Recombinant protein and application thereof in preparation of fish vaccine

Abstract

The invention belongs to the technical field of biological medicines, and discloses a recombinant protein and application thereof in preparation of fish vaccines. According to the invention, CP4 protein of nervous necrosis virus of grouper is taken as antigen for vaccine, recombinant Escherichia coli BL21 (pET32a-CP4) capable of expressing the protein is constructed by means of genetic engineering technology, recombinant protein rCP4 is obtained through induced expression, and oxidized single-walled carbon nanotubes are taken as carriers to prepare the grouper immersion vaccine (namely SWCNT-rCP4 vaccine). According to the SWCNT-rCP4 vaccine provided by the invention, the immunization of the grouper can be realized through soaking immunization, the survival rate of the grouper reaches 85% after the grouper is soaked and immunized for 4w and then is infected with NNV, and the immune protection effect is remarkable.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to a recombinant protein and an application thereof in preparing a vaccine for fish. Background Art

[0002] Grouper is a general term for fish of the order Perciformes and genus Epinephelus, mainly distributed in tropical and subtropical warm waters. Grouper is susceptible to a variety of diseases, such as tail rot, ulcer disease, iridovirus disease, viral nervous necrosis, etc. Among them, viral nervous necrosis (VNN) of grouper caused by nervous necrosis virus (NNV) is the most serious. NNV belongs to the family Nodaviridae and the genus β-Nodavirus. It is an icosahedron with a diameter of 25-30nm and no capsule. It can infect a variety of hosts including grouper. VNN is generally prevalent from April to November, with a long epidemic period. The peak of the epidemic is concentrated in the high temperature season in summer and autumn, mainly from May to August. The water temperature of the disease is mostly between 24 and 33℃. It mainly harms juveniles and young fish, and the mortality rate after infection can reach more than 90%. Symptoms of sick fish include uncoordinated behavior, spiral swimming, sometimes swollen swim bladder, decreased vitality, floating on the water surface, etc. Typical pathological changes are necrosis and cavitation of central nervous system and retinal tissue.

[0003] Vaccination has been widely recognized and studied as a greener, safer and more effective method to replace antibiotics and chemical drugs for the prevention and control of aquatic animal diseases. At present, genetically engineered vaccines based on the screening of protective antigens with strong immunogenicity and high conservation are gaining more and more attention. Capsid protein (CP) is the only structural protein of NNV. Previous studies have shown that genetically engineered subunit vaccines with CP as antigen have good immune protection effects on grouper against NNV infection. However, the preparation of vaccines is still in the laboratory stage. How to screen and prepare commercial vaccines with excellent immune protection effects against NNV based on capsid protein is still a huge challenge facing the grouper farming industry. Summary of the invention

[0004] In order to effectively prevent and control viral neuronecrosis (VNN) in grouper farming, reduce the economic losses caused by it to grouper farming, and promote the healthy development of aquaculture, the present invention aims to provide a nanocarrier soaked vaccine for neuronecrosis virus (NNV). The present invention uses the CP4 protein of NNV as a vaccine antigen, and uses genetic engineering technology to construct a recombinant genetically engineered bacterium capable of expressing the protein. The recombinant protein rCP4 is obtained by inducing expression, and then combined with oxidized single-walled carbon nanotubes (SWCNT) to prepare a nanocarrier soaked vaccine, which provides a better immune protection effect for grouper against NNV infection. In order to achieve this technical purpose, the present invention provides the following detailed technical scheme.

[0005] The present invention first provides a recombinant protein, wherein the recombinant protein is rCP4, and the amino acid sequence of rCP4 is shown in SEQ ID NO:1.

[0006] In a second aspect, the present invention also provides a gene encoding the above-mentioned recombinant protein, and the nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0007] In a third aspect, the present invention further provides an expression vector for expressing the above-mentioned recombinant protein, wherein the expression vector contains the nucleotide sequence shown in SEQ ID NO:2.

[0008] In a fourth aspect, the present invention further provides a recombinant genetically engineered bacterium for expressing the above-mentioned recombinant protein, wherein the recombinant genetically engineered bacterium contains the above-mentioned expression vector.

[0009] In a fifth aspect, the present invention also provides applications of the above-mentioned recombinant protein. Specifically, the recombinant protein is used to prepare fish vaccines.

