Aquatic animal antigen system and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses an aquatic animal antigen system and application thereof. The preparation method comprises the following steps: carrying out carboxylation treatment on bacterial cellulose to prepare carboxylated bacterial nano-cellulose, and then carrying out grafting reaction on the carboxylated bacterial nano-cellulose and an aldehyde group polymer to prepare surface modified nano-cellulose (namely poly (N-isopropylacrylamide) grafted carboxylated bacterial nano-cellulose). The surface-modified nanocellulose is used as a carrier and is chemically connected with a virus antigen protein (namely recombinant G protein) to prepare an antigen system (namely BNC-G) carrying the virus antigen protein. According to the antigen system, virus antigen protein can be delivered into aquatic animal bodies through soaking immunization, the effect of preventing and controlling aquatic animal virus diseases is achieved, and the antigen system is suitable for production of aquatic animal SPF seedlings and preparation of aquatic animal immune protection preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an aquatic animal antigen system and application thereof. Background Art

[0002] Although the immune effect of aquatic animal virus vaccines is limited by factors such as the large coefficient of virus variation, the key technical bottleneck that prevents the large-scale promotion and application of fishery vaccines lies in the immunization route and efficiency. Commonly used fishery vaccines can be divided into three immunization routes: injection immunization, oral immunization and immersion immunization. Among them, the protection rate of injection immunization can reach 40~90%, but it is only suitable for larger-sized aquatic animals, and this immunization method is difficult to meet the epidemic prevention needs of large-scale aquatic animals; the antigens of vaccines used in oral immunization are easily decomposed by digestive enzymes, resulting in reduced immune effect, and the protection rate is generally 10~50%; immersion immunization is suitable for small-sized aquatic animals, with simple operation and low cost, and is also suitable for large-scale immunization, but the protective barriers such as the skin and mucous membranes of aquatic animals reduce their immune effect, and the immune protection rate is only 30~60%. Therefore, the construction of an antigen delivery system that can break through the barrier system of organisms will be of great significance for the prevention and control of viral diseases in aquatic animals and the production of specific pathogen free (SPF) aquatic seedlings.

[0003] Cellulose is a natural high molecular polymer with a wide range of sources, abundant resources and low price. Natural cellulose can be processed into microcrystalline cellulose, nanocellulose, etc. through crushing, acid-base or enzyme hydrolysis. Compared with powdered cellulose and microcrystalline cellulose, nanocellulose has the advantages of high biocompatibility, degradability, safety and non-toxicity. It has good application prospects in the construction of drug and vaccine delivery systems, drug sustained release and tissue engineering scaffolds. In the medical field, the research on nanocellulose mainly focuses on anticancer drugs, hydrogels and wound dressings, and there is still a lack of technical support in aquatic animal antiviral drugs, especially in the preparation of aquatic animal SPF seedlings.

[0004] In addition, in production practice, a large number of wild virus-free parents are usually screened over multiple generations, and then the wild virus-free parents are used for several generations to breed offspring, and then virus testing is performed to finally obtain SPF seedlings. However, this method of producing SPF seedlings for aquatic animals has the disadvantages of long production cycle, high cost, and difficulty in isolating viruses, which limits the production and promotion of SPF seedlings for aquatic animals. Summary of the invention

[0005] In view of the above technical status, in order to further improve the immune effect of aquatic animal virus vaccines, solve the technical defects of traditional SPF seedling production methods, and achieve the prevention and control of aquatic animal viral diseases, the present invention uses surface-modified nanocellulose as a carrier of viral antigen proteins to prepare an antigen system. The antigen system can transport viral antigen proteins into aquatic animals through immersion immunization to achieve the technical purpose of preventing and controlling aquatic animal diseases. To this end, the present invention provides the following detailed technical solutions.

[0006] First, the present invention provides an antigen system, which is formed by chemically connecting poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose and recombinant G protein; the recombinant G protein is a fusion protein containing the amino acid sequence of the largemouth bass rhabdovirus G protein and a His tag, and the amino acid sequence of the largemouth bass rhabdovirus G protein is shown in SEQ ID NO: 1.

[0007] Further, in the above antigen system, the chemical connection method is: The poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose was ultrasonically treated with a 2-morpholineethanesulfonic acid buffer having a pH of 5.6-6.2 for 0.2-0.5 h, and 500 mL of the 2-morpholineethanesulfonic acid buffer was used for every 3 g of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-carbonylsuccinimide were added, and ultrasonic treatment was performed at 40kHz and 500W for 1-2h. For every 3g of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, 0.2g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.3g of N-carbonylsuccinimide were used. Solid-liquid separation: the solid product was mixed with PBS buffer at pH 7.2-7.4, with 500 mL PBS buffer used for every 3 g poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose; Add recombinant G protein, the amount of which is 0.5-1.5 times the mass of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, and react at 20-25°C for 6-48h; After the reaction is completed, the dialyzate is placed in pure water for dialyzing, the dialyzate is centrifuged to separate the solid and the liquid, and the obtained solid matter is freeze-dried.

