A grouper NNV and TGIV dual-injection nano vaccine and its preparation method and application

Abstract

The present invention relates to a grouper NNV and TGIV dual immersion nano vaccine and a preparation method and application thereof, belonging to the field of biomedicine technology. The vaccine grouper neural necrosis virus dominant antigen NNV-MCP and grouper iridovirus dominant antigen TGIV-MCP are connected in series through a connecting peptide and then fused with the N-terminus of the grouper ferritin H subunit. The vaccine of the present invention can produce protective antibodies in the fish body by immersion, improves the immune efficacy, and can effectively prevent grouper neural necrosis disease and iridovirus disease.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and relates to a grouper NNV and TGIV dual-injection nano vaccine, and a preparation method and application thereof. Background Art

[0002] my country is the world's largest grouper farming country, accounting for about 80% of the world's total grouper farming output. In 2022, my country's grouper farming output was 206,000 tons, an increase of nearly 70% over 10 years ago, with good development momentum and huge market prospects. Grouper farming is one of the major national strategies to achieve a strong marine country and a blue granary, especially as a dozen artificially cultivated species in coastal areas such as Guangdong, with an annual output value of more than 10 billion yuan. However, over the years, its neuronecrosis virus disease and iris virus disease have frequently broken out, causing serious economic losses and threatening the healthy development of the grouper industry. Research on organic nanocarrier immersion vaccines provides technical support and guarantee for the effective prevention and control of diseases and the protection of aquatic food safety.

[0003] Vaccines have become the mainstream product in the field of aquatic animal disease prevention and control, and are also an inevitable choice to ensure the green development of aquaculture. Since my country successfully prepared a grass carp native vaccine in 1969, after years of development, it has made some breakthroughs in the preparation of native vaccines, live vaccines, inactivated vaccines, subunit vaccines and DNA vaccines for various aquatic pathogens, and successfully approved the first fishery vaccine (grass carp hemorrhagic disease live vaccine) in 2011. However, there are still some theoretical and technical bottlenecks in the large-scale industrial application of fishery vaccines, especially in terms of immunization pathways and immunization effects. Among them, injection immunization has the best protective effect, but it is difficult to meet the requirements of large-scale industrialization due to operating technology and production costs; although immersion and oral immunization have simple operating technology and low production cost, they also have a series of difficult-to-overcome technical problems such as skin and intestinal barrier restriction, enzymatic inactivation, and low immune protection rate. A large number of studies have shown that nanomaterials as carriers of fishery vaccines can effectively improve the immune protection effect of vaccines. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a grouper neuronecrosis virus and iridovirus dual immersion nano vaccine and its preparation and application. The present invention uses fish's own ferritin and antigen protein sequences to perform tandem prokaryotic expression in Escherichia coli to prepare nano spherical particles with antigen protein displayed on the surface. The immersion method can induce the fish to produce protective antibodies, improve the immune efficacy, and effectively prevent grouper neuronecrosis virus disease and iridovirus disease. The vaccine preparation method of the present invention is safe and simple, and is suitable for rapid large-scale production.

[0005] The present invention is achieved through the following technical solutions:

[0006] A grouper neural necrosis virus and iridovirus dual-injection nano vaccine, wherein the grouper neural necrosis virus dominant antigen NNV-MCP and the grouper iridovirus dominant antigen TGIV-MCP are connected in series through a connecting peptide and then fused with the N-terminus of the grouper ferritin H subunit.

[0007] Preferably, the sequence of the connecting peptide between the dominant antigen NNV-MCP of grouper neural necrosis virus and the dominant antigen protein of grouper iris virus is GGGGGGGSGGGGGG; the connecting peptide between the dominant antigen protein of grouper iris virus and the H subunit of grouper ferritin is GGGGSGGGGS.

[0008] Preferably, the amino acid sequence of the grouper neuronecrosis virus dominant antigen NNV-MCP is shown in SEQ ID NO.1, the amino acid sequence of the grouper iris virus dominant antigen protein is shown in SEQ ID NO.2, the amino acid sequence of the grouper ferritin H subunit is shown in SEQ ID NO.3, and the amino acid sequence of the vaccine is shown in SEQ ID NO.4.

[0009] The present invention also provides application of the vaccine in preparing medicine for preventing grouper neuronecrosis virus and / or iris virus.

