CN110734478B

CN110734478B - Foot-and-mouth disease A type synthetic peptide vaccine and preparation method and application thereof

Info

Publication number: CN110734478B
Application number: CN201911085560.0A
Authority: CN
Inventors: 吴冬荀; 蔡薇; 肖进; 向王震
Original assignee: China Animal Husbandry Industry Co Ltd
Current assignee: China Animal Husbandry Industry Co Ltd
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2020-12-01
Anticipated expiration: 2039-11-08
Also published as: CN110734478A

Abstract

The invention provides a synthetic peptide vaccine for preparing foot-and-mouth disease A type, a preparation method and application thereof. The aftosa A type synthetic peptide vaccine comprises an antigen polypeptide composition, wherein the antigen polypeptide composition comprises polypeptides shown in a sequence 1 and a sequence 2. The aftosa synthetic peptide vaccine provided by the invention has good immune efficacy, and the antigen of the vaccine is chemically synthesized polypeptide, does not contain aftosa virus nucleic acid particles, and has high safety. Therefore, the vaccine of the invention can effectively cope with the current aftosa epidemic strains, has no biological safety, is easy to synthesize in a large scale and has good application prospect. The invention also provides a preparation method of the polypeptide and the peptide vaccine and a pharmaceutical application thereof.

Description

Foot-and-mouth disease A type synthetic peptide vaccine and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a foot-and-mouth disease synthetic peptide vaccine and a preparation method thereof.

Background

Foot and Mouth Disease (FMD) is an acute, highly contact and febrile infectious disease of artiodactyl and is widely distributed worldwide. Foot-and-mouth disease is highly contagious and rapidly spread, which causes death of young livestock and sharply decreases production capacity of adult animals after infecting livestock such as pigs, sheep and the like, thereby seriously harming the development of animal husbandry and the production and supply of meat and livestock products thereof. At present, foot-and-mouth disease causes the market circulation and international trade of animals and animal products to be greatly blocked and limited, and causes huge economic loss to animal husbandry production in epidemic countries and regions.

Foot-and-mouth disease is caused by foot-and-mouth disease virus (FMDV) infection. Foot-and-mouth disease virus belongs to picornavirus and has the characteristics of polymorphism, changeability and the like. Currently, there are 7 serotypes of foot and mouth disease virus known worldwide: A. o, C, Sat1 (south African type I), Sat2 (south African type II), Sat3 (south African type III) and Asia I (Asia type I). Each of these major types is divided into several subtypes, and more than 70 subtypes have been found. The foot and mouth disease viruses of serotype A, O, C and Asia I are most common, with serotype A viruses being the most varied, having more than 30 subtypes. The results of the study showed that the capsid protein of foot-and-mouth disease virus is composed of four structural proteins VP1, VP2, VP3 and VP4, 60 each. VP1-VP3 constitute capsid protein subunits, located outside the capsid protein, whereas VP4 is located inside the viral particle.

The foot and mouth disease viruses have different antigenicity among different types and cannot be mutually immunized. Furthermore, the degree of antigenic variation within the same serotype is so great that a foot and mouth disease vaccine that is effective against one subtype may not be protective against another subtype within the same serotype. In addition, the antigenicity of the foot-and-mouth disease strain is continuously changed, and the efficacy of the original vaccine is weakened or even disappears with the lapse of time, thereby bringing great difficulty to the prevention and treatment work of the foot-and-mouth disease.

At present, the foot-and-mouth diseases popular in the swinery of China are mainly O-type and A-type foot-and-mouth diseases. The foot-and-mouth disease is forcibly immunized in China, and vaccine immunization is a main means for preventing and treating the foot-and-mouth disease. However, the foot-and-mouth disease vaccine currently used in China is mainly a virus inactivated vaccine, and has the problems of poor biological safety, large side effect, unstable product quality and the like. In contrast, many countries in the world today have stopped using inactivated vaccines, and import of animal products from countries where inactivated vaccines are used is prohibited. Therefore, in the aspect of preventing and treating the foot-and-mouth disease, China lags behind the world development situation.