[0008] Furthermore, in the above antigen system, the preparation method of the poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose is: Dissolve the aldehyde polymer in pure water, using 0.6 mL of pure water for every 100 mg of aldehyde polymer; Add the carboxylated nanocellulose solution, and then add isocyanocyclohexane while stirring. The amount of carboxylated nanocellulose solution and isocyanocyclohexane is as follows: 2.5 mL of 10 mg / mL carboxylated nanocellulose solution and 22 mg of isocyanocyclohexane are used for every 100 mg of aldehyde polymer, and stir at room temperature for 24 hours. The reaction product was centrifuged to obtain a precipitate, and then dialyzed against 5 L of pure water for 24 h and 2 L of methanol for 24 h, respectively. The methanol was distilled off under reduced pressure, and the product was freeze-dried.

[0009] Further, in the preparation method of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, the preparation method of the aldehyde polymer is: Potassium hydroxide is dissolved in ethylene glycol, bromoacetaldehyde dimethyl acetal is slowly added while stirring, and the mixture is stirred at 115°C for 72 hours. After cooling to room temperature, pure water is added, and the solution is extracted with chloroform. The extract is dried with magnesium sulfate, and then the magnesium sulfate is removed by filtration and the filtrate is retained. After reduced pressure distillation, a yellow 2-(2,2-dimethoxyethoxy)ethanol liquid is obtained; wherein, in terms of molar ratio, potassium hydroxide: bromoacetaldehyde dimethyl acetal = 1.5-2.5:1; Take 2-(2,2-dimethoxyethoxy)ethanol, dissolve it and 3-[[(benzylthio)thiocarbonyl]thio]propionic acid (BSPA) in dichloromethane at 0°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and continue stirring at 0°C for 2 hours, stir at room temperature for 40 hours, wash, dry and distill under reduced pressure, and purify by column chromatography to obtain 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propanoate; wherein the molar ratio is 2-(2,2-dimethoxyethoxy)ethanol:3-[[(benzylthio)thiocarbonyl]thio]propionic acid:1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride:4-dimethylaminopyridine=1:0.2~0.3:0.4~0.7:0.2~0.3; Dissolve 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate, N-isopropylacrylamide, and azobisisobutyronitrile in tetrahydrofuran, wherein the molar ratio of 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate:N-isopropylacrylamide:azobisisobutyronitrile is 1:90-120:0.08-0.12; place in a sealed container and remove oxygen, stir at 65°C for 120h; terminate the reaction by ice bath for 10min, dialyze with pure water for 24h and methanol for 24h, respectively, and then perform reduced pressure distillation to collect the product; Take 160 mg of the product obtained by vacuum distillation, dissolve it in a mixture of 2.4 mL of tetrahydrofuran and 2 mL of hydrochloric acid, both with a concentration of 1 mol / L, and stir it at room temperature for 72 hours for deprotection; dialyze the obtained sample with pure water for 24 hours and methanol for 24 hours, and remove the solvent by vacuum distillation to obtain poly(N-isopropylacrylamide) with aldehyde groups, which is an aldehyde polymer.

[0010] Further, in the preparation method of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, the preparation method of the carboxylated nanocellulose comprises: preparation of nanocellulose and carboxylation of nanocellulose, wherein: Preparation of nanocellulose: Wash bacterial cellulose with 0.1 mol / L sodium hydroxide solution to remove surface impurities, then wash repeatedly with distilled water until the pH of the bacterial cellulose surface is 8; put bacterial cellulose into 68% sulfuric acid solution, use 20 mL sulfuric acid solution for every 0.5 g of bacterial cellulose, heat at 50°C for 2 h, then add ice-water mixture to the reaction system to terminate the reaction, dialyze in pure water with a 14 kDa dialysis bag until the pH is neutral, and freeze-dry to obtain nanocellulose; Carboxylation of nanocellulose: Take 1g of nanocellulose, add 40mL of deionized water and stir, then add 0.05~2g of iron oxide with a particle size of 1μm, and heat to 80~85℃ while stirring after ultrasonic dispersion, add 30~40mL of 20% hydrogen peroxide solution and continue stirring and heating. After 3h, add 30~40mL of 20% hydrogen peroxide solution, continue stirring and heating for 3h; cool the reaction system to room temperature, add 0.5M sodium hydroxide to adjust the pH to 7.8~8.8 and stir; filter the mixed solution to obtain the filtrate, centrifuge the filtrate, collect the supernatant and add 20~40mL of ethanol for precipitation, filter the precipitated product, dialyze it in pure water with a 14kDa dialysis bag for 2~3 days, and freeze-dry the liquid in the dialysis bag to obtain carboxylated nanocellulose powder.

[0011] In a second aspect, the present invention seeks to protect the use of the above-mentioned antigen system in the preparation of SPF aquatic seedlings. Specifically, in the antigen system, the concentration of the viral antigen protein is 2-30 mg / L.

[0012] In a third aspect, the present invention further requests to protect the use of the above antigen system in the preparation of an immune protection preparation for aquatic animals. Specifically, the immune protection preparation for aquatic animals adopts immersion immunization, and the immersion immunization time is 1 to 5 hours.

