Foot and mouth disease virus effective antigen inactivation protective agent and application thereof

Abstract

The invention discloses a foot and mouth disease virus effective antigen inactivation protective agent and application thereof. The protective agent is prepared from the following components in percentage by mass: 0.05 to 20 percent of a structure curing agent, 0.01 to 5 percent of an ion regulator, 0.005 to 1.0 percent of an antioxidant and the balance of water, the protective agent does not influence the inactivation effect in the process of acting on nucleic acid, inhibits folding and unfolding of a protein physical structure, prevents change of a protein space physical structure, improves free energy of protein denaturation, enhances the stability of a virus space physical structure, maintains the integrity of virions, and has a good application prospect. The effects of preserving the immune biological activity of the protein and reducing the loss of viruses in the inactivation process are achieved, so that the foot-and-mouth disease inactivation antigen liquid with high immunogenicity is obtained. Compared with the conventional antigen protection method, the method has the advantages that the virus is protected before the inactivation process is implemented, the production process is advanced, the stability of the effective antigen can be protected to the greatest extent, and support is provided for obtaining an effective antigen solution with high immunocompetence.

Description

Technical Field

[0001] The invention belongs to the technical field of animal biological products, and in particular relates to an effective antigen inactivation protective agent for foot-and-mouth disease virus and application thereof. Background Art

[0002] Foot-and-mouth disease is an acute, febrile, highly contagious and rapidly long-distance animal disease caused by the foot-and-mouth disease virus (FMDV). It is highly prevalent in even-toed ungulates and can cause animal production performance declines, severely restricting international trade in animals and animal products. Due to the high infectivity of the foot-and-mouth disease virus and the huge direct economic losses it can cause, it is listed as the first animal disease of Category A by the OIE. The foot-and-mouth disease virus belongs to the microRNA virus family and the foot-and-mouth disease virus genus. The virus has seven serotypes: A, O, C, SATⅠ, SATⅡ, SATⅢ, and Asia1. The foot-and-mouth disease virus is spherical, without an envelope, and the particle diameter is 28 to 30 nanometers. The complete foot-and-mouth disease virus consists of a molecule of RNA wrapped in a capsid. The capsid is icosahedral and is composed of 60 asymmetric protomers composed of four structural proteins (VP1 to VP4). The complete foot-and-mouth disease virus particle is the immune antigen that determines the effectiveness of the immune response. Its quantity and stability determine the effectiveness of the vaccine. The intact FMD virus particles have a significant structural instability. When the isoelectric point, pH value or temperature changes, they will depolymerize into monomers, and their immune effect will be greatly reduced. Currently, the vaccines widely used for FMD prevention and immunization are mainly inactivated virus vaccines, which are prepared by emulsifying the inactivated FMD whole virus antigen with an oil adjuvant.

[0003] Virus inactivation is to denature and inactivate the virus, thereby killing it. Commonly used inactivators include formaldehyde, glutaraldehyde, alkylating agents, crystal violet, phenol, β-propiolactone, and thimerosal. The most widely used inactivator in foot-and-mouth disease vaccines is diethyleneimine (BEI) among alkylating agents. Its inactivation mechanism is to use the ethylene oxide group to act on the foot-and-mouth disease virus nucleic acid to cause the loss of toxicity. The basic method of the process is to add BEI with a final concentration of 0.02-0.05% to the virus solution, shake it at 30°C and inactivate it for 8-24 hours. After the inactivation is completed, 2% sodium thiosulfate is added to block the inactivation effect. In the inactivation process currently used, although BEI inactivators and other substances do not directly act on the virus capsid protein, the virus has the natural instability of protein and is easily affected by external conditions such as temperature, vibration, shear force, buffer system and ionic strength, and even changes in its own protein concentration or purity. The addition of inactivators and blockers during the inactivation process also changes the buffer system and ionic strength of the virus solution to a certain extent. At the same time, some changes in physical conditions during the inactivation process, such as the required higher temperature and shaking for a certain period of time, also have a certain degree of impact on the protein structure. The above factors will cause protein aggregation, denaturation, and degradation, and there is inevitably a risk of loss of effective antigens. At the same time, after inactivation, the virus antigens are more sensitive to environmental conditions and are more unstable, and there is a risk of quality decline, thus affecting the quality of the vaccine.