In the research aspect of the novel aftosa vaccine, the research reports of a genetic engineering subunit vaccine, an aftosa virus vector vaccine and an aftosa genetic engineering modified vaccine exist in sequence, but the research reports have many problems in the aspects of immune effect and biological safety, and the use of the novel vaccine is influenced. In addition, these vaccines tend to be less effective against circulating strains that have become mutated at present and do not protect animals effectively. Thus, there remains a need in the art for a safe and effective novel vaccine for foot-and-mouth disease.

Disclosure of Invention

The invention aims to provide an antigenic polypeptide for preparing a aftosa A type synthetic peptide vaccine, a polypeptide composition and a vaccine containing the polypeptide composition.

In order to achieve the purpose, 2 polypeptide sequences consisting of amino acids are designed in the laboratory according to the epitope of the foot-and-mouth disease type A epidemic virus, wherein the polypeptide sequences are respectively shown as a sequence 1 and a sequence 2 in a sequence table.

Synthesizing by solid phase method, and mixing with ISA50V adjuvant to obtain oil emulsion vaccine. Proved by efficacy tests, the foot-and-mouth disease type A synthetic peptide vaccine aims at PD attacked by AF/72 virulent virus₅₀The values exceed 6, the protection requirement is met, and the ability of protecting the test animal pigs from virus attack is achieved. Safety tests prove that the aftosa synthetic peptide vaccine has no side reaction on guinea pigs, mice and pigs, has high safety and no toxin-dispersing danger, so the aftosa synthetic peptide vaccine has good popularization prospect and market value.

The synthetic peptide vaccine greatly improves the safety and specificity, and the preparation process is simpler, the production cost is lower, the vaccine quality is more reliable, and the large-scale production is facilitated. As a novel foot-and-mouth disease vaccine, the vaccine has the advantages of bringing new competitiveness and economic growth points for enterprises and markets and having huge market space.

The invention adopts the following technical scheme:

the invention provides an antigen polypeptide for preparing a foot-and-mouth disease synthetic peptide vaccine, which is a polypeptide shown as a sequence 1 or a sequence 2 in a sequence table.

The antigen polypeptide composition for preparing the foot-and-mouth disease synthetic peptide vaccine comprises a composition of a polypeptide described by a sequence 1 in a sequence table and a polypeptide described by a sequence 2 in the sequence table.

The polypeptide fragment of the present invention may be a peptide substance obtained by solid phase organic synthesis.

When two polypeptide compositions are selected to form the vaccine, the molar ratio of the two polypeptide compositions is (0.5-1.5): 0.5-1.5); most preferably, they are present in a molar ratio of 1: 1.

The application of the polypeptide composition in preparing the aftosa synthetic peptide vaccine also belongs to the protection scope of the invention.

The invention provides a foot-and-mouth disease synthetic peptide vaccine which comprises the polypeptide composition.

The peptide vaccine further comprises an adjuvant.

The invention provides a preparation method of the aftosa synthetic peptide vaccine, which comprises the following steps:

(1) diluting the polypeptide composition with water for injection to a concentration of 10-100. mu.g/ml (preferably 50. mu.g/ml) to obtain a polypeptide antigen aqueous phase;

(2) sterilizing the adjuvant (30 minutes at 121 ℃);

(3) under the condition of 20-28 ℃, according to the volume ratio of the polypeptide antigen water phase to the adjuvant of 1:1, the adjuvant is firstly added into an emulsification tank, stirred for 1.5-3 minutes at 90-150 rpm, then the polypeptide antigen water phase is slowly added, stirred for 20-30 minutes, then stirred for 15-30 minutes at 8000-10000 rpm, kept stand for 3-10 minutes (preferably 5 minutes), and subpackaged.

