CN115851623A - Construction of African swine fever MGF505-2R gene deletion attenuated strain and application of attenuated strain as vaccine - Google Patents

Abstract

The invention belongs to the technical field of biological engineering, and particularly relates to an MGF505-2R gene deletion attenuated strain and a vaccine. The invention firstly discovers that the gene knockout of the African swine fever virus can weaken the toxicity of the African swine fever virus, and the preparation method isA novel target gene of an attenuated swine fever vaccine; the African swine fever virus MGF505-2R gene is deleted in the II-type African swine fever virus strain ASFV CN/GS 2018 by adopting a genetic engineering means, so that the toxicity of a parent strain is reduced, and an attenuated African swine fever candidate vaccine strain with high safety is obtained; after the attenuated African swine fever candidate vaccine strain is used for immunizing pigs, the pigs are completely weakened and the immunized pigs are healthy, and the attenuated African swine fever candidate vaccine strain can challenge the virulent strain of the II-type African swine fever virus strain ASFV CN/GS 2018 (10) ² HAD ₅₀ Dosage) provides good immune protection effect, can be used as a safe and effective candidate vaccine for preventing and controlling the epidemic situation of the African swine fever, and has great social value.

Description

Construction of African swine fever MGF505-2R gene deletion attenuated strain and application of attenuated strain as vaccine

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to construction of an African swine fever MGF505-2R gene deletion attenuated strain and application of the attenuated strain as a vaccine.

Background

African Swine Fever (African Swine Fever) is an acute severe highly contagious disease caused by infection with African Swine Fever virus (African Swine Fever virus), and is characterized by Fever and organ bleeding of the whole body, and the death rate of the domestic pig is as high as 100%. Due to the lack of commercial effective vaccines for African swine fever, the outbreak of epidemic can be controlled only by killing and burying and the like. The vaccine is the most economic and effective means for preventing and controlling infectious diseases and is important for preventing and controlling African swine fever. Due to the fact that the African Swine Fever Virus (ASFV) is complex in structure, huge in genome, most of functions are unknown, the infection and pathogenic mechanism are not clear, the theory of vaccine development is lacked, and the like, the difficulty of developing the African swine fever vaccine is increased, and although the virus is already popular for over a century, no commercial vaccine is still available.

The development of an effective commercial vaccine is currently an urgent task. Currently, the African swine fever vaccines mainly comprise inactivated vaccines, attenuated live vaccines (natural attenuated vaccines, gene knockout vaccines and the like), genetic engineering vaccines and the like (nucleic acid vaccines, subunit vaccines, live vector vaccines and the like). The genetic engineering vaccine has obvious advantages in the aspects of safety, differential diagnosis and the like, and has great research potential in the genetic engineering vaccines such as ASFV subunit vaccines, nucleic acid vaccines, live vector vaccines and the like, but because the ASFV genome is huge and the immune escape mechanism is complex, the genetic engineering vaccine needs to be deeply researched on the basis of fully analyzing the main protein structure and function of the ASFV, the infection and immune mechanism and the identification of the main immunogen and the vaccine target spot. The naturally separated or virulence gene knocked-out ASFV attenuated strain has a good prospect in developing attenuated live vaccines, but the safety is an important factor influencing the practical application of the ASFV attenuated live vaccine, the safety risks of virulent return, adverse reactions, persistent infection and the like of the strain need to be comprehensively evaluated, and a cell line suitable for ASFV culture needs to be developed for producing vaccine antigens. The gene knocked out by the gene knockout vaccine mainly aims at harmful virulence genes, immune suppression genes and the like of an organism. For the knockout of the virulence gene of the African swine fever virus, the virulence gene is usually considered to have immune response and protection to the body, and also has pathogenicity and safety performance, and in addition, the virulence gene needs to be considered: (1) Whether the knocked-out virulence genes carry viruses or not and whether the carried viruses and the toxin are eliminated or not, whether tissue damage and production performance are reduced or not and the like are important indexes of the safety of the gene deletion vaccine; (2) The different virus strains have different effects of deleting the same gene, the insufficient gene deletion may cause toxicity reduction and pathogenicity enhancement, multi-gene knockout weakens the virus titer, and the immunogenicity or protective action of the attenuated strain is reduced; (3) Although the whole genome sequencing work of the African swine fever is completed, the number of the regulatory genes and structural genes which form the African swine fever virus is huge (> 160), the functions are not clear, and the genes are important for the pathogenesis of the African swine fever and the vaccine development.