[0010] In a sixth aspect, the present invention further provides a fish vaccine, wherein the active ingredient of the fish vaccine is the above-mentioned recombinant protein.

[0011] Furthermore, the fish vaccine provided by the present invention also includes components of: an oxidized single-walled carbon nanotube carrier.

[0012] Furthermore, for the fish vaccine provided by the present invention, applicable fish include grouper.

[0013] Furthermore, for the fish vaccine provided by the present invention, the fish vaccine is inoculated by immersion, and the vaccine content of the immersion inoculation is not less than 20 mg / L.

[0014] In a seventh aspect, the present invention also provides a method for preparing the fish vaccine, comprising: obtaining recombinant protein, modifying oxidized single-walled carbon nanotubes, and preparing the vaccine.

[0015] Obtaining the recombinant protein: constructing an expression vector containing the above recombinant protein encoding gene, and transferring it into genetically engineered bacteria for induced expression. After the expression is completed, the bacteria are broken, and the recombinant protein rCP4 is collected and purified; Modification of oxidized single-walled carbon nanotubes: Concentrated H2SO4: concentrated HNO3 are mixed in a volume ratio of 3:1, wherein concentrated H2SO4 is added first to obtain a mixed acid; 2 g of oxidized single-walled carbon nanotubes are placed in 140 mL of the mixed acid and acidified and stirred for 72 hours; after the treatment, the mixture is diluted with pure water, filtered using a 0.22 μm filter membrane until the pH is neutral, the solid part is dried to constant weight, ground into powder, and then sieved through a 300-mesh sieve to obtain modified oxidized single-walled carbon nanotubes; Preparation of the vaccine: Take 0.5 g of modified oxidized single-walled carbon nanotubes, add them into 200 mL of 0.1 mol / L, pH=5.6 2-(N-morpholino)ethanesulfonic acid (MES) buffer, disperse them evenly, and treat them ultrasonically for 30 min; add 5.0 g of ethyldimethylaminopropylcarbodiimide (EDAC) and 7.5 g of N-carbonylsuccinimidyl (NHS), treat them ultrasonically for 2 h, then centrifuge at 6000 g for 5 min, and retain the centrifugal precipitate; 5 g of the centrifugal precipitate was added to 2000 mL PBS buffer at pH = 7.4 and dispersed evenly, and then 3.0 g of the purified recombinant protein rCP4 was added, ultrasonically dispersed for 2 hours, and stirred at room temperature for reaction for 48 hours; the reaction product was dialyzed in pure water for 3 days and then filtered with a circulating water vacuum pump. The obtained precipitate was the oxidized single-walled carbon nanotube carrier soaked vaccine system SWCNT-rCP4, which was freeze-dried to prepare a lyophilized powder to obtain the fish vaccine.

[0016] Compared with the prior art, the present invention "a recombinant protein and its use in preparing fish vaccines" has the following beneficial effects: First, the present invention utilizes the coding gene of NNV virus capsid protein CP4 A recombinant expression vector was constructed and further transferred into genetically engineered bacteria to express the recombinant protein rCP4. The recombinant protein rCP4 was used as an active ingredient and oxidized single-walled carbon nanotubes were used as a carrier to prepare a fish soaking vaccine. After soaking the vaccine in immunized grouper for 4 weeks and then infecting NNV, the survival rate of the grouper reached 85%, with significant results.

[0017] Secondly, the oxidized single-walled carbon nanotube carrier immersion vaccine system SWCNT-rCP4 constructed by the present invention can achieve the goal of large-scale, simple and rapid seedling immunization through immersion immunization, greatly improve the immersion immunization effect, help realize the industrialization of commercial fish vaccines represented by grouper NNV vaccine, and also lay a theoretical foundation for the research and application of nanocarrier vaccines for other aquatic animals, provide new research ideas, and are of great significance to the sustainable development of fisheries and the safe production of aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results of agarose gel electrophoresis of PCR (a) and single and double restriction enzyme digestion (b) of the recombinant plasmid pET32a-CP4. Lane M is DNA Marker; a Figure 1 Lane 1 is the PCR result of plasmid pET32a-CP4; Lane 2 is the PCR result of negative control; b Figure 1 Lane 1 shows the single enzyme digestion result of plasmid pET32a-CP4; Lane 2 shows the double enzyme digestion result of plasmid pET32a-CP4.

