptoms related to African swine fever such as coughing, skin bleeding or ulcers were observed at the inoculation dose of this example. It is non-lethal and has the potential to be made into an attenuated live vaccine.

[0115] Example 5: Study on immunogenicity of ASFV HLJ / 18ΔD250R strain

[0116] To evaluate the immunogenicity of the HLJ / 18ΔD250R strain, blood was collected from the inoculated pigs and the control pigs in Example 4 every 5 days after inoculation to prepare serum, and the p22 protein blocking ELISA antibody detection method was used to determine the p22 antibody blocking rate. The detection procedure is as follows:

[0117] 1. Coating: Dilute the prokaryotic expressed African swine fever virus p22 protein antigen into 4 μg / ml antigen dilution with antigen coating solution (carbonate buffer), add to 96-well plate, 50 μl / well, and coat overnight at 4°C.

[0118] 2. Washing: Wash 5 times with 300 μl / well of PBST (phosphate buffered saline containing 0.05 v / v% Tween 20), each time for 1 minute.

[0119] 3. Blocking: Block with 5 w / v% skim milk, 200 μl / well, incubate at 37°C for 2 hours.

[0120] 4. Washing: Same as 2.

[0121] 5. Sample addition: dilute all the sera from the pigs in Example 4 with PBS at a volume ratio of 1:1, add to a 96-well plate, 50 μl / well, and incubate at 37° C. for 30 minutes.

[0122] 6. Washing: Same as 2.

[0123] 7. Enzyme-labeled antibody: Use secondary antibody diluent (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., catalog number: HRP-SD-001) to dilute the enzyme-labeled antibody (mouse anti-African swine fever virus p22 protein pig IgG monoclonal antibody labeled with horseradish peroxidase) at a volume ratio of 1:2000, 50 μl / well, and incubate at 37°C for 30 minutes.

[0124] 8. Washing: Same as 2.

[0125] 9. Color development: Add 50 μl TMB substrate to each well for color development and react at 37°C in the dark for 10 minutes.

[0126] 10. Stop: Add 50 μl stop solution (2M H2SO4) to each well.

[0127] 11. Reading: Determine OD 450nm value.

[0128] According to the following formula, the blocking rate % = [(negative control average OD 450nm - Sample OD 450nm ) / mean OD of negative control 450nm ] × 100. The judgment criteria are: a blocking rate higher than 47% is positive, and a blocking rate lower than 47% is negative.

[0129] The results show that for 10 3 TCID 50 In the pig group, no pigs had positive antibody conversion on days 5, 10, and 15 after vaccination (one pig was between the positive and negative thresholds on day 15), and all pigs had positive antibody conversion on days 20 and 25. 5 TCID 50In the pig group, the antibody conversion ratios were 0 / 5, 1 / 5, and 4 / 5 on days 5, 10, and 15 after vaccination, respectively, and the antibody conversion was positive on days 20 and 25; the antibody of the non-immunized control group was always negative (not shown in the figure). Figure 6 The results showed that the ASFV HLJ / 18ΔD250R strain has strong immunogenicity and is valuable for manufacturing a live vaccine against African swine fever virus.

[0130] Example 6: Animal challenge test

[0131] After 28 days of immunization of the pigs in Example 4, the immunized group and the control group were challenged with the homologous virulent HLJ / 18 strain as shown in Table 1, with a dose of 100 PLD 50 The route of infection was intramuscular injection. After infection, the animals' mental state, feeding conditions, and body temperature were continuously observed. On the 21st day after ASFV infection, all surviving pigs were euthanized.

[0132] The results showed that after the strong strain of African swine fever virus was infected, 3 TCID 50 One pig in the immunization group developed fever on the third day after the challenge ( Figure 7 D) Until death on the 10th day ( Figure 7 A), the body temperatures of other pigs were normal ( Figure 7 D) 10 5 TCID 50 / No abnormal phenomenon such as elevated body temperature was observed in the immunized pigs in the whole monitoring period ( Figure 7 E), all pigs survived, with a protection rate of 100% ( Figure 7 B); The blank control group began to develop fever on the 4th day after the virus attack ( Figure 7 F); Pigs began to die on the 6th day, and all pigs in the blank control group died on the 8th day after the challenge ( Figure 7 C).

