CN106986925A - Ox aftosa is O-shaped, A type divalence synthetic peptide vaccines and its preparation method and application - Google Patents
Ox aftosa is O-shaped, A type divalence synthetic peptide vaccines and its preparation method and application Download PDFInfo
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- CN106986925A CN106986925A CN201710316780.4A CN201710316780A CN106986925A CN 106986925 A CN106986925 A CN 106986925A CN 201710316780 A CN201710316780 A CN 201710316780A CN 106986925 A CN106986925 A CN 106986925A
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- C—CHEMISTRY; METALLURGY
- 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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- 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
- A61K39/12—Viral antigens
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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
- A61K2039/55—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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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 present invention, which is provided, a kind of is used to preparing that ox aftosa to be O-shaped, antigen polypeptide composition and vaccine of A type divalence synthetic peptide vaccines.Antigen polypeptide composition includes the polypeptide shown in sequence 1 and sequence 2.The vaccine of the present invention includes peptide composition.Ox aftosa that the present invention is provided is O-shaped, A type divalence synthetic peptide vaccines have good immune efficacy, and the antigen of vaccine is chemically synthesized polypeptide, safe without foot and mouth disease virus nucleic acid particle.Therefore the vaccine of the present invention can successfully manage the antigenic variation of current foot and mouth disease virus, in the absence of biological safety, it is easy to extensive synthesis, have a good application prospect.Present invention also offers the polypeptide and the preparation method of peptide vaccine and its pharmaceutical applications.
Description
Technical field
The invention belongs to pharmaceutical technology field, specifically, the present invention relates to a kind of ox aftosa is O-shaped, the synthesis of A types divalence
Peptide vaccine, with and preparation method thereof.
Background technology
Aftosa (foot and mouth disease, abbreviation FMD) be a kind of artiodactyl occur it is acute, highly connect
Property, infectious fever are touched, it is worldwide widely distributed.The infectiousness of aftosa is high, propagates rapid, infected pigs, ox, sheep
Deng cub will be caused after livestock dead, adults production capacity drastically declines, therefore seriously endangers development and the meat of animal husbandry
The production and supply of food and its livestock products.At present, aftosa make the market circulation and international trade of animal and animal's products by
Greatly block and limitation, huge economic loss is caused to popular countries and regions Animal husbandry production.
Aftosa is caused by foot and mouth disease virus (FMDV) infection.Foot and mouth disease virus belongs to picornavirus,
The features such as with polymorphism, mutability.At present it is known that there is the foot and mouth disease virus of 7 kinds of serotype in the whole world：A, O, C, Sat1 (south
Non- I types), Sat2 (South Africa II types), Sat3 (South Africa type III) and Asia I (Asia I type).Each in these principal modes is divided again
Some hypotypes, the existing kind more than 70 of the hypotype having now been found that.Serotypes A, O, C and Asia I types foot and mouth disease virus it is most commonly seen,
The mutation of wherein serotypes A virus is most, with more than 30 kinds hypotypes.Result of study shows, the capsid protein of foot and mouth disease virus
It is made up of four kinds of Structural protein VP1s, VP2, VP3 and VP4, every kind of each 60.VP1-VP3 constitutes capsomere,
Positioned at the outside of capsid protein, and VP4 is located at the inside of virion.
Antigenicity between foot and mouth disease virus is various is different, and Immunogenicity is unable to each other.Moreover, in same serum
The degree of antigenic difference is also very big in type so that can be reasonably resistant to a kind of aftosa vaccine of hypotype may be directed to it is same
Another hypotype in serotype does not have protectiveness.In addition, aftosa strain antigenicity also constantly changing, with when
Between passage, the efficacy wanes of original vaccine even disappear, therefore bring very big difficulty to the preventing and controlling of aftosa.
