An aquatic animal antigen system and its application

Abstract

The present invention belongs to the field of biomedical technology, and discloses an aquatic animal antigen system and its application. In the present invention, bacterial cellulose is carboxylated to obtain carboxylated bacterial nanocellulose, and then it is grafted with an aldehyde polymer to obtain surface-modified nanocellulose (i.e., poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose). Using the surface-modified nanocellulose as a carrier, it is chemically linked with a viral antigen protein (i.e., recombinant G protein) to prepare an antigen system carrying the viral antigen protein (i.e., BNC-G). The antigen system can deliver the viral antigen protein into the body of aquatic animals through immersion immunization, achieving the effect of preventing and controlling viral diseases of aquatic animals, and is applicable to the production of SPF fry of aquatic animals and the preparation of immunoprotective agents for aquatic animals.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to an antigen system for aquatic animals and its application. Background Art

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

[0003] Cellulose is a natural polymer with a wide source, rich resources, and low price. Natural cellulose can be processed to obtain microcrystalline cellulose, nanofibrillated cellulose, etc. through processes such as crushing, acid-base or enzymatic hydrolysis. Compared with powdered cellulose and microcrystalline cellulose, nanofibrillated cellulose has the advantages of high biocompatibility, biodegradability, and safety and non-toxicity, and has good application prospects in constructing drug and vaccine delivery systems, drug sustained release, and scaffolds for tissue engineering. In the medical field, the research on nanofibrillated cellulose mainly focuses on anti-cancer drugs, hydrogels, and wound dressings, etc., and there is still a lack of technical support in antiviral drugs for aquatic animals, especially in the preparation of SPF fry of aquatic animals.

[0004] In addition, in production practice, a large number of wild virus-free parents are usually screened for multiple generations, and then the wild virus-free parents are used to breed offspring for several generations and virus detection is carried out, and finally SPF fry are obtained. However, this method for producing SPF fry of aquatic animals has the disadvantages of a long production cycle, high cost, and great difficulty in isolating viruses, which limits the production and popularization and application of SPF fry of aquatic animals. Summary of the Invention

[0005] In view of the above technical status quo, in order to further improve the immune effect of aquatic animal virus vaccines, and at the same time solve the technical defects of traditional SPF seed production methods and achieve the prevention and control of viral diseases in aquatic animals, the present invention uses surface-modified nanocellulose as a carrier for virus antigen proteins to prepare an antigen system. The antigen system can transport virus antigen proteins into aquatic animals through immersion immunization to achieve the technical purpose of preventing and controlling diseases in aquatic animals. For this reason, 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 with 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, and the amino acid sequence of the G protein of the largemouth bass rhabdovirus is shown in SEQ ID NO:1.

[0007] Furthermore, in the above antigen system, the chemical connection method is as follows:

[0008] Ultrasonically treat poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose with 2-(N-morpholino)ethanesulfonic acid buffer solution with a pH of 5.6-6.2 for 0.2-0.5 h, and use 500 mL of 2-(N-morpholino)ethanesulfonic acid buffer solution for every 3 g of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose;

[0009] Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-carbonyldiimidazole, and ultrasonically treat at 40 kHz and 500 W for 1-2 h. Use 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.3 g of N-carbonyldiimidazole for every 3 g of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose;

[0010] Perform solid-liquid separation, mix the solid product with PBS buffer solution with a pH of 7.2-7.4, and use 500 mL of PBS buffer solution for every 3 g of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose;

[0011] Add recombinant G protein, and the addition amount of recombinant G protein is 0.5-1.5 times the mass of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, and react at 20-25 °C for 6-48 h;

[0012] After the reaction is completed, dialyze in pure water, centrifuge the dialysis solution to separate the solid and liquid, and freeze-dry the obtained solid substance.

[0013] Furthermore, in the above antigen system, the preparation method of the poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose is as follows:

[0014] Dissolve the aldehyde group polymer in pure water, and use 0.6 mL of pure water for every 100 mg of the aldehyde group polymer;

[0015] Add the carboxylated nanocellulose solution, and then add isocyanocyclohexane while stirring. The dosages of the carboxylated nanocellulose solution and isocyanocyclohexane are: use 2.5 mL of a carboxylated nanocellulose solution with a concentration of 10 mg / mL and 22 mg of isocyanocyclohexane for every 100 mg of the aldehyde group polymer, and stir at room temperature for 24 h;

[0016] After centrifuging the reaction product to obtain the precipitate, dialyze it with 5 L of pure water for 24 h and then with 2 L of methanol for 24 h, remove methanol by vacuum distillation, and freeze-dry to obtain the product.

