Foot-and-mouth disease purified antigen resuspension

Abstract

The invention discloses an inorganic base and alkaline earth metal ion resuspension liquid applied to a foot-and-mouth disease purification antigen, which is prepared from the following substances in percentage by weight: 0.005-5mol / L of alkali metal salt, 0.005-5mol / L of alkaline earth metal salt and the balance of water for injection. The designed inorganic base and alkaline-earth ion resuspension can effectively resuspend the foot-and-mouth disease purified antigen, the foot-and-mouth disease antigen can be stored for a long time, the immunocompetence of the foot-and-mouth disease antigen can be protected, and meanwhile, no additional organic component is added into the resuspension, so that animals can obtain good immune protection, and meanwhile, the occurrence of animal side reactions can be effectively reduced.

Description

Technical Field

[0001] The present invention relates to a purified foot-and-mouth disease antigen resuspension solution, and particularly to an inorganic base and alkaline earth metal ion liquid resuspending agent for purified foot-and-mouth disease antigen, which is composed of an inorganic base and an alkaline earth metal ion liquid. The present invention belongs to the field of veterinary medicine. Background Art

[0002] Foot-and-mouth disease is an acute, febrile, highly contagious disease of cloven-hoofed animals such as cattle, pigs, and sheep caused by Foot and Mouth Disease Virus (FMDV). It can reduce the production performance of animals, causing huge direct economic losses, and is listed as the first category A animal disease by the OIE. Foot-and-mouth disease virus belongs to the Picornaviridae family and the Foot-and-mouth disease virus genus. This virus has seven serotypes: type A, type O, type C, SAT1 type, SAT2 type, SAT3 type, and Asia1 type. The antigenicity of foot-and-mouth disease virus is weak, and there is no cross-protection reaction between different serotypes. The prevention and control measures for foot-and-mouth disease mainly focus on immunoprophylaxis. Safe and effective vaccines are the key conditions for successfully preventing and controlling and ultimately eradicating foot-and-mouth disease. In recent years, with the progress of vaccine production technology, purifying foot-and-mouth disease virus antigen is an inevitable choice for vaccine production enterprises. The purified antigen resuspension solution used in the purification process is a very cautious choice for production enterprises because the intact foot-and-mouth disease virus particles (146S) in inactivated foot-and-mouth disease antigen are the immune antigens that determine the immune effectiveness. Their quantity and stability determine the efficacy of the vaccine. Due to the natural significant instability of the intact foot-and-mouth disease virus particles (146S) caused by the protein's own structure, choosing a suitable resuspension solution plays a crucial role in the stability, solubility of the virus protein, and maintaining the structure of the immune sites. Improper use of the resuspension solution may lead to aggregation, precipitation, or insolubility of the virus protein. If the precipitated and concentrated antigen obtained by the polyethylene glycol precipitation purification method of foot-and-mouth disease antigen is simply resuspended with PBS or TRIS solution, the individual antigens aggregated into groups after centrifugation cannot be fully suspended and separated. The physical form of the antigen in the resuspension solution is not sufficiently stretched, which will affect the stability of the antigen and result in the immune effect not meeting the actual requirements. Adding additional organic components such as sugars, amino acids, and protein substances as antigen protection components in the resuspension solution often has an impact on subsequent process inspection and animal side reactions.