Preferably, the adjuvant is one or more selected from white oil, 50V, 50VII (montainide ISA50V, 50VII adjuvant (SEPPIC corporation, france)).

The invention also provides a preparation method of the polypeptide in the polypeptide polymer, which comprises the following steps:

(1) taking amino resin as an initial raw material, taking amino acid protected by 9-fluorenylmethyloxycarbonyl as a monomer, sequentially condensing and connecting the amino acid according to the amino acid sequence to synthesize the polypeptide, and sealing an unreacted amino end by using acetyl imidazole after each step of condensation reaction;

(2) after the synthesis is finished, adding a cracking reagent so as to crack the polypeptide from the amino resin;

(3) precipitating the polypeptide using diethyl ether;

(4) the polypeptide is purified by ultrafiltration and then subjected to aseptic processing.

In the above preparation method, the step (1) specifically includes the steps of:

(1-a) deprotection reaction: putting amino resin into N-methyl pyrrolidone (NMP) solution of hexahydropyridine with the volume percentage of 15-30%, and reacting for 25-40 minutes at the temperature of 20-28 ℃, so as to remove 9-fluorenylmethoxycarbonyl protective groups on the amino resin;

(1-b) washing: blowing the amino resin by nitrogen, and then washing the amino resin by N-methylpyrrolidone;

(1-c) condensation reaction: adding 1-hydroxy benzotriazole (HOBT), Diisopropylcarbodiimide (DIC) and amino acid protected by 9-fluorenylmethoxycarbonyl, and reacting at 20-28 ℃ for 0.5-2.5 hours;

(1-d) washing: blowing the amino resin by nitrogen, and then washing the amino resin by N-methylpyrrolidone;

(1-e) blocking reaction: adding 1.5-4% by weight of N-methylpyrrolidone (NMP) solution of the acetyl imidazole, and reacting for 20-40 minutes at the temperature of 20-28 ℃.

In the preparation method, the components of the cracking reagent in the step (2) are trifluoroacetic acid, triisopropylsilane, phenol and water in a volume ratio of 85:8: 6: 1; and the cracking time of the step (2) is 1-4 hours.

In the above preparation method, the step (3) specifically includes:

(3-a) precipitating the polypeptide using diethyl ether, followed by washing with dimethylformamide;

(3-b) forming a disulfide bond between two cysteines in the amino acid sequence of the precipitated polypeptide with the addition of 10% DMSO; or, reacting head and tail amino acid residues of the amino acid sequence of the polypeptide to form a covalent linkage; preferably, the carboxyl group and the amino group of the head and tail amino acid residues of the amino acid sequence of the polypeptide are reacted with each other under the condition of 1% DIC and 0.5% HOBT addition, or are reacted under the condition of 0.1M H₂SO₄In which case the carboxyl group reacts with the hydroxyl group to form a covalent linkage.

In the above preparation method, the step (4) specifically includes the steps of:

(4-a) ultrafiltering the polypeptide by using a tangential filtration membrane at the temperature of 20-28 ℃ so as to remove small molecular impurities;

(4-b) sterilized and stored by using a 0.2 μm filter.

In still another aspect, the invention provides the use of the above polypeptide or polypeptide vaccine in the preparation of a biological product for preventing swine type a foot-and-mouth disease.

Specifically, the invention researches the variation condition of main antigen sites of the foot-and-mouth disease by sequence determination of recent epidemic strains of the foot-and-mouth disease in China and combining with the sequence of a foot-and-mouth disease vaccine strain, counts the variation frequency of the main variation amino acid sites, and simultaneously analyzes and predicts the foot-and-mouth disease antigen sites by combining with the assistance of a computer, and chemically synthesizes possible antigen site peptide segments, namely uses different amino acids at the sites according to the statistical variation frequency aiming at the easy variation sites, so as to obtain a plurality of candidate polypeptide antigens covering all the current possible variation sites. Furthermore, the candidate polypeptide antigens are screened through a large number of animal experiments to obtain the polypeptide antigen which can cause the immune response of animals, has high immune response level and can well protect the animals from being attacked by the aftosa epidemic strains. In addition, the inventor optimizes the foot-and-mouth disease virus antigen sites according to screening experiment results, and a large amount of screening is carried out to obtain the polypeptide with excellent immune effect, so that the immune effect of the polypeptide antigen combination is enhanced.

