CN115948473A

CN115948473A - Pseudo rabies virus vector for expressing exogenous SVA capsid protein and construction method and application thereof

Info

Publication number: CN115948473A
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: 2023-04-11
Anticipated expiration: 2042-12-07
Also published as: CN115948473B

Abstract

The invention discloses a pseudorabies virus vector for expressing exogenous SVA capsid protein and a construction method and application thereof, belonging to the technical field of biology. The construction method comprises the following steps: (1) Inserting the sequence of SEQ ID NO.1 into pEGFP-gI28K eukaryotic expression plasmid containing pseudorabies gI and 28K gene sequence homology arms to obtain a pEGFP-gI28K-VP4-1 vector; (2) Transfecting the BHK-21 cells with pEGFP-gI28k-VP4-1 and psgRNA-gE plasmids, and then inoculating a pseudorabies virus vector PRVXJ deleted with TK genes; (3) Repeatedly freezing and thawing the cells after 80 percent of cytopathic effect is inoculated, centrifuging and taking supernatant, wherein the supernatant contains PRV eukaryotic expression vectors, which are abbreviated as rPRVXJ-delta gE/gI/TK-VP4-2-3-1; and (4) purifying the carrier.

Description

Pseudo rabies virus vector for expressing exogenous SVA capsid protein and 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 Seneca Valley Virus (SVV), is a membrane-free single-stranded positive-strand RNA virus belonging to the picornaviridae family, the genus Senecavirus, the only member of this genus. The genome is about 7300nt in length, a single Open Reading Frame (ORF), and can encode about 2180 amino acids, with the 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, blisters appear on hoofs of the pigs, and the symptoms are similar to the symptoms of the swine vesicular disease caused by foot-and-mouth disease, vesicular virus and the like. SVA can infect pigs alone, can also be mixed with other viruses, and when mixed with other vesicular viruses, the vesicular ulceration symptoms are more severe than when infected alone. The pathological changes of the vesicular ulcer caused by SVA mainly occur in sows and fattening pigs, the course of disease lasts for 1-2 weeks, and the vesicular ulcer can be self-healed, but the production performance and the feed conversion rate are seriously influenced. SVA can also produce transient epidemic neonatal injury (ETNL) to 1-7 days old newborn piglets, and the clinical manifestations are continuous diarrhea, dehydration and nervous symptoms, the death rate of 1-3 days old piglets reaches 40% -80%, and the death rate of 4-7 days old piglets reaches 0-30%. At present, SVA becomes a common swine vesicular disease pathogen in various domestic provinces, an effective treatment method and a vaccine which can control SVA are not available at present, and once the SVA is attacked, huge economic losses are 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 sequence of SEQ ID NO.1 into pEGFP-gI28K eukaryotic expression plasmid containing pseudorabies gI and 28K gene sequence homology arms to obtain a pEGFP-gI28K-VP4-1 vector;

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

(3) And after the inoculated cytopathic effect is 80%, repeatedly freezing and thawing the cells, centrifuging to obtain a 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 abbreviated as rPRVXJ-delta gE/gI/TK-VP4-2-3-1.

Preferably, the SEQ ID NO.1 sequence in the step (1) is inserted between the restriction recognition sites of EcoRI and Mlu I of pEGFP-gI28k eukaryotic expression plasmid.

More preferably, the step (2) is specifically operated as follows: adding the solution (2) into the solution (1) to obtain a mixed solution, dripping the mixed solution into a 12-hole plate in which BHK-21 cells grow, uniformly mixing, transfecting, inoculating 5 mu L of the PRVXJ with the TK gene deleted pseudorabies virus vector 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 DMEM70 mu L + Lipofectamine TM30007.5 mu L; solution (2) is DMEM70 μ L + P3000TM5 μ L +5 μ g plasmid, where the plasmid is pEGFP-gI28k-VP4-1 and CRISPR-CasgE each 2.5 μ g.

Preferably, the method also comprises the step (4) of purifying the rPRVXJ-delta gE/gI/TK-VP4-2-3-1.

