CN106986925B - O-type and A-type bivalent synthetic peptide vaccine for bovine foot and mouth disease and preparation method and application thereof - Google Patents
O-type and A-type bivalent synthetic peptide vaccine for bovine foot and mouth disease and preparation method and application thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention provides an antigen polypeptide composition for preparing a bivalent synthetic peptide vaccine of O-type and A-type foot-and-mouth diseases of cattle and a vaccine. The antigen polypeptide composition comprises polypeptides shown in sequence 1 and sequence 2. The vaccines of the present invention include polypeptide compositions. The bivalent synthetic peptide vaccine for the bovine foot-and-mouth disease O-type and A-type has good immune efficacy, and the antigen of the vaccine is chemically synthesized polypeptide, does not contain foot-and-mouth disease virus nucleic acid particles, and has high safety. Therefore, the vaccine of the invention can effectively cope with the antigen variation of the existing foot-and-mouth disease virus, 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
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an O-type and A-type bivalent synthetic peptide vaccine for bovine foot-and-mouth disease 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, cattle, sheep, etc., 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 cattle flocks in 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 bivalent synthetic peptide vaccines of O type and A type of bovine foot-and-mouth disease, a polypeptide composition and a vaccine containing the polypeptide composition.
In order to achieve the purpose, the laboratory designs 2 polypeptide sequences consisting of amino acids according to the epitope of O-type and A-type epidemic viruses of foot and mouth disease, 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 bivalent synthetic peptide vaccine against O-type and A-type bovine foot-and-mouth disease is used for treating PD attacked by AF/72 virulent virus or OS/99 virulent virus50The values exceed 6, the protection requirement is met, and the capability of protecting test animal cattle from virus attack is realized. Safety tests prove that the O-type and A-type bivalent synthetic peptide vaccines of the foot-and-mouth disease of the cattle do not have side reactions to guinea pigs, mice and cattle, have high safety and no toxin-dispersing danger, and therefore have 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 O-type and A-type bivalent synthetic peptide vaccines of bovine foot-and-mouth disease, which is a polypeptide shown as a sequence 1 or a sequence 2 in a sequence table.
The antigen polypeptide composition for preparing the bivalent synthetic peptide vaccine of the bovine foot-and-mouth disease type O and A comprises the polypeptide described by a sequence 1 in a sequence table and the polypeptide described by a sequence 2 in the sequence table.
Wherein, the peptide bond connection is formed between the carboxyl of the 17 th amino acid and the amino of the 18 th amino acid from the amino terminal of the polypeptide segment shown in the sequence 1 (the sequence is from the amino terminal to the carboxyl terminal from left to right).
And (3) the polypeptide fragment shown in the sequence 2, wherein the peptide bond connection is formed between the carboxyl of the 19 th amino acid and the amino of the 20 th amino acid from the amino terminal (the sequence is from the amino terminal to the carboxyl terminal from left to right).
The 5 th amino acid (X1aa) from the amino terminal in the sequence 1 in the sequence table is Ala or Lys; the 13 th (X2aa) amino acid from the amino terminus is Tyr or Gly; the 7 th amino acid (X1aa) from the amino terminal in the sequence 2 in the sequence table is Ala or Thr; the 11 th (X2aa) amino acid from the amino terminus is Val or Tyr; the 16 th (X3aa) amino acid from the amino terminus is Gly or Arg.
The polypeptide fragment of the present invention may be a peptide substance obtained by solid phase organic synthesis.
The specific sequences of the polypeptides are shown as follows:
polypeptide 1 (polypeptide shown as sequence 1 in a sequence table):
RMEAX1KTGIEIAX2DERR-K-VYNGNCKYTGGSAPNVRGDLQVLAPKAAR CLPTSFNYGAIK
X1＝A/K，X2＝Y/G
polypeptide 2 (polypeptide shown in sequence 2 in the sequence table):
KKGAGLX1GVMX2TESVX3FRK-K-VYSGTCKYSAPQNRRGDLGPLAARLA ACLPASFNFGAIR
X1＝A/T X2＝V/Y X3＝G/R
as described above, when the polypeptide represented by SEQ ID No. 1 and the polypeptide represented by SEQ ID No. 2 have a polymorphic site (i.e., two optional amino acids exist at one amino acid site), these polypeptides having different sequences due to the site polymorphism may be present alone or in combination in the polypeptide composition. In one embodiment of the invention, during the synthesis of the polypeptide, two amino acids are loaded simultaneously at positions having two alternative amino acids, and are randomly synthesized.