[0004] In view of the above-mentioned problems existing in actual production, the present invention proposes to add a certain proportion of virus protective agent before inactivating the foot-and-mouth disease virus to prevent changes in the virus protein structure caused during the inactivation process, strengthen the physical spatial structural elasticity of the virus, and adjust the aqueous solution environment where the virus is located to prevent cross-linking of virus capsid proteins or aggregation and denaturation of virus particles, so as to maintain the integrity, stability and immunogenicity of virus particles during the inactivation process, and do not change the biological characteristics of the virus protein and nucleic acid itself, do not affect the inactivation process and the final inactivation effect of the currently used inactivator, and do not affect the subsequent production process and the immune effect of the final product vaccine. Summary of the invention

[0005] The first purpose of the present invention is to provide a virus protein protective agent used in the inactivation process of foot-and-mouth disease virus, specifically, the protective agent is used to inhibit the folding and unfolding of the protein structure, prevent the change of the spatial structure of the protein, improve the free energy of protein denaturation, enhance the stability of the spatial physical structure of the virus, maintain the integrity of the virus particles, preserve the immune biological activity of the protein, and reduce the loss of the virus during the inactivation process, so as to obtain a highly immunogenic foot-and-mouth disease inactivated antigen liquid. Compared with the addition of antigen protective agents to the inactivated antigen in current production and related patents, the production process is advanced, the effective antigen can be protected in advance, and the destruction of its spatial structure by inactivators and blockers can be prevented, and the stability of the final antigen can be improved.

[0006] In order to achieve the above-mentioned purpose, the protective agent of the present invention may contain polymer substances such as povidone, ethylene vinyl acetate copolymer, polyvinyl acetaldehyde diethylamine ethyl ester, acrylates and copolymers thereof, polyvinyl alcohol, polyvinyl amines, polyglycolic acid, polyacrylamide, lactic acid glycolic acid copolymer, polyethylene glycol, polyacrylic acid sodium salt, carboxyethylene copolymer (carbomer), acrylic acid-acrylamide copolymer, polythiamine, sodium alginate, hyaluronic acid, sodium starch phosphate, propylene glycol alginate, sodium hydroxymethyl cellulose, polydextrose, polyaspartic acid, polyasparagine, polyglutamic acid, poly-L-lysine, etc., which are used to solidify the physical structure of viral proteins. By increasing the viscosity of the viral liquid, due to the presence of high-viscosity polymers, the extended molecular chains hinder the spatial structural changes of the virus and prevent the movement and aggregation of protein molecules. At the same time, the polymer can also form a certain hydration layer on the surface of the virus, hindering the collision and aggregation of viral particles and improving the stability of the physical form of viral proteins.

[0007] Furthermore, the protective agent may include ion regulators such as salts, amines, sugars, etc., which are used to form hydrogen bonding substances with viral proteins to meet the needs of charged groups on the protein surface to form hydrogen bonds, which has a significant inhibitory effect on the extension and aggregation of protein polypeptide chains. At the same time, it adjusts the ion concentration in the solution to prevent buffer salt crystallization from causing changes in pH value and thus preventing protein precipitation.

[0008] Further, the protective agent may contain a surfactant, such as polyoxyethylene sorbitan fatty acid ester, polyoxyethylene maltitol fatty acid ester, polyoxyethylene xylitol fatty acid ester, polyoxyethylene sucrose fatty acid ester, polyglycerol fatty acid ester, polyethylene oxide polypropylene glycol ether (polyether), hydroxylated lecithin, sodium alkyl sulfate, alkyl amine oxide, and coconut oil ethanolamide. Surfactants stabilize viral proteins mainly through interfacial competition, and their surface activity is much higher than that of proteins. Surfactants can competitively occupy interfacial positions, inhibit viral proteins from being adsorbed to the gas-liquid interface, reduce oxidation or aggregation caused by contact with air, and effectively prevent viral proteins from being unfolded at the interface during inactivation, purification, and emulsification and storage. At the same time, surfactants can also interact with viral proteins to increase the colloid stability of viral proteins. Surfactants interact with hydrophobic groups on the surface of viral proteins, inhibiting the tendency that viral proteins may form aggregates through their intermolecular hydrophobic interactions, thereby preventing the aggregation of viral proteins and even the generation of particles. By selecting surfactants with high biocompatibility, non-toxicity, high hydrophile-lipophile balance (HLB) and low critical micelle (CMC) (1.4*10-2g / L), effective physical protection of viral proteins can be achieved at lower concentrations (less than 0.1-1.0mg / ml) and sufficient spatial stability of viral proteins.