[0003] The foot-and-mouth disease antigen protein particles carry a large number of negative charges on their surfaces, and the interaction between the particles generally conforms to the DLVO theory. The stability of the antigen protein resuspension depends not only on the magnitude of the repulsive potential energy and attractive potential energy of the particles in the suspension, but also on the ion-specific effect of the reaction between the ions in the suspension and the surface of the charged antigen particles. Ion hydration is not only an important reason for the ion-specific effect, but also the non-classical polarization of ions is an important reason for the ion-specific effect. The regulation of different ions plays a crucial role in the stability, solubility, and structural maintenance of the antigen protein. Inappropriate ions may cause antigen protein aggregation, precipitation, or insolubility, thus seriously affecting its immunogenicity. Alkali metals refer to the elements in Group 1 of the periodic table, including lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and francium (Fr). These elements exist in the form of ions in nature, that is, they have lost one or more electrons to form positively charged ions. Alkali metal ions have unique properties and applications. Alkali metal ions have high ionic conductivity. Since their positively charged ions can move freely in the solution, alkali metal ion solutions have high conductivity. Alkali metal ions are difficult to form weak electrolytes or non-electrolytes, and it is difficult to form complex cations or complex anions in the solution, so it is difficult to form insoluble stable complexes with other substances. Alkaline earth metals refer to all the elements in Group IIA, including beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), and radium (Ra). The nitrates, chlorides, perchlorates, and acetates of alkaline earth metals are easily soluble in water, and the halides (except fluorides) of alkaline earth metals are also easily soluble in water. The vast majority of alkali and alkaline earth metal salts belong to typical ionic compounds. Summary of the Invention

[0004] Starting from the mechanism of antigen charge interaction and ion-specific effect, the present invention selects a safe and effective inorganic alkali and alkaline earth metal salt ionic liquid to form a resuspension to effectively disperse and resuspend the precipitated antigen in the actual purification production process of the foot-and-mouth disease vaccine, achieving the purpose of uniform antigen distribution, complete morphological structure, stable antigen state, and safe and effective antigen. The resuspension provided by the present invention does not contain substances such as proteins, amino acids, and sugars, has a simple structure, clear components, and controllable quality, does not have an adverse impact on subsequent processes, and is conducive to large-scale production applications.

[0005] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0006] A foot-and-mouth disease purified antigen resuspension is composed of the following substances in mass percentage: alkali metal salt 0.005 - 5 mol / L, alkaline earth metal salt 0.005 - 5 mol / L, and the balance is injection water.

[0007] The alkali metal salt includes one or more inorganic salts of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and francium (Fr);

[0008] The alkaline earth metal salt includes one or more inorganic salts of beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), etc.

[0009] Compared with the application of the prior art, the beneficial effects of the present invention are as follows: to provide a purified foot-and-mouth disease antigen resuspension solution, which can promote the successful resuspension of antigen precipitates in the purification process, with the antigen evenly distributed, the morphological structure intact, and the antigen state stable, thereby protecting the integrity of the foot-and-mouth disease antigen, inhibiting the degradation of its active ingredient 146S, ensuring its immunogenicity, and being suitable for the long-term preservation of the foot-and-mouth disease resuspended antigen. The components of the resuspension solution used are all inorganic salts of alkali and alkaline earth metals, without organic components such as sugars, amino acids, and proteins. These components do not interfere with the determination of total protein by the Lowry method for protein determination, do not affect the evaluation of the purity of the vaccine, meet national standards and GMP standards, and the resuspension solution has clear components, reasonable design, simple preparation, and safe and efficient use.

[0010] The present invention verifies through the 37°C accelerated aging experiment and the 4°C long-term preservation experiment that the resuspension solution provided by the present invention can effectively disperse the purified antigen, protect the antigen integrity, and inhibit the degradation of 146S, and is suitable for the long-term preservation of the resuspended purified antigen. The present invention proves through animal experiments that the provided foot-and-mouth disease antigen resuspension solution does not affect the immunogenicity of the antigen protein and has a positive effect on the immune effect of animals.

[0011] Based on the above actual needs and principles, the resuspension solution provided by the present invention contains alkali metal salts as one or more inorganic salts of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs), and francium (Fr), and the alkaline earth metal salts contained in the present invention are one or more inorganic salts of beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), and radium (Ra).