[0017] Furthermore, in the preparation method of poly(N-isopropylacrylamide)-grafted carboxylated bacterial nanocellulose, the preparation method of the aldehyde group polymer is as follows:

[0018] Dissolve potassium hydroxide in ethylene glycol, slowly add bromoacetaldehyde dimethyl acetal while stirring and stir at 115 °C for 72 h. After cooling to room temperature, add pure water, then extract the solution with chloroform, dry the extract with magnesium sulfate, then filter to remove magnesium sulfate and retain the filtrate, and obtain a yellow liquid of 2-(2,2-dimethoxyethoxy)ethanol after vacuum distillation; where in terms of molar ratio, potassium hydroxide:bromoacetaldehyde dimethyl acetal = 1.5 - 2.5:1;

[0019] Take 2-(2,2-dimethoxyethoxy)ethanol, dissolve it with 3-[[(benzylthio)thiocarbonyl]thio]propionic acid (BSPA) in dichloromethane at 0 °C, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and then continue to stir at 0 °C for 2 h and at room temperature for 40 h. Wash, dry and vacuum distill, and purify by column chromatography to obtain 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate; where in terms of molar ratio, 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;

[0020] Dissolve 2-(2,2-dimethoxyethoxy)ethyl 3-(benzylthiocarbonylthio)propionate, N-isopropylacrylamide, and azobisisobutyronitrile in tetrahydrofuran, where the molar ratio is 2-(2,2-dimethoxyethoxy)ethyl 3-(benzylthiocarbonylthio)propionate:N-isopropylacrylamide:azobisisobutyronitrile = 1:90 - 120:0.08 - 0.12; place it in a sealed container and remove oxygen, stir at 65 °C for 120 h; terminate the reaction with an ice bath for 10 min, dialyze with pure water for 24 h and then with methanol for 24 h, and then perform vacuum distillation to collect the product;

[0021] Take 160 mg of the vacuum-distilled product and 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 at room temperature for 72 h for deprotection; dialyze the obtained sample with pure water for 24 h and then with methanol for 24 h, remove the solvent by vacuum distillation to obtain poly(N-isopropylacrylamide) with aldehyde groups, which is the aldehyde group polymer.

[0022] Furthermore, in the preparation method of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, the preparation method of the carboxylated nanocellulose includes: the preparation of nanocellulose and the carboxylation of nanocellulose, where:

[0023] Preparation of nanocellulose: Wash bacterial cellulose with 0.1 mol / L sodium hydroxide solution to remove surface impurities, and then wash repeatedly with distilled water until the pH of the bacterial cellulose surface is 8; put the bacterial cellulose into 68% sulfuric acid solution, use 20 mL of 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 with a 14 kDa dialysis bag in pure water until the pH is neutral, and freeze-dry to obtain nanocellulose;

[0024] Carboxylation of nanocellulose: Take 1 g of nanocellulose, add 40 mL of deionized water and stir, then add 0.05 - 2 g of iron oxide with a particle size of 1 μm, disperse ultrasonically and heat to 80 - 85 °C while stirring, add 30 - 40 mL of 20% hydrogen peroxide solution and continue stirring and heating, add another 30 - 40 mL of 20% hydrogen peroxide solution after 3 h, and continue stirring and heating for 3 h; cool the reaction system to room temperature, add 0.5 M sodium hydroxide to adjust the pH to 7.8 - 8.8 and stir; filter the mixture to obtain the filtrate, centrifuge the filtrate, collect the supernatant and add 20 - 40 mL of ethanol for precipitation, filter to obtain the precipitate product, dialyze with a 14 kDa dialysis bag in pure water for 2 - 3 days, and freeze-dry the liquid in the dialysis bag to obtain carboxylated nanocellulose powder.

[0025] In a second aspect, the present invention claims the use of the above antigen system in the preparation of SPF aquatic fry, specifically, in the antigen system, the concentration of the viral antigen protein is 2 to 30 mg / L.

[0026] In a third aspect, the present invention also claims 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 is used for immersion immunization, and the immersion immunization time is 1 to 5 h.

[0027] Compared with the prior art, the "antigen system for aquatic animals and its application" of the present invention has at least the following beneficial effects:

[0028] The present invention carboxylates 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). Compared with ordinary nanocellulose, the surface-modified nanocellulose prepared by the present invention has good water solubility and can penetrate the body surface of aquatic animals in water and enter tissues and cells.

[0029] Using the surface-modified nanocellulose as a carrier, chemically connect it with a viral antigen protein (i.e., recombinant G protein) to prepare an antigen system carrying the viral antigen protein (i.e., BNC-G). The antigen system can deliver the viral antigen protein into aquatic animals through immersion treatment, achieving the effect of preventing and controlling diseases of aquatic animals, and is applicable to the production of SPF fry 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%.

[0030] The antigen system provided by the present invention has a simple usage method and low preparation cost, and has good practical application prospects in the aquatic industry. Description of the Drawings

[0031] Figure 1 It is a scanning electron microscope image of carboxylated nanocellulose.

