Porcine group A rotavirus DNA vaccine and its application

Abstract

The present invention discloses a porcine group A rotavirus DNA vaccine and its application. The DNA vaccine is composed of a eukaryotic expression plasmid containing a G5 genotype VP7 protein gene sequence, a eukaryotic expression plasmid containing a G9 genotype VP7 protein gene sequence, a eukaryotic expression plasmid containing a P[23] genotype VP4 protein gene sequence and a TNFα adjuvant, wherein the G5 genotype VP7 protein gene sequence is shown in SEQ ID NO:1, the G9 genotype VP7 protein gene sequence is shown in SEQ ID NO:2, the P[23] genotype VP4 protein gene sequence is shown in SEQ ID NO:3, and the TNFα gene sequence is shown in SEQ ID NO:4. The DNA vaccine prepared by the present invention can induce significant humoral immunity and cellular immunity, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of animal vaccines, and in particular relates to a porcine group A rotavirus DNA vaccine and an application thereof. Background Art

[0002] Rotavirus is an 11-segment double-stranded RNA virus with a diameter of about 65-75 nm and a genome length of about 18.5 kb. It is a member of the genus Rotavirus in the family Reoviridae and is a zoonotic pathogen. It is a serious hazard to infected infants and young animals, with the main clinical manifestations being vomiting, diarrhea and dehydration. It poses a serious threat to human health and the development of the breeding industry.

[0003] According to the difference in VP6 protein antigenicity, rotavirus can be divided into 9 different serogroups (A~D, FJ). Among them, Group A Rotavirus (RVA) is the main cause of acute dehydration diarrhea in humans and various animals. Based on the differences in the sequences of the genes encoding VP7 and VP4, rotavirus is divided into different G genotypes and P genotypes. Currently, there are 58 P genotypes and 42 G genotypes of RVA found in human and animal populations.

[0004] Group A rotavirus infection is very common in pigs. Currently, there is no other good treatment for group A rotavirus infection in pigs except vaccination. Epidemiological surveys on porcine viral diarrhea in recent years have shown that the G9, G5, G4, G3, G11, and G12 genotypes currently exist simultaneously, with G9 and G5 being the main prevalent genotypes, which are usually prevalent in combination with P

[23] .

[0005] Vaccination is an effective measure to prevent and control porcine rotavirus infection. Currently, inactivated vaccines, attenuated vaccines, subunit vaccines, DNA vaccines and mRNA vaccines have been developed for rotavirus. Compared with other types of vaccines, DNA vaccines have the advantages of being easy to prepare, highly stable, and able to induce humoral and cellular immunity. However, previous studies have shown that naked DNA immunization induces limited humoral immunity and weak immunogenicity. Therefore, DNA vaccines need to be optimized to meet clinical needs. Summary of the invention

[0006] In view of the shortcomings of the prior art, the object of the present invention is to provide a porcine group A rotavirus DNA vaccine and its application.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In the first aspect, the present invention protects a porcine group A rotavirus DNA vaccine, which is composed of a recombinant plasmid containing a nucleotide sequence encoding a G5 genotype VP7 protein, a recombinant plasmid containing a nucleotide sequence encoding a G9 genotype VP7 protein, a recombinant plasmid containing a nucleotide sequence encoding a P

[23] genotype VP4 protein, and a mouse tumor necrosis factor α (TNFα) protein adjuvant, wherein the nucleotide sequence encoding the G5 genotype VP7 protein is as shown in SEQ ID NO:1, the nucleotide sequence encoding the G9 genotype VP7 protein is as shown in SEQ ID NO:2, the nucleotide sequence encoding the P

[23] genotype VP4 protein is as shown in SEQ ID NO:3, and the TNFα protein encoding nucleotide sequence is as shown in SEQ ID NO:4.