Therefore, in the research and development process of the attenuated vaccine for African swine fever, the selection of the target gene determines the safety and effectiveness of the vaccine strain, identifies the function of the virus gene and the influence of the virus gene on pathogenicity and immune response, and provides a theoretical basis for the selection of the target gene. However, the inventors found in literature investigations That MGF360-16R, which is in The same multigene family, although able to react with Host Proteins SERTAD3 and SDCBP, does not change The replication capacity of The recombinant Virus after deletion (E Rami i rez-medina, et al, "The MGF360-16R ORF of African Swine Virus Strain street nucleic acid Gene foreign genes with Host Proteins SERTAD3 and SDCBP." Virus 12.1 (2020): 60-75.); the Strain GZ201801 lacking MGF100-1R, replication-competent similar to the parental Virus GZ201801, failed as an attenuated vaccine (Yl, A, et al, "Development and In vivo Evaluation of MGF100-1R Deletion mutation In an animal wind turbine Virus Chinese Strain (2021)); the rate of viral replication and clinical symptoms following loss of MGF110-1L and MGF360-1L are similar to those of the parental virus, independent of viral virulence (Ramirezmendina, E., et. Development and In Vivo Evaluation of a MGF110-1L Deletion Mutant In African Swine Fever series 13.2 (2021): 286.; ra mirez-Medina, E., et. Evaluation In Swine rotor Viruses splicing the MGF-360-1L Gene. Videos 12.10 (2020): 1193.); after deletion of MGF360-13L and MGF360-14L simultaneously in ASFV-G, there was no change in virus replication (Borca, M.V., et al, "Development of a fluor plasmid ASFV strain that genes their ability to cause diseases in the virus," Scientific Reports 7 (2017): 46747.).

In the research of gene function and a regulation mechanism thereof, the invention discovers for the first time that the African swine fever virus MGF505-2R gene has stronger immune suppression effect and is a newly discovered target gene in the genome of the II-type African swine fever virus strain ASFV CN/GS 2018; according to the invention, the pathogenicity of the newly identified target gene African swine fever virus MGF505-2R coding protein is further reduced, the safety of the African swine fever virus MGF505-2R coding protein is improved, and a virus strain which is completely weakened to pigs is obtained; after the low virulent strain obtained by the gene deletion method is used for immunizing animals, pigs living with the immunized pigs are not horizontally infected, the immunized pigs are anatomically observed, each tissue organ is healthy, and the further detection nucleic acid is negative, so that the safe and reliable attenuated African swine fever virus strain is obtained by the deletion; the virus attacking experiment shows that the attenuated African swine fever virus can attack the virulent strain of the II-type African swine fever virus strain ASFV CN/GS 2018 and has good immune protection function; therefore, the MGF505-2R is knocked out and then can be used as a vaccine for controlling the epidemic situation of the African swine fever, and the vaccine has extremely high social value.

Disclosure of Invention

Aiming at the technical problems, the invention firstly discovers that in the II type African swine fever virus strain ASFV CN/GS 2018, MGF505-2R gene has stronger immunosuppressive action and is a newly discovered target gene for weakening the African swine fever virus. Based on the technical scheme, the invention aims to provide a strategy for constructing the attenuated strain of African swine fever, which is realized by the following technical scheme:

in a first aspect, the invention provides an application of preparing an African swine fever attenuated strain by deleting MGF-505-2R gene in a type II African swine fever virus strain ASFV CN/GS 2018, wherein the type II African swine fever virus strain ASFV CN/GS 2018 is collected in China center for type culture Collection with the collection number of CCTCC NO: v202096. The function of the encoded protein can be lost by the deletion of the whole nucleotide sequence of the African swine fever virus MGF505-2R gene, or the function of the encoded protein can be lost by the deletion of a partial nucleotide sequence (MGF 505-2R gene promoter sequence) of the African swine fever virus MGF505-2R gene, so that the protein encoded by the African swine fever virus MGF505-2R gene cannot be expressed. According to the common general knowledge of the skilled person, besides the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the function of the protein encoded by the African swine fever virus MGF-505-2R gene, such as: frame shift mutations, point mutations, frame shift deletions, insertion of nucleotide sequences, and the like.

Preferably, the MGF505-2R gene sequence is shown in SEQ ID NO. 1; the deleted MGF-505-2R gene is the 33122-34702 th position of the full-length sequence of the II type African swine fever virus strain ASFV CN/GS 2018.