[0032] Figure 2 It is an appearance image of a suspension of carboxylated nanocellulose dispersed in water.

[0033] Figure 3 It is a particle size distribution diagram of carboxylated nanocellulose.

[0034] Figure 4 It is the dissolution situation of carboxylated nanocellulose and poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose in pure water.

[0035] Figure 5 Particle size distribution diagrams of carboxylated nanocellulose and poly(N-isopropylacrylamide)-grafted carboxylated bacterial nanocellulose.

[0036] Figure 6 Standard curve of aldehyde polymer content / absorbance.

[0037] Figure 7 SDS-PAGE analysis results of total proteins induced and expressed in Escherichia coli BL21. Lane M is the protein molecular weight standard, and lane 1 is the total proteins.

[0038] Figure 8 SDS-PAGE analysis results of total proteins purified by Ni-NTA agarose purification resin. Lane M is the protein molecular weight standard, and lane 1 is the purified recombinant G protein.

[0039] Figure 9 After 21 days of immersion immunization IgM Gene expression levels.

[0040] Figure 10 After 21 days of immersion immunization TCR-α Gene expression levels.

[0041] Figure 11 After 21 days of immersion immunization IL-12 Gene expression levels.

[0042] Figure 12 Survival rate changes of the recombinant G protein immunization group and the blank control group after virus challenge.

[0043] Figure 13 Survival rate changes of the BNC-G immunization group and the blank control group after virus challenge. Specific implementation manners

[0044] The present invention will be described below in conjunction with the embodiments. The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0045] The information of test materials in the embodiments is as follows:

[0046] Main reagents or kits: Bacterial cellulose was purchased from Hainan Yide Food Co., Ltd.; dialysis bags (14 kDa) were purchased from Sangon Biotech (Shanghai) Co., Ltd.; 1,4-butanediol diglycidyl ether, 2-morpholinoethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-carbonyldiimidazole were purchased from Sigma-Aldrich Co., LLC in the United States. The pET-32a(+) vector (product number LM1216) was purchased from Shanghai Lianmai Biotechnology Co., Ltd.; competent strains Top10 and BL21(DE3) were purchased from Beijing ComWin Biotech Co., Ltd.; the pMD19-T vector was purchased from TaKaRa Bio Inc. The viral RNA extraction kit, DM2000 DNA Marker, PCR amplification kit, chromogenic agent DAB, and BCA protein assay kit were purchased from Beijing ComWin Biotech Co., Ltd.; the reverse transcription kit, restriction endonucleases (EcoRI, Hind III), and T4 DNA ligase were purchased from TaKaRa Bio Inc.; the DNA purification kit was purchased from Axygen; primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the protein Marker was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; murine anti-histidine monoclonal antibody and horseradish peroxidase-labeled goat anti-mouse IgG were purchased from Abcam; the ELISA detection kit was purchased from Beijing ComWin Biotech Co., Ltd.; and sheep red blood cells were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0047] Main test instruments: Thermo Multiskan MK3 microplate reader, Thermo Fisher Scientific Inc. in the United States; PCR instrument, Bio-Rad Laboratories, Inc. in the United States. MALVERN nanolaser particle size analyzer, Malvern Instruments Ltd. in the United Kingdom; FEI field emission scanning electron microscope, FEI Company in the United States. Liquid Zeta particle size / potential analyzer, Anton Paar GmbH in Austria.

[0048] Test materials: The Novirhabdovirus largemouth bass strain was isolated and preserved by the Aquatic Science Laboratory of the College of Animal Science and Technology, Northwest A&F University; the test animals were juvenile largemouth bass (body length: 4 - 6 ± 0.5 cm) purchased from Yongsheng Fisheries Development Co., Ltd.

[0049] Example 1

[0050] This example describes the preparation of carboxylated bacterial nanocellulose.

[0051] 1. Preparation of nanocellulose

[0052] Wash the bacterial cellulose with 0.1 mol / L sodium hydroxide solution to remove surface impurities, and then wash it repeatedly with distilled water until the pH of the bacterial cellulose surface is 8; put the bacterial cellulose into a 68% mass fraction sulfuric acid solution after being broken by a blender, with 20 mL of sulfuric acid solution used for every 0.5 g of bacterial cellulose, stir and heat at 50 °C for 2 h (stirring speed 800 r / min), then add 10 times the volume of ice-water mixture to the reaction system to terminate the reaction; then dialyze with a 14 kDa dialysis bag in pure water until the pH is neutral, and place the liquid in the dialysis bag in a freeze dryer for freeze drying to obtain nanocellulose.