The results of efficacy experiments and safety experiments of the synthetic peptide vaccine show that the synthetic peptide vaccine for foot-and-mouth disease provided by the invention has good immune efficacy and is safe to guinea pigs, mice and domestic pigs, so that the vaccine provided by the invention can effectively cope with the antigen variation of the current foot-and-mouth disease virus, has no biological safety, is easy to synthesize in a large scale and has good application prospect.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

Example 1 solid phase Synthesis of polypeptide antigen for synthetic peptide vaccine for foot-and-mouth disease

The invention researches the variation condition of the main antigen site of the foot-and-mouth disease by determining the sequence of the recent epidemic strain of the foot-and-mouth disease in China and combining the sequence of the vaccine strain of the foot-and-mouth disease, counts the variation frequency of the main variation amino acid site, and analyzes and predicts the antigen site of the foot-and-mouth disease by combining the assistance of a computer, and chemically synthesizes possible antigen site peptide segments, namely uses different amino acids at the sites according to the statistical variation frequency aiming at the easily variable sites, so as to obtain a plurality of candidate polypeptide antigens covering all the possible variation sites at present. Furthermore, the candidate polypeptide antigens are screened through a large number of animal experiments to obtain the polypeptide antigen which can cause the immune response of animals, has high immune response level and can well protect the animals from being attacked by the aftosa epidemic strains. In addition, the inventor optimizes the foot-and-mouth disease virus antigen site according to the screening experiment result, effectively combines T cell epitope and B cell epitope, and enhances the immune effect of the polypeptide antigen.

The synthetic peptide antigen of the present invention, which was screened and identified, was prepared using a Merrifield solid phase synthesis method using a fully automated peptide synthesizer, USA 433A, in which an amino acid modified with 9-fluorenylmethyloxycarbonyl (Fmoc) was used and the solid phase carrier was Rink Amide MBHA resin from Sigma, USA. The production process generally comprises solid phase synthesis of polypeptide antigen, cleavage of polypeptide, purification of antigen and aseptic storage.

Solid phase synthesis of synthetic peptide antigens

1.1.1 Synthesis Material preparation

The sequence of the synthetic polypeptide antigen is as follows:

the polypeptide shown in sequence 1 in the sequence table and the polypeptide shown in sequence 2 in the sequence table.

Depending on the sequence of the antigen and the synthetic scale of 1mmol, the appropriate Fmoc-modified amino acids [ purchased from gill, shanghai biochemistry ] were prepared and added to the corresponding amino acid vials. Rink Amide MBHA resin was also weighed as required, placed in the reaction chamber, the upper and lower caps were tightened, the label was attached, and the name of the synthesized peptide, the batch number, the tare weight of the reaction chamber, and the weight of the resin were recorded. The reaction chamber was loaded into the synthesizer. Preparing synthetic reagents, including N-methylpyrrolidone (NMP), Acetyl Imidazole (AIM), piperidine (PIP), methanol and the like, and placing the synthetic reagents into corresponding reagent bottles.

1.1.2 synthesizer State detection

Checking whether the polypeptide synthesizer is normalAnd (5) operating. After the computer is started, a Run Self Test program is operated, and the instrument Self-checks whether each index is normal. Check N additionally₂Whether the gauge pressure is normal or not. The flow rate of each synthesis reagent is measured because the performance of the instrument is known prior to synthesis. Sending Flow Rate1-18 to synthesizer, selecting Main Menu-Module Test, finding ModuleA, ModuleD, ModuleI, ModuleA according to Pres or next, measuring or observing according to more, if the Flow is not proper, adjusting the pressure of lower valve until reaching the requirement.

