Recombinant bivalent live vaccine of African swine fever and classical swine fever and its preparation method and application

Abstract

The present invention discloses a genetically engineered virus strain, which uses a gene-deficient weak strain of African swine fever virus of gene type II as a genetically engineered vector, and has a classical swine fever virus E2 protein gene that can be expressed in the genome. The present invention also discloses a preparation method of the aforementioned genetically engineered virus strain and a vaccine composition containing the aforementioned genetically engineered virus strain. Inoculating pigs with the genetically engineered virus strain as an immunogen can produce antibodies against classical swine fever virus and African swine fever virus, and can provide complete protection against attacks by both classical swine fever strong strains and African swine fever strong strains, and can be used as a recombinant bivalent live vaccine for the prevention and control of classical swine fever and African swine fever.

Description

Technical Field

[0001] The present invention belongs to the field of preventive veterinary medicine, and relates to a recombinant bivalent live vaccine for African swine fever and classical swine fever, and a preparation method and application thereof, and in particular to a recombinant African swine fever virus attenuated strain expressing the E2 protein of the classical swine fever virus. The present invention also relates to a method for constructing the recombinant African swine fever virus strain, and an application thereof in the preparation of a recombinant bivalent live vaccine for preventing and controlling African swine fever virus and classical swine fever virus infection. Background Art

[0002] African swine fever (ASF) is an acute, highly lethal disease caused by infection with the African swine fever virus (ASFV). The World Organization for Animal Health (WOAH) lists it as a legally reportable animal disease, and my country lists it as a Class I animal infectious disease.

[0003] ASFV is the only member of the genus ASFV of the family ASFVidae, and is also the only insect-borne DNA virus. The hosts of ASFV include domestic pigs, African warthogs, jungle pigs, and soft ticks. ASFV-infected pigs show high fever, increased heart rate, difficulty breathing, cyanosis of the skin, edema and bleeding of immune organs such as the spleen and lymph nodes. Infection with strains of different virulence will lead to different clinical manifestations, usually death within 3 to 10 days after infection, and the mortality rate of domestic pigs is close to 100%. ASFV can form a persistent infection in soft ticks without showing obvious clinical symptoms. The full length of the ASFV genome is 170 to 193 kb, containing 150 to 167 open reading frames (ORFs), encoding 150 to 200 proteins, including more than 50 structural proteins and more than 100 non-structural proteins. More than half of the proteins encoded by the ASFV gene are proteins of unknown function.

[0004] In August 2018, ASFV was first reported and isolated in my country. The whole genome sequence analysis showed that it was gene II, and the pathogenicity test showed that it was a strong strain. The inoculated pigs showed an acute onset of disease and 100% mortality. There is an urgent need to carry out ASF vaccine research. At present, domestic researchers have selected a 7-gene deletion strain rASFVHLJ / 18-7GD (abbreviated as HLJ / 18-7GD) based on the African swine fever virus isolate Pig / HLJ / 2018 as the skeleton, which can effectively protect the lethal attack of the strong toxic African swine fever virus and is an ideal vaccine candidate.

[0005] Classical swine fever is a highly contagious and pathogenic disease caused by Classical Swine Fever Virus (CSFV), which is currently spread all over the world. Classical swine fever virus belongs to the genus Pestivirus of the family Flaviviridae, and its genome is a single-stranded positive-strand RNA virus. CSFV is composed of four structural proteins: C, E0, E1 and E2. Among them, E2 protein is the only protective antigen in the structural proteins of CSFV virus that can induce the body to produce neutralizing antibodies.

[0006] The combination of live attenuated vaccines and subunit vaccines is considered an effective genetic engineering vaccine that can induce long-lasting and effective immune protection with a single injection without the need for adjuvants. At present, poxviruses, baculoviruses, adenoviruses and pseudorabies viruses are often used as viral vectors for live attenuated vaccines. Adenoviruses, pseudorabies viruses and poxviruses have been used as live vectors in CSFV vaccine research. However, there are no reports on the use of ASFV as a vector to express CSFV-related proteins for vaccine development. Therefore, the development of a recombinant African swine fever vaccine strain expressing CSFV E2 protein is of groundbreaking significance and can be used to simultaneously prevent ASFV and CSFV, two highly harmful infectious diseases, with huge economic and social benefits. Summary of the invention

[0007] In order to improve the deficiencies in the prior art, the first aspect of the present invention provides a genetically engineered virus strain, wherein the genetically engineered virus strain uses an African swine fever virus vector strain as a genetically engineered vector, and the genome of the genetically engineered virus strain has an expressible classical swine fever virus E2 protein gene.