In a second aspect, the present invention provides an application of an attenuated african swine fever vaccine prepared by deleting MGF-505-2R gene in type II african swine fever virus strain ASFV CN/GS 2018, which is deposited in the chinese type culture collection with the collection number of CCTCC NO: v202096. The function of the encoded protein can be lost by the deletion of the whole nucleotide sequence of the African swine fever virus MGF505-2R gene, or the function of the encoded protein can be lost by the deletion of a partial nucleotide sequence (MGF 505-2R gene promoter sequence) of the African swine fever virus MGF505-2R gene, so that the protein encoded by the African swine fever virus MGF505-2R gene cannot be expressed. According to the common general knowledge of the skilled person, besides the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the function of the protein encoded by the African swine fever virus MGF-505-2R gene, such as: frameshift mutations, point mutations, frameshift deletions, insertion of nucleotide sequences, and the like.

Preferably, the MGF505-2R gene sequence is shown in SEQ ID NO. 1; the deleted MGF-505-2R gene is the 33122-34702 th position of the full-length sequence of the II type African swine fever virus strain ASFV CN/GS 2018.

In a third aspect, the invention provides a gene deletion attenuated African swine fever virus strain, which is a type II African swine fever virus strain ASFV CN/GS 2018 with MGF-505-2R gene deletion, wherein the type II African swine fever virus strain ASFV CN/GS 2018 is collected in China center for type culture Collection with the collection number of CCTCC NO: v202096. The function of the encoded protein can be lost by the deletion of the whole nucleotide sequence of the African swine fever virus MGF505-2R gene, or the function of the encoded protein can be lost by the deletion of a partial nucleotide sequence (MGF 505-2R gene promoter sequence) of the African swine fever virus MGF505-2R gene, so that the protein encoded by the African swine fever virus MGF505-2R gene cannot be expressed. According to the common knowledge of those skilled in the art, besides the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the function of the protein encoded by the African swine fever virus MGF-505-2R gene, such as: frame shift mutations, point mutations, frame shift deletions, insertion of nucleotide sequences, and the like.

Preferably, the MGF505-2R gene sequence is shown in SEQ ID NO. 1; the deleted MGF-505-2R gene is the 33122-34702 th position of the full-length sequence of the II type African swine fever virus strain ASFV CN/GS 2018.

In a fourth aspect, the present invention provides an attenuated african swine fever vaccine, wherein the attenuated african swine fever vaccine is the attenuated african swine fever virus strain with the gene deletion described in the third aspect.

In a fifth aspect, the invention provides a method for preparing a gene-deleted attenuated African swine fever virus strain, which is characterized in that the gene sequence of MGF-505-2R of the II-type African swine fever virus strain ASFV CN/GS 2018 is deleted by genetic engineering means, and the nucleotide sequence of the MGF-505-2R gene is shown as SEQ ID NO. 1; the II type African swine fever virus strain ASF V CN/GS 2018 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: v202096. The function of the encoded protein can be lost by the deletion of the whole nucleotide sequence of the African swine fever virus MGF505-2R gene, or the function of the encoded protein can be lost by the deletion of a partial nucleotide sequence (MGF 505-2R gene promoter sequence) of the African swine fever virus MGF505-2R gene, so that the protein encoded by the African swine fever virus MGF505-2R gene cannot be expressed. According to the common general knowledge of the skilled person, besides the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the function of the protein encoded by the African swine fever virus MGF-505-2R gene, such as: frame shift mutations, point mutations, frame shift deletions, insertion of nucleotide sequences, and the like.

Preferably, the method is a homologous recombination technique.

Preferably, the MGF-505-2R gene sequence deletion method comprises the following steps:

(1) Designing 1.5kb of upstream and downstream sequences of MGF-505-2R gene respectively as left and right homologous recombination arms, and cloning the left and right homologous recombination arm genes and eGFP gene screening expression cassette gene fragment p72-eGFP-S virus 40polyA to a pUC57 vector at the same time to obtain a recombinant plasmid p52-eGFP;

(2) The recombinant plasmid p52-eGFP is transfected into a BMDM cell infected with a II type African swine fever virus strain ASFV CN/GS 2018, and the African swine fever virus strain with the deletion of the MGF-505-2R gene of the African swine fever virus is obtained by purifying and screening by a plaque cloning method.

In a sixth aspect, the present invention provides a gene-deleted attenuated african swine fever virus strain produced by the method of the fifth aspect.

In a seventh aspect, the invention provides a use of the attenuated african swine fever virus strain with gene deletion in the third and sixth aspects in the preparation of an african swine fever vaccine.