[0009] The present invention constructs two VP7 protein eukaryotic expression plasmids of different genotypes, and performs co-immunization with the VP4 protein eukaryotic expression plasmid. Although co-immunization can improve the broad spectrum of the vaccine, it may also lead to competition or interference between antibody responses, thereby affecting the strength of the immune response. However, the experimental results show that the trivalent vaccine of the present invention can effectively maintain the independent immune response of each antigen, and has no negative impact on the induction of individual antibodies.

[0010] In a specific embodiment, the eukaryotic vector used in the construction of the recombinant plasmid is pcDNA3.4, wherein the recombinant plasmid containing the nucleotide sequence encoding the G5 genotype VP7 protein is named pcDNA3.4-VP7-G5, the recombinant plasmid containing the nucleotide sequence encoding the G9 genotype VP7 protein is named pcDNA3.4-VP7-G9, and the recombinant plasmid containing the nucleotide sequence encoding the P

[23] genotype VP4 protein is named pcDNA3.4-VP4-P

[23] .

[0011] Compared with naked plasmids, TNFα protein and eukaryotic expression plasmids were co-immunized into mice by intramuscular injection to induce significantly higher levels of specific IgG antibodies against antigens. Cytokine analysis showed that the DNA vaccine developed by the present invention can effectively induce Th1 and Th2 immune responses.

[0012] In a specific embodiment, the TNFα protein adjuvant is mixed with the recombinant plasmids pcDNA3.4-VP7-G5, pcDNA3.4-VP7-G9, pcDNA3.4-VP4-P

[23] at a mass ratio of 10 µg: 100 µg: 100 µg: 100 µg to obtain the DNA vaccine.

[0013] In a specific embodiment, the TNFα protein adjuvant is obtained by inserting the mouse TNFα protein encoding gene into the prokaryotic expression vector pGEX-4T-1, introducing it into Escherichia coli, and inducing expression to obtain the TNFα protein.

[0014] The present invention uses TNFα protein as a porcine rotavirus DNA vaccine adjuvant for the first time. When a dose of 10 µg is injected into each mouse, the DNA vaccine of the present invention does not cause adverse reactions or excessive immune activation, and can effectively enhance the immune response while ensuring safety.

[0015] In a specific embodiment, the vaccine further contains a pharmaceutically acceptable carrier or excipient.

[0016] In a specific embodiment, the vaccine is administered by intramuscular injection.

[0017] In a second aspect, the present invention also protects the use of any of the porcine group A rotavirus DNA vaccines described above in the preparation of drugs for preventing diseases caused by porcine rotavirus.

[0018] The positive beneficial effects of the present invention are:

[0019] 1. The present invention uses prokaryotically expressed mouse TNFα protein as the DNA vaccine adjuvant developed by the present invention. The prepared DNA vaccine can induce a comprehensive immune response (including humoral immunity and cellular immunity) and has excellent vaccine application value.

[0020] 2. The DNA vaccine of the present invention is simple to prepare and easy to produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 1 shows the results of PCR identification of eukaryotic recombinant plasmids; 1: DL5000 Marker; 2-4: PCR identification of G5, G9, P

[23] eukaryotic expression plasmids.

[0022] Figure 2 IFA was used to verify the expression of the eukaryotic recombinant plasmid in 293T cells.

[0023] Figure 3 SDS-PAGE and Western-blot were used to verify the purified TNFα protein.

[0024] Figure 4 The immune enhancement effect of TNFα adjuvant was verified by indirect ELISA to detect the level of IgG antibody specific to the protein encoded by the target gene of rotavirus in serum; the ELISA coating antigens were G5 genotype VP7 protein ( Figure 4 Figure A), G9 genotype VP7 protein ( Figure 4 Figure B), P

[23] genotype VP4 protein ( Figure 4 Figure C in the figure).