[0008] According to the embodiments of the present invention, it can be undoubtedly confirmed that the genetically engineered recombinant virus strains formed by transferring the E2 protein gene of the classical swine fever virus into different African swine fever virus strains can produce neutralizing antibodies against the E2 protein of the classical swine fever virus when inoculated into pigs. They are immune to infection with swine fever viruses, and can also produce antibodies against African swine fever viruses that are close to the strains of African swine fever viruses, and are immune to infection with African swine fever viruses. The recombinant virus can be used as an immunogen for a two-in-one vaccine against African swine fever virus and classical swine fever virus. The present invention proves that the mixture of immunogens such as the E2 protein of the classical swine fever virus and all proteins of the African swine fever virus has no visible adverse interactions, and can comprehensively play a role in stimulating the immunity of pigs and synergistically prevent these two diseases of pigs.

[0009] In some embodiments, the amino acid sequence of the classical swine fever virus E2 protein is shown as SEQ ID NO.3.

[0010] In some embodiments, the nucleic acid coding sequence of the classical swine fever virus E2 protein is as shown in SEQ ID NO.2.

[0011] In some embodiments, the promoter of the classical swine fever virus E2 protein gene is the African swine fever virus p72 promoter.

[0012] In some embodiments, the African swine fever virus vector strain is a genotype II African swine fever virus strain.

[0013] In some embodiments, the African swine fever virus vector strain is a weak strain of African swine fever virus.

[0014] In some embodiments, in the genome of the African swine fever virus vector strain, any one, any two, any three, any four, any five, any six or seven of the MGF_505-1R gene, MGF_505-2R gene, MGF_505-3R gene, MGF_360-12L gene, MGF_360-13L gene, MGF_360-14L gene and EP402R gene are inactivated genes.

[0015] In some embodiments, the coding sequence of the MGF_505-1R gene is shown in the sequence of positions 27747-29342 of the genome of the African swine fever virus Pig / HLJ / 2018 strain;

[0016] The coding sequence of the MGF_505-2R gene is shown in the sequence of positions 33133-34713 of the genome of the African swine fever virus Pig / HLJ / 2018 strain;

[0017] The coding sequence of the MGF_505-3R gene is shown in the sequence of positions 34800-35642 of the genome of the African swine fever virus Pig / HLJ / 2018 strain;

[0018] The coding sequence of the MGF_360-12L gene is shown in the sequence of positions 29395-30447 of the genome of the African swine fever virus Pig / HLJ / 2018 strain;

[0019] The coding sequence of the MGF_360-13L gene is shown in the sequence of positions 30608-31669 of the genome of the African swine fever virus Pig / HLJ / 2018 strain;

[0020] The coding sequence of the MGF_360-14L gene is shown in the sequence of positions 31854-32927 of the genome of the African swine fever virus Pig / HLJ / 2018 strain;

[0021] The coding sequence of the EP402R gene is shown in the sequence of positions 73394-74476 of the genome of the African swine fever virus Pig / HLJ / 2018 strain;

[0022] The Genbank sequence number of the Pig / HLJ / 2018 strain is MK333180.1.

[0023] In some embodiments, the inactivated gene fragment is a deletion mutation, an insertion mutation, or a substitution mutation.

[0024] In some embodiments, the microbial preservation number of the African swine fever virus vector strain is: CCTCC NO: V201924.

[0025] In some embodiments, the African swine fever virus vector strain is obtained by genetically engineering the African swine fever virus with a microbial preservation number of CCTCC NO: V202403.

[0026] 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 is to transfer the E2 protein gene of the classical swine fever virus into the African swine fever virus vector strain to obtain the genetically engineered virus strain.