[0009] Furthermore, the protective agent may contain antioxidants, such as ascorbic acid, ascorbyl palmitate, vitamin E, thioglycerol, tea polyphenols, lipoic acid, glutathione, etc., for oxidative degradation of viral protein antigens.

[0010] The above-mentioned substances do not participate in the chemical inactivation process of viral nucleic acid, nor do they participate in the blocking process of sodium thiosulfate, and do not affect the final inactivation effect.

[0011] The second object of the present invention is to provide a foot-and-mouth disease virus inactivation process using a protective agent, which is simple to operate and easy to implement without changing the existing inactivation process parameters.

[0012] Based on the above objectives, the present invention is achieved through the following technical solutions:

[0013] 1. Preparation of inactivation protective agent

[0014] The protective agent for effective antigens used in the inactivation process of foot-and-mouth disease virus provided by the present invention comprises a structural curing agent, an ion regulating agent, a surfactant and an antioxidant. The above substances all provide support and protection for the viral protein structure, do not change the chemical structure and physical spatial form of the viral protein, do not affect the inactivation of viral nucleic acid and do not reduce the immune effect of the inactivated antigen. The curing agent and the ion regulating agent mainly adjust the spatial structure between viruses and viruses, the surfactant and the antioxidant mainly maintain the stability of the chemical properties of the virus, and the above substances promote each other and play a synergistic role.

[0015] The structural curing agent is a high molecular polymer, which can be one or any combination of two or more of polyvidone (PVP), ethylene vinyl acetate copolymer, polyvinyl acetaldehyde diethylamine ethyl ester, acrylate and its copolymer, polyvinyl alcohol, polyvinyl amine, polyglycolic acid, polyacrylamide, lactic acid glycolic acid copolymer (PLGA), polyethylene glycol, polyacrylic acid sodium salt, carboxyethylene copolymer (carbomer), acrylic acid-acrylamide copolymer, polythiamine, sodium alginate, hyaluronic acid, sodium starch phosphate, propylene glycol alginate, sodium hydroxymethyl cellulose, polydextrose, polyaspartic acid, polyasparagine (PHEA), polyglutamic acid, poly-L-lysine, etc., and the proportion composition can be 0.05-20% of the total mass percentage.

[0016] The ion regulator is one or any combination of two or more of sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium octanoate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium citrate, sodium acetate, sodium stearate, zinc gluconate, urea, ethanolamine, diethanolamine, triethanolamine, glucose, fructose, sucrose, trehalose, lactose, sorbitol, dextran, maltose, mannose, cellobiose, etc., and the proportion composition can be 0.01-5% of the total mass percentage.

[0017] The surfactant is polyoxyethylene sorbitan fatty acid ester (such as Tween 21, Tween 61, Tween 81, Tween 85, etc.), polyoxyethylene maltitol fatty acid ester, polyoxyethylene xylitol fatty acid ester, polyoxyethylene sucrose fatty acid ester, polyglycerol fatty acid ester (such as decaglycerol monooleate, hexaglycerol monooleate), polyethylene oxide polypropylene oxide propylene glycol ether (polyether) (such as poloxamer F188, F407, etc.), hydroxylated lecithin, alkyl sodium sulfate (such as sodium lauryl sulfate, etc.), alkyl amine oxide (such as lauryl amine oxide, etc.), coconut oil ethanolamide, etc., or any combination of two or more thereof, and the proportion composition is 0.01-1.0% of the total mass percentage.

[0018] The antioxidant is one or any combination of ascorbic acid, ascorbyl palmitate, vitamin E, coenzyme Q10, lutein, carotenoids, tert-butylhydroquinone, uric acid, disodium EDTA, thioglycerol, tea polyphenols, lipoic acid, glutathione, etc., and the proportion is 0.005-1.0% of the total mass percentage.