[0053] 2. Carboxylation of nanocellulose

[0054] Take 1 g of the dried nanocellulose prepared above, add 40 mL of deionized water and stir, then add 0.05 - 2 g of iron oxide with a particle size of 1 μm, disperse ultrasonically at 40 kHz for 10 - 20 min, stir and heat to 80 - 85 °C while stirring, add 30 - 40 mL of 20% hydrogen peroxide solution and continue to stir and heat, add another 30 - 40 mL of 20% hydrogen peroxide solution after 3 h, and continue to stir and heat for 3 h; cool the reaction system to room temperature, add 0.5 M 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 12000 rpm for 30 min, collect the supernatant and add 30 mL of ethanol for precipitation, filter to obtain the precipitate product, dialyze with a 14 kDa dialysis bag in 5 L of pure water for 2 - 3 days, and freeze dry the liquid in the dialysis bag to obtain carboxylated nanocellulose powder.

[0055] 3. Characterization of nanocellulose

[0056] 1) Scanning electron microscope detection

[0057] 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 magnification of 100,000 times under a high voltage of 5 kV ( Figure 1 )

[0058] 2) Dynamic light scattering (DLS) analysis

[0059] Take the carboxylated nanocellulose prepared in 1.2.2, dissolve it in pure water and disperse ultrasonically for 10 min, add it to a quartz cuvette for DLS analysis ( Figure 3 )

[0060] 4. Results and analysis

[0061] Figure 1 It is the scanning electron microscope image of carboxylated nanocellulose. It can be seen that the particle size of carboxylated bacterial nanocellulose is all below 1000 nm and the size is relatively uniform.Figure 2 Figure 1 shows the appearance of a suspension of carboxylated nanocellulose in water. There are no obvious particles in the suspension, and no obvious precipitation occurs after standing for half an hour. Figure 3 Figure 2 shows the DLS analysis results used to characterize the particle size distribution of carboxylated nanocellulose. Figure 3 It can be seen that the size of carboxylated bacterial nanocellulose shows a normal distribution, with an average particle size of 215 nm.

[0062] Example 2

[0063] This example describes the preparation of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, including: the preparation of aldehyde group polymer, and the grafting reaction of aldehyde group polymer with carboxylated bacterial nanocellulose to obtain poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose.

[0064] 1. Preparation of aldehyde group polymer

[0065] Add 20 g (0.36 mol) of potassium hydroxide to 37.5 mL of ethylene glycol, stir and reflux at 115 °C. After the potassium hydroxide is completely dissolved, slowly add 15.6 mL of bromoacetaldehyde dimethyl acetal 21.6 mL (0.18 mol) within 30 min, and continuously stir at 115 °C for 72 h to obtain suspension a. Cool suspension a to room temperature, add 150 mL of pure water to dissolve the precipitate, then extract with 5×50 mL of chloroform, dry the extract with magnesium sulfate, then filter to remove magnesium sulfate and retain the filtrate, and perform vacuum distillation to obtain a yellow liquid of 2-(2,2-dimethoxyethoxy)ethanol;

[0066] Dissolve 3.3 g (22 mmol) of 2-(2,2-dimethoxyethoxy)ethanol and 1.8 g (6.6 mmol) of 3-[[(benzylthio)thiocarbonyl]thio]propionic acid (BSPA) in 30 mL of dichloromethane at 0 °C, then add 2.8 g (14.6 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.69 g (5.6 mmol) of 4-dimethylaminopyridine, continue to stir at 0 °C for 2 h, and then stir at room temperature for 40 h to obtain mixture b; wash mixture b 3 - 5 times with 50 mL of pure water, then dry with magnesium sulfate, filter to remove magnesium sulfate, distill off the solvent under reduced pressure, and further purify by column chromatography (the mobile phase used is: hexane:ethyl acetate = 1:1 by volume ratio) to obtain the product 2-(2,2-dimethoxyethoxy)ethyl-3-(benzylthiocarbonylthio)propionate;

[0067] 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 it in a sealed container and remove oxygen, stir at 65 °C for 120 h; terminate the reaction in an ice bath for 10 min, dialyze with pure water for 24 h and then with methanol for 24 h respectively, and then perform vacuum distillation to collect the product;

[0068] Take 160 mg of the vacuum-distilled product, 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 at room temperature for 72 h to deprotect; dialyze the obtained sample with pure water for 24 h and then with methanol for 24 h, remove the solvent by vacuum distillation to obtain poly(N-isopropylacrylamide) with aldehyde groups, which is the aldehyde group polymer.

[0069] 2. Grafting reaction of aldehyde group polymer with carboxylated bacterial nanocellulose

[0070] Dissolve 100 mg of the aldehyde group polymer in 0.6 mL of pure water, add 2.5 mL of a carboxylated nanocellulose solution with a concentration of 10 mg / mL, and then add 22 mg of isocyanocyclohexane while stirring, and stir at room temperature for 24 h;

[0071] After centrifuging the reaction product to obtain the precipitate, dialyze it with 5 L of pure water for 24 h and then with 2 L of methanol for 24 h respectively, remove methanol by vacuum distillation, and freeze-dry to obtain poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose.