[0010] The present invention also provides a method for preparing the vaccine, wherein the vaccine protein is synthesized according to the sequence described in SEQ ID NO.4, and then self-assembled into a nano-spherical structure under neutral conditions.

[0011] The beneficial effects of the present invention compared with the prior art are as follows: the vaccine of the present invention is a dual immersion vaccine of grouper neuronecrosis virus and iridovirus based on ferritin nanoparticles, and nano-spherical particles with surface-display antigenic proteins are prepared by prokaryotic expression in Escherichia coli, which can induce fish to produce protective antibodies by immersion, thereby improving the immune efficacy and effectively preventing grouper neuronecrosis and iridovirus diseases. The vaccine preparation method of the present invention is safe, simple, and suitable for rapid large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 A is the identification diagram of the purified GMM-FeH recombinant protein; A is the SDS-PAGE detection result diagram of the purified GMM-FeH recombinant protein. Lane M is the protein marker; Lane 1 is the IPTG-induced E.coli BL21 (DE3) (pET32a-GMM-FeH) protein; Lane 2 is the purified pET32a-GMM-FeH protein; B is the Western Blot analysis result of NNV, and C is the Western Blot analysis result of MCP;

[0013] Figure 2 This is the transmission electron microscopy observation result of GMM-FeH nanovaccine.

[0014] Figure 3 The changes in antibody levels of groupers in each group after vaccine immersion immunization. The data are expressed as mean ± standard error. Different lowercase letters between groups indicate P < 0.05.

[0015] Figure 4 is the immune protection rate of grouper after being infected with nervous necrosis virus and iris virus. A is the immune protection rate of grouper after being infected with nervous necrosis virus; B is the immune protection rate of grouper after being infected with iris virus. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with specific embodiments, but these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0017] Example 1. Preparation of self-assembled soaked vaccine

[0018] 1 Materials and methods

[0019] 1.1 Materials

[0020] 1.1.1 Vectors and strains

[0021] The pET-32a(+) vector was preserved in the laboratory. Escherichia coli DH5α and BL21(DE3) strains were products of Beijing Kangwei Century Biotechnology Co., Ltd. The pET32a-GMM-FeH(BL21(DE3)) strain (containing the dominant antigen protein NNV-MCP of the neuronecrosis virus, the antigen protein TGIV-MCP of the iridovirus, and the sequence of the grouper ferritin H subunit FeH) was synthesized by Wuhan Jinkairui Biological Co., Ltd.

[0022] 1.1.2 Reagents

[0023] The colorimetric reagent ECL and BCA protein content determination kits were products of Beijing Kangwei Century Biotechnology Co., Ltd. Protein Marker was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Mouse polyclonal antibodies against MCP protein of neuronecrosis virus and mouse polyclonal antibodies against MCP protein of iridovirus were prepared and stored by our laboratory. Mouse histidine monoclonal antibody and horseradish peroxidase-labeled goat anti-mouse IgG were purchased from Abcam. Other conventional reagents were domestically produced and of analytical grade.

[0024] 1.1.3 Test instruments

[0025] ALC-1100.2 electronic balance, Beijing Sartorius Instrument System Co., Ltd.; HH-4 digital constant temperature water bath, Shanghai Techeng Machinery Equipment Co., Ltd.; H1650-W desktop high-speed microcentrifuge, Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; 1-15K high-speed refrigerated centrifuge, Sigma, USA; SY-360 ultrasonic extractor, Shanghai Ningshang Ultrasonic Instrument Co., Ltd.; Classic ultrapure water instrument, Veolia Water Treatment Technology (Shanghai) Co., Ltd.; ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.; DYCZ-24DN mini dual vertical electrophoresis instrument and WD-9413A gel imaging analysis system were purchased from Beijing Liuyi Instrument Factory; Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific, USA; transmission electron microscope FEI Tecnai F20, FEI Company, USA.

