阅读说明：本技术 一种鲈鱼弹状病毒亚单位疫苗及其制备方法 (Perch rhabdovirus subunit vaccine and preparation method thereof ) 是由 王高学 朱斌 凌飞 郭孜饶 焦铁军 于 2021-09-13 设计创作，主要内容包括：本发明选取MSRV糖蛋白的部分蛋白片段G2,通过原核重组表达获得G2蛋白,并对G2蛋白进行甘露糖化修饰,制备得到了鲈鱼弹状病毒纳米载靶向性疫苗,研究表明,该亚单位疫苗免疫鲈鱼21天后攻毒MSRV FJ985毒株,鲈鱼免疫保护率为94%-96%,该疫苗安全、有效,免疫鲈鱼后可产生较好的保护作用,并能使鲈鱼有效抵御MSRV强毒的攻击。(The invention selects a partial protein fragment G2 of MSRV glycoprotein, obtains G2 protein through prokaryotic recombinant expression, and carries out mannosylation modification on G2 protein to prepare the weever rhabdovirus nano-carrier targeting vaccine, and researches show that the subunit vaccine attacks the MSRV FJ985 strain 21 days after the weever is immunized, the weever immune protection rate is 94-96 percent, the vaccine is safe and effective, can generate better protection effect after the weever is immunized, and can enable the weever to effectively resist the attack of MSRV virulent virus.)

1. The bass rhabdovirus subunit vaccine is characterized in that the subunit vaccine is prepared from G2 protein and oxidized single-walled carbon nanotubes, wherein the amino acid sequence of the G2 protein is SEQ ID No: 2, in the preparation process, carrying out mannosylation modification on the G2 protein.

2. The rhabdovirus subunit vaccine of claim 1, wherein said mannosylation modification is by the following method: dissolving 1- (4-isocyanatephenol) -alpha-D-mannose into PBS (phosphate buffer solution) to 1mg/ml to obtain a functionalized mannose solution, diluting a G2 protein solution into the target protein of 360 mu G/ml by PBS, mixing the functionalized mannose solution and the G2 protein solution at the ratio of = 1: 30 (V/V), stirring at room temperature of 80r/min for 24 hours, dialyzing the obtained product, and freeze-drying to obtain the mannose-glycosylated G2 protein.

3. The rhabdovirus subunit vaccine of said weever according to claim 1 or 2, wherein said G2 protein concentration in said vaccine is 5 mg/ml.

4. The method of preparing the rhabdovirus subunit vaccine of weever of claim 1 or 2, comprising the steps of:

step one, preparing and purifying recombinant protein;

step two, preparing a carbon nano tube delivery system: carrying out mannosylation modification on the G2 protein, mixing the protein with the oxidized single-walled carbon nanotube for reaction, centrifuging, taking the precipitate for dissolution, dissolving the precipitate again to prepare a protein suspension, and adding formaldehyde for inactivation;

step three, emulsification: mixing the protein suspension and glycerol at a certain proportion to obtain a mixed solution, and stirring at 100r/min for 30 minutes to obtain a vaccine;

step four, subpackaging: quantitatively subpackaging, capping and labeling.

5. The method for preparing the rhabdovirus subunit vaccine of the weever according to claim 4, wherein the carbon nanotube delivery system in the second step is prepared by the following steps:

(1) dissolving the functionalized mannose into 1mg/ml by PBS, taking a G2 protein solution, diluting the G2 protein solution by PBS until the target protein is 360 mu G/ml, then mixing the functionalized mannose solution and the G2 protein solution according to the volume ratio of 1: 30, stirring the mixture at room temperature for 24 hours at 80r/min, dialyzing and freeze-drying the obtained product to obtain a mannose-glycosylated G2 protein crude product, and carrying out gel filtration and purification on the crude product for later use;

(2) adding 6g of functionalized carbon nano tube into 800mL of 0.1M 2- (N-morpholine) ethanesulfonic acid buffer solution with pH =5.6 by adopting a condensation acylation method, uniformly stirring the mixed solution by using a glass rod, and simultaneously carrying out ultrasonic treatment on the mixed solution for 20 min;

(3) respectively weighing 18g of ethyl dimethyl amine propyl carbodiimide and 24g N-carbonyl succinimide, adding the ethyl dimethyl amine propyl carbodiimide and the 24g N-carbonyl succinimide into the uniformly dispersed carbon nano tube mixed solution, stirring the mixture by using a glass rod during adding to fully mix the mixture, simultaneously carrying out ultrasonic treatment for 2 hours, pausing for 10 minutes every 0.5 hour of ultrasonic treatment during ultrasonic treatment, and timely replacing water in an ultrasonic cleaner to avoid over-serious heating of the water;

(4) centrifuging the dispersed solution at 12000g for 30min, discarding the supernatant, washing the precipitate with a PBS solution with pH =7.4, washing 3 times, and dissolving the precipitate in 2000mL of PBS solution with pH = 7.4;

(5) weighing 5G of mannose glycosylation G2 protein, adding into the solution obtained in the step (4), stirring while avoiding generating bubbles during stirring, wherein the stirring amplitude is not too large, and then carrying out ultrasonic dispersion for 1.5 h;

(6) after ultrasonic treatment, adding a rotor, and stirring the mixed solution in a rotary stirrer for 48 hours in a dark environment;

(7) transferring the reaction product into a 10 ten thousand dialysis bag for dialysis for 2d, centrifuging the product for 30min at 12000g after dialysis, removing supernatant, and freeze-drying the obtained solid precipitate to obtain the targeting carbon nanotube-loaded vaccine system, and storing in a refrigerator at-20 ℃;

(8) mixing 50mg of solid precipitate with 1ml of PBS to obtain protein suspension, and storing at-15 deg.C;

(9) protein inactivation: adding formaldehyde into the protein suspension to a final concentration of 0.07%, and inactivating the protein suspension for 72 hours at a temperature of 2-8 ℃.

6. The method of preparing the rhabdovirus subunit vaccine of claim 4, wherein said emulsifying is: preparing a mixed solution according to the volume ratio of the protein suspension to the glycerol of 2: 3, and stirring for 30 minutes at 100r/min to prepare the vaccine with the target protein concentration of 5 mg/ml.

7. The method of preparing the rhabdovirus subunit vaccine of said weever according to claim 4, wherein said first step is: amplifying to obtain a G2 target gene, and constructing recombinant Escherichia coli E, coli BL21/pET32a-G2 for expressing G protein; the recombinant Escherichia coli E, coli BL21/pET32a-G2 is fermented, expressed and purified to prepare the G2 protein.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a weever rhabdovirus subunit vaccine.

