African swine fever virus attenuated strain, preparation method therefor and use thereof

Abstract

Provided are an African swine fever virus attenuated strain, a preparation method therefor, and a use thereof in preparation of drugs (such as vaccines) for preventing African swine fever virus infection. The African swine fever virus attenuated strain has deletion of A137R and CD2V genes or functional fragments thereof in a genome relative to a wild-type strain; compared with a wild-type virus strain, the African swine fever virus attenuated strain has greatly reduced virulence, can induce high-level humoral immune response against African swine fever virus, can protect experimental animals from attack by lethal doses of highly virulent strains of African swine fever virus, and thus has excellent application prospects in preventing African swine fever virus infection.

Description

An attenuated African swine fever virus strain, preparation method and application thereof

[0001] Cross-references

[0002] This application claims priority to the Chinese patent application filed on September 18, 2023, with application number 202311203922.8 and invention name “A attenuated strain of African swine fever virus, its preparation method and application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application belongs to the field of bioengineering technology, and specifically relates to an attenuated strain of African swine fever virus, a preparation method thereof, and an application thereof. Background Art

[0004] African swine fever (ASF) is an acute, febrile, highly contagious disease of swine caused by the African swine fever virus (ASFV). It is listed as a notifiable disease by the World Organization for Animal Health (OIE) and as a Category I animal disease in my country. ASF was first reported in Kenya in 1921 and has since become endemic primarily in African countries. It first appeared outside of Africa in Portugal in 1957 and has since spread throughout Europe. In 2007, an outbreak in Georgia in the Caucasus marked the third phase of ASF's outbreak. In August 2018, my country reported its first outbreak of ASF, which subsequently spread rapidly throughout the country, causing significant losses to the country's swine industry. Currently, ASF remains a persistent threat to the swine industry in my country and globally.

[0005] However, due to limited basic research on the functions of ASFV-related genes and proteins, as well as immunology, there is no commercially available vaccine for ASFV in China. Research over the past few decades has shown that while inactivated ASFV vaccines can induce antibody production in immunized animals, they do not effectively protect against virulent infection. Compared to inactivated vaccines, subunit vaccines can increase the effective antigen content and are safer. However, while subunit vaccines can stimulate targeted humoral immunity, their effects on cellular immunity are limited. Furthermore, it is difficult to fully and effectively protect animals against virulent infection with a single protein antigen as a subunit vaccine; effective use requires the combined use of multiple protein antigens.

[0006] Genetic engineering to knock out genes associated with viral virulence and construct gene-deficient strains of African swine fever is an effective means of studying virus-host interactions, pathogenic mechanisms, immune escape, and vaccine development. Existing technologies have been used to construct strains with gene deletions, such as I177L and MGF. However, due to the difficulty in achieving a good balance between reducing virulence and maintaining immunogenicity, with the exception of Vietnam, which launched an attenuated African swine fever vaccine with I177L deletion in 2022, no other country has approved the marketing of an African swine fever vaccine.

[0007] In view of the above, there is an urgent need for an African swine fever virus vaccine that is both highly safe and has sufficient immune protection.

[0008] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the application and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0009] Summary of the Invention

[0010] Purpose of the Invention

[0011] In response to the defects in the above-mentioned prior art, the purpose of this application is to provide an attenuated strain of African swine fever virus with greatly reduced virulence and sufficient immune protection, a preparation method thereof, and its use in the preparation of drugs (especially vaccines) for preventing African swine fever virus infection.

[0012] Solution

[0013] To achieve the purpose of this application, this application provides the following technical solutions:

[0014] In a first aspect, the present application provides an attenuated strain of African swine fever virus, which lacks the following genes or fragments thereof in its genome relative to the wild-type African swine fever virus:

[0015] (1) A137R gene or a functional fragment thereof; and

[0016] (2) CD2V gene or its functional fragment.