Wherein K is a connecting arm between polypeptide fragments, namely a peptide bond is formed by amino at the site of lysine, and the specific principle is as follows:
each amino acid comprising at least one "-COOH" (carboxyl) and one "-NH group3"(amino group), the carboxyl group and amino group attached to α carbon atoms are referred to as" α carboxyl group "and" α amino group ". The typical peptide synthesis reaction is the reaction of the" α amino group "of the previous amino acid with the" α carboxyl group "of the next amino acid to form an amide bond(peptide bond).
Lys (lysine) is a basic amino acid with an amino group in place in addition to the normal "alpha amino" and "alpha carboxy" groups, as shown in the following formula.
K is particularly specified to refer to the amino group participating in peptide formation reaction as an "amino group" rather than an "alpha amino group", and is used for connecting epitopes which need to be used together but do not affect each other due to the long arm structure.
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 bivalent synthetic peptide vaccines of O type and A type of bovine foot-and-mouth disease also belongs to the protection scope of the invention.
The invention provides a bivalent synthetic peptide vaccine for O-type and A-type bovine foot-and-mouth disease, which comprises the polypeptide composition.
The peptide vaccine further comprises an adjuvant.
The invention provides a preparation method of the bivalent synthetic peptide vaccine for O-type and A-type bovine foot-and-mouth disease, 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 polypeptide or the polypeptide vaccine in the preparation of biological products for preventing O-type and A-type foot-and-mouth diseases of cattle.
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 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 results of efficacy experiments and safety experiments of the synthetic peptide vaccine show that the O-type and A-type bivalent synthetic peptide vaccines for the foot-and-mouth disease of the cattle have good immune efficacy and are safe to guinea pigs, mice and the cattle, so that the vaccine can effectively deal 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 antigens of bivalent synthetic peptide vaccine for bovine foot-and-mouth disease type O and type A
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.
1.1 solid phase Synthesis of synthetic peptide antigens
1.1.1 Synthesis Material preparation
The sequence of the synthetic polypeptide antigen is as follows:
polypeptide 1 (polypeptide shown as sequence 1 in a sequence table):
RMEAX1KTGIEIAX2DERR-K-VYNGNCKYTGGSAPNVRGDLQVLAPKA ARCLPTSFNYGAIK
X1＝A/K，X2＝Y/G
polypeptide 2 (polypeptide shown in sequence 2 in the sequence table):
KKGAGLX1GVMX2TESVX3FRK-K-VYSGTCKYSAPQNRRGDLGPLAAR LAACLPASFNFGAIR
X1＝A/T X2＝V/Y X3＝G/R
wherein K is a connecting arm between polypeptide fragments, namely a peptide bond is formed by amino at the site of lysine. Namely, the peptide bond connection is formed between the carboxyl of the 17 th amino acid and the amino of the 18 th amino acid from the amino terminal of the polypeptide fragment shown in the sequence 1 (the sequence is from the amino terminal to the carboxyl terminal from left to right). And (3) the polypeptide fragment shown in the sequence 2, wherein the peptide bond connection is formed between the carboxyl of the 19 th amino acid and the amino of the 20 th amino acid from the amino terminal (the sequence is from the amino terminal to the carboxyl terminal from left to right). (in the following synthetic process, the reaction solution is alkaline, the purchased product lysine has its amino group at the site protected by an alkali sensitive group and its amino group at the alpha site protected by an acid sensitive group
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
And (5) checking whether the polypeptide synthesizer normally operates. 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 MainMenu-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 1.0 Sol 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. This gives a polypeptide loop structure that mimics the native conformation of the virion.
