Construction of R298L gene deleted African swine fever virus strain and application of R298L gene deleted African swine fever virus strain as vaccine

Abstract

The invention belongs to the technical field of biology, and particularly relates to construction of an R298L gene deleted and attenuated African swine fever virus strain and application of the R298L gene deleted and attenuated African swine fever virus strain as a vaccine. Firstly, the invention provides a recombinant virus with R298L gene deletion, and the recombinant virus utilizes a parent strain ASFV CN / GS / 2018 to recombine a recombinant strain with R298L gene deletion through a genetic engineering means; secondly, the invention provides an African swine fever virus vaccine candidate strain, after pigs are immunized by the ASFV recombinant virus constructed by the invention, the pathogenicity of the pigs is remarkably reduced, clinical symptoms such as death do not occur, the survival rate is 100%, the generation of neutralizing antibodies can be induced, and the African swine fever virus vaccine candidate strain can be used as a safe and effective African swine fever vaccine candidate strain, and has good application prospects. And the method has great social application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to the construction of an African swine fever virus strain with a R298L gene deletion and its application as a vaccine. Background Art

[0002] African swine fever (ASF) is an acute, febrile, highly contagious animal disease of pigs caused by infection with African swine fever virus (ASFV). The World Organization for Animal Health has listed it as a notifiable Class A animal disease. The disease is characterized by a short course, rapid spread, and a mortality rate of up to 100%. Due to the huge viral genome, encoding more than one hundred viral proteins, and complex structure, nearly 50% of the interaction mechanisms between viral proteins and the host are still unclear, which increases the difficulty of vaccine research. So far, no safe and effective vaccine is available. Therefore, exploring the interaction mechanism between African swine fever virus proteins and host proteins can provide a deep understanding of the pathogenic mechanism of ASFV, explore the virulence genes of the virus, and provide a theoretical basis for the design of efficient vaccines.

[0003] ASFV is the only member of the African swine fever virus family and the genus African swine fever virus. Its genome is a double-stranded DNA of 170-193kb and a diameter of about 260nm. It consists of five layers of structures: nucleoid, core shell, inner membrane, capsid and outer envelope. It contains 150-167 open reading frames and can encode more than 150 viral proteins, including more than 50 structural proteins and more than 100 non-structural proteins. The African swine fever virus protein R298L is a serine / threonine protein kinase. This type of kinase regulates the activity and level of key adaptor proteins in various intracellular pathways through post-transcriptional modifications including phosphorylation, controls the activation of intracellular pathways, and further regulates cell metabolism, inflammatory response, and even apoptosis, necrosis and other processes that determine cell fate, thereby affecting the replication of the virus. At present, there are no relevant research reports on R298L gene deletion strains.

[0004] To date, there is still no effective commercial vaccine available. Traditional inactivated vaccines do not have ideal protective effects. MLV vaccines have safety issues and can easily cause chronic infection. The development of the most promising genetically engineered attenuated live vaccines, subunit vaccines, and DNA vaccines requires a comprehensive understanding of viral virulence, replication, or innate immune response-related genes. The candidate virulence genes that have been discovered include DP96R, multigene family MGF, DP71L, A238L, A224L, I177L, B646L, CP204L, E183L, CP530R, etc.

[0005] Both L83L and R298L can inhibit the expression of the interferon pathway, but existing literature shows that the virulence of the African swine fever virus knocked out by L83L is as strong as that of the wild strain, and is not suitable for the preparation of attenuated strains. The present invention deletes the R298L gene in the African swine fever parent strain ASFV / CN / GS / 2018 through genetic engineering technology, and obtains a highly safe attenuated strain of the African swine fever candidate vaccine, which reduces the pathogenicity of the virus to pigs, can significantly induce immune pigs to produce antibody protection, and can be used as a safe and effective candidate vaccine for African swine fever, which has great social application value. Summary of the invention

[0006] In view of the above technical problems, the object of the present invention is to provide a strategy for constructing an attenuated strain of African swine fever, which is achieved through the following technical solutions:

[0007] In the first aspect, the present invention provides an application for preparing an African swine fever attenuated strain by deleting the R298L gene in the type II African swine fever virus strain ASFV CN / GS2018. The type II African swine fever virus strain ASFV CN / GS2018 was deposited in the China Center for Type Culture Collection on December 21, 2020, with the preservation number CCTCC NO: V202096.