Background

Perch in large mouth (Micropterus salmoides) The method is commonly called as Micropterus salmoides, belongs to the genus Perpterus of the family Lateolaceae of Perciformes, introduces Perpterus salmoides in 1983 in Guangdong Foshan, succeeds in artificial propagation in 1985, and widely breeds the fingerlings, achieves good economic benefits and is well popular with vast breeders and consumers. However, weever is susceptible to various diseases, such as gill rot disease, ulcer disease, nocardiosis, trichodiniasis, and the like. Wherein the disease is caused by weever rhabdovirus (a)Micropterus salmoides rhabdovirus， MSRV) The resulting rhabdovirus disease of weever is the most serious. The method has wide epidemic regions, long morbidity time and morbidity mortality rate of more than 85 percent, and brings huge economic loss to weever culture.

The rhabdovirus disease of weever is reported in the main culture areas (Jiangsu, Jiangxi, Hunan, Hubei, Zhejiang, Guangdong and the like) in China. The disease starts at 4 months per year and lasts for 11 months, and the main epidemic season is 4-5 months per year. When the water temperature reaches 25-28 ℃, the dissolved oxygen and the transparency are reduced, and the water quality is deteriorated, the weever species is most susceptible to and erupts of weever rhabdovirus diseases, so that the weever is killed in a large scale. The main symptoms of diseased fish are rotten body, rotten fins and stopping eating, the dying fish roam on the water surface, and the body color of severe fish is blackened. Serious liver swelling and congestion of the dissected diseased fish can be seen; splenomegaly, hyperemia; swollen kidney, etc.

MSRV is single-stranded RNA virus, the size of virus particle is (100-430) nm x (45-100) nm, and the shape is rod-like or bullet-like. The MSRV genome encodes mainly 5 structural proteins, nucleoprotein N, phosphoprotein P, matrix M, glycoprotein G and RNA polymerase L. The G protein, namely glycoprotein, is mainly responsible for binding with host cell receptors and is the main antigen protein of the virus, and the G protein forms a trimeric envelope particle on the surface of the virus, is bound with the receptors on the cells and mediates the endocytosis of the virus. The G protein is used as the main antigen of the virus, induces the body to produce neutralizing antibodies and stimulates cellular immunity, and determines the serological characteristics of the virus.

Currently, there is still a lack of effective therapeutic approaches for the diseases associated with MSRV. The research shows that the Chinese herbal medicine as the medicine for preventing and treating the rhabdovirus disease of the weever has the advantages of small toxic and side effect, rich resources, medicinal property and the like, and can play a certain role in preventing and treating the fish disease. Vaccines have also been reported to prevent MSRV as the most effective means of preventing viral infection. Chen et al (The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus,Veterinary Microbiology,157 (2012) 264–275) The glycoprotein G plasmid of the rhabdovirus is constructed, and after the injection of the glycoprotein G plasmid into a fish body, the glycoprotein plasmid can trigger I-type IFN (interferon) antiviral immune response, remarkably reduce the damage degree of muscle tissues after virus infection and inhibit virus replication, so that the study on the G protein of MSRV is particularly important; sun-jian LYU et al (Zhejiang Univ-Sci B (Biomed & Biotechnol) 2019 20(9):728-739) A baculovirus expression system of MSRV virus glycoprotein (G protein) is constructed, high-abundance virus glycoprotein is obtained by utilizing the baculovirus expression system, and the virus glycoprotein can be used for the research and development of subsequent oral vaccines by combining silkworms, but the development of the vaccines and the verification of immune effects are not carried out; Zi-Rao Guo et al (Fish and Shellfish Immunology 99 (2020) 548-554) An impregnated single-walled carbon nanotube-loaded subunit vaccine compounded by MSRV glycoprotein (G) is reported, the protective effect of the vaccine on largemouth bass is evaluated, the single-walled carbon nanotube-glycoprotein is prepared by combining a prokaryotic expressed G protein complete sequence and a single-walled carbon nanotube, and the result shows that the single-walled carbon nanotube-glycoprotein (SWCNTs-G) is a promising candidate of the immersed subunit vaccine, can resist death caused by MSRV, and under the help of the SWCNTs, the immune protection rate of the SWCNTs-G group (40 mg/L) reaches 70.1 percent and is improved by 30.6 percent compared with the single G protein. Zhang Chen (northwest agriculture and forestry science and technology university) and Zhao (northwest agriculture and forestry science and technology university) respectively carry out structure modification on mannose and single-walled carbon nanotubes under the guidance of the inventor, and thenThrough a chemical connection technology, a targeted carbon nanotube vaccine-carried system (SWCNTs-MG) is constructed and is respectively used for preventing and treating spring viremia of carp and infectious spleen and kidney necrosis virus disease of mandarin fish, and good prevention and treatment effects are obtained.

At present, an effective treatment mode aiming at relevant diseases caused by the MSRV is still lack, and the vaccine serving as the most effective mode for preventing virus infection has a certain report on the prevention of the MSRV, however, no MSRV subunit vaccine is on the market at present. Therefore, safe and efficient subunit vaccines aiming at the MSRV are developed as soon as possible, so that the predicament of the weever aquaculture industry and the market can be solved, and the healthy development of the aquaculture industry is promoted. At present, the fishing vaccine still has some theoretical and technical bottlenecks in the aspect of large-scale industrial application, particularly in the aspect of immune approach and immune effect. The injection immune protection effect is the best, but the large-scale industrialization requirements are difficult to meet due to the operation technology, the production cost and the like; although the soaking and oral immunization are simple in operation technology and low in production cost, a series of difficult-to-overcome technical problems of body surface skin and intestinal barrier restriction, enzymolysis inactivation, low immune protection rate and the like exist at the same time. Therefore, how to further improve the immune effect of the vaccine is a problem to be solved urgently.

Disclosure of Invention

In order to solve the dilemma of weever breeding industry and market and promote the healthy development of aquaculture industry, the invention aims to provide a weever rhabdovirus subunit vaccine and a preparation method thereof.

In order to solve the technical problems, the invention adopts the following technical means:

the bass rhabdovirus subunit vaccine is characterized in that the subunit vaccine is prepared from G2 protein and oxidized single-walled carbon nanotubes, wherein the amino acid sequence of the G2 protein is SEQ ID No: 2, in the preparation process, carrying out mannosylation modification on the G2 protein.

