CN113373119A

CN113373119A - Three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus, construction method and application thereof

Info

Publication number: CN113373119A
Application number: CN202110628837.0A
Authority: CN
Inventors: 唐玉新; 叶昱; 顾俊; 宋德平; 黄冬艳
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2021-06-02
Filing date: 2021-06-02
Publication date: 2021-09-10

Abstract

The invention discloses a three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus, a construction method and application thereof, belonging to the technical field of biological medicine, wherein the invention utilizes CRISPR/Cas9 technology to quickly edit PRV virus strain genome, deletes three non-essential virulence genes TK, gI and gE of PRV, and then utilizes homologous recombination to repair, and inserts CD2v and p72 of ASFV or protein expression cassettes of p54 and p30 at the positions of TK and gI/gE genes respectively at fixed points. The invention does not need to utilize and remove the screening marker, edits three genes and inserts two exogenous genes at the same time, the method is simple, quick and efficient, the constructed recombinant strain provides a vaccine candidate strain for preventing PRV and ASFV infection, and the established method provides a new method and thought for the research and development of novel vaccines.

Description

Three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus, construction method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus, a construction method and application thereof.

Background

African Swine Fever (ASF) is a virulent infectious disease of domestic and wild pigs caused by African Swine Fever Virus (ASFV) infection, which can cause 100% morbidity and mortality of infected pigs. However, there are currently no vaccines and drugs available in the world for the prevention and control of african swine fever, and only through strict biosafety measures. The prevention and control measure has high cost and limited effect, and the vaccine is the most economic and effective means for preventing and controlling infectious diseases. At present, the vaccine for African swine fever in the world mainly comprises inactivated vaccine, subunit vaccine, DNA vaccine, attenuated live vaccine and the like.

And (3) inactivated vaccine: early vaccine research focused primarily on inactivated vaccines. The inactivated vaccine is safe, but has weak cellular immune response, is difficult to induce organisms to generate effective neutralizing antibodies, has no effective protection effect on ASFV virulent attack, and may have antibody-mediated infection enhancement.

Subunit vaccines: the CD2v, p72, p54 and p30 proteins can all induce neutralizing antibodies, and can be used as candidate antigens for subunit vaccine development. Researchers use baculovirus vectors to express related proteins to immunize swinery, and the baculovirus vectors have a certain effect of protecting the swinery from strong virus attack. However, the ASFV protective antigen currently used in subunit vaccines is not sufficient to provide complete protection. A few ASFV proteins, even if neutralizing antibodies are present, are difficult to provide effective immunoprotection as antigens.

DNA vaccine: the DNA vaccines reported so far can induce high levels of specific T cell responses in the host, producing humoral and cellular immunity, but still do not provide effective protection. The goal of providing effective immune protection through DNA vaccines remains a long way.

Attenuated live vaccines: (1) natural attenuated vaccine or attenuated vaccine by conventional technology: the ASFV low virulent strain obtained by screening natural low virulent strain from the natural stage as vaccine candidate strain or by continuous passage of cells can induce protective immune response aiming at parent strain. The vaccine may have cross protection, but the natural low virulent strain is easy to have strong virulence and has serious side effect; (2) genetic engineering attenuated vaccines: gene engineering technology is adopted to artificially delete genes so as to obtain attenuated vaccine strains. China reports a gene deletion virus strain (HLJ/18-7GD) constructed by Harbin veterinary research institute, and preliminary tests prove that the seven-gene deletion vaccine has better safety and effectiveness. However, the attenuated vaccine is a strong pathogenic or even lethal vaccine for animals with normal immunity if the animals have low immunity caused by immunosuppressive diseases or other basic diseases; in addition, different attenuated vaccine strains have the risk of generating new pathogenic strains through recombination; furthermore, there is a potential for reversion of all the attenuated seedlings.

Live virus vector vaccines: a large number of researches show that the virus vector vaccine can induce the pig to generate strong humoral response and cellular immunity in vivo and provide partial effective protection against ASFV infection. However, the immune response and protective efficacy induced by viral vector vaccines still need to be further evaluated.

At present, the research on the African swine fever vaccine at home and abroad mainly utilizes a gene deletion technology to research on an attenuated live vaccine, and the strategy for researching the attenuated live vaccine has at least two restriction bottlenecks: firstly, the safety of the vaccine is difficult to ensure; and the other is that no cell line is available for commercial production of attenuated live vaccines. The chimeric vector vaccine produced by using the porcine pseudorabies virus (PRV) which is the large DNA virus (the existing mature vaccine) as a framework can solve the two restriction factors, and simultaneously can become a multi-connection and multi-valence vaccine, and one vaccine can prevent two or even three diseases.

ASFV is a large double-stranded DNA virus, the genome size is 170-193kbp, contains 175 open reading frames 160-200, and can encode 150-200 proteins. It is now known that viral proteins p72(B646L), p54(E183L) and p30(CP204L) can be targets for neutralizing antibodies, and that expression of CD2v (EP402R) can also induce partial protection. Therefore, the gene can be inserted into a PRV vector to create a novel recombinant PRV African swine fever virus vaccine.

Pseudorabies Virus (PRV) belongs to the family of herpesviridae, the subfamily of alphaherpesviridae, the genus varicella. The virus can cause Porcine pseudorabies (Porcine pseudorabies), and the infection of newborn piglets is mainly manifested as nervous symptoms and digestive system symptoms such as diarrhea; infection in pregnant sows can cause abortion, stillbirth, mummy and respiratory symptoms. The PRV genome is more than 143kbp, can hold longer exogenous genes, and is an ideal chimeric seedling construction vector. Thus, by deleting non-essential genes of the virus, the virulence of the virus is reduced. On the basis, the insertion of exogenous target genes to construct the recombinant pseudorabies virus chimeric vector vaccine expressing the African swine fever gene is an ideal way.