The invention has the beneficial effects that: the invention obtains an attenuated African swine fever virus strain by losing the function of MG F505-2R gene coding protein of II type African swine fever virus strain ASFV CN/GS 2018, and the attenuated African swine fever virus strain has obvious attenuated virulence and good safety performance. The toxicity of the parent strain is further reduced by losing the function of MGF505-2R gene coding protein in the parent strain (II type African swine fever virus strain ASFV CN/GS 2018), and the obtained attenuated African swine fever virus can provide good immune protection effect on the attack of the African swine fever virus parent strain, has high safety, and is suitable for serving as a vaccine candidate strain for preventing the African swine fever.

Drawings

FIG. 1 is a schematic diagram showing the construction of an MGF505-2R gene deletion vector (expressing green fluorescence);

FIG. 2 shows the results of fluorescence detection;

FIG. 3 shows the result of purification and identification of an attenuated African swine fever virus strain lacking MGF505-2R gene, wherein Δ 47 is MGF505-2R gene lacking African swine fever virus; ASFV represents type II African swine fever virus strain ASFV CN/GS 2018;

FIG. 4 shows the results of body temperature changes after immunization of experimental pigs;

FIG. 5 survival of experimental pigs after immunization;

FIG. 6 results of measurement of IFN-. Beta.expression in serum of experimental swine after immunization.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.

The experiments described in the following examples obtain biosafety permits and african swine fever laboratory activity permits:

according to the related requirements of biological safety 3-level laboratory (BSL-3) and related biological safety of African swine fever, the Lanzhou veterinary research institute of the Chinese agricultural academy reports the biological safety committee, the ethical committee of experimental animals, the biological safety committee of the Chinese agricultural academy of sciences, the ethical committee of experimental animals of the Lanzhou veterinary research institute and the biological safety committee of the Lanzhou veterinary research institute step by step, and the permission of developing the highly pathogenic African swine fever virus pathogen and animal research is obtained by the Ministry of agriculture and rural area and meets the requirements of the national biological safety level.

Experimental cell, viral and plasmid sources described in the following examples:

primary Porcine Alveolar Macrophages (PAM) and primary bone marrow macrophages (BMDM) were taken from 2-4 month old healthy SPF Bama minipigs, aseptically harvested, lysed with red blood cell lysate (purchased from Biosharp), red blood cells were removed, centrifuged at low speed, the supernatant was discarded, the cell pellet was resuspended in RPMI 1640 complete medium (purchased from Gibco) containing 10% FBS (purchased from PAN), and the medium was incubated at 37 ℃ for 5% CO ₂ Culturing in an incubator. BMDM cell culture requires the additional addition of 10ng/mL final concentration of recombinant porcine GM-CSF (purchased from R) to RPMI 1640 complete medium&D Systems Co.), at 37 ℃ and 5% CO ₂ Inducing in an incubator, washing once every 2-3 days, centrifuging the nonadherent cells, adding into a new cell dish again, changing the liquid for continuous induction, and freezing for 3-7 days or using. And (3) amplifying the African swine fever virus by using the PAM cells, titrating the virus content, and using the BMDM cells for plasmid transfection and virus recombination experiments.

The type II African swine fever virus strain ASFV CN/GS 2018 is an African swine fever regional laboratory isolate of Lanzhou veterinary research institute of Chinese agricultural science, belongs to the type II gene, is preserved in a China center for type culture Collection in 12 months and 21 days in 2020, and has a preservation number of CCTCC NO: v202096; the preservation address is as follows: wuhan, wuhan university, china; and (3) contact telephone: 027-68752319. The virus titer is 5 × 10 ⁷ TCID ₅₀ and/mL, which is the 4 th generation virus after PAM cell propagation, and is subpackaged and stored at-80 ℃ for later use.

The peGFP-N1 vector and the pUC57 vector are purchased from Riborley Biotechnology, inc. of Lanzhou; endotoxin-free plasmid extraction kit, purchased from OMEGA.

The procedures in the experiments are those known in the art unless otherwise specified.

Definition of

The term "loss of function of a protein" refers to the loss of function of a protein encoded by a gene by knocking out, mutating or inserting a part of the gene in a gene segment encoding the protein, so that the protein encoded by the gene is subjected to frame shift mutation. The invention aims to knock out the MGF505-2R gene in the II type African swine fever virus strain ASFV CN/GS 2018 to cause the loss of the function of the MGF505-2R gene coding protein, thereby constructing the attenuated African swine fever virus with the MGF505-2R gene coding protein with the loss of function and being used for producing the African swine fever vaccine; one skilled in the art can also delete the MGF505-2R gene promoter sequence to disable the expression of the MGF505-2R gene encoded protein, resulting in the loss of the function of the encoded protein. According to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the function of the protein encoded by the MGF505-2R gene, such as: frame shift mutations, point mutations, frame shift deletions, insertion of nucleotide sequences, and the like.