Preferably, the mannosylation modification is performed by the following method: dissolving 1- (4-isocyanatephenol) -alpha-D-mannose into 1mg/ml by PBS to obtain a functionalized mannose solution, diluting the G2 protein solution by PBS until the target protein is 360 mu G/ml, then mixing the functionalized mannose solution and the G2 protein solution = 1: 30 (V/V) in a ratio, stirring the mixture at room temperature of 80r/min for 24 hours, dialyzing and freeze-drying the obtained product to obtain a mannose glycosylated G2 protein crude product, and purifying the crude product by gel filtration for later use.

Preferably, the subunit vaccine is prepared by the following method, which comprises the following steps:

step one, preparing and purifying recombinant protein;

step two, preparing a carbon nano tube delivery system: carrying out mannosylation modification on the G2 protein, mixing the protein with the oxidized single-walled carbon nanotube for reaction, centrifuging, taking the precipitate for dissolution, dissolving the precipitate again to prepare a protein suspension, and adding formaldehyde for inactivation;

step three, emulsification: mixing the protein suspension and glycerol at a certain proportion to obtain a mixed solution, and stirring at 100r/min for 30 minutes to obtain a vaccine;

step four, subpackaging: quantitatively subpackaging, capping and labeling.

Preferably, the concentration of the protein of interest in the vaccine is 5 mg/ml.

Preferably, the carbon nanotube delivery system in the second step is prepared by the following steps:

(1) dissolving the functionalized mannose into 1mg/ml by PBS, taking a G2 protein solution, diluting the G2 protein solution by PBS until the target protein is 360 mu G/ml, then mixing the protein solution and the G2 protein solution in a ratio of = 1: 30 (V/V), stirring the mixture at room temperature for 24 hours at 80r/min, dialyzing and freeze-drying the obtained product to obtain a mannose-glycosylated G2 protein crude product, and carrying out gel filtration and purification on the crude product for later use;

(2) adding 6g of functionalized carbon nano tube into 800mL of 2- (N-morpholine) ethanesulfonic acid buffer solution (0.1M, pH = 5.6) by adopting a condensation acylation method, uniformly stirring the mixed solution by using a glass rod, and simultaneously carrying out ultrasonic treatment on the mixed solution for 20 min;

(3) respectively weighing 18g of ethyl dimethyl amine propyl carbodiimide and 24g N-carbonyl succinimide, adding the ethyl dimethyl amine propyl carbodiimide and the 24g N-carbonyl succinimide into the uniformly dispersed carbon nano tube mixed solution, stirring the mixture by using a glass rod during adding to fully mix the mixture, simultaneously carrying out ultrasonic treatment for 2 hours, pausing for 10 minutes every 0.5 hour of ultrasonic treatment during ultrasonic treatment, and timely replacing water in an ultrasonic cleaner to avoid over-serious heating of the water;

(4) the dispersed solution was centrifuged at 12000g for 30min, and the supernatant was discarded. Washing the precipitate with a PBS solution of pH =7.4, after 3 washes, dissolving the precipitate in 2000mL of a PBS solution of pH = 7.4;

(5) weighing 5G of mannose glycosylation G2 protein, adding into the solution obtained in the previous step, stirring while avoiding generating bubbles during stirring, wherein the stirring amplitude is not too large, and then performing ultrasonic dispersion for 1.5 h;

(6) after ultrasonic treatment, adding a rotor, and stirring the mixed solution in a rotary stirrer for 48 hours in a dark environment;

(7) the reaction product is transferred into a dialysis bag (10 ten thousand) for dialysis for 2 d. After dialysis, the product was centrifuged at 12000g for 30min and the supernatant discarded. Freezing and drying the obtained solid precipitate to obtain a targeting carbon nanotube vaccine-loaded system (SWCNTs-MG), and storing in a refrigerator at-20 deg.C;

(8) mixing 50mg of solid precipitate with 1ml of PBS to obtain protein suspension, and storing at-15 deg.C;

(9) protein inactivation: adding formaldehyde into the protein suspension to a final concentration of 0.07%, and inactivating the protein suspension for 72 hours at a temperature of 2-8 ℃.

Preferably, the emulsifying is: preparing a mixed solution according to the ratio of the protein suspension to the glycerol = 2: 3 (V/V), and stirring for 30 minutes at 100r/min to prepare the vaccine with the target protein concentration of 5 mg/ml.

The invention also claims a method for preparing the weever rhabdovirus subunit vaccine, which is characterized by comprising the following steps:

step one, preparing and purifying recombinant protein;

step two, preparing a carbon nano tube delivery system: carrying out mannosylation modification on the G2 protein, mixing the protein with the oxidized single-walled carbon nanotube for reaction, centrifuging, taking the precipitate for dissolution, dissolving the precipitate again to prepare a protein suspension, and adding formaldehyde for inactivation;

step three, emulsification: mixing the protein suspension and glycerol at a certain proportion to obtain a mixed solution, and stirring at 100r/min for 30 minutes to obtain a vaccine;

step four, subpackaging: quantitatively subpackaging, capping and labeling.

Preferably, the first step is: amplifying to obtain a G2 target gene, and constructing recombinant Escherichia coli E, coli BL21/pET32a-G2 for expressing G protein; the recombinant Escherichia coli E, coli BL21/pET32a-G2 is fermented, expressed and purified to prepare the G2 protein.

Preferably, the emulsifying is: preparing a mixed solution according to the ratio of the protein suspension to the glycerol = 2: 3 (V/V), and stirring for 30 minutes at 100r/min to prepare the vaccine with the target protein concentration of 5 mg/ml.

Based on the technical scheme, the invention has the following advantages and beneficial effects:

firstly, on the basis of a large amount of analysis and experiments, a G2 protein fragment with good immune raw materials in glycoprotein is obtained by screening, the fragment has better immunogenicity and immune protection performance compared with the whole G protein and other fragments, 21 days after the weever is immunized by a subunit vaccine prepared based on the G2 protein, the MSRV FJ985 strain is attacked, the immune protection rate of the weever is 94%, and is higher than the protection rate reported in the prior art.

Secondly, on the basis of the constructed carbon nano tube-carried vaccine system, the characteristic of a mannose receptor is combined, the targeted carbon nano tube-carried vaccine system is constructed, the system can realize the aim of large-scale, simple and quick seedling targeted immunization through soaking immunization, the effect of the soaking immunization is greatly improved, the industrialization of commercial fishing vaccines represented by weever rhabdovirus nano-carried targeted vaccines is realized, a theoretical basis is laid for the research and application of other aquatic animal nano-carried targeted genetic engineering vaccines, a new research thought is provided, and the system has important significance for the sustainable development of fishery and the safe production of aquatic food.

On the basis of a large amount of analysis and tests, a partial protein fragment G2 of MSRV glycoprotein is selected, a G2 protein is obtained through prokaryotic recombinant expression, mannose modification is adopted, and the weever rhabdovirus nano-carrier targeting vaccine is prepared.