[0017] In a feasible embodiment, the wild-type African swine fever virus is a type II African swine fever virus, preferably selected from: HuB 2019, Pig / CN / HLJ / 2018 (GeneBank accession number is MK333180.1), ASFV-SY18, Georgia 2008 / 1, Georgia 2007 / 1, ASFV GZ2018, and most preferably HuB 2019.

[0018] In a feasible embodiment, the A137R gene or a functional fragment thereof is shown in SEQ ID NO: 1:

[0019] In a feasible embodiment, the CD2V gene or a functional fragment thereof is shown in SEQ ID NO: 2:

[0020] In a second aspect, the present application provides a method for preparing an attenuated African swine fever virus strain as described in the first aspect above, comprising the following steps:

[0021] Through genetic engineering, the A137R gene or its fragment and the CD2V gene or its fragment of the wild-type African swine fever virus are deleted.

[0022] In a feasible embodiment, the genetic engineering method is CRISPR-Cas9 technology combined with homologous recombination technology, which specifically includes the following steps:

[0023] S1) constructing CRISPR-Cas9 plasmids containing guide RNA sequences for the CD2v gene or its functional fragment and the A137R gene or its functional fragment to be knocked out, respectively;

[0024] S2) constructing a homologous recombination plasmid comprising a homologous recombination cassette of the CD2v gene and a homologous recombination cassette of the A137R gene, respectively; and,

[0025] S3) The CRISPR-Cas9 plasmid and homologous recombination plasmid constructed above are co-transfected into PAM cells, and then the wild-type African swine fever virus strain is inoculated. After screening, an attenuated African swine fever virus strain that simultaneously lacks the CD2V gene or its functional fragment and the A137R gene or its functional fragment is obtained.

[0026] Preferably, in step S1), there are three Guide RNA sequences for the CD2v gene to be knocked out or its functional fragment, as shown in SEQ ID NOs: 3, 4, and 5, respectively;

[0027] and / or, there are three Guide RNA sequences for the A137R gene to be knocked out or its functional fragment, as shown in SEQ ID NOs: 6, 7, and 8, respectively;

[0028] Preferably, the CRISPR-Cas9 plasmid uses the PX458 vector as a backbone, in which two nuclear localization signal sequences are removed.

[0029] Preferably, in step S2), the homologous recombination frame of the CD2v gene includes: a homologous recombination arm 1065 bp upstream of the CD2v open reading frame, a first LoxP recombination site, a p72 promoter sequence, an enhanced green fluorescent protein, a second LoxP recombination site, and a homologous recombination arm 974 bp downstream of the CD2v open reading frame;

[0030] And / or, the homologous recombination frame of the A137R gene includes: a homologous recombination arm 1000 bp upstream of the A137R open reading frame, a first LoxP recombination site, a p72 promoter sequence, a red fluorescent protein, a second LoxP recombination site, and a homologous recombination arm 1000 bp downstream of the A137R open reading frame.

[0031] In a third aspect, the present application provides an African swine fever virus-expressed protein, which is a protein expressed by the attenuated African swine fever virus strain as described in the first aspect above, preferably a full protein expressed thereby.

[0032] In a fourth aspect, the present application provides the use of the attenuated African swine fever virus strain as described in the first aspect above or the African swine fever virus expression protein as described in the third aspect above in the preparation of a drug for preventing African swine fever virus infection.

[0033] Preferably, the drug is a vaccine, more preferably a live attenuated vaccine, a whole virus inactivated vaccine or a protein vaccine;

[0034] Further preferably, the vaccine is in the form of an oral preparation or a parenteral preparation;

[0035] Optionally, the oral preparation is selected from tablets, powders, granules and oral liquids; further optionally, the granules are fine granules;

[0036] Optionally, the parenteral formulation is an injectable or bolus formulation.