The term "gene deletion" refers to the phenomenon that a certain segment on a chromosome and a gene carried by the segment are lost together to cause mutation, and the invention aims to obtain an attenuated African swine fever virus strain by deleting MGF505-2R gene in the II-type African swine fever virus strain ASFV CN/GS 2018 and reduce the toxicity of parent strains. One skilled in the art can also delete the MGF505-2R gene promoter sequence to disable the expression of the MGF505-2R gene encoded protein, resulting in the loss of the function of the encoded protein. According to the common knowledge of those skilled in the art, in addition to the above-mentioned gene editing means, other gene editing means can be used to simultaneously lose the function of the protein encoded by the MGF505-2R gene, such as: frame shift mutations, point mutations, frame shift deletions, insertion of nucleotide sequences, and the like.

The term "gene mutation" refers to a gene mutation, which means that the base pair composition or arrangement sequence of the gene is changed in structure, i.e., a new gene suddenly appears at a site, instead of the original gene, and the gene is called a mutant gene, and the gene mutation causes the expression of a progeny to suddenly appear with a new character which never exists. On the basis of deleting MGF505-2R gene in II type African swine fever virus strain ASFV CN/GS 2018 to cause the function of MGF505-2R gene coding protein to be deleted, obtaining the attenuated African swine fever virus strain and reducing the toxicity of parent strains, the technical personnel in the field can also cause the original performance of the MGF505-2R gene coding protein to disappear by mutating the MGF505-2R gene or inserting gene segments to cause the code shift mutation of the gene coding protein, thereby realizing the construction of the African attenuated swine fever virus strain.

The gene deletion method generally refers to gene knockout, and is an exogenous DNA introduction technology in which a DNA fragment containing a certain known sequence is subjected to homologous recombination with a gene having the same or similar sequence in a recipient cell genome, is integrated into the recipient cell genome, and is expressed. Methods of gene knockout generally include: homologous recombination technology, random insertion mutation technology and RNA interference technology; wherein, the homologous recombination technique is also called gene targeting, which means that the recombination occurs between the homologous sequence on the exogenous DNA and the chromosome DNA of the receptor cell, and the homologous sequence is integrated to a predetermined position, thereby changing some genetic characteristics, and the recombination aims at knocking out a certain gene; random insertion mutation technology is that certain viruses, bacteria or other gene vectors capable of randomly inserting gene sequences are utilized to carry out random insertion mutation on a cell bank in a target cell genome, and then corresponding gene knockout cells are obtained by screening through corresponding markers; RNA interference technology refers to a technology in which target mRNA degradation specifically initiated by double-stranded RNA homologous to target gene mRNA endogenous to an organism leads to silencing of target gene expression.

Although the MGF505-2R gene in the II type African swine fever virus strain ASFV CN/GS 2018 is knocked out only by the homologous recombination technology, the MGF505-2R gene of the African swine fever virus can be knocked out by the random insertion mutation technology and the RNA interference technology.

The MGF505-2R gene is positioned at 33122-34702 of the whole gene sequence of the II type African swine fever virus strain ASFV CN/GS 2018, the invention knocks out the whole nucleotide sequence of MGF505-2R in the II type African swine fever virus strain ASFV CN/GS 2018 by a homologous recombination technology, so that the function of the MGF505-2R gene coding protein is lost, and the attenuated African swine fever virus strain with the deletion of the African swine fever virus MGF-505-2R gene is successfully constructed and is used as a vaccine candidate strain.

The term "vaccine" refers to a biological agent capable of providing a protective response in an animal, wherein the vaccine has been delivered and is not capable of causing serious disease. The vaccine of the invention is a genetically engineered gene deletion attenuated virus vaccine, wherein the deleted gene is MGF505-2R gene of type II African swine fever virus strain ASFV CN/GS 2018. It is understood that the recombinant mutant obtained by mutation of MGF505-2R gene of African swine fever virus strain type II ASFV CN/GS 2018 can also be used as attenuated virus vaccine.

The attenuated african swine fever virus of the present invention, further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, chemical immune adjuvants such as aluminum hydroxide, freund's adjuvant, mineral oil, span, etc.; microbial immune adjuvants such as mycobacteria, lipopolysaccharide, muramyl dipeptide, cytopeptide, lipid soluble waxy D, short corynebacterium; the plant immunologic adjuvant is polysaccharides extracted from plant or large fungi, such as pachyman, carthamus tinctorius polysaccharide, chinese herbal medicine, etc. And biochemical immune adjuvants such as thymosin, transfer factor, interleukin, etc. Preferred adjuvants may be nano-adjuvant biological adjuvants, interleukins, interferons, etc.