The CRISPR/Cas9 system is the latest gene editing tool, gaining the annual nobel prize in 2020. The basic working principle of the CRISPR/Cas9 system is that sgRNA is used for guiding Cas9 protein to combine with a target gene locus, DNA double-strand break is generated at the specific gene locus, and then homologous sequences are provided for homologous recombination repair, so that a specific sequence can be inserted at the target gene locus, and the purpose of gene editing is achieved. The CRISPR/Cas9 is used as a high-efficiency gene editing tool, and the CRISPR/Cas9 system is widely applied to the field of biomedicine.

At present, CRISPR/Cas9 is utilized to construct PRV polygene deletion strain for expressing African swine fever protein, and the following reports mainly exist: feng et al, backbone a strain of pseudorabies virus with a deletion of the gE/gI double gene, inserted the expression cassette of CD2v after cleavage by a single sgRNA at the TK gene. The recombination fragment is inserted between 108 th and 109 th bases at the N end of the TK gene. Wherein, the N-terminal homology arm is 492bp, and the C-terminal homology arm is 466 bp. EGFP is driven by CMV promoter, Flag-CD2v-Flag is driven by EF1 alpha promoter, and the EGFP is provided with green fluorescence and Flag labels, but the selection marker is not removed, and refer to the description in figure 5.

The yellow sword also carries out the construction of recombinant viruses on the basis of a gE/gI double-gene-deleted PRV strain, uses double sgRNA strategy-mediated homologous recombination to insert p72, p54, p30, CD2v and pEP153R expression cassettes of ASFV containing CMV promoters into PRV genomes respectively and replace TK genes of PRV, and obtains recombinant viruses rPRV-p72, rPRV-p54, rPRV-p30, rPRV-CD2v and rPRV-pEP153R through screening and purification, and the scheme does not use a screening marker.

The technical route of the German scholars is as follows: screening out a cell line expressing Cas9 protein, inserting Bartha strain genome deletion gG gene into BAC vector to construct infectious clone plasmid pPrV-Ba delta gG, further deleting promoter and initial sequence of gD gene on the basis of the infectious clone plasmid pPrV-Ba delta gG to construct pPrV-Ba delta gGD, limiting the virus growth of the deletion gD gene, facilitating screening of recombinant virus, removing BAC plasmid sequence through homologous repair vector pUC-BAKJCAGPLnew, replacing the ASFV protein expression cassette and gD gene deletion part, and obtaining the normally replicated recombinant virus. The process is very complicated (refer to the attached figure 6 in the specification), but the CAG promoter is utilized to enhance the protein expression capacity. Therefore, the prior art has the following problems:

(1) in the prior art, gene editing is not carried out on the basis of PRV wild virus variant strains, but the construction of recombinant viruses is carried out after BAC plasmids and double-gene deletion strains are constructed, and the process is complex.

(2) The prior art partially carries the screening marker, and does not remove the screening marker, thereby having influence on clinical application.

(3) The prior art only inserts a single ASFV gene into the PRV genome.

The above references:

[1]FENG Z,CHEN J,LIANG W,et al.The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice[J].Virol J,2020,17(1):180.

[2] yellow sword, expression African swine fever virus structural protein recombinant pseudorabies virus construction and biological characteristic analysis [ D ]. Fujian agriculture and forestry university, 2020.

[3]

A,KEIL G M,KABUUKA T,et al.Efficient transgene insertion in a pseudorabies virus vector by CRISPR/Cas9 and marker rescue-enforced recombination[J].Journal of Virological Methods,2018,262: 38-47.

Disclosure of Invention

The invention aims to at least solve one of the technical problems in the prior art and provides a three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus, a construction method and application thereof.

The technical solution of the invention is as follows:

the invention relates to a three-gene deletion recombinant virus strain for expressing African swine fever virus, which is deposited by an agency approved by a patent and has the deposit numbers as follows: CCTCC NO: v202139; the classification is named as: porcine herpesvirus type 1 PRV TK^-/gI^-/gE^--p54⁺/p30⁺. The preservation unit: china center for type culture Collection; the preservation time was 2021, 05 and 10 days. The address of the depository: wuhan, Wuhan university, China.

The preservation number is as follows: CCTCC NO: v202128; the classification is named as: porcine herpesvirus type 1 PRV TK^-/gI^-/gE^--CD2v⁺/p72⁺. The preservation unit: china center for type culture Collection; the preservation time was 2021, 05 and 10 days. The address of the depository: wuhan, Wuhan university, China.

A recombinant virus strain expressing three gene deletions of African swine fever virus is characterized in that CD2v and p72 are respectively inserted into the TK gene and the gI/gE gene at fixed points, and the TK gene and the gI/gE gene are completely deleted.

A recombinant virus strain expressing African swine fever virus with three gene deletions is characterized in that p54 and p30 are respectively inserted into the sites of the TK and gI/gE genes of pseudorabies virus at fixed points, and the TK and gI/gE genes are completely deleted.

The invention also discloses a construction method for expressing the African swine fever virus three-gene deletion recombinant virus strain, which comprises the steps of editing a PRV virus strain genome by using CRISPR/Cas9, deleting TK and gI/gE genes of PRV, repairing by using homologous recombination, and respectively inserting CD2v and p72 of ASFV at fixed points at the TK and gI/gE genes, or inserting p54 and p30 at fixed points, wherein the TK and the gI/gE genes are all deleted.