[0027] In some embodiments, the classical swine fever virus E2 protein gene is transferred into the African swine fever virus vector strain by homologous recombination.

[0028] The third aspect of the present invention provides a genetically engineered vaccine composition, which uses 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 as the only immunogen, an immunogen in a multivalent vaccine, or an immunogen in a combined vaccine.

[0029] In some embodiments, the genetically engineered vaccine composition is a live virus vaccine composition.

[0030] In some embodiments, the genetically engineered vaccine composition further comprises an adjuvant.

[0031] The fourth aspect of the present invention provides the use of the genetically engineered virus strain described in the first aspect of the present invention, or the genetically engineered vaccine composition described in the third aspect of the present invention, in the preparation of a preparation for use alone, in combination with other immune preparations and / or drugs, or as a component of a compound preparation composed of other immune preparations and / or drugs to prevent, mitigate and / or control African swine fever and / or classical swine fever.

[0032] The advantages of the present invention over the prior art are: using the gene-deleted recombinant virus HLJ / 18-7GD as a vector to express the immunogenic swine fever virus E2 protein, preparing a genetic engineering vaccine strain rASFV-HLJ / 18-7GD-E2 (abbreviated as HLJ / 18-7GD-E2) that combines the attenuated live vaccine of African swine fever virus with the subunit vaccine of classical swine fever, which can express the E2 protein and has good immunogenicity, and at the same time produces antibodies against African swine fever virus and swine fever virus, and can completely protect against the attack of African swine fever virus and swine fever virus. The present invention confirms that HLJ / 18-7GD can be used as a safe and effective live virus vector to construct a multi-unit vaccine, and the recombinant virus HLJ / 18-7GD-E2 can be used as a recombinant two-unit live vaccine for the prevention and control of African swine fever-swine fever. The present invention supports a new genetic engineering vaccine vector model, which can be used to transfer the main immunogens of other swine pathogens into the gene-deleted recombinant virus HLJ / 18-7GD to prevent African swine fever and other swine diseases at the same time. It can also be used to transfer multiple pathogen antigens to prepare multi-vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A schematic diagram of the vector construction for eGFP gene replacement is shown.

[0034] Figure 2 A schematic diagram of PCR identification of recombinant virus with E2 gene inserted is shown.

[0035] Figure 3 The figure shows the PCR identification results of the recombinant virus inserted into the E2 gene.

[0036] Figure 4 The Western blot results of the recombinant virus strain HLJ / 18-7GD-E2 are shown.

[0037] Figure 5 It shows that the African swine fever virus vaccine strain lacks the eGFP gene but expresses the mCherry fluorescent protein.

[0038] Figure 6 The in vitro growth curves of the recombinant virus HLJ / 18-7GD-E2 and the parental strain HLJ / 18-7GD are shown.

[0039] Figure 7 Shown are the results of body temperature monitoring after piglets were immunized with the HLJ / 18-7GD-E2 recombinant virus.

[0040] Figure 8 Shown are the production of ASFV P30-specific antibodies in piglets at different time points after immunization.

[0041] Fig. 9Shown are the production of CSFV E2-specific antibodies in piglets at different time points after immunization.

[0042] Fig.10 Shown are the body temperature results of piglets challenged with classical swine fever virus after being immunized with the recombinant virus HLJ / 18-7GD-E2.

[0043] Fig.11 The results of survival rate of piglets challenged with classical swine fever virus after being immunized with the recombinant virus HLJ / 18-7GD-E2 are shown.

[0044] Fig.12 Shown are the body temperature results of African swine fever virus challenge in piglets immunized with the recombinant virus HLJ / 18-7GD-E2.

[0045] Fig.13 The results of the survival rate of piglets challenged with African swine fever virus after being immunized with the recombinant virus HLJ / 18-7GD-E2 are shown.

[0046] Fig.14 The figure shows the African swine fever virus content in various tissues and organs after infection. DETAILED DESCRIPTION

[0047] 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.

[0048] The materials and instruments not described in the present invention are conventional materials and instruments in the art, and the operation details not described in the present invention are conventional operations in the art.