[0012] Further, the alkali and alkaline earth metal salts are sulfates, phosphates, nitrates, chlorates, hydrogen phosphates, carbonates, hydrogen sulfates, sulfites, etc. For example, the alkali metal ion salts can be lithium chloride (LiCl), potassium chloride (KCl), potassium hydrogen sulfate (KHSO 4 ), potassium sulfate (K 2 SO 4 ), potassium nitrate (KNO 3 ), potassium carbonate (K 2 CO 3 ), potassium bicarbonate KHCO 3 , potassium sulfite (K 2 SO 3) Potassium thiosulfate (K 2 S 2 O 3 ) Dipotassium hydrogen phosphate (K 2 HPO 4 ) Potassium dihydrogen phosphate (K H 2 PO 4 ) Sodium chloride (NaCl), Sodium bisulfate (NaHSO 4 ) Sodium sulfate (Na 2 SO 4 ) Sodium nitrate (NaNO 3 ) Sodium sulfite (Na 2 SO 3 ) Sodium thiosulfate (Na 2 S 2 O 3 ) Sodium carbonate (Na 2 CO 3 ) Sodium bicarbonate (NaHCO 3 ) Disodium hydrogen phosphate (Na 2 HPO 4 ) Sodium dihydrogen phosphate (NaH 2 PO 4 ) Cesium chloride (CsCl), Cesium sulfate (Cs 2 SO 4 ) Cesium nitrate (CsNO 3 ) Rubidium chloride ((RbCl), Rubidium nitrate (RcNO 3 ) and Rubidium sulfate (Rb 2 SO 4 ) in any combination of one or more of them.

[0013] The alkaline earth metal ion salt is magnesium chloride (MgCl 2 ), Magnesium sulfate (MgSO 4 ), Magnesium bisulfate (Mg(HSO 4 )) 2 ) Magnesium nitrate (Mg(NO 3 )) 2 ) Calcium chloride (CaCl 2 ), Calcium nitrate

[0014] (Ca(NO 3 )) 2 ) Calcium bisulfate (Ca(HSO 4 )) 2 ) Calcium sulfite (CaSO 3 ), Barium chloride (BaCl 2 ), Barium nitrate (Ba(NO 3 )) 2 ) Strontium chloride (SrCl 2) and strontium nitrate (SrNO 3 ) 2 ) or any combination of two or more of them.

[0015] Preferably, the alkali metal ion salt can be: potassium chloride ((KCl)), potassium bisulfate (KHSO 4 ), potassium sulfate (K 2 SO 4 ), potassium carbonate (K 2 CO 3 ), potassium bicarbonate (KHCO 3 ), potassium sulfite (K 2 SO 3 ), potassium thiosulfate (K 2 S 2 O 3 ), sodium chloride (NaCl), sodium sulfate (Na 2 SO 4 ), sodium bisulfate (NaHSO 4 ), sodium sulfite (Na 2 SO 3 ), sodium thiosulfate (Na 2 S 2 O 3 ), sodium carbonate (Na 2 CO 3 ), sodium bicarbonate (NaHCO 3 ), cesium chloride (CsCl), cesium sulfate (Cs 2 SO 4 ), cesium nitrate (CsNO 3 ), rubidium chloride ((RbCl) and rubidium nitrate (RbNO 3 ), or any combination of two or more of them.

[0016] The alkaline earth metal ion salt can be: magnesium chloride (MgCl 2 ), magnesium sulfate (MgSO 4 ), magnesium nitrate (Mg(NO 3 )) 2 ), calcium chloride (CaCl 2 ), calcium nitrate (Ca(NO 3 )) 2 ), calcium bisulfate (Ca(HSO 4 )) 2 ), calcium sulfite (CaSO 3 ), barium chloride (BaCl 2 ), and strontium chloride (SrCl 2 ), or any combination of two or more of them.