Preferably, the method comprises the following steps:

s1, acquiring a full-length sequence of a PRV genome of a wild epidemic variant strain;

s2, designing sgRNAs for guiding Cas9 protein to specifically cut a target gene according to the sequencing sequences of the genes of the mutant strain PRV TK and gI/gE, wherein the sgRNAs are sgRNA-TK and sgRNA-gI/gE respectively; designing and synthesizing a forward and reverse sgRNA single-chain sequence on the sgRNA, connecting the annealed double-chain sgRNA to a PX459 plasmid, and transforming and extracting PX459-TK and PX459-gI/gE plasmid vectors for expressing the sgRNA sequence and Cas9 protein;

s3, respectively amplifying TKhmL, TKhmR, gI/gEhmL and gI/gEhmR of the left and right homologous arms of the TK gene and the gI/gE gene in vitro, and constructing homologous arm plasmids pUC19-TKhm and pUC19-gI/gEhm by enzyme digestion connection; inserting CAG-bGHpA elements between the left and right homologous arms to construct intermediate plasmids pUC19-TKhm-CAG-bGHpA and pUC 19-gI/gEhm-CAG-bGHpA;

s4, chemically synthesizing CD2v, p30, p54 and p72 gene sequences of ASFV;

s5, further constructing homologous repair donor plasmids pUC19-TKhm-CAG-CD2v-bGHpA, pUC19-TKhm-CAG-p54-bGHpA, pUC19-gI/gEhm-CAG-p72-bGHpA and pUC19-gI/gEhm-CAGp30-bGHpA by utilizing a method of enzyme digestion connection or seamless cloning; linearizing the four homologous repair donor plasmids by using ScaI or EcoRI restriction enzymes;

PRV genomic DNA, PX459-TK prepared in S2, PX459-gI/gE, pUC19-TKhm-CAG-CD2v-bGHpA linearized in S5, pUC19Co-transfecting gI/gEhm-CAG-p72-bGHpA to PK15 cells simultaneously to obtain the recombinant pseudorabies virus PRV TK expressing CD2v and p72 proteins^-/gI^-/gE^--CD2v⁺/p72⁺；

Or; PRV genome DNA, PX459-TK prepared in S2, PX459-gI/gE, and pUC19-TKhm-CAG-p54-bGHpA linearized in S5, pUC19-gIgEhm-CAG-p30-bGHpA are co-transfected into PK15 cells at the same time to obtain the recombinant pseudorabies virus PRV TK expressing p54 and p30 proteins^-/gI^-/gE^--p54⁺/p30⁺。

Preferably, the nucleotide sequences of the sgRNA-TK and the sgRNA-gI/gE are shown in SEQ ID No.1 and No. 2;

the nucleotide sequence of the TKhmL is shown in SEQ ID No. 3;

the nucleotide sequence of the TKhmR is shown in SEQ ID No. 4;

the nucleotide sequence of the gI/gEhmL is shown in SEQ ID No. 5;

the nucleotide sequence of the gI/gEhmR is shown in SEQ ID No. 6;

the nucleotide sequence of the CAG-bGHpA is shown in SEQ ID No. 7;

the nucleotide sequence of the CD2v is shown in SEQ ID No. 8;

the nucleotide sequence of the p30 is shown in SEQ ID No. 9;

the nucleotide sequence of the p54 is shown in SEQ ID No. 10;

the nucleotide sequence of the p72 is shown in SEQ ID No. 11.

The invention also discloses an application of the three-gene deletion recombinant virus strain for expressing the African swine fever virus in preparation of a medicament or a vaccine for treating or preventing PRV and ASFV infection.

The invention has at least one of the following beneficial effects: the invention provides a construction method for expressing a three-gene deletion recombinant virus strain of African swine fever virus. Compared with the traditional method of single gene editing and introducing a fluorescent screening marker and then removing the screening marker, the method provided by the invention has the advantages that three virulence genes of PRV are knocked out in one step by virtue of high editing efficiency of CRISPR/Cas9, and two recombinant PRV strains expressing ASFV proteins are constructed by respectively inserting two expression cassettes of the ASFV proteins. The process does not introduce fluorescence labeling genes, and the fluorescence labeling genes are deleted without screening and constructing successfully. The method is novel, efficient, rapid and accurate, and greatly reduces the operation flow and time. The invention provides a vaccine candidate strain, a novel vaccine research and development method and a novel vaccine research and development idea for preventing ASFV and PRV infection.

The literature in the simultaneous and background art has the following advantages:

(1) in the prior art, gene editing is not carried out on the basis of PRV wild virus variant strains, but the construction of recombinant viruses is carried out after BAC plasmids and double-gene deletion strains are constructed, and the process is complex. The invention directly operates PRV wild virus variant isolates.

(2) In order to facilitate screening of recombinant viruses, a screening marker is added in the prior art, and after the screening marker is added, the screening marker needs to be removed. And when the antibody aiming at the foreign protein is lacked, a protein label is added to facilitate the verification of whether the virus expresses the inserted foreign gene or not after construction, but the addition of the label belongs to a redundant sequence, the risk still needs to be evaluated in clinical application, and certain safety risk is realized. The method directly introduces the ASFV protein without any label and screening marker by utilizing the high-efficiency editing of the CRISPR/Cas9 system, thereby avoiding the step of label removal.

(3) In the prior art, only a single ASFV gene is inserted into a PRV genome, and in one transfection, editing and insertion of two sites are performed simultaneously, so that the efficiency is not high, and whether the editing and insertion can be completed simultaneously or not needs to be evaluated. The construction method of the invention can insert two genes of ASFV while deleting three genes directly, and obtain PRV three-gene deletion strains expressing two ASFV proteins rapidly.