[0049] The nucleic acid sequences shown in the present invention are all written from left to right in the 5' to 3' direction.

[0050] 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.

[0051] Material:

[0052] (1) Pig / HLJ / 2018 strain

[0053] 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.

[0054] The isolated African swine fever virus HLJ / 18 strain was submitted to a patent procedure approved depository for deposit. 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 culture name is African swine fever virus ASFV HLJ / 18 African Swine Fever Virus ASFVHLJ / 18; the Chinese classification name is: African swine fever virus; the English classification name is: African Swine Fever Virus; the deposit time is January 5, 2024; the identified survival time is January 11, 2024.

[0055] (2)HLJ / 18-7GD strain

[0056] HLJ / 18-7GD is an attenuated strain of type II African swine fever virus obtained by genetic engineering at the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences using the Pig / HLJ / 2018 strain (Genbank sequence number: MK333180.1).

[0057] The transformation process of the HLJ / 18-7GD strain of this application is recorded in the patent document of Chinese patent application number 201910348878.7, in which the corresponding name is rASFVΔCD2V / 360-eGFP-mCherry strain. The depository of this strain is China Center for Type Culture Collection; the address is Wuhan University, Wuhan, China; the deposit number is: CCTCC NO: V201924; the culture name is African swine fever virus rASFVΔCD2V / 360-eGFP-mCherry, the Chinese classification name is: African swine fever virus; the English classification name is: African Swine Fever Virus; the deposit date is April 24, 2019.

[0058] Compared with the Pig / HLJ / 2018 strain, the HLJ / 18-7GD strain lacked two large fragments. A total of six genes corresponding to the nucleotides 27942-35500 of the Pig / HLJ / 2018 strain genome, including MGF_505-1R (abbreviated as MGF505-1R), MGF_505-2R (abbreviated as MGF505-2R), MGF_505-3R (abbreviated as MGF505-3R), MGF_360-12L (abbreviated as MGF360-12L), MGF_360-13L (abbreviated as MGF360-13L), and MGF_360-14L (abbreviated as MGF360-14L), were replaced by homologous recombination with the red fluorescent protein mCherry gene. The EP402R gene (name of the encoded protein: CD2v) corresponding to nucleotides 73394-74476 of the Pig / HLJ / 2018 strain genome was replaced by the green fluorescent protein eGFP gene by homologous recombination.

[0059] The coding sequence of the MGF_505-1R gene is shown in the sequence of positions 27747-29342 of the genome of the African swine fever virus Pig / HLJ / 2018 strain; the coding sequence of the MGF_505-2R gene is shown in the sequence of positions 33133-34713 of the genome of the African swine fever virus Pig / HLJ / 2018 strain; the coding sequence of the MGF_505-3R gene is shown in the sequence of positions 34800-35642 of the genome of the African swine fever virus Pig / HLJ / 2018 strain; the coding sequence of the MGF_360-12L gene is shown in the sequence of positions 34800-35642 of the genome of the African swine fever virus Pig / HLJ / 2018 strain; / HLJ / 2018 strain genome sequence as shown in the 29395-30447 position; the coding sequence of the MGF_360-13L gene is shown in the 30608-31669 position sequence of the African swine fever virus Pig / HLJ / 2018 strain genome; the coding sequence of the MGF_360-14L gene is shown in the 31854-32927 position sequence of the African swine fever virus Pig / HLJ / 2018 strain genome; the coding sequence of the EP402R gene is shown in the 73394-74476 position sequence of the African swine fever virus Pig / HLJ / 2018 strain genome.

[0060] Compared with the Pig / HLJ / 2018 strain, the HLJ / 18-7GD strain (rASFVΔCD2V / 360-eGFP-mCherry strain) lacked seven genes, namely MGF_505-1R, MGF_505-2R, MGF_505-3R, MGF_360-12L, MGF_360-13L, and EP402R, while retaining the immunogenicity of the Pig / HLJ / 2018 strain and losing the pathogenicity of the Pig / HLJ / 2018 strain.

[0061] The African swine fever virus strain obtained by inactivating the above-mentioned seven genes using a method different from the patent document with Chinese patent application number 201910348878.7 (for example, knocking out by CRISPR / CAS9 technology rather than homologous recombination) is still applicable to the present invention.