[0133] The above experimental results prove that after piglets are immunized with the recombinant African swine fever virus HLJ / 18ΔD250R, it can provide complete protection against attacks from homologous strong strains of African swine fever, and can be used as an economical and efficient African swine fever attenuated live vaccine.

[0134] Example 7: Preparation of vaccine

[0135] Preparation of lyophilized protective agent: an aqueous solution containing 8w / w% gelatin and 40w / w% sucrose is sterilized by high pressure at 116°C for 20 minutes and then placed at 2-8°C for use.

[0136] Virus culture: ASFV HLJ / 18ΔD250R strain was diluted to 10000 TCID using RPMI Medium 1640 (purchased from Thermo Fisher Biochemicals (Beijing) Co., Ltd., catalog number: C11875500BT) complete culture medium (containing 10 v / v% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin). 50 / ml. The diluted virus strain was inoculated into primary PAMs cells. The culture flask was placed in an incubator at 37°C and 5% CO2 and the virus was harvested when the CPE reached 80%.

[0137] Mixing: Add African swine fever virus HLJ / 18ΔD250R culture medium (virus content not less than 10 6.0 TCID 50 / ml) and freeze-dried protective agent were mixed evenly at a volume ratio of 8.5:1.

[0138] Freeze-drying: freeze-dry the mixture at the following conditions: -5°C, 1 hour; -40°C, 2 hours; -16°C, 14-18 hours; -6°C, 3 hours; 5°C, 2 hours; 28°C, 6 hours.

[0139] Characterization of physical properties: The freeze-dried vaccine obtained is loose and porous, sponge-like, and dissolves quickly after adding water.

[0140] Example 8: Pathogenicity analysis of ASFV JS / LG / 21 strain

[0141] To evaluate the virulence and transmissibility of ASFV JS / LG / 21 strain to pigs, 7-week-old SPF pigs were selected from the Experimental Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences and injected intramuscularly for 10 3 HAD 50 with 10 6 HAD 50 ASFV JS / LG / 21 strain (PAMs culture of ASFV JS / LG / 21 strain diluted with RPMI Medium 1640 culture medium), 6 pigs were inoculated with each dose.

[0142] Starting from the first day of infection, two SPF pigs were selected from each group to be housed with infected pigs to evaluate the transmission ability of ASFV JS / LG / 21 strain. The survival and clinical signs of all pigs were monitored daily.

[0143] In 10 6 HAD 50 In the group, all 6 vaccinated pigs started to have fever on day 4 pi (post-vaccination) and died between days 5 and 8 pi; the 2 contact pigs started to have fever on day 9 after contact and died on day 12.

[0144] In 10 3 HAD 50 In the group, all pigs died between days 6 and 15; two contact pigs developed fever on days 9 and 10 after infection and died on days 12 and 14 after inoculation, respectively. These results indicate that the recombinant virus isolate ASFV JS / LG / 21 is highly lethal and transmissible.

[0145] Example 9: Construction of genetically engineered virus strains

[0146] It can be seen from Example 8 that the ASFV JS / LG / 21 strain has high virulence and high lethality. In order to study the effect of inactivation of the D250R gene on the virulence of the ASFV JS / LG / 21 strain, the present invention prepared a D250R gene-deficient strain of the ASFV JS / LG / 21 strain.

[0147] The full-length genome sequence of the ASFV JS / LG / 21 strain in the present invention is based on the genome sequence recorded by Genbank sequence number OQ504956.1.

[0148] 1. Construction of homologous recombination vector

[0149] The gene sequence to be replaced corresponds to the 133420-134172th position of the full-length genome sequence of the JS / LG / 21 strain. It is planned to use the DNA sequence of the 132420-133419th position corresponding to the full-length genome sequence of the JS / LG / 21 strain upstream of the D250R gene as the homologous recombination left homology arm (LR). It can be seen by comparison that the sequence is the same as the homologous recombination left homology arm sequence for the HLJ / 18 strain in Example 2. It is planned to use the DNA sequence of the 134173-135172th position corresponding to the full-length genome sequence of the JS / LG / 21 strain downstream of the D250R gene as the homologous recombination right homology arm (RR). It can be seen by comparison that the sequence is the same as the homologous recombination right homology arm sequence for the HLJ / 18 strain in Example 2. Therefore, pUC57-P72EGFP constructed using Example 2 is used as the homologous recombination transfer vector of this embodiment.