[0026] 1.2 Test methods

[0027] 1.2.1 GMM-FeH protein design and inducible expression

[0028] The sequence of GMM-FeH protein is designed as follows: the C-terminus of the dominant antigen protein NNV-MCP of the neuronecrosis virus is connected in series through a connecting peptide (GGGGGGGSGGGGGG) and the N-terminus of the iridovirus antigen protein TGIV-MCP, and the C-terminus of the connected GMM is connected in series to the N-terminus of the grouper ferritin H subunit FeH through a connecting peptide (GGGGSGGGGS). The designed sequence was commissioned to Wuhan Jinkairui Biological Co., Ltd. for synthesis, and the pET32a-GMM-FeH plasmid containing the above protein gene was synthesized, and then the pET32a-GMM-FeH plasmid was transformed into the BL21 (DE3) competent state by heat shock. E. coli BL21 (DE3) containing the pET32a-GMM-FeH recombinant plasmid was cultured at 37°C for 1 h with shaking, then spread on LB plates (containing ampicillin) and cultured at 37°C overnight. A single colony was picked and cultured in LB liquid medium (containing ampicillin) at 37°C with shaking overnight. Another 1% of the bacterial solution was taken and expanded in new medium until OD 600 When the concentration of β-D-thiogalactopyranoside was 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) was added at a final concentration of 0.6 mmol / L, and the culture was induced for 4-6 hours, centrifuged, and the supernatant was discarded. The centrifuged precipitate was washed twice with 1×PBS, and an equal volume of sample buffer was added, mixed, boiled for 5 minutes, and electrophoresed by 10% polyacrylamide gel. The purified protein lane was transferred to nitrocellulose membrane, and the anti-neuronecrosis virus MCP protein mouse polyclonal antibody or anti-iridovirus MCP protein mouse polyclonal antibody was used as the primary antibody, and the horseradish peroxidase-labeled goat anti-mouse IgG was used as the secondary antibody for incubation. After washing, it was treated with an ECL kit and analyzed by Western Blot.

[0029] 1.2.2 Purification of GMM-FeH protein

[0030] Collect a large amount of induced expression bacterial liquid, centrifuge at 12000r / min and 4℃ for 10min in a 50mL centrifuge tube, collect the precipitate, resuspend the precipitate with ultrasonic disruption buffer, ultrasonicate for 40min in an ice bath, centrifuge at 12000r / min for 20min, take the precipitate, wash with deionized water, add denaturing solution containing 8mol / L urea to dissolve, centrifuge at 12000r / min and 4℃ for 10min, take the supernatant, dialyze in PBS containing 6.0, 4.0, 2.0, and 1.0mol / L urea, respectively, and finally dialyze in 1×PBS overnight. Purify the protein by His-tagged nickel column kit, collect the purified protein liquid, freeze it and extract it into lyophilized powder for storage. Dissolve it in sterile water when using, and determine the protein concentration with BCA protein content assay kit.

[0031] 1.2.3 Observation of the morphology of GMM-FeH vaccine

[0032] The renatured GMM-FeH fusion protein was sent to the electron microscopy experimental platform of the Institute of Zoology, Chinese Academy of Sciences for transmission electron microscopy observation. Negative staining: Take the dialyzed renatured protein sample, dilute it, and drop the sample on the plastic film in sequence. Use tweezers to clamp the copper mesh, and place the side with the carbon support film on the sample droplet. Wait for the copper mesh membrane to adsorb the protein for 3-5 minutes, and use absorbent paper to absorb excess water along the edge of the copper mesh, and rinse it with sterile water. The absorbent paper absorbs excess water. Then place the copper mesh on the uranium acetate stain solution for 5 minutes, and finally place the copper mesh on the clean bench to dry, ready for observation under a transmission electron microscope.

[0033] 2 Results and analysis

[0034] 2.1 Expression and detection results of GMM-FeH protein

[0035] The recombinant expression plasmid pET32a-GMM-FeH was expressed in E. coli BL21. After 4 hours of induction, the relative molecular mass of the expressed fusion protein was about 82 kDa ( Figure 1 The results of Western Blot analysis showed that there was an obvious recognition band at 82KDa ( Figure 1 B and C), which confirmed that it had good antigenic reactivity. GMM-FeH protein was successfully prepared.

[0036] 2.2 Morphological observation results of self-assembled nanovaccines

[0037] From the results of transmission electron microscopy (see Figure 2), GMM-FeH protein can self-assemble into a nano-spherical structure with a particle size of about 30nm under neutral conditions, confirming the successful preparation of the self-assembled nano-vaccine.