[0013] Compared with the prior art, the present invention "an aquatic animal antigen system and its application" has at least the following beneficial effects: The present invention performs carboxylation treatment on bacterial cellulose to obtain carboxylated bacterial nanocellulose, and then performs grafting reaction on the bacterial nanocellulose with an aldehyde polymer to obtain surface-modified nanocellulose (i.e., poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose). Compared with ordinary nanocellulose, the surface-modified nanocellulose prepared by the present invention has good water solubility and can penetrate the surface of aquatic animals in water and enter tissues and cells.

[0014] The surface-modified nanocellulose is used as a carrier, and is chemically connected to the viral antigen protein (i.e., recombinant G protein) to prepare an antigen system (i.e., BNC-G) carrying the viral antigen protein. The antigen system can deliver the viral antigen protein to aquatic animals through immersion treatment, thereby achieving the effect of preventing and controlling aquatic animal diseases, and is suitable for the production of SPF seedlings of aquatic animals and the preparation of immune protection preparations for aquatic animals. It has been verified that BNC-G has a stronger immune protection effect than recombinant G protein. The immune protection rate of 10 mg / L BNC-G is 42%, and the immune protection rate of 30 mg / L BNC-G reaches 83%.

[0015] The antigen system provided by the invention has a simple method of use, low preparation cost and good practical application prospect in the aquatic products industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Scanning electron microscopy image of carboxylated nanocellulose.

[0017] Figure 2 This is the appearance of a suspension of carboxylated nanocellulose dispersed in water.

[0018] Figure 3 This is the particle size distribution diagram of carboxylated nanocellulose.

[0019] Figure 4 The dissolution of carboxylated nanocellulose and poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose in pure water.

[0020] Figure 5 Figure 2 shows the particle size distribution of carboxylated nanocellulose and poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose.

[0021] Figure 6 This is the standard curve of aldehyde polymer content / absorbance.

[0022] Figure 7 The results of SDS-PAGE analysis of total protein induced and expressed in E. coli BL21 are shown in Figure 1. Lane M is the protein relative molecular mass standard, and lane 1 is the total protein.

[0023] Figure 8The results of SDS-PAGE analysis of total protein after purification by Ni-NTA agarose purification resin, where lane M is the protein relative molecular mass standard and lane 1 is the purified recombinant G protein.

[0024] Fig. 9 21 days after immersion immunization IgM Gene expression levels.

[0025] Fig.10 21 days after immersion immunization TCR-α Gene expression levels.

[0026] Fig.11 21 days after immersion immunization IL-12 Gene expression levels.

[0027] Fig.12 The figure shows the changes in survival rate of the recombinant G protein immunized group and the blank control group after virus attack.

[0028] Fig.13 The figure shows the changes in survival rate of the BNC-G immunized group and the blank control group after virus attack. DETAILED DESCRIPTION

[0029] 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.

[0030] The test material information in the examples is as follows: Main reagents or kits: Bacterial cellulose was purchased from Hainan Yide Food Co., Ltd.; dialysis bag (14kDa) was purchased from Shenggong Biotechnology (Shanghai) Co., Ltd.; 1,4-butanediol diglycidyl ether, 2-morpholineethanesulfonic acid (MES), ethyl dimethylaminopropyl carbodiimide, and N-carbonyl succinic acid imide were purchased from Sigma, USA. pET-32a (+) vector (catalog number LM1216) was purchased from Shanghai Lianmai Biotechnology Co., Ltd.; competent strains Top10 and BL21 (DE3) were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; pMD19-T vector was purchased from TaKara. Viral RNA extraction kit, DM2000 DNAMarker, PCR amplification kit, colorimetric agent DAB, and BCA protein content determination kit were purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; reverse transcription kit, restriction endonucleases (EcoRI, Hind Ⅲ), and T4 DNA ligase were purchased from TaKara; DNA purification kit was purchased from Axygen; primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.; protein marker was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; mouse histidine monoclonal antibody and horseradish peroxidase-labeled goat anti-mouse IgG were purchased from Abcam; Elisa detection kit was purchased from Beijing Kangwei Century Biotechnology Co., Ltd.; Mianyang red blood cells were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0031] Main test instruments: Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific, USA; PCR instrument, Bio-Rad, USA; MALVERN nano-laser particle size analyzer, Malvern Instruments, UK; FEI field emission scanning electron microscope, FEI, USA; Liquid Zeta particle size / potential analyzer, Anton Paar, Austria.

[0032] Experimental materials: The rhabdovirus strain of largemouth bass was isolated and preserved by the Fisheries Science Laboratory, College of Animal Science and Technology, Northwest Agriculture and Forestry University; the experimental animals were largemouth bass summer flower seedlings (body length: 4~6±0.5cm) purchased from Yongsheng Fishery Development Co., Ltd.

[0033] Example 1 This example describes the preparation of carboxylated bacterial nanocellulose.

[0034] 1. Preparation of Nanocellulose The bacterial cellulose was washed with 0.1 mol / L sodium hydroxide solution to remove surface impurities, and then repeatedly washed with distilled water until the surface pH of the bacterial cellulose was 8; the bacterial cellulose was crushed in a blender and put into a 68% mass fraction sulfuric acid solution, and 20 mL of sulfuric acid solution was used for every 0.5 g of bacterial cellulose, and stirred and heated at 50°C for 2 h (stirring speed 800 r / min), and then 10 times the volume of ice-water mixture was added to the reaction system to terminate the reaction; then dialyzed in pure water with a 14 kDa dialysis bag until the pH was neutral, and the liquid in the dialysis bag was placed in a freeze dryer for freeze drying to obtain nanocellulose.