[0025] Figure 5 The immunogenicity of monovalent DNA vaccine and trivalent DNA vaccine was verified by indirect ELISA to detect the level of IgG antibody specific to the protein encoded by the target gene of rotavirus in serum; the ELISA coating antigens were G5 genotype VP7 protein ( Figure 5 Figure A), G9 genotype VP7 protein ( Figure 5 Figure B), P

[23] genotype VP4 protein ( Figure 5 Figure C in the figure).

[0026] Figure 6 The virus neutralization test was used to measure the antibody titer of rotavirus in serum 28 days after immunization. The target viruses used were G5P

[23] genotype rotavirus ( Figure 6 Figure A) and G9P

[23] genotype rotavirus ( Figure 6 Figure B in the figure).

[0027] Figure 7 ELISA was used to measure the secretion of IFN-γ in the serum of mice 28 days after the first immunization. Figure 7 Figure A) and IL-4 ( Figure 7 Figure B in the figure).

[0028] Figure 8 Proliferation of mouse splenic lymphocytes.

[0029] Fig. 9 Flow cytometric analysis of CD3 in splenocytes isolated from different immune groups + CD4 + The proportion of T lymphocytes ( Fig. 9 Figure A) and CD3 + CD8 + The proportion of T lymphocytes ( Fig. 9 Figure B in the figure). DETAILED DESCRIPTION

[0030] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0031] Example 1 Construction and identification of porcine group A rotavirus eukaryotic expression plasmid

[0032] 1. Construction of porcine rotavirus group A eukaryotic expression plasmid

[0033] The target gene was obtained by three rounds of PCR amplification using laboratory isolated G9P

[23] genotype and G5P

[23] genotype porcine group A rotavirus as templates. The amplification primers are shown in Table 1, which are the nucleotide sequence encoding the G9 genotype VP7 protein (SEQ ID NO:1) and the G5 genotype VP7 protein (SEQ ID NO:2) and the P

[23] genotype VP4 protein (SEQ ID NO:3). By PCR amplification, the vector homology arm, Kozak sequence (GCCACC) and VP7 protein signal peptide (SEQ ID NO:5) or VP4 protein signal peptide (SEQ ID NO:6) were added to the 5' end of the target gene, and the flexible protein (SEQ ID NO:7), strep tag sequence (SEQ ID NO:8) and vector homology arm (SEQ ID NO:9) were added to the 3' end of the target gene. Figure 1 ). The three-round PCR amplification steps are as follows;

[0034] One round of PCR amplification:

[0035] (1) Using the isolated G5P

[23] genotype strain as a template, primers pcDNA3.4-QYSP-G5, G9-VP7-Fn and pcDNA3.4-G5-VP7-Rn were used to amplify the VP7-G5 first-round PCR product;

[0036] (2) Using the isolated G9P

[23] genotype strain as a template, primers pcDNA3.4-QYSP-G5, G9-VP7-Fn and pcDNA3.4-G9-VP7-Rn were used to amplify the VP7-G9 first-round PCR product;

[0037] (3) Using the isolated G9P

[23] genotype strain as a template, primers pcDNA3.4-IL-4-P

[23] -VP4-Fn and pcDNA3.4-P

[23] -VP4-Rn were used to amplify the VP4-P

[23] first-round PCR product.

[0038] Second round of PCR amplification:

[0039] (1) The first-round PCR products of VP7-G5 and VP7-G9 were used as templates, and the second-round PCR products of VP7-G5 and VP7-G9 were amplified using primers pcDNA3.4-QYSP-Fz and pcDNA3.4-Rz respectively;

[0040] (2) Using the first-round PCR product of VP4-P

[23] as a template, primers pcDNA3.4-IL-4-Fz and pcDNA3.4-Rz were used to amplify the second-round PCR product of VP4-P

[23] .