The vaccines of the present invention may also be used in vaccines, such as other vaccines with swine, but emphasis is placed on live attenuated vaccines, particularly in the integration of viral genes, such as bivalent, trivalent, etc. The vaccine may comprise a plurality of attenuated non-swine fever viruses of different genotypes, such that a cross-protective immune response is induced against the plurality of non-swine fever virus genotypes.

The administration of the vaccines of the present invention may be by any convenient route, for example, intramuscular injection, intranasal, oral, subcutaneous, transdermal and vaginal routes. The attenuated vaccines of the present invention are preferably administered intramuscularly. The vaccine may be administered after a prime-boost regimen. For example, after a first vaccination, the subject may receive a second booster administration after a period of time (e.g., about 7, 14, 21, or 28 days). Typically, the booster is administered at the same or a lower dose than the prime dose. In addition, a third booster immunization may be performed, for example 2-3 months, 6 months or a year after immunization.

Example 1 construction and purification identification of an attenuated Virus Strain Δ 47 with deletion of the MGF505-2R Gene

1. Screening expression cassette construction

To facilitate screening, expression cassettes for the selectable marker genes were constructed.

Construction of enhanced Green fluorescent protein (eGFP) gene screening expression cassette: reference is made to the literature (O' Donnell V.African Swine Fever Virus Georgia 2007with a Deletion of Virus ce-Associated Gene 9GL (B119L), where added identified at Low Doses, heads to Virus attachment in Swine and indeces an Effective Protection against native Horolous channel. JVirol.2015;89 (16): 8556-66), amplification of the p72 promoter (sequence from-196 nt upstream of the p72 Gene to + 17), for use by PCR; the amplification primers are as follows: a forward primer of 5; using a peGFP-N1 vector as a template to amplify an eGFP gene for later use, wherein amplification primers are as follows: a forward primer of 5 'ATGGTGAGCAAAGGGCGAGGAG-3' (shown in SEQ ID NO. 4) and a reverse primer of 5 'ACCACAACTAGAATGCAGTG-3' (shown in SEQ ID NO. 5); reference is made to the literature (Borca MV, holinka LG, berggren KA, gladeu DP. CRISPR-Cas9, a tool to effect in the amplification of the gene of recombinant African swine viruses. Sci Rep.2018;8 (1): 3154.) the gene fragment of eGFP screening expression cassette, named p72-eGFP-S virus 40polyA, obtained by the process of fusion PCR, contains S virus 40polyA termination sequence.

2. Construction of homologous recombination transfer vector

Constructing a homologous recombination transfer vector for knocking out MGF505-2R gene in II type African swine fever virus strain ASFV CN/GS 2018 by using a pUC57 vector as a skeleton vector, wherein the nucleotide sequence of the MGF505-2R gene is shown as SEQ ID NO. 1; the deleted gene sequence is positioned at 33122-34702 of the whole gene sequence of the II type African swine fever virus strain ASFV CN/GS 2018, and the construction strategy is shown in figure 1.

The method comprises the following specific steps: designing an upstream sequence 1.5kb of an MGF505-2R gene fragment in an African swine fever virus strain II ASFV CN/GS 2018 as a homologous recombination Left arm (shown by Left arm and SEQ ID NO. 6) and a downstream sequence 1.5kb as a homologous recombination Right arm (shown by Right arm and SEQ ID NO. 7), and respectively cloning into a pUC57 vector to obtain a recombination transfer vector p72-mEGFP-NOSpolyA of the MGF505-2R gene fragment; after the sequencing is correct, the homologous recombination transfer vector is named as P47-eGFP; extracting DNA with endotoxin-free plasmid extraction kit, measuring concentration, and storing at-20 deg.C; 3. cell transfection and recombinant virus screening

Homologous recombination transfer vectors P47-eGFP and

the-Macrophage DNA transfection reagent is fully mixed, co-transfected to well-conditioned bone marrow Macrophage (BMDM) cells taken from 2-4 month-old healthy SPF small healthy pigs, directly infected with 1MOI type II African swine fever virus strain ASFV CN/GS 2018 purified virus strains, then p72-eGFP-SV40polyA plasmids are transfected to BMDM cells infected with type II African swine fever virus strain ASFV CN/GS 2018, recombinant African swine fever virus viruses with MGF505-2R deletion are obtained through recombination, and the MGF505-2R gene deletion attenuated African swine fever virus strains are obtained through purifying the virus strains.