Drawings

FIG. 1 is an electrophoretogram of PCR amplified CO cell cultures. In FIG. 1, M represents a DNA Marker 2000; FIG. 1 shows 1 CO cell cultures; 2 in FIG. 1 represents MSRV positive plasmid samples; in FIG. 1, 3 represents a negative control.

FIG. 2 is a diagram showing the identification result of recombinant plasmid pET32 a-G2. In FIG. 2, a is the PCR amplification product of the G2 gene: m represents DL2000 DNA Marker, 1 represents G2 gene, and 2 represents negative control. In FIG. 2, b is the double digestion analysis of recombinant expression plasmid: m represents DL5000 DNA Marker; 1 represents pET32a-G2 recombinant plasmid; 2 represents double digestion of pET32a-G2 recombinant plasmid.

FIG. 3 is an SDS-PAGE detection of recombinant protein G2. In FIG. 3, M represents a protein Marker; FIG. 3, 1, shows the uninduced plasmid containing pET32a-G2E. coliBL21 mycoprotein; FIG. 3, 2, shows IPTG-induced plasmid containing pET32a-G2E. coliMycoprotein in BL21 supernatant; FIG. 3 shows IPTG-inducible plasmid containing pET32a-G2E. coliAnd (3) bacterial protein in the BL21 precipitate.

FIG. 4 is a Western blot assay of recombinant protein G2. In fig. 4, M denotes Marker; in FIG. 4, 1 represents a negative control; the purified G2 protein is shown at 2 in FIG. 4.

FIG. 5 is a graph showing the results of antibody level detection after different protein fragments are immunized.

FIG. 6 is a liver and spleen slice. In FIG. 6, A represents a normal liver; in fig. 6B represents a diseased liver; c in FIG. 6 represents normal spleen; in FIG. 6, D represents a diseased spleen.

FIG. 7 shows the survival (protection) after challenge for different protein fragment subunit vaccines.

Detailed Description

Example 1: recombinant Escherichia coliE. coliConstruction and identification of BL21/pET32a-G2 Strain

Amplification of G2 target Gene

By referring to the specification of a virus RNA extraction kit, MSRV FJ985 strain virus liquid cultured by CO cells (the MSRV FJ985 strain is provided by northwest agriculture and forestry science and technology university, particularly Fei Yang and the like,Evaluation on the antiviral activity of ribavirin against Micropterus salmoides rhabdovirus (MSRV) in vitro and in vivo，Aquaculture，Volume 543network disclosure time: 2021, 5 months and 27 days) as material, and extracting MSRV virus RNA. Agarose gel electrophoresis was used to check the integrity of RNA, and a micro nucleic acid analyzer was used to determine the concentration and quality of sample RNA. And inverting the extracted RNA sample into cDNA using a reverse transcription kit. Amplifying the G2 gene by using a MSRV-G-F/R and VP7-F/R primer pair, wherein the ratio of MSRV-G-F: 5'-GAACTGTGGATGGAATAACG-3' (SEQ ID No: 6); MSRV-G-R: 5'-GTCTGACGGCGTACCCAA-3' (SEQ ID No: 7).

And (3) carrying out agarose gel electrophoresis identification on the PCR product, wherein the electrophoresis conditions are as follows: voltage of 120V for 30 minutes. Cutting gel of electrophoresis product, recovering, purifying DNA product by gel recovery kit, connecting the purified product with pMD19-T vector, and naming the recombinant product as pMD 19T-G2. And transforming the transformant into a competence of Escherichia coli DH5 alpha, screening by using a blue-white spot to obtain a positive strain, and extracting a plasmid to perform PCR amplification detection and sequencing identification. The nucleotide sequence of the G2 protein is shown in SEQ ID NO. 1. The results of the PCR amplification CO cell culture electropherogram are shown in FIG. 1.

Recombinant Escherichia coli expressing G protein (E.coli)E. coli BL21/pET32 a-G2) construction

Construction of prokaryotic expression vector pET32 a-G2: the recombinant pMD19T-G2 plasmid is subjected to double enzyme digestion and cloned between corresponding enzyme digestion sites on a pET-32a expression vector to obtain a recombinant plasmid pET32 a-G2.

Transformation and screening of the recombinant plasmid pET32 a-G2: the recombinant plasmid pET32a-G2 was transformed intoE. coliBL21 (DE 3) was applied to LB solid medium containing ampicillin and cultured overnight at 37 ℃ in an inverted state. Then, single colonies were picked for colony PCR detection identification against G2. After the PCR product is electrophoresed through 1% agarose gel, observing the size of the target band of the amplified product under a gel imager, as shown in a in FIG. 2The colonies corresponding to the expected G2414 bp are shown to be recombinantE. coli BL21/ pET 32a-G2。

Identification of the recombinant plasmid pET32 a-G2: inoculating the colony with positive PCR to LB culture solution containing 100 mug/ml ampicillin, culturing at 37 deg.C for 12-16 h at 180r/min, extracting plasmid according to the Omega plasmid extraction kit, measuring the plasmid concentration, taking 8 mug plasmid solution, 1 mug L10 Xenzyme digestion Buffer and 0.5 mug restriction enzyme, mixing, and cutting at 37 deg.C for 5 min. And detecting the enzyme digestion product by 1% agarose gel electrophoresis. The result is shown in fig. 2 b.

Inducible expression of recombinant bacterium E, coli BL21/pET32a-G2

Selecting a small amount of bacterial liquid from a strain for production by using an inoculating loop, streaking and inoculating the bacterial liquid to an LB solid culture medium plate, standing and culturing at 37 ℃ for 12-16 hours, selecting a single colony and inoculating the single colony to an LB liquid culture medium, and culturing at 37 ℃ for 12-16 hours at 180r/min to serve as a primary seed; inoculating the primary seeds into LB liquid culture medium at 1% (V/V), and culturing at 37 deg.C and 180r/min for 12-16 hr to obtain secondary seeds. Secondary seeds were inoculated at 1% (V/V) into modified LB medium, while ampicillin was added to a final concentration of 100. mu.g/ml. Ventilating, fermenting and culturing for 5-7 hours at 37 ℃, and dissolving 30% -40% of oxygen until the OD of the bacterial liquid₆₀₀When the value is 1.1-1.3, IPTG is added to the final concentration of 0.001mol/L, the induction culture is carried out for 6 hours at 37 ℃, and the fermentation is stopped.