1.4 purification and Sterilization of synthetic peptide antigens
The synthetic peptide antigen is ultrafiltered by a circulating tangential filtration membrane at the temperature of 20-28 ℃ (Tangentialflow Device circulating tangential filtration membrane and a peristaltic pump matched with the Tangentialflow Device circulating tangential filtration membrane), 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 synthesis process and the later 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 bivalent synthetic peptide vaccine for O-type and A-type bovine 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.
Example 3 antigen screening test for bivalent synthetic peptide vaccine of bovine foot-and-mouth disease type O and type A
1. Materials and methods
1.1 materials
1.1.1 synthetic peptide vaccines
Screening antigens of the bivalent synthetic peptide vaccine were prepared according to the method of example 1, and then single component vaccines containing the screening antigens were prepared separately according to example 2. The vaccine prepared aiming at the alternative antigen of the O-type foot-and-mouth disease virus has the corresponding batch number: NO1301, NO1302, NO1303, NO 1304. The vaccine prepared aiming at the alternative antigen of the A-type foot-and-mouth disease virus has the corresponding batch number: NA1401, NA1402, NA1403, NA1404, NA 1405.
1.1.2 test animals
Selecting 45 healthy cattle 45 heads (purchased from cattle farms in Lanzhou region) with negative foot-and-mouth disease antibodies (the neutralizing antibody titer of suckling mice is less than or equal to 1:4) at 6 months of age.
1.1.3 foot-and-mouth disease virus seeds AF/72 strain, seed virus OS/99 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
Healthy susceptible cattle 45 are randomly divided into 9 groups, and each group has 5 heads, and is immunized with NO1301, NO1302, NO1303, NO1304, NA1401, NA1402, NA1403, NA1404 and NA1405 respectively. The dose of immunization is 1ml per head, and neck intramuscular injection is adopted during immunization. On days 14 and 21 after inoculation, venous blood is collected respectively, serum is separated, and the O-type and A-type foot-and-mouth disease antibody titer is determined by using a foot-and-mouth disease O-type antibody liquid phase blocking ELISA detection kit and a foot-and-mouth disease A-type antibody liquid phase blocking ELISA detection kit respectively.
The cattle of the groups 1, 2, 3 and 4 are attacked with OS/99 virulent virus, the cattle of the groups 5, 6, 7, 8 and 9 are attacked with AF/72 virulent virus, 4 control cattle are treated, wherein two points on two sides of the upper surface of the tongue of 2 cattle are injected with AF/72 virulent virus in the skin, and the other 2 cattle are injected with OS/99 virulent virus. Each spot was 0.1ml (total 0.2ml, 10%4ID50) The observation was continued for 10 days.
1.3 determination of results
The control cattle should have lesions (blisters or ulcers) on more than 3 hooves, the immune cattle only have blisters or ulcers on the lingual surface, and the immune cattle is judged to be protected when no lesions exist on other parts, and is judged to be unprotected when a typical foot-and-mouth disease lesion (blisters or ulcers) exists on any part except the lingual surface. The 6 groups of vaccines to be detected are subjected to the challenge protection test according to the method.
2. Test results and discussion
2.1 test results
The 6 batches of laboratory vaccines are used for immunizing animals according to a method of 1.2, serum antibody titer is detected on 14 days and 21 days respectively, AF/72 virulent strain and OS/99 virulent strain are immediately attacked after the antibody titer is detected on 21 days, and the result is judged after 10 days. Antibody levels and challenge protection results are shown in table 1.