[0150] 2. Construction and identification of the recombinant African swine fever virus JS / LG / 21ΔD250R strain

[0151] The plasmid pUC57-P72EGFP was homologously recombined with the JS / LG / 21 strain using the same method and operating parameters as in Example 2. The recombinant African swine fever virus was named JS / LG / 21ΔD250R.

[0152] The primers F, R1 and R2 in Example 2 were used to identify the homologous recombination results.

[0153] Two pairs of primers (F / R1, F / R2) were used to amplify the genomic templates of JS / LG / 21 and JS / LG / 21ΔD250R, respectively. The electrophoresis results of the amplified products were shown in Figure 8 , where lane 1 is the amplification result of JS / LG / 21ΔD250R genome template F / R1; lane 2 is the amplification result of JS / LG / 21 genome template F / R1; lane 5 is the amplification result of JS / LG / 21ΔD250R genome template F / R2; lane 6 is the amplification result of JS / LG / 21 genome template F / R2; lanes 3 and 7 are the amplification results of the negative control (distilled water); lane 4 is not loaded. The above results show that the JS / LG / 21ΔD250R recombinant virus has been successfully constructed.

[0154] JS / LG / 21 and the recombinant virus strain JS / LG / 21ΔD250R infected PAMs cells for 48 hours and took photos using an inverted fluorescence microscope. The photo results are shown in Fig. 9 , The left picture is a white light photograph of JS / LG / 21ΔD250R infection, and the right picture is a fluorescent photograph of JS / LG / 21ΔD250R infection.

[0155] It can be seen that the construction of JS / LG / 21ΔD250R was in line with expectations, and a genetically recombinant strain in which the D250R gene was replaced by the EGFP gene was obtained.

[0156] Example 10: Animal Immunization Experiment

[0157] The experiment was conducted in the ABSL-3 animal room of the National Animal Disease Prevention and Control High-Level Biosafety Laboratory of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. 14 7-week-old Large White and Landrace inbred SPF pigs (purchased from the Animal Center of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) were grouped and inoculated as shown in Table 2. The experimental group was inoculated with the recombinant virus JS / LG / 21ΔD250R strain by intramuscular injection in the neck, and the control group was not inoculated. After immunization, the animals' mental state, feeding conditions, and body temperature were continuously observed.

[0158] Table 2. Design of pig immune challenge experiment

[0159]

[0160] All JS / LG / 21ΔD250R immunized pigs showed no abnormal phenomena such as elevated body temperature and reduced food intake within 28 days after immunization, and all immunized pigs survived normally ( Fig.10 ). Fig.10 In the figure, A is the survival rate of pigs on each day after vaccination, and B is the body temperature statistics of pigs on each day after vaccination.

[0161] It can be seen that JS / LG / 21ΔD250R has low toxicity and high safety, is non-lethal at the vaccination dose of this example, and has the potential to be used as a live vaccine.

[0162] Example 11: Study on immunogenicity of ASFV JS / LG / 21ΔD250R strain

[0163] To evaluate the immunogenicity of the JS / LG / 21ΔD250R strain, blood was collected from the inoculated pigs and the control pigs in Example 10 every 5 days after inoculation to prepare serum, and the p22 antibody blocking rate was determined using the same p22 protein blocking ELISA antibody detection method as in Example 5. The results of immunization time and blocking rate are shown in Fig.11 .

[0164] according to Fig.11 It can be seen that for 10 6 TCID 50 In the pig group, 8 / 10 pigs turned positive 10 days after vaccination, and all the vaccinated pigs turned positive 15 days after vaccination. The antibody titer was high 25 days after vaccination, and the blocking rate was more than 80%; the antibody of the non-immunized control group was always negative (not shown in the figure). The results show that the ASFV JS / LG / 21ΔD250R strain has strong immunogenicity and is valuable for manufacturing a live attenuated vaccine against African swine fever virus.