[0041] Three rounds of PCR amplification:

[0042] (1) Using the second-round PCR products of VP7-G5 and VP7-G9 as templates, primers pcDNA3.4-QYSP-Fw and pcDNA3.4-Rw were used to amplify the third-round PCR products of VP7-G5 and VP7-G9;

[0043] (2) Using the second-round PCR product of VP4-P

[23] as a template, primers pcDNA3.4-IL-4-Fz and pcDNA3.4-Rw were used to amplify the third-round PCR products of VP7-G5, VP7-G9 (same primers) and VP4-P

[23] .

[0044] The gene fragments obtained above (three-round PCR products) were connected to the pcDNA3.4 vector (double-digested with Xba I and ECoR V) by homologous recombination, and then transformed and verified by PCR amplification and sequencing ( Figure 1 ), and finally three recombinant plasmids carrying different target genes were obtained.

[0045] 2. IFA Identification of Eukaryotic Expression Plasmid of Porcine Group A Rotavirus

[0046] 293T cells were plated in a 48-well cell culture plate. When the cell density reached 80%, the recombinant plasmid constructed above was transfected with PEI transfection reagent, and the pcDNA3.4 vector was transfected as a negative control. Indirect immunofluorescence identification was performed 48 hours after transfection. The specific steps are as follows: discard the culture medium, wash once with PBS, add acetone methanol fixative (commercial propanol solution and methanol solution mixed in a ratio of 1:1), and stand at room temperature for 30 min. Discard the fixative and wash twice. Add the diluted strep antibody and place at 37 °C for 1 h. Discard the solution and wash five times with PBS, add FITC-labeled goat anti-mouse secondary antibody, and place at 37 °C for 1 h. After washing five times, add DAPI, shake gently at room temperature for 10 min, and observe under a fluorescence microscope. The results showed that specific green fluorescence was observed in 293T cells transfected with recombinant plasmids pcDNA3.4-VP7-G5, pcDNA3.4-VP7-G9, and pcDNA3.4-VP4-P

[23] , while the negative control showed no green fluorescence, indicating that the three target proteins were expressed in the transfected 293T cells ( Figure 2 ).

[0047]

[0048] Example 2 Preparation of TNFα adjuvant for porcine group A rotavirus DNA vaccine

[0049] Using mouse spleen cDNA as a template, the mouse TNFα nucleotide sequence (SEQ ID NO: 4) was amplified by PCR. The amplification primers are shown in Table 2, and it was cloned into the pGEX-4T-1 vector to obtain the ligation product pGEX-4T-1-mouse-TNFα, which was transformed into the competent cells BL21 (DE3). The specific steps are as follows: Take out the BL21 (DE3) competent cells from the -80 ℃ refrigerator and place them on ice to thaw. Take 5 μL of the ligation product and add it to 50 μL of competent cells. After incubation on ice for 30 min, immediately place it in a 42 ℃ water bath for 60 s, and then continue to cool on ice for 3 min. Add 1 mL of LB medium and place it in a 37 ℃ shaker at 100 rpm for 45 min. Pipette 100 μL of the culture and evenly spread it on a solid LB plate with ampicillin resistance. Invert the plate and culture it in a 37 ℃ incubator for 16 h. Pick a single clone and culture it in 1 mL of LB liquid culture medium with ampicillin resistance. After culturing at 37°C and 220rpm for 5-6 hours, perform PCR identification and send the bacterial solution with the correct band size for testing. The clone with the correct sequencing is the recombinant bacterium that subsequently expresses mouse TNFα protein.

[0050]