[0017] More preferably, the alkali metal ion salt may be: potassium chloride (KCl), potassium bicarbonate (KHCO 3 ), potassium sulfate (K 2 SO 4 ), potassium bisulfate (KHSO 4 ), potassium sulfite (K 2 SO 3 ), potassium thiosulfate (K 2 S 2 O 3 ), sodium chloride (NaCl), sodium bicarbonate (NaHCO 3 ), sodium sulfate (Na 2 SO 4 ), sodium bisulfate (NaHSO 4 ), sodium sulfite (Na 2 SO 3 ), sodium thiosulfate (Na 2 S 2 O 3 ), cesium chloride (CsCl), and rubidium chloride ((RbCl), or any combination of two or more thereof.

[0018] The alkaline earth metal ion salt may be: magnesium chloride (MgCl 2 ), magnesium sulfate (MgSO 4 ), magnesium nitrate (Mg(NO 3 ) 2 ), calcium chloride (CaCl 2 ), calcium nitrate (Ca(NO 3 ) 2 ), calcium bisulfate (Ca(HSO 4 ) 2 ), calcium sulfite (CaSO 3 ), and strontium chloride (SrCl 2 ), or any combination of two or more thereof.

[0019] In a specific embodiment of the present invention, the foot-and-mouth disease purified antigen resuspension is composed of the components described in any one of the following:

[0020] 1) 0.1 mol / L KCl, 0.1 mol / L RbCl, 0.005 mol / L K 2 SO 4 0.005 mol / L Mg(NO 3 ) 2

[0021] 0.005 mol / L CaCl 2 0.005 mol / L SrCl 2 0.005 mol / L, and the balance is water for injection;

[0022] 2) 0.1 mol / L of NaCl, 0.1 mol / L of RbCl, K 2 SO 3 0.005 mol / L, KHCO 3

[0023] 0.005 mol / L, Na 2 SO 4 0.005 mol / L, Na 2 SO 3 0.005 mol / L, MgCl 2 0.005 mol / L, Ca(NO 3 ) 2 0.005 mol / L, SrCl 2 0.005 mol / L, with the balance being water for injection;

[0024] 3) 0.1 mol / L of NaCl, 0.1 mol / L of RbCl, K 2 SO 4 0.005 mol / L, Na 2 SO 3

[0025] 0.005 mol / L, Na 2 S 2 O 3 0.005 mol / L, MgSO 4 0.005 mol / L, Mg(NO 3 ) 2

[0026] 0.005 mol / L, CaSO 3 0.005 mol / L, SrCl 2 0.005 mol / L, with the balance being water for injection;

[0027] 4) 0.1 mol / L of KCl, 0.1 mol / L of NaCl, 0.1 mol / L of RbCl, K 2 SO 4 0.005 mol / L, K 2 SO 3 0.005 mol / L, Na 2 S 2 O 3 0.005 mol / L, Mg(NO 3 ) 2 0.005 mol / L, Ca(HSO 4 )2 0.005 mol / L, CaSO 3 0.005 mol / L, SrCl 2 0.005 mol / L, with the balance being water for injection;

[0028] 5) NaCl 0.1 mol / L, RbCl 0.1 mol / L, K 2 SO 3 0.005 mol / L, K 2 S 2 O 3

[0029] 0.005 mol / L, Na 2 SO 3 0.005 mol / L, MgSO 4 0.005 mol / L, Ca(HSO 4 ) 2

[0030] 0.005 mol / L, SrCl 2 0.005 mol / L, with the balance being water for injection;

[0031] 6) KCl 0.1 mol / L, RbCl 0.1 mol / L, KHSO 4 0.005 mol / L, KHCO 3

[0032] 0.005 mol / L, Na 2 SO 4 0.005 mol / L, CsCl 0.005 mol / L, Ca(HSO 4 ) 2

[0033] 0.005 mol / L, CaSO 3 0.005 mol / L, SrCl 2 0.005 mol / L, with the balance being water for injection.