1.1.3 Synthesis initiation of synthetic peptide antigens

The method Std Fmoc 1.0Sol DIC90 required for synthesis was sent to the synthesizer in the program of the synthesizer. File-New-Sequence-editing the Sequence of the synthetic peptide, and storing. File-New-Run, check Chemistry; whether Sequence is the stored name; setting Cycles; and (5) storing. And finally sending the data to a synthesizer.

Main Menu-Cycle Monitor-begin, run.

1.1.4 Synthesis of synthetic peptide antigens

The polypeptide sequence is synthesized by starting from the C-terminal to the N-terminal, and repeating the following steps in sequence according to a given sequence:

(1) deprotection reaction: placing the amino resin in NMP solution of piperidine with the volume percentage of 15-30%, and reacting at 20-28 ℃ for 25-40 minutes to remove the Fmoc protective group on the amino resin;

(2) washing: drying with nitrogen, and washing amino resin with NMP;

(3) condensation reaction: adding HOBT, DIC and Fmoc protected amino acid, and reacting at 20-28 deg.C for 0.5-2.5 hr;

(4) washing: drying with nitrogen, and washing amino resin with NMP;

(5) and (3) blocking reaction: adding 1.5-4% by weight of an NMP solution of acetyl imidazole according to volume percentage, and reacting for 20-40 minutes at 20-28 ℃.

1.1.5 synthetic peptide antigen Synthesis completion

The synthesizer will automatically stop after the synthesis of the antigen is finished. And then taking the reactor off the polypeptide synthesizer, washing the polypeptide resin for 3 times by using 100% methanol, then drying the polypeptide resin in a fume hood, transferring the polypeptide resin into a brown bottle, putting the brown bottle into a refrigerator with the temperature of 20 ℃ below zero, and sealing a sealing film for later use.

1.2 cleavage and identification of synthetic peptide antigens

1.2.1 cleavage of synthetic peptide antigens

Preparing a lysate according to a volume ratio (trifluoroacetic acid (TFA)/Triisopropylsilane (TIS)/phenol/water (85/8/6/1)), taking out the synthesized polypeptide resin from a refrigerator, placing the polypeptide resin into a round-bottom flask, adding the prepared lysate and a magnetic stirrer into the flask in a fume hood, then stably placing the flask on a magnetic stirrer, and continuously stirring for 1 hour at room temperature until the reaction is completed. After the reaction is finished, the TFA in the crude product is removed by continuous evaporation for 30-120 minutes by using a rotary evaporator with a cold trap. And collecting and precipitating the polypeptide by using ether, washing a crude product of the polypeptide antigen for multiple times by using Dimethylformamide (DMF), and finally filtering the mixed resin by using a sand core funnel to obtain the polypeptide antigen.

1.2.2 identification of synthetic peptide antigens

After the antigen synthesis is finished, the qualitative and quantitative analysis is carried out by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC).

1.3 conformation formation of synthetic peptide antigens

Preparing polypeptide antigen into a polypeptide solution with the concentration of 2mg/ml by using 15% DMSO, adjusting the pH value of the primarily separated polypeptide solution to be 5.5-7.5 by using 0.1N NaOH or 0.1N HCl, and placing the primarily separated polypeptide solution on a shaking table with the rotating speed of 110rpm for 48 hours at the temperature of 25 ℃ to form a disulfide bond and form a ring structure.

Alternatively, the following head-to-tail cyclization is performed:

-COOH' and-NH of head and tail amino acids₂"cyclization method is described in Mengfen et al Peptide Protein Reserch 1996.48: 229-239; for methods of reacting the "-COOH" and "-OH" of the head-to-tail amino acid to form a cyclic structure, see Mmenhofer et al, chem. Soc 1970.92: 3771-3777. Thereby obtaining a native conformation that mimics the viral particleThe polypeptide of (1) is a cyclic structure.