[0019] Preferred:

[0020] The structural curing agent is one or any combination of two or more of povidone, ethylene vinyl acetate copolymer, polyethylene acetaldehyde diethylamine ethyl ester, polyethylene glycol, polyacrylic acid sodium salt, carboxyethylene copolymer (carbomer), acrylic acid-acrylamide copolymer, polydextrose, polyaspartic acid, polyasparagine, polyglutamic acid, poly-L-lysine, etc., and the proportion composition can be 0.1 to 15% of the total mass percentage.

[0021] The ion regulator is one or any combination of two or more of sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium caprylate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ethanolamine, diethanolamine, triethanolamine, glucose, sucrose, trehalose, lactose, dextran, maltose, and mannose, and the proportion composition can be 0.05 to 3% of the total mass percentage.

[0022] The surfactant is one or any combination of two or more of polyoxyethylene sorbitan fatty acid esters (such as Tween 21, Tween 61, Tween 81, Tween 85, etc.), polyglycerol fatty acid esters (such as decaglycerol monooleate, hexaglycerol monooleate), polyethylene oxide polypropylene oxide propylene glycol ether (polyether) (such as poloxamer F188, F407, etc.), hydroxylated lecithin, alkyl amine oxide (dodecyl amine oxide), coconut oil ethanolamide, etc., and the proportion is 0.05-1% of the total mass percentage.

[0023] The antioxidants are ascorbyl palmitate, thioglycerol, disodium EDTA, tea polyphenols, lipoic acid and glutathione, and the proportion of the total weight percentage is 0.01-1%.

[0024] More preferred:

[0025] The structural curing agent is one or any combination of two or more of polyvidone (PVP), polyethylene glycol, sodium polyacrylic acid, carboxyethylene copolymer (carbomer), acrylic acid-acrylamide copolymer, polydextrose, polyaspartic acid, polyasparagine (PHEA), polyglutamic acid, poly-L-lysine, etc., and the proportion composition can be 0.1 to 10% of the total mass percentage.

[0026] The ion regulator is one or any combination of two or more of sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ethanolamine, diethanolamine, triethanolamine, glucose, sucrose, trehalose, dextran, and mannose, and the proportion composition can be 0.05 to 1% of the total mass percentage.

[0027] The surfactants include Tween 21, Tween 61, Tween 81, decaglycerol monooleate, hexaglycerol monooleate, poloxamer F188, F407, hydroxylated lecithin, dodecylamine oxide, etc., and the proportion of the total mass percentage is 0.1-1%.

[0028] The antioxidants are ascorbyl palmitate, thioglycerol, lipoic acid and glutathione, and the proportion of the total weight percentage is 0.01-0.5%.

[0029] 2. Inactivation production process using inactivation protective agent

[0030] To achieve the second purpose of the present invention, the foot-and-mouth disease virus inactivation process used is: add the above-mentioned protective agent to the virus cell culture fluid, the amount of the protective agent added is 0.01-2% of the volume of the virus liquid, after stirring, dispersing and dissolving, let it stand for 1-12 hours, and the temperature of the environment and the virus liquid during the standing period is 4-30° C. After the standing period is completed, add the inactivator BEI, inactivate at 30° C. for 8-24 hours, add 2% sodium thiosulfate to stop the inactivation, and complete the virus inactivation process.

[0031] Preferably, the amount of the protective agent is 0.05-1% of the virus volume, the static action time is 1-4 hours, and the temperature of the environment and the virus solution during the static action is 4-25°C.

[0032] More preferably, the amount of the protective agent is 0.1-1% of the virus volume, the static action time is 2 hours, and the temperature of the environment and the virus solution during the static action is 4-8° C., the storage environment of the current virus solution.

[0033] Beneficial effects of the present invention:

[0034] Compared with the previous antigen protection methods, the present invention protects the virus before the inactivation process is implemented, and the production process is advanced, which can protect the stability of the effective antigen to the greatest extent and provide support for obtaining an effective antigen liquid with high immune activity.

[0035] The protective agent of the present invention does not affect the inactivation effect during the process of acting on nucleic acid, while inhibiting the folding and unfolding of protein structure, preventing the change of the spatial structure of protein, increasing the free energy of protein denaturation, enhancing the stability of the spatial physical structure of virus, maintaining the integrity of virus particles, preserving the immunobiological activity of protein, and reducing the loss of virus during the inactivation process, thereby obtaining a foot-and-mouth disease inactivated antigen liquid with high immunogenicity. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific examples. The following examples will help those skilled in the art to further understand the present invention. The listed examples are all implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples. The raw materials used in the present invention can be obtained from commercial channels or conventional methods unless otherwise specified.