The microscopic observation results are shown in fig. 2, and the results show that the transfected and purified gene-deleted virus expresses green fluorescence when infecting BMD M cells, which indicates that the recombinant virus is successfully constructed and is named as Δ 47.

And (3) detecting the purity: selecting MGF 505-2R-check-F/R primer pair (MGF 505-2R-F: GCGGGAGAGGA AGAACAA (shown in SEQ ID NO. 8)) and MGF 505-2R-R: TTTACGGCTTGGGTCAGGAC (shown in SEQ ID NO. 9)); and (5) carrying out purity detection. Meanwhile, P72 in the virus genome is selected as an internal reference gene to ensure the quality of the detected virus genome, and primers for detecting the P72 are respectively P72-F: TGTAATGAACATTACGTCTTAT GTC (shown in SEQ ID NO. 10) and p72-R: CGATCCCGAACCCACTTTGA (shown in SEQ ID NO. 11). Selecting MGF 505-2R-insert-F/R primer pair (MGF 505-2R-IF: ACTTTCAAGAACCCTTA TCCCT (shown in SEQ ID NO. 12)) and MGF 505-2R-IR: CGATGCTGCTACTTTGAGTGC (shown in SEQ ID NO. 13)); and (4) carrying out insertion identification of the sequence.

As shown in FIG. 3, the primer (MGF 505-2R-insert-F/R primer pair) set in the internal African swine fever virus MGF505-2R is used to detect whether the gene deletion attenuated African swine fever virus is successfully constructed, wherein ASFV is type II African swine fever virus ASFV CN/GS 2018, and Delta 47 represents MGF505-2R gene deletion attenuated African swine fever virus. The result shows that MGF505-2R gene deletion attenuated African swine fever virus genome has no detected MGF505-2R gene, which indicates that MGF505-2R gene knockout is successful; the above results indicate that the attenuated African swine fever virus with gene deletion has been successfully constructed, purified and has the correct sequence size, and is named as delta 47 (ASFV-delta MGF 505-2R).

EXAMPLE 2MGF 505-2R Gene deletion attenuated Virus Strain Δ 47 determination of Virus titres

The titer of African swine fever virus was determined using half the blood cell adsorption (50%; haemadsorption ₅₀ ) Denotes, HAD ₅₀ Specific procedures for the experiments are described in the literature (Borca MV, ramirez-Medina E, silva E, vulo E, rai A, pruitt S, holinka LG, velazquez-Salinas L, zhu J, gladue DP. Development of a high effective African swith vaccine by deletion of the I177L gene responses in stereo immunity acquisition of the current epidemic European strain JVirol. Pi: I.02017-19), with appropriate manipulations being performedAdjusting: in 96-well plates at about 1X 10 ⁵ Inoculating primary PAM cells to the cells per well, continuously diluting the recombinant virus to be detected by 10-fold gradient for 7 dilutions, adding the diluted virus to PAM in a 96-well plate according to 100 mu l/well, immediately adding red blood cells, and repeating for three times. The virus infection can be determined according to rosette formed by erythrocyte aggregation around infected cell, continuously observing for 6 days, counting number of positive holes, and calculating half number of blood cell adsorption amount (HAD) ₅₀ ). And (4) determining the titer to be qualified, and evaluating the pathogenicity.

Example 3 evaluation of virulence in a MGF505-2R Gene-deleted attenuated Virus Strain, Δ 47

In order to detect the toxicity of the gene deletion strain delta 47, 10 is used in the experiment ² HAD ₅₀ The dose was evaluated for virulence by intramuscular injection to piglets.

The 16 African swine fever antigen antibody negative healthy long-white piglets used for the experiment are divided into 3 groups in total, wherein the gene deletion strain delta 47 is used for immunizing 8 groups, the homozygote is used for immunizing 4 groups, and the positive control group (parent strain ASFV CN/GS 2018) is used for immunizing 4 groups. Body temperature changes were measured daily after immunization, peripheral blood and saliva collected, and reference literature (Donald PKing, scott MReid, geoffre HHutchings, sylvia SGrierson, philip JWilkinson, linda K Dixon, armanda D.S. Bastos, tr or WDrow. Development of a

PCR assay with internal amplification control for the detection of African swine virus Methods,2003.107 (1): 53-60.) and determination of the ASFV virus content in blood by fluorescent quantitative PCR method, detection was terminated by 31 days. And (4) counting the lethality of the immunized group and the homozygote group animals. The result of the change of the body temperature is shown in fig. 4 (immune part) and the survival rate is shown in fig. 5 (immune part), after intramuscular injection of the gene deletion strain delta 47, the positive control group died completely after 7 days, except that one pig died in the later period of the experiment in the immune group, the body temperatures of the rest immune group pigs and the pigs in the same living group were normal, and no continuous high temperature appeared, which indicates that the gene deletion strain delta 47 has good safety and no toxin expelling phenomenon.