[0034] Furthermore, the present invention verifies that the resuspension solution provided by the present invention can effectively preserve antigens through 37°C accelerated aging experiments and 4°C long-term storage experiments. Through the evaluation of the protection against virus challenge in animal immunization experiments, it is shown that the resuspension provided by the present invention has a positive effect on the immune effect of the vaccine. The alkali and alkaline earth metal ion salts used in the present invention are all commercially available products. After being formulated, they can resuspend the purified antigen and inactivated antigen, which is very convenient for large-scale production, safe and reliable to use, and has broad application prospects. The inorganic alkali and alkaline earth ion resuspension solution designed by the present invention can not only effectively resuspend the purified product, but also provide long-term preservation for foot-and-mouth disease antigens and protect their immune activity. At the same time, no additional organic components such as sugars, amino acids, and proteins are added to the resuspension solution. Therefore, animals can obtain good immune protection while effectively reducing the occurrence of animal side reactions. **Detailed Description of the Invention**

[0035] The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0036] In the following experimental methods of the examples, unless otherwise specified, they are all conventional methods. The reagent materials used in the following examples, unless otherwise specified, are all commercially purchased products.

[0037] Is there an optimization of the ratio and components of the alkali metal salt and alkaline earth metal salt of the present invention to highlight the creativity of the present invention?

[0038] Example Preparation:

[0039] Potassium chloride (KCl), potassium bicarbonate (KHCO 3 ), potassium bisulfate (KHSO 4 ), potassium sulfate (K 2 SO 4 ), potassium sulfite (K 2 SO 3 ), potassium thiosulfate (K 2 S 2 O 3 ), sodium chloride (NaCl), sodium bicarbonate (NaHCO 3 ), sodium bisulfate (NaHSO 4 ), sodium sulfate (Na 2 SO 4 ), sodium sulfite (Na 2 SO 3 ), sodium thiosulfate (Na 2 S 2 O 3 ), cesium chloride (CsCl), rubidium chloride (RbCl); magnesium chloride (MgCl 2 ), magnesium sulfate (MgSO4 ) Magnesium nitrate (Mg(NO 3 ) 2 ) Calcium chloride (CaCl 2 ) Calcium nitrate (Ca(NO 3 ) 2 ) Calcium bisulfate (Ca(HSO 4 ) 2 ) Calcium sulfite (CaSO 3 ) Strontium chloride (SrCl 2 ) were each prepared into a 1 mol / L stock solution and stored at 4 °C for later use.

[0040] Example 1

[0041] Take 100 mL each of the stock solutions of KCl and RbCl in the above-mentioned preparation solution for implementation. For K 2 SO 4 , Mg(NO 3 ) 2 , CaCl 2 , SrCl 2 in the above stock solution, take 5 mL each and add them to 800 mL of water for injection. Shake well and finally make up the volume to 1000 mL. After sterilizing and filtering with a 0.22 - μm filter membrane, store it at 4 °C. According to the concentration required by the production process, add the resuspension to the purified antigen precipitate for resuspension to obtain a resuspended purified antigen solution and store it at 4 °C.

[0042] Example 2

[0043] Take 100 mL each of the stock solutions of NaCl and RbCl in the above-mentioned preparation solution for implementation. For the stock solutions of K 2 SO 3 , KHCO 3 , Na 2 SO 4 , Na 2 SO 3 , MgCl 2 , Ca(NO 3 ) 2 , SrCl 2 in the above stock solution, take 5 mL each and add them to 800 mL of water for injection. Shake well and finally make up the volume to 1000 mL. After sterilizing and filtering with a 0.22 - μm filter membrane, store it at 4 °C. According to the concentration required by the production process, add the resuspension to the purified antigen precipitate for resuspension to obtain a resuspended purified antigen solution and store it at 4 °C.

[0044] Example 3

[0045] Take 100 mL each of the stock solutions of NaCl and RbCl in the above-mentioned preparation solution for implementation. For K 2 SO 4, Na 2 SO 3 , Na 2 S 2 O 3 , MgSO 4 , Mg(NO 3 ) 2 , CaSO 3 , SrCl 2 Take 5 mL of each, add them to 800 mL of water for injection, shake well and finally make up the volume to 1000 mL. After sterilizing and filtering with a 0.22 - um filter membrane, store at 4°C. According to the concentration required by the production process, add the resuspension to the purified antigen precipitate for resuspension, and store the resuspended purified antigen solution at 4°C.