[0019] Figure 2 This is the SDS-PAGE result of the recombinant protein rCP4. Lane M1 is the protein marker; Lane 1 is the bacteria without IPTG induction; Lane 2 is the supernatant of the bacteria induced by IPTG; Lane 3 is the broken precipitate of the bacteria induced by IPTG.

[0020] Figure 3 This is the Western blot detection result of recombinant protein rCP4.

[0021] Figure 4 It is the detection of serum antibody level of grouper. Different shoulder letters indicate significant difference ( p <0.05).

[0022] Figure 5 is the survival rate of each group of grouper after NNV infection. DETAILED DESCRIPTION

[0023] The present invention is explained below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1 This example provides the construction and identification of recombinant Escherichia coli 1. Construction of recombinant Escherichia coli BL21 (pET32a-CP4) expressing recombinant protein rCP4 The recombinant plasmid pET32a-CP4 containing the CP4 protein coding gene was synthesized by a biological company (Beijing Qingke Biotechnology Co., Ltd.) and transformed into Escherichia coli BL21 (DE3), spread on LB solid medium containing kanamycin, and cultured inverted at 37°C overnight. Then a single colony was picked and tested for CP4Colony PCR detection of genes: Inoculate the colonies with positive colony PCR into LB culture medium containing kanamycin and culture at 37°C and 180r / min for 12-16h.

[0025] The recombinant plasmid pET32a-CP4 was extracted according to the instructions of the Omega plasmid extraction kit, and PCR and double enzyme digestion were performed for identification. The PCR primers were: CP4-F: 5'-ATGCTGTCCGAAGTTTCCCTGATCT-3' (SEQ ID NO: 3); CP4-R: 5'-CTCGAGTTACAGGATTGGGAAACC-3' (SEQ ID NO: 4).

[0026] The PCR and double enzyme digestion products were subjected to agarose gel electrophoresis for identification. The electrophoresis conditions were: 120V, 30min. Figure 1 The results of agarose gel electrophoresis of PCR (a) and single and double restriction enzyme digestion (b) for the recombinant plasmid pET32a-CP4. Figure 1 The results showed that the single and double enzyme digestion results of the recombinant plasmid pET32a-CP4 were correct.

[0027] 2. Recombinant E. coli BL21 (pET32a-CP4) induced expression of recombinant protein rCP4 Use an inoculation loop to pick up a small amount of BL21 (pET32a-CP4) bacterial liquid, streak inoculate on LB solid medium plate, incubate at 37℃ for 12~16h, pick a single colony and inoculate it in LB liquid medium, incubate at 37℃, 180r / min for 12~16h as seed liquid, then inoculate it into LB medium at 1% (V / V), and add kanamycin to a final concentration of 50μg / mL. Incubate at 37℃ for 5~7h until the bacterial liquid OD 600 When the value was 1.1-1.3, IPTG was added to a final concentration of 0.001 mol / L, and the culture was induced at 37 °C for 6 h to terminate the fermentation.

[0028] 3. Isolation and purification of recombinant protein rCP4 The fermentation product was centrifuged at 10000r / min at room temperature, the cells were collected, washed 3 times with PBS (0.01mol / L, pH=7.2), and the wet cells were collected. The cells were resuspended in a ratio of 10mL PBS buffer per 1g of wet cells and ultrasonically disrupted under ice bath conditions. The disrupted bacterial solution was centrifuged at 4℃ and 12000r / min, the bacterial protein was collected, and the bacterial protein was dissolved with 8M urea to obtain a mixed protein solution containing the recombinant protein rCP4.

[0029] Dissolve the protein precipitate in a ratio of 10 mL of dissolving solution for every 1 g of protein precipitate. The dissolving conditions are as follows: after 2 hours of shaking and dissolving at room temperature at 200 r / min, centrifuge at 4°C and 10,000 r / min for 30 minutes, and collect the supernatant. After chelating the affinity chromatography column with the equilibration buffer, load the sample at 2 times the column volume, equilibrate with the equilibration buffer, and then elute with the elution buffer to collect the protein solution. Place the purified 200 mL protein solution in a dialysis bag (SnakeSkin™) with a molecular weight cutoff (MWCO) of 10K, and immerse it completely in 10 L of refolding solution, and dialyze it at 4°C for 12 hours. During this period, the refolding solution was replaced every 6 hours. After the dialysis, the dialyzed protein solution was collected and stored at 4°C for later use.