Example 4 measurement of expression of serum IFN-. Beta.after immunization of animals with Virus-deleted-. DELTA.47

Are respectively 10 ² HAD ₅₀ After the immunized animal is infected by the gene deletion virus strain delta 47 and the parent strain in the dosage, the expression level of IFN-beta in the serum of the immunized animal is detected by using an ELISA kit. The results are shown in FIG. 6, and the IFN-beta expression level is increased but not increased greatly after the parental virus infects animals; the level of IFN-beta induced by recombinant virus delta 47 after deletion of MGF505-2R gene after immunization is obviously higher than the expression of IFN-beta induced by parental virus after immunization. The above results indicate that the function of the protein encoded by the ASFV MGF-505-2R gene in the gene-deleted virus strain Δ 47 is lost, and the immunosuppressive effect is released.

In conclusion, the attenuated African swine fever virus with MGF505-2R gene deleted is constructed by deleting ASFV MGF-505-2R gene from parent ASFV CN/GS/2018 isolate through homologous recombination technology, wherein the ASFV-delta MGF-505-2R gene of the MGF505-2R gene deleted encodes protein with loss of function. The invention is not limited to the homologous recombination technology, and on the basis of the invention, the attenuated African swine fever virus with the MGF505-2R gene encoding protein with lost function can be obtained by losing the MGF505-2R gene encoding protein with other technical means.

Claims (10)

1. The application of preparing the African swine fever attenuated strain by deleting MGF505-2R gene in II type African swine fever virus strain ASFV CN/GS 2018 is characterized in that the II type African swine fever virus strain ASFV CN/GS 2018 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: v202096.

2. The application of preparing the attenuated African swine fever vaccine by deleting MGF505-2R gene in II type African swine fever virus strain ASFV CN/GS 2018 is characterized in that the II type African swine fever virus strain ASFV CN/GS 2018 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: v202096.

3. The gene deletion attenuated African swine fever virus strain is MGF-505-2R gene deletion type II African swine fever virus strain ASFV CN/GS 2018, the type II African swine fever virus strain ASFV CN/GS 2018 is preserved in China center for type culture collection with the preservation number of CCTCC NO: v202096.

4. The gene deletion attenuated african swine fever virus strain of claim 3, wherein the MGF505-2R gene sequence is as set forth in SEQ ID No. 1; the deleted MGF505-2R gene is the 33122-34702 th position of the full-length sequence of the II type African swine fever virus strain ASF V CN/GS 2018.

5. Use of the attenuated African swine fever virus strain of claim 3 or 4 having a gene deletion for the preparation of an African swine fever vaccine.

6. An attenuated African swine fever vaccine, comprising the attenuated African swine fever virus strain of claim 3 or 4, wherein the gene deletion is attenuated.

7. A method for preparing a gene-deleted attenuated African swine fever virus strain is characterized in that the method is characterized in that a MGF-505-2R gene sequence of a II-type African swine fever virus strain ASFV CN/GS 2018 is deleted by a genetic engineering means, and the nucleotide sequence of the MGF-505-2R gene is shown as SEQ ID NO. 1; the II type African swine fever virus strain ASFV CN/GS 2018 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: v202096.

8. The method of claim 7, wherein the deletion of the MGF-505-2R gene sequence comprises the steps of:

(1) Designing 1.5kb of upstream and downstream sequences of MGF-505-2R gene respectively as left and right homologous recombination arms, and cloning the left and right homologous recombination arm genes and eGFP gene screening expression cassette gene fragment p72-eGFP-S virus 40polyA to a pUC57 vector at the same time to obtain a recombinant plasmid p52-eGFP;

(2) The recombinant plasmid p52-eGFP is transfected into a BMDM cell infected with a II type African swine fever virus strain ASFV CN/GS 2018, and the African swine fever virus strain with the deletion of the MGF-505-2R gene of the African swine fever virus is obtained by purifying and screening by a plaque cloning method.

9. The attenuated african swine fever virus strain with a deleted gene prepared according to the method of claim 7 or 8.

10. Use of the gene deleted attenuated african swine fever virus strain of claim 9 in the preparation of an african swine fever vaccine.

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