Drawings

FIGS. 1(a), (b) are homologous arm plasmids pUC19-TKhm, pUC19-gI/gEhm, respectively, according to the present invention;

FIGS. 2(a), (b) are the intermediate plasmids pUC19-TKhm-CAG-bGHpA, pUC19-gI/gEhm-CAG-bGHpA, respectively, according to the present invention;

FIGS. 3(a), (b) are homologous repair donor plasmids pUC19-TKhm-CAG-CD2v-bGHpA, pUC19-gI/gEhm-CAG-p72-bGHpA, respectively, of the present invention;

FIGS. 4(a), (b) are homologous repair donor plasmids pUC19-TKhm-CAG-p54-bGHpA, pUC19-gI/gEhm-CAG-p30-bGHpA, respectively, of the present invention;

FIG. 5 is a construction strategy diagram of Feng et al in the background art;

fig. 6 is a technical route diagram of a german scholars in the background art.

Detailed Description

In order to further specifically illustrate the present invention, the technical solutions in the embodiments of the present invention will be specifically and clearly described below, the embodiments described below are only a part of the embodiments in the present invention, and the present disclosure is not limited to the following experimental examples. The examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The following experimental methods are all conventional methods unless otherwise specified. Note that gI/gE is expressed as two genes of gI and gE at adjacent positions, and the two genes are deleted as a whole in editing.

The invention provides a TK/gI/gE triple gene deletion PRV vector for expressing CD2v (or p54) and p72 (or p30) proteins of ASFV, a recombinant PRV strain and a construction method thereof, and specifically comprises the following steps: fig. 1-4 may be combined.

1. A field epidemic variant PRV strain is separated and identified by using the conventional virology and molecular biology techniques. Specifically, diseased organs of diseased pigs with typical PRV clinical symptoms and pathological changes are collected, total DNA is extracted, and PCR identification is carried out. And homogenizing and filtering the positive disease material, inoculating PK15 cells, conducting blind transmission for three generations, and performing three-round plaque purification after characteristic lesions appear to obtain a PRV strain.

2. And (3) carrying out ultra-separation purification on the PRV produced by separation by utilizing sucrose density gradient centrifugation, extracting PRV genome DNA by utilizing a phenol chloroform method, and obtaining a PRV genome full-length sequence by utilizing a Nanopore sequencing technology.

3. Design and plasmid construction of sgRNA of targeting pseudorabies virus gI/gE gene and TK gene

(1) According to the TK gene and gI/gE gene sequences obtained by sequencing, an online sgRNA design software (https:// zlab. bio/guide-design-resources) is used for designing sgRNA sequences which are named as sgRNA-TK and sgRNA-gI/gE respectively. On the basis of the sequence, forward and reverse sgRNA single-stranded sequences (table 1) were synthesized, and were subjected to phosphorylation modification using T4 PNK, followed by annealing to form a double-stranded DNA. The specific reaction conditions are as follows: carrying out reaction at 37 ℃ for 30min for phosphorylation; reacting at 95 ℃ for 5min to inactivate enzyme and fully denature the nucleic acid single strand; the temperature is reduced by 5 ℃ per minute, annealing is slowly carried out until the temperature reaches 25 ℃, double strands of the sgRNA with sticky ends are formed, and the sgRNA is temporarily stored at the temperature of 4 ℃.

TABLE 1

(2) After the sgRNA annealing product is diluted by 200 times, T4 DNA ligase is used for carrying out ligation reaction with PX459 vector linearized by BbsI restriction endonuclease, and the reaction conditions are as follows: 25 ℃ for 30 min. The ligation products were transformed into Top10 competent cells, plated with LB plates containing ampicillin, and single colonies were picked for sequencing validation. Successfully identified positive clone is amplified and cultured, a plasmid is extracted by using an endotoxin-free plasmid extraction kit, and vectors PX459-TK and PX459-gI/gE of the sgRNA and Cas9 protein co-plasmid are prepared.

4. Construction of homologous repair Donor plasmids

(1) Construction of intermediate plasmids pUC19-TKhm-CAG-bGHpA, pUC19-gI/gEhm-CAG-bGHpA

Based on the TK and gI/gE gene sequences and the upstream and downstream sequences thereof, primers (see Table 2) are designed to amplify the upstream and downstream homologous arm sequences. The specific process is as follows, PRV genome DNA is used as a template, and NEB Q5 Hot-Start super fidelity DNA polymerase and TKhmL-F/R, TKhmR-F/R, gI/gEhmL-F/R, gI/gEhmR-F/R four pairs of primers are used for PCR amplification (refer to NEB official instruction). And (3) carrying out gel recovery and purification after electrophoresis, wherein the obtained TK gene left and right homologous arm fragments are named as TKhmL and TKhmR, and the obtained gI/gE gene left and right homologous arm fragments are named as gI/gEhmL and gI/gEhmR. The pUC19 vector and each of the homologous arm fragments were subjected to double digestion. The three-fragment ligation reaction of pUC19, TKhmL and TKhmR was performed using T4 DNA ligase to obtain the homologous arm plasmid pUC 19-TKhm. The three-fragment ligation reaction of pUC19, gI/gEhmL and gI/gEhmR was performed using T4 DNA ligase to obtain the homologous arm plasmid pUC 19-gI/gEhm. The CAG-bGHpA element is cut off from a eukaryotic expression plasmid preserved in a laboratory by double enzyme digestion reaction and is connected to homologous arm plasmids pUC19-TKhm and pUC19-gI/gEhm to construct intermediate plasmids pUC19-TKhm-CAG-bGHpA and pUC 19-gI/gEhm-CAG-bGHpA.