[0037] Example 1

[0038] (I) Preparation of inactivation protective agent

[0039] The structural curing agent totals 7.72%, including:

[0040]

[0041] Ion regulators total 0.64%, including:

[0042]

[0043]

[0044] Surfactants total 0.7%, including:

[0045]

[0046] The total amount of antioxidants is 0.02%, including:

[0047] Ascorbyl palmitate, monothioglycerol 0.01% each.

[0048] The above components are added into pure water for injection respectively, dispersed and dissolved by stirring, and then sterilized by filter membrane for later use.

[0049] (II) Inactivation production process using inactivation protective agents

[0050] The prepared protective agent was added to the virus cell culture fluid, the amount of the protective agent added was 1% of the volume of the virus fluid, after stirring, dispersing and dissolving, it was allowed to stand for 4 hours, and the temperature of the environment and the virus fluid during the standing period was 4° C. After the standing period, the inactivator BEI was added, inactivated at 30° C. for 8 hours, and 2% sodium thiosulfate was added to terminate the inactivation, completing the virus inactivation process.

[0051] Example 2

[0052] (I) Preparation of inactivation protective agent

[0053] The total amount of structural curing agent is 10%, of which:

[0054]

[0055] Ion regulators total 0.27%, including:

[0056]

[0057] Surfactants total 0.4%, including:

[0058]

[0059] The total amount of antioxidants is 0.02%, including:

[0060] Thioglycerol and lipoic acid 0.01% each.

[0061] The above components are added into pure water for injection respectively, dispersed and dissolved by stirring, and then sterilized by filter membrane for later use.

[0062] (II) Inactivation production process using inactivation protective agents

[0063] The prepared protective agent is added to the virus cell culture fluid, the amount of the protective agent added is 0.01-2% of the volume of the virus fluid, after stirring, dispersing and dissolving, it is allowed to stand for 2 hours, and the temperature of the environment and the virus fluid during the standing period is 8° C. After the standing period is completed, the inactivator BEI is added, and the inactivation is carried out at 30° C. for 8 hours, and 2% sodium thiosulfate is added to terminate the inactivation, completing the virus inactivation process.

[0064] Example 3

[0065] (I) Preparation of inactivation protective agent

[0066] The total amount of structural curing agent is 0.23%, of which:

[0067]

[0068]

[0069] Ion regulators total 0.26%, including:

[0070]

[0071] Surfactants total 0.5%, including:

[0072]

[0073] The total amount of antioxidants is 0.02%, including:

[0074] Monothioglycerol, glutathione 0.01% each.

[0075] The above components are added into pure water for injection respectively, dispersed and dissolved by stirring, and then sterilized by filter membrane for later use.

[0076] (II) Inactivation production process using inactivation protective agents

[0077] The prepared protective agent was added to the virus cell culture solution, the amount of the protective agent added was 0.1% of the volume of the virus solution, and after stirring, dispersing and dissolving, it was allowed to stand for 6 hours, and the temperature of the environment and the virus solution during the standing period was 8° C. After the standing period, the inactivator BEI was added, and the inactivation was performed at 30° C. for 8 hours, and 2% sodium thiosulfate was added to terminate the inactivation, completing the virus inactivation process.

[0078] Example 4

[0079] (I) Preparation of inactivation protective agent

[0080] The total amount of structural curing agent is 0.1%, wherein:

[0081]

[0082] The total amount of ion regulators is 0.07%, including:

[0083] Triethanolamine, 0.05%,

[0084] Sucrose 0.01%,

[0085] Mannose 0.01%;

[0086] Surfactants total 0.5%, including:

[0087]

[0088] The total amount of antioxidants is 0.03%, including:

[0089] Ascorbyl palmitate, thioglycerol, lipoic acid 0.01% each,

[0090] The above components are added into pure water for injection respectively, dispersed and dissolved by stirring, and then sterilized by filter membrane for later use.