[0072] 3. Characterization of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose

[0073] Perform solubility tests on carboxylated bacterial nanocellulose and poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, and perform DLS analysis on both in a neutral aqueous environment. The DLS analysis method is shown in Example 1. Due to the π→π* transition of the C=S bond of the aldehyde group polymer prepared in 1.2.1, there is a characteristic absorption peak at 305 nm, so the grafting situation of the aldehyde group polymer on the surface of carboxylated nanocellulose can be analyzed by ultraviolet-visible spectrophotometry. According to the absorbance of aldehyde group polymers with different concentrations at 305 nm, draw a standard curve of aldehyde group polymer content / absorbance, and then calculate the grafting efficiency.

[0074] Grafting efficiency = (amount of aldehyde group polymer grafted onto nanocellulose / amount of initially added aldehyde group polymer) × 100%

[0075] 4. Results and analysis

[0076] The dissolution of the same amount of carboxylated bacterial nanocellulose and poly(N-isopropylacrylamide)-grafted carboxylated bacterial nanocellulose in pure water is as follows Figure 4 shown. The dispersion solution of the former is relatively turbid, while the latter is relatively clear, indicating that the water solubility of carboxylated bacterial nanocellulose is enhanced after surface modification.

[0077] The results of DLS analysis are as follows Figure 5 shown. After surface modification, the average particle size of carboxylated bacterial nanocellulose increases from 215 nm to 338 nm, but the particle size distribution range becomes smaller, and the polydispersity index (PDI) decreases from 0.34 to 0.26, indicating that the particle size distribution of poly(N-isopropylacrylamide)-grafted carboxylated bacterial nanocellulose is more uniform. Based on the analysis by ultraviolet-visible spectrophotometry, the standard curve of aldehyde group polymer content / absorbance is as follows Figure 6 shown. Substituting the absorbance of poly(N-isopropylacrylamide)-grafted carboxylated bacterial nanocellulose into the standard curve, the grafting efficiency is calculated to be 56%.

[0078] Example 3

[0079] This example describes the preparation of an antigen system, including: expression and purification of recombinant G protein, and chemical connection of recombinant G protein with a carrier.

[0080] 1. Expression and purification of recombinant G protein

[0081] Referring to the instruction manual of the viral RNA extraction kit, total RNA of largemouth bass rhabdovirus was extracted, and the RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0082] According to the G protein gene sequence of largemouth bass rhabdovirus in the GenBank database (Accession NO.: KF146308.1), primers were designed:

[0083] G-F (EcoR I): 5'-GAGCTCCTATGACAAGCGCACTCAGAG-3' (SEQ ID NO:2);

[0084] G-R (Hind Ⅲ): 5'-GAATTCTCAGTGGAATGAGTCGGAGTC-3' (SEQ ID NO:3)

[0085] PCR amplification reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min. Take the PCR product for agarose gel electrophoresis identification, electrophoresis conditions: constant voltage of 120 V for 20 min. Cut the gel of the electrophoresis product, recover it, purify it with a DNA purification kit, ligate the purified product with the pMD19-T vector after double digestion with EcoR I and Hind III to obtain the recombinant plasmid pMD19T-G, transform it into Escherichia coli Top10 competent cells, and obtain positive strains through blue-white screening. At the same time, extract the recombinant plasmid pMD19T-G for PCR and sequencing identification.

[0086] Digest the recombinant plasmids pMD19T-G and pET-32a(+) with EcoR I and Hind III respectively, perform agarose gel electrophoresis, cut the gel, recover, purify with a DNA purification kit, ligate with T4 ligase to obtain the recombinant plasmid pET32a-G, and transform it into Escherichia coli BL21(DE3) competent cells. At the same time, extract the recombinant plasmid pET32a-G for PCR and sequencing identification.

[0087] Cultivate Escherichia coli BL21(DE3) containing the recombinant plasmid pET32a-G at 37°C with shaking for 1 h, spread it on an LB plate (containing ampicillin) for cultivation, overnight at 37°C, pick a single colony into an LB liquid medium (containing ampicillin), and cultivate overnight at 37°C with shaking. Take another 1% of the bacterial solution from it for enlarged cultivation in a new medium until the OD 600 reaches 0.6, add IPTG (isopropyl-β-D-thiogalactopyranoside) to make its final concentration 1.0 mmol / L, induce cultivation for 4 - 6 h, centrifuge to discard the supernatant, wash the precipitate 2 times with 1×PBS, add an equal volume of SDS loading buffer, mix well and boil for 5 min, and perform 10% SDS-PAGE electrophoresis verification.