[0062] (3) CSFV challenge strain:

[0063] The Shimen strain of classical swine fever virus was purchased from the China Veterinary Microbiological Culture Collection Center, with the virus strain number: CVCCAV1411.

[0064] (4) Primary porcine alveolar macrophages

[0065] Primary porcine alveolar macrophages (PAM) were obtained from 30-50 day old healthy SPF pigs and routinely cultured in 1640 medium containing 10% FBS in a 37° C., 5% CO 2 incubator.

[0066] Example 1. Construction of genetically engineered virus strains

[0067] 1. Construction of homologous recombination vector

[0068] In order to replace the eGFP gene of the HLJ / 18-7GD strain with the classical swine fever virus CSFV E2 gene by homologous recombination, the pCAGGS vector was used as a backbone vector to construct a homologous recombination transfer vector for eGFP gene knockout, and the replaced gene sequence corresponded to positions 73394-74476 of the full-length genome sequence of the Pig / HLJ / 2018 strain.

[0069] The DNA sequence at positions 72394-73393 corresponding to the full-length genome sequence of Pig / HLJ / 2018 strain upstream of eGFP was used as the left homologous arm (LR) of homologous recombination, and the DNA sequence at positions 74477-75476 corresponding to the full-length genome sequence of Pig / HLJ / 2018 strain downstream of eGFP was used as the right homologous arm (RR) of homologous recombination. The CSFV E2 expression cassette (the promoter is the p72 promoter of the African swine fever virus p72 gene, and the coding sequence is the CSFV E2 gene) was inserted between the left homologous arm gene sequence and the right homologous arm gene sequence of the recombinant transfer vector. The CSFV E2 gene expression cassette sequence containing the viral P72 promoter sequence was synthesized by Jilin Kumei Biotechnology Co., Ltd. The three fragments of the left homologous arm, the CSFV E2 expression cassette, and the right homologous arm were cloned into the pCAGGS vector using a one-step cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). After the DNA sequencing was correct, the homologous recombination transfer vector was named pCAGGS-E2; the construction strategy is shown in Figure 1 .

[0070] p72 promoter of African swine fever virus (sequence from -74nt to +1 upstream of the p72 gene of Pig / HLJ / 2018 strain, SEQ ID NO.1):

[0071] TTGTTATTATCAAGATCCTTCGCATAAACCGCCATATTTAATAAAAACAATAAATTATTTTTATAACATTATATA

[0072] The E2 gene sequence of CSFV is available in GenBank No. AAV40685.2.

[0073] The E2 gene coding sequence is (SEQ ID NO.2):

[0074]

[0075] The protein sequence of the E2 gene is (SEQ ID NO.3):

[0076] MKVLRGQIVQGVIWLLLVTGAQGRLACKEDYRYALSSTNEIGLLGAGGLTTTWEEYSHDLQLNDGTVKAICVAGSFKVTALNVVSRRYLASLHKGALLTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRRE KPFPHRMDCVTTTVENEDLFYCKLGGNWTCVKGEPVVYTGGQVKQCKWCGFDFNEPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISAEGSHECLIGNTTVKVHASDERLGPMPCRPKEIVSSAGPVRKTSCTFNYAKTLKNKYYEPRDSYFQQYMLKGEYQYWFDLDVTDRHSDYFA

[0077] One-step cloning kit: purchased from Nanjing Novezan Biotechnology Co., Ltd., catalog number: C113-02.

[0078] 2. Construction and identification of recombinant African swine fever virus HLJ / 18-7GD-E2

[0079] The plasmid pCAGGS-E2 was transfected into PAM cells infected with HLJ / 18-7GD (MOI=1) using Trans IT-LT1 transfection reagent (purchased from Mirus Bio, USA, catalog number MIR 2300) according to the instructions of the kit, and the recombinant African swine fever virus lacking eGFP and expressing the E2 protein of the swine fever virus was purified by the plaque cloning method. Specifically, plaques with red fluorescence and no green fluorescence were picked, and the CSFV E2 gene fragment with a fragment size of 1432 bp was detected by PCR and the CSFV E2 protein was detected by the conventional Western Blot method to identify the positive.