1.4 purification and Sterilization of synthetic peptide antigens

The synthetic peptide antigen is subjected to ultrafiltration (Tangential Flow Device circulating Tangential filtration membrane and a peristaltic pump matched with the Tangential Flow Device circulating Tangential filtration membrane) at the temperature of 20-28 ℃, the polypeptide antigen is a filtration membrane through which macromolecules can not pass through a certain aperture, and micromolecular impurities formed or introduced in the early-stage synthesis process and the later-stage cyclization reaction can pass through the filtration membrane. Then sterilizing through a filter with the pore diameter of 0.2 mu m, subpackaging the finally obtained solution into sterile plastic bottles, and labeling. The name, serial number, production lot number, concentration, production date, storage life and storage condition of the polypeptide are marked on the label, and the label is subpackaged and stored at-20 ℃ or-40 ℃ for later use.

For ease of transport and long term storage requirements, the polypeptide antigen is freeze-dried to obtain the polypeptide in a solid state. And taking out the pre-frozen polypeptide antigen, and drying on a Labconco freeze dryer to obtain the polypeptide antigen in a solid state. And simultaneously labeling. The name, number, production lot number, concentration, production date, shelf life and storage conditions of the polypeptide are indicated on the label.

Example 2 preparation of A-type synthetic peptide vaccine for foot-and-mouth disease

1.1 preparation of the aqueous phase of the antigen

Synthetic peptide antigens represented by polypeptide 1 or polypeptide 2 prepared according to example 1 were weighed and mixed at a molar ratio of 1:1, respectively, and then the total concentration of the synthetic peptide antigens was diluted to 50. mu.g/ml with sterile water for injection. The resulting antigen solution was filtered through a filter having a pore size of 0.2 μm and sterilized.

1.2 oil phase adjuvant preparation

Sterilizing the oil phase adjuvant 50V at 121 deg.C for 30 min.

1.3 emulsification of synthetic peptide vaccines

Cleaning IKA emulsification equipment for 3 times by using 2000ml of sterilized distilled water, adding an oil phase into an emulsification tank at the temperature of 20-28 ℃ according to the volume ratio of 1:1 of an oil phase adjuvant to an antigen water phase, starting a motor to rotate at a low speed of 90-150 rpm and stir for 2min, simultaneously slowly adding an aqueous phase antigen, stirring for 30min after the addition is finished, stirring at a high speed of 10000rpm for 20min, standing for 5min, and emulsifying the vaccine into a water-in-oil single-phase vaccine to obtain a polypeptide 1 and polypeptide 2 mixed vaccine with the molar ratio of 1: 1.

A vaccine containing only a single component, at an antigen concentration of 50. mu.g/ml, was formulated in the same manner.

Examples 3,Efficacy test of aftosa type a synthetic peptide vaccine

1. Materials and methods

1.1 materials

1.1.1 synthetic peptide vaccines

Polypeptide antigens selected in example 3, such as polypeptide 1 (seq id No. 1) and polypeptide 2 (seq id No. 2), were prepared according to example 1, and then a synthetic peptide vaccine for foot and mouth disease type a containing mixed components of polypeptide 1 and polypeptide 2 was formulated according to example 2, corresponding to lot nos: a1901, A1902 and A1903, and the vaccine-corresponding lot A1904 made of polypeptide 1 (SEQ ID NO: 1) and the vaccine-corresponding lot A1905 made of polypeptide 2 (SEQ ID NO: 2).

1.1.2 test animals

Selecting 27 healthy mount pigs (silver pig farm in Lanzhou) with the same variety, age of 4 months, weight of about 40Kg and negative foot-and-mouth disease neutralizing antibody.