[0051] The correctly sequenced recombinant bacteria were inoculated into 8 mL of LB medium containing ampicillin resistance, cultured in a shaker at 37°C and 220 rpm for 6-8 h, added to 300 mL of LB medium containing ampicillin resistance at a ratio of 1:100, and cultured in a shaker at 37°C and 220 rpm for 2-3 h. When the OD 600nm When the concentration reaches 0.6-0.8, add IPTG with a final concentration of 1mM, induce and culture for 16 hours at 16 ℃ and 130 rpm, centrifuge at 8000 ×g for 2 min, and collect the bacterial precipitate. Resuspend the precipitate in PBS and perform ultrasonic disruption. Centrifuge the ultrasonically disrupted bacterial solution at 8000 rpm and 4 ℃ for 15 min to obtain the lysate supernatant and lysate precipitate, respectively. Take the corresponding supernatant and precipitate samples for SDS-PAGE electrophoresis. The results show that TNFα protein can be expressed in both the precipitate and the supernatant. Take the protein in the supernatant for purification. According to the instructions of the GST-tag Purification Resin purification column, absorb 1 mL of the GST column into the affinity chromatography column, wash it twice with PBS, and then add it to the protein supernatant. The GST beads and the target protein are combined in a shaker overnight. After flow-through removal, the target protein is eluted with 13 mM GSH. Use an ultrafiltration tube to replace the buffer of the purified TNFα protein from GSH to PBS, and concentrate the protein. The concentrated target protein was subjected to SDS-PAGE electrophoresis and Western Blot verification ( Figure 3 ) At the same time, the concentration of the concentrated TNFα protein was determined by BCA, and the aliquots were stored at -80°C.

[0052] Example 3 Safety Assessment of TNFα Protein

[0053] After immunizing mice with the expressed TNFα protein at different concentrations (0.5, 1.0, 2.5, 5.0, 7.5 and 10 µg), the effect of TNFα protein on the physiological state of mice was evaluated by observing the clinical manifestations of mice. The results showed that the mice had smooth fur, normal food and water intake, good mental state, and no abnormal manifestations at different concentrations during the observation period. The TNFα protein of the present invention has good safety below 10 µg, so 10 µg TNFα protein was selected as a DNA vaccine adjuvant.

[0054] Example 4 Preparation of porcine group A rotavirus DNA vaccine

[0055] The TNFα adjuvant was mixed with the recombinant plasmids pcDNA3.4-VP7-G5, pcDNA3.4-VP7-G9, and pcDNA3.4-VP4-P

[23] at a ratio of 10 µg:100 µg, respectively, to obtain the monovalent DNA vaccines G5+TNFα, G9+TNFα, and P

[23] +TNFα for porcine group A rotavirus. At the same time, the TNFα adjuvant was mixed with the recombinant plasmids pcDNA3.4-VP7-G5, pcDNA3.4-VP7-G9, and pcDNA3.4-VP4-P

[23] at a ratio of 10 µg:100 µg:100 µg:100 µg to obtain the trivalent DNA vaccine G5+G9+P

[23] +TNFα for porcine group A rotavirus.

[0056] Example 5 Evaluation of the effect of TNF-α protein on DNA vaccine-induced humoral immunity

[0057] Six-week-old SPF BALB / c female mice were selected and divided into recombinant plasmid groups (G5, G9 and P

[23] ), DNA vaccine groups (G5+TNFα, G9+TNFα and P

[23] +TNFα), control groups (Mock group, immunized with pcDNA3.4 and 10 µg TNFα protein), and blank control group (immunized with PBS). The mice were vaccinated by intramuscular injection. After the first immunization, booster immunization was performed at intervals of 2 weeks. At the same time, blood samples were collected by orbital bleeding 14 days and 28 days after the first immunization. The collected blood was centrifuged in a centrifuge to separate the serum. The target gene-encoded protein was used as the coating antigen (encoding the VP7 protein of the G9 genotype and the G5 genotype and the VP4 protein of the P

[23] genotype), and the specific IgG antibody level in the serum was determined by indirect ELISA. The specific operation steps are as follows: VP7 protein encoding G9 genotype and G5 genotype and VP4 protein encoding P

[23] genotype were used as coating antigens, the coating concentration of the three antigens was 2.5 μg / ml, and the serum dilution was 1:100. The protein was diluted with coating solution, 100 μL per well, and coated overnight at 4 ℃. The coating solution was discarded, washed with PBS 3 times, and blocked with 5% skim milk at 37 ℃ for 2 hours. Discarded, washed 5 times with PBST. Dilute the serum to be tested, 100 μL per well, incubated at 37 ℃ for 1 hour, and set up negative and positive serum controls. Add 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG, 100 μL per well, incubated at 37 ℃ for 1 hour. Discard the secondary antibody, wash 5 times with PBST. Add 100 μL TMB colorimetric solution, and color for 15 minutes in the dark. Add 50 µL of stop solution 2M H2SO4 to each well, OD 450nm Read the value.