[0038] The dominant antigen protein sequence of grouper neural necrosis virus is (SEQ ID NO.1): MATIVPDLLPRLGHAARIFQRYAVETLEFEIQPMCPANTGGGYVAGFLPDPTDNDHTFDALQATRGAVVAKWWESRTVRPQYTRTLLWTSSGTRTLLWTSSGKEQRLTSPGRLILLCVGNNTDVVNVSVLCRWSVRLSVPSLETPEETTAPIMTQGSLYNDSLSTNDFKSILLGSTPLDIAPDGAVFQLDRPLSIDYSLGTGDVDRAVYWHLKKFAGNAGTPAGWFRWGIWDNFNKTFTDGVAYYSDEQPRQILLPVGTVCTRVDSEN. The dominant antigen protein sequence of grouper iris virus is (SEQ ID NO.2): VSVSFNDLVAQTLTSEFLDFWNACMMPGSKQSGYNKMIGMRSDLVGGITNGQTMPAAYLNLPIPLFFTRDTGLALPTVSLPYNEVRIHFKLRRWEDLLISQSTQADMAISTVTLANIGNVA

[0039] The sequence of the grouper ferritin H subunit is (SEQ ID NO. 3): MSSQVRQNYHQDCEAAINRQINLELYASYVYLSMGYYFDRDDQALHNFAKFFRHQSHEEREHAEKLMKLQNQRGGRIFLQDVKKPERDEWGSGVEALECALQLEKSVNQSLLDLHKLCSEHNDPHLCDFIETHYLDEQVKSIKELADWVTNLRRMGAPQNGLAEYLFDKHTMGKESS

[0040] The sequence of the two-combination soaked nanovaccine is ((SEQ ID NO.4)):MATIVPDLLPRLGHAARIFQRYAVETLEFEIQPMCPANTGGGYVAGFLPDPTDNDHTFDALQATRGAVVAKWWESRTVRPQYTRTLLWTSSGTRTLLWTSSGKEQRLTSPGRLILLCVGNNTDVVNVSVLCRWSVRLSVPSL ETPEETTAPIMTQGSLYNDSLSTNDFKSILLGSTPLDIAPDGAVFQLDRPLSIDYSLGTGDVDRAVYWHLKKFAGNAGTPAGWFRWGIWDNFNKTFTDGVAYYSDEQPRQILLPVGTVCTRVDSENGGGGGGGSGGGGGGVSVSFNDLV AQTLTSEFLDFWNACMMPGSKQSGYNKMIGMRSDLVGGITNGQTMPAAYLNLPIPLFFTRDTGLALPTVSLPYNEVRIHFKLRRWEDLLISQSTQADMAISTVTLANIGNVAGGGGSGGGGSMSSQVRQNYHQDCEAAINRQINLELYA SYVYLSMGYYFDDRDDQALHNFAKFFRHQSHEEREHAEKLMKLQNQRGGRIFLQDVKKPERDEWGSGVEALECALQLEKSVNQSLLDLHKLCSEHNDPHLCDFIETHYLDEQVKSIKELADWVTNLRRMGAPQNGLAEYLFDKHTMGKESS

[0041] Example 2: Experimental study on the preventive effect of the self-assembled ferritin carrier-free injection vaccine on grouper neural necrosis and iridovirus disease

[0042] 1.1 Materials

[0043] 1.1.1 Experimental animals

[0044] About 1000 groupers (average weight: 5.0±1.0 g) were purchased from Haikou grouper farm.

[0045] 1.1.2 Viruses, cells, bacteria and reagents

[0046] Neuronecrosis virus NNV (RGNNV genotype) and iridovirus TGIV were isolated and preserved by our laboratory. SSN-1 cells were preserved by our laboratory; grouper soaked nano-vaccine GMM-FeH was prepared and preserved by our laboratory; NaCl was from Sinopharm Chemical Reagent Co., Ltd.; mouse histidine monoclonal antibody and horseradish peroxidase-labeled goat anti-mouse IgG were purchased from Abcam; 96-well ELISA plate was from Corning, USA; Elisa detection kit was from Beijing Kangwei Century Biotechnology Co., Ltd.

[0047] 1.1.3 Test instruments

[0048] Refrigerated low-temperature centrifuge, Thermo Corporation, USA; ultra-low temperature refrigerator, Thermo Corporation, USA; Classic ultrapure water instrument, Veolia Water Treatment Technology (Shanghai) Co., Ltd.; KQ-500DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd.; tissue crusher and homogenizer, Jintan Chengdong Xinrui Instrument Factory; Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific, USA.