[0030] In the above steps, the dissolving solution includes: 5mmol / L imidazole, 0.5mmol / L sodium chloride, 8M urea, 20mmol / LTirs-HCl, pH=7.9.

[0031] The equilibration buffer solution includes: 5 mmol / L imidazole, 0.5 mmol / L sodium chloride, 8 M urea, 20 mmol / L Tirs-HCl, pH=7.9.

[0032] The elution buffer solution includes: 0.5 mol / L imidazole, 0.5 mmol / L sodium chloride, 8 M urea, 20 mmol / L Tirs-HCl, pH=7.9.

[0033] The refolding solution includes: 150mmol / L NaCl, 2.5mmol / L KCl, 10mmol / L Na2HPO4, 2mmol / LKH2PO4, 1% Tween-20, 10mmol / L β-cyclodextrin, 1mol / L L-cysteine, 3mmol / L reduced glutathione, 1mmol / L oxidized glutathione, pH=7.9.

[0034] 4. Detection and identification of recombinant proteins (1) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection The SDS-PAGE test experiment is divided into 3 lanes, as follows: Lane M1 is the protein marker; Lane 1 is the bacteria without IPTG induction; Lane 2 is the supernatant of the bacteria induced by IPTG; Lane 3 is the broken precipitate of the bacteria induced by IPTG. The acrylamide concentration of the separation gel is 12%; Coomassie brilliant blue staining, decolorization with decolorization solution until the bands are clear. The SDS-PAGE test results are as follows Figure 2 As shown in the results of each group, IPTG induction significantly increased the expression of recombinant protein rCP4, and the recombinant protein rCP4 was mainly present in the precipitate after bacterial cell disruption ( Figure 2 3 lanes in the figure).

[0035] (2) Western-blot identification The recombinant protein rCP4 obtained by the above SDS-PAGE was used for Western-blot test. The above SDS-PAGE film was placed on the transfer instrument and transferred at 200mA for 1h. The PVDF membrane loaded with protein was placed in a plastic washing box, and 25mL of 5% skim milk was added to block at room temperature for 2h, the skim milk was poured out, and after washing with TBST 3 times, 30mL of rabbit anti-NNV antibody (primary antibody, diluted with antibody diluent, ratio 1:1000) was added, and the reaction was slowly shaken at room temperature for 2h. After the reaction was completed, the primary antibody was poured out, and after washing with TBST 5 times, 30mL of horseradish peroxidase-labeled goat anti-rabbit antibody (secondary antibody, diluted with antibody diluent, ratio 1:2000) was added, and the reaction was slowly shaken at room temperature for 2h, the secondary antibody was poured out, and after washing with TBST 5 times, the membrane was placed in a flat dish and placed in a dark room. First add 1mL of deionized water, then add DAB colorimetric solution (1 drop each of solution A and solution B), and rinse the membrane surface repeatedly with a pipette for 1 minute. After color development is complete, rinse with deionized water, dry, and image. After color development, there is a specific band at about 30kDa, indicating that the recombinant protein rCP4 ( Figure 3 ).

[0036] Example 2 This example provides the preparation of nanocarrier vaccines.

[0037] 1. Modification of oxidized single-walled carbon nanotubes (SWCNTs): Concentrated H2SO4:concentrated HNO3 are mixed in a volume ratio of 3:1, wherein concentrated H2SO4 is added first to obtain a mixed acid; 2 g of SWCNTs are placed in 140 mL of the mixed acid and acidified and stirred for 72 h; after the treatment, the mixture is diluted with pure water and filtered using a 0.22 μm filter membrane until the pH is neutral (or the mixture is dialyzed until the pH no longer changes), the solid part is dried at 80°C to constant weight (or freeze-dried), ground into powder in a mortar, and passed through a 300-mesh sieve to obtain modified oxidized single-walled carbon nanotubes.

[0038] 2. Take 0.5g of modified SWCNTs, add it to 200mL of 0.1mol / L, pH=5.6 2-(N-morpholine)ethanesulfonic acid (MES) buffer, disperse it evenly, and treat it ultrasonically for 30min; add 5.0g of ethyldimethylaminopropylcarbodiimide (EDAC) and 7.5g of N-carbonylsuccinic acid imide (NHS), treat it ultrasonically for 2h, then centrifuge at 6000g for 5min, and retain the centrifugal precipitate.