[0058] Example 6 Immunological Evaluation of Porcine Group A Rotavirus DNA Vaccine in Mice

[0059] Similarly, 6-week-old, SPF-grade BALB / c female mice were selected and inoculated with DNA vaccines by intramuscular injection. The porcine group A rotavirus monovalent DNA vaccine group (G5+TNFα, G9+TNFα and P

[23] +TNFα) and trivalent DNA vaccine group (G5+G9+P

[23] +TNFα) were set up. The mice were immunized with pcDNA3.4 vector and 10 µg TNFα protein as the control group, and PBS immunization was used as the blank control group. All mice were boosted 14 days after the first immunization. Blood was collected from the eye sockets before the first immunization and 14 and 28 days after the first immunization. The serum was collected after centrifugation and stored in a -40 ℃ refrigerator. After the blood was collected on the 28th day, all mice were euthanized and their spleen cells were isolated to evaluate their cellular immune response.

[0060] 1. Levels of rotavirus protein-specific IgG antibodies in sera of immunized mice

[0061] The levels of IgG antibodies specific to the corresponding proteins in the serum before DNA vaccine immunization and 14 and 28 days after immunization were determined by indirect ELISA. The specific operation steps are shown in Example 5.

[0062] 2. Levels of rotavirus neutralizing antibodies in mouse serum 28 days after first immunization

[0063] Serum antibody neutralization levels were measured in 96-well cell culture plates. 50 The isolated porcine rotavirus strain was treated with 20 μg / mL pancreatin at 37°C for 1 h and then mixed with an equal amount of twice-diluted immune serum (the lowest serum dilution gradient was 1:2 4 ), add it to the MA104 cells that have grown monolayers after incubation at 37 ℃ for 1 h, 100 μL / well, incubate at 37 ℃ for 1 h, discard the solution, and add DMEM maintenance solution containing trypsin at a final concentration of 3.0 μg / mL, 100 μL / well. After culturing at 37 ℃ and 5% CO2 for 72 h, observe the pathological changes of the cells. For the uncertain cell wells, the indirect immunofluorescence method is used for determination. The highest dilution of cells without fluorescent signal labeling is the maximum dilution of the serum that can neutralize the virus after dilution. The titer of neutralizing antibodies is expressed as the reciprocal of the highest dilution of serum.

[0064] 3. Serum IFN-γ and IL-4 concentrations in mice 28 days after first immunization

[0065] The serum of mice 28 days after immunization was obtained and diluted twice, and the concentrations of IFN-γ and IL-4 in the serum were measured using IFN-γ and IL-4 enzyme-linked immunosorbent assay kits.

[0066] 4. Lymphocyte Proliferation Assay

[0067] On the 28th day after the first immunization, the mice were euthanized and their spleen cells were obtained. Splenocytes were inoculated into 96-well cell culture plates, with 2 × 10 5 cells / 100 μL. Five replicate wells were set up for each sample, and three replicate wells were stimulated with 5 μg / mL ConA. The 96-well plate was incubated in a 37 °C 5% CO2 incubator for 72 h, and lymphocyte proliferation was assessed using the Cell Counting Kit-8 (CCK8). The average OD of the stimulated wells was 450nm The average OD of the unstimulated wells 450nm The ratio is the lymphocyte proliferation index.