(2) Construction of pUC19-TKhm-CAG-CD2v-bGHpA, pUC19-TKhm-CAG-p54-bGHpA, pUC19-gI/gEhm-CAG-p72-bGHpA, pUC19-gI/gEhm-CAG-p30-bGHpA

ASFV CD2v, p72, p54 and p30 gene sequences are synthesized, CD2v and p72 sequences, and p54 and p30 sequences are respectively inserted into pUC19-TKhm-CAG-bGHpA, pUC19-gI/gEhm-CAG-bGHpA by using enzyme digestion connection or seamless cloning technology to obtain four homologous repair donor plasmids pUC19-TKhm-CAG-CD2v-bGHpA, pUC19-TKhm-CAG-p54-bGHpA and pUC19-gI/gEhm-CAG-p72-bGHpA, pUC19-gI/gEhm-CAG-p30-bGHpA, and non-endotoxin plasmids are extracted.

TABLE 2

4. Porcine herpesvirus type 1 PRV TK^-/gI^-/gE^--CD2v⁺/p72⁺Construction of

(1) Linearized homologous repair donor plasmid: pUC19-TKhm-CAG-CD2v-bGHpA and pUC19-gI/gEhm-CAG-p72-bGHpA were linearized using ScaI restriction enzymes.

(2) Transfection: the total amount of the mixed DNA is 5 mug, and the mixed DNA comprises PRV genome DNA, linearized pUC19-TKhm-CAG-CD2v-bGHpA, linearized pUC19-gI/gEhm-CAG-P72-bGHpA, PX459-TK, PX459-gI/gE according to the mass ratio of 1: 1: 1: 1: 1, use Lipofectamine ^TM3000 Reagent was co-transfected into PK15 cells and after 48 hours of culture, lesions were observed.

(3) And (3) plaque purification: harvesting lesion cells, and carrying out multiple rounds of plaque purification, wherein each round of detection is carried out on CD2v, p72, TK and gI/gE genes by using a PCR method; until the CD2v and p72 genes can be stably detected, and the TK and gI/gE genes cannot be detected.

5. Porcine herpesvirus type 1 PRV TK^-/gI^-/gE^--p54⁺/p30⁺Construction of

(1) Linearized homologous repair donor plasmid: pUC19-TKhm-CAG-p54-bGHpA, pUC19-gI/gEhm-CAG-p30-bGHpA were linearized using EcoRI restriction enzymes.

(2) Transfection: the total amount of the mixed DNA is 5 mug, and the mixed DNA comprises PRV genome DNA, linearized pUC19-TKhm-CAG-p54-bGHpA, linearized pUC19-gI/gEhm-CAG-p30-bGHpA, PX459-TK, PX459-gI/gE according to the mass ratio of 1: 1: 1: 1: 1, use Lipofectamine ^TM3000 Reagent was co-transfected into PK15 cells and after 48 hours of culture, lesions were observed.

(3) And (3) plaque purification: and (3) harvesting diseased cells, and carrying out multiple rounds of plaque purification, wherein p54, p30, TK and gI/gE genes are detected by using a PCR method in each round until the p54 and p30 genes can be stably detected, but the TK and gI/gE genes cannot be detected.

In the embodiment, three nonessential virulence genes TK, gI and gE of PRV are deleted, the toxicity is reduced, homologous recombination is used for repairing, ASFV CD2v and p72 with inducible neutralizing antibodies or protein expression cassettes of p54 and p30 are respectively inserted at fixed points at the TK and gI/gE genes, a vaccine candidate strain is provided for preventing PRV and ASFV infection, and the established method provides a new method and thought for the development of a novel vaccine.

The embodiments were chosen and described in detail to help explain the invention in an extensive manner, not all embodiments, and not all details of the invention are described in an exhaustive manner. Further modifications and variations are possible in light of the above teachings. But all such modifications and improvements that do not depart from the gist of the invention are intended to be within the scope of the invention as claimed.

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cctcgtctcg taccaggtgg tgcccccgcc cgtctccgcg tggtactttg tgcagcgccc 480

gcgcgagcgc ccgcgcctct cggggccgcc ctcgggcgcg gagctcgtgg ccttcgacgc 540

gcccggcgtc cggcgcacgt acaccacggc ggcggtgtgg cccgcggagg tggccgtcct 600

cgcggacgcg gaggcgcgct gccccgcggc cgtcttcaac gtgacgctgg gcgaggcctt 660

cctcggcctg cgcgtcgcgc tgcgctcctt cctgccgctg gaggtcatca tctccgccga 720

gcggatgcgc atgatcgcgc ccccggcgct cggctcggac ctggagccgc cgggcccgcc 780

cgcgggccgc ttccacgtgt acacgctcgg cttcctctcc gacggggcca tgcaccagac 840

gatgcgcgac gtggccgcct acgtgcacga gagcgacgac tacctcgccc agctgtcggc 900

ggcgcacgcg gccgccctgg ccgccgtggt gcagcccggg ccgtactact tttaccgcgc 960

ggcggtgcgc ctcggcgtgg ccgccttcgt cttctccgag gcgg 1004

<210> 5

<211> 1123

<212> DNA

<213> Artificial Sequence 5(Artificial Sequence5)

