CN105802921B

CN105802921B - Recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein and construction method and application thereof

Info

Publication number: CN105802921B
Application number: CN201610236150.1A
Authority: CN
Inventors: 仇华吉; 孙元; 雷建林; 罗玉子
Original assignee: Harbin Veterinary Research Institute of CAAS
Current assignee: Harbin Veterinary Research Institute of CAAS
Priority date: 2016-04-15
Filing date: 2016-04-15
Publication date: 2020-08-18
Anticipated expiration: 2036-04-15
Also published as: CN105802921A

Abstract

The invention discloses a recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein and a construction method and application thereof. The invention firstly discloses a recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein, which is constructed by taking a recombinant pseudorabies virus variant strain with deletion of three genes of gE, gI and TK as a carrier and expressing classical swine fever virus E2protein, wherein the microorganism preservation number is as follows: CGMCC No. 12038. The recombinant pseudorabies virus variant strain for expressing the classical swine fever virus E2protein has genetic stability, can stably express the E2protein, has high safety to piglets, can simultaneously induce high-level pseudorabies virus and classical swine fever virus specific antibodies and neutralizing antibodies, can completely protect the classical swine fever virus and the pseudorabies virus variant strain from being attacked, and can be used as a bivalent genetic engineering vaccine candidate strain for preventing and controlling the current epidemic pseudorabies virus variant strain and classical swine fever virus mixed infection.

Description

Recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein and construction method and application thereof

Technical Field

The invention relates to a recombinant pseudorabies virus variant strain, in particular to a recombinant pseudorabies virus variant strain expressing classical swine fever virus E2protein, and also relates to a construction method of the recombinant pseudorabies virus variant strain and application of the recombinant pseudorabies virus variant strain in preparation of a bivalent genetic engineering vaccine for preventing and controlling common infection of PRV variant strains and classical swine fever viruses.

Background

Classical Swine Fever (CSF) is a virulent swine fever disease caused by CSFV. CSF is listed by the world animal health Organization (OIE) in the OIE epidemic list (OIE-iisted diseases). In China, "national middle and long term animal epidemic prevention and control program (2012-2020)," swine fever "is listed as one of the major animal epidemics with priority for prevention and control.

In the middle of the 50 th century, Chinese scientists successfully cultured a hog cholera lapinized attenuated vaccine (C strain) by continuously transmitting hog cholera virulent virus to rabbits for hundreds of generations, and the vaccine plays an important role in preventing and controlling the Chinese hog cholera. However, other comprehensive control measures are ignored due to the one-sided dependence on vaccination for a long time, and the C strain vaccine lacks a marking function (namely, the vaccine immunity and the CSFV infection cannot be distinguished) and other reasons, so that the vaccine is not completely controlled although being applied to China for more than half a century, swine fever still occurs in some areas, and still forms a great threat to the pig industry in China so far. Therefore, there is an urgent need to develop a novel swine fever vaccine which is safe, effective and has a labeling function.

Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease of various domestic and wild animals such as pigs, sheep, cattle, etc. caused by pseudorabies virus (PRV) and is characterized mainly by fever, extreme itching, encephalomyelitis, respiratory and nervous system disorders. After the pregnant sow is infected, abortion, dead fetus and mummy fetus occur, wherein the pregnant sow is mainly characterized by the production of the dead fetus; the piglets after the infection of the suckling piglets have nervous symptoms, paralysis and exhaustion death, the death rate is almost up to 100 percent, and the piglets are greatly damaged. Since 2011, China has more than 20 provinces of pig farms, including large-scale pig farms immunized with the attenuated pseudorabies vaccine Bartha-K61 strain, and the outbreak of the pseudorabies epidemic situation is continued. Studies have shown that new circulating strains of PRV are significantly altered and have significantly enhanced pathogenicity compared to previous strains, and that existing vaccines do not completely protect the infection of the new circulating strains (Wang CH, et al. A novel gE-deleted vaccines viruses (PRV) pro-viruses and complete protection from circulating strains with the PRV variant implementation in Bartha-K61-vaccine deletion in China. Vacine. 2014; 32(27): 3379-85; Luo Y, et al. nutritional analysis of a pathogenic virus variant in Bartha-K61-vaccine mutation. 2014; 7. 2014. 7. Micro. 1. 15. A. novel strain is a novel strain of A. vaccine virus variant).

The lack of one or several virus replication non-essential genes in PRV gene can reduce virus virulence without affecting its proliferation and immunogenicity, so that PRV as carrier carrying exogenous antigen gene into organism can induce organism to produce immune reaction to PRV and immune response to exogenous antigen. Therefore, these gene-deleted PRVs are often used as vectors to express foreign genes to construct multivalent vaccines (Luominghua, et al, Pseudorabies virus vector vaccine research progress. animal medicine progress. 2013; 34(6): 136-. PRV different types of gene deletion viruses are widely applied to the development of vaccines for viral animal diseases such as porcine reproductive and respiratory syndrome, rabies, foot and mouth disease, porcine circovirus, porcine parvovirus, Japanese encephalitis, swine fever virus, swine influenza and the like (Tian ZJ, ethylene. A receptor pseudovirus infection the HA gene from H3N2 subtype viral infection detection from viral infection change. vector immunological detection. 2006; 111(3-4): 8; Zhang K, et al. recombinant pseudovirus infection P12A and 3C of FMDV splice virus infection strain DVage. Res Vet; 2011 1.2011.2011.4).