[0091] (II) Inactivation production process using inactivation protective agents

[0092] The prepared protective agent is added to the virus cell culture fluid, the amount of the protective agent added is 2% of the volume of the virus liquid, after stirring, dispersing and dissolving, it is allowed to stand for 1 to 12 hours, and the temperature of the environment and the virus liquid during the standing period is 8° C. After the standing period is completed, the inactivator BEI is added, and the inactivation is carried out at 30° C. for 8 hours, and 2% sodium thiosulfate is added to terminate the inactivation, completing the virus inactivation process.

[0093] Example 5

[0094] (I) Preparation of inactivation protective agent

[0095] The total amount of structural curing agent is 0.14%, including:

[0096]

[0097] The total amount of ion regulator is 0.06%, including:

[0098] Triethanolamine 0.05%,

[0099] Trehalose 0.01%;

[0100] The surfactants total 0.5%, of which:

[0101]

[0102] The total amount of antioxidants is 0.03%, including:

[0103] Ascorbyl palmitate, thioglycerol, glutathione 0.01% each,

[0104] The above components are added into pure water for injection respectively, dispersed and dissolved by stirring, and then sterilized by filter membrane for later use.

[0105] (II) Inactivation production process using inactivation protective agents

[0106] The prepared protective agent was added to the virus cell culture fluid, the amount of the protective agent added was 1% of the volume of the virus fluid, after stirring, dispersing and dissolving, it was allowed to stand for 8 hours, and the temperature of the environment and the virus fluid during the standing period was 4° C. After the standing period, the inactivator BEI was added, inactivated at 30° C. for 8 hours, and 2% sodium thiosulfate was added to terminate the inactivation, completing the virus inactivation process.

[0107] Example 6

[0108] (I) Preparation of inactivation protective agent

[0109] The total amount of structural curing agent is 0.1%, wherein:

[0110] Polyaspartic acid 0.05%,

[0111] Polyglutamic acid 0.05%;

[0112] The total amount of ion regulator is 0.25%, including:

[0113] Sodium chloride 0.2%,

[0114] Triethanolamine 0.05%;

[0115] Surfactants total 0.2%, including:

[0116] Dodecylamine oxide 0.1%

[0117] Hydroxylated lecithin 0.1%;

[0118] The total amount of antioxidants is 0.02%, including:

[0119] Thioglycerol, glutathione 0.01% each,

[0120] The above components are added into pure water for injection respectively, dispersed and dissolved by stirring, and then sterilized by filter membrane for later use.

[0121] (II) Inactivation production process using inactivation protective agents

[0122] The prepared protective agent is added to the virus cell culture fluid, the amount of the protective agent added is 0.01-2% of the volume of the virus fluid, after stirring, dispersing and dissolving, it is allowed to stand for 2 hours, and the temperature of the environment and the virus fluid during the standing period is 4° C. After the standing period is completed, the inactivator BEI is added, and the inactivation is carried out at 30° C. for 8 hours, and 2% sodium thiosulfate is added to terminate the inactivation, completing the virus inactivation process.

[0123] Comparison Example

[0124] In accordance with the foot-and-mouth disease production procedures, the inactivator BEI was added to the virus cell culture medium and inactivated at 30°C for 8 hours. 2% sodium thiosulfate was added to terminate the inactivation and complete the virus inactivation process.

[0125] Experimental test example

[0126] (I) Test on the protective effect of protective agent on effective antigens in the process of foot-and-mouth disease inactivation

[0127] The sucrose density centrifugation ultraviolet measurement method was used to determine the content of effective antigen (146S) after foot-and-mouth disease inactivation. The specific data are shown in Table 1 below.

[0128] Table 1. 146S content in different groups

[0129] Group 146S content (μg / ml) Example 1 18.1 Example 2 16.6 Example 3 17.4 Example 4 15.8 Example 5 17.6 Example 6 18.2 Comparison Example 11.5

[0130] (II) Virus inactivation safety test

[0131] The inactivated foot-and-mouth disease virus solution was subcutaneously inoculated into 8 suckling mice of 3-5 days old in each group, for a total of 7 groups, 0.2 mL per mouse, and 2 healthy suckling mice were set as controls. 48 hours after inoculation, 2 inoculated suckling mice were taken from each group, and 1:10 tissue fluid was prepared after death, and the supernatant was obtained by centrifugation; then 4 suckling mice were inoculated in each group, 0.2 ml per mouse, and 2 healthy suckling mice were set as controls. All inoculated suckling mice were observed for 7 days, and none of them showed symptoms of foot-and-mouth disease or died.