[0008] Preferably, the R298L gene sequence is shown as SEQ ID NO.1.

[0009] In the second aspect, the present invention provides an application of preparing an African swine fever attenuated vaccine by deleting the R298L gene in the type II African swine fever virus strain ASFV CN / GS2018. The type II African swine fever virus strain ASFV CN / GS2018 was deposited in the China Center for Type Culture Collection on December 21, 2020, with the preservation number CCTCC NO: V202096.

[0010] Preferably, the R298L gene sequence is shown as SEQ ID NO.1.

[0011] In the third aspect, the present invention provides a gene-deleted attenuated African swine fever virus strain, which is a type II African swine fever virus strain ASFV CN / GS2018 with a R298L gene deletion. The type II African swine fever virus strain ASFV CN / GS2018 has been deposited in the China Center for Type Culture Collection on December 21, 2020, with the preservation number CCTCC NO: V202096.

[0012] Preferably, the R298L gene sequence is shown as SEQ ID NO.1.

[0013] In a fourth aspect, the present invention provides the use of the gene-deleted attenuated African swine fever virus strain described in the third aspect in the preparation of an African swine fever vaccine.

[0014] In a fifth aspect, the present invention provides an attenuated African swine fever vaccine, which comprises the gene-deleted attenuated African swine fever virus strain described in the third aspect above.

[0015] In a sixth aspect, the present invention provides a method for preparing the attenuated African swine fever virus strain described in the third aspect, the method comprising: deleting all or part of the nucleotide sequence of the R298L gene of the parental African swine fever virus strain by genetic engineering means to thereby lose the function of the protein encoded by the R298L gene.

[0016] Preferably, the method is a homologous recombination technique, and the method comprises the following steps:

[0017] (1) Design the upstream and downstream sequences of the ASFV R298L gene as the left and right homologous recombination arms and clone them into the pUC19 vector respectively to obtain the recombinant transfer vector;

[0018] (2) inserting the screening expression cassette gene fragment into the left and right arm gene sequences of the recombinant transfer vector in step (1) to obtain a homologous recombinant transfer vector;

[0019] (3) Transfecting the homologous recombination transfer vector described in step (2) into BMDM cells infected with the parental African swine fever virus strain, purifying and screening to obtain an attenuated African swine fever virus strain in which the protein encoded by the R298L gene has lost its function.

[0020] The beneficial effects of the present invention are as follows: the present invention obtains an attenuated African swine fever virus strain by losing the function of the protein encoded by the R298L gene of the type II African swine fever virus strain ASFV CN / GS2018, and the virulence of the attenuated African swine fever virus strain is significantly weakened, and has good safety performance. By losing the function of the protein encoded by the R298L gene in the parent strain (type II African swine fever virus strain ASFV CN / GS2018), the toxicity of the parent strain is reduced, and the obtained attenuated African swine fever virus can provide good immune protection against the attack of the parent strain of African swine fever virus, and has high safety, and is suitable as a candidate strain for a vaccine to prevent African swine fever. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the construction of a homologous recombination transfer vector used to prepare an African swine fever virus strain with a R298L gene deletion;

[0022] Figure 2 Fluorescence detection results of the recombinant virus of the African swine fever virus strain ASFVΔR298L with R298L gene deletion;

[0023] Figure 3 The results of PCR identification of the gene deletion of the African swine fever virus strain ASFVΔR298L with R298L gene deletion;

[0024] Figure 4 Animal temperature monitoring results after challenge with African swine fever virus strain ASFVΔR298L and parental strains;

[0025] Figure 5 Animal survival after challenge with African swine fever virus strain ASFVΔR298L and the parental strain;

[0026] Figure 6 Pathological changes in animal tissues after infection with African swine fever virus strain ASFVΔR298L and the parental strain;

[0027] Figure 7 Comparison of viral copy numbers in animal sera after challenge with African swine fever virus strain ASFVΔR298L and the parental strain;

[0028] Figure 8 Comparison of neutralizing antibody levels in animal serum after infection with African swine fever virus strain ASFVΔR298L and the parental strain. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0030] Example 1 Construction, purification and identification of an attenuated African swine fever virus strain with loss of function of the protein encoded by the R298L gene

[0031] The Chinese epidemic strain of African swine fever virus ASFV / CN / GS / 2018 was isolated and preserved by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Lungs were taken from healthy pigs aged 30 to 50 days. The lavage fluid obtained by lavaging the lungs with sterile PBS was centrifuged at low speed to obtain porcine alveolar macrophages. The cells were resuspended in RPMI 1640 medium (purchased from ThermoScientific) containing 10% FBS (purchased from BI) and cultured in a 37°C, 5% CO2 incubator.