The E2protein is located on the surface of CSFV cyst membrane, participates in the virus infection process, is the main protective antigen protein of CSFV, can induce organism to generate neutralizing antibody, and protect organism from CSFV virulent attack, and the protein becomes the main antigen for developing new vaccines for preventing and controlling CSFV, such as DNA vaccine, subunit vaccine and live virus vector vaccine. However, many of these vaccines still have problems such as failure to provide complete protection of CSFV, failure to produce neutralizing antibodies prior to challenge, and failure to fully convert positive antibodies specific to E2 induced by immunized pigs.

In order to prevent the current PRV and CSFV mixed infection, reduce the production cost and overcome the defect that the C vaccine lacks the marking function, the development of a new bivalent genetic engineering vaccine candidate strain with better safety and immunogenicity is urgently needed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein, the recombinant pseudorabies virus variant strain has genetic stability, can stably express E2protein, has good safety and immunogenicity, can simultaneously induce high-level PRV and CSFV specific antibodies and neutralizing antibodies, and can be used as a bivalent genetic engineering vaccine candidate strain for preventing and controlling the common infection of the current popular PRV variant strain and classical swine fever virus;

the invention also provides a construction method of the recombinant pseudorabies virus variant strain for expressing the classical swine fever virus E2 protein.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention firstly discloses a recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein, which takes a recombinant pseudorabies virus variant strain rPRVTJ-delgE/gI/TK with deletion of genes gE, gI and TK as a carrier, and inserts a CSFV E2 gene into a gE/gI deletion part of the recombinant virus rPRVTJ-delgE/gI/TK to construct a recombinant PRV variant strain rPRVTJ-delgE/gI/TK-E2 for expressing the classical swine fever virus E2protein with immunogenicity.

PCR, IFA and Western blotting identification show that the existence of CSFV E2 gene, the deletion of TK and gE/gI gene can be detected in the recombinant PRV variant rPRVTJ-delgE/gI/TK-E2, and the CSFV E2protein can be stably expressed after the recombinant virus rPRVTJ-delgE/gI/TK-E2 is inoculated with PK-15 cells.

The invention carries out plaque purification on recombinant virus rPRVTJ-delgE/gI/TK-E2, after each clone is continuously passed for 20 generations, CSFV E2 gene is still existed in genome of all virus strains and can be stably expressed, and the sequencing result of E2 gene amplified by each virus strain is consistent with the theoretical sequence, which indicates that exogenous gene can be stably existed in recombinant virus and has genetic stability. The comparison result of the one-step growth curve shows that the one-step growth curve trends of the recombinant virus rPRVTJ-delgE/gI/TK-E2 and the vector virus rPRVTJ-delgE/gI/TK are consistent, which indicates that the insertion of the exogenous fragment CSFV E2 has no influence on the propagation property of the vector virus but is obviously weaker than that of the parent strain PRV TJ.

The safety and immune efficacy evaluation results show that the recombinant virus rPRVTJ-delgE/gI/TK-E2 has high safety for piglets, and all immunized pigs have no clinical symptoms. After pigs are immunized by recombinant virus rPRVTJ-delgE/gI/TK-E2 with different doses, PRV specific antibodies and neutralizing antibodies with higher levels are generated, and the antibody level of the PRV specific antibodies and the neutralizing antibodies have no significant difference with the rPRVTJ-delgE/gI/TK; meanwhile, higher levels of CSFV specific antibody and neutralizing antibody are generated, and the antibody titer is basically consistent with that of the C strain. After challenge, rPRVTJ-delgE/gI/TK-E2 (10)⁶Or 10⁵TCID₅₀/head) immune pigs are able to completely resist the attack of CSFV virulent strains and PRV variant strains.

The invention deposits the above mentioned recombinant pseudorabies virus variant rPRVTJ-delgE/gI/TK-E2 expressing CSFV E2protein by submitting patent approved organization, and the microorganism deposit numbers are: CGMCC No. 12038; the classification is named as: a recombinant mutant strain pseudorabies virus expressing classical swine fever virus E2 protein. The preservation unit: china general microbiological culture Collection center; the preservation time was 2016, 02 months, 01 days: and (4) storage address: west road No.1 institute 3, north kingdom rising area, china academy of sciences, and the institute of microbiology.

The main reason for developing live virus vector vaccines is that the inserted foreign gene does not affect the proliferation characteristics and immunogenicity of the vector virus, and can be stably present and normally expressed (Souza AP, et al. recombinant viruses as vaccines and viral diseases. Braz J Med Biol Res 2005; 38(4): 509-22.). The identification result shows that the recombinant virus rPRVTJ-delgE/gI/TK-E2 has good genetic stability, the proliferation characteristic of the recombinant virus is not influenced by the expression of the E2 gene, and the growth kinetics of the recombinant virus is consistent with that of a vector virus PRVTJ-delgE/gI/TK; moreover, the recombinant virus has good safety and immunogenicity, can simultaneously induce high-level PRV and CSFV specific antibodies and neutralizing antibodies, and can provide complete protection against the attack of classical swine fever virus and pseudorabies virus variant strains.

The recombinant pseudorabies virus variant strain expressing the classical swine fever virus E2protein can be applied to preparation of vaccines for preventing and treating classical swine fever or pseudorabies, and particularly can be applied to preparation of bivalent genetic engineering vaccines for preventing and treating joint infection of classical swine fever and pseudorabies.

The invention further discloses a vaccine for preventing swine fever and pseudorabies simultaneously, or a vaccine for preventing swine fever or pseudorabies, which comprises the following components in percentage by weight: a prophylactically effective amount of the recombinant pseudorabies virus variant strain expressing classical swine fever virus E2 protein. The vaccine can also be added with a pharmaceutically acceptable adjuvant to enhance the antigenicity of the vaccine.