[0039] 3. Add 5 g of the centrifugal precipitate into 2000 mL PBS buffer with pH = 7.4 and disperse evenly, then add 3.0 g of purified recombinant protein rCP4, ultrasonically disperse for 2 hours, and react at room temperature with stirring for 48 hours; the reaction product is dialyzed in pure water for 3 days and then filtered with a circulating water vacuum pump. The resulting precipitate is the oxidized single-walled carbon nanotube carrier soaked vaccine system SWCNT-rCP4, which is freeze-dried to prepare a lyophilized powder to obtain the fish vaccine (i.e., SWCNT-rCP4 vaccine), which is stored at low temperature.

[0040] Example 3 This example provides an evaluation of the immune effect of SWCNT-rCP4 vaccine.

[0041] 1. ELISA test of serum antibody levels 200 healthy groupers weighing 5.0-6.0 g were selected and divided into 4 groups, namely PBS group, SWCNTs group, rCP4 group and SWCNT-rCP4 group, 50 fish / group, and the treatment methods were as follows: PBS group: grouper were immersed in phosphate buffer solution with pH = 7.4 for 8 h and then transferred to normal culture water; SWCNTs group: The grouper was immersed in 20 mg / L SWCNTs solution for 8 h and then transferred to normal culture water; rCP4 group: Prepare an aqueous solution with a recombinant protein rCP4 content of 20 mg / L, soak the grouper in it for 8 hours and then transfer it to normal breeding water; SWCNT-rCP4 group: Prepare immunization water with SWCNT-rCP4 vaccine content of 20 mg / L, soak the grouper for 8 hours and then transfer to normal breeding water.

[0042] Blood was collected from each group of groupers at 1 week, 2 weeks, 3 weeks, and 4 weeks after immunization, and the collected blood was left to stand at room temperature for 2 hours and placed in a refrigerator at 4°C overnight. The next day, it was centrifuged at 5000g for 15 minutes in a low-temperature refrigerated centrifuge, and the upper serum was taken and stored at -20°C.

[0043] Serum antibody levels were determined by indirect ELISA: serum was diluted 1:200 with coating diluent, 100 μL per well was added to a 96-well ELISA plate, three replicates per sample were placed at 4°C for overnight coating. After completion, the liquid in the well was discarded, 5% calf serum was added and blocked at 37°C for 1 hour. After blocking, the wells were washed 3 times with TBST, each for 3 minutes. After the recombinant protein rCP4 was diluted to 0.1 mg / mL, 100 μL per well was added to the ELISA reaction wells, and incubated at 37°C for 40-60 minutes. Then, TBST was washed 3 times, each for 3 minutes. Mouse His-tag monoclonal antibody (1:1000) was diluted, 100 μL per well was added to the ELISA reaction wells, incubated at 37°C for 30-60 minutes, and then washed 3 times with TBST, each for 3 minutes. Dilute the goat anti-mouse IgG (1:5000) labeled with horseradish peroxidase and add 100 μL per well to the enzyme-labeled reaction wells, incubate at 37°C for 30-60 min, then wash 3 times with TBST, 3 min each time. Add 100 μL of tetramethylbenzidine (TMB) colorimetric solution to each well, place at 37°C in the dark for 5-10 min, add 50 μL of stop solution to each well to stop the reaction, and measure the absorbance at 450 nm within 20 min.

[0044] The results showed that the serum antibody levels of each group reached the highest level 4 weeks after immunization, and the antibody levels of the SWCNT-rCP4 group were significantly higher at each week after immunization ( p <0.05) were higher than those in the PBS group, SWCNTs group, and rCP4 group. Figure 4 shown.

[0045] 2. Post-infection immune protection rate detection The groupers in the above groups were infected by intraperitoneal injection of NNV virus suspension 4 weeks after immunization, with an injection dose of 100 μL / tail. The groupers were observed continuously for 14 days after infection, and the mortality of the groupers was recorded and the survival rate was calculated.