More preferably, the purification operation of the rPRVXJ-delta gE/gI/TK-VP4-2-3-1 of the step (4) adopts 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 invention also aims to provide the application of the pseudorabies virus vector for expressing the exogenous SVA capsid protein in the development of a porcine Seneca virus subunit vaccine.

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

(1) The whole capsid protein of SVV is difficult to be integrated on a pseudorabies vector by a transfer vector by utilizing self-cleavage peptide to be connected in series, and the whole capsid protein is close to 2500bp in series, has longer fragment and is not easy to be connected on the pseudorabies vector. The invention linearizes the annular transfer vector by using MscI restriction endonuclease, and increases the probability that the transfer vector integrates the foreign gene into the pseudorabies vector through a homology arm.

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

Drawings

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

FIG. 2 shows the first observation of rPRVXJ- Δ gE/gI/TK-VP4-2-3-1 in example 1.

FIG. 3 shows rPRVXJ- Δ gE/gI/TK-VP4-2-3-1 after purification in example 1.

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

Detailed Description

Example 1 construction and immunogenicity study of Pseudorabies Virus vector expressing exogenous SVA capsid protein

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

According to the nucleotide sequence and the arrangement sequence of the capsid protein of the porcine SVA virus, a nucleic acid sequence which can express the complete capsid protein of the SVA is artificially constructed, and is shown as SEQ ID NO. 1. According to the sequence of the SVA capsid protein, the SVA capsid protein is connected through different self-cleavage peptides, and the sequence is E2A-VP4-T2A-VP2-F2A-VP3-P2A-VP1, as shown in SEQ ID NO. 1. Wherein 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 Nanjing Kingsry Biotechnology GmbH, the synthesized sequence is inserted between the EcoRI and Mlu I enzyme digestion recognition sites of pEGFP-gI28K eukaryotic expression plasmid (constructed by the animal biotechnology center of Sichuan university of agriculture) containing pseudorabies gI and 28K gene sequence homology arms by adopting a seamless cloning kit, namely, the SEQ ID NO.1 replaces a small segment between the EcoRI and Mlu I enzyme digestion recognition sites of the pEGFP-gI28K vector, and other sequences of the pEGFP-gI28K vector are kept unchanged to obtain a virus vector expressing SVA capsid protein, and the constructed vector is named as pEGFP-gI 28K-E2A-4-VP 2A-VP 2-F2A-3-P2A-gA-1, namely, and the constructed vector is named as pEGFP-gI 28K-E2A-4-VP 2A-4 (abbreviated as GFP-VP 4-gI).

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

The BHK-21 cells are passaged to a 12-hole plate by a conventional method, and Lipofectamine is used when the cells grow to 70% -80% ^TM 3000 transfection reagent (Invitrogen corporation) Instructions BHK-21 cells were transfected with the transfer vectors pEGFP-gI28k-VP4-1 and psgRNA-gE plasmid obtained in step 1. The principle is that pEGFP-gI28K-VP4-1 contains partial homologous sequences of PRVgI and 28K genes, the original sequence between gI and 28K (containing PRV gE genes) is replaced by SEQ ID NO.1 by utilizing homologous substitution, psgRNA-gE plasmid is a CRISPR-Cas9 system for directionally 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 solution (1) and solution (2), wherein the solution (1) is DMEM70 mu L + Lipofectamine ^TM 30007.5 μ L; solution (2) is DMEM70 mu L + P3000 ^TM mu.L + 5. Mu.g plasmid (pEGFP-gI 28k-VP4-1 and CRISPR-CasgE 2.5. Mu.g each). Shaking and mixing the solution (1) and the solution (2) respectively, adding the solution (2) into the solution (1), shaking for several times to obtain a mixed solution, and standing and incubating for 15min at room temperature. The mixed solution obtained in the previous step is dripped into a 12-hole plate in which BHK-21 cells grow, the mixed solution is uniformly mixed for transfection, green fluorescence can be observed after 12h (see figure 1), the fact that the BHK cells express SVV capsid protein containing green fluorescence labels (EGFP) is shown, 5 mu L of pseudorabies virus vector PRVXJ without TK gene (nucleotide sequence shown by SEQ ID NO.4 in a sequence table) is inoculated into the transfected cells, and the cells are placed in a 37 ℃ 5% carbon dioxide incubator for continuous culture. Observing the cell state every day, repeatedly freezing and thawing the cells at-80 ℃ for three times when the inoculated cells are diseased by 80%, centrifuging for 10min at 30min,4000rpm each time, and taking the 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 abbreviated as rPRVXJ-delta gE/gI/TK-VP4-2-3-1 (see figure 2). The supernatant was subjected to a 96-well plate limiting dilution method and a 6-well plate viral plaque purification method to obtain a purified expression vector, which was stored at-80 ℃.