[0132] 3. Inspection of finished vaccine products

[0133] Vaccine safety testing

[0134] Guinea pigs weighing 350-450g were used in each group, with 2 ml of vaccine for each of the example group and the control group injected intraperitoneally; mice weighing 18-22g were used in each group, with 0.5 ml of vaccine for each of the 6 groups of the invention example and the control group injected subcutaneously. The observation was continued for 7 days, and no abnormal clinical reaction was observed.

[0135] Two healthy susceptible piglets (no FMD neutralizing antibodies were detected by suckling mouse neutralization test) aged 30 to 40 days were injected intramuscularly at points behind the ears of both sides with 2 doses of the vaccines of the 6 groups of the invention and the control group, a total of 7 groups, for each pig, and observed daily for 14 consecutive days. No symptoms of FMD or obvious abnormal toxic reactions caused by vaccine injection occurred.

[0136] The FMDV antigen inactivated by the above method and the oil adjuvant required by the regulations were used to prepare the vaccine for a total of 7 groups, each group had 3 healthy susceptible cattle at least 6 months old (the titer of FMD cell neutralizing antibodies was less than or equal to 1:8), and 2 mL of the vaccine was injected into 20 points on the upper surface of the tongue of each cattle, 0.1 mL per point, and observed daily for at least 4 days. Afterwards, each cattle was injected with 3 doses of the vaccine intramuscularly, and continued to be observed daily for 6 days. All cattle did not show symptoms of FMD or toxic reactions caused by the injection of the vaccine.

[0137] Vaccine immune effect test

[0138] The above-mentioned preparation method prepared 6 groups of vaccines in the invention example, the control group vaccine and the blank group, selected piglets weighing about 40kg, and the neutralization test of suckling mice determined that there was no neutralizing antibody against foot-and-mouth disease, a total of 7 groups of 38 pigs, respectively injected with 2ml / head of the embodiment, a total of 6 groups and the control group, each group was 5 pigs, a total of 35 pigs, and a blank group control of 3 pigs was set. Blood was collected from the piglets 0 week before vaccination and every week after vaccination to separate serum, and the antibody level was determined using a liquid phase blocking ELISA detection kit for foot-and-mouth disease antibodies, and blood was collected 6 times a week. The data are shown in Table 2 below.

[0139] Table 2 Foot-and-mouth disease antibody level test data table

[0140] Group No. 0 Weeks 1 week 2 weeks 3 weeks 4 weeks 5 weeks Example 1 <8 16 32 256 512 512 Example 2 <8 16 32 256 512 512 Example 3 <8 16 32 256 512 512 Example 4 <8 16 32 256 512 512 Example 5 <8 16 32 256 512 512 Example 6 <8 16 32 256 512 512 Comparison Example <8 8 16 128 128 128 Blank control group <8 <8 <8 <8 <8 <8

[0141] It can be seen from Table 2 that the antibody levels of each group in the embodiment are relatively high, and better protection can be obtained; while the antibody levels of the control group are relatively low, and a good immune effect cannot be obtained.

[0142] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A foot-and-mouth disease virus effective antigen inactivation protective agent, consisting of the following components in percentage by mass: Structural curing agent 0.05~20%, Ion regulator 0.01~5%, Surfactant 0.01~1.0%, Antioxidant 0.005~1.0%, and the balance of water; The structural curing agent is a high molecular polymer; it can be one or any combination of two or more of povidone, ethylene vinyl acetate copolymer, polyvinyl acetaldehyde diethylamine ethyl ester, acrylate and its copolymer, polyvinyl alcohol, polyvinyl amine, polyglycolic acid, polyacrylamide, lactic acid glycolic acid copolymer, polyethylene glycol, polyacrylic acid sodium salt, carboxyethylene copolymer, acrylic acid-acrylamide copolymer, polythiamine, sodium alginate, hyaluronic acid, sodium starch phosphate, propylene glycol alginate, sodium hydroxymethyl cellulose, polydextrose, polyaspartic acid, polyasparagine, polyglutamic acid and poly-L-lysine; The ion regulator is one or any combination of two or more of sodium chloride, potassium chloride, ammonium chloride, sodium sulfate, sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium octanoate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium citrate, sodium acetate, sodium stearate, zinc gluconate, urea, ethanolamine, diethanolamine, triethanolamine, glucose, fructose, sucrose, trehalose, lactose, sorbitol, dextran, maltose, mannose, and cellobiose; The surfactant is one or any combination of two or more of polyoxyethylene sorbitan fatty acid ester, polyoxyethylene maltitol fatty acid ester, polyoxyethylene xylitol fatty acid ester, polyoxyethylene sucrose fatty acid ester, polyglycerol fatty acid ester, polyethylene oxide polypropylene oxide propylene glycol ether, hydroxylated lecithin, sodium alkyl sulfate, alkyl amine oxide and coconut oil ethanolamide; The antioxidant is one or any combination of two or more of ascorbic acid, ascorbyl palmitate, vitamin E, coenzyme Q10, lutein, carotenoids, tert-butylhydroquinone, uric acid, disodium EDTA, thioglycerol, tea polyphenols, lipoic acid and glutathione.