The invention further discloses a construction method of the recombinant pseudorabies virus variant for expressing the classical swine fever virus E2protein, which comprises the following steps: (1) carrying out enzyme digestion on a recombinant virus rPRVTJ-delgE/gI/TK genome, then co-transfecting a cell with pOK-LR-EGFP plasmid, and carrying out plaque screening and purification to obtain a recombinant virus rPRVTJ-delTK/gE/gI-EGFP; (2) carrying out enzyme digestion on the genome of the recombinant virus rPRVTJ-delTK/gE/gI-EGFP, then co-transfecting cells with pOK-LR-CMV-E2 plasmid, and screening and purifying to obtain the recombinant virus rPRVTJ-delTK/gE/gI-EGFP.

Wherein, the enzyme digestion in the step (1) or the step (2) is carried out by PacI and Pme I double enzyme digestion; the cell is a Vero cell. The recombinant virus rPRVTJ-delgE/gI/TK in the step (1) is constructed according to the literature (Cong X, et al.2016.); the construction of plasmid pOK-LR-EGFP (containing homologous recombination left and right arms flanking the gE/gI gene and the EGFP gene) is described in the literature (Wang CH, et al 2014.). The construction of the pOK-LR-CMV-E2 plasmid (plasmid vector containing the homologous recombination left and right arms flanking the gE/gI gene and the CSFV E2 gene) described in step (2) is described in the literature (Wang Y, et al 2015.).

In the process of constructing recombinant virus rPRVTJ-delgE/gI/TK-E2, the CMV promoter is arranged at the upstream of the E2 gene, and the PRV promoter is not used, so that the expression quantity of foreign proteins can be greatly increased. In the construction method, the invention adopts a method of combining reverse screening of the indicator virus and introduction of single enzyme cutting sites (Pac I and Pme I), thereby greatly accelerating the acquisition and purification of the recombinant virus.

The vector virus used in the present invention, rPRVTJ-delgE/gI/TK, is 10⁶TCID₅₀After the vaccine is inoculated to mice, sheep and piglets, no PRV specific clinical symptoms (including pruritus, fever and the like) exist, the safety is high, and the immunized piglets can completely resist the attack of the variant PRV TJ strain (Cong et al, 2016), so that the PRV TJ strain can still normally proliferate in a host body after the artificial deletion of the main virulence genes of the PRV TJ strain, and the immunogenicity of the PRV TJ strain is not influenced. The invention proves that the recombinant virus rPRVTJ-delgE/gI/TK can be used as a safe live vaccine vector to express antigen genes with immunogenicity of pathogens such as bacteria, parasites or other viruses and the like, and construct bi-or multi-valent genetic engineering vaccines.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the invention takes PRV three-gene-deleted recombinant virus rPRVTJ-delgE/gI/TK as a vector to express classical swine fever virus E2protein with immunogenicity, and the constructed recombinant virus rPRVTJ-delgE/gI/TK-E2 has genetic stability, can stably express E2protein, has good safety, can simultaneously induce high-level PRV and CSFV specific antibody and neutralizing antibody, and can provide complete protection for attack of CSFV strong virus and pseudorabies virus variant strains. The recombinant virus rPRVTJ-delgE/gI/TK-E2 can be used as a bivalent genetic engineering vaccine candidate strain for preventing and controlling the common infection of the current epidemic PRV variant strain and the classical swine fever virus.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The terms-vaccine "or-vaccine composition" used interchangeably refer to a pharmaceutical composition that includes at least one immunogenic composition that induces an immune response in an animal. The vaccine or vaccine composition may protect the animal from disease or possible death due to infection and may or may not include one or more additional components that enhance the immunological activity of the active component. The vaccine or vaccine composition may additionally comprise further components typical for vaccines or vaccine compositions, including for example adjuvants or immunomodulators. The immunologically active components of the vaccine may include the fully live organism in its original form or as an attenuated organism in a modified live vaccine, or an organism inactivated by suitable means in a killed or inactivated vaccine, or a subunit vaccine comprising one or more immunogenic components of the virus, or a genetically engineered, mutated or cloned vaccine prepared by methods known to those skilled in the art. The vaccine or vaccine composition may comprise one or more than one of the above components at the same time.

Drawings

FIG. 1 shows PCR identification of E2 and TK gene of recombinant virus rPRVTJ-delgE/gI/TK-E2; wherein, 1-5 and 9-13: recombining different plaque clone strains of the virus; 6: a CSFV control; 14: PRV TJ control; 7. 15: PK-15 cell control; 8. 16: h₂O control; m: DL 2000;

FIG. 2 shows the result of IFA identification of CSFV E2protein expression;

FIG. 3 is a Western blotting identification of recombinant virus rPRVTJ-delgE/gI/TK-E2;

FIG. 4 shows PCR identification of different clones of recombinant virus; wherein, 1: a CSFV control; 2-15: recombining different clone strains of the virus; 16: PK-15 cell control; m: DL 2000;

FIG. 5 shows IFA identification of recombinant virus rPRVTJ-delgE/gI/TK-E2 genetic stability and CAFV E2protein expression;

FIG. 6 is a one-step growth curve of recombinant virus rPRVTJ-delgE/gI/TK-E2;

FIG. 7 shows the production of CSFV E2-specific antibodies at different time points after rPRVTJ-delgE/gI/TK-E2 immune challenge;

FIG. 8 shows gB-specific antibodies produced after immunization of piglets and PRV challenge;

