Infectious herpes virus DNA vaccine and application thereof

Abstract

The invention provides a sheep infectious herpes virus DNA vaccine and application thereof. The sheep infectious herpes virus DNA vaccine can effectively prevent and treat sheep infectious herpes viruses. Wherein the nucleotide sequence of the used nucleic acid fragment is SEQ ID NO: 11, and the amino acid sequence of the encoded protein is SEQ ID NO: 12. Animal experiments prove that the DNA vaccine can induce sheep to generate a high-level specific antibody and strong cellular immune response, has a remarkable protection effect, has no toxic or side effect on experimental sheep, and can be popularized and applied as a novel vaccine.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products, and particularly relates to an ovine infectious herpesvirus DNA vaccine and its application. Background Art

[0002] Ovine Herpesvirus (OvHV) is a pathogen that seriously endangers the sheep industry. It belongs to the Herpesviridae family and can cause various clinical symptoms in sheep, such as respiratory diseases, reproductive disorders, and neurological diseases. OvHV infection not only affects the health and productivity of sheep but also causes serious economic losses. Currently, the prevention and control of OvHV mainly rely on inactivated vaccines and attenuated vaccines, but these traditional vaccines have the following problems: Although inactivated vaccines have high safety, their immunogenicity is weak, and multiple immunizations are required to achieve the protection effect, and the immune duration is short. Although attenuated vaccines have good immune effects, there is a risk of reversion to virulence, which may lead to the spread of the vaccine strain in the flock and cause diseases.

[0003] In recent years, DNA vaccines, as a new vaccine technology, have received extensive attention due to their high safety, simple preparation, and ability to induce a comprehensive immune response. DNA vaccines insert gene fragments encoding target antigens into eukaryotic expression vectors, and after being introduced into host cells, express antigen proteins, thereby stimulating the body's immune response. However, traditional DNA vaccines usually use complete viral antigen genes and have the following limitations:

[0004] 1) Low antigen expression efficiency: The complete antigen gene may contain some sequences that inhibit expression, resulting in insufficient expression of antigen proteins.

[0005] 2) Weak immunogenicity: The complete antigen protein may contain some immunosuppressive regions, reducing the immunogenicity of the vaccine. Safety hazards: The complete antigen protein may have potential pathogenicity, increasing the safety risk of the vaccine. Summary of the Invention

[0006] The purpose of the present invention is to provide an ovine infectious herpesvirus DNA vaccine and its application, which can effectively prevent and control ovine infectious herpesvirus, thus making up for the deficiencies of the prior art.

[0007] The present invention first provides a nucleic acid fragment that can be used to prepare an ovine infectious herpesvirus DNA vaccine, and its nucleotide sequence is SEQ ID NO:11;

[0008] For the nucleic acid fragment described above, the amino acid sequence of the encoded protein is SEQ ID NO:12;

[0009] The present invention also provides a use of the provided nucleic acid fragment, which is used to prepare a DNA vaccine.

[0010] In another aspect of the present invention, there is also provided a DNA vaccine against ovine infectious herpesvirus, which uses the above nucleic acid fragment as an antigen;

[0011] Furthermore, the nucleic acid fragment is inserted into a eukaryotic expression vector;

[0012] As a specific description of an embodiment, the eukaryotic expression vector is a pVAX1 expression vector.

[0013] The present invention provides a DNA vaccine against ovine infectious herpesvirus. It has been confirmed by animal experiments that it can induce sheep to produce high levels of specific antibodies and strong cellular immune responses, has a significant protective effect, and has no toxic or side effects on experimental sheep, and can be promoted and applied as a new vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Electrophoresis detection diagram of recombinant expression proteins of each fragment;

[0015] Figure 2 : Immunogenicity of each antigen fragment;

[0016] Figure 3 : Recombinant expression electrophoresis detection diagram of the fusion antigen;

[0017] Figure 4 : Immunogenicity detection diagram of the recombinant protein;

[0018] Figure 5 : Diagram of the change level of cytokines in experimental sheep. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] After analyzing the gB protein of ovine infectious herpesvirus OvHV, the present invention screens, truncates and modifies it to obtain a fragment with stronger antigenicity, and applies it to the construction of a DNA vaccine to improve the immune effect and safety of the vaccine.

[0020] The present invention will be described in detail below with reference to the embodiments and the drawings.