[0068] 5. T lymphocyte analysis

[0069] For each sample, 1 × 10 6 Splenocytes were resuspended in 100 μL HBSS containing CD3-FITC / CD4-PE / CD8-APC antibodies and incubated at 4 °C in the dark for 30 min. The cells were then washed twice with PBS and resuspended in 1 mL PBS. The number of CD3 per 20,000 cells was counted by flow cytometry. + CD4 + and CD3 + CD8 + The number of T cells was counted and the percentage of cells was determined.

[0070] 6. Statistical Analysis

[0071] GraphPad Prism 9 was used for statistical analysis, and the experimental data were processed by one-way analysis of variance and two-way analysis of variance. The means marked with different lowercase letters were significantly different (P < 0.05). The data are the mean ± standard deviation (SD) of each group of mice.

[0072] Test results

[0073] 1. TNFα protein in DNA vaccine-induced humoral immunity

[0074] The role of TNFα protein in the process of DNA vaccine-induced humoral immunity was evaluated by indirect ELISA. Figure 4 As shown. On the 14th and 28th days after the first vaccination with 100 µg naked plasmid, the immune response to the corresponding protein was relatively weak. However, when TNFα protein was used as an immune adjuvant, it significantly enhanced the IgG antibody level of the DNA vaccine against the corresponding protein in mice compared with the naked plasmid alone. In addition, we also observed a certain degree of cross-reactivity between the G9 monovalent DNA vaccine and the G5 monovalent DNA vaccine ( Figure 4 ).

[0075] 2. Mouse serum specific IgG antibody detection

[0076] The levels of IgG antibodies specific to G9-VP7, G5-VP7, and P

[23] -VP4 proteins in serum were detected by indirect ELISA to evaluate the immunogenicity of the DNA vaccine. The results showed that immunization with the trivalent DNA vaccine in mice did not affect the expression of the respective target proteins in vivo and the production of corresponding IgG antibodies, and there was no significant difference with the results of vaccination with the corresponding monovalent DNA vaccine. Whether used alone or in combination, mice produced a significant immune response to the vaccine 14 days after the first immunization. The same DNA vaccine was used to boost immunization of each group of mice 14 days after the first immunization, and the IgG antibody levels at 28 days were significantly increased compared with those at 14 days. In addition, when G9-VP7 and G5-VP7 proteins were used as coating antigens, the trivalent DNA vaccine induced the highest IgG antibody levels after the first immunization and booster immunization, which may be due to the cross-reaction between the G9 and G5 genotype VP7 proteins ( Figure 5 ).

[0077] Neutralizing antibody (NAb) titers were measured in serum on day 28 after the first immunization. When porcine group A rotavirus of genotype G5P

[23] was used as the target virus, the average NAb titer of most mice immunized with G9 monovalent DNA vaccine was 2 6 , while the NAb titer of mice immunized with G5 and P

[23] monovalent DNA vaccines was 2 7 In contrast, when the G9P

[23] genotype porcine group A rotavirus was used as the target virus, the NAb titer of mice immunized with the G9 monovalent DNA vaccine was approximately 2 7 The average NAb titer of mice vaccinated with G5 and P

[23] monovalent DNA vaccines was 2 6 In addition, when facing the target virus of the same genotype, the NAb titer induced by the trivalent DNA vaccine was not significantly different from that induced by the monovalent DNA vaccine ( Figure 6 ).

[0078] 3. Detection of cytokines IL-4 and IFN-γ in mouse serum

[0079] IL-4 and IFN-γ detection kits were used to detect the IL-4 and IFN-γ levels in the mouse serum at 28 days after DNA vaccine immunization. The results showed that the IFN-γ level induced by the trivalent DNA vaccine (range 243-345 pg / mL) was significantly higher than that of other monovalent vaccine groups (range 112-171 pg / mL) and the control group (69-106 pg / mL). Similarly, among all immunization groups, the trivalent DNA vaccine induced the highest IL-4 level (197-226 pg / mL), and the PBS control group had the lowest IL-4 level (120-144 pg / mL), with a significant difference ( Figure 7 ).