[0046] like Figure 5 As shown, grouper immunized with SWCNT-rCP4 vaccine were immersed in it for 4 weeks and then artificially infected with NNV. The survival rate of grouper 14 days after infection was significantly higher than that of PBS group, SWCNTs group and rCP4 group, reaching 85%, indicating that the SWCNT-rCP4 vaccine provided by the present invention has an immune protection effect against NNV.

[0047] In summary, the present invention uses the CP4 protein of grouper neural necrosis virus as a vaccine antigen, and uses genetic engineering technology to construct a recombinant Escherichia coli BL21 (pET32a-CP4) capable of expressing the protein, obtains the recombinant protein rCP4 by inducing expression, and uses modified SWCNTs as a carrier to prepare a grouper soaking vaccine (i.e., SWCNT-rCP4 vaccine). The SWCNT-rCP4 vaccine provided by the present invention can achieve immunization of grouper by soaking immunization. After 4 weeks of soaking immunization, the grouper is infected with NNV, and the survival rate of the grouper reaches 85%, and the immune protection effect is significant.

[0048] The embodiments described above are only some embodiments of the present invention, not all embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A recombinant protein, characterized in that The recombinant protein is rCP4, and the amino acid sequence of rCP4 is shown in SEQ ID NO:

1.

2. The gene encoding the recombinant protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. An expression vector for expressing the recombinant protein according to claim 1, characterized in that: The expression vector contains the nucleotide sequence shown in SEQ ID NO:

2.

4. A recombinant genetically engineered bacterium expressing the recombinant protein according to claim 1, characterized in that: The recombinant genetically engineered bacteria contains the expression vector according to claim 3.

5. The use of the recombinant protein according to claim 1, characterized in that: The recombinant protein is used for preparing fish vaccine.

6. A vaccine for fish, characterized in that: The active ingredient of the fish vaccine is the recombinant protein according to claim 1.

7. The fish vaccine according to claim 6, characterized in that: The components of the fish vaccine also include: an oxidized single-walled carbon nanotube carrier.

8. The fish vaccine according to claim 6, characterized in that: Suitable fish for the fish vaccine include grouper.

9. The fish vaccine according to claim 6, characterized in that: The fish vaccine is inoculated by immersion, and the vaccine content of the immersion inoculation is not less than 20 mg / L.

10. The method for preparing the fish vaccine according to claim 6, characterized in that: include: Obtaining recombinant proteins, modifying oxidized single-walled carbon nanotubes, and preparing vaccines; Obtaining the recombinant protein: constructing an expression vector containing the recombinant protein encoding gene of claim 1, and transferring it into genetically engineered bacteria for induced expression, breaking the bacteria after the expression is completed, and collecting and purifying the recombinant protein rCP4; Modification of oxidized single-walled carbon nanotubes: Concentrated H2SO4: concentrated HNO3 are mixed in a volume ratio of 3:1, wherein concentrated H2SO4 is added first to obtain a mixed acid; 2 g of oxidized single-walled carbon nanotubes are placed in 140 mL of the mixed acid and acidified and stirred for 72 hours; after the treatment, the mixture is diluted with pure water, filtered using a 0.22 μm filter membrane until the pH is neutral, the solid part is dried to constant weight, ground into powder, and then sieved through a 300-mesh sieve to obtain modified oxidized single-walled carbon nanotubes; Preparation of the vaccine: Take 0.5 g of the modified oxidized single-walled carbon nanotubes, add them into 200 mL of 0.1 mol / L, pH=5.6 2-(N-morpholine)ethanesulfonic acid buffer, disperse them evenly, and treat them ultrasonically for 30 min; add 5.0 g of ethyldimethylaminopropylcarbodiimide and 7.5 g of N-carbonylsuccinic acid imide, treat them ultrasonically for 2 h, then centrifuge at 6000 g for 5 min, and retain the centrifugal precipitate; 5 g of the centrifugal precipitate was added to 2000 mL PBS buffer at pH = 7.4 and dispersed evenly, and then 3.0 g of the purified recombinant protein rCP4 was added, ultrasonically dispersed for 2 hours, and stirred at room temperature for reaction for 48 hours; the reaction product was dialyzed in pure water for 3 days and then filtered with a circulating water vacuum pump. The obtained precipitate was the oxidized single-walled carbon nanotube carrier soaked vaccine system SWCNT-rCP4, which was freeze-dried to prepare a lyophilized powder to obtain the fish vaccine.

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