The specific experimental operation method comprises the following steps:

limiting dilution method of 96-well plate: the BHK-21 cells are passaged to a 96-pore plate by a conventional method, when the cells grow to a compact monolayer, the supernatant which is preserved at the temperature of minus 80 ℃ and contains the rPRVXJ-delta gE/gI/TK-VP4-2-3-1 expression vector is taken and diluted by a serum-free DMEM cell culture solution in a 10-fold gradient manner to obtain 10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 Four dilutions of the gradient dilutions were plated in 96-well plates at 100uL per well, with 24 replicates per gradient. After adding the virus diluent, the 96-well plate was incubated at 37 ℃ in a 5% carbon dioxide incubator for daily observation. Selecting the wells with more green fluorescent spots and higher dilution, repeatedly freezing and thawing at-80 ℃ for 3 times, centrifuging at 12000rpm for 2min, and storing the supernatant at-80 ℃.

The 6-pore plate virus plaque purification method comprises the following steps: the BHK-21 cells are passaged to a 6-pore plate by a conventional method, when the cells grow to a compact monolayer, the supernatant fluid which is harvested from a 96-pore plate and contains the rPRVXJ-delta gE/gI/TK-VP4-2-3-1 expression vector is taken and diluted by 10 times of gradient and multiple ratio by using serum-free DMEM cell culture fluid to obtain 10 ^-3 、10 ^-4 、10 ^-5 Three dilutions of gradient dilution, 10 ^-3 -10 ^-5 The diluted recombinant virus solution was inoculated in 6-well plates at 200uL,37 ℃ C. And 5% CO per well ₂ The virus solution was discarded after 1h of adsorption in the incubator, 2 × DMEM was mixed with 2% low melting agarose 1, and then 6 well plates were added to reduce the fluidity of the medium, which was placed at 37 ℃,5% CO ₂ The culture is carried out in an incubator, and the observation is carried out every day, after plaques appear, single virus plaques emitting green fluorescence are picked out under a fluorescence microscope as much as possible, and then the single virus plaques are subjected to scale-up culture in a 12-well plate. The 96-well plate limiting dilution method and the 6-well plate viral plaque purification method were repeated until the purified expression vector rPRVXJ-. DELTA.gE/gI/TK-VP 4-2-3-1 was obtained (see FIG. 3).

3. Expression of foreign protein after purification of rPRVXJ-delta gE/gI/TK-VP4-2-3-1

Passaging BHK-21 cells to 96-well plates according to conventional methods, 37 ℃,5% in CO2 incubator to dense monolayer. Taking purified F1, F10, F20 and F21 rPRVXJ-delta gE/gI/TK-VP4-2-3-1, and diluting to 10 times of gradient by using DMEM containing 2% serum ^-8 Yield 10 ^-1 -10 ^-9 Attached to 96 well plates, 24 wells per gradient were repeated. 37 ℃ after inoculation, 5% CO ₂ Culturing in an incubator. Placing the cultured 96-well plate under an inverted fluorescence microscope to observe whether each dilution hole has cytopathy and simultaneously generates green fluorescence, recording the quantity of the green fluorescence of each dilution hole, and calculating the TCID of rPRVXJ-delta gE/gI/TK-VP4-2-3-1 according to a Reed-Muench method ₅₀ Are respectively 10 ⁷ TCID ₅₀ /ml、10 ^6.8 TCID ₅₀ /ml、10 ^6.8 TCID ₅₀ /ml、10 ⁷ TCID ₅₀ /ml。