1.1.3 foot-and-mouth disease Virus species AF/72 strain [ Zhongmu industry Co., Ltd ]

Measuring and adjusting the toxicity value of a suckling mouse with the age of 3-4 days, and freezing and storing at-70 ℃ for later use.

1.2 test methods

90 healthy susceptible pigs are randomly divided into 6 groups, each group comprises 15 pigs, group 1 immunization A1901, group 2 immunization A1902, group 3 immunization A1903, group 4 immunization A1904, group 5 immunization A1905, group 6 immunization congener product control vaccine foot-and-mouth disease type A inactivated vaccine (AF/72 strain). Each component comprises 3 dose groups, and the specific immunization dose is as follows: 1 part (5 heads), 1/3 parts (5 heads) and 1/9 parts (5 heads), wherein each part of A1901, A1902, A1903, A1904 and A1905 is 1ml, and each part of a similar product control seedling is 3 ml. The immunization was performed by intramuscular injection into the neck, and the injection dose and the specific grouping are shown in Table 1. 28 days after inoculation, 2 nd,4. Pigs in groups 6, 8, 10, and 12 challenged AF/72 virulent. Control pigs 2, wherein the two sides of the upper surface of tongue of 2 pigs are injected with AF/72 virulent drugs at two points, each point is 0.1ml (total 0.2ml, containing 10)³ID₅₀) The observation was continued for 10 days.

1.3 determination of results

The control pig should have lesion (blister or ulcer) on more than 3 hooves, and the immune pig is judged to be unprotected when any foot-and-mouth disease symptom appears. According to the protection number of the immune pig, the PD of the detected vaccine is calculated according to the Read-Muench method₅₀. The 12 groups of vaccines to be tested were tested for efficacy as described above.

2. Test results and discussion

2.1 test results

The 3 batches of laboratory vaccines and the control vaccine are used for immunizing animals according to a 1.2 method, AF/72 virulent strain is attacked after 28 days, and the result is judged after 10 days, and the result is shown in table 1.

TABLE 1 results of efficacy test of aftosa type A synthetic peptide vaccine

2.2 discussion of results

From the above results, it can be seen that: three batches of aftosa A type synthetic peptide vaccines A1901, A1902 and A1903 are used for immunizing animals respectively, and PD is attacked by AF/72 virulent virus 21 days later₅₀The values are respectively: 9.00, PD of the same kind of preparation after AF/72 challenge compared with vaccine₅₀The value was 7.49. While the single polypeptide immunizes the PD after the strong virus attack of OA1904 of groups 7-8 and OA1905 of groups 9-10₅₀The value also reaches 7.6 or more.

The above results show that: PD of foot-and-mouth disease A type synthetic peptide vaccine aiming at AF/72 virulent virus and virulent virus attack₅₀The values exceed 6, and the protection requirement is met. The polypeptide vaccine is proved to have the capability of protecting the test animal pigs from the virus attack. And the foot-and-mouth disease A type synthetic peptide vaccine aims at the PD attacked by AF/72 virulent virus₅₀PD with value higher than that of inactivated vaccine of pig foot-and-mouth disease₅₀The values show that the polypeptide vaccine has better protection effect compared with the conventional inactivated vaccine.The experiment proves that the synthetic peptide vaccine not only meets the requirements of disease prevention, but also meets the requirements of food safety, and the market space is very wide.

Example 5 safety test of aftosa type A synthetic peptide vaccine

1. Materials and methods

1.1 synthetic peptide vaccines

The same as in example 3.

1.2 test animals [ self reproduction ]

350-450 g of guinea pigs; 18-22 g of mice; healthy susceptible pigs at least 6 months of age.

1.3 test methods

1.3.1 15 guinea pigs weighing 350-450 g were injected subcutaneously with 2ml vaccine per animal; 15 mice weighing 18-22 g were injected with 0.5ml of vaccine per mouse. After continuously observing for 7 days, death or obvious local adverse reaction or systemic reaction caused by vaccine injection cannot occur.