[0165] Example 12: Animal challenge test

[0166] (I) Homologous virus challenge test

[0167] After 28 days of immunization of the pigs in Example 10, the immunized group and the control group were challenged with ASFV virulent virus (JS / LG / 21 strain) at a dose of 100 PLD 50 The route of challenge was intramuscular injection. After challenge, the animals' mental state, feeding habits, and body temperature were continuously observed.

[0168] In order to determine the protective effect of JS / LG / 21ΔD250R recombinant virus against the strong strain of African swine fever virus, the present invention immunized the test pigs with JS / LG / 21ΔD250R recombinant virus and then challenged them with the strong strain of African swine fever virus JS / LG / 21. On the 28th day after ASFV challenge, all surviving pigs were euthanized. The results showed that after the strong strain of African swine fever virus was challenged, only one immunized pig in the immunized group had a slight increase in body temperature during the entire monitoring period ( Fig.12 B) and both survived ( Fig.12 A), the protection rate is 100% ( Fig.12 A); The blank control group began to develop fever on the third day after the virus attack, and all of them developed fever on the fourth day ( Fig.12D); Pigs began to die on the 7th day, and all pigs in the blank control group died on the 11th day after the challenge ( Fig.12 C).

[0169] The experimental results show that after piglets are immunized with the recombinant African swine fever virus JS / LG / 21ΔD250R, it can provide good protection against attacks from the homologous strong strain of African swine fever virus JS / LG / 21, and can be used as an economical and efficient African swine fever attenuated live vaccine.

[0170] (II) Heterologous virus challenge test

[0171] In order to test whether JS / LG / 21ΔD250R strain inoculation can protect against heterologous virus attack, the virulent virus used for the challenge was replaced by the heterologous gene type II virulent HLJ / 18 strain instead of the homologous gene I / II recombinant virulent JS / LG / 21 strain. Other materials, dosages and operating parameters were the same as those in the aforementioned "homologous virus challenge test" of this example. The results are shown in Fig.13 .

[0172] In order to determine whether the JS / LG / 21ΔD250R recombinant virus can protect against the attack of heterologous viruses. After immunizing the test pigs with the JS / LG / 21ΔD250R recombinant virus, the virulent HLJ / 18 strain of heterologous African swine fever virus was used for virus attack. On the 28th day after the virus attack, all surviving pigs were euthanized. The results showed that the body temperature of the immunized pigs was normal during the entire monitoring period, and only one pig had a transient slightly high body temperature (40.1°C), while the body temperature of the other pigs was normal ( Fig.13 B), all pigs survived, with a protection rate of 100% ( Fig.13 A); The blank control group (unvaccinated pigs were challenged with HLJ / 18 strain) began to develop fever on the 4th day after the challenge ( Figure 7 F), pigs began to die on the 6th day, and all pigs in the blank control group died on the 8th day after the infection ( Figure 7 C).

[0173] The experimental results showed that after piglets were immunized with the recombinant African swine fever virus JS / LG / 21ΔD250R strain, it could provide good protection against attacks from the heterologous virulent African swine fever virus HLJ / 18 strain, and could be used as an economical and efficient attenuated live African swine fever vaccine.

[0174] Example 13: Preparation of vaccine

[0175] Preparation of lyophilized protective agent: an aqueous solution containing 8w / w% gelatin and 40w / w% sucrose is sterilized by high pressure at 116°C for 20 minutes and then placed at 2-8°C for use.

[0176] Virus culture: ASFV HLJ / 18ΔD250R strain was diluted to 10000 TCID using RPMI Medium 1640 (purchased from Thermo Fisher Biochemicals (Beijing) Co., Ltd., catalog number: C11875500BT) complete culture medium (containing 10 v / v% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin). 50 / ml. The diluted virus strain was inoculated into primary PAMs cells. The culture flask was placed in an incubator at 37°C and 5% CO2 and the virus was harvested when the CPE reached 80%.