[0035] 2. Carboxylation of Nanocellulose Take 1g of the dry nanocellulose prepared above, add 40mL of deionized water and stir, then add 0.05~2g of iron oxide with a particle size of 1μm, disperse by 40kHz ultrasound for 10~20min, and heat to 80~85℃ while stirring, add 30~40mL of 20% hydrogen peroxide solution and continue stirring and heating, add 30~40mL of 20% hydrogen peroxide solution after 3h, continue stirring and heating for 3h; cool the reaction system to room temperature, add 0.5M sodium hydroxide to adjust the pH to 7.8~8.8 and stir; filter the mixed solution to obtain the filtrate, centrifuge the filtrate at 12000rpm for 30min, collect the supernatant and add 30mL of ethanol for precipitation, filter the precipitated product, dialyze it in 5L of pure water with a 14kDa dialysis bag for 2~3 days, and freeze-dry the liquid in the dialysis bag to obtain carboxylated nanocellulose powder.

[0036] 3. Characterization of Nanocellulose 1) Scanning electron microscope detection Take the carboxylated nanocellulose prepared in 1.2.2, fix it on the sample stage with conductive glue, observe it after gold spraying, and take a scanning electron microscope image at a high voltage of 5kV and a magnification of 100,000 times ( Figure 1 ).

[0037] 2) Dynamic light scattering (DLS) analysis Take the carboxylated nanocellulose prepared in 1.2.2, dissolve it in pure water and ultrasonically disperse it for 10 minutes, add it into a quartz cuvette for DLS analysis ( Figure 3 ).

[0038] 4. Results and Analysis Figure 1 This is a scanning electron microscope image of carboxylated bacterial nanocellulose. It can be seen that the particle size of carboxylated bacterial nanocellulose is below 1000nm and the size is relatively uniform. Figure 2 This is the appearance of a suspension of carboxylated nanocellulose dispersed in water. No obvious particles are seen in the suspension, and no obvious precipitation is produced after standing for half an hour. Figure 3 DLS analysis results are used to characterize the particle size distribution of carboxylated nanocellulose. Figure 3 It can be seen that the size of carboxylated bacterial nanocellulose is normally distributed, with an average particle size of 215nm.

[0039] Example 2 This example describes the preparation of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, including: preparation of aldehyde polymer, grafting reaction of aldehyde polymer with carboxylated bacterial nanocellulose to obtain poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose.

[0040] 1. Preparation of aldehyde-based polymers 20 g (0.36 mol) of potassium hydroxide was added to 37.5 mL of ethylene glycol, and the mixture was stirred and refluxed at 115°C. After the potassium hydroxide was completely dissolved, 15.6 mL of bromoacetaldehyde dimethyl acetal and 21.6 mL (0.18 mol) were slowly added within 30 min, and the mixture was continuously stirred at 115°C for 72 h to obtain a suspension a. The suspension a was cooled to room temperature, 150 mL of pure water was added to dissolve the precipitate, and then the suspension was extracted with 5×50 mL of chloroform, and the extract was dried with magnesium sulfate. The magnesium sulfate was then removed by filtration, and the filtrate was retained, and the suspension was distilled under reduced pressure to obtain a yellow 2-(2,2-dimethoxyethoxy)ethanol liquid. 3.3 g (22 mmol) of 2-(2,2-dimethoxyethoxy)ethanol and 1.8 g (6.6 mmol) of 3-[[(benzylthio)carbonylthio]propionic acid (BSPA) were dissolved in 30 mL of dichloromethane at 0°C, and then 2.8 g (14.6 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.69 g (5.6 mmol) of 4-dimethylaminopyridine were added, and the mixture was stirred at 0°C for 2 h, and then stirred at room temperature for 40 h to obtain a mixed solution b; the mixed solution b was washed with 50 mL of pure water 3 to 5 times, and then dried with magnesium sulfate, the magnesium sulfate was filtered off, the solvent was removed by reduced pressure distillation, and the product 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate was further purified by column chromatography (the mobile phase used was: hexane:ethyl acetate = 1:1 by volume ratio) to obtain the product; Dissolve 40.0 mg (98.7 μmol) of 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate, 1.1 g (9.7 mmol) of N-isopropylacrylamide, and 1.5 mg (9.1 μmol) of azobisisobutyronitrile in 2 mL of tetrahydrofuran; place in a sealed container and remove oxygen, stir at 65 °C for 120 h; terminate the reaction by ice bath for 10 min, dialyze with pure water for 24 h and methanol for 24 h, respectively, and then perform vacuum distillation to collect the product; Take 160 mg of the product obtained by vacuum distillation, dissolve it in a mixture of 2.4 mL of tetrahydrofuran and 2 mL of hydrochloric acid, both with a concentration of 1 mol / L, and stir it at room temperature for 72 hours for deprotection; dialyze the obtained sample with pure water for 24 hours and methanol for 24 hours, and remove the solvent by vacuum distillation to obtain poly(N-isopropylacrylamide) with aldehyde groups, which is an aldehyde polymer.