<400> 5

cgtacaccga gtcgtggcag ctgacgctga cgacggtccc ctcgcccttc gtcggccccg 60

cggacgtcta ccacacgcgc ccgctggagg acccgtgcgg ggtggtggcg ctgatctccg 120

acccgcaggt ggaccggctg ctgaacgagg cggtggccca ccggcggccc acgtaccgcg 180

cccacgtggc ctggtaccgc atcgcggacg ggtgcgcgca cctgctgtac tttatcgagt 240

acgccgactg cgaccccagg cagatctttg ggcgctgccg gcgccgcacc acgccgatgt 300

ggtggacccc gtccgcggac tacatgttcc ccacggagga cgagctgggg ctgctcatgg 360

tggccccggg gcggttcaac gagggccagt accggcgcct ggtgtccgtc gacggcgtga 420

acatcctcac cgacttcatg gtggcgctcc ccgaggggca agagtgcccg ttcgcccgcg 480

tggaccagca ccgcacgtac aagttcggcg cgtgctggag cgacgacagc ttcaagcggg 540

gcgtggacgt gatgcgattc ctgacgccgt tctaccagca gcccccgcac cgggaggtgg 600

tgaactactg gtaccgcaag aacggccgga cgctcccgcg ggcctacgcc gccgccacgc 660

cgtacgccat cgaccccgcg cggccctcgg cgggctcgcc gaggcccagg ccccggcccc 720

ggcccaggcc ccggccgaag cccgagcccg ccccggcgac gcccgcgccc cccggccgcc 780

tgcccgagcc ggcgacgcgg gaccacgccg ccggggggcg ccccacgccg cgacccccga 840

ggcccgagac gccgcaccgc cccttcgccc cgccggccgt cgtgcccagc gggtggccgc 900

agcccgcgga gccgttcccg ccccggacca ccgccgcgcc gggcgtctcg cgccaccgct 960

cggtgatcgt cggcacgggc accgcgatgg gcgcgctcct ggtgggcgtg tgcgtctaca 1020

tcttcttccg cctgaggggg gcgaaggggt atcgcctcct gggcggtccc gcggacgccg 1080

acgagctaaa agcgcagccc ggtccgtagc ctccgcagta ccg 1123

<210> 6

<211> 1329

<212> DNA

<213> Artificial Sequence 6(Artificial Sequence6)

<400> 6

catggacacg tttgacccca gcgcccccgt cccgacgagc gtctcgaacc cggccgccga 60

cgtcctgctg gcccccaagg gaccccgctc cccgctgcgc ccccaggacg actcggactg 120

ctactacagc gagagcgaca acgagacgcc cagcgagttc ctgcgccgcg tgggacgccg 180

gcaggcggcg cgtcggagac gccgccgctg cctgatgggc gtcgcgatca gcgccgccgc 240

gctggtcatc tgctcgctgt ccgcgctact cgggggcatc gtcgccaggc acgtgtagcg 300

agcgagcgag cgaacgggag cgggggcccg cccccatccg ccgcgcccag gagagggggg 360

agagagcggg gggttgggcg cgccacgtgg tgtgggcacg gactcggact tgtcacaata 420

aatgggcccc ggcgtgtccg ggcgcacaca gcagccttcc tctcctccgc gtctctgttc 480

cgcccgtctc tcgccggact cttcttctcc accgcctcca ccgtcgcagt tgtcgcgagc 540

gcgttcgcac catgggggtg acggccatca ccgtggtcac gctgatggac ggggccgggc 600

gcatccccgc cttcgtgggc gaggcgcacc cggacctgtg gaaggtgctc accgagtggt 660

gctacgcgtc gatggtgcag cagcggcgcg ccgccgacga gaactcgccg cggcagcacg 720

tggtgctgcg ctcctcggag atctcccccg gctcgctggc cctgctgccg cgcgccgtgc 780

gccccgtcgt gcggacgcgg tccgacccca cggcgccgtt ctacatcacc accgagacgc 840

acgagctgac gcggcgcccc ccggcggacg gctcgaagcc cggggagccc ctcaggatca 900

gcccaccccc gcggctggac acggagtggt cgtccgtcct gaacgggatc cagtacctga 960

actcgggggc ccggggcacg gcccccgtcc acctgtggat cctgggcgcc gccgacctct 1020

gcgaccaggt gctcctggcc gcctcccgca gcaccgccgc cggagcctcc cacgcccaga 1080

cgggcgcgcg cctgacccgg cgccggcccg ggctgacgga cgccgacgcc ctggacgtga 1140

tcgtcgccgg gatccaggcg acccgcgcca tgttcgcgcg ggtccacaac cgctcctggc 1200

gccacgccgg cgagtggacg gaggccctgc actcccagat cgtgacccgg ggcgacgtgc 1260

gccggcgccg aggcgggcgc ggcaacggac gcgagcgcgc cccgcgatgt accatctcct 1320

agacggcag 1329

<210> 7

<211> 1058

<212> DNA

<213> Artificial Sequence 7(Artificial Sequence7)

<400> 7

tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga 60

cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggt 120

ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 180

gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac 240

cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 300

cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat 360

tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg 420

gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc 480

ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg 540

gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg 600

ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt 660

actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agctgagcaa 720

gaggtaaggg tttaagggat ggttggttgg tggggtatta atgtttaatt acctggagca 780

cctgcctgaa atcacttttt ttcaggttgg accggtacgc tagcagctcg ctgatcagcc 840

tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg 900

accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat 960

tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag 1020

gattgggaag agaatagcag gcatgctggg gaatcgat 1058

<210> 8

<211> 1083

<212> DNA

<213> Artificial Sequence 8(Artificial Sequence8)

