CN115948473B

CN115948473B - Pseudorabies virus vector for expressing exogenous SVA capsid protein, construction method and application thereof

Info

Publication number: CN115948473B
Application number: CN202211565866.8A
Authority: CN
Inventors: 朱玲; 陶倩; 徐志文
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2022-12-07
Filing date: 2022-12-07
Publication date: 2024-03-08
Anticipated expiration: 2042-12-07
Also published as: CN115948473A

Abstract

The invention discloses a pseudorabies virus vector for expressing exogenous SVA capsid protein, and a construction method and application thereof, and belongs to the technical field of biology. The construction method comprises the following steps: (1) Inserting the SEQ ID NO.1 sequence into a pEGFP-gI28K eukaryotic expression plasmid containing homologous arms of pseudorabies gI and 28K gene sequences to obtain a pEGFP-gI28K-VP4-1 vector; (2) Transfecting pEGFP-gI28k-VP4-1 and psgRNA-gE plasmids into BHK-21 cells, and then inoculating a pseudorabies virus vector PRVXJ with deleted TK genes; (3) Repeatedly freezing and thawing cells in 80% of cytopathy after inoculation, centrifuging to obtain supernatant, wherein the supernatant contains PRV eukaryotic expression vector, which is called rPRVXJ-delta gE/gI/TK-VP4-2-3-1 for short; (4) purification of the vector.

Description

Pseudorabies virus vector for expressing exogenous SVA capsid protein, construction method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a pseudorabies virus vector for expressing exogenous SVA capsid protein, and a construction method and application thereof.

Background

Senecavirus A (SVA), also known as Senecavirus (Seneca valley virus, SVV), is a capless single-stranded positive-strand RNA virus belonging to the family Picornaviridae, genus Senecavirus, which is the only member of this genus. The genome is about 7300nt in length, a single open reading frame (open reading frame, ORF) can encode about 2180 amino acids, with a typical L-4-3-4 genomic layout of picornaviruses, whose capsid proteins are produced by cleavage of the intermediate protein P1, VP2, VP3, and VP4, respectively. The main clinical symptoms of SVA are acute fever reaction of pigs, and the foot and mouth part is soaked, which is similar to the symptoms of swine vesicular disease caused by foot and mouth disease, vesicular virus and the like. SVA can infect pigs alone or in combination with other viruses, and when in combination with other vesicular viruses, vesicular ulceration symptoms are more severe than infection alone. The pathological changes of the vesicular ulcer caused by SVA mainly occur in sows and fattening pigs, the disease course lasts for 1-2 weeks, the disease can be self-healed, but the production performance and the feed conversion rate can be seriously affected. SVA can also produce transient epidemic neonatal injury (ETNL) to 1-7 day-old newborn piglets, and the clinical manifestation is continuous diarrhea, dehydration and neurological symptoms, the death rate of 1-3 day-old piglets is up to 40% -80%, and the death rate of 4-7 day-old piglets is 0-30%. At present, SVA becomes a pathogen of the pig-origin vesicular disease commonly existing in various provinces in China, no effective treatment method and vaccine can be controlled at present, and once the disease occurs, huge economic loss can be caused to a pig farm.

Disclosure of Invention

One of the purposes of the invention is to provide a construction method of a pseudorabies virus vector for expressing exogenous SVA capsid protein, which comprises the following steps:

(1) Inserting the SEQ ID NO.1 sequence into a pEGFP-gI28K eukaryotic expression plasmid containing homologous arms of pseudorabies gI and 28K gene sequences to obtain a pEGFP-gI28K-VP4-1 vector;

(2) Transfecting pEGFP-gI28k-VP4-1 and psgRNA-gE plasmids into BHK-21 cells, and then inoculating a pseudorabies virus vector PRVXJ with deleted TK genes;

(3) And (3) repeatedly freezing and thawing the cells after cytopathy 80% of inoculation, centrifuging to obtain supernatant, wherein the supernatant contains PRV eukaryotic expression vectors which express SVV capsid proteins VP4, VP2, VP3 and VP1 and lack gE, gI and TK, and the PRV eukaryotic expression vectors are called rPRVXJ-delta gE/gI/TK-VP4-2-3-1 for short.

Preferably, the sequence of SEQ ID NO.1 of step (1) is inserted between the cleavage recognition sites of the pEGFP-gI28k eukaryotic expression plasmids EcoRI and MluI.