Bacterial liquid treatment and ultrasonic crushing

Centrifuging the fermentation product at room temperature of 12000r/min by a tube centrifuge, collecting thalli, washing the thalli for 2 times by PBS (0.015 mol/l, pH7.2), resuspending the collected thalli according to the ratio of wet thalli to PBS solution (0.015 mol/l, pH7.2) plastid to 1: 9, crushing bacteria by a 2-8 ℃ high-pressure homogenizer, smearing the crushed bacteria liquid after the bacteria continuously pass through the homogenizer twice, dyeing for 0.5 min by using 0.1% crystal violet solution, taking 3-5 visual fields under a microscope for observation, completely crushing the bacteria in the visual fields to obtain cell fragments and not seeing complete thalli until the crushing rate of the bacteria is not changed any more. Centrifuging at 12000r/min with tubular centrifuge, and collecting protein precipitate.

Purification of proteins

Dissolving the protein precipitate with 10ml dissolving solution (5 mmol/L imidazole, 0.5mmol/L sodium chloride, 8M urea, 20mmol/L Tirs-HCl, pH 7.9) at room temperature at 200r/min for 2 hr, centrifuging at 4 deg.C at 10000r/min for 30min, and collecting supernatant. The metal (Ni-Ni) was fully equilibrated with equilibration buffer (5 mmol/L imidazole, 0.5mmol/L sodium chloride, 8M urea, 20mmol/L Tirs-HCl, pH 7.9)^2＋) Chelating affinity chromatographic column, loading 2 times of column volume, balancing with balance buffer solution, eluting with elution buffer solution (0.5 mol/L imidazole, 0.5mmol/L sodium chloride, 8M urea, 20mmol/L Tirs-HCl, pH7.9), and collecting protein. The purified 200ml protein was placed in a Snakeskin-dialysis bag (10K MWCO) and directly and completely immersed in 10L renaturation solution (150 mM NaCl, 2.5 mM KCl, 10mM Na)₂HPO₄、2 mM KH₂PO₄1% Tween-20, 10mM beta-cyclodextrin, 1 ML-cysteine, 3 mM reduced form and 1mM oxidized form glutathione, pH 7.9), dialyzing for 12 h at 4 ℃, changing the solution once every 6h in the dialysis period, and collecting the dialyzed protein solution for storage at 4 ℃ after the dialysis is finished.

Endotoxin removal

Triton X-114 was added to the dialyzed protein solution to a final concentration of 1.5%, and the mixture was stirred at 4 ℃ for 1 hour. After treatment, the temperature of the sample is restored to 30 ℃ and maintained for 40 minutes, a high-speed centrifuge is used for 17000r/min, precipitates are removed by centrifugation, and supernatant is collected. The supernatant was treated in triplicate as described above.

Detection of recombinant proteins

SDS-PAGE: adding equal volume of 2 Xgel loading buffer solution into the supernatant after bacteria breaking and the solution after heavy suspension precipitation, and directly adding equal volume of 2 Xgel loading buffer solution without ultrasonic wave breaking. Boiling for 10min, and detecting by SDS-PAGE electrophoresis, the acrylamide concentration of the separation gel is 15%. Dyeing with Coomassie brilliant blue, and decolorizing with decolorizing solution until bands are clear. A control E.coli BL21/pET32a-G2 culture induced without IPTG was also set up. The results are shown in FIG. 3.

And (3) identification: the recombinant protein was subjected to SDS-PAGE in the same manner as above. After electrophoresis, a piece of film is taken and placed on a film rotating instrument, and the film is rotated for 1 hour under the condition of 200 mA. Putting the PVDF membrane carrying the protein into a plastic washing box, adding 25ml of 5% skim milk, sealing at room temperature for 2 hours, pouring off the skim milk, washing with TBST for 3 times, adding 30ml of primary anti-mouse histidine monoclonal antibody (diluted by antibody diluent in a ratio of 1: 1000), slowly oscillating at room temperature for 2 hours, pouring off the primary antibody, washing with TBST for 5 times, adding 30ml of horseradish peroxidase-labeled goat anti-mouse secondary antibody (diluted by antibody diluent in a ratio of 1: 2000), slowly oscillating at room temperature for 2 hours, pouring off the secondary antibody, washing with TBST for 5 times, putting the membrane into a flat dish, and putting the flat dish in a dark room. Adding 1ml of deionized water, adding DAB color development solution (1 drop of each of solution A and solution B), repeatedly washing the surface of the membrane for 1 minute by using a pipettor until the color development is complete, washing with the deionized water, airing and imaging, wherein a specific band exists at about 30KDa after the color development, which indicates that the G2 protein with correct expression exists in the sample. The results are shown in FIG. 4.

Example 2: evaluation of the immunopotency of recombinant proteins

In the development process, different fragments are designed, different glycoprotein fragments and complete glycoprotein are respectively prepared by adopting the method of example 1, and the immune effectiveness of the glycoprotein fragments is respectively verified by adopting glycoprotein fragment G1 (the amino acid sequence is SEQ ID No: 3), glycoprotein fragment G3 (the amino acid sequence is SEQ ID No: 4) protein and complete glycoprotein G (the amino acid sequence is SEQ ID No: 5). Specifically, the following immunopotency evaluation test was used: taking 300 healthy weever tails, randomly dividing into 6 groups including a control group (PBS), a G1 protein group (40 mg/L), a G2 protein group (40 mg/L), a G3 protein group (40 mg/L), a G protein group (40 mg/L) and a G protein group (80 mg/L), and inoculating corresponding vaccines to all weever through bath solution and soaking for 6 hours. Subsequently, the vaccinated weever was transferred to a different water tank and monitored daily. In each group, three fish were selected once a week for sampling and plasma preparation, which was stored at-20 ℃ until the fourth week, and incubated at 37 ℃ for about 1.5 hours with serum diluted in PBS containing 3% skim milk as the primary antibody. Then, purified G protein (containing his-tag) was used as an antigen, followed by 1:1Determination of OD Using TMB as colorimetric substrate, anti-His tag antibody and goat-mouse IgG antibody diluted at 500₄₅₀Mean antibody levels in serum were analyzed. The specific results are shown in FIG. 5. Based on the results shown in fig. 5, the highest antibody level was achieved at day 21 after immunization by both the glycoprotein and its fragments, wherein the recombinant protein of G2 fragment selected in the present invention has better immunogenicity than the G1 protein fragment, the G3 protein fragment and the intact G protein, and thus the recombinant protein of G2 of the present invention is a relatively optimal antigenic protein of subunit vaccine.

Example 3: preparation of intermediate for vaccine production

Preparation of seed bacteria liquid

Recombinant Escherichia coliE. coli Diluting a basic strain BL21/pET32a-G2 strain with physiological saline, selecting a small amount of bacterial liquid by using an inoculating loop, inoculating the bacterial liquid into an LB liquid culture medium, culturing at 37 ℃ for 12-16 hours at 180r/min, harvesting the bacterial liquid, and storing at the temperature below 20 ℃.