<400> 8

atggtaatac ttattttttt aatattttct aacatagttt taagtattga ttattgggtt 60

agttttaata aaacaataat tttagatagt aatattacta atgataataa tgatataaat 120

ggagtatcat ggaatttttt taataattct tttaatacac tagctacatg tggaaaagca 180

ggtaactttt gtgaatgttc taattatagt acatcaatat ataatataac aaataattgt 240

agcttaacta tttttcctca taatgatgta tttgatacaa catatcaagt agtatggaat 300

caaataatta attatacaat aaaattatta acacctgcta ctcccccaaa tatcacatat 360

aattgtacta attttttaat aacatgtaaa aaaaataatg gaacaaacac taatatatat 420

ttaaatataa atgatacttt tgttaaatat actaatgaaa gtatacttga atataactgg 480

aataatagta acattaacaa ttttacagct acatgtataa ttaataatac aattagtaca 540

tctaatgaaa caacacttat aaattgtact tatttaacat tgtcatctaa ctatttttat 600

acttttttta aattatatta tattccatta agcatcataa ttgggataac aataagtatt 660

cttcttatat ccatcataac ttttttatct ttacgaaaaa gaaaaaaaca tgttgaagaa 720

atagaaagtc caccacctga atctaatgaa gaagaacaat gtcagcatga tgacaccact 780

tccatacatg aaccatctcc cagagaacca ttacttccta agccttacag tcgttatcag 840

tataatacac ctatttacta catgcgtccc tcaacacaac cactcaaccc atttccctta 900

cctaaaccgt gtcctccacc caaaccatgt ccgccaccca aaccatgtcc tccacctaaa 960

ccatgtcctt cagctgaatc ctattctcca cccaaaccac tacctagtat cccgctacta 1020

cccaatatcc cgccattatc tacccaaaat atttcgctta ttcacgtaga tagaattatt 1080

taa 1083

<210> 9

<211> 606

<212> DNA

<213> Artificial Sequence 9(Artificial Sequence9)

<400> 9

atggatttta ttttaaatat atccatgaaa atggaggtca tcttcaaaac ggatttaaga 60

tcatcttcac aagttgtgtt tcatgcgggt agcctgtata attggttttc tgttgagatt 120

atcaatagcg gtagaattgt tacgaccgct ataaaaacat tgcttagtac tgttaagtat 180

gatattgtga aatctgctcg tatatatgca gggcaagggt atactgaaca tcaggctcaa 240

gaagaatgga atatgattct gcatgtgctg tttgaagagg agacggaatc ctcagcatct 300

tcggagaaca ttcatgaaaa aaatgataat gaaaccaatg aatgcacatc ctcctttgaa 360

acgttgtttg agcaagagcc ctcatcggag gtacctaaag actccaagct gtatatgctt 420

gcacaaaaga ctgtgcaaca tattgaacaa tatggaaagg cacctgattt taacaaggtt 480

attagagcac ataattttat tcaaaccatt tatggaaccc ctctaaagga agaagaaaaa 540

gaggtggtaa gactcatggt tattaaactt ttaaaaaaaa taagctttta tctcacctac 600

atttaa 606

<210> 10

<211> 555

<212> DNA

<213> Artificial Sequence10 (Artificial Sequence10)

<400> 10

atggattctg aattttttca accggtttat ccgcggcatt atggtgagtg tttgtcacca 60

gtcactacac caagcttctt ctccacacat atgtatacta ttctcattgc tatcgtggtc 120

ttagtcatca ttatcatcgt tctaatctat ctattctctt caagaaagaa aaaagctgct 180

gctattgagg aggaagatat acagtttata aatccttatc aagatcagca gtgggtagaa 240

gtcactccac aaccaggtac ctctaaacca gctggagcga ctacagcaag tgtaggcaag 300

ccagtcacgg gcagaccggc aacaaacaga ccagcaacaa acaaaccagt tacggacaac 360

ccagttacgg acagactagt catggcaact ggcgggccgg cggccgcacc tgcggccgcg 420

agtgctcctg ctcatccggc tgagccttac acgacagtca ctactcagaa cactgcttca 480

caaacaatgt cggctattga aaatttacga caaagaaaca cctatacgca taaagaccta 540

gaaaactcct tgtaa 555

<210> 11

<211> 1941

<212> DNA

<213> Artificial Sequence11 (Artificial Sequence11)