FIG. 9 shows gE-specific antibodies produced before and after PRV challenge in immunized piglets;

fig. 10 shows the survival rate of immunized piglets after PRV challenge.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 recombinant Pseudorabies Virus variant expressing classical Swine fever Virus E2protein

Construction of rPRVTJ-delgE/gI/TK-E2, and evaluation of safety and immune efficacy

1. Materials and methods

1.1 plasmids, viruses and Biochemical Agents

Plasmid pOK-LR-EGFP (containing homologous recombination left and right arms and EGFP genes on both sides of gE/gI gene) (WangcH, et al. A novel gE-deleted plasmids virus (PRV) provided and complete protection from free passage change with the PRV variant embodiment in Bartha-K61-modified plasmid delivery in China. vaccine.2014; 32(27):3379-85.) and pOK-CMV-E2 (plasmid vector containing homologous recombination left and right arms and CSE 2 gene on both sides of gE/gI gene) (Wang Y, Yuan J, Cong X, HY Qin, Wang CH, Li Y, genetic engineering. circulation of preservation of Vaccine. 9. expression and 9. expression of Vaccine strain 1121, 2. expression of Vaccine strain 1121; recombinant virus rPRVTJ-delgE/gI/TK was constructed by the present inventors (Cong X, Lei JL, Xia SL, Wang YM, Li Y, Li S, et al. Pathology and diagnosis of a gE/gI/TK gene-deleted pseudonoise virus variant microorganism. vet Microbiol 2016; 182: 170-77.); the PRV TJ strain is separated, identified and stored in the laboratory of the inventor; strain C vaccine was purchased from Harbin Vitaceae (batch No.: 2014001); PK-15 and Vero cells are preserved in a laboratory where the inventor is located; the transfection reagent X-treme GENE HP was purchased from Roche; the Tissue DNA Kit (batch No. D3396-01) is a product of Omega.

1.2 obtaining of recombinant viruses

Mu.g of the rPRVTJ-delgE/gI/TK genome (double digested with PacI and pmei) and 7. mu.g of the pOK-LR-EGFP plasmid were co-transfected into a monolayer of Vero cells with a confluence of around 80%. Harvesting a transfected cell product when the cytopathic effect reaches about 50%, repeatedly freezing and thawing at-80 ℃/37 ℃ for 2 times, centrifuging, taking a supernatant, inoculating PK-15 cells, carrying out plaque screening, selecting green fluorescent plaques with cytopathic effect, and carrying out about 6 rounds of purification to obtain the virus named as rPRVTJ-delTK/gE/gI-EGFP. Extracting genome of recombinant virus rPRVTJ-delTK/gE/gI-EGFP, carrying out double enzyme digestion treatment on the genome and the genome after PacI and PmeI, co-transfecting Vero cells with pOK-LR-CMV-E2, picking out plaques which have cytopathic effect but do not emit green fluorescence according to the method, and purifying the plaques.

1.3 identification of recombinant viruses

Plaques with cytopathic effect but no green fluorescence were cloned and purified 6 times, and then subjected to PCR, immunofluorescent assay (IFA), Western blotting, growth kinetics and stability characterization.

1.3.1PCR identification

PK-15 cells are inoculated with the virus plaque clone to be identified, tissue DNA is extracted when cytopathic effect reaches about 60%, and corresponding fragments are amplified by using a CSFV E2 gene specific primer (P1S/P1R) and a TK gene deletion identifying primer (P2S/P2R) respectively (Table 1).

TABLE 1 primers used for recombinant virus identification

1.3.2IFA

The PK-15 cells were infected with the correct viral plaque clones initially identified by PCR at a dose of 0.1MOI, while rPRVTJ-delgE/gI-E2(Wang Y, Yuan J, Cong X, Qin HY, Wang CH, Li Y, et al, Generation and efficacy evaluation of a recombinant plasmid viruses variable expression of the E2protein of a structural dish virus in pixels vaccine 2015,22(10):1121-9.), CSSHIMEN strain as FV positive control and rPRVTJ-delgE/gI/TK and PK-15 cells without any treatment as negative control. Discarding the supernatant after 24h, washing the cells 3 times with 4 ℃ precooled Phosphate Buffer (PBS), then fixing the cells for 20min with-20 ℃ precooled absolute ethanol, adding CSFV E2protein monoclonal antibody HQ06, acting for 2h at 37 ℃, washing 3 times with PBS and PBS (PBST) containing 0.05% Tween 20, respectively, adding 1:100 diluted FITC labeled goat anti-mouse IgG (Sigma company), acting for 45min at 37 ℃ in a wet box, washing 3 times with PBS/PBST, placing under an inverted fluorescence microscope for observation, and analyzing the expression condition of CSFV E2 protein.

1.3.3Western blotting analysis

The correct viral plaque clones identified by PCR and IFA were infected with PK-15 cells at a dose of 0.1MOI, while rPRVTJ-delgE/gI-E2(Wang et al 2015,22(10):1121-9.), CSFV Shimen strain as positive control and rPRVTJ-delgE/gI/TK and PK-15 cells without any treatment as negative control. After 36h of infection, discarding the supernatant, washing the cells for 3 times by PBS precooled at 4 ℃, then acting on WB/IP cell lysate on ice for 1h, collecting cell lysate, centrifuging at 12,000r/min at 4 ℃ for 20min, collecting the supernatant, adding a proper proportion of sample buffer solution, boiling for 10min, and carrying out SDS-PAGE electrophoresis to detect the expression condition of the E2protein by using a monoclonal antibody HQ06 specific to CSFV E2 protein.