[0021] Example 1: Screening and modification of the target antigen fragment

[0022] The gB protein of OvHV was selected as the target antigen. The gB protein is one of the major envelope glycoproteins of herpesviruses, plays a key role in the process of virus invading host cells, and has high immunogenicity. The gB protein (GenBank accession number: NC_007646.1) was analyzed using the DNASTAR Protean module to predict its antigenic epitopes, hydrophilicity, flexibility and other characteristics, and potential antigenic regions were screened out. According to the analysis results, truncated fragments of the gB protein were designed and synthesized. MKSELTEEDFTACLSFFNRPFPQIIDTSAANLRTLRDTKGPLQQL

[0023] Table 1: Information table of antigen fragmentation of gB protein

[0024]

[0025] Each truncated fragment was cloned into the prokaryotic expression vector pET-28a(+), and then transformed into Escherichia coli BL21(DE3). A single colony was inoculated into LB liquid medium and cultured with shaking at 37 °C until the OD600 reached 0.6 - 0.8. IPTG was added to a final concentration of 1 mM, and induction expression was carried out at 16 °C for 12 hours. The bacterial cells were collected, sonicated, and the supernatant was taken after centrifugation. The recombinant protein was purified using a Ni-NTA affinity chromatography column, and the purity was detected by SDS-PAGE. The results showed that fragments 3 - 5 were significantly expressed, while the expression efficiency of fragments 1 and 2 was relatively low ( Figure 1 ), so the immune effects of fragments 3 - 5 were detected.

[0026] The reactivity of the recombinant proteins of fragments 3 to 5 with OvHV positive serum was detected by ELISA: The purified recombinant proteins were diluted to 1 μg / mL and coated on a 96-well plate overnight at 4 °C. The coating solution was discarded, and 5% skim milk was added for blocking and incubated at 37 °C for 1 hour. Diluted OvHV positive serum was added and incubated at 37 °C for 1 hour. HRP-labeled goat anti-IgG secondary antibody was added and incubated at 37 °C for 1 hour. TMB chromogenic solution was added, and the reaction was carried out at room temperature in the dark for 15 minutes. The stop solution was added, and the OD450 value was measured. It was found that fragments 3 and 5 had antigenicity ( Figure 2 ), but the effect was not prominent.

[0027] To improve the immunogenicity of the antigen, fragments 3 and 5 were linked by a linker as a fusion fragment. The nucleotide sequence of the fusion fragment is as follows:

[0028] ATGGACAAGCTCTACACGCTATGTATAGCTCTGTCTGTTCTCCATGCCT

[0029] CCCGGGCTTTTCCTCTGGCCGCGGTAGACAGCTCTGATCTGAATTACTCCG

[0030] GAGAAGAAGGTACAGATAGTCCTCTGCTGGGTGGTGGTGGTAGCGGTGGT

[0031] GGTGGTAGCGGTGGTGGTGGTAGCGAGGTTCTCACCAGGGTGCTGAAAAT

[0032] TATAGTGGGGGAGATAGACGGAGATGGAGAAAGCGGCTCCGGCGACGACC

[0033] CCAACTTTGACTGGTCCACGTACCTCACAGACGCCTATGAGGACTCCCTA

[0034] (SEQ ID NO:11)。

[0035] The amino acid sequence encoding the antigen polypeptide is as follows:

[0036] MDKLYTLCIALSVLHASRAFPLAAVDSSDLNYSGEEGTDSPLLGGGGSG GGGSGGGGSEVLTRVLKIIVGEIDGDGESGSGDDPNFDWSTYLTDAYEDSL(SEQ ID NO:12)。

[0037] The nucleotide sequence corresponding to the fusion fragment was synthesized by the company, and the synthesized fragment was cloned into the prokaryotic expression vector pET-28a(+), and then transformed into Escherichia coli BL21(DE3) for fusion expression. Figure 3 It can be seen that the fusion antigen protein was successfully expressed, and the recombinant fusion protein Lin-gB was obtained. Then, the immunogenicity of the protein Lin-gB was detected by ELISA, and the results showed that the antigenicity of the recombinant protein after fusion was significantly improved( Figure 4 )。

[0038] Example 2: Construction of DNA vaccine

[0039] The nucleotide fragment of the fusion protein was ligated into the pVAX1 vector and transformed into Escherichia coli DH5α, and then spread on an LB plate containing kanamycin (50 μg / mL) and cultured at 37 °C for 16 hours. Single colonies were picked and inoculated into an LB liquid medium and cultured overnight with shaking at 37 °C. The plasmid DNA of the positive clone was extracted and verified by sequencing. The sequencing results confirmed that the Lin-gB gene fragment was correctly inserted into the pVAX1 vector.

[0040] The recombinant plasmid pVAX1-Lin-gB was transformed into Escherichia coli DH5α by heat shock method or electroporation method, and spread on LB agar plates containing kanamycin (50 μg / mL), and cultured at 37 °C for 16 - 18 hours. Single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL), and cultured with shaking at 37 °C overnight (12 - 16 h). The recombinant plasmid was extracted using Qiagen Plasmid MaxiKit, and the concentration and purity of the plasmid (A260 / A280 ratio) were determined using an ultraviolet spectrophotometer. An A260 / A280 ratio of 1.9 indicated a relatively high plasmid purity. The plasmid was aliquoted and stored at -20 °C for later use.