[0177] Mix: Add African swine fever virus JS / LG / 21ΔD250R culture medium (virus content not less than 10 6.0 TCID 50 / ml) and freeze-dried protective agent were mixed evenly at a volume ratio of 8.5:1.

[0178] Freeze-drying: freeze-dry the mixture at the following conditions: -5°C, 1 hour; -40°C, 2 hours; -16°C, 14-18 hours; -6°C, 3 hours; 5°C, 2 hours; 28°C, 6 hours.

[0179] Characterization of physical properties: The freeze-dried vaccine obtained is loose and porous, sponge-like, and dissolves quickly after adding water.

[0180] Summary: After the D250R gene of the genotype II virulent strain HLJ / 18 and the genotype I / II natural recombinant strain JS / LG / 21 was inactivated, the immunogenicity remained basically unchanged, the toxicity and pathogenicity basically disappeared, and antibodies against the African swine fever virus were produced. After inoculation, the pigs were able to resist the attack of the homologous virus. It can be seen that knocking out the D250R gene can effectively attenuate the African swine fever virus and has the potential to prepare a live vaccine.

[0181] It can be known or reasonably expected that after the D250R gene is inactivated in the African swine fever virus strains listed below, or after the gene is inactivated in other African swine fever virus strains that also contain the D250R gene listed below, the virulence can be significantly reduced, and it has the potential to prepare live vaccines.

[0182] (1) GenBank accession number is NC001659.2, strain name is BA71V, SEQ ID NO.5

[0183] MDTAMQLKTSIGLITCRMNTQNNQIETILVQKRYSLAFSEFIHCHYSINANQGHLIKMFNNMTINERLLVKTLDFDRMWYHIWIETPVYELYHKKYQKFRKNWLLPDNGKKLISLINQAKGSGTL LWEIPKGKPKEDESDLTCAIREFEEETGITREYYQILPEFKKSMSYFDGKTEYKHIYFLAMLCKSLEEPNMNLSLQYENRIAEISKISWQNMEAVRFISKRQSFNLEPMIGPAFNFIKNYLRYKH

[0184] (2) GenBank accession number is MK333180.1, strain name is Pig / HLJ / 2018, SEQ ID NO.6

[0185] MDTAMQLKTSIGLITCRMNTQNNQIETILVQKRYSLAFSEFIHCHYSINANQGHLIKMFNNMTINERLLVKTLDFDRMWYHIWIETPVYELYHKKYQKFRKNWLLPDNGKKLISLINQAKGSGTL LWEIPKGKPKEDESDLTCAIREFEEETGITREYYQILPEFKKSMSYFDGKTEYKHIYFLAMLCKSLEEPNMNLSLQYENRIAEISKISWQNMEAVRFISKRQSLNLEPIIGPAFNFIKNYLRYKH

[0186] (3) The protein sequence encoded by the D250R gene of the JS / LG / 21 strain with GenBank accession number OQ504956.1 is identical to the sequence of the Pig / HLJ / 2018 strain mentioned above.

[0187] (4) GenBank accession number is KM111294.1, strain name is Ken05 / Tk1, SEQ ID NO.7

[0188] MDTAMQLKTSIGLITCRMNTQSNQIETILVQKRYSLAFSEFIHCHYSINSNHSHLIKMFNNMTINERLLIKTLDFDRMWYHIWIETPVYELYHKKYQKFKKNWLIPDNGKKLISLINQAKGSGTL LWEIPKGKPKENESDLACAIREFEEETGIAREDYQILPAFKKSMSYFEGKTEYKHIYFLAVLCKSLEEPNMNLSLQYETRIAEISKISWQNMEAVRFISKHQSLNLEPIIGPAFNFIKNYLRYKH

[0189] (5) GenBank accession number is MN630494.2, strain name is Zaire, SEQ ID NO.8

[0190] MDTAMQLKTSIGLITCRMNTQNNQIETILVQKRYSLAFSEFIHCHYSINANQGHLIKMFNNMTINERLLVKTLDFDRMWYHIWIETPVYELYHKKYQKFRKNWLLPDNGKKLISLINQAKGSGTL LWEIPKGKPKEDESDLTCAIREFEEETGITREYYQILPEFKKSMSYFNGKTEYKHIYFLAMLCKSLEEPNMNLSLQYENRIAEISKISWQNMEAVRFISKRQSLNLEPIIGPAFNFIKNYLRYKH