[0032] 1. Construction of homologous recombination transfer vector

[0033] The ASFV / CN / GS / 2018R298L gene sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. About 1 kb of the upstream and downstream sequences of the R298L gene were used as left and right homologous recombination arms. The left arm (Left arm, shown in SEQ ID NO.3) and the right arm (Right arm, shown in SEQ ID NO.4) and the PCR fragment of eRFP were cloned into the pUC57 vector using a homologous recombination kit, wherein the eRFP gene was expressed using the p30 promoter to obtain the plasmid pUC57-LRΔR298L-eRFP. The construction strategy is as follows: Figure 1 shown.

[0034] 2. Screening, purification and identification of R298L gene-deleted recombinant viruses

[0035] Homologous recombination transfer plasmid pUC57-LRΔR298L-eRFP was transfected into porcine BMDM cells (cell number was about 10) using lipo3000 transfection reagent. 6 After 6 hours of transfection, the cells were directly infected with ASFV / CN / GS / 2018 (MOI=0.01) without changing the medium. The number of fluorescent cells was observed under a fluorescence microscope for 48 hours after infection. The cells were digested, and the fluorescent cells in all single wells were picked and carefully dispersed in a new culture dish. After settling for 1 hour, all single fluorescent cells were picked. After repeated freezing and thawing, the pre-plated 96-well plate BMDM cells were inoculated, and the fluorescent cell wells were observed. After marking, the observation was continued for 72 hours. If the proportion of fluorescent cells was high, such as Figure 2 As shown, this indicates that the construction of the recombinant virus was basically successful.

[0036] The fully positive wells were expanded by 10 times of limited dilution culture, and the 13th generation recombinant virus wells were picked and digested into single cells. 10 fluorescent cells were carefully aspirated and inoculated into pre-laid 96-well plate PAM cells respectively, and continued to grow for 72 hours. Cells with more RFP fluorescence were picked, and the genomes of ASFV and gene-deficient ASFV were extracted using a viral genome extraction kit (purchased from Beijing Tiangen Biotechnology Co., Ltd.). PCR identification was performed using primers for R298L-F / R to confirm whether the deletion was successful, where R298L-F: 5'-ATGTATTTCATTGCGACGA-3' (SEQ ID NO.5), R298L-R: 5'-CTTCGGTTTCCTCAAGTTT-3' (SEQ ID NO.6). The results are as follows: Figure 3 As shown, the parental strain ASFV WT can detect the R298L gene, while the gene-deficient strain ASFVΔR298L cannot detect the R298L gene, indicating that ASFVΔR298L was successfully constructed and purified.

[0037] Example 2 Virus titer determination of ASFV wild strains and recombinant strains

[0038] The virus was adsorbed to red blood cells (HAD) using a 50% haemadsorption test. 50 ) Determine the titer of African swine fever virus. Take 100 μl of the above wild strain and recombinant virus respectively for 10-fold gradient dilution, inoculate the pre-laid 96-well monolayer of primary porcine PAM cells, add 25 μl of 1% red blood cell suspension to each well, and the virus infection can be determined by the rosette formed by the aggregation of red blood cells around the infected cells. The first plate reading can be carried out after 12-16 hours of culture, and the observation is continued for 7 days. The number of positive wells is counted, and the HAD of the virus is calculated according to the Reed and Muench method. 50 .