[0041] 2. Grafting reaction of aldehyde polymer and carboxylated bacterial nanocellulose 100 mg of aldehyde polymer was dissolved in 0.6 mL of pure water, and 2.5 mL of 10 mg / mL carboxylated nanocellulose solution was added, followed by 22 mg of isocyanocyclohexane while stirring, and stirred at room temperature for 24 h; The reaction product was centrifuged to obtain a precipitate, and then dialyzed with 5L pure water for 24h and 2L methanol for 24h, respectively. The methanol was removed by vacuum distillation, and the product was freeze-dried to obtain poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose.

[0042] 3. Characterization of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose The solubility test of carboxylated bacterial nanocellulose and poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose was carried out, and the DLS analysis of the two was carried out in a neutral water environment. The DLS analysis method is shown in Example 1. Since the π→π* transition of the C=S bond of the aldehyde polymer prepared in 1.2.1 has a characteristic absorption peak at 305nm, the grafting of the aldehyde polymer on the surface of the carboxylated nanocellulose can be analyzed by UV-visible spectrophotometry. According to the absorbance of aldehyde polymers of different concentrations at 305nm, the aldehyde polymer content / absorbance standard curve is drawn, and then the grafting efficiency is calculated.

[0043] Grafting efficiency = (the amount of aldehyde-based polymer grafted onto nanocellulose / the amount of aldehyde-based polymer initially added) × 100% 4. Results and Analysis The dissolution of the same amount of carboxylated bacterial nanocellulose and poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose in pure water is shown in Figure 2. Figure 4 As shown, the dispersed solution of the former is turbid, while that of the latter is clear, indicating that the water solubility of carboxylated bacterial nanocellulose is enhanced after surface modification.

[0044] DLS analysis results are as follows Figure 5As shown in the figure, the average particle size of carboxylated bacterial nanocellulose increased from 215nm to 338nm after surface modification, but the particle size distribution range became smaller, and the aggregation index (PDI) decreased from 0.34 to 0.26, indicating that the particle size distribution of poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose was more uniform. Based on UV-visible spectrophotometry analysis, the aldehyde polymer content / absorbance standard curve is shown in Figure 6 The absorbance of poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose was substituted into the standard curve, and the grafting efficiency was calculated to be 56%.

[0045] Example 3 This example describes the preparation of the antigen system, including: expression and purification of recombinant G protein, and chemical connection of the recombinant G protein to a vector.

[0046] 1. Expression and purification of recombinant G protein According to the instructions of the virus RNA extraction kit, the total RNA of largemouth bass rhabdovirus was extracted, and the RNA was reversed into cDNA using a reverse transcription kit.

[0047] Primers were designed based on the G protein gene sequence of largemouth bass rhabdovirus in the GenBank database (Accession NO.: KF146308.1): GF (EcoR I): 5'-GAGCTCCTATGACAAGCGCACTCAGAG-3' (SEQ ID NO: 2); GR (Hind III): 5'-GAATTCTCAGTGGAATGAGTCGGAGTC-3' (SEQ ID NO: 3) PCR amplification reaction conditions: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 56℃ annealing for 30s, 72℃ extension for 1min, 30 cycles; 72℃ extension for 10min. The PCR product was identified by agarose gel electrophoresis, and the electrophoresis conditions were: 120V constant voltage for 20min. The electrophoresis product was cut, recovered, and purified by a DNA purification kit. The purified product was double-digested with EcoR I and Hind Ⅲ and connected to the pMD19-T vector to obtain the recombinant plasmid pMD19T-G, which was transformed into Escherichia coli Top10 competent cells, and the positive strain was obtained by blue-white screening. At the same time, the recombinant plasmid pMD19T-G was extracted for PCR and sequencing identification.

[0048] The recombinant plasmid pMD19T-G and pET-32a (+) plasmid were digested with EcoR I and Hind III, agarose gel electrophoresis, gel cutting, recovery, DNA purification kit purification, T4 ligase ligation, to obtain the recombinant plasmid pET32a-G, which was transformed into Escherichia coli BL21 (DE3) competent cells. At the same time, the recombinant plasmid pET32a-G was extracted for PCR and sequencing identification.

[0049] E. coli BL21 (DE3) containing the recombinant plasmid pET32a-G was cultured at 37°C for 1 hour with shaking, then spread on LB plates (containing ampicillin) and cultured at 37°C overnight. A single colony was picked and placed in LB liquid medium (containing ampicillin) and cultured at 37°C with shaking overnight. Another 1% of the bacterial solution was taken and expanded in a new medium until the OD 600 When the p-value was 0.6, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to make the final concentration 1.0mmol / L, and the culture was induced for 4-6h. The supernatant was discarded after centrifugation, and the precipitate was washed twice with 1×PBS. An equal volume of SDS loading buffer was added, and the mixture was mixed and boiled for 5min. The result was verified by 10% SDS-PAGE electrophoresis.