[0080] The obtained recombinant strain was named HLJ / 18-7GD-E2. Compared with the full-length sequence of the African swine fever virus Chinese epidemic strain Pig / HLJ / 2018, the corresponding nucleotides at positions 27942-35500 were replaced with the red fluorescent protein mCherry gene, and the positions of nucleotides 73394-74476 were replaced with the gene expression elements composed of the P72 promoter and the CSFV E2 gene.

[0081] The recombinant virus strain HLJ / 18-7GD-E2 and the parental strain HLJ / 18-7GD were extracted using a viral genome extraction kit (purchased from Beijing Tiangen Biotechnology Co., Ltd.), and primer pairs F (SEQ ID NO.4) and R (SEQ ID NO.5) were designed outside the eGFP coding sequence for PCR identification to confirm whether the deletion was successful. The principle is shown in Figure 2 .

[0082] F: 5'-GAGTCAGTACTATTACGTGATAGTG-3'.

[0083] R: 5'-GAATCACATGATGTTCTCGATGATC-3'.

[0084] The parent strain HLJ / 18-7GD amplified a 985 bp fragment, while the recombinant virus strain HLJ / 18-7GD-E2 amplified a 1432 bp fragment ( Figure 3 ). The above results showed that the HLJ / 18-7GD-E2 recombinant virus had been successfully constructed.

[0085] Conventional Western blot experiments were performed using monoclonal antibodies against the E2 protein of classical swine fever virus as primary antibodies against the E2 protein and monoclonal antibodies against the P30 protein of African swine fever virus as primary antibodies against the P30 protein. The results are shown in Figure 4 , wherein the first lane is a protein marker, the second lane is a negative control (PAM cells), the third and fourth lanes are cultures of the recombinant virus strain HLJ / 18-7GD-E2, and the fifth lane is a positive control (CSFV-E2 protein solution stored in the applicant's laboratory).

[0086] The recombinant virus strain HLJ / 18-7GD-E2 can express P30 protein, indicating that it is modified based on African swine fever virus and can express classical swine fever E2 protein, indicating that the virus modification of the present invention is successful.

[0087] The recombinant virus strain HLJ / 18-7GD-E2 and the parental virus strain HLJ / 18-7GD were infected with PAM cells for 48 hours, and then photographed using an inverted fluorescence microscope. The photograph results are shown in Figure 5 .

[0088] HLJ / 18-7GD can express green light produced by eGFP and red light produced by mCherry, indicating that the parental strain expresses protein normally.

[0089] HLJ / 18-7GD-E2 can express mCherry, indicating that the recombinant strain survives normally and expresses protein, but cannot express eGFP, indicating that the eGFP gene is inactivated, thus confirming that the E2 gene replacement is successful.

[0090] Example 2. Titration of virus titer

[0091] The titration of African swine fever virus was performed using the 50% tissue culture infectious dose (TCID 50 ) for characterization.

[0092] TCID 50 The titration was performed according to the following steps: the ASFV cell supernatant was diluted 10 times in serum-free 1640 medium, inoculated into PAM cells cultured in a 96-well culture plate with a cell density of 90-100%, and 8 wells were inoculated in parallel for each dilution, with 0.02 mL per well. The cells were cultured at 37°C and 5% CO2, and observed for 3-7 days. The cells were judged to be infected based on the red fluorescence, and the half-cell infection dose (TCID) was calculated by the Reed and Muench method. 50 ).

[0093] TCID of the embodiment of the present invention 50 All were measured using this method.

[0094] Analysis of biological characteristics of the recombinant virus strain HLJ / 18-7GD-E2 and the parental strain HLJ / 18-7GD:

[0095] The recombinant virus strain HLJ / 18-7GD-E2 and the parental virus strain HLJ / 18-7GD were inoculated into PAM cells at a virus dose of 0.1 MOI, and the cell supernatant was collected at different time points (0h, 24h, 48h, 72h, 96h and 120h) after infection, and the TCID 50 Determination, according to the determination results draw a curve ( Figure 6 ). The results showed that the growth trends of HLJ / 18-7GD-E2 and the parental virus HLJ / 18-7GD were basically the same, which indicates that the gene recombination operation did not significantly affect the replication and proliferation ability of the ASFV strain.