[0037] In a fifth aspect, the present application provides a live attenuated African swine fever virus vaccine, comprising the attenuated African swine fever virus strain described in the first aspect as an active ingredient;

[0038] Preferably, in the attenuated live vaccine, the virus content of the attenuated African swine fever virus strain is ≥10 TCID 50 / ml;

[0039] Preferably, the live attenuated vaccine is in the form of an oral preparation or a parenteral preparation;

[0040] Optionally, the oral preparation is selected from tablets, powders, granules and oral liquids; further optionally, the granules are fine granules;

[0041] Optionally, the parenteral formulation is an injectable or bolus formulation.

[0042] In a sixth aspect, the present application provides a method for preparing a live attenuated African swine fever virus vaccine as described in the fifth aspect above, comprising the following steps:

[0043] 1) inoculating the attenuated African swine fever virus strain described in the first aspect above into primary PAM cells, expanding the culture, and harvesting the virus liquid;

[0044] 2) The virus content is ≥10TCID 50 / ml of virus liquid can be made into vaccine directly or with adjuvant.

[0045] In a seventh aspect, the present application provides an inactivated whole virus vaccine of an attenuated African swine fever virus strain, comprising as an active ingredient an inactivated whole virus obtained by inactivating the attenuated African swine fever virus strain as described in the first aspect above;

[0046] Preferably, the inactivated vaccine is in the form of a parenteral formulation;

[0047] Optionally, the parenteral formulation is an injectable or bolus formulation;.

[0048] In an eighth aspect, the present application provides an attenuated whole protein vaccine of African swine fever virus, comprising the African swine fever virus expressed protein as described in the third aspect above as an active ingredient;

[0049] Preferably, the whole protein vaccine is in the form of a parenteral formulation;

[0050] Optionally, the parenteral formulation is an injectable or bolus formulation.

[0051] In the ninth aspect, the present application provides a method for preventing African swine fever virus disease, comprising: administering to a subject in need thereof a preventively effective amount of the attenuated strain of African swine fever virus as described in the first aspect above, or the African swine fever virus expressed protein as described in the third aspect above, or the attenuated live vaccine of African swine fever virus as described in the fifth aspect above, or the whole virus inactivated vaccine of the attenuated strain of African swine fever virus as described in the seventh aspect above, or the attenuated whole protein vaccine of African swine fever virus as described in the eighth aspect above.

[0052] The "preventively effective amount" can be determined based on the local African swine fever epidemic situation, the type of dosage form, the method of administration, etc., according to the judgment of the veterinarian. Beneficial effects

[0053] This application uses genetic engineering to delete both the A137R and CD2v genes of wild-type African swine fever virus to construct an attenuated African swine fever virus strain; the constructed attenuated African swine fever virus strain has the following advantages:

[0054] (1) Fully attenuated; its virulence is greatly reduced compared to the wild-type virus, therefore, it has extremely high safety;

[0055] (2) The characteristics are stable and the genetic stability is good, so the possibility of reverting to the wild-type virus is extremely low;

[0056] (3) It can induce an effective immune protective response in animals, thereby protecting them from attack by a lethal dose of the parental African swine fever virus virulent strain;

[0057] (4) When constructing the attenuated African swine fever virus strain, a loxP element can be inserted into the strain genome, and the fluorescent label gene introduced during gene knockout can be removed if necessary.

[0058] In view of the above, the attenuated African swine fever virus strain of the present application is a very potential candidate strain for African swine fever virus vaccine (especially attenuated live vaccine), and has good application prospects in preventing African swine fever virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] One or more embodiments are exemplarily illustrated by the accompanying figures, and these exemplary illustrations do not limit the embodiments. The word "exemplary" is used herein to mean "serving as an example, example, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0060] Figure 1 is a schematic diagram of the construction of the ASFV mutant strain ASFVΔCD2v / A137R with double gene deletions of A137R and CD2v described in Example 1 of the present application.

[0061] FIG2 shows the PCR identification results of the recombinant virus strains with single gene deletion of CD2v and A137R and double gene deletion of CD2v / A137R described in Example 1 of the present application.