<400> 11

atggcatcag gaggagcttt ttgtcttatt gctaacgatg ggaaggccga caagattata 60

ttggcccaag acttgctgaa tagcaggatc tctaacatta aaaatgtgaa caaaagttat 120

gggaaacccg atcccgaacc cactttgagt caaatcgaag aaacacattt ggtgcatttt 180

aatgcgcatt ttaagcctta tgttccagta gggtttgaat acaataaagt acgcccgcat 240

acgggtaccc ccaccttggg aaacaagctt acctttggta ttccccagta cggagacttt 300

ttccatgata tggtgggcca tcatatattg ggtgcatgtc attcatcctg gcaggatgct 360

ccgattcagg gcacgtccca gatgggggcc catgggcagc ttcaaacgtt tcctcgcaac 420

ggatatgact gggacaacca aacaccctta gagggcgccg tttacacgct tgtagatcct 480

tttggaagac ccattgtacc cggcacaaag aatgcgtacc gaaacttggt ttactactgc 540

gaataccccg gagaacgact ttatgaaaac gtaagattcg atgtaaatgg aaattcccta 600

gacgaatata gttcggatgt cacaacgctt gtgcgcaaat tttgcatccc aggggataaa 660

atgactggat ataagcactt ggttggccag gaggtatcgg tggagggaac cagtggccct 720

ctcctatgca acattcatga tttgcacaag ccgcaccaaa gcaaacctat tcttaccgat 780

gaaaatgata cgcagcgaac gtgtagccat accaacccga aatttctttc acagcatttt 840

cccgagaact ctcacaatat ccaaacagca ggtaaacaag atattactcc tatcacggac 900

gcaacgtatc tggacataag acgtaatgtt cattacagct gtaatggacc tcaaacccct 960

aaatactatc agccccctct tgcgctctgg attaagttgc gcttttggtt taatgagaac 1020

gtgaaccttg ctattccctc agtatccatt cccttcggcg agcgctttat caccataaag 1080

cttgcatcgc aaaaggattt ggtgaatgaa tttcctggac tttttgtacg ccagtcacgt 1140

tttatagctg gacgccccag tagacgcaat atacgcttta aaccatggtt tatcccagga 1200

gtcattaatg aaatctcgct cacgaataat gaactttaca tcaataacct gtttgtaacc 1260

cctgaaatac acaacctttt tgtaaaacgc gttcgctttt cgctgatacg tgtccataaa 1320

acgcaggtga cccacaccaa caataaccac cacgatgaaa aactaatgtc tgctcttaaa 1380

tggcccattg aatatatgtt tataggatta aaacctacct ggaacatctc cgatcaaaat 1440

cctcatcaac accgagattg gcacaagttc ggacatgttg ttaacgccat tatgcagccc 1500

actcaccacg cagagataag ctttcaggat agagatacag ctcttccaga cgcatgttca 1560

tctatatctg atattagccc cgttacgtat ccgatcacat tacctattat taaaaacatt 1620

tccgtaactg ctcatggtat caatcttatc gataaatttc catcaaagtt ctgcagctct 1680

tacataccct tccactacgg aggcaatgcg attaaaaccc ccgatgatcc gggtgcgatg 1740

atgattacct ttgctttgaa gccacgggag gaataccaac ccagtggtca tattaacgta 1800

tccagagcaa gagaatttta tattagttgg gacacggatt acgtggggtc tatcactacg 1860

gctgatcttg tggtatcggc atctgctatt aactttcttc ttcttcagaa cggttcagct 1920

gtgctgcgtt acagtaccta a 1941

Claims

1. A three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus is characterized in that: preserved in China Center for Type Culture Collection (CCTCC) at 2021, 5 months and 10 days, and porcine herpesvirus type 1 PRV TK^-/gI^-/gE^--p54⁺/p30⁺The preservation number is: CCTCC NO: v202139; porcine herpesvirus type 1 PRV TK^-/gI^-/gE^--CD2v⁺/p72⁺The preservation number is: CCTCC NO: v202128.

2. A three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus is characterized in that: CD2v and p72 are respectively inserted into the sites of TK and gI/gE genes of pseudorabies virus at fixed points, and the TK and gI/gE genes are totally deleted.

3. A three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus is characterized in that: the p54 and the p30 are respectively inserted into the sites of the TK and gI/gE genes of the pseudorabies virus at fixed points, and the TK and gI/gE genes are totally deleted.

4. A construction method for expressing three-gene deletion recombinant pseudorabies virus strains of African swine fever virus is characterized by comprising the following steps: editing PRV virus strain genome by using CRISPR/Cas9, deleting TK and gI/gE genes of PRV, repairing by using homologous recombination, and respectively inserting CD2v and p72 of ASFV at the positions of TK and gI/gE genes, or inserting p54 and p30 at fixed points, wherein the TK and gI/gE genes are completely deleted.

5. The method for constructing the African swine fever virus three-gene deletion recombinant pseudorabies virus strain according to claim 4, wherein the method comprises the following steps: the method comprises the following steps:

s1, obtaining a full-length sequence of a PRV genome of a variant strain;

s2, designing sgRNAs for guiding Cas9 protein to specifically cut a target gene according to TK and gI/gE gene sequencing sequences of a variant PRV, wherein the sgRNAs are sgRNA-TK and sgRNA-gI/gE respectively; designing and synthesizing a forward and reverse sgRNA single-chain sequence on the sgRNA single-chain sequence, connecting a double-chain sgRNA obtained after annealing the sgRNA to a PX459 plasmid, and transforming and extracting PX459-TK and PX459-gI/gE plasmid vectors for expressing the sgRNA sequence and Cas9 protein;

s4, chemically synthesizing CD2v, p30, p54 and p72 gene sequences of ASFV;

s5, further constructing homologous repair donor plasmids pUC19-TKhm-CAG-CD2v-bGHpA, pUC19-TKhm-CAG-p54-bGHpA, pUC19-gI/gEhm-CAG-p72-bGHpA and pUC19-gI/gEhm-CAG-p30-bGHpA by utilizing a method of enzyme digestion connection or seamless cloning; linearizing the four homologous repair donor plasmids by using ScaI or EcoRI restriction enzymes;

s6, simultaneously co-transfecting PRV genome DNA, PX459-TK prepared in S2, PX459-gI/gE and linearized pUC19-TKhm-CAG-CD2v-bGHpA and UC19-gI/gEhm-CAG-p72-bGHpA in S5 into PK15 cells to obtain the recombinant pseudorabies virus PRV TK expressing CD2v and p72 proteins^-/gI^-/gE^--CD2v⁺/p72⁺；

6. The method for constructing the African swine fever virus three-gene deletion recombinant pseudorabies virus strain according to claim 5, wherein the method comprises the following steps: the nucleotide sequences of the sgRNA-TK and the sgRNA-gI/gE are shown in SEQ ID No.1 and No. 2;

the nucleotide sequence of the TKhmL is shown in SEQ ID No. 3;

the nucleotide sequence of the TKhmR is shown in SEQ ID No. 4;

the nucleotide sequence of the gI/gEhmL is shown in SEQ ID No. 5;

the nucleotide sequence of the gI/gEhmR is shown in SEQ ID No. 6;

the nucleotide sequence of the CAG-bGHpA is shown in SEQ ID No. 7;

the nucleotide sequence of the CD2v is shown in SEQ ID No. 8;

the nucleotide sequence of the p30 is shown in SEQ ID No. 9;

the nucleotide sequence of the p54 is shown in SEQ ID No. 10;

the nucleotide sequence of the p72 is shown in SEQ ID No. 11.

7. Use of a recombinant pseudorabies virus strain expressing the deletion of the three genes of african swine fever virus according to any one of claims 1 to 3 for the preparation of a medicament or vaccine for the treatment or prevention of PRV and ASFV infections.