Preparation of the semifinished products

Preparing first-class seeds: selecting a small amount of bacterial liquid from a strain for production by using an inoculating loop, streaking and inoculating the bacterial liquid to an LB solid culture medium plate, standing and culturing at 37 ℃ for 12-16 hours, selecting a single colony and inoculating the single colony in an LB liquid culture medium, and culturing at 37 ℃ for 12-16 hours at 180 r/min.

Preparing secondary seeds: inoculating the first-class seeds into LB liquid culture medium at 1% (V/V), and culturing at 37 ℃ for 12-16 hours at 180 r/min.

And (3) fermentation and expression: secondary seeds were inoculated at 1% (V/V) into modified LB medium, while ampicillin was added to a final concentration of 100. mu.g/ml. Ventilating, fermenting and culturing for 5-7 hours at 37 ℃, and dissolving 30% -40% of oxygen until the OD of the bacterial liquid₆₀₀When the value is 1.1-1.3, IPTG is added to the final concentration of 0.001mol/L, the induction culture is carried out for 6 hours at 37 ℃, and the fermentation is stopped.

Bacterial disruption: centrifuging the fermentation product at room temperature of 12000r/min by a tube centrifuge, collecting thalli, washing the thalli for 2 times by PBS (0.015 mol/L, pH 7.2), carrying out resuspension on the collected thalli according to the ratio of wet bacteria to PBS solution (0.015 mol/L, pH 7.2) to the plastid ratio of 1: 9, crushing the bacteria by a 2-8 ℃ high-pressure homogenizer, smearing the crushed bacteria liquid after the bacteria continuously pass through the homogenizer twice, dyeing for 0.5 min by using 0.1% crystal violet solution, taking 3-5 visual fields under a microscope for observation, completely crushing the bacteria in the visual fields to obtain cell fragments and not seeing complete thalli until the crushing rate of the bacteria is not changed any more. Centrifuging at 12000r/min with tubular centrifuge, and collecting protein precipitate.

And (3) protein purification: dissolving the protein precipitate with 10ml dissolving solution (5 mmol/L imidazole, 0.5mmol/L sodium chloride, 8M urea, 20mmol/L Tirs-HCl, pH 7.9) at room temperature at 200r/min for 2 hr, centrifuging at 4 deg.C at 10000r/min for 30min, and collecting supernatant. The metal (Ni-Ni) was fully equilibrated with equilibration buffer (5 mmol/L imidazole, 0.5mmol/L sodium chloride, 8M urea, 20mmol/L Tirs-HCl, pH 7.9)^2＋) Chelating affinity chromatographic column, loading 2 times of column volume, balancing with balance buffer solution, eluting with elution buffer solution (0.5 mol/L imidazole, 0.5mmol/L sodium chloride, 8M urea, 20mmol/L Tirs-HCl, pH7.9), and collecting protein. The purified 200ml protein was placed in a Snakeskin-dialysis bag (10K MWCO) and directly and completely immersed in 10L renaturation solution (150 mM NaCl, 2.5 mM KCl, 10mM Na)₂HPO₄、2 mM KH₂PO₄1% Tween-20, 10mM beta-cyclodextrin, 1 ML-cysteine, 3 mM reduced form and 1mM oxidized form glutathione, pH 7.9), dialyzing for 12 h at 4 ℃, changing the solution once every 6h in the dialysis period, and collecting the dialyzed protein solution for storage at 4 ℃ after the dialysis is finished.

And (3) removing endotoxin: triton X-114 was added to the dialyzed protein solution to a final concentration of 1.5%, and the mixture was stirred at 4 ℃ for 1 hour. After treatment, the temperature of the sample is restored to 30 ℃ and maintained for 40 minutes, a high-speed centrifuge is used for 17000r/min, precipitates are removed by centrifugation, and supernatant is collected. The supernatant was treated in triplicate as described above.

Inspection of intermediate products

Protein concentration and purity determination: and (4) taking the protein from which the endotoxin is removed, and measuring the concentration and the purity of the target protein by using a densitometry method. The protein concentration per ml should be more than or equal to 600 mug, and the protein purity should be more than or equal to 60%.

And (3) identification: and (4) taking the protein with endotoxin removed, and carrying out Western-blot detection. The destination band should be visible around 30 KDa.

And (3) detecting endotoxin: the content of endotoxin in the intermediate product per ml is less than or equal to 5000EU according to the appendix of the first part of the existing Chinese veterinary pharmacopoeia.

Example 4: synthesis of targeting carbon nanotube vaccine-loaded system

According to the method of Zhang morning (Zhang morning, research on carp spring viremia by targeting carbon nanotube vaccine-loaded system [ D ]. university of agriculture and forestry, 2019.) to chemically modify mannose (appendix 1), modify a single-walled carbon nanotube structure (page 11, 2.2.3 single-walled carbon nanotube structure modification) and prepare the targeting carbon nanotube vaccine-loaded system.

Chemical modification of functionalized mannose:

(1) adding 240mL of acetic anhydride and 200mL of pyridine into a 1000mL round-bottom flask, cooling the flask to-10 ℃ in an ice salt bath, slowly adding 25g of D-mannose while stirring, and stirring for 5 hours until white powder disappears gradually to obtain a colorless clear solution. Shaking the solution, slowly pouring into 4L water containing crushed ice, stirring until no solid is precipitated, vacuum filtering, washing the obtained white solid with water, and drying to obtain white powder (pentoxyacetyl-beta-D-mannose);

(2) 10g of p-nitrophenol, 10g of pentoxyacetyl-D-mannopyranose and 50mL of toluene solution are added into a 500mL three-necked flask, 10mL of boron trifluoride ether solution is slowly dropped into the flask under the protection of nitrogen, and the mixture is stirred and reacted for 2 hours at normal temperature. After the reaction is finished, extracting the reaction solution by using saturated sodium carbonate solution and anhydrous Na₂SO₄The organic phase toluene was dried overnight, filtered under suction, and the toluene removed by rotary evaporation to give a yellow liquid. The column chromatography purification conditions were hexane: ethyl acetate = 3: and 2, recrystallizing by using hot ethanol to obtain the p-nitrophenol-tetraoxyacetyl-mannopyranose.

(3) Adding 5.4g of p-nitrophenol-tetraoxyacetyl-D-mannopyranose into 60mL of anhydrous methanol, stirring to obtain a suspension, adding 30mM of sodium methoxide, stirring at room temperature for 5h, removing part of methanol by rotary evaporation, standing at-20 ℃ overnight, precipitating a large amount of crystals, and filtering to obtain white powder, i.e. p-nitrophenol-D-mannopyranose.