[0088] Collect a large amount of the induced expression bacterial solution, centrifuge at 12000 r / min at 4°C for 10 min in a 50 mL centrifuge tube, collect the precipitate and resuspend it, ultrasonicate in an ice bath for 30 min, centrifuge at 12000 r / min at 4°C for 20 min, collect the precipitate, dissolve it with a denaturing solution containing 8 mol / L urea, centrifuge at 12000 r / min at 4°C for 10 min, discard the supernatant, purify it with Ni-NTA agarose purification resin, and perform the purification operation according to the instructions. Then dialyze the purified protein solution in PBS containing 6.0, 4.0, 2.0, 1.0 mol / L urea respectively, and finally dialyze overnight in 1×PBS, collect the dialysate, and obtain the recombinant G protein after freeze-drying for storage for later use. The recombinant G protein is a fusion protein containing the amino acid sequence of the G protein of Micropterus salmoides rhabdovirus (SEQ ID NO:1) and a His tag.

[0089] 2. Chemically link the recombinant G protein with the vector

[0090] Add 3 g of poly(N-isopropylacrylamide)-grafted carboxylated bacterial nanocellulose to 500 mL of 2-(N-morpholino)ethanesulfonic acid buffer with a pH of 5.6 and a concentration of 0.1 mol / L, and sonicate at 40 kHz and 500 W for 0.5 h;

[0091] Add 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.3 g of N-carbonyldiimidazole, and sonicate at 40 kHz and 500 W for 2 h;

[0092] Centrifuge at 6000 rpm for 5 min to separate the solid and liquid phases, and mix all the centrifuged precipitates with 500 mL of PBS buffer at pH = 7.4;

[0093] Add 3 g of the recombinant G protein and react at 25 °C for 48 h;

[0094] After the reaction, dialyze with a dialysis bag with a cut-off molecular weight of 100 kDa in pure water for 72 h. Centrifuge the dialysis solution at 3000 rpm for 10 min to separate the solid and liquid phases. The obtained centrifuged precipitate is the poly(N-isopropylacrylamide)-grafted carboxylated bacterial nanocellulose-recombinant G protein complex (BNC-G). After freeze-drying, store it at 4 °C.

[0095] Accurately weigh 1.0 g of the BNC-G freeze-dried powder, dilute it to 10 mL with pure water, and disperse it evenly by sonication. Refer to the BCA protein concentration assay kit instruction manual, dilute the dispersed solution in gradients, and use a Thermo Multiskan MK3 microplate reader to measure the content of the recombinant G protein. Calculate the loading capacity of the recombinant G protein in BNC-G according to the recombinant G protein concentration. The formula for the loading capacity is as follows:

[0096] Protein loading capacity = (mass of recombinant G protein in the complex / mass of the complex) × 100%

[0097] 3. Results and analysis

[0098] The recombinant plasmid pET32a-G was expressed in Escherichia coli BL21. After 4 h of induction, SDS-PAGE electrophoresis was performed. As shown in the SDS-PAGE electrophoresis Figure 7 , the expression of the recombinant G protein (with a relative molecular mass of approximately 75.6 kDa) was visible in the lane, which was consistent with the expectation. After purification with Ni-NTA agarose purification resin, a single target band was observed ( Figure 8). The protein loading of poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose-recombinant G protein complex (BNC-G) was determined to be 23.8% by a BCA protein concentration assay kit.

[0099] Example 4

[0100] This example describes the evaluation of the antiviral effect of the antigen system.

[0101] 1. Immunization of Micropterus salmoides

[0102] The juvenile Micropterus salmoides were randomly divided into 9 groups, with 300 fish in each group, and were separately reared in aquaculture tanks filled with 30 L of aerated tap water. Recombinant G protein freeze-dried powder and BNC-G were used, and 8 groups were respectively soaked and immunized at 5.0, 10.0, 20.0, and 30.0 mg / L (calculated based on the concentration of recombinant G protein). After 2 h of soaking immunization, they were transferred to a conventional aquaculture tank for rearing. A heating rod was used to adjust the water temperature to maintain it at 28 ± 0.5 °C, and aeration was used to increase the dissolved oxygen content in the water to more than 6 mg / L. The white / black light cycle was 14:10 h. Feed was given once a day in the morning, and the feed input was limited to be eaten within 10 min. The residual feed at the bottom of the tank was removed 1 h after feeding, and 1 / 3 of the water was changed. The aquaculture tank was cleaned once a week. The blank control group was not treated with soaking immunization, and the remaining aquaculture conditions were the same as those of the experimental groups.