More preferably, the specific operation of the step (2) is as follows: adding the solution (2) into the solution (1) to obtain a mixed solution, dripping the mixed solution into a 12-pore plate growing BHK-21 cells, uniformly mixing for transfection, inoculating 5 mu L of the pseudorabies virus vector PRVXJ with the TK gene deleted into the transfected cells when green fluorescence is observed, and placing the cells in a 5% carbon dioxide incubator at 37 ℃ for continuous culture; the solution (1) is DMEM 70. Mu.L+Lipofectamine TM30007.5. Mu.L; solution (2) was DMEM 70. Mu.L+P3000. Mu.M 5. Mu.L+5. Mu.g plasmid, where the plasmid was pEGFP-gI28k-VP4-1 and CRISPR-Cas gE each 2.5. Mu.g.

Preferably, the purification of step (4) rPRVXJ-. DELTA.gE/gI/TK-VP 4-2-3-1 is also included.

More preferably, the purification procedure of step (4) rPRVXJ-. DELTA.gE/gI/TK-VP 4-2-3-1 employs a 96-well plate limiting dilution method or a 6-well plate virus plaque purification method.

The second purpose of the invention is to provide the pseudorabies virus vector which is constructed by the construction method and expresses the exogenous SVA capsid protein.

The third purpose of the invention is to provide the application of the pseudorabies virus vector expressing the exogenous SVA capsid protein in the development of porcine Sesinkavirus subunit vaccine.

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

(1) It is difficult to integrate the whole capsid proteins of SVV into a pseudorabies vector by transfer vectors using self-cleaving peptides in tandem, because the whole capsid proteins are closer to 2500bp in tandem, the fragment is longer, and it is not easy to ligate to the pseudorabies vector. The invention uses MscI restriction endonuclease to linearize the ring-shaped transfer vector, and increases the probability of the transfer vector integrating exogenous genes onto the pseudorabies vector through a homology arm.

(2) According to the invention, the SVV capsid protein is expressed by taking the pseudorabies virus as a live vector, the whole capsid protein of the SVV is expressed completely, the neutralizing antibody can appear only on the 7 th day after immunization, and the result of taking the mouse as an experimental animal shows that the SVV capsid protein expressed by taking the pseudorabies virus as the live vector is used for immunizing the mouse, so that the virus expelling time of the SVV can be greatly shortened, and the virus load of the SVV in each tissue of the mouse can be reduced.

Drawings

FIG. 1 shows expression of VPs proteins with EGFP green fluorescence 24h after co-transfection of plasmids CRISPR/Cas-gE and pEGFP-gI28K-SVA-VP4-2-3-1 in example 1.

FIG. 2 is the first observation of rPRVXJ-. DELTA.gE/gI/TK-VP 4-2-3-1 in example 1.

FIG. 3 shows rPRVXJ-. DELTA.gE/gI/TK-VP 4-2-3-1 after purification in example 1.

FIG. 4 shows the expression of the foreign proteins VP1, VP2, VP3, VP4 in the rPRVXJ- ΔgE/gI/TK-VP4-2-3-1F21 protein sample of example 1.

Detailed Description

EXAMPLE 1 construction and immunogenicity Studies of pseudorabies virus vector expressing exogenous SVA capsid protein

1. Design and synthesis of exogenous SVA capsid protein expression transfer vector

According to the nucleotide sequence and arrangement sequence of the capsid protein of the SVA virus, the nucleic acid sequence capable of expressing the complete capsid protein of the SVA is artificially constructed, and is shown as SEQ ID NO. 1. According to the sequence order of SVA capsid protein, connecting SVA capsid protein by different self-cleaving peptides, the sequence is E2A-VP4-T2A-VP2-F2A-VP3-P2A-VP1, and is shown in SEQ ID NO. 1. The VP1 protein sequence is shown in SEQ ID NO.2, the VP2 sequence is shown in SEQ ID NO.3, the VP3 sequence is shown in SEQ ID NO.4, the VP4 sequence is shown in SEQ ID NO.5, the designed sequence is synthesized by Nanjin St Biotechnology Inc., the synthesized sequence is obtained by adopting a seamless cloning kit according to the sequence of a pEGFP-gI28K eukaryotic expression plasmid (constructed by the university of Sichuan agricultural animal biotechnology center) containing homologous arms of pseudorabies gI and 28K gene sequences, the small fragment between EcoRI and MluI cleavage recognition sites of the pEGFP-gI28K vector is replaced by SEQ ID NO.1, and other sequences of the pEGFP-gI28K vector are kept unchanged, so that the virus vector expressing SVA capsid protein is obtained, and the constructed vector is named pEGFP-gI28K-E2A-VP4-T2A-VP2-F2A-VP 3-P2A-1, and GFP-2-1 (named as pEGFP-gI 28-4-VP 4-gI) which is called pEGFP-gI-2A-2-VP 4-1-gI.