[0050] Collect a large amount of induced expression bacterial liquid, centrifuge at 12000r / min and 4°C for 10min in a 50mL centrifuge tube, collect the precipitate and resuspend, ultrasonicate in an ice bath for 30min, centrifuge at 12000r / min and 4°C for 20min, collect the precipitate, add a denaturing solution containing 8mol / L urea to dissolve, centrifuge at 12000r / min and 4°C for 10min, remove the supernatant, purify with Ni-NTA agarose purification resin, perform purification according to the instructions, and then dialyze the purified protein solution in PBS containing 6.0, 4.0, 2.0, and 1.0mol / L urea, respectively, and finally dialyze in 1×PBS overnight, collect the dialyzate, freeze-dry to obtain the recombinant G protein, and store it for later use. The recombinant G protein is a fusion protein containing the amino acid sequence of the largemouth bass rhabdovirus G protein (SEQ ID NO: 1) and a His tag.

[0051] 2. Chemical connection of recombinant G protein and vector 3 g of poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose was added to 500 mL of 2-morpholineethanesulfonic acid buffer with a pH of 5.6 and a concentration of 0.1 mol / L, and ultrasonicated at 40 kHz and 500 W for 0.5 h; Add 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.3 g of N-carbonylsuccinimide, and perform ultrasonic treatment at 40 kHz and 500 W for 2 h; Centrifuge at 6000 rpm for 5 min to separate the solid and liquid, and mix all the centrifugal precipitates with 500 mL PBS buffer at pH 7.4; Add 3 g of recombinant G protein and react at 25°C for 48 h; After the reaction, the dialyzate was dialyzed in pure water for 72 h using a dialysis bag with a cutoff of 100 kDa. The dialyzate was centrifuged at 3000 rpm for 10 min to separate the solid and liquid. The resulting centrifugal precipitate was the poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose-recombinant G protein complex (BNC-G), which was freeze-dried and stored at 4°C.

[0052] Accurately weigh 1.0g of BNC-G lyophilized powder, dilute to 10mL with pure water, disperse evenly by ultrasonication, refer to the instructions of the BCA protein concentration determination kit, gradient dilute the dispersed solution, and use the Thermo Multiskan MK3 microplate reader to determine the content of recombinant G protein. Calculate the carrying capacity of recombinant G protein in BNC-G according to the concentration of recombinant G protein, where the calculation formula of the carrying capacity is as follows: Protein loading capacity = (mass of recombinant G protein in the complex / mass of the complex) × 100% 3. Results and Analysis The recombinant plasmid pET32a-G was expressed in E. coli BL21 and after 4 h of induction, the results of SDS-PAGE electrophoresis showed that Figure 7 As shown, the expression of recombinant G protein (relative molecular weight of about 75.6kDa) can be seen in the lane, which is consistent with expectations. After purification by Ni-NTA agarose purification resin, a single target band can be observed ( Figure 8 ). The protein loading capacity of poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose-recombinant G protein complex (BNC-G) was 23.8% as determined by BCA protein concentration assay kit.

[0053] Example 4 This example describes the evaluation of the antiviral effect of the antigen system.

[0054] 1. Largemouth Bass Immunity The summer flower seedlings of largemouth bass were randomly divided into 9 groups, 300 in each group, and were raised in a culture tank filled with 30L aerated tap water. Recombinant G protein freeze-dried powder and BNC-G were used, and 8 groups were immersed and immunized at 5.0, 10.0, 20.0 and 30.0 mg / L (in terms of recombinant G protein concentration). After immersion and immunization for 2 hours, they were transferred to a conventional culture tank for breeding. The heating rod adjusted the water temperature to maintain 28±0.5℃, and the aeration and oxygenation maintained the dissolved oxygen content in the water above 6mg / L. The white / black light cycle was 14:10h. Feed was fed once a day in the morning. The feed was eaten within 10min. After 1h of feeding, the residual bait at the bottom of the tank was removed, 1 / 3 of the water was changed, and the culture tank was cleaned once a week. The blank control group was not treated with immersion immunization, and the other breeding conditions were the same as those of the experimental group.

[0055] On the 14th, 21st, 28th, 49th and 70th days after immunization, 10 fish were taken from each group, the spine was cut and sacrificed, 2.0 mL of 0.6% saline was added, homogenized with a tissue homogenizer at 12000 rpm for 5 min, centrifuged with a high-speed refrigerated centrifuge at 3000 rpm for 10 min, and the supernatant was transferred to a new tube and stored in a -80°C refrigerator. Antibody detection was performed by indirect agglutination method, and the sensitized carrier selected was sheep red blood cells prepared by glutaraldehyde one-step method.

[0056] After 21 days of immersion immunization, kidney tissues of largemouth bass were collected, 3 per group, placed in 1.5 mL centrifuge tubes, and preserved with Trizol. Tissue RNA was extracted using the Trizol method. The concentration and purity of the extracted RNA were measured using a spectrophotometer. The RNA was reverse transcribed into cDNA using a reverse transcription kit. The reverse transcribed cDNA was used to measure the expression of immune factor genes, including: immunoglobulin M gene ( IgM ), T cell receptor gene ( TCR-α )、Interleukin-12 gene ( IL-12 The sequences of the primers for the immune factor genes are shown in Table 1, and the primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd. β-actin The gene was used as the internal reference gene. The amplification conditions of RT-qPCR were: 95°C for 10 min; 95°C for 1 min, 53°C for 30 s, 72°C for 10 s, and 30 cycles. The expression of immune factor genes was calculated by the ΔΔCt method using the Ct value given by the Bio-IQ5 real-time quantitative PCR instrument (Bio-Rad, USA), and the obtained data were analyzed.