[0046] Example 4

[0047] Take 100 mL of each of the mother liquors KCl, NaCl, and RbCl in the above - mentioned implementation preparation solution. For the K 2 SO 4 , K 2 SO 3 , Na 2 S 2 O 3 , Mg(NO 3 ) 2 , Ca(HSO 4 ) 2 , CaSO 3 , SrCl 2 Take 5 mL of each, add them to 800 mL of water for injection, shake well and finally make up the volume to 1000 mL. After sterilizing and filtering with a 0.22 - um filter membrane, store at 4°C. According to the concentration required by the production process, add the resuspension to the purified antigen precipitate for resuspension, and store the resuspended purified antigen solution at 4°C.

[0048] Example 5

[0049] Take 100 mL of each of the mother liquors NaCl and RbCl in the above - mentioned implementation preparation solution. For the K 2 SO 3 ,, K 2 S 2 O 3 , Na 2 SO 3 , MgSO 4 , Ca(HSO 4 ) 2 , SrCl 2Take 5 mL each and add them to 800 mL of water for injection. Shake well and finally make up the volume to 1000 mL. After sterilizing and filtering with a 0.22-μm filter membrane, store it at 4°C. According to the concentration required by the production process, add the resuspension to the purified antigen precipitate for resuspension to obtain the resuspended purified antigen solution and store it at 4°C.

[0050] Example 6

[0051] Take 100 mL each of the mother liquors KCl and RbCl in the above-mentioned preparation solution for implementation, and take KHSO 4 , KHCO 3 , Na 2 SO 4 , CsCl, Ca(HSO 4 ) 2 , CaSO 3 , SrCl 2 Take 5 mL each and add them to 800 mL of water for injection. Shake well and finally make up the volume to 1000 mL. After sterilizing and filtering with a 0.22-μm filter membrane, store it at 4°C. According to the concentration required by the production process, add the resuspension to the purified antigen precipitate for resuspension to obtain the resuspended purified antigen solution and store it at 4°C.

[0052] Control example:

[0053] Using a conventional PBS resuspension (0.02 mol / L PBS, pH 8.0), according to the concentration required by the production process, add the PBS to the purified antigen precipitate for resuspension to obtain the PBS-resuspended purified antigen solution and store it at 4°C.

[0054] Experimental example 1

[0055] Under aseptic conditions, aliquot the resuspended purified antigen solutions in Examples 1 to 6 and the PBS-resuspended purified antigen solution in the control example, and place them in a water bath at 37°C for an accelerated test. The sampling times are 0 day, 7 days, 14 days, 21 days, and 28 days. The content (μg / ml) of foot-and-mouth disease virus 146S in the resuspended purified antigen was determined by sucrose density gradient ultracentrifugation. The results are shown in Table 1 below:

[0056] Table 1: Determination values of antigen 146S in the 37°C accelerated test

[0057]

[0058] Calculate the preservation rate of antigen 146S in the 37°C accelerated test according to the data in Table 1 as shown in Table 2 below

[0059] Table 2: Preservation rate of antigen 146S in the 37°C accelerated test

[0060]

[0061]

[0062] Under the test under the 37°C accelerated condition, for the resuspension of the purified antigen with the resuspension solution provided by the present invention, the decline of antigen 146S from the 7th day to the 28th day does not exceed 3%, and there is basically no degradation. However, for the conventional PBS resuspension solution used in the control example, after the 7-day accelerated test, the antigen loss can reach more than 50%, and the remaining antigen 146S at the 28th day is less than 5% (as shown in Tables 1 and 2). This shows that the resuspension solution provided by the present invention can improve the thermal stability of antigen 146S compared with conventional PBS, and can significantly improve the thermal stability of the purified resuspended antigen.