2. Construction of pseudorabies virus vector for expressing exogenous SVA capsid protein

Conventional methods are used for passaging BHK-21 cells to 12-hole plates, and Lipofectamine is used when the cells grow to 70% -80% ^TM 3000 transfection reagent (Invitrogen) Instructions the transfer vectors pEGFP-gI28k-VP4-1 and psgRNA-gE plasmid obtained in step 1 were transfected into BHK-21 cells. The principle is that pEGFP-gI28K-VP4-1 contains PRVGI and 28K gene partial homologous sequences, the original sequence between gI and 28K (including PRV gE gene) is replaced by SEQ ID NO.1 by homologous substitution, the psgRNA-gE plasmid is a CRISPR-Cas9 system with oriented shearing gE, and the probability of pEGFP-gI28K-VP4-1 homologous substitution is increased by shearing gE. The specific implementation steps are as follows: preparing a solution (1) and a solution (2), wherein the solution (1) is DMEM 70 mu L+Lipofectamine ^TM 30007.5. Mu.L; the solution (2) was DMEM 70. Mu.L+P3000 ^TM 5. Mu.L+5. Mu.g of plasmid (pEGFP-gI 28k-VP4-1 and CRISPR-Cas gE each 2.5. Mu.g). And (3) respectively shaking and uniformly mixing the solution (1) and the solution (2), adding the solution (2) into the solution (1), performing spot shaking for several times to obtain a mixed solution, standing at room temperature, and incubating for 15min. The mixture obtained in the previous step is dripped into a 12-well plate growing BHK-21 cells to be evenly mixed for transfection, green fluorescence can be observed 12 hours after transfection (see figure 1), which indicates that the BHK cells already express SVV capsid protein containing green fluorescence label (EGFP), and then 5 mu L of deleted TK gene (nucleotide sequence shown in SEQ ID NO.4 in a sequence table is inoculated in the transfected cells) The cells were placed in a 5% carbon dioxide incubator at 37℃for continuous culture. The state of cells was observed daily, and the cells were repeatedly freeze-thawed at-80℃three times, each time at 30min and 4000rpm, and centrifuged for 10min to obtain a supernatant containing PRV eukaryotic expression vectors expressing SVV capsid proteins VP4, VP2, VP3, VP1 and lacking gE, gI and TK, abbreviated as rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 (see FIG. 2). The supernatant is subjected to 96-well plate limiting dilution method and 6-well plate virus plaque purification method to obtain purified expression vector, and the purified expression vector is stored at-80 ℃.

The specific experimental operation method comprises the following steps:

96-well plate limiting dilution method: passaging BHK-21 cells into a 96-well plate by a conventional method, taking supernatant containing rPRVXJ-delta gE/gI/TK-VP4-2-3-1 expression vector stored at-80 ℃ when the cells grow to a compact monolayer, and carrying out 10-fold ratio gradient dilution by using serum-free DMEM cell culture solution to obtain 10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Four dilutions of the gradient dilutions were plated in 96-well plates, 100uL per well, and 24 wells repeated per gradient. After the addition of the virus dilutions, 96-well plates were placed in a 5% carbon dioxide incubator at 37℃for daily observation. Wells with more green spots and higher dilution were selected, freeze thawing was repeated 3 times at-80℃and centrifugation at 12000rpm for 2min, and the supernatant was stored at-80 ℃.

6 well plate virus plaque purification method: passaging BHK-21 cells into a 6-well plate by a conventional method, taking supernatant containing rPRVXJ-delta gE/gI/TK-VP4-2-3-1 expression vector obtained from the 96-well plate when the cells grow to a compact monolayer, and performing 10-fold gradient ratio dilution by using serum-free DMEM cell culture solution to obtain 10 ^-3 、10 ^-4 、10 ^-5 Three dilutions of the gradient dilutions, 10 ^-3 -10 ^-5 The diluted recombinant virus solution was inoculated into 6-well plates with 200uL at 37℃and 5% CO per well ₂ Adsorbing in incubator for 1 hr, discarding virus solution, mixing 2 XDMEM and 2% low melting point agarose 1:1, adding 6-well plate to reduce mobility of culture medium, standing at 37deg.C, and 5% CO ₂ Culturing in incubator, observing daily, and after plaque appearance, performing fluorescence microscopeSingle viral plaques emitting green fluorescence were picked and then grown in 12-well plates. The 96-well plate limiting dilution method and the 6-well plate virus plaque purification method were repeated until purified expression vector rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 was obtained (see FIG. 3).