[0057] Table 1. Primer sequences for RT-qPCR detection

[0058] 2. Largemouth bass rhabdovirus (MSRV) infection challenge experiment Largemouth bass (100 per group) were taken 21 days after immunization, and the virus solution was diluted with 0.6% sterile saline containing double antibodies (penicillin and streptomycin) to TCID 50 of 10 1.5 times, 50μL per tail, injected at the base of the pectoral fin, controlled the water temperature (28±0.5℃), checked and recorded the disease situation regularly, and calculated the survival rate and immune protection rate of the experimental group and the control group.

[0059] Immune protection rate = 1-mortality rate of immunized group / mortality rate of control group.

[0060] 3. Results and Analysis 1) Immune titer determination The recombinant G protein immunization group and the BNC-G immunization group were immersed in 5.0, 10.0, 20.0 and 30.0 mg / L (in terms of recombinant G protein concentration) concentrations to immunize largemouth bass summer flower seedlings, and the antibody titers of different groups were determined by indirect agglutination method. The results are shown in Table 2. When the immersion dose of the recombinant G protein immunization group was lower than 10.0 mg / L, the immune titer was not significantly different from that of the control group. At 20.0 mg / L, the immune titer could only reach 1:4~1:8 on the 14th day after immunization. When the immersion dose of the BNC-G immunization group was 5.0 mg / L, the immune titer reached 1:32~1:64 on the 14th day after immunization, and the immune titer could still be maintained at 1:2~1:4 on the 70th day after immunization. When the immersion dose was 10.0 mg / L, the immune titer could reach 1:64~1:128 on the 14th day after immunization, which is a more ideal immersion immunization dose.

[0061] Table 2. Indirect agglutination test results of antibody titers in each group

[0062] 2) Determination of immune factor gene expression The expression levels of immune factor genes after 21 days of immersion immunization Figures 9-11 As shown, Figures 9-11 It can be seen that compared with the control group, the expression levels of immune factor genes in the recombinant G protein immunization group and the BNC-G immunization group were higher, and the expression levels of immune factor genes in the BNC-G immunization group were significantly higher than those in the recombinant G protein immunization group when the immersion concentration was 10, 20, and 30 mg / L. With the increase of the immersion concentration, the expression level of each immune factor gene also increased.

[0063] 3) Mortality and immune protection rate After 21 days of immersion immunization, the largemouth bass rhabdovirus (MSRV) was used to challenge the virus. The survival rate changes of the recombinant G protein immunization group, BNC-G immunization group and blank control group after the virus challenge were as follows Figures 12-13 As shown. Figures 12-13It can be seen that the recombinant G protein has a certain immune protection effect compared with the blank control group, among which the immune protection rate of the recombinant G protein at a concentration of 30 mg / L is 32%, and the immune protection effect of BNC-G is stronger than that of the recombinant G protein, with an immune protection rate of 42% corresponding to a 10 mg / L immersion concentration and an immune protection rate of 83% corresponding to a 30 mg / L immersion concentration. The cumulative relative protection rate on the 14th day after the challenge is shown in Table 3.

[0064] Table 3. Relative protection rate of each group on the 14th day after challenge

[0065] In summary, the present invention performs carboxylation treatment on bacterial cellulose to obtain carboxylated bacterial nanocellulose, and then grafts it with an aldehyde polymer to obtain surface-modified nanocellulose (i.e., poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose). The surface-modified nanocellulose is used as a carrier, and is chemically connected with a viral antigen protein (i.e., a recombinant G protein) to prepare an antigen system (i.e., BNC-G) carrying the viral antigen protein. The antigen system can deliver the viral antigen protein to aquatic animals by soaking immunization, thereby achieving the effect of preventing and controlling viral diseases in aquatic animals, and is suitable for the production of SPF seedlings of aquatic animals and the preparation of immune protection preparations for aquatic animals.

[0066] 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. An antigen system, characterized in that: The antigen system is formed by chemically linking poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose and recombinant G protein; The recombinant G protein is a fusion protein containing the amino acid sequence of the G protein of the largemouth bass rhabdovirus and a His tag. The amino acid sequence of the G protein of the largemouth bass rhabdovirus is shown in SEQ ID NO:

1.

2. The antigen system according to claim 1, characterized in that The chemical connection method is: The poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose was ultrasonically treated with a 2-morpholineethanesulfonic acid buffer having a pH of 5.6-6.2 for 0.2-0.5 h, and 500 mL of the 2-morpholineethanesulfonic acid buffer was used for every 3 g of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-carbonylsuccinimide were added, and ultrasonic treatment was performed at 40kHz and 500W for 1-2h. For every 3g of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, 0.2g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.3g of N-carbonylsuccinimide were used. Solid-liquid separation: the solid product was mixed with PBS buffer at pH = 7.2-7.4, and 500 mL PBS buffer was used for every 3 g of poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose; Add recombinant G protein, the amount of which is 0.5-1.5 times the mass of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, and react at 20-25°C for 6-48h; After the reaction is completed, the dialyzate is dialyzed in pure water, the dialyzate is centrifuged to separate the solid and the liquid, and the obtained solid matter is freeze-dried.