1.3.4 one-step growth Curve

The recombinant virus identified correctly by PCR, IFA and Western blotting is named as rPRVTJ-delgE/gI/TK-E2, the recombinant virus, the rPRVTJ-delgE/gI/TK and the parent virus PRV TJ strain are inoculated to a single-layer PK-15 cell in a 24-hole cell culture plate at the dose of 10MOI, after the cells are infected on ice for 1h, culturing in a cell maintenance solution at 37 deg.C for 1h, inactivating extracellular virus with acetic acid solution (Metalleiter, T.C. glycoprotein gIII dispersions of microorganisms infected in virus entry. virology.1989; 171(2):623-5.), collecting cells and culture supernatant at 0h, 4h, 8h, 12h, 16h, 20h, 24h, 32h, 36h and 40h after virus inoculation, repeatedly freezing and thawing at 37 deg.C/80 deg.C for 2 times, centrifuging to remove cell debris, inoculating to PK-15 cells, and performing toxicity value determination. Repeating for 3 times, calculating the average value and standard deviation, drawing a one-step growth curve, and comparing whether the recombinant virus and the parent virus have difference in growth kinetics.

1.4 safety and immunogenicity of recombinant virus rPRVTJ-delgE/gI/TK-E2 to target animal pigs

1.4.1rPRVTJ-delgE/gI/TK-E2 immunization of pigs for the protection against CSFV

30 healthy piglets of 20 days old with CSFV and PRV antigens and antibodies negative are selected and randomly divided into 6 groups, and each group has 5 piglets. The first 3 groups were inoculated with 1mL of different doses (10) separately via the neck muscle⁶、10⁵Or 10⁴TCID₅₀) rPRVTJ-delgE/gI/TK-E2, and rPRVTJ-delgE/gI/TK, hog cholera lapinized virus vaccine strain C and DMEM were inoculated into the last 3 groups as controls (Table 2). All pigs were boosted 3w after immunization with the same vaccine, same dose and route. 1w after the boost, all pigs were challenged with CSFV Shimen strain at a challenge dose of 10⁶TCID₅₀After attacking, all live pigs are euthanized 16d, and pathological change observation and pathological histological examination are carried out on each tissue organ; after immunization and challenge, all pigs were subjected to daily body temperature measurements and observed for clinical response.

TABLE 2 pig immunization challenge experiment design

Note: the immunization and toxin counteracting approaches are neck intramuscular injection; the interval time refers to the time from the prime.

1.4.2 challenge protection of rPRVTJ-delgE/gI/TK-E2 immune pigs against PRV

25 healthy piglets of 20 days old, which were negative for both CSFV and PRV antigens and antibodies, were randomly selected and randomized into 5 groups (table 3), each of which had 5 piglets. The first 3 groups were inoculated with different doses (10) through the neck muscle⁶、10⁵And 10⁴TCID₅₀) Is/are as followsrPRVTJ-delgE/gI/TK-E2, and the last 2 groups were inoculated with rPRVTJ-delgE/gI/TK, respectively (10)⁶TCID₅₀) And DMEM as a control. Immunization 14d followed by 10⁶TCID₅₀The strong-toxicity PRV TJ with the dosage is dripped into the nose for counteracting the toxicity, all live pigs are euthanized 15 days after counteracting the toxicity, and lesion observation and examination are carried out on each tissue organ; after immunization and challenge, all pigs were subjected to daily body temperature measurements and observed for clinical symptoms including fever, itching, anorexia, lassitude, bedridden, diarrhea, etc.

TABLE 3PRV challenge protection experimental design

1.4.3 blocking ELISA and Virus neutralization assays

Before immunization, after immunization and after challenge, every 3d, all pig anterior vena cava were sampled and serum was isolated. PRV gB (IDEXX, lot No. DJ358), gE (IDEXX, lot No. CJ291) and hog cholera antibody detection kit (IDEXX, lot No. C281) were used for antibody detection, and the specific procedures were as described in the specification. Neutralizing antibodies in serum were also tested at each time point using a virus neutralization assay.

1.4.4 toxin expulsion assays

After challenge, anal swabs of all pigs were collected every day, diluted with PBS, added with 1000IU penicillin and 1000 μ g streptomycin per ml, filtered through a 0.45 μm filter, inoculated to PK-15 cells, observed for cytopathic effect, blindly passed for 3 generations if the first generation had no cytopathic effect, and then extracted with OMEGA Tissue DNA Kit for genome, followed by detection of the presence of PRV using primers specific for amplification of gE gene.

1.4.5 fluorescent quantitative RT-PCR

After CSFV 15D, all live pigs were euthanized, and all tissue organs of the test pigs were harvested, ground, extracted for total viral RNA, and reverse transcribed, and then tested for CSFV content in each tissue organ by the reported fluorescent quantitative RT-PCR method (ZHao JJ, Cheng D, LiN, et al. evaluation of multiple real-time RT-PCR for quantitative analysis and C-strain vaccine of classic wire-type viruses and V-sample Microbiol. 2008; 126(1-3):1-10.) to determine the copy number of CSFV nucleic acids in each tissue organ of the test pigs.

1.5 statistical analysis

Statistical analysis was performed on all data using SPSS statistical software to compare differences between groups. Wherein, the difference is not significant when p is more than or equal to 0.05, the difference is significant when p is less than 0.05, and the difference is very significant when p is less than 0.01.