[0062] Figure 3 shows the qPCR identification results of the recombinant virus strain with double CD2v / A137R gene deletion described in Example 1 of the present application; wherein A is the qPCR amplification curve of the p72 gene, B is the qPCR amplification curve of the CD2v gene, and C is the qPCR amplification curve of the A137R gene.

[0063] Figure 4 is a survival rate curve of piglets in the ASFVΔCD2v / A137R vaccination group and the control group described in Example 4 of the present application.

[0064] Figure 5 is the body temperature change curve (upper figure) and clinical symptom score (lower figure) of the ASFVΔCD2v / A137R inoculated group and the control group piglets after virus infection described in Example 4 of the present application. DETAILED DESCRIPTION

[0065] To make the purpose, technical solutions, and advantages of the embodiments of this application more clear, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of this application, not all of them. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0066] In addition, in order to better illustrate the present application, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present application can be implemented without certain specific details. In some embodiments, raw materials, components, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present application.

[0067] Unless expressly stated otherwise, throughout the specification and claims, the term "comprise" or variations such as "include" or "comprising", etc., will be understood to include the stated elements or components but not to exclude other elements or other components.

[0068] Biosafety License and African Swine Fever Laboratory Activity License: In accordance with the relevant requirements of Biosafety Level 3 Laboratory (ABSL-3) and African Swine Fever-related biosafety, after review by the Biosafety Management Committee and the Laboratory Animal Welfare Ethics Committee of the China Veterinary Drug Administration, the China Veterinary Drug Administration has obtained a license from the Ministry of Agriculture and Rural Affairs to carry out laboratory activities for highly pathogenic animal pathogens including ASFV, and has filed it with the Ministry of Agriculture and Rural Affairs. The ASFV test activities related to this application were carried out in the ABSL-3 laboratory of the National Veterinary Microbiology Center of the China Veterinary Drug Administration, which meets the corresponding national biosafety level requirements.

[0069] Example 1. Construction and identification of ASFV recombinant strains with single gene deletion of CD2v and A137R and double gene deletion of CD2v / A137R

[0070] In this example, based on the ASFV HuB 2019 strain, genetic engineering technologies such as CRISPR-Cas9 and homologous recombination were used to construct and identify ASFV recombinant strains with a single CD2v gene deletion (hereinafter referred to as ASFVΔCD2v), a single A137R gene deletion (hereinafter referred to as ASFVΔA137R), and a double CD2v and A137R gene deletion (hereinafter referred to as ASFVΔCD2v / A137R).

[0071] The schematic diagram of the construction of the ASFV recombinant strain ASFVΔCD2v / A137R with double gene deletions of CD2v and A137R is shown in Figure 1.

[0072] 1.1 Construction of single or double gene deletion strains

[0073] 1.1.1 Construction of CRISPR-Cas9 plasmid

[0074] Specifically, the PX458 vector (Nanjing GenScript Biotech Co., Ltd.) was used as the backbone, its two nuclear localization NLS sequences were removed, and three different guide RNA sequences targeting the CD2v gene and the A137R gene were inserted, respectively (see Table 1), thereby constructing three CRISPR-Cas9 plasmids targeting the CD2v and A137R genes.

[0075] Table 1. Guide RNA sequences for knockout of CD2v and A137R genes

[0076] 1.1.2 Construction of homologous recombination plasmid

[0077] Specifically, the plasmid pBlueScriptSK-2 (Shanghai Sangon Biotech Co., Ltd.) was used as a backbone, and the homologous recombination cassettes of the CD2v and A137R genes were inserted respectively to construct homologous recombination plasmids of the two.