[0096] Example 3. Animal Immunization Experiment

[0097] 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. Eighteen 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 according to Table 1. The experimental group was inoculated with the recombinant virus HLJ / 18-7GD-E2 by intramuscular injection in the neck, and the control group was not inoculated. After immunization, the mental state and feeding of the animals were continuously observed, and the body temperature of the animals was monitored. Procoagulant blood was collected from the anterior vena cava on days 0, 7, 14, 21, and 28 after immunization for serum separation.

[0098] Table 1. Design of pig immune challenge experiment

[0099]

[0100] All HLJ / 18-7GD-E2 immunized pigs had no abnormal mental state, feeding status, etc. within 28 days after immunization, and all survived normally, with no difference from the control group; all immunized pigs had no significant increase at different time points after immunization ( Figure 7 ), body temperature was below 40°C, indicating that HLJ / 18-7GD-E2 vaccination was safe for pigs.

[0101] Antibody detection was performed using the Classical Swine Fever Virus ELISA Antibody Detection Kit (Harbin Guosheng Biotechnology Co., Ltd., batch number 20220523) and the African Swine Fever Virus ELISA Antibody Detection Kit (Luoyang Putai Biotechnology, batch number 202103). The specific operation method is shown in the instructions. The results showed that after the recombinant virus HLJ / 18-7GD-E2 was immunized in pigs, both ASFV and CSFV specific antibodies were produced at a high level ( Figure 8 , Fig. 9 ), showing good immunogenicity. Figure 8 The S / P calculation formula is: S / P value = sample A value / average A value of positive control; the judgment standard is: S / P value ≥ 0.5 is positive for African swine fever virus antibodies. Fig. 9 The S / P calculation formula is: S / P value = (sample OD 450nm - Negative control serum OD 450nm Average value) / (positive control serum OD 450nm Mean value - negative control serum OD 450nm The judgment standard is: S / P value>0.2 is positive.

[0102] Example 4. Animal challenge test

[0103] After 28 days of immunization of the pigs in Example 3, the immunized group and the control were challenged with ASFV virulent strain (Pig / HLJ / 2018 strain) according to Table 1, with a dose of 10 2.5HAD 50 and CSFV virulent strain Shimen (Shimen strain), the dose was 10 5 TCID 50 The route of infection was intramuscular injection. After infection, the animals' mental state, feeding habits, and body temperature were continuously observed. On the 16th day after CSFV infection and the 28th day after ASFV infection, all surviving pigs were euthanized, and spleen, kidney, tonsil, submandibular lymph node, inguinal lymph node and other tissues and organs were collected for ASFV virus load determination and classical swine fever virus isolation.

[0104] In order to determine the protective effect of HLJ / 18-7GD-E2 recombinant virus against classical swine fever virus, the present invention immunized the test pigs with HLJ / 18-7GD-E2 recombinant virus and then challenged them with the highly virulent strain of classical swine fever virus, Shimen. The results showed that after the challenge with the highly virulent strain of classical swine fever virus, the immunized group developed fever (body temperature above 40°C) on the 2nd to 6th day after the challenge, and basically returned to normal on the 7th day ( Fig.10 A), survived throughout the monitoring period, with a protection rate of 100% ( Fig.11 ); in the blank control group, 3 / 4 pigs developed fever on the second day, and 4 / 4 pigs developed fever on the third day ( Fig.10 B), dead pigs appeared on the 5th day, and all died on the 6th day ( Fig.11 All tissues and organs in the immunized group were negative for virus isolation, while all tissues of all pigs in the blank control group were positive for virus isolation (Table 2).

[0105] Table 2. Results of isolation of swine fever virus from tissues and organs after lethal challenge with CSFV Shimen strain

[0106]

[0107] Note: - indicates that the virus isolation is negative; + indicates that the virus isolation is positive.