An F21 generation rPRVXJ-delta gE/gI/TK-VP4-2-3-1 is inoculated to BHK-21 cells, a cell protein sample is collected after 36h, and the detection of foreign proteins VP1, VP2, VP3 and VP4 is carried out through WB (the VP1, VP2, VP3 and SVV whole virus polyclonal antibody is provided by animal biotechnology of Sichuan university of agriculture), and the result is shown in figure 4.

rPRVXJ- Δ gE/gI/TK-VP4-2-3-1 safety test

Female BALB/c mice (average body weight 18 + -2 g) at 6-8 weeks of age were from Beijing Huafukang Biotech, inc. Randomly dividing mice into 4 groups, the first 3 groups are experimental groups, each group comprises 8 mice, and each group comprises 10 mice ⁷ TCID ₅₀ 、10 ⁶ TCID ₅₀ 、10 ⁵ TCID ₅₀ The dosage of rPRVXJ-delta gE/gI/TK-VP4-2-3-1 virus solution is injected. Group 4 mice were selected as a control group and were injected intramuscularly with 0.2mL DMEM. Mice survival was recorded for 15 consecutive days. After 15 days, the mouse brain tissue was fixed with 4% paraformaldehyde for histopathological observation, and the safety of rPRVXJ- Δ gE/gI/TK-VP4-2-3-1 to the mouse was evaluated.

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

Immunogenicity study of rPRVXJ-delta gE/gI/TK-VP4-2-3-1 with mouse as animal model

6-8 week old female BALB/c mice (average body weight 18 + -2 g) were from Beijing Huafukang Biotech GmbH. Mice were randomly divided into two groups (immunization group and pair)Control group, 30 per group), immunization group each mice was immunized 10 μ L by nasal drip ⁷ TCID ₅₀ Perml rPRVXJ-delta gE/gI/TK-VP4-2-3-1, control group each mouse nasal drop immunization 50u LDMEM, two weeks after the initial immunization. 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, with isolation of 14 and 28dpv mouse splenocytes.

5.1 measurement of cellular immune Effect of rPRVXJ- Δ gE/gI/TK-VP4-2-3-1 Using mouse as animal model

(1) The secretion of 14 and 28dpv IL-4 and IFN-gamma in peripheral blood of mice in the immunization group and the control group was measured 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) Splenocytes were isolated from 14 and 28dpv immunized and control mice, triplicates were set for each group. Counting by using a cell counting plate, and diluting the splenocytes by 1640 culture medium until the number of cells is 5 multiplied by 10 ⁶ at/mL, the cells 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) UV inactivated 10 ⁶ TCID ₅₀ 100 μ L/ml SVV virus solution; (2) 10 μ g/ml of Canavalia gladiata protein A (MP Biomedicals, LLC: 195283) (Concanavalin A, conA), 100 μ L per well; (3) 1640 medium, 100. Mu.L per well. Triplicate replicates per well were averaged. After 72h of culture, the OD450nm absorbance of each well was measured by using a CCK-8 (Beyotime, china) detection kit, and then the stimulation index was calculated to evaluate the proliferation of spleen lymphocytes. The Stimulation Index (SI) is calculated as: SI = (immune OD value-control OD value)/(negative control OD value-control OD value), results are shown in table 2, SI values of the vaccinated rpvxj- Δ gE/gI/TK-VP4-2-3-1 mouse group were significantly higher than those of the vaccinated DMEM mouse group when stimulated with inactivated SVV virus solution and ConA.