[0078] Among them, the homologous recombination frame for knocking out the CD2v gene includes: a homologous recombination arm 1065bp upstream of the CD2v open reading frame (refer to the fragment between 72279nt and 73343nt of the ASFV MK333180.1 strain in GenBank), the first Loxp recombination site, the p72 promoter sequence (located between 105559nt and 105597nt of the negative chain of the above-mentioned reference strain), enhanced green fluorescent protein (eGFP), the second loxp recombination site, and a 974bp homologous recombination arm downstream of the CD2v open reading frame (located between 74443nt and 75416nt of the above-mentioned reference strain); the homologous recombination frame for knocking out the A137R gene includes: a homologous recombination arm 1000bp upstream of the A137R open reading frame (refer to the ASFV in GenBank). The fragment consists of a fragment between 53566nt and 54565nt of the MK333180.1 strain), the first Loxp recombination site, the p72 promoter sequence, red fluorescent protein (mCherry), the second loxp recombination site, and a homologous recombination arm 1000bp downstream of the A137R open reading frame (located between 54985nt and 55984nt of the above-mentioned reference strain).

[0079] 1.1.3 Plasmid transfection

[0080] Specifically, according to the instructions of the lipofectamine 3000 transfection kit produced by Thermo Fisher Scientific, the above-constructed CRISPR-Cas9 plasmid and homologous recombination plasmid were co-transfected into PAM cells (for the construction of a single-gene deleted virus strain, the CRISPR-Cas9 plasmid and the homologous recombination plasmid targeting the single gene can be co-transfected into PAM cells; for the construction of a double-gene deleted virus strain, the CRISPR-Cas9 plasmid and the homologous recombination plasmid targeting these two genes can be co-transfected into PAM cells), and then the ASFV HuB 2019 strain was inoculated and cell culture was performed; culture wells containing green and / or red fluorescent cells were selected, and after about 12 consecutive generations of limiting dilution, the purified recombinant virus strain was obtained.

[0081] 1.2 PCR identification of single-gene or double-gene deletion strains

[0082] Specifically, a pair of primers were designed on the left and right homologous recombination arms of CD2v and A137R, respectively (see Table 2). DNA was extracted from the virus cultured in PAM cells, and PCR amplification and electrophoresis detection were performed according to conventional methods. The results are shown in Figure 2.

[0083] Table 2. Primers used for conventional PCR detection of deletion strains

[0084] For the ASFV HuB 2019 parental strain, the amplified CD2v fragment is expected to be 2189bp; for the ASFVΔCD2v and ASFVΔCD2v / A137R strains, the amplified CD2v fragment is expected to be 1916bp; for the ASFV HuB 2019 parental strain, the amplified A137R fragment is expected to be 564bp; for the ASFVΔA137R and ASFVΔCD2v / A137R strains, the amplified A137R fragment is expected to be 963bp.

[0085] As can be seen from Figure 2, after PCR identification, ASFVΔCD2v, ASFVΔA137R and ASFVΔCD2v / A137R all amplified a single gene fragment of the expected size, which indicates that the recombinant virus strains with single gene deletions of CD2v and A137R and double gene deletions of CD2v / A137R were successfully constructed and relatively pure.

[0086] 1.3 qPCR identification of double gene deletion strains

[0087] Specifically, qPCR primers and probes were designed with the goal of amplifying p72, CD2v, and A137R genes. DNA was extracted from the CD2v / A137R double-gene deleted recombinant virus cultured in PAM cells, and qPCR detection was performed using the primers and probes in the table. The results are shown in Figure 3.

[0088] Table 3. Primers and probes for qPCR identification of ASFVΔCD2v / A137R strain

[0089] It is expected that the parental strain should be able to detect three genes, p72, CD2v and A137R, while the CD2v / A137R double gene deletion strain can detect the p72 gene, but neither the CD2v nor the A137R genes can be detected.

[0090] As can be seen from Figure 3, the qPCR identification results are consistent with the above expectations, which indicates that the recombinant virus strain with double gene deletion of CD2v / A137R was successfully constructed.