The results show that the antibody titers of the O-type vaccine antigens OP1 and OP2 are higher than those of the other two antigens at 14 days and 21 days after immunization, and the challenge protection rate is 5/5 full protection, so that the antigens are selected as the O-type antigens of the bivalent synthetic peptide vaccine (when X1aa (amino acid 5 th position from sequence 1) ═ Ala or Lys, and X2aa (amino acid 13 th position from sequence 1) ═ Tyr or Gly, the antibody levels are highest and are higher than OP3 and OP4, and the challenge protection effect rate is 5/5). The antibody titer of the A-type vaccine antigens AP2, AP3 and AP4 is higher than that of the other two antigens at 14 days and 21 days after immunization, and the challenge protection rate is 5/5 full protection, so the A-type vaccine antigens are selected as the A-type antigens of the bivalent synthetic peptide vaccine (when X1aa (the 7 th amino acid from the sequence 2) ═ Ala or Thr, X2aa (the 11 th amino acid from the sequence 2) ═ Val or Tyr, and X3aa (the 16 th amino acid from the sequence 2) ═ Gly or Arg, the antibody titer is highest at 14 days and 21 days after immunization, and is higher than that of AP1 and AP5, and the challenge protection rate is 5/5 full protection).
TABLE 1 results of vaccine candidate antigen screening assays
Example 4 efficacy test of bivalent synthetic peptide vaccine for bovine foot-and-mouth disease type O and A
1. Materials and methods
1.1 materials
1.1.1 synthetic peptide vaccines
Polypeptide antigens selected in example 3, such as polypeptide 1 and polypeptide 2, were prepared according to example 1, and then bivalent synthetic peptide vaccines of bovine foot-and-mouth disease type O, type a containing mixed components of polypeptide 1 and polypeptide 2 were formulated according to example 2, corresponding to lot numbers: NOA1601, NOA1602, NOA 1603.
1.1.2 test animals
Selecting 124 healthy cattle with 6 months age (purchased from cattle farms in Lanzhou region) which are negative in foot-and-mouth disease antibodies (the neutralizing antibody titer of suckling mice is less than or equal to 1: 4).
1.1.3 foot-and-mouth disease virus seeds AF/72 strain, seed virus OS/99 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
Healthy susceptible cattle 120 are randomly divided into 8 groups, each group comprises 15 groups, 1-2 groups of immune NOA1601, 3-4 groups of immune NOA1602, 5-6 groups of immune NOA1603, and 7-8 groups of immune congeners of control vaccine type-O, type-A and type-Asia type-1 trivalent inactivated vaccine (O/HB/HK/99 strain + AF/72 strain + Asia-1/XJ/KLMY/04 strain). Each component comprises 3 dose groups, and the specific immunization dose is as follows: 1 part (5 heads), 1/3 part (5 heads) and 1/9 part (5 heads), wherein each part of NOA1601, NOA1602 and NOA1603 is 1ml, and each part of similar products control vaccine 1607002 (commercial trivalent inactivated vaccine of bovine foot and mouth disease type O, A and Asia 1) 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. 21 days after inoculation, cattle of groups 1, 3, 5, and 7 were challenged with AF/72 virulent strain, and cattle of groups 2, 4, 6, and 8 were challenged with OS/99 virulent strain. 4 control cattle, wherein 2 cattle have AF/72 virulent drugs injected into the tongue at two points at two sides, and the other 2 cattle have OS/99 virulent drugs injected into the tongue at 0.1ml (total 0.2ml, containing 10) at each point4ID50) The observation was continued for 10 days.
1.3 determination of results
The control cattle should have lesions (blisters or ulcers) on more than 3 hooves, the immune cattle only have blisters or ulcers on the lingual surface, and the immune cattle is judged to be protected when no lesions exist on other parts, and is judged to be unprotected when a typical foot-and-mouth disease lesion (blisters or ulcers) exists on any part except the lingual surface. According to the protection number of the immune cattle, the PD of the vaccine to be detected is calculated according to the Read-Muench method50. The 8 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 and OS/99 virulent strain are attacked after 21 days, the results are judged after 10 days, and the results are shown in a table 2.
TABLE 2 results of efficacy test of bivalent synthetic peptide vaccine for O-type and A-type bovine foot and mouth disease
2.2 discussion of results
From the above results, it can be seen that: three batches of bivalent synthetic peptide vaccines NOA1601, NOA1602 and NOA1603 of bovine foot-and-mouth disease type O and type A are used for immunizing animals respectively, and PD is attacked by AF/72 virulent virus 21 days later50The values are respectively: 9.00, and the like products control vaccine 1607002 used AF/72 post-challenge PD50The value was 7.49. Three batches of polypeptide vaccine used PD after OS/99 virulent challenge50The values are respectively: 10.81, 10.32, 9.00, preparation of same type control vaccine 1607002 PD after challenge with OS/99 virulent50The value was 7.05.