1.3.2 healthy susceptible pigs (the titer of cell neutralizing antibodies is not higher than 1:8) with the age of at least 6 months are used for 9 pigs, 20 points of vaccine are injected into the back of the tongue of each pig at the intradermal position, each point is 0.1ml, after observing for 4 days, each pig is injected with 6ml of vaccine at the muscle, and observing for 6 days continuously. The foot-and-mouth disease symptoms or obvious toxic reaction caused by vaccine injection are not required to appear.

2. Test results

2.1 safety of vaccines against guinea pigs and mice

15 guinea pigs, 2ml of vaccine per subcutaneous injection; 15 mice were injected subcutaneously with 0.5ml each. No death or obvious local adverse reaction or systemic reaction caused by vaccine injection occurs after 7 days of continuous observation, and the specific results are shown in the following table 3.

TABLE 3 vaccine safety test results in guinea pigs and mice

2.2. Safety of vaccines against healthy susceptible pigs

After taking the synthetic peptide vaccine out to equilibrate to room temperature, 2ml of the vaccine was injected into each pig tongue at 20 spots, 0.1ml per spot, with at least 4 days observed day by day. Thereafter, 9ml of vaccine was injected intramuscularly to each pig, and observation was continued for 6 days one by one. The specific results are shown in Table 4.

TABLE 4 vaccine safety test results for healthy susceptible pigs

The results show that the aftosa A-type synthetic peptide vaccine has high safety to guinea pigs, mice and pigs, has no toxin-dispersing risk, and therefore has good popularization prospect and market value.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

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Claims

1. An antigen polypeptide composition for preparing a foot-and-mouth disease A type synthetic peptide vaccine consists of a polypeptide shown in a sequence 1 and a polypeptide shown in a sequence 2.

2. The antigenic polypeptide composition of a synthetic peptide vaccine against foot-and-mouth disease type A according to claim 1, wherein the molar ratio of the polypeptide represented by SEQ ID No. 1 to the polypeptide represented by SEQ ID No. 2 is (0.5-1.5): (0.5-1.5).

3. The antigenic polypeptide composition of a synthetic peptide vaccine against foot-and-mouth disease type A according to claim 2, wherein the molar ratio of the polypeptide represented by SEQ ID No. 1 to the polypeptide represented by SEQ ID No. 2 is 1: 1.

4. A synthetic peptide vaccine for foot-and-mouth disease type A, comprising the antigen polypeptide composition of the synthetic peptide vaccine for foot-and-mouth disease type A according to any one of claims 1 to 3.

5. The aftosa type a synthetic peptide vaccine according to claim 4, further comprising a pharmaceutically acceptable adjuvant.

6. Use of the antigenic polypeptide composition according to any one of claims 1 to 3 for the preparation of a synthetic peptide vaccine for foot and mouth disease type a or for the preparation of a biological product for the prevention of foot and mouth disease type a.

7. The use according to claim 6, wherein said foot and mouth disease is porcine type A foot and mouth disease.

8. The preparation method of the aftosa A-type synthetic peptide vaccine is characterized by comprising the following steps:

(1) diluting the polypeptide composition of any one of claims 1-3 with water for injection to a concentration of 10-100 μ g/ml, thereby obtaining a polypeptide antigen aqueous phase;

(2) sterilizing the adjuvant;

(3) under the condition of 20-28 ℃, according to the volume ratio of the polypeptide antigen water phase to the adjuvant of 1:1, the adjuvant is firstly added into an emulsification tank, stirred for 1.5-3 minutes at 90-150 rpm, then the polypeptide antigen water phase is slowly added, stirred for 20-30 minutes, then stirred for 15-30 minutes at 8000-10000 rpm, kept stand for 3-10 minutes, and subpackaged.