[0191] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A genetically engineered virus strain of African swine fever virus, wherein the D250R gene is an inactivated gene in the genome of the genetically engineered virus strain; The parent used to prepare the genetically engineered virus strain is selected from the following strains: The GenBank accession number of the genome sequence is OQ504956.1, and the microbial deposit number is CCTCCNO: V 202318; The GenBank accession number of the genome sequence is OQ504954.1; The GenBank accession number of the genome sequence is OQ504955.

1.

2. The genetically engineered virus strain according to claim 1, characterized in that: The genetically engineered virus strain is a genetically inactivated attenuated strain of African swine fever virus; and / or The D250R gene is obtained by point mutation, deletion mutation, insertion mutation or replacement mutation to obtain the genetically engineered virus strain.

3. The method for preparing the genetically engineered virus strain according to claim 1 or 2, wherein the method comprises inactivating the D250R gene of the parent strain of the genetically engineered virus strain through genetic manipulation.

4. The preparation method according to claim 3, characterized in that: The D250R gene of the parent strain of the genetically engineered virus strain is inactivated by gene coding sequence fragment or base deletion mutation, gene coding sequence fragment or base insertion mutation, gene coding sequence point mutation, gene coding sequence fragment replacement mutation, gene regulatory region mutation or gene transcriptional regulation.

5. The preparation method according to claim 3 or 4, characterized in that: The preparation method comprises: knocking out the D250R gene of the parent strain of the genetically engineered virus strain by homologous recombination method.

6. The preparation method according to any one of claims 3 to 5, characterized in that: The steps of the preparation method are: P1: constructing a homologous recombination vector, wherein the homologous recombination vector contains a marker gene expression cassette, wherein the upstream sequence of the marker gene expression cassette is homologous to a sequence on one side of the D250R gene coding sequence, and the downstream sequence of the marker gene expression cassette is homologous to a sequence on the other side of the D250R gene coding sequence; P2: Infecting African swine fever virus susceptible cells with the parent strain of the genetically engineered virus strain, and then transfecting African swine fever virus susceptible cells infected with the parent strain of the genetically engineered virus strain with the homologous recombination vector to obtain double genetically manipulated susceptible cells; P3: performing monoclonal plaque culture on the susceptible cells subjected to the dual genetic manipulation; P4: Selecting monoclonal plaques capable of detecting marker gene products to obtain the genetically engineered virus strain.

7. The preparation method according to claim 6, characterized in that: The marker gene is a fluorescent protein gene; and / or The African swine fever virus susceptible cells are primary porcine alveolar macrophages, Vero cells, primary porcine peripheral blood mononuclear cells or primary porcine bone marrow cells.

8. A method for culturing the genetically engineered virus strain according to claim 1 or 2 or the genetically engineered virus strain prepared by the preparation method according to any one of claims 3 to 7, the culturing method comprising: inoculating the genetically engineered virus strain into cultured cells or susceptible animals, and performing proliferation culture of the genetically engineered virus strain to obtain a propagated genetically engineered virus strain.

9. The culture method according to claim 8, characterized in that The cultured cells are primary porcine alveolar macrophage cells, Vero cells, primary porcine peripheral blood mononuclear cells or primary porcine bone marrow cells.

10. A biomaterial, wherein the biomaterial is selected from any one or a combination of the following P1, P2, P3 and P4: P1: homologous recombination vector The homologous recombination vector contains a marker gene expression cassette, the upstream sequence of the marker gene expression cassette is homologous to a sequence on one side of the D250R gene coding sequence of the African swine fever virus JS / LG / 21 strain, and the downstream sequence of the marker gene expression cassette is homologous to a sequence on the other side of the D250R gene coding sequence of the African swine fever virus JS / LG / 21 strain; The GenBank accession number of the African swine fever virus JS / LG / 21 strain is OQ504956.1; the microbial preservation number of the African swine fever virus JS / LG / 21 strain is CCTCC NO: V 202318; The backbone of the genetic engineering vector can be transfected into cells susceptible to African swine fever virus and express the gene in the marker gene expression cassette; P2: Recombinant cells The recombinant cell contains the homologous recombination vector described in P1; P3: Composition The composition contains the homologous recombination vector described in P1 or the recombinant cell described in P2; P4: Test kit The kit contains the homologous recombination vector described in P1 or the recombinant cell described in P2.