[0103] On the 14th, 21st, 28th, 49th, and 70th days after immunization, 10 fish were taken from each group, the spinal columns were cut to death, 2.0 mL of 0.6% normal saline was added, and they were homogenized with a tissue homogenizer at 12000 rpm for 5 min and centrifuged at 3000 rpm for 10 min with a high-speed refrigerated centrifuge. The supernatant was transferred to a new tube and stored frozen in a -80 °C refrigerator. Indirect agglutination was used for antibody detection, and the sensitized carrier selected was sheep red blood cells prepared by the one-step method with glutaraldehyde.

[0104] Twenty-one days after soaking immunization, the kidney tissues of Micropterus salmoides were collected, 3 fish from each group were placed in 1.5 mL centrifuge tubes, Trizol was added for preservation, and tissue RNA was extracted using the Trizol method. The concentration and purity of the extracted RNA were measured with a spectrophotometer. The RNA was reverse transcribed into cDNA using a reverse transcription kit. The expression levels of immune factor genes were determined using the reverse transcribed cDNA, including: immunoglobulin M gene ( IgM ), T cell receptor gene ( TCR-α ), interleukin 12 gene ( IL-12 ). The primer sequences of the immune factor genes are shown in Table 1, and the primers were synthesized by Shanghai Sangon Biotech Co., Ltd. Using β-actinThe gene was used as an internal reference gene. The amplification conditions for RT-qPCR were as follows: 95°C for 10 min; 95°C for 1 min, 53°C for 30 s, 72°C for 10 s, for 30 cycles. The Ct values given by the Bio-IQ5 real-time quantitative PCR instrument (Bio-Rad, USA) were used to calculate the expression levels of immune factor genes by the ΔΔCt method, and the obtained data were analyzed.

[0105] Table 1. Primer sequences for RT-qPCR detection

[0106]

[0107] 2. Infection challenge experiment with Micropterus salmoides rhabdovirus (MSRV)

[0108] Take Micropterus salmoides (100 tails per group) 21 days after immunization. Dilute the virus solution to 10 50 times of TCID 1.5 with 0.6% sterilized normal saline containing double antibiotics (penicillin and streptomycin), and inject at a dose of 50 μL per tail at the base of the pectoral fin. Control the water temperature (28 ± 0.5°C), regularly check and record the disease occurrence, and count the survival rate and immune protection rate of the experimental group and the control group.

[0109] Immune protection rate = 1 - mortality rate of the immunized group / mortality rate of the control group.

[0110] 3. Results and analysis

[0111] 1) Determination of immune titer

[0112] The recombinant G protein immunized group and the BNC-G immunized group were used to soak and immunize Micropterus salmoides fry at concentrations of 5.0, 10.0, 20.0, and 30.0 mg / L (calculated based on the concentration of recombinant G protein), respectively. The antibody titers of different groups were determined by the indirect agglutination method. The results are shown in Table 2. When the soaking dose of the recombinant G protein immunized group was lower than 10.0 mg / L, there was no significant difference in the immune titer compared with the control group. When the concentration was 20.0 mg / L, the immune titer on the 14th day after immunization only reached 1:4 - 1:8. When the soaking dose of the BNC-G immunized group was 5.0 mg / L, the immune titer reached 1:32 - 1:64 on the 14th day after immunization, and still remained at 1:2 - 1:4 on the 70th day after immunization. When the soaking dose was 10.0 mg / L, the immune titer reached 1:64 - 1:128 on the 14th day after immunization, which was an ideal soaking immunization dose.

[0113] Table 2. Detection results of indirect agglutination reaction of antibody titers in each group

[0114]

[0115] 2) Determination of the expression level of immune factor genes

[0116] The expression levels of immune factor genes after 21 days of immersion immunization were as Figures 9 - 11 shown Figures 9 - 11 It can be seen from Figures 9 - 11 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 levels of various immune factor genes also increased

[0117] 3) Mortality rate and immune protection rate

[0118] After 21 days of immersion immunization, the largemouth bass rhabdovirus (MSRV) was used for challenge. The survival rate changes of the recombinant G protein immunization group, the BNC-G immunization group, and the blank control group after challenge were as Figures 12 - 13 shown Figures 12 - 13 It can be known from Figures 12 - 13 that the recombinant G protein has a certain immune protection effect compared with the blank control group. The immune protection rate of the recombinant G protein at an immersion concentration of 30 mg / L was 32%, while the immune protection effect of BNC-G was stronger than that of the recombinant G protein. The immune protection rate corresponding to an immersion concentration of 10 mg / L was 42%, and the immune protection rate corresponding to an immersion concentration of 30 mg / L reached 83%. The cumulative relative protection rate on the 14th day after challenge is shown in Table 3

[0119] Table 3. Relative protection rate of each group on the 14th day after challenge

[0120]