(3) Spleen cells from 14 and 28 dpv-isolated immunized and control mice were diluted to 2X 10 with PBS ⁴ mL, 500. Mu.L were taken and treated 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 Staining for 30min in the dark at room temperature, detecting with a flow cytometer, and analyzing the data with flowjo10 software. The results are shown in Table 3, and at day 28 after immunization, mouse splenic lymphocytes CD3 were measured ⁺ 、CD3 ⁺ CD4 ⁺ (humoral immune-related), CD3 ⁺ CD8 ⁺ (cell immunity related) T cell ratio, and finding out the CD3 of the immunized mice ⁺ CD4 ⁺ The percentage of T cells was significantly higher than the control, CD ³⁺ CD ⁸⁺ The percentage of T cells 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 mouse spleen lymphocytes CD3+, CD4+, CD8+

5.2 rPRVXJ-delta gE/gI/TK-VP4-2-3-1 humoral immune effect determination using mouse as animal model

Sera of 0, 1, 3, 5, 7, 14, 21, 28, 35, 42dpv immunized and control mice were collected and the levels of mouse gE, gB antibodies were tested with reference to the french id. The indirect ELISA method of SVVVP1 protein established in the laboratory is used for detecting the VP1 specific antibody level. Serum neutralizing antibody levels were determined by plaque reduction.

Specific steps of ELISAThe method comprises the following steps: (1) coating: coating antigen with prokaryotic expression VP1 protein, diluting VP1 prokaryotic expression protein to 31.25ng/ml and 100 μ L/hole with CBS coating solution, performing ELISA 96-well plate (corning) coating, coating at 37 ℃ for 2h, discarding coating solution, washing with 200 μ L/hole PBST for 5 times and 5 min/time; (2) and (3) sealing: sealing 5% skimmed milk at 37 deg.C and 200 μ L/hole for 1.5h, discarding sealing solution, and washing 200 μ L/hole PBST for 5 times and 5 min/time; (3) a first antibody: the collected mouse sera were expressed as 1: diluting with 400, setting positive serum control (VP 1 protein murine polyclonal antibody) and negative serum control (serum of mice without any treatment), incubating at 37 deg.C for 1.5h with 100 μ L/well, discarding serum, and washing with 200 μ L/well PBST for 5 times, 5 min/time; (4) secondary antibody: HRP-labeled goat anti-mouse IgG (1 diluted in 5000), 100 μ L/well, incubated at 37 ℃ for 1h, secondary antibody discarded, and 200 μ LPBST washed 5 times, 5 min/time; (5) color development: adding 100 mu LTMB substrate developing solution into each hole, and incubating for 15min at room temperature; finally 50. Mu.L of 2M H was added to each well ₂ SO ₄ Stopping the reaction, and detecting OD within 15min by using an enzyme-labeling instrument _450nm And (4) light absorption value.

Preparation of VP1 prokaryotic expression protein: (1) constructing a prokaryotic expression vector pCold-VP1 and transforming the prokaryotic expression vector pCold-VP1 into BL21 (DE 3) competent cells to obtain the recombinant expression strain BL21-pCold-VP1. (2) The recombinant expression bacterium 1 _600nm At 0.6, IPTG was added to a final concentration of 1mM to induce protein expression, and BL21 (DE 3) expressing bacteria containing the empty vector pCold-TF were set up as controls. (3) After the expression of the recombinant protein is determined by SDS-PAGE, expression conditions such as IPTG concentration, induction temperature, induction time and the like are optimized, and the solubility of the recombinant protein is analyzed. (4) After the recombinant protein is expressed in large quantities under the optimal expression condition, the recombinant protein is purified and concentrated according to the operation instruction of a Ni-NTA protein purification kit (Productivity: C600332-0001), and the concentration of the purified protein is detected by a nucleic acid protein instrument (Scandrop 100 nucleic acid protein instrument ANALYTIKKENA (Germany)).

The assay results showed that the gE antibody was negative at all mice at each time point, 21dpv (1 week after the second immunization), the gB antibody, VP1 antibody were positive in the immunized mice, and the antibody levels increased with time (see tables 4, 5, 6).