Expression of foreign proteins after purification of rPRVXJ- ΔgE/gI/TK-VP4-2-3-1

BHK-21 cells were passaged to 96 well plates according to conventional methods and incubated in a 5% CO2 incubator at 37℃until dense monolayers were obtained. Purified F1, F10, F20, F21 rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 was diluted 10-fold with 2% serum DMEM to 10 ^-8 Obtaining 10 ^-1 -10 ^-9 The wells were plated in 96-well plates, 24 wells per gradient. Inoculation is carried out at 37 ℃ after inoculation, 5% CO ₂ Culturing in an incubator. Placing the cultured 96-well plate under an inverted fluorescence microscope to observe whether cytopathy appears in each dilution well and simultaneously generate green fluorescence, recording the green fluorescence quantity of each dilution cell, and calculating the TCID of rPRVXJ-delta gE/gI/TK-VP4-2-3-1 according to a Reed-Muench method ₅₀ Respectively 10 ⁷ TCID ₅₀ /ml、10 ^6.8 TCID ₅₀ /ml、10 ^6.8 TCID ₅₀ /ml、10 ⁷ TCID ₅₀ /ml。

BHK-21 cells were inoculated with F21 rPRVXJ- ΔgE/gI/TK-VP4-2-3-1, and after 36 hours, cell protein samples were collected, and the foreign proteins VP1, VP2, VP3, and VP4 were examined by WB (VP 1, VP2, VP3, SVV whole virus polyclonal antibody was supplied by animal biotechnology from Sichuan university of agriculture), and the results were shown in FIG. 4.

rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 Security test

Female BALB/c mice (average body weight 18.+ -.2 g) of 6-8 weeks old were obtained from Fukang Biotech Co., ltd. The mice were randomly divided into 4 groups, the first 3 groups being experimental groups of 8 mice each, each group being according to 10 respectively ⁷ TCID ₅₀ 、10 ⁶ TCID ₅₀ 、10 ⁵ TCID ₅₀ Is injected with rPRVXJ-delta gE/gI/TK-VP4-2-3-1 virus solution. Group 4 mice were selected as control group and were given intramuscular injections of 0.2mL DMEM. Mice survival was recorded by continuous observation for 15 days. After 15 days, fixation with 4% paraformaldehydeThe brain tissue of the mice was subjected to histopathological observation to evaluate the safety of rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 on the mice.

Experimental results show that the rPRVXJ-delta gE/gI/TK-VP4-2-3-1 has no death, and the tissue sections have no pathological changes, and have no obvious difference compared with the control group. Each concentration gradient was safe for mice.

Immunogenicity Studies of rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 in mice as animal models

Female BALB/c mice (average body weight 18.+ -.2 g) of 6-8 weeks old were obtained from Fukang Biotech Co., ltd. The mice were randomly divided into two groups (immunized and control groups of 30 mice each), and 50 μl of each mouse was immunized by nasal drip ⁷ TCID ₅₀ Each mouse of the control group was immunized by nasal drip with 50. Mu. LDMEM per ml rPRVXJ-. DELTA.gE/gI/TK-VP 4-2-3-1, and was two weeks after the initial priming. Blood was collected by tail vein blood collection on days 0, 1, 3, 5, 7, 14, 21, 28, 35, 42 (dpv) from the primary immunization, while 14 and 28dpv mouse spleen cells were isolated.

Measurement of cellular immune Effect of rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 Using mice as animal model

(1) The secretion of 14 and 28dpv IL-4 and IFN-gamma in the peripheral blood of mice in the immunized group and the control group was detected by using a cytokine detection kit (Xinbo Sheng: IL-4 (EMC 003.96); IFN-gamma (EMC 101g. 96)), and the results are shown in Table 1.

(2) Spleen cells were isolated from 14 and 28dpv immunized and control mice, each with three mice in duplicate. Spleen cells were diluted to a cell number of 5X 10 in 1640 medium using cell counting plate ⁶ at/mL, the wells were plated in 96-well plates (100. Mu.L per well). After 2h incubation in an incubator at 37 ℃, splenocyte proliferation was measured for each mouse under three treatment conditions: (1) 10 after UV inactivation ⁶ TCID ₅₀ Per ml SVV virus solution, 100. Mu.L per well; (2) 10 μg/ml of Canavalia ectenes A (MP Biomedicals, LLC: 195283) (ConcanavalinA, conA), 100 μl per well; (3) 1640 medium, 100 μl per well. The average was taken in triplicate per well. After 72h incubation, the stimulation index was calculated to assess spleen lymphocyte proliferation after detection of absorbance at OD450nm per well using CCK-8 (Beyotime, china) detection kit. Stimulation(s)The index (SI) calculation formula is: si= (immune group OD value-control group OD value)/(negative control group OD value-control group OD value), results are shown in table 2, and SI values of the vaccinated rPRVXJ- Δge/gI/TK-VP4-2-3-1 mice group were significantly higher than that of the vaccinated DMEM mice group, with significant differences when stimulated with inactivated SVV virus solution and ConA.