[0121] In summary, in the present invention, bacterial cellulose is carboxylated to obtain carboxylated bacterial nanocellulose, and then it is grafted 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 chemically linked with a viral antigen protein (i.e., recombinant G protein) to prepare an antigen system carrying the viral antigen protein (i.e., BNC-G). The antigen system can deliver the viral antigen protein into aquatic animals through immersion immunization, achieving the effect of preventing and controlling viral diseases of aquatic animals, and is applicable to the production of SPF fry of aquatic animals and the preparation of immune protection preparations for aquatic animals

[0122] The embodiments described above are only a part of the embodiments of the present invention, rather than all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art under the premise of not making creative efforts through relevant deductions and substitutions made under the conditions of the inventive concept of the present invention fall within 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 with recombinant G protein; The recombinant G protein is a fusion protein containing the amino acid sequence of the G protein of Micropterus salmoides rhabdovirus and a His tag, and the amino acid sequence of the G protein of Micropterus salmoides rhabdovirus is as shown in SEQ ID NO:1; The preparation method of the poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose is as follows: Dissolve the aldehyde group polymer in pure water, and use 0.6 mL of pure water for every 100 mg of the aldehyde group polymer; Add the carboxylated nanocellulose solution, and then add isocyanocyclohexane while stirring. The dosages of the carboxylated nanocellulose solution and isocyanocyclohexane are: use 2.5 mL of a carboxylated nanocellulose solution with a concentration of 10 mg / mL and 22 mg of isocyanocyclohexane for every 100 mg of the aldehyde group polymer, and stir at room temperature for 24 h; After the reaction product is centrifuged to obtain the precipitate, dialyze it with pure water for 24 h and then with methanol for 24 h, remove methanol by reduced pressure distillation, and freeze-dry it to obtain the product; The aldehyde group polymer is poly(N-isopropylacrylamide) with aldehyde groups.

2. The antigen system according to claim 1, characterized in that, The chemical linking method is as follows: Ultrasonically treat the poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose with a 2-morpholinoethanesulfonic acid buffer solution with a pH of 5.6 - 6.2 for 0.2 - 0.5 h, and use 500 mL of the 2-morpholinoethanesulfonic acid buffer solution for every 3 g of the poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose; Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-carbonyldiimidazole, and ultrasonically treat at 40 kHz and 500 W for 1 - 2 h. Use 0.2 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 0.3 g of N-carbonyldiimidazole for every 3 g of the poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose; Perform solid-liquid separation, mix the solid product with a PBS buffer solution with a pH of 7.2 - 7.4, and use 500 mL of the PBS buffer solution for every 3 g of the poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose; Add the recombinant G protein, and the addition amount of the recombinant G protein is 0.5 - 1.5 times the mass of the poly(N-isopropylacrylamide) grafted carboxylated bacterial nanocellulose, and react at 20 - 25 °C for 6 - 48 h; After the reaction is completed, dialyze it in pure water, centrifuge the dialysis solution to perform solid-liquid separation, and freeze-dry the obtained solid substance to obtain the product.

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

4. The antigen system according to claim 1, characterized in that, The preparation method of the carboxylated nanocellulose includes: the preparation of nanocellulose and the carboxylation of nanocellulose, wherein: Preparation of nanocellulose: Wash bacterial cellulose with 0.1 mol / L sodium hydroxide solution to remove surface impurities, and then wash repeatedly with distilled water until the pH of the bacterial cellulose surface is 8; put the bacterial cellulose into 68% sulfuric acid solution, use 20 mL of sulfuric acid solution for every 0.5 g of bacterial cellulose, heat at 50 °C for 2 h, then add an ice-water mixture to the reaction system to terminate the reaction, dialyze with a 14 kDa dialysis bag in pure water until the pH is neutral, and freeze-dry to obtain nanocellulose; Carboxylation of nanocellulose: Take 1 g of nanocellulose, add 40 mL of deionized water and stir. Then add 0.05 - 2 g of iron oxide with a particle size of 1 μm. While ultrasonically dispersing, stir and heat to 80 - 85 °C. After adding 30 - 40 mL of 20% hydrogen peroxide solution, continue to stir and heat. After 3 h, add another 30 - 40 mL of 20% hydrogen peroxide solution and continue to stir and heat for 3 h. Cool the reaction system to room temperature, add 0.5 M sodium hydroxide to adjust the pH to 7.8 - 8.8 and stir. Filter the mixture to obtain the filtrate, centrifuge the filtrate, collect the supernatant and add 20 - 40 mL of ethanol for precipitation. Filter to obtain the precipitate product, dialyze it in pure water with a 14 kDa dialysis bag for 2 - 3 days, and freeze-dry the liquid in the dialysis bag to obtain carboxylated nanocellulose powder.

5. Use of the antigen system according to claim 1 in the preparation of an immunoprotective preparation against largemouth bass rhabdovirus.

6. The application according to claim 5, characterized in that, The immunoprotective preparation uses immersion immunization, and the immersion immunization time is 1 - 5 h.

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