[0108] In order to determine the protective effect of HLJ / 18-7GD-E2 recombinant virus against the strong strain of African swine fever virus, the present invention immunized the test pigs with HLJ / 18-7GD-E2 recombinant virus and then challenged them with the strong strain of African swine fever virus Pig / HLJ / 2018. The results showed that after the strong strain of African swine fever virus was challenged, two pigs in the immunized group had transient fever (body temperature above 40°C) on the 4th day after the challenge, one pig had fever on the 7th to 9th day after the challenge, and one pig had fever on the 6th to 11th day after the challenge. The body temperatures of other pigs and other times were normal. On the 12th day after the challenge, all pigs had normal body temperatures ( Fig.12 A). All immunized pigs survived throughout the monitoring period, with a protection rate of 100% ( Fig.13 ); The blank control group began to develop fever on the third day after the challenge ( Fig.12B); Pigs began to die on the 6th day, and all pigs in the blank control group died on the 11th day after the challenge ( Fig.13 ). Referring to the method recommended by WOAH, the qPCR method was used to detect the African swine fever virus p72 gene in tissues and organs. The samples included heart, liver, lung, spleen, kidney, tonsil, intestinal lymph node (LN1), inguinal lymph node (LN2), submandibular lymph node (LN3), bronchial lymph node (LN4), hepatogastric lymph node (LN5), mediastinal lymph node (LN6)). The results showed that after the immunization group was challenged with the virus, only low levels of virus content were detected in individual organs (CT values ​​were all >31). After the blank control group was challenged with the virus, high levels of virus content were detected in all organs (CT values ​​were all <26) ( Fig.14 ).

[0109] The above experimental results prove that the weak strain of African swine fever virus HLJ / 18-7GD can be used as a safe and effective live virus vector to construct a recombinant multi-unit vaccine. The recombinant African swine fever virus HLJ / 18-7GD-E2 expressing CSFV E2 has good safety and immunogenicity. After immunizing piglets, it can provide complete protection against attacks of both strong strains of swine fever and strong strains of African swine fever, and can be used to develop an economical and efficient recombinant two-unit live vaccine of African swine fever-swine fever.

[0110] 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, wherein the genetically engineered virus strain uses an African swine fever virus vector strain with a microbial collection number of CCTCC NO: V201924 as a genetically engineered vector, and the genome of the genetically engineered virus strain has an expressible classical swine fever virus E2 protein gene; the position of nucleotides 73394-74476 corresponding to the genome sequence shown in Genbank sequence number MK333180.1 in the genome of the genetically engineered virus strain is replaced with a gene expression element consisting of an African swine fever virus p72 promoter and the classical swine fever virus E2 gene.

2. The genetically engineered virus strain according to claim 1, characterized in that: The amino acid sequence of the classical swine fever virus E2 protein is shown in SEQ ID NO.

3.

3. The genetically engineered virus strain according to claim 2, characterized in that: The nucleic acid coding sequence of the classical swine fever virus E2 protein is shown in SEQ ID NO.

2.

4. A method for preparing the genetically engineered virus strain according to any one of claims 1 to 3, wherein the preparation method comprises transferring the E2 protein gene of the classical swine fever virus into the African swine fever virus vector strain to obtain the genetically engineered virus strain.

5. The preparation method according to claim 4, characterized in that: The E2 protein gene of the classical swine fever virus is transferred into the African swine fever virus vector strain through homologous recombination method.

6. A genetically engineered vaccine composition, wherein the genetically engineered virus strain described in any one of claims 1 to 3 or the genetically engineered virus strain prepared by the preparation method described in claim 4 or 5 is the only immunogen, an immunogen in a multivalent vaccine, or an immunogen in a combined vaccine.

7. The genetically engineered vaccine composition according to claim 6, characterized in that: The genetically engineered vaccine composition is a live virus vaccine composition; And / or, the genetically engineered vaccine composition further comprises excipients.

8. Use of the genetically engineered virus strain described in any one of claims 1 to 3, or the genetically engineered vaccine composition described in 6 or 7, in the preparation of a preparation for use alone, in combination with other immune preparations and / or drugs, or as a component of a compound preparation composed of other immune preparations and / or drugs to prevent, mitigate and / or control African swine fever and / or classical swine fever.

Images