[0091] Example 2. Virulence test of CD2v and A137R double gene deletion ASFV strain

[0092] In this example, 4 weaned SPF British Large White piglets (hereinafter referred to as SPF piglets, purchased from Beijing SPF Pig Breeding Management Center) at about 25 days of age were used. The inoculation volume was 1 ml per pig, and the pigs were injected intramuscularly with the cervical follicle for 10 minutes. 5 The CD2v / A137R double gene deletion strain ASFVΔCD2v / A137R was used with a TCID50 / ml concentration, and the piglet condition was observed for 28 consecutive days.

[0093] The results are shown in Table 4 below.

[0094] Table 4. Safety of ASFVΔCD2v / A137R strain in SPF piglets

[0095] As shown in Table 4, during the 28 days of observation, only a brief slight fever (not exceeding 41°C) and mental depression occurred on the 14th day. The other observation days were normal and all four piglets survived. This shows that the virulence of the double-gene deletion strain is significantly weakened compared with the parental strain and has better safety.

[0096] Example 3. Immunogenicity detection of CD2v and A137R double gene deleted ASFV strain

[0097] Referring to Example 2, 4 piglets were fed with 10 5The double-gene deletion strain ASFVΔCD2v / A137R was inoculated with TCID50 / head through neck muscle immunization, and the ASFV P30 antibody detection kit (purchased from Beijing Jinnuo Biotech Co., Ltd.) was used to measure the ASFV P30 specific antibody level in the serum within 27 days after vaccination according to the instructions of the kit.

[0098] The results are shown in Table 5 below.

[0099] Table 5. ELISA test results of ASFV P30-specific antibodies in the serum of vaccinated animals

[0100] Note: The values ​​in the table are the S / P values ​​of the samples; the calculation method of the S / P value is as follows:

[0101] Among them, NC represents negative control, PC represents positive control, both of which come with the kit; and,

[0102] The criteria are: S / P ≥ 0.4 for positive, 0.4 > S / P > 0.3 for questionable, and S / P ≤ 0.3 for negative. The shaded areas in the table show antibody-positive test results.

[0103] Table 5 shows that ASFV-specific antibodies were detected in one piglet starting from the 10th day after vaccination. By the 18th day, ASFV-specific antibodies were detected in all piglets. During the observation period, the antibody level generally increased with the extension of the immunization time.

[0104] Example 4. Immune protection test of CD2v and A137R double gene deletion ASFV strain

[0105] First, the virulence of the parental virulent strain ASFV HuB2019 was determined; specifically, the virulent strain ASFV HuB2019 blood virus was graded diluted with PBS to 1HAD50, 10HAD50, and 100HAD50 / ml, and the SPF susceptible British Large White piglets purchased from the Beijing SPF Pig Breeding Management Center (6 pigs per group, untreated piglets served as the control group) were injected through the neck muscle at a dose of 1 ml per pig. The piglets were observed for 21 consecutive days, and the symptoms were observed and the rectal temperature was measured every day. If the body temperature rose by 41°C for 2 consecutive days or more and there were obvious symptoms such as depression, loss of appetite, and lying down, or the animals were dying, they were euthanized.

[0106] The fever standard is a body temperature exceeding 41°C. The results of the toxin test are shown in Table 6 below.

[0107] Table 6. Virulence of the virulent strain ASFV HuB2019 in SPF pigs

[0108] The results in Table 6 show that 6 / 6 of the piglets in the 100HAD50 vaccination group died from the 4th to the 9th day. Among them, except for the piglet that died on the 4th day, no fever was detected in the other piglets. All of the other piglets had fever, with body temperatures exceeding 41°C. In the 10HAD50 vaccination group, except for one animal whose body temperature did not exceed 41°C and survived, the other 5 / 6 piglets had fever exceeding 41°C and died. All the piglets in the 1.0HAD50 vaccination group and the control group showed no obvious symptoms and all survived.

[0109] In view of the above results, the following 100HAD 50 As a lethal challenge dose, it was used to test the protective effect of the double-gene deletion strain ASFVΔCD2v / A137R.