[0080] 4. Mouse Splenic Lymphocyte Proliferation

[0081] 28 days after the first immunization, the spleen cells of the mice were isolated to study the proliferation of spleen lymphocytes after immunization. The results showed that the trivalent DNA vaccine group induced stronger lymphocyte proliferation than the monovalent DNA vaccine group (Figure 8).

[0082] 5. Ratio of mouse spleen lymphocyte subtypes

[0083] Flow cytometry was used to evaluate the distribution of spleen lymphocyte subsets after vaccination. The results showed that there was a significant difference between the trivalent DNA vaccine group and the monovalent DNA vaccine group in CD3 + CD4 + and CD3 + CD8 + Significant differences in T cell levels. CD3 + CD4 + and CD3 + CD8 + The percentages of the subgroups were 33.70% and 19.40%; the G5 monovalent DNA vaccine group was 14.8% and 11.27%; the G9 monovalent DNA vaccine group was 17.70% and 10.60%; P

[23] the monovalent DNA vaccine group was approximately 20.50% and 12.8%, which was significantly different ( Fig. 9 ).

[0084] 6. Changes in physical signs of mice after immunization

[0085] During the test, the mice in each group were in good spirits, their fur was smooth and soft, they ate and drank water normally, and there was no local reaction such as redness, swelling, pain or nodules at the inoculation site, indicating that the porcine rotavirus DNA vaccine of the present invention has good safety.

[0086] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. A porcine rotavirus DNA vaccine, characterized in that: The DNA vaccine is composed of a recombinant plasmid containing a nucleotide sequence encoding a G5 genotype VP7 protein, a recombinant plasmid containing a nucleotide sequence encoding a G9 genotype VP7 protein, a recombinant plasmid containing a nucleotide sequence encoding a P[23] genotype VP4 protein, and a mouse TNFα protein adjuvant, wherein the nucleotide sequence encoding the G5 genotype VP7 protein is shown in SEQ ID NO: 1, the nucleotide sequence encoding the G9 genotype VP7 protein is shown in SEQ ID NO: 2, the nucleotide sequence encoding the P[23] genotype VP4 protein is shown in SEQ ID NO: 3, and the nucleotide sequence encoding the mouse TNFα protein is shown in SEQ ID NO: 4; The vector used in the construction of the recombinant plasmid is pcDNA3.4, wherein the recombinant plasmid containing the nucleotide sequence encoding the G5 genotype VP7 protein is named pcDNA3.4-VP7-G5, the recombinant plasmid containing the nucleotide sequence encoding the G9 genotype VP7 protein is named pcDNA3.4-VP7-G9, and the recombinant plasmid containing the nucleotide sequence encoding the P[23] genotype VP4 protein is named pcDNA3.4-VP4-P[23].

2. The porcine rotavirus DNA vaccine according to claim 1, characterized in that the TNFα protein adjuvant is mixed with the recombinant plasmids pcDNA3.4-VP7-G5, pcDNA3.4-VP7-G9, pcDNA3.4-VP4-P[23] in a mass ratio of 10 µg: 100 µg: 100 µg: 100 µg to obtain a DNA vaccine.

3. The porcine rotavirus DNA vaccine according to claim 1, characterized in that The mouse TNFα protein coding gene was inserted into the prokaryotic expression vector pGEX-4T-1, introduced into Escherichia coli, and TNFα protein was obtained after inducing expression.

4. The porcine rotavirus DNA vaccine according to claim 1, characterized in that The vaccine also contains a pharmaceutically acceptable carrier or excipient.

5. The porcine rotavirus DNA vaccine according to any one of claims 1 to 4, characterized in that: The vaccine is administered by intramuscular injection.

6. Use of the porcine rotavirus DNA vaccine according to any one of claims 1 to 4 in the preparation of a medicament for preventing diseases caused by porcine rotavirus.

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