3. The antigen system according to claim 2, characterized in that: The preparation method of the poly (N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose is: Dissolve the aldehyde polymer in pure water, using 0.6 mL of pure water for every 100 mg of aldehyde polymer; Add the carboxylated nanocellulose solution, and then add isocyanocyclohexane while stirring. The amount of carboxylated nanocellulose solution and isocyanocyclohexane is as follows: 2.5 mL of 10 mg / mL carboxylated nanocellulose solution and 22 mg of isocyanocyclohexane are used for every 100 mg of aldehyde polymer, and stir at room temperature for 24 hours. The reaction product is centrifuged to obtain a precipitate, and then dialyzed with pure water for 24 hours and methanol for 24 hours, respectively. The methanol is distilled off under reduced pressure, and the product is freeze-dried.

4. The antigen system according to claim 3, characterized in that The preparation method of the aldehyde polymer is: Potassium hydroxide is dissolved in ethylene glycol, bromoacetaldehyde dimethyl acetal is slowly added while stirring, and the mixture is stirred at 115°C for 72 hours. After cooling to room temperature, pure water is added, and the solution is extracted with chloroform. The extract is dried with magnesium sulfate, and then the magnesium sulfate is removed by filtration and the filtrate is retained. After reduced pressure distillation, a yellow 2-(2,2-dimethoxyethoxy)ethanol liquid is obtained; wherein, in terms of molar ratio, potassium hydroxide: bromoacetaldehyde dimethyl acetal = 1.5-2.5:1; Take 2-(2,2-dimethoxyethoxy)ethanol, dissolve it and 3-[[(benzylthio)thiocarbonyl]thio]propionic acid in dichloromethane at 0°C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and continue stirring at 0°C for 2 hours, stir at room temperature for 40 hours, wash, dry and distill under reduced pressure, and purify by column chromatography to obtain 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propanoate; wherein the molar ratio is 2-(2,2-dimethoxyethoxy)ethanol:3-[[(benzylthio)thiocarbonyl]thio]propionic acid:1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride:4-dimethylaminopyridine=1:0.2~0.3:0.4~0.7:0.2~0.3; Dissolve 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate, N-isopropylacrylamide, and azobisisobutyronitrile in tetrahydrofuran, wherein the molar ratio of 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate:N-isopropylacrylamide:azobisisobutyronitrile is 1:90-120:0.08-0.12; place in a sealed container and remove oxygen, stir at 65°C for 120h; terminate the reaction by ice bath for 10min, dialyze with pure water for 24h and methanol for 24h, respectively, and then perform reduced pressure distillation to collect the product; Take 160 mg of the product obtained by vacuum distillation, dissolve it in a mixture of 2.4 mL of tetrahydrofuran and 2 mL of hydrochloric acid, both with a concentration of 1 mol / L, and stir it at room temperature for 72 hours for deprotection; dialyze the obtained sample with pure water for 24 hours and methanol for 24 hours, and remove the solvent by vacuum distillation to obtain poly(N-isopropylacrylamide) with aldehyde groups, which is an aldehyde polymer.

5. The antigen system according to claim 3, characterized in that: The method for preparing the carboxylated nanocellulose comprises: preparing the nanocellulose and carboxylating the nanocellulose, wherein: Preparation of nanocellulose: Wash bacterial cellulose with 0.1 mol / L sodium hydroxide solution to remove surface impurities, then wash repeatedly with distilled water until the pH of the bacterial cellulose surface is 8; put bacterial cellulose into 68% sulfuric acid solution, use 20 mL sulfuric acid solution for every 0.5 g of bacterial cellulose, heat at 50°C for 2 h, then add ice-water mixture to the reaction system to terminate the reaction, dialyze in pure water with a 14 kDa dialysis bag until the pH is neutral, and freeze-dry to obtain nanocellulose; Carboxylation of nanocellulose: Take 1g of nanocellulose, add 40mL of deionized water and stir, then add 0.05~2g of iron oxide with a particle size of 1μm, and heat to 80~85℃ while stirring after ultrasonic dispersion, add 30~40mL of 20% hydrogen peroxide solution and continue stirring and heating. After 3h, add 30~40mL of 20% hydrogen peroxide solution, continue stirring and heating for 3h; cool the reaction system to room temperature, add 0.5M sodium hydroxide to adjust the pH to 7.8~8.8 and stir; filter the mixed solution to obtain the filtrate, centrifuge the filtrate, collect the supernatant and add 20~40mL of ethanol for precipitation, filter the precipitated product, dialyze it in pure water with a 14kDa dialysis bag for 2~3 days, and freeze-dry the liquid in the dialysis bag to obtain carboxylated nanocellulose powder.

6. Use of the antigen system according to claim 1 in the preparation of SPF aquatic seedlings.

7. The use according to claim 6, characterized in that: In the antigen system, the concentration of the viral antigen protein is 2-30 mg / L.

8. Use of the antigen system according to claim 1 in the preparation of immune protection preparations for aquatic animals.

9. The use according to claim 8, characterized in that: The immune protection preparation for aquatic animals adopts immersion immunization, and the immersion immunization time is 1~5h.

Images