[0110] Specifically, 10 5 Three piglets (numbered 1#, 2#, and 4# in Figure 5 ) 28 days after immunization with the double-gene deletion strain ASFV△CD2v / A137R at TCID50 / head and two piglets in the control group (numbered 13# and 33# in Figure 5 ) were respectively challenged with a lethal dose of 100HAD50 of virulent ASFV HuB2019 and observed for 21 days. The results are shown in Table 7, Figures 4 and 5 .

[0111] Table 7. Protective efficacy of the double gene deletion strain ASFVΔCD2v / A137R against a lethal dose of ASFV HuB2019 in SPF pigs

[0112] Figure 4 shows the survival rate of the ASFVΔCD2v / A137R vaccination group and the control group; it shows that all the piglets in the ASFVΔCD2v / A137R vaccination group survived during the observation period, while the piglets in the control group died within 10 days; this shows that the piglets in the ASFVΔCD2v / A137R vaccination group were well immune protected and could resist the attack of a lethal dose of the virulent strain ASFV HuB2019.

[0113] Figure 5 shows the changes in body temperature (upper figure) and clinical symptom scores (lower figure) of the ASFVΔCD2v / A137R vaccination group and the control group after infection; the results in Figure 5 show that none of the three piglets in the ASFV△CD2v / A137R vaccination group showed ASF-related symptoms, and their body temperatures did not exceed 40°C; while the two piglets in the control group showed symptoms of ASF such as body temperature rising above 41°C, depression and loss of appetite.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the present application. Industrial Applicability

[0115] The attenuated strain of African swine fever virus provided in this application has the following advantages: 1) It is fully attenuated and its virulence is greatly reduced compared to the wild-type virus, so it has extremely high safety; 2) It has stable characteristics and good genetic stability, so the possibility of reverting to the wild-type virus is extremely low; 3) It can induce animals to produce an effective immune protection response, thereby protecting animals from attack by a lethal dose of the parental African swine fever virus virulent strain; 4) loxp elements can be inserted into the strain genome, and the fluorescent label gene introduced during gene knockout can be removed if necessary. In view of the above, the attenuated strain of African swine fever virus in this application is a very potential candidate strain for attenuated live vaccine of African swine fever virus, and has a good application prospect in preventing African swine fever virus infection.

Claims

1. An attenuated strain of African swine fever virus, which lacks the following genes or fragments thereof in its genome relative to the wild-type African swine fever virus: (1) A137R gene or a functional fragment thereof; and (2) CD2V gene or its functional fragment.

2. The attenuated African swine fever virus strain according to claim 1, characterized in that: The wild-type African swine fever virus is a type II African swine fever virus, preferably selected from: HuB 2019, Pig / CN / HLJ / 2018, ASFV-SY18, Georgia 2008 / 1, Georgia 2007 / 1, ASFV GZ2018, and most preferably HuB 2019; And / or, the A137R gene or a functional fragment thereof is as shown in SEQ ID NO: 1; And / or, the CD2V gene or a functional fragment thereof is as shown in SEQ ID NO:

2.

3. A method for preparing an attenuated African swine fever virus strain according to any one of claims 1 to 2, comprising the following steps: Through genetic engineering, the A137R gene or its fragment and the CD2V gene or its fragment of the wild-type African swine fever virus are deleted.

4. The preparation method according to claim 3, characterized in that: The genetic engineering method is CRISPR-Cas9 technology combined with homologous recombination technology, which specifically includes the following steps: S1) constructing a CRISPR-Cas9 plasmid containing Guide RNA sequences for the CD2v gene or its functional fragment to be knocked out and the A137R gene or its functional fragment; S2) constructing a homologous recombination plasmid comprising a homologous recombination frame of the CD2v gene and a homologous recombination frame of the A137R gene, respectively; and, S3) The CRISPR-Cas9 plasmid and homologous recombination plasmid constructed above are co-transfected into PAM cells, and then the wild-type African swine fever virus strain is inoculated. After screening, an attenuated African swine fever virus strain that simultaneously lacks the CD2V gene or its functional fragment and the A137R gene or its functional fragment is obtained.