The above results show that: bovine foot-and-mouth disease type-O and type-A bivalent synthetic peptide vaccine against PD attacked by AF/72 virulent virus or OS/99 virulent virus50The values exceed 6, and the protection requirement is met. The polypeptide vaccine is proved to have the capability of protecting test animal cattle from virus attack. And the bivalent synthetic peptide vaccine of O type and A type of bovine foot-and-mouth disease aims at the PD attacked by AF/72 virulent virus and OS/99 virulent virus50PD with value higher than that of inactivated vaccine of foot-and-mouth disease of cattle50The 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 bivalent synthetic peptide vaccine for bovine foot-and-mouth disease type O and type A
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 cattle at least 6 months old.
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 cattle (the titer of cell neutralizing antibodies is not higher than 1:8) with the age of at least 6 months are used for 9 heads, 20 points of vaccine are injected into the back of the tongue of each cattle, 0.1ml of vaccine is injected into each point, after 4 days of daily observation, 6ml of vaccine is injected into the muscle of each cattle, and 6 days of daily observation are continued. 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 cattle
After the synthetic peptide vaccine was allowed to equilibrate to room temperature, 2ml of the vaccine was injected at 20 spots per bovine tongue, 0.1ml per spot, with at least 4 days observed daily. Thereafter, 9ml of vaccine was injected intramuscularly to each cow, 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 cattle
The results show that the bivalent synthetic peptide vaccines of O type and A type of the bovine foot-and-mouth disease have high safety to guinea pigs, mice and cattle, have no toxin-dispersing danger, and have 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.
Sequence listing
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<120> O-type and A-type bivalent synthetic peptide vaccine of bovine foot-and-mouth disease and preparation method and application thereof
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Claims (8)
1. An antigen polypeptide composition for preparing bivalent synthetic peptide vaccines of O type and A type of bovine foot-and-mouth disease comprises a polypeptide shown as a sequence 1 in a sequence table and a polypeptide shown as a sequence 2 in the sequence table.
2. The antigenic polypeptide composition for preparing bivalent synthetic peptide vaccine of bovine foot-and-mouth disease type O and type A according to claim 1, wherein the polypeptide fragment shown in sequence 1 is formed by peptide bond connection of carboxyl of 17 th amino acid from amino terminal and amino of 18 th amino acid.
3. The antigenic polypeptide composition for preparing bivalent synthetic peptide vaccine of bovine foot-and-mouth disease type O and type A according to claim 1, wherein the polypeptide fragment shown in sequence 2 is formed by peptide bond connection of carboxyl of 19 th amino acid from amino terminal and amino of 20 th amino acid.
4. The antigenic polypeptide composition for preparing bivalent synthetic peptide vaccine of type O and type A of the bovine foot-and-mouth disease according to claim 1, which consists of the polypeptide shown in the sequence 1 and the polypeptide shown in the sequence 2.
5. The antigenic polypeptide composition of bivalent synthetic peptide vaccine against bovine foot-and-mouth disease type O and type A according to claim 4, 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).
6. The antigenic polypeptide composition of bivalent synthetic peptide vaccine against bovine foot-and-mouth disease type O and type A according to claim 4, 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.
7. Use of the antigenic polypeptide composition of any one of claims 1-6 in the preparation of bivalent synthetic peptide vaccine for type O and A of bovine foot-and-mouth disease or in the preparation of biological products for preventing type O and A of bovine foot-and-mouth disease.
8. The preparation method of the bivalent synthetic peptide vaccine for the O-type and A-type foot-and-mouth diseases of cattle is characterized by comprising the following steps:
(1) diluting the polypeptide composition of any one of claims 1-6 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.
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| CN1541710A (en) * | 2003-11-07 | 2004-11-03 | 复旦大学 | Bivalence polypeptide vaccine for resisting A type and O type foot-and-mouth disease virus and its preparation |
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