[0063] Experimental Example 2

[0064] The resuspended purified antigen solutions in Examples 1 to 6 and the resuspended purified antigen solution in the control example were aseptically aliquoted and stored at 4°C for 12 months. Then, the content (μg / ml) of foot-and-mouth disease virus 146S in the resuspended purified antigen was determined by sucrose density gradient ultracentrifugation. The results are shown in Table 3 below:

[0065] Table 3 Determination values and preservation rates of antigen 146S stored at 4°C for 12 months

[0066]

[0067] It can be seen from Table 3 that for the antigen solution resuspended with the control PBS buffer, the decline of 146S after being stored at 4°C for 12 months is more than 50%, while for each of the examples provided by the present invention, the decline of 146S after 12 months is less than 1%. This shows that the resuspension solution of the present invention has the effect of reducing the degradation of 146S antigen and maintaining the thermal stability of the resuspended antigen solution, and is suitable for the long-term preservation of the purified antigen after resuspension.

[0068] Experimental Example 3 Animal testing experiment

[0069] a. Vaccine preparation:

[0070] Production diluent was added to the resuspended purified antigen solutions in Examples 1 to 6 and the control example to make the final concentration of antigen 146S 25 μg / ml to form the vaccine aqueous phase. According to the mass ratio of aqueous phase:oil adjuvant = 1:1, the vaccine was emulsified according to the requirements of the production process. After emulsification, the vaccine was aliquoted and stored refrigerated at 4°C.

[0071] b. Animal immunization experiment:

[0072] The animals used were 107 healthy piglets about 40 kg in weight and four months old. They were diagnosed as negative by a liquid-phase blocking ELISA kit, with serum neutralizing antibody not higher than 1:8 and diagnosed as negative by a 3ABC monoclonal antibody detection kit. 105 pigs were randomly divided into 7 groups, including 7 groups of vaccines of Examples 1 to 6 and the control group, with 15 pigs in each group. 5 pigs in each group were immunized with 1 dose, 1 / 3 dose, and 1 / 9 dose respectively, and at the same time, two pigs were set as the blank group. 28 days after immunization, the virus strain used was the OS99 standard virulent strain, and the challenge dose was 1000 ID 50 / pig, and the challenge method was intramuscular injection in the neck. The pigs were continuously observed for 10 days after challenge, and the morbidity was recorded. According to the number of protected immunized pigs, the PD of the vaccine was calculated by the Reed-Muench method 50 . The results are shown in Table 4

[0073] Table 4: Results of PD50 of animal immunization

[0074]

[0075]

[0076] According to the challenge results shown in Table 4, the PD of each group of vaccines 50 values all reached the requirements of the regulations (>6 PD 50 ). The blank control group got sick, and the challenge results were valid. Among them, the PD 50 values of each embodiment of the present invention were better than those of the control vaccine group. All animals in each group of the vaccines of the embodiments of the present invention were protected, reaching the theoretical limit value of 15.59, indicating that compared with the conventional PBS resuspension, the potency test value of the purified antigen resuspension provided by the present invention is not lower than the existing level, and has a positive impact on the animal immunization effect

[0077] The above Examples 1 to 6 are only the preferred embodiments of the present invention. It should be noted that: the embodiments of the present invention are not limited to the above examples. Any other changes, substitutions, and combinations made without departing from the essential principles or component compositions of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention. For those of ordinary skill in the relevant technical fields of the present invention, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as within the protection scope of the present invention

Claims

1. A foot-and-mouth disease purified antigen resuspension, consisting of the following components: 0.005-5 mol / L of alkali metal salt, 0.005-5 mol / L of alkaline earth metal salt, and the balance is water for injection; the alkali metal salt is selected from one or more inorganic salts of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) and francium (Fr); the alkaline earth metal salt is selected from one or more inorganic salts of beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra).

2. The foot-and-mouth disease purified antigen resuspension according to claim 1, characterized in that The alkali metal salt or alkaline earth metal salt can be sulfate, phosphate, nitrate, chloride, carbonate or sulfite.