[0041] Example 3: DNA Vaccine Immunization

[0042] Healthy sheep (6 months old) with similar body weights were selected and randomly divided into an experimental group and a control group, with 10 sheep in each group. The sheep in the experimental group were intramuscularly injected with pVAX-Lin-gB DNA vaccine (100 μg / sheep), and the sheep in the control group were injected with an equal amount of gB-3 vaccine (100 μg / sheep). The immunization schedule was to immunize once at 0 day, 14 days, and 28 days. Serum was collected regularly after immunization (at 0 day, 14 days, 28 days, 42 days, and 56 days) for antibody detection and analysis of cellular immune responses.

[0043] The specific antibody levels in the sera of immunized sheep were detected by the indirect ELISA method. The purified Lin-gB protein was diluted to 1 μg / mL and coated on a 96-well plate overnight at 4 °C. The coating solution was discarded, and 5% skim milk was added for blocking, and incubated at 37 °C for 1 hour. Diluted serum samples (starting from 1:100, serially diluted) were added and incubated at 37 °C for 1 hour. HRP-labeled goat anti-IgG secondary antibody was added and incubated at 37 °C for 1 hour. TMB chromogenic solution was added, and the reaction was carried out in the dark at room temperature for 15 minutes. The stop solution was added, and the OD450 value was measured.

[0044] Peripheral blood mononuclear cells (PBMC) of immunized sheep were isolated, stimulated with ConA or Lin-gB protein, and the lymphocyte proliferation was detected using a CCK-8 kit. Venous blood was collected, and PBMC were isolated using Ficoll density gradient centrifugation. PBMC were seeded in 96-well plates, and ConA (5 μg / mL) or Lin-gB protein (10 μg / mL) was added, and incubated at 37 °C, 5% CO 2Cultivate for 72 hours. Add 10 μL of CCK-8 solution to each well and continue to cultivate for 4 hours, then measure the OD450 value. Use an ELISA kit to detect the levels of cytokines such as IFN-γ and IL-4 in the culture supernatant of PBMC to evaluate the immune response. Operate according to the kit instructions, coat the antibody, add the standard product and the sample, add the detection antibody and the enzyme-labeled substance after incubation, and measure the OD450 value after color development.

[0045] On the 56th day after immunization, each sheep was inoculated intranasally with 1×10 6 TCID50 of OvHV, observe the clinical symptoms and mortality of the sheep in the experimental group and the control group, collect nasal swabs, and use qPCR to detect the viral load to evaluate the protective effect of the DNA vaccine.

[0046] Table 2 Specific antibody levels

[0047]

[0048] Table 3 Table of lymphocyte proliferation

[0049]

[0050] Table 4 Table of results of the challenge experiment

[0051]

[0052] As can be seen from Table 2, specific antibodies were detected in the experimental group starting from 14 days after immunization, and the antibody level increased significantly with the prolongation of the immunization time, which was significantly higher than that of the control group (P<0.05). Low levels of antibodies were also produced in the control group after immunization, but the antibody titer was significantly lower than that of the experimental group. The lymphocyte proliferation of the experimental animals is shown in Table 3. The OD450 values of the ConA and Lin-gB stimulation groups in the experimental group were significantly higher than those of the control group (P<0.05), indicating that the pVAX-gB vaccine induced a weak cellular immune response, while the pVAX-Lin-gB vaccine induced a strong specific cellular immune response. The IFN-γ level in the experimental group was significantly higher than that of the control group, and the IL-4 level was slightly higher than that of the control group ( Figure 5 ), indicating that the pVAX-Lin-gB vaccine induced a strong Th1-type immune response and also induced a partial Th2-type immune response. The clinical symptoms of the sheep in the experimental group were mild, the morbidity and mortality were significantly lower than those of the control group (P<0.05), and the viral load was significantly reduced (Table 4), indicating that the DNA vaccine has a good protective effect against OvHV infection.

[0053] The present invention provides a DNA vaccine against ovine infectious herpesvirus and a preparation method thereof. This vaccine uses a truncated gB antigen linked with a linker, which has stronger antigenicity than the original gB protein fragment and can induce a higher level of antibody and cellular immune responses in the body. The DNA vaccine provided by the present invention has high safety, a simple preparation method, is easy to produce on a large scale, and can effectively prevent and control the infection of ovine infectious herpesvirus.

Claims

1. A nucleic acid fragment, characterized in that The nucleic acid fragment can be used to prepare a sheep infectious herpes virus DNA vaccine, and the nucleotide sequence thereof is SEQ ID NO:

11.

2. The nucleic acid fragment according to claim 1, wherein The amino acid sequence of the protein encoded by the nucleic acid fragment is SEQ ID NO:

12.

3. Use of the nucleic acid fragment according to claim 1 in the preparation of a DNA vaccine.

4. A sheep herpes virus DNA vaccine, characterized in that: The DNA vaccine is prepared using the nucleic acid fragment described in claim 1 as an antigen.

5. The DNA vaccine according to claim 4, characterized in that The nucleic acid fragment is inserted into a eukaryotic expression vector.

6. The DNA vaccine according to claim 5, characterized in that The eukaryotic expression vector is a pVAX1 expression vector.

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