2. The protective agent according to claim 1, characterized in that It is composed of the following components in percentage by mass: Structural curing agent 0.1~15%, Ion regulator 0.05~3%, Surfactant 0.05~1.0%, Antioxidant 0.01~1.0%, and the balance of water; The structural curing agent is one or any combination of two or more of povidone, ethylene vinyl acetate copolymer, polyvinyl acetaldehyde diethylamine ethyl ester, polyethylene glycol, polyacrylic acid sodium salt, carboxyethylene copolymer, acrylic acid-acrylamide copolymer, polydextrose, polyaspartic acid, polyasparagine, polyglutamic acid and poly-L-lysine; The ion regulator is one or any combination of two or more of sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium octanoate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ethanolamine, diethanolamine, triethanolamine, glucose, sucrose, trehalose, lactose, dextran, maltose and mannose. The surfactant is one or any combination of two or more of polyoxyethylene sorbitan fatty acid ester, polyglycerol fatty acid ester, polyethylene oxide polypropylene oxide propylene glycol ether, hydroxylated lecithin, alkyl amine oxide and coconut oil ethanolamide; The antioxidant is one or any combination of two or more of ascorbyl palmitate, thioglycerol, disodium EDTA, tea polyphenols, lipoic acid and glutathione.

3. The protective agent according to claim 1, characterized in that It is composed of the following components in percentage by mass: Structural curing agent 0.1~10%, Ion regulator 0.05~1.0%, Surfactant 0.1~1.0%, Antioxidant 0.01~0.5%, and the balance of water; The structural curing agent is one or any combination of two or more of povidone, polyethylene glycol, sodium polyacrylic acid, carboxyethylene copolymer, acrylic acid-acrylamide copolymer, polydextrose, polyaspartic acid, polyasparagine, polyglutamic acid and poly-L-lysine; The ion regulator is one or any combination of two or more of sodium chloride, potassium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ethanolamine, diethanolamine, triethanolamine, glucose, sucrose, trehalose, dextran and mannose; The surfactant is one or any combination of two or more of Tween 21, Tween 61, Tween 81, decaglycerol monooleate, hexaglycerol monooleate, poloxamer F188, F407, hydroxylated lecithin and dodecylamine oxide; The antioxidant is one or any combination of two or more of ascorbyl palmitate, thioglycerol, lipoic acid and glutathione.

4. A method for inactivating foot-and-mouth disease virus, comprising the following steps: The protective agent described in any one of claims 1 to 3 is added to the foot-and-mouth disease virus cell culture fluid, the amount of the protective agent added is 0.01-2% of the volume of the virus liquid, after stirring, dispersing and dissolving, standing for 1-12 hours, the temperature of the environment and the virus liquid during the standing period is 4-30° C.; after the standing period is completed, the inactivator BEI is added, inactivated at 30° C. for 8-24 hours, and 2% sodium thiosulfate is added to terminate the inactivation, thereby completing the virus inactivation process.

5. The method according to claim 4, characterized in that The dosage of the protective agent is 0.05-1% of the volume of the virus, the static action time is 1-4 hours, and the temperature of the environment and the virus liquid during the static action is 4-25°C.

6. The method according to claim 4, characterized in that The dosage of the protective agent is 0.1-1% of the virus volume, the static action time is 2 hours, and the temperature of the environment and the virus solution during the static action is 4-8°C, the storage environment of the current virus solution.

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