2. Results of the experiment

2.1 rescue and screening of recombinant viruses

The genome of the recombinant virus rPRVTJ-delgE/gI/TK and a transfer vector pOK-LR-EGFP are subjected to cotransfection on Vero cells, 2d later, a large number of cells are diseased, a small number of diseased cells emit green fluorescence under a fluorescence microscope, after the cell culture is repeatedly frozen and thawed for 3 times, 6 rounds of plaque screening are carried out on PK-15 cells until the screened virus plaques are inoculated to the PK-15 cells, the cytopathic effect in a visual field is all green fluorescence, and the recombinant virus is identified by PCR and named as rPRVTJ-delTK/gE/gI-EGFP. The rPRVTJ-delTK/gE/gI-EGFP genome was co-transfected with pOK-LR-CMV-E2 into Vero cells and subjected to 7 rounds of plaque purification on PK-15 cells and the purified viral plaques were identified as described above.

The PCR identification and sequencing result shows that the TK and the gE/gI genes are deleted, and the CSFV E2 gene is detected in the recombinant virus (figure 1). IFA results show that the recombinant virus can express CSFV E2protein after being inoculated into PK-15 cells (figure 2). Western blotting analysis showed that a specific band was detected in the PK-15 sample lane inoculated with the recombinant virus to be identified, and the size of the band was consistent with that of the positive control (FIG. 3). The invention names the recombinant virus identified as correct by PCR, IFA and Westernblotting as rPRVTJ-delgE/gI/TK-E2.

In order to identify whether rPRVTJ-delgE/gI/TK-E2 has genetic stability and can stably express CSFVE 2protein, the obtained recombinant virus rPRVTJ-delgE/gI/TK-E2 is subjected to multiple rounds of plaque purification on PK-15 cells, more than 10 clones are randomly picked, and PCR identification, sequencing and IFA identification are carried out on each generation of virus after each clone continuously passes 20 generations; the PCR and sequencing results show that the CSFV E2 gene amplified fragment is consistent with the expected size (figure 4), and the E2 gene has no gene mutation or deletion after being compared with the theoretical sequence; IFA results showed that CSFV E2 gene was able to be stably expressed in each generation of clones, and that the proliferation properties of each generation of recombinant virus on PK-15 cells were not altered compared to the vector virus rPRVTJ-delgE/gI/TK (FIG. 5).

The invention submits the recombinant virus rPRVTJ-delgE/gI/TK-E2 with good genetic stability to the China general microbiological culture Collection center for preservation, and the preservation numbers of the microorganisms are as follows: CGMCC No. 12038.

2.2 one-step growth Curve

Inoculating the obtained recombinant virus rPRVTJ-delgE/gI/TK-E2, rPRVTJ-delgE/gI/TK and the parent virus PRV TJ strain thereof to PK-15 cells, collecting virus at different time points, measuring the virus price, drawing respective one-step growth curves (namely the curve of the relationship between the virus proliferation titer and the time, reflecting the virus growth and proliferation rules), and comparing. The result shows that the trend of the one-step growth curves of the recombinant virus rPRVTJ-delgE/gI/TK-E2 and the rPRVTJ-delgE/gI/TK is basically consistent, which indicates that the insertion of the exogenous fragment CSFV E2 has no influence on the proliferation property of the virus; but significantly lower than the parental virulent PRV TJ strain, the differences were very significant at 4h, 8h, 12h, 24h, 28h, and 36h after inoculation (p <0.001), and at the remaining time points (p <0.05) (fig. 6).

2.3 safety of rPRVTJ-delgE/gI/TK-E2 for pigs

rPRVTJ-delgE/gI/TK-E2 at different doses (10)⁶、10⁵And 10⁴TCID₅₀) After healthy piglets aged 20 days are immunized, all immunized pigs have no clinical symptoms, the body temperature is maintained within a normal physiological range, the appetite is normal, and the piglet immunizing method has no significant difference from a DMEM control pig.

2.4 neutralizing antibodies specific for E2 and against CSFV induced after immunization of pigs with rPRVTJ-delgE/gI/TK-E2

Different dosages (10)⁶、10⁵And 10⁴TCID₅₀) The recombinant disease rPRVTJ-delgE/gI/TK-E2, 3w later, a booster immune of 10⁶TCID⁵⁰After the dose of recombinant virus rPRVTJ-delgE/gI/TK-E2 is boosted by the virus, the CSFV E2 specific antibody induced by all piglets at 1w turns positive (figure 7), and the statistical analysis result shows that the antibody induced by the group of piglets has no significant difference with the C strain immune group piglets (P is P)>0.05), but the blocking rate of the 3d hog cholera antibody after the challenge is temporarily reduced, then all the immune pig antibodies gradually rise, and the blocking rate reaches about 80% after the 15d of the challenge. 10⁵TCID₅₀The antibody of the piglet immunized by the dose does not change the positive before the challenge, the antibody rapidly rises 6 days after the challenge, and 15 days and 10 days after the challenge⁶TCID₅₀The piglet induced antibody production of the dose immunization group and the C strain immunization group has no significant difference (P)>0.05), but 10⁴TCID₅₀The antibody produced by the piglet immunized by the dose is obviously lower than that of the piglet C strain.

The detection result of the neutralizing antibody shows that 21d 10 is after immunization⁶TCID₅₀The piglets of the rPRVTJ-delgE/gI-E2 immune group and the C strain immune group produced neutralizing antibodies at the earliest, and the level of the neutralizing antibodies between the two groups was not significantly different (P)>0.05). 1 before attacking toxin⁶TCID₅₀The neutralizing antibody of the rPRVTJ-delgE/gI-E2 immune group can reach 40 +/-8, and the C strain immune group is 60 +/-6. 6d recombinant virus rPRVTJ-delgE/gI-E2 immunization group all doses after challenge (10)⁶、10⁵、10⁴TCID₅₀) Neutralizing antibodies were produced and neutralizing antibody titers gradually increased over the time of challenge. After immunization and challenge, the neutralizing antibodies of all immunization groups of rPRVTJ-delgE/gI-E2 and the immunization group of the C strain are subjected to statistical analysis; results show that 10⁶TCID₅₀The dosage and the C strain have no obvious difference after immunization and challenge (P)>0.05), after attacking 10⁵And 10⁴TCID₅₀The average level of neutralizing antibodies induced by the rPRVTJ-delgE/gI-E2 immune group is significantly different from that of the C strain (p)<0.05) (table 4).