[0049] 1.2 Test methods

[0050] The vaccines GMM-FeH, FeH (grouper ferritin H subunit), and GMM (neural necrosis virus dominant antigen protein NNV-MCP C-terminus connected in series via a connecting peptide (GGGGGGGSGGGGGG) and the N-terminus of iridovirus antigen protein TGIV-MCP) prepared in this laboratory were dissolved and diluted into a suspension with distilled water, and immersion immunization was performed at a concentration of 20 mg / L for 6 hours. 100 fish were included in each concentration, and a blank control group was set up at the same time. After immunization, 20 ml of water was placed in each group. 3 In large and small breeding ponds, aeration was added, and 100 fish were stocked in each breeding pond. The water temperature was controlled at 25-28°C. Three fish were randomly collected from the treatment group on the 7th, 14th, 21st and 28th days after immunization for antibody titer determination. The fish were killed by cutting the spine, and the blood was collected. The blood was left to stand at room temperature for 2 hours and placed in a 4°C refrigerator overnight. The next day, the fish were centrifuged at a speed of 5000g in a low-temperature refrigerated centrifuge for 15 minutes, and the upper serum was taken. The antibody detection was performed by enzyme-linked immunosorbent assay: the purified vaccine was used as the antigen, the above-mentioned sampled serum was used as the antibody, the mouse histidine monoclonal antibody was used as the primary antibody, and the horseradish peroxidase-labeled goat anti-mouse IgG was used as the secondary antibody. The dilution ratio of the primary antibody and the secondary antibody was 1:1000. The color reaction was processed using a DAB horseradish peroxidase colorimetric kit. After color development, the absorbance at a wavelength of 450nm was measured by a ThermoMultiskan MK3 microplate reader (Molecular Devices Corp., Palo Alto, CA).

[0051] The method for detecting the virus titer is as follows: add a density of 1×10 5 100 μL of SSN-1 cells were added to each well and cultured at 25°C for 16-24 hours. When the cells grew to 80%-90%, 10 1 ~10 10 100 μL of virus solution / well, 8 parallel wells for each dilution, and cultured in a 25°C incubator for 2 hours. After incubation, the virus solution was recovered, the cells in the wells were rinsed twice with L-15 culture medium, and 100 μL of cell maintenance medium was added to continue culture for 96 hours. The experiment was set up in 3 parallel groups, and the CPE phenomenon of the monolayer cells at each dilution was observed and recorded every 24 hours, and the number of corresponding lesion wells was recorded. The tissue culture infective dose (TCID) of SVCV was calculated according to the Reed-Muench method. 50 ).

[0052] 28 days after immunization, 65 fish were randomly selected from each group of the test fish and placed in a new fish tank for the challenge test. In addition, the water temperature in the fish tank was controlled at 25.0±1.0℃ during the challenge test, and other breeding conditions remained unchanged. The challenge method was injection, and the injection site was the abdominal cavity of the fish. Each fish was injected with 20μL 5.0×10 8.3 TCID 50 / mL of NNV virus solution or 20μL of 1.0×10 7.6 TCID 50 / mL of TGIV virus liquid. Observe the test fish regularly and record the disease situation in time. Preserve the dead fish in time and test the virus. The formula for calculating the immune protection is: relative protection rate = (1-mortality rate of the immune group / mortality rate of the control group) × 100%.

[0053] 2 Results and analysis

[0054] Depend on Figure 3 It can be seen that the antibody titer of grouper after immunization will increase significantly with the growth of time. It will reach a peak level around 28 days, and the serum antibody level of the self-assembled nano-vaccine soaked group will be significantly higher than that of other groups (P < 0.05).

[0055] from Figure 4 It can be seen that after attacking with grouper neural necrosis virus and iris virus respectively, the protection rates of the self-assembled immersion nanovaccine group were as high as 78.46% and 86.08%, respectively, which were significantly higher than those of other groups, indicating that the grouper dual immersion nanovaccine provided by the present invention can effectively prevent diseases caused by grouper neural necrosis virus and iris virus.

[0056] Self-assembled immersion nanovaccines have broad application prospects in the prevention of grouper neuronecrosis virus disease and iridovirus.

[0057] The above specific implementations do not constitute a limitation on the protection scope of the present invention. It should be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions may occur depending on design requirements and other factors. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO.

4.

2. Use of the fusion protein according to claim 1 in the preparation of drugs for preventing grouper neuronecrosis virus and iris virus.

3. A dual-injection nano vaccine of grouper neuronecrosis virus and iridovirus, characterized in that: The fusion protein of claim 1 is synthesized according to the sequence of SEQ ID NO. 4, and the fusion protein self-assembles into a nano-spherical structure under neutral conditions.

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