Current aftosa popular in China cows is mainly O-shaped and A type aftosas.China carries out aftosa and forced
Property it is immune, vaccine immunity is the Main Means for preventing and treating aftosa.But, the existing aftosa vaccine used of China is mainly virus
Inactivated vaccine, there is that biological safety is poor, side reaction big, unstable product quality the problems such as.By contrast, the current world
Upper many countries have stopped using inactivated vaccine, also forbid from the national import livestock products using inactivated vaccine.As can be seen here,
In terms of aftosa preventing and treating, China has lagged behind world development situation.
In terms of the research of aftosa new generation vaccine, successively there are genetic engineering subunit vaccine, hoof-and-mouth disease poisonous carrier epidemic disease
Seedling, foot-and-mouth disease gene engineering modification vaccine research report, but its all existed in terms of immune effect, biological safety it is all
Many problems, have impact on the use of these new generation vaccines.In addition, these vaccines are past for the popular strain for currently having occurred and that variation
It is poor toward effect, it is impossible to effectively to protect animal.Therefore, this area is still had for the new epidemic disease of safe and effective aftosa
The demand of seedling.
The content of the invention
It is used to preparing that ox aftosa to be O-shaped, A type divalence synthetic peptide vaccines antigen is more it is an object of the invention to provide a kind of
Peptide, peptide composition, and the vaccine containing the peptide composition.
To achieve the above object, this laboratory devises 2 amino according to the epitope that aftosa is O-shaped, A types prevalence is malicious
The peptide sequence of acid composition, sequence 1 and the polypeptide shown in sequence 2 respectively in sequence table.
Using Solid phase synthesis, oil emulsion vaccine is mixed and made into ISA50V adjuvants.Proved by potency test, ox mouthful hoof
Epidemic disease is O-shaped, A type divalence synthetic peptide vaccine is for the strong poison of AF/72 or the PD of OS/99 strong virus attacks50Value reaches that protection will more than 6
Ask, with ability of the protection test animal ox from virus attack.Safety testing proof ox aftosa is O-shaped, the synthesis of A types divalence
Peptide vaccine occurs to cavy, mouse and ox without side reaction, safe, dangerous without poison is dissipated, so with good promotion prospect
And market value.
Synthetic peptide vaccine greatly improves security and specificity, and its preparation technology simpler, production cost is more
Cheap, vaccine quality is relatively reliable, beneficial to large-scale production.As a kind of Vaccine for hoof-and-mouth disease, its advantage can be enterprise
New competitiveness and point of economic increase is brought with market, the market space is huge.
Present invention employs following technical scheme：
The present invention provide it is a kind of be used to preparing that ox aftosa to be O-shaped, A type divalence synthetic peptide vaccines antigen polypeptide, be sequence
Sequence 1 or the polypeptide shown in sequence 2 in table.
Provided by the present invention for preparing, ox aftosa is O-shaped, A type divalence synthetic peptide vaccines antigen polypeptide composition, bag
Include the composition of polypeptide described in sequence 2 in polypeptide described in sequence 1 and sequence table in sequence table.
Wherein, the polypeptide fragment shown in sequence 1, from the carboxyl and the ε of the 18th amino acids of the amino acids of aminoterminal the 17th
Bit amino formation peptide bond connection (sequence is from left to right aminoterminal to c-terminus).
Polypeptide fragment shown in sequence 2, from the carboxyl and the ε bit aminos of the 20th amino acids of the amino acids of aminoterminal the 19th
Form peptide bond connection (sequence is from left to right aminoterminal to c-terminus).
In sequence 1 from the 5th (X1aa) amino acid of aminoterminal it is Ala or Lys in above-mentioned sequence table；From the of aminoterminal
13 (X2aa) amino acid are Tyr or Gly；In sequence table in sequence 2 from the 7th (X1aa) amino acid of aminoterminal for Ala or
Thr；It is Val or Tyr from the 11st (X2aa) amino acid of aminoterminal；It is Gly from the 16th (X3aa) amino acid of aminoterminal
Or Arg.
Heretofore described polypeptide fragment can be the peptide material obtained by Solid-phase organic synthesis.