5. The preparation method according to claim 4, characterized in that: In step S1), there are three Guide RNA sequences for the CD2v gene to be knocked out or its functional fragment, as shown in SEQ ID NO: 3, 4, and 5 respectively; and / or, there are three Guide RNA sequences for the A137R gene to be knocked out or its functional fragment, as shown in SEQ ID NOs: 6, 7, and 8, respectively; Preferably, the CRISPR-Cas9 plasmid is based on the PX458 vector, in which two nuclear localization signal sequences are removed.

6. The preparation method according to claim 4 or 5, characterized in that: In step S2), the homologous recombination frame of the CD2v gene includes: a homologous recombination arm of 1065 bp upstream of the CD2v open reading frame, a first Loxp recombination site, a p72 promoter sequence, an enhanced green fluorescent protein, a second loxp recombination site, and a homologous recombination arm of 974 bp downstream of the CD2v open reading frame; And / or, the homologous recombination frame of the A137R gene includes: a homologous recombination frame 1000 bp upstream of the A137R open reading frame arm, the first loxP recombination site, the p72 promoter sequence, the red fluorescent protein, the second loxP recombination site, and the homologous recombination arm 1000 bp downstream of the A137R open reading frame.

7. An African swine fever virus expression protein, characterized in that: It is a protein expressed by the attenuated African swine fever virus strain as described in claim 1 or 2, preferably a full protein expressed thereby.

8. Use of the attenuated African swine fever virus strain according to any one of claims 1 to 2 or the African swine fever virus expression protein according to claim 7 in the preparation of a medicament for preventing African swine fever virus infection.

9. The use according to claim 8, characterized in that: The drug is a vaccine, preferably a live attenuated vaccine, a whole virus inactivated vaccine or a protein vaccine; Preferably, the vaccine is in the form of an oral formulation or a parenteral formulation; Optionally, the oral preparation is selected from tablets, powders, granules and oral liquids; further optionally, the granules are fine particles; Optionally, the parenteral formulation is an injectable or bolus formulation.

10. A live attenuated African swine fever virus vaccine, comprising the attenuated African swine fever virus strain according to any one of claims 1 to 2 as an active ingredient; Preferably, in the attenuated live vaccine, the virus content of the attenuated strain of African swine fever virus is ≥ 10 TCID 50 / ml; Preferably, the live attenuated vaccine is in the form of an oral preparation or a parenteral preparation; Optionally, the oral preparation is selected from tablets, powders, granules and oral liquids; further optionally, the granules are fine particles; Optionally, the parenteral formulation is an injectable or bolus formulation.

11. The method for preparing the live attenuated African swine fever virus vaccine according to claim 10, characterized in that: The preparation method comprises the following steps: 1) inoculating the attenuated African swine fever virus strain according to any one of claims 1 to 2 into primary PAM cells, expanding the culture, and harvesting the virus liquid; 2) The virus content is ≥10TCID 50 / ml of virus liquid can be made into vaccine directly or with adjuvant.

12. An inactivated whole virus vaccine of an attenuated African swine fever virus strain, comprising as an active ingredient an inactivated whole virus obtained by inactivating the attenuated African swine fever virus strain according to any one of claims 1 to 2; Preferably, the inactivated vaccine is in the form of a parenteral formulation; Optionally, the parenteral formulation is an injectable or bolus formulation.

13. An attenuated whole protein vaccine of African swine fever virus, comprising the African swine fever virus expression protein as claimed in claim 7 as an active ingredient; Preferably, the whole protein vaccine is in the form of a parenteral formulation; Optionally, the parenteral formulation is an injectable or bolus formulation.

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