TABLE 4 CSFV neutralizing antibodies generated after rPRVTJ-delgE/gI/TK-E2 immunization of pigs

Note:^*：rPRVTJ-delgE/gI/TK-E2 immunization group (10)⁵And 10⁴TCID₅₀) The neutralizing antibody produced by the C strain immunity group is obviously different (p)<0.05)。

2.5rPRVTJ-delgE/gI/TK-E2 immunization of pigs against CSFV-generated challenge protection

After the booster immunization, the number of 1w of the pigs is 10⁶TCID₅₀The virulent CSFV Shimen strain with the dosage is used for counteracting the toxin, 10⁶TCID₅₀Piglets in the rPRVTJ-delgE/gI/TK-E2 immune group and the C strain immune group do not have any clinical symptoms after being attacked by toxin, such as fever, anorexia, lassitude and the like; 10⁵TCID₅₀The piglet in the dose group has no obvious clinical symptoms, and has no obvious specific CSFV clinical symptoms after only transient fever; 10⁴TCID₅₀The piglets in the dose group showed obvious CSF specific clinical symptoms, and 2 deaths occurred after 15 days of toxicity attack; piglets in the rPRVTJ-delgE/gI/TK immune group and the DMEM negative control group begin to have specific swine fever clinical symptoms such as high fever, anorexia, depression, diarrhea, dyspnea, purple ears and the like at 1d after challenge, and two groups of pigs die at 15d after challenge (Table 5). Collecting anticoagulation of all pigs every 3d after challenge, and detecting the virus content in the anticoagulation by using fluorescence quantitative RT-PCR (ZHao et al, 2008) established in the laboratory of the inventor to determine whether virology symptoms are generated after challenge of the immune pigs; results show that 10⁶And 10⁵TCID₅₀The piglets of the rPRVTJ-delgE/gI/TK-E2 immune group and the C strain immune group do not detect the existence of CSFV nucleic acid after challenge, the piglets of the rPRVTJ-delgE/gI/TK immune group and the DMEM injection group detect virus nucleic acid after 6 days of challenge, and can reach the maximum value of 10 after 12 days⁵Copies/. mu.L (Table 6), 10⁴TCID₅₀Lower levels of viral nucleic acid copy number were detected 6d and 9d after immunization challenge. Collecting anal swabs of all pigs every day after the toxin is attacked, filtering and sterilizing, and then carrying out an indirect immunofluorescence experiment to determine whether the immune pigs expel toxin after the toxin is attacked; results show that 10⁶And 10⁵TCID₅₀CSFV was not detected in the anal swabs of all piglets in rPRVTJ-delgE/gI/TK-E2 group and C strain immunization group after challenge, whereas 10⁴TCID₅₀rPRVTJ-delgE/gI/TK-E2 immunizationGroups also detected the presence of CSFV in post-challenge anal swabs 6d and 9 d; CSFV is detected three days after the piglets of the rPRVTJ-delgE/gI/TK immune group and the DMEM control group are attacked.

TABLE 5 clinical statistics of immunized pigs challenged with CSFV

Note: -: no heat was generated.

All live pigs were euthanized 15d after challenge and all pigs were necropsied for ocular virological examination. The results show that the vaccine immunization group of the C strain and 10⁶And 10⁵TCID₅₀No obvious pathological changes appear in all organs (heart liver, spleen, lung, kidney, mandibular lymph node, inguinal lymph node, tonsil, bladder and ileocecal valve) of rPRVTJ-delgE/gI/TK-E2 immune pigs, and 10⁴TCID₅₀Pathological changes of rPRVTJ-delgE/gI/TK-E2 immune pig viscera include lymph node enlargement, bleeding points with pinpoint size of kidney and bladder, and the like; all the pig organs of the rPRVTJ-delgE/gI/TK immune group and the DMEM control group have obvious pathological changes, including severe bleeding and swelling of lymph nodes and tonsils, massive bleeding points and dark color of kidneys, massive bleeding and blackening of bladder, massive bleeding and blackening of spleen and the like.

TABLE 6 quantification of CSFV RNA in anticoagulation at different time points after immune pig challenge

Note: -: no CSFV RNA; /: and death.

2.6rPRVTJ-delgE/gI/TK-E2 induces production of gB specific antibodies and neutralizing antibodies against PRV in piglets

Different dosages (10)⁶、10⁵、10⁴TCID₅₀) After immunizing 14d piglets with the recombinant virus rPRVTJ-delgE/gI/TK-E2, the piglets are detoxified with a virulent PRV TJ strain, 10⁶TCID₅₀Dosage formThe rPRVTJ-delgE/gI/TK-E2 and the rPRVTJ-delgE/gI/TK immune pigs are all positive-converted by the gB antibody at 6d after immunization, and the results of statistical analysis show that the antibody levels of the two groups have no significant difference (P>0.05). 9d, 10 post-immunization⁵TCID₅₀And 10⁴TCID₅₀The gB antibodies of the pigs in the dose are all positive, and the gB antibodies generated by the immunized pigs in different doses of rPRVTJ-delgE/gI/TK-E2 are not significantly different from the gB antibodies generated by the immunized pigs in different doses of rPRVTJ-delgE/gI/TK 12d after immunization (P)>0.05). At 3d post challenge, there was a downward trend for the immune group gB antibodies, but rapidly increased after 6d, with a peak at 15d (fig. 8). No gE-specific antibodies were detected before immunization and before challenge, and all porcine gE antibodies turned positive 12 days after challenge (fig. 9).