The sequence of each specific above-mentioned polypeptide is as follows：
Polypeptide 1 (polypeptide in sequence table shown in sequence 1)：
RMEAX1KTGIEIAX2DERR-εK-VYNGNCKYTGGSAPNVRGDLQVLAPKAAR CLPTSFNYGAIK
X1=A/K, X2=Y/G
Polypeptide 2 (polypeptide in sequence table shown in sequence 2)：
KKGAGLX1GVMX2TESVX3FRK-εK-VYSGTCKYSAPQNRRGDLGPLAARLA ACLPASFNFGAIR
X1=A/T X2=V/Y X3=G/R
Wherein, as described above, including the polypeptide shown in sequence 1, there is pleomorphism site (i.e. in the polypeptide shown in sequence 2
There are two kinds of optional amino acid in one amino acid sites) when, these not homotactic polypeptides brought due to loci polymorphism can
With individually or be mixed in peptide composition.In an embodiment of the invention, in the synthesis of polypeptide, with
Two kinds of optional amino acid sites are loaded simultaneously to two kinds of amino acid, random synthesis.
Wherein, ε K are the ε bit aminos formation peptide bond of the linking arm between polypeptide fragment, i.e. lysine, and concrete principle is as follows
It is shown：
Each amino acid comprises at least "-COOH " (carboxyl) and "-a NH3" (amino), it is connected to alpha-carbon atom
On carboxyl and amino be referred to as " α carboxyls " and " α amino ".General peptide symthesis reaction for a upper amino acid " α amino " and
" α carboxyls " reaction of next amino acid forms amido link (peptide bond) and formed.
Lys (lysine) is a kind of basic amino acid, in addition to normal " α amino " and " α carboxyls ", on ε positions also
There are an amino, such as following formula.
The amino that ε K refer in particular to participate in peptide formation is " ε amino " rather than " α amino ", because of its longer arm structure, because
This, be used to connect the epitope that needs are used in conjunction with but do not interacted.
When choosing the composition vaccine of the peptide composition described in two kinds of sequences, their mol ratio is (0.5-1.5):
(0.5-1.5)；Most preferably, their mol ratio is 1:1.
Described peptide composition is preparing that ox aftosa is O-shaped, the application in A type divalence synthetic peptide vaccines falls within this hair
Bright protection domain.
A kind of ox aftosa that the present invention is provided is O-shaped, A type divalence synthetic peptide vaccines, includes above-mentioned peptide composition.
The peptide vaccine also includes adjuvant.
The invention provides described ox aftosa is O-shaped, A type divalence synthetic peptide vaccines preparation method, the preparation side
Method comprises the following steps：
(1) peptide composition is diluted to 10-100 μ g/ml (preferably 50 μ g/ml) concentration with water for injection, from
And obtain polypeptide antigen aqueous phase；
(2) adjuvant is sterilized and (sterilized 30 minutes under the conditions of 121 DEG C)；
(3) under the conditions of 20~28 DEG C, according to the polypeptide antigen aqueous phase and the adjuvant 1:1 volume ratio, first high-ranking officer
Agent is added in emulsion tank, is stirred 1.5~3 minutes under 90~150rpm, is then slowly added into polypeptide antigen aqueous phase, then stirs
20~30 minutes, then stirred 15-30 minutes under 8000~10000rpm, stand 3-10 minutes (preferably 5 minutes), packing.
Preferably, the adjuvant is selected from white oil, 50V, 50VII (MONTANIDE ISA 50V, 50VII adjuvant (France
SEPPIC companies)) in one or more.
Present invention also offers the preparation method of the polypeptide in aforementioned polypeptides polymer, the preparation method includes following step
Suddenly：
(1) using amino resins as initiation material, using the amino acid protected by 9-fluorenylmethyloxycarbonyl as monomer, according to the ammonia
Base acid sequence is condensed successively connects amino acid to synthesize the polypeptide, often walks unreacted with acetyl imidazole closing after condensation reaction
Aminoterminal；
(2) synthesis adds lytic reagent after finishing, so that the polypeptide be cleaved from amino resins；
(3) polypeptide is precipitated using ether；
(4) ultrafiltration purification is carried out to the polypeptide, then carries out aseptic process.