(4) Adding 0.8g of p-nitrophenol-D-mannopyranose into a 250mL round-bottom flask containing 50mL of methanol, stirring for 15min, adding 100mg of palladium carbon, introducing H2, stirring at room temperature for 6H, filtering, and concentrating under reduced pressure until white crystals precipitate. Separating and purifying the solid by a silica gel column to obtain the p-aminophenol-alpha-D-mannopyranose.

(5) To a 100mL round bottom flask, 30mL of a mixture of absolute ethanol and water (9: 1) and 1mM (271 mg) of p-aminophenol-D-mannopyranose were added, and CSCl was slowly added dropwise under ice-bath conditions₂ 0.4mL, KOUTONG N₂Stirring and reacting for 3h, adjusting the pH value of the solution to 6.0 by using 0.1M NaOH under the ice bath condition, distilling the solution under reduced pressure to obtain a yellow solid, separating and purifying the yellow solid on a column (chloroform: methanol = 5: 1), and distilling the solvent under reduced pressure to obtain a brown yellow oily liquid, namely the required product 1- (4-isocyanate phenol) -alpha-D-mannose.

Single-walled carbon nanotube structure modification

The functionalized modification of the single-walled carbon nanotube adopts a mixed acid treatment method:

(1) and cleaning the required beaker, glass rod and measuring cylinder, and drying in an oven at 55 ℃.

(2) In a fume hood, measuring concentrated sulfuric acid and concentrated nitric acid with corresponding volumes, wherein the volume ratio is 3: adding commercial single-walled carbon nanotubes into mixed acid, stirring while adding, wherein a glass rod cannot touch the bottom and the side wall of a beaker during stirring, adding a rotor, and stirring for 48 hours in a magnetic stirrer.

(3) Diluting the solution obtained in the last step with pure water, performing ultrasonic treatment until the solution is neutral, performing suction filtration with a 0.22 filter membrane, and finally washing the precipitate obtained after suction filtration with absolute ethyl alcohol.

(4) And (3) placing the washed oxidized single-walled carbon nanotube precipitate in an oven to dry, grinding the precipitate into powder in a mortar, sieving the powder with a 300-mesh sieve to obtain the oxidized single-walled carbon nanotubes (o-SWCNTs), and subpackaging and sealing for later use.

Carbon nanotube delivery system preparation

(1) Dissolving the functionalized mannose prepared in the previous step to 1mg/ml by PBS, taking G2 protein solution to be diluted by PBS until the target protein is 360 mu G/ml, then mixing the functionalized mannose solution and the G2 protein solution = 1: 30 (V/V), stirring the mixture at room temperature of 80r/min for 24 hours, dialyzing and freeze-drying the obtained product to obtain a crude product of the mannose-glycosylated G2 protein, and purifying the crude product by gel filtration for subsequent reaction.

(2) By the condensation acylation method, 6g of functionalized carbon nanotubes were added to 800mL of 2- (N-morpholino) ethanesulfonic acid buffer (0.1M, pH = 5.6), and the mixture was stirred with a glass rod and subjected to ultrasonic treatment for 20 min.

(3) Respectively weighing 18g of ethyl dimethyl amine propyl carbodiimide and 24g N-carbonyl succinimide, adding the ethyl dimethyl amine propyl carbodiimide and the 24g N-carbonyl succinimide into the uniformly dispersed carbon nano tube mixed solution, stirring the mixture by using a glass rod when adding the mixture to fully mix the mixture, carrying out ultrasonic treatment for 2 hours simultaneously, pausing for 10 minutes every 0.5 hour of ultrasonic treatment, and timely replacing water in an ultrasonic cleaner to avoid over-serious water heating.

(4) The dispersed solution was centrifuged at 12000g for 30min, and the supernatant was discarded. The precipitate was washed with a PBS solution of pH =7.4, and after 3 times of washing, the precipitate was dissolved in 2000mL of a PBS solution of pH = 7.4.

(5) Weighing 5G of mannose glycosylation G2 protein, adding into the solution obtained in the previous step, stirring while avoiding bubble generation, wherein the stirring amplitude is not too large, and then performing ultrasonic dispersion for 1.5 h.

(6) After the ultrasonic treatment, a rotor was added, and the mixture was stirred in a rotary stirrer for 48 hours in a dark environment.

(7) The reaction product is transferred into a dialysis bag (10 ten thousand) for dialysis for 2 d. After dialysis, the product was centrifuged at 12000g for 30min and the supernatant discarded. And freeze-drying the obtained solid precipitate to obtain the targeting carbon nanotube vaccine-loaded system (SWCNTs-MG), and storing in a refrigerator at-20 ℃.

Preparing a semi-finished product:

mixing 50mg dried solid precipitate with 1ml PBS, making into protein suspension, and storing at below-15 deg.C.

Protein inactivation: adding formaldehyde into the protein suspension to a final concentration of 0.07%, and inactivating the protein suspension for 72 hours at a temperature of 2-8 ℃.

Inspection of semi-finished product

The sterility test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the growth is carried out aseptically.

The endotoxin detection is carried out according to the appendix of the first part of the existing Chinese veterinary pharmacopoeia, and the endotoxin content of each milliliter of semi-finished products is less than or equal to 5000 EU.

Vaccine preparation

Emulsification: preparing a mixed solution according to the ratio of the protein suspension to the glycerol = 2: 3 (V/V), and stirring for 30 minutes at 100r/min to prepare the vaccine with the target protein concentration of 5 mg/ml.

Subpackaging: quantitatively subpackaging, capping and labeling.

EXAMPLE 5 inspection of finished product

The characteristics are as follows: black suspension. After a long time, a small amount of precipitate is at the bottom, and a uniform suspension is formed after shaking.

And (4) checking the loading quantity: the inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the regulations are required to be met.

And (4) sterile inspection: the bacteria-free growth is carried out according to the examination of the appendix of the current Chinese veterinary pharmacopoeia.

And (3) detecting endotoxin: the content of endotoxin in each ml of vaccine is less than or equal to 5000EU according to the inspection of the appendix of the first part of the existing Chinese veterinary pharmacopoeia.