(3) Spleen cells of 14 and 28 dpv-isolated immunized and control mice were diluted to 2X 10 with PBS ⁴ mu.L was taken per mL, stained with FITC-CD3, APC-CD4 and PE-CD8 antibodies (FITC anti-mouse CD3 Antibody:100204; APC anti-mouse CD4Antibody:100412; PE anti-mouse CD8a Antibody: 100708; biolegend, using a concentration of 1:250) at room temperature for 30min and then examined by flow cytometry, and the data was analyzed using flowjo10 software. The results are shown in Table 3, and on day 28 post-immunization, mouse spleen lymphocytes CD3 were assayed ⁺ 、CD3 ⁺ CD4 ⁺ (humoral immune-related), CD3 ⁺ CD8 ⁺ T cell ratio (cell immunity related) found immunized group mouse CD3 ⁺ CD4 ⁺ The percentage of T cells is significantly higher than that of the control group, CD ³⁺ CD ⁸⁺ The T cell percentage was slightly higher than the control group.

TABLE 1 detection results of IL-4 and IFN-gamma cytokines in peripheral blood of mice

TABLE 2 mouse spleen lymphocyte proliferation assay

Table 3 percentage of spleen lymphocytes cd3+, cd4+, cd8+ in mice

5.2 measurement of humoral immune Effect of rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 Using mice as animal model

Serum from mice of immunized and control groups of 0, 1, 3, 5, 7, 14, 21, 28, 35, 42dpv was collected, and the levels of the mice gE, gB antibodies were detected with reference to french id.vet innvative dignose porcine pseudorabies virus gE, gB antibody detection kit instructions. VP 1-specific antibody levels were detected by a laboratory-established SVVVP1 protein indirect ELISA method. Serum neutralizing antibody levels were determined using plaque reduction.

The ELISA comprises the following specific steps: (1) coating: coating antigen with prokaryotic expression VP1 protein, diluting VP1 prokaryotic expression protein to 31.25ng/ml with CBS coating liquid, coating ELISA 96-well plate (Corning) at 37deg.C for 2 hr, discarding coating liquid, and cleaning 200 μl/well PBST for 5 times and 5 min/time; (2) closing: 5% skimmed milk 200 μl/well at 37deg.C for 1.5h, discarding the blocking solution, and cleaning 200 μl/well with PBST 5 times for 5 min/time; (3) an antibody: collected mouse serum was collected at 1:400 dilution, simultaneously setting positive serum control (VP 1 protein murine polyclonal antibody) and negative serum control (serum of untreated mice), incubating for 1.5h at 37 ℃ with 100 mu L/well, discarding serum, washing for 5 times with 200 mu L/well PBST, and 5 min/time; (4) and (2) secondary antibody: HRP-labeled goat anti-mouse IgG (1:5000 dilution), 100. Mu.L/well, incubation at 37℃for 1h, discarding secondary antibody, washing with 200. Mu.LPBST 5 times, 5 min/time; (5) color development: adding 100 mu LTMB substrate color development liquid into each hole, and incubating for 15min at room temperature; finally 50 mu L of 2M H are added to each well ₂ SO ₄ Terminating the reaction, and detecting OD (optical density) within 15min by using an enzyme-labeled instrument _450nm Absorbance values.

Preparation of VP1 prokaryotic expression protein: (1) the prokaryotic expression vector pCold-VP1 is constructed and transformed into BL21 (DE 3) competent cells to obtain recombinant expression bacteria BL21-pCold-VP1. (2) Amplifying recombinant expression bacteria 1:100 to OD _600nm At 0.6, IPTG was added at a final concentration of 1mM to induce protein expression, while BL21 (DE 3) expressing bacteria containing empty vector pCold-TF was established as a control. (3) After determining the expression of the recombinant protein by SDS-PAGE, optimizing the expression conditions such as IPTG concentration, induction temperature, induction time and the like, andit was subjected to a solubility analysis. (4) After a large amount of recombinant protein was expressed under the optimal expression conditions, the recombinant protein was purified and concentrated according to the protocol of Ni-NTA protein purification kit (manufacturing: C600332-0001), and the concentration of the purified protein was measured by a nucleic acid protein meter (Scandrop 100 nucleic acid protein meter ANALYTIKJENA (Germany)).

The results showed that gE antibodies were negative at each time point in all mice, 21dpv (after 1 week of secondary immunization), both gB antibodies and VP1 antibodies of immunized mice turned positive, and the antibody levels increased with time (results shown in tables 4, 5, and 6).