And (3) detecting a neutralizing antibody by a plaque reduction method: (1) test sera (control and immune groups)) The dilution by fold was (1: 2-1:64 100PFU of SVV virus solution and exposed to 37 ℃ for 1 hour. (2) The density of BHK-21 cells cultured in the 6-well plate is up to 80%. Serum-virus neutralization was 200. Mu.L/well, and 100PFU of SVV virus solution was separately inoculated to each well. The inoculated cells were adsorbed at 37 ℃ for 1h. (3) The well liquid was discarded, 2 × DMEM was mixed with 2% low melting agarose 1, and the mixture was added to a 6-well plate to reduce the fluidity of the medium, which was placed at 37 ℃,5% CO ₂ Culturing in an incubator, observing every day, after the occurrence of plaques, fixing and dyeing by using formalin-crystal violet fixing dyeing liquid, automatically reading the number of the plaques by using IPP6.0 software, calculating the serum dilution with the exact plaques reduced by half according to a proportional method, and calculating the serum dilution, namely the serum neutralization titer.

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

Establishing a standard curve for the fluorescent quantitative PCR detection of SVV by taking SVV conserved gene 3D as a template, wherein y = -3.4653x +36.194 ² =0.9993. 50ul 10 infection of control group and immune group ⁶ TCID ₅₀ SVV, collecting the brain, heart, liver, spleen, lung, kidney, large intestine, small intestine, intestinal stranguria and feces of mice 1, 3, 5, 7, 10 and 14 days (dpi) after infection, and detecting the virus load of SVV in the tissues of the control group and the immune group.

The detection result shows that SVV is in a transient infection in a mouse, the heart, liver, spleen, lung, intestinal stranguria and feces virus load of a control group inoculated SVV mouse is higher, the peak value is reached at 3-6dpi, and 10dpi basically finishes detoxification (the result is shown in a table 8). When the viral loads of heart, liver, spleen, lung, intestinal gonorrhea and feces of 3 and 6dpi immune groups are compared with those of a control group, the SVV 3D gene copy value is obviously reduced (the result is shown in a table 9).

TABLE 4gB antibody assay results

Note: the positive result is that S/N% is less than or equal to 40%; the sample is suspicious when S/N% is more than 40% and more than 50%; S/N% > 50% was negative.

TABLE 5 gE antibody assay results

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

TABLE 6 mouse VP1 protein antibody assay results

Note: OD _450nm More than or equal to 0.286 is positive; OD _450nm <0.251 is negative.

TABLE 7 SVV neutralizing antibody assay results

TABLE 8 control SVV viral load assay results

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TABLE 9 immune group SVV viral load assay results

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-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A construction method of a pseudorabies virus vector for expressing exogenous SVA capsid protein is characterized by comprising the following steps:

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

2. The method for constructing a recombinant plasmid as claimed in claim 1, wherein the sequence of SEQ ID NO.1 in the step (1) is inserted between EcoRI and Mlu I cleavage recognition sites of pEGFP-gI28k eukaryotic expression plasmid.

3. The construction method according to claim 2, wherein the step (2) is specifically operated as follows: adding the solution (2) into the solution (1) to obtain a mixed solution, dripping the mixed solution into a 12-hole plate in which BHK-21 cells grow, uniformly mixing, transfecting, inoculating 5 mu L of a pseudorabies virus vector PRV XJ without TK genes 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 DMEM70 mu L + Lipofectamine TM30007.5 mu L; solution (2) is DMEM 70. Mu.L + P3000TM 5. Mu.L + 5. Mu.g plasmid, wherein plasmid pEGFP-gI28k-VP4-1 and CRISPR-CasgE are each 2.5. Mu.g.

4. The method of constructing according to any one of claims 1 to 3, further comprising the step (4) of purifying rPRVXJ- Δ gE/gI/TK-VP4-2-3-1.

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

6. The pseudorabies virus vector which is constructed by the construction method of any one of claims 1-3 and 5 and expresses the exogenous SVA capsid protein.

7. The pseudorabies virus vector which is constructed by the construction method of claim 4 and expresses the exogenous SVA capsid protein.

8. The use of the pseudorabies virus vector expressing the exogenous SVA capsid protein according to claim 6 in the development of a porcine Seneca virus subunit vaccine.

9. The use of the pseudorabies virus vector expressing the exogenous SVA capsid protein according to claim 7 in the development of a porcine Seneca virus subunit vaccine.