The result of the neutralizing antibody determination shows that before the challenge (14 d after immunization) 10⁶TCID₅₀Neutralizing antibodies were generated in rPRVTJ-delgE/gI/TK-E2 and rPRVTJ-delgE/gI/TK immunized pigs, 10⁵TCID₅₀And 10⁴TCID₅₀The dose immunization groups respectively detect neutralizing antibodies 3d and 6d after the challenge, and the neutralizing antibodies of all the immunized pigs continuously rise along with the prolongation of the challenge time; different doses (10) 15 days after challenge⁶、10⁵And 10⁴TCID₅₀) Neutralizing antibodies induced by rPRVTJ-delgE/gI/TK-E2 immune pigs have no significant difference from rPRVTJ-delgE/gI/TK (P>0.05) (table 7).

TABLE 7 neutralizing antibodies against PRV induced before and after PRV challenge in immunized pigs

Note:

^*: rPRVTJ-delgE/gI/TK-E2 immune group (10)⁵TCID₅₀) The neutralizing antibody generated by the rPRVTJ-delgE/gI/TK immune group is remarkably different (p)<0.05)；

^**: rPRVTJ-delgE/gI/TK-E2 immune group (10)⁴TCID₅₀) The difference of the neutralizing antibody generated by the rPRVTJ-delgE/gI/TK immune group is very significant (p)<0.001)。

2.7 challenge protection of immune pigs against PRV by rPRVTJ-delgE/gI/TK-E2

Variant PRV TJ (10) at 14d after immunization⁶TCID₅₀) Virulent toxin attacking all pigs in a nasal drip mode, 10⁶TCID₅₀The rPRVTJ-delgE/gI/TK-E2 and the rPRVTJ-delgE/gI/TK immune pigs have no PRV specific clinical symptoms after being challenged, including anorexia, pruritus and the like, and PRV is not detected in the nasal and anal swabs (Table 8); 10⁵TCID₅₀The immunized pigs only showed transient fever (4d), showed no other clinical symptoms, and were not detected for detoxification. 10⁴TCID₅₀Mild clinical symptoms appeared in the dose groups, including transient fever (5 days), anorexia, depression, pruritus, etc., PRV was detected in nasal swabs of 2 pigs 1d after challenge (table 8), and 1 pig died 7d after challenge (fig. 10); at 7 days after challenge, all immunized surviving pigs showed no clinical symptoms, and body temperature and appetite were recovered to normal, in contrast to control pigs which showed severe clinical symptoms such as persistent fever, itching, anorexia, bedridden condition, shivering, dyspnea and diarrhea, PRV was detected in nasal swabs of all pigs at 1 day after challenge, and 4 pigs died at 5-7 days after challenge (fig. 10).

TABLE 8 detection results of immune pig detoxification before and after PRV challenge

Note: -: toxin expelling detection is negative; +: positive toxin expelling detection; /: death was caused by death

After 15 days of toxin attack, all live pigs are euthanized, and tissue organs of all pigs are collected for eye pathology examination; 10⁶Or 10⁵TCID₅₀The rPRVTJ-delgE/gI/TK-E2 and the rPRVTJ-delgE/gI/TK immune pigs have no pathological change of each organ after only a caesarean examination, 10⁵TCID₅₀The pig in the dose group has slight hyperemia in the brain, bleeding spots in the lung and no obvious pathological changes in other organs. All pig viscera in the DMEM group have obvious pathological changes, including massive cerebral hemorrhage, severe hemorrhagic plaques in lung, severe damage to tonsil, heart, kidney, liver and spleen。

Claims

1. A recombinant pseudorabies virus variant strain for expressing classical swine fever virus E2protein is characterized in that: the microorganism preservation number of the recombinant pseudorabies virus variant strain is as follows: CGMCC No. 12038.

2. Use of the recombinant pseudorabies virus variant of claim 1 in the preparation of a vaccine for the prevention of hog cholera or pseudorabies.

3. Use of the recombinant pseudorabies virus variant of claim 1 in the preparation of a bivalent genetic engineering vaccine for the prevention of swine fever and pseudorabies.

4. A vaccine for preventing swine fever and pseudorabies simultaneously, comprising: a prophylactically effective amount of the recombinant pseudorabies virus variant of claim 1.

5. A method for constructing the recombinant pseudorabies virus variant strain of claim 1, comprising the following steps:

(1) carrying out enzyme digestion on a recombinant virus rPRVTJ-delgE/gI/TK genome, then co-transfecting a cell with pOK-LR-EGFP plasmid, and carrying out plaque screening and purification to obtain a recombinant virus rPRVTJ-delTK/gE/gI-EGFP; (2) carrying out enzyme digestion on the genome of the recombinant virus rPRVTJ-delTK/gE/gI-EGFP, then co-transfecting cells with pOK-LR-CMV-E2 plasmid, screening and purifying to obtain the recombinant virus rPRVTJ-delTK/gE/gI-EGFP.

6. The construction method according to claim 5, wherein: the enzyme in the step (1) or the step (2) is cut intoPacI andPmei, double enzyme digestion.

7. The construction method according to claim 5, wherein: the cells in the step (1) or the step (2) are Vero cells.