11. The biomaterial according to claim 10, characterized in that Any one or a combination thereof selected from the following B1, B2, B3, B4, B5, B6 and B7; B1: The backbone of the homologous recombination vector is a pUC57 vector; B2: The marker gene is a fluorescent protein gene; B3: The promoter in the marker gene expression cassette is the promoter of the African swine fever virus P72 gene; B4: The African swine fever virus susceptible cells are primary porcine alveolar macrophages, Vero cells, primary porcine peripheral blood mononuclear cells or primary porcine bone marrow cells; B5: The upstream sequence of the marker gene expression cassette is the sequence of positions 132420-133419 of the full-length genome sequence shown in Genbank sequence number OQ504956.1; the downstream sequence of the marker gene expression cassette is the sequence of positions 134173-135172 of the full-length genome sequence shown in Genbank sequence number OQ504956.1; B6: The host cell of the recombinant cell is an Escherichia coli cell; B7: The coding sequence encoded by the D250R gene is shown in the sequence of positions 133420-134172 of the full-length genome sequence shown in Genbank sequence number OQ504956.

1.

12. Use of the biological material according to claim 10 or 11 in preparing a genetically engineered virus strain of African swine fever virus, wherein in the genome of the genetically engineered virus strain, the D250R gene is an inactivated gene.

13. A composition, wherein the active biological material in the composition comprises the genetically engineered virus strain according to claim 1 or 2, the genetically engineered virus strain prepared by the preparation method according to any one of claims 3 to 7, or the genetically engineered virus strain cultured by the culture method according to claim 8 or 9.

14. The composition according to claim 13, characterized in that The composition uses the genetically engineered virus strain described in claim 1 or 2, the genetically engineered virus strain prepared by the preparation method described in any one of claims 3-7, or the genetically engineered virus strain cultured by the culture method described in claim 8 or 9 as the sole immunogen, an immunogen in a multivalent vaccine, or an immunogen in a combined vaccine.

15. The composition according to claim 14, characterized in that The composition is a live African swine fever virus vaccine composition; and / or The composition also includes auxiliary materials.

16. The composition according to claim 15, characterized in that The auxiliary material is a lyophilization protective agent; in the raw materials of the vaccine composition, the lyophilization protective agent is an aqueous solution containing 4-12w / w% gelatin and 30-50w / w% sucrose; and / or In the raw materials of the vaccine composition, the dry weight ratio of the African swine fever virus strain to the lyophilized protective agent is 1-20×10 6.0 TCID 50 :1g; TCID of the African swine fever virus strain 50 It is calculated based on primary porcine alveolar macrophage culture using immunofluorescence analysis according to the Reed-Muench method.

17. A method for preparing the composition according to any one of claims 13 to 16, the method comprising: using the genetically engineered virus strain as an immunogen to prepare a vaccine composition.

18. The preparation method according to claim 17, characterized in that: The preparation method comprises: mixing the genetically engineered virus strain with the freeze-drying protective agent to obtain a mixture, and freeze-drying the mixture to obtain the vaccine composition.

19. The genetically engineered virus strain according to claim 1 or 2, the genetically engineered virus strain prepared by the preparation method according to any one of claims 3-7, or the genetically engineered virus strain cultured by the culture method according to claim 8 or 9, in the preparation of a preparation for single use, for use in combination with other immune preparations and / or drugs, as a component of a compound preparation composed of other immune preparations and / or drugs, or as a parent virus of a genetically engineered virus to prevent, mitigate and / or control African swine fever, or in the preparation of a preparation for distinguishing and detecting wild African swine fever virus strains and African swine fever vaccine virus strains.

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