And (4) safety inspection: taking 150 tails of 0.5-1 g (more than 60 days old) healthy weever, and dividing the weever into an immune group 1, an immune group 2 and a control group, wherein each group has 50 tails. The immunization group has an immunization dose of 2ml/L, 20180121 and 20180125 batches of vaccines are respectively adopted for immunization, a control group uses normal saline to replace the vaccines, the vaccines are transferred into normal culture water after being soaked for 6 hours of immunization, and the test weever is continuously observed for 14 days and needs to be completely healthy and alive. If non-specific death occurs, the number of deaths in the immune group should not exceed 5 and the sum of the number of deaths in the control group should not exceed 10. The results are shown in Table 1.

TABLE 1 clinical observations of two batches of Vaccination trial Perch

Batch number	Food intake	Move about	Mental state	Clinical response	Observation of each part
						20180121	50/50 Normal	50/50 Normal	50/50 Normal	50/50 has no adverse side effects	50/50 has no abnormal reaction
20180125	50/50 Normal	50/50 Normal	50/50 Normal	50/50 has no adverse side effects	50/50 has no abnormal reaction
						Control group	49/50 Normal	49/50 Normal	49/50 Normal	49/50 has no adverse side effects	49/50 has no abnormal reaction

The measurement of the residual amount of formaldehyde is carried out according to the appendix of the current Chinese veterinary pharmacopoeia and is in accordance with the regulations.

Example 6: vaccine immunopotency assessment

Efficacy test-immune challenge protection: taking 100 tails of 0.5-1 g (more than 60 days old) healthy weever, and dividing into 2 groups (50 tails/group), wherein 1 group is an immune group, and 1 group is a control group. The immunity dose of the test group is 1ml/L (recombinant protein content is 5 mg/L), the control group uses normal saline to replace vaccine, the control group is transferred into normal aquaculture water after soaking and immunizing for 6 hours, and after continuously observing for 21 days, the enterotoxin challenge is respectively carried out by intraperitoneal injection by using weever rhabdovirus FJ985 strain, 15 mul (virus content is 100 TCID)₅₀/ml)/tail. Continuously observing for 14 days after the toxin is attacked, and regularly checking and recording the disease occurrence condition; and c, dissecting and inspecting dead weever at any time till the observation period is finished, dissecting and killing all weever, and observing pathological changes of organs.

After the challenge, the continuous observation is carried out for 14 days after the challenge, the death condition of the disease is regularly checked and recorded every day, the result shows that the virus-challenge MSRVFJ985 strain is carried out after the protein is soaked and immunized into the weever for 21 days, the immune protection rate of the weever is 94 percent, and the observation record is shown in table 2.

TABLE 2 protective results for the immunotoxin-challenge MSRVFJ985 strain

And c, dissecting and killing dead fishes at any time and marking the dead fishes, dissecting and killing all the test fishes after the observation period is finished, and observing the pathological changes of all the organs. The results show that the organs of the weever in the immunization group have obvious pathological changes, and the organs of the weever in the control group have obvious pathological changes. The section picture shows that the normal liver is closely arranged and has a complete structure, the cytoplasma of the pathological liver is dissolved, the intercellular space is enlarged, the arrangement is disordered and the bleeding phenomenon exists; clear capsule and splenic cord can be seen in normal spleen, incomplete capsule and vacuolization of diseased spleen can be seen. See table 3 and fig. 6 for details.

TABLE 3 Observation and examination table for immunity attack protection test

Therefore, the prepared weever rhabdovirus genetic engineering subunit inactivated vaccine (E.coli-G2 strain) can protect weever against the attack of a virulent MSRV strain.

Evaluation of the immunopotency of vaccines prepared with different protein fragments

In the vaccine development process, different fragments are designed, different glycoprotein fragments and complete glycoprotein are respectively prepared, namely G1 (amino acid sequence is SEQ ID NO: 3), G2 (amino acid sequence is SEQ ID NO: 2), G3 (amino acid sequence is SEQ ID NO: 4) protein and glycoprotein (SEQ ID NO: 5), and the carbon nanotube vaccine based on the glycoprotein fragments is respectively prepared according to the above method, wherein the protein of G2 corresponds to the vaccine of the above example 2, and the vaccines prepared from G1, G3 and the G protein are respectively named as G1 vaccine, G3 vaccine and G vaccine, and are respectively subjected to efficacy immune evaluation.

Taking 300 tails of 0.5-1 g (more than 60 days old) healthy weever, and dividing into 5 groups (50 tails/group), wherein the 1 st group to the 5 th group are immune groups, and the 6 th group is a control group. The immunity dose of the test group is 1ml/L (recombinant protein content is 5 mg/L), the control group uses normal saline to replace vaccine, the control group is transferred into normal aquaculture water after soaking and immunizing for 6 hours, and after continuously observing for 21 days, the enterotoxin challenge is respectively carried out by intraperitoneal injection by using weever rhabdovirus FJ985 strain, 15 mul (virus content is 100 TCID)₅₀/ml)/tail. Continuously observing for 15 days after challenge, and regularly checking and recording the mortality of 3, 6, 9, 12 and 15 days after challenge. The results are shown in FIG. 7.

Based on the above tests, it can be seen that, from the evaluation of the immune potency of the vaccines prepared from the truncated G protein fragment and the intact G protein, the protective rate of the G2 protein group after challenge is the highest, and the survival rate thereof after challenge is as high as 96%, while the protective rates of the vaccines prepared from the white fragments of G1 and G3 and the intact G protein are relatively low, the protective rates of the intact G protein and the G3 fragment are higher than 80%, and the protective rates of the G1 fragment on day 12 and day 15 are less than 70%, so that the vaccine prepared by the invention based on the G2 protein fragment has the highest protective rate against challenge, and the protective rate thereof on fish fries is as high as 96%.

The above description is only for the preferred embodiment of the present invention, and the scope of the present invention is not limited by the above description, and therefore, the equivalent changes made according to the claims of the present invention are still included in the scope of the present invention.

Sequence listing

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Met Ser Pro Phe Ser Glu Gly Ile Gly Lys Val Tyr Arg Ile His Arg

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Val Ile Glu Tyr Leu Pro Thr Thr Pro Glu Met Cys Lys Glu Ala Lys

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Arg Ala Ser Asp Arg Gly Glu Ser Leu Ala Pro His Phe Pro Thr Glu

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Thr Ser Asn Thr Leu Lys Glu Leu Ala Glu His Val Asp Glu Ile Ala

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Leu Asn Ala Ile Cys Leu Gln Glu Val Arg Arg Ala Arg Glu Thr Lys

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Ala Val Ser Asp Trp Leu Leu Ser Met Met Ser Pro Phe Ser Glu Gly

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Ile Gly Lys Val Tyr Arg Ile His Arg Gly Met Leu Glu Ser Thr Val

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Gly Phe Tyr Arg Lys Val Val Leu Glu Gly Asp Gly Thr Pro Glu Arg

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