3. The foot-and-mouth disease purified antigen resuspension according to claim 2, characterized in that The alkali metal ion salt can be lithium chloride (LiCl), potassium chloride (KCl), potassium hydrogen sulfate (KHSO4), potassium sulfate (K2SO4), potassium nitrate (KNO3), potassium carbonate (K2CO3), potassium bicarbonate (KHCO3), potassium sulfite (K2SO3), potassium thiosulfate (K2S2O3), dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), sodium bisulfate (NaHSO4), sodium sulfate (Na2SO4), sodium nitrate (NaNO3), sodium sulfite (Na2SO3), sodium thiosulfate (Na2S2O3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), cesium chloride (CsCl), cesium sulfate (Cs2SO4), cesium nitrate (CsNO3), rubidium chloride ((RbCl), rubidium nitrate (RcNO3) and rubidium sulfate (Rb2SO4) One or any combination of two or more; The alkaline earth metal ion salt is magnesium chloride (MgCl2), magnesium sulfate (MgSO4), magnesium hydrogen sulfate (Mg(HSO 42 ), magnesium nitrate (Mg(NO3)2), calcium chloride (CaCl2), calcium nitrate (Ca(NO3)2), calcium hydrogen sulfate (Ca(HSO4)2), calcium sulfite (CaSO3), barium chloride (BaCl2), barium nitrate (Ba(NO3)2), strontium chloride (SrCl2) and strontium nitrate (Sr(NO3)2) One or any combination of two or more thereof.

4. The foot-and-mouth disease purified antigen resuspension according to claim 3, characterized in that The alkali metal ion salt is potassium chloride (KCl), potassium bisulfate (KHSO4), potassium sulfate (K2SO4), potassium carbonate (K2CO3), potassium bicarbonate (KHCO3), potassium sulfite (K2SO3), potassium thiosulfate (K2S2O3), sodium chloride (NaCl), sodium sulfate (Na2SO4), sodium bisulfate (NaHSO4), sodium sulfite (Na2SO3), sodium thiosulfate (Na2S2O3), sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), cesium chloride (CsCl), cesium sulfate (Cs2SO4), cesium nitrate (CsNO3), rubidium chloride (RbCl) and rubidium nitrate (RbNO3) or any combination of two or more thereof; The alkaline earth metal ion salt is magnesium chloride (MgCl2), magnesium sulfate (MgSO4), magnesium nitrate (Mg(NO3)2), calcium chloride (CaCl2), calcium nitrate (Ca(NO3)2, calcium hydrogen sulfate (Ca(HSO4)2), calcium sulfite (CaSO3), barium chloride (BaCl2) and strontium chloride (SrCl2) or any combination of two or more thereof.

5. The foot-and-mouth disease purified antigen resuspension according to claim 4, characterized in that The alkali metal ion salt is potassium chloride (KCl), potassium bicarbonate (KHCO3), potassium sulfate (K2SO4), potassium bisulfate (KHSO4), potassium sulfite (K2SO3), potassium thiosulfate (K2S2O3), sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium sulfate (Na2SO4), sodium bisulfate (NaHSO4), sodium sulfite (Na2SO3), sodium thiosulfate (Na2S2O3), cesium chloride (CsCl) and rubidium chloride ((RbCl)) or any combination of two or more thereof; The alkaline earth metal ion salt is one or any combination of two or more of magnesium chloride (MgCl2), magnesium sulfate (MgSO4), magnesium nitrate (Mg(NO3)2), calcium chloride (CaCl2), calcium nitrate (Ca(NO3)2), calcium hydrogen sulfate (Ca(HSO4)2), calcium sulfite (CaSO3) and strontium chloride (SrCl2).

6. Use of the foot-and-mouth disease virus purified antigen resuspension according to any one of claims 1 to 5 in suspending foot-and-mouth disease virus antigen to prepare a suspension.

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