[0039] Example 4 Animal challenge experiment with the recombinant African swine fever virus strain ASFVΔR298L

[0040] The pig challenge experiment was conducted in the biosafety level 3 experimental animal room of the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and 8 healthy Landrace piglets aged 7 weeks were introduced. After the pigs adapted for 2-3 days, they were divided into 3 groups, as shown in Table 1, and the same infection dose of attenuated African swine fever virus strain ASFVΔR298L and parent strain ASFV / CN / GS / 2018 were injected intramuscularly for challenge, and PBS injection was used as an uninfected control. After the challenge, the mental state of the animals was continuously observed, the body temperature of the animals was monitored, anticoagulated blood and non-anticoagulated blood were collected and serum was separated, and the observation was terminated after 21 days.

[0041] Table 1 Animal challenge experimental plan

[0042]

[0043] like Figure 4 and 5 As shown, animals in group 2 began to develop persistent high fever on the third day after infection with the wild-type virus, and died successively from the ninth to the eleventh day. Animals in group 3 had a slight fever after infection with ASFVΔR298L, and the survival rate was 100% until the 21st day of monitoring. Figure 6 The results of pathological examination showed that the heart, liver, spleen, lung, kidney and lymph nodes of animals in Group 2 were enlarged, and some organs suffered severe bleeding. The pathological changes of organs of animals in Group 3 were not significant after infection with the recombinant virus ASFVΔR298L.

[0044] Figure 7The results showed that the serum virus content after ASFVΔR298L infection was maintained at a low level and was significantly lower than that after infection with the wild-type virus. In addition, ASFVΔR298L was able to significantly induce the production of neutralizing antibody p30 starting from the eleventh day after infection, providing effective protection for animals. Figure 8 shown.

[0045] The above results show that compared with the wild strain, the ASFVΔR298L recombinant strain is sufficiently weak in pigs, its pathogenicity is significantly reduced, and it can significantly induce the body to produce neutralizing antibodies, so it can be used as a candidate target for gene-deficient vaccines.

[0046] The above-mentioned embodiments are only embodiments of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, other improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. The use of preparing an attenuated African swine fever strain by deleting the R298L gene in a type II African swine fever virus strain ASFV CN / GS2018, characterized in that: The type II African swine fever virus strain ASFV CN / GS2018 is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: V202096.

2. The use according to claim 1, characterized in that The R298L gene sequence is shown in SEQ ID NO.

1.

3. The use of an attenuated African swine fever vaccine prepared by deleting the R298L gene in a type II African swine fever virus strain ASFV CN / GS2018, characterized in that: The type II African swine fever virus strain ASFV CN / GS2018 is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: V202096.

4. The use according to claim 3, characterized in that The R298L gene sequence is shown in SEQ ID NO.

1.

5. A gene-deleted attenuated African swine fever virus strain, characterized in that: The gene-deficient attenuated African swine fever virus strain is a type II African swine fever virus strain ASFV CN / GS2018 with a R298L gene deletion. The type II African swine fever virus strain ASFV CN / GS2018 is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: V202096.

6. The attenuated African swine fever virus strain according to claim 5, characterized in that The sequence of the protein encoded by the R298L gene is shown in SEQ ID NO.2; the sequence of the R298L gene is shown in SEQ ID NO.

1.

7. Use of the gene-deleted attenuated African swine fever virus strain as described in claim 5 or 6 in the preparation of African swine fever vaccine.

8. An attenuated African swine fever vaccine, characterized in that: The attenuated African swine fever vaccine comprises the gene-deleted attenuated African swine fever virus strain described in claim 5 or 6.

9. The method for preparing the attenuated African swine fever virus strain according to claim 5 or 6, characterized in that: The method comprises: deleting all or part of the nucleotide sequence of the R298L gene of the parental African swine fever virus strain by genetic engineering means so as to lose the function of the protein encoded by the R298L gene.

10. The method according to claim 9, characterized in that The method is a homologous recombination technique, and the method comprises the following steps: (1) Design the upstream and downstream sequences of the ASFV R298L gene as the left and right homologous recombination arms and clone them into the pUC19 vector respectively to obtain the recombinant transfer vector; (2) inserting the screening expression cassette gene fragment into the left and right arm gene sequences of the recombinant transfer vector in step (1) to obtain a homologous recombinant transfer vector; (3) Transfecting the homologous recombination transfer vector described in step (2) into BMDM cells infected with the parental African swine fever virus strain, purifying and screening to obtain an attenuated African swine fever virus strain in which the protein encoded by the R298L gene has lost its function.

Images