Plaque reduction assay of neutralizing antibodies: (1) the serum to be tested (control group and immune group) is diluted to (1:2-1:64) times and added with 100PFU SVV virus liquid, and the mixture is placed at 37 ℃ for 1h. (2) BHK-21 cells were cultured in 6-well plates to a density of 80%. Serum-virus neutralization solution 200. Mu.L/well, wells were additionally placed to be inoculated with 100PFU SVV virus solution alone. The inoculated cells were adsorbed at 37℃for 1h. (3) Removing liquid in the well, mixing 2 XDMEM with 2% low melting point agarose 1:1, adding into 6-well plate to reduce fluidity of culture medium, and standing at 37deg.C and 5% CO ₂ Culturing in an incubator, observing every day, after plaque appears, fixing and staining by formalin-crystal violet fixing staining solution, automatically reading the plaque number by IPP6.0 software, calculating the serum dilution by halving the exact plaque according to a proportion method, and calculating the serum dilution, namely the serum neutralization titer.

The assay results showed that the plaque number gradually decreased from 7dpv (after 1 week of initial priming) in the immunized mice, and that the immune mice began to produce SVV neutralizing antibodies, and the antibody levels increased with time (see table 7 for results).

Using SVV conserved gene 3D as a template, establishing a fluorescent quantitative PCR detection standard curve about SVV, wherein y= -3.4653x+36.194, R ² =0.9993. Infection of control and immune groups with 50ul 10 ⁶ TCID ₅₀ SVV, 100mg each of brain, heart, liver, spleen, lung, kidney, large intestine, small intestine, intestinal strangles, feces of mice 1, 3, 5, 7, 10, 14 days after infection (dpi) were collected, and the viral loads of SVV in the tissues of mice in the control group and the immunized group were examined.

The detection result shows that SVV presents a overinfection in mice, the heart, liver, spleen, lung, intestinal stranguria and fecal virus load of the control group inoculated SVV mice are higher, peak value is reached at 3-6dpi, and basic detoxification at 10dpi is completed (the result is shown in Table 8). The viral loads of 3, 6dpi immunized groups of heart, liver, spleen, lung, stranguria and feces were significantly reduced with the SVV 3D gene copy number compared to the control group (results are shown in Table 9).

TABLE 4gB antibody assay results

Note that: S/N is less than or equal to 40 percent and is positive; more than or equal to 50 percent of S/N percent of more than 40 percent is suspicious; S/N% > 50% is negative.

Table 5 results of gE antibody assay

Note that: S/N is less than or equal to 60 percent and is positive; more than or equal to 70 percent of S/N percent of more than 60 percent is suspicious; S/N% > 70% was negative.

TABLE 6 determination of mouse VP1 protein antibody

Note that: OD (optical density) _450nm More than or equal to 0.286 is positive; OD (optical density) _450nm <0.251 is negative.

TABLE 7 determination of SVV neutralizing antibodies

TABLE 8 results of SVV viral load determination for control group

TABLE 9 results of the determination of the viral load of the immune group SVV

E2A-VP4-T2A-VP2-F2A-VP3-P2A-VP1(SEQ ID NO.1)：

ATGGGGTCCGGCCAATGTACTAACTACGCTTTGTTGAAACTCGCTGGCGATGTTGAAAGTAACCCCGGT CCTGGTAATGTTCAGACAACCTCAAAGAATGACTTTGATTCCCGCGGCAATAATGGTAACATGACCTTCAATTACTACGCAAACACTTACCAGAATTCAGTAGACTTCTCGACCTCCTCGTCGGCGTCAGGCGCCGGACCCGGGAACTCCCGGGGCGGACTAGCGGGTCTCCTCACAAATTTCAGTGGAATCTTGAACCCTCTTGGCTACCTCAAAGGCAGTGGAGAGGGC AGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGATCAC

AATACCGAAGAAATGGAAAACTCTGCTGATCGAGTCATAACGCAAACGGCGGGCAACA

CTGCCATAAACACGCAATCATCACTGGGTGTGTTGTGTGCCTACGTTGAAGACCCGACC

AAATCTGACCCTCCGTCCAGCAGCACAGATCAACCCACCACCACTTTTACTGCCATCGA

CAGGTGGTATACTGGACGCCTCAATTCTTGGACAAAAGCTGTAAAAACCTTCTCTTTTCA

GGCCGTCCCGCTCCCTGGAGCCTTCCTGTCTAGACAGGGAGGCCTCAACGGGGGGGCC

TTCACGGCTACCCTACATAGACACTTCTTAATGAAGTGCGGGTGGCAGGTGCAGGTCCA

ATGCAATTTGACGCAATTCCACCAAGGTGCTCTTCTTGTTGCCATGGTCCCTGAGACCAC

CCTTGATGTCAAACCTGACGGCAAGGCAAAGAGCTTACAGGAGCTGAATGAAGAACAG

TGGGTAGAAATGTCTGACGATTACCGGACCGGGAAAAACATGCCTTTTCAGTCTCTTGG

CACATACTATCGGCCCCCCAACTGGACTTGGGGTCCCAATTTTATCAACCCCTATCAAGT

AACAGTTTTCCCACACCAAATTCTGAACGCGAGAACCTCTACCTCGGTAGACATAAGTG

TCCCATACATCGGGGAGACTCCTACACAATCCTCAGAGACACAGAACTCCTGGACCCTC

CTCGTTATGGTGCTTGTCCCCTTAGACTACAAGGAGGGAGCCACAACTGACCCAGAAAT

TACATTCTCTGTAAGGCCTACAAGTCCTTACTTCAATGGGCTTCGTAACCGTTTCACGAC

CGGGACGGACGAGGAACAGGGTTCTGCCGTGAAACAGACTTTGAATTTTGACCTTCTC

AAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCGGGCCCATTCCCACAGCACCCA

GAGAAAACTCGCTTATGTTTCTCTCGACCATCCCCGACGACACTGTCCCTGCTTACGGG

AATGTGCGTACCCCTCCCGTCAATTACCTCCCCGGTGAAATAACCGACCTCTTACAACTG

GCCCGTATACCCACTCTTATGGCGTTTGGGCGGGTGTCTGAACCCGAGCCTGCCTCAGA

CGCTTATGTGCCCTACGTTGCTGTTCCTGCTCAGTTCGACGACAAGCCTCTCATCTCCTT

CCCGATCACCCTTTCAGATCCTGTCTACCAGAACACCCTGGTAGGCGCCATCAGTTCGA

ACTTCGCTAACTACCGGGGGTGTATCCAAATCACTCTGACATTTTGTGGACCCATGATGG

CAAGAGGGAAATTCCTGCTCTCGTATTCTCCCCCAAATGGAGCACAACCACAGACCCTT

TCTGAAGCTATGCAGTGCACATACTCTATCTGGGACATAGGCTTGAACTCTAGTTGGACC

TTTGTCATCCCCTACATCTCGCCCAGTGATTACCGTGAAACTCGGGCTATTACTAACTCA

GTTTATTCTGCTGATGGTTGGTTTAGCTTACACAAGCTGACCAAAATCACTCTACCACCT

GACTGCCCACAGAGTCCCTGTATTCTTTTTTTCGCCTCTGCTGGTGAGGATTACACCCTC

CGTCTCCCTGTTGATTGTAATCCTTCCTATGTGTTCCACGGTTCTGCCGCCACGAACTTCT

CTCTGTTAAAGCAAGCAGGAGACGTGGAAGAAAACCCCGGTCCTTCCACCGACAACGC

CGAGACTGGTGTTATTGAGGCAGGTAACACTGACACCGATTTCTCTGGTGAACTGGCGG

CTCCTGGCTCTAACCATACTAATGTCAAATTTCTTTTTGATCGATCTCGACTACTGAATGT

AATTAAGGTACTGGAGAAGGACGCTGTCTTCCCCCGTCCTTTCCCCACAGCAACAGGTG

CACAGCAGGACGATGGTTACTTTTGTCTTCTGACACCCCGCCCAACAGTCGCTTCCCGA

CCCGCCACCCGTTTCGGCCTGTACGTCAACCCGTCTGACAGTGGCGTTCTCGCTAACAC

TTCACTGGATTTCAATTTTTACAGTTTGGCCTGTTTCACTTACTTTAGATCAGACCTTGAA

GTCACGGTAGTCTCACTGGAGCCAGATCTGGAATTCGCCGTGGGGTGGTTCCCCTCTGG

CAGTGAGTACCAGGCTTCTAGCTTTGTCTACGACCAACTGCATGTACCCTACCACTTTAC

TGGGCGCACTCCCCGCGCTTTCACCAGCAAGGGCGGAAAGGTATCTTTCGTGCTCCCTT

GGAACTCTGTCTCTTCCGTGCTTCCCGTGCGCTGGGGGGGCGCTTCCAAGCTTTCTTCT

GCCACGCGGGGTCTGCCGGCTCATGCTGACTGGGGGACCATTTACGCCTTCATCCCCCG

TCCTAACGAGAAGAAAAGCACCGCTGTAAAGCACGTGGCGGTGTACGTTCGGTACAAG

AACGCGCGTGCTTGGTGCCCCAGCATGCTTCCCTTTCGCAGCTACAAGCAGAAGATGCTGATGCAA。

VP1(SEQ ID NO.2)：

GPIPTAPRENSLMFLSTIPDDTVPAYGNVRTPPVNYLPGEITDLLQLARIPTLMAFGRVSEPEPASDAYVPYVAVPAQFDDKPLISFPITLSDPVYQNTLVGAISSNFANYRGCIQITLTFCGPMMARGKFLLSYSPPNGAQPQTLSEAMQCTYSIWDIGLNSSWTFVVPYISPSDYRETRAITNSVYSADGWFSLHKLTKITLPPDCPQSPCILFFASAGEDYTLRLPVDCNPSYVFH。

VP2(SEQ ID NO.3)：

DHNTEEMENSADRVITQTAGNTAINTQSSLGVLCAYVEDPTKSDPPSSSTDQPTTTFTAIDR

WYTGRLNSWTKAVKTFSFQAVPLPGAFLSRQGGLNGGAFTATLHRHFLMKCGWQVQVQC

NLTQFHQGALLVAMVPETTLDVKPDGKAKSLQELNEEQWVEMSDDYRTGKNMPFQSLGT

YYRPPNWTWGPNFINPYQVTVFPHQILNARTSTSVDISVPYIGETPTQSSETQNSWTLLVMVLVPLDYKEGATTDPEITFSVRPTSPYFNGLRNRFTTGTDEEQ。

VP3(SEQ ID NO.4)：

GPIPTAPRENSLMFLSTIPDDTVPAYGNVRTPPVNYLPGEITDLLQLARIPTLMAFGRVSEPEP

ASDAYVPYVAVPAQFDDKPLISFPITLSDPVYQNTLVGAISSNFANYRGCIQITLTFCGPMMA

RGKFLLSYSPPNGAQPQTLSEAMQCTYSIWDIGLNSSWTFVVPYISPSDYRETRAITNSVYSADGWFSLHKLTKITLPPDCPQSPCILFFASAGEDYTLRLPVDCNPSYVFH。

VP4(SEQ ID NO.5)：

GNVQTTSKNDFDSRGNNGNMTFNYYANTYQNSVDFSTSSSASGAGPGNSRGGLAGLLTNF SGILNPLGYLK。

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A method for constructing a pseudorabies virus vector expressing exogenous SVA capsid protein, comprising the steps of:

(2) Transfecting pEGFP-gI28k-VP4-1 and psgRNA-gE plasmids into BHK-21 cells, and inoculating a pseudorabies virus vector PRV XJ with deleted TK genes;

2. The method according to claim 1, wherein the sequence of SEQ ID NO.1 is inserted between EcoRI and MluI cleavage recognition sites of the pEGFP-gI28k eukaryotic expression plasmid in step (1).

3. The construction method according to claim 2, wherein the step (2) specifically comprises the following steps: adding the solution (2) into the solution (1) to obtain a mixed solution, dripping the mixed solution into a 12-pore plate growing BHK-21 cells, uniformly mixing for transfection, inoculating 5 mu L of pseudorabies virus vector PRV XJ with deleted TK genes into transfected cells when green fluorescence can be observed, and placing the cells in a 37 ℃ and 5% carbon dioxide incubator for continuous culture; the solution (1) is DMEM 70 [ mu ] L+Lipofectamine TM 3000.7.5 [ mu ] L; solution (2) is DMEM 70 [ mu ] L+P3000 [ mu ] L+5 [ mu ] g plasmid, wherein the plasmids are pEGFP-gI28k-VP4-1 and CRISPR-Cas gE 2.5 [ mu ] g respectively.

4. A method of construction according to any one of claims 1-3, further comprising the purification of step (4) rPRVXJ- Δge/gI/TK-VP4-2-3-1.

5. The construction method according to claim 4, wherein the purification operation of step (4) rPRVXJ- ΔgE/gI/TK-VP4-2-3-1 is performed by 96-well plate limiting dilution or 6-well plate virus plaque purification.

6. A pseudorabies virus vector expressing exogenous SVA capsid protein constructed by the construction method of any one of claims 1-3, 5.

7. The pseudorabies virus vector expressing exogenous SVA capsid protein constructed by the construction method of claim 4.

8. Use of the pseudorabies virus vector expressing exogenous SVA capsid protein of claim 6 in the preparation of porcine sai virus subunit vaccine.

9. Use of the pseudorabies virus vector expressing exogenous SVA capsid protein of claim 7 in the preparation of porcine saint virus subunit vaccine.