In above-mentioned preparation method, the step (1) specifically includes following steps：
(1-a) deprotection reaction：Amino resins is placed in the N- first for the hexahydropyridine that percent by volume is 15%~30%
In base pyrrolidones (NMP) solution, reacted 25~40 minutes under the conditions of 20~28 DEG C, so as to remove the 9- fluorenes on amino resins
Methoxycarbonyl group blocking group；
(1-b) is washed：Nitrogen is dried up, and then washs amino resins with 1-METHYLPYRROLIDONE；
(1-c) condensation reaction：Add 1- hydroxyls phenylpropyl alcohol triazole (HOBT), DIC (DIC) and by 9-
The amino acid of fluorenylmethyloxycarbonyl protection, then reacts 0.5~2.5 hour under the conditions of 20~28 DEG C；
(1-d) is washed：Nitrogen is dried up, and then washs amino resins with 1-METHYLPYRROLIDONE；
(1-e) capping：Add N- crassitude of the percent weight in volume for 1.5%~4% acetyl imidazole
Ketone (NMP) solution, reacts 20~40 minutes under the conditions of 20~28 DEG C.
In above-mentioned preparation method, the component of lytic reagent is that volume ratio is 85 in the step (2):8::6:1 trifluoro
Acetic acid:Tri isopropyl silane:Phenol:Water；Also, the pyrolysis time of the step (2) is 1-4 hours.
In above-mentioned preparation method, the step (3) specifically includes：
(3-a) precipitates the polypeptide using ether, is then washed with dimethylformamide；
(3-b) makes two and half Guang ammonia in the amino acid sequence for the polypeptide that precipitation obtains in the case of 10% DMSO is added
Disulfide bond is formed between acid；Or, make the polypeptide amino acid sequence head and the tail amino acid residue between reaction form covalent
Connection；Preferably, the head and the tail amino of the amino acid sequence of the polypeptide is made in the case of 1% DIC and 0.5% HOBT is added
The carboxyl of sour residue is with amino or in 0.1M H2SO4In the case of between carboxyl and hydroxyl reaction formed be covalently attached.
In above-mentioned preparation method, the step (4) specifically includes following steps：
(4-a) uses tangential flow filtration film bag polypeptide described in ultrafiltration under the conditions of 20~28 DEG C, so as to remove small molecular weight impurity；
(4-b) is preserved using 0.2 μm of filter is degerming.
Further aspect, is being prepared for preventing cattle O type, A type aftosas the invention provides aforementioned polypeptides or polypeptide vaccine
Biological products in purposes.
Specifically, the present invention by the sequencing of popular strain and combining aftosa vaccine recently to domestic aftosa
Strain sequence, studies the variation situation of aftosa major antigenic sites, and its variation is counted for the amino acid sites mainly made a variation
Frequency, the analysis for carrying out foot-and-mouth disease antigen site in combination with area of computer aided is predicted, possible antigen site peptide fragment is entered
Row chemical synthesis, i.e., for variation frequency of the easy variant sites according to statistics, use different amino acid in these sites, obtain
Cover a variety of candidate polypeptide antigens of currently be possible to variant sites.And then, by substantial amounts of animal experiment to these candidates
Polypeptide antigen is screened, and obtains that the immune response of animal can be caused, and immune response level is high, can be good at protection
Polypeptide antigen of the animal from the popular strain attack of aftosa.In addition, the present inventor according to screening experiment result to hoof-and-mouth disease
Malicious antigen site is optimized, and efficient combination t cell epitope and B cell epitope, enhances the immune effect of polypeptide antigen
Really.
Synthetic peptide vaccine potency test, safety experiment result show that the ox aftosa that the present invention is provided is O-shaped, A type divalence
Synthetic peptide vaccine has good immune efficacy, and safe to cavy, mouse, this animal ox, therefore the vaccine of the present invention
The antigenic variation of current foot and mouth disease virus can be successfully managed, in the absence of biological safety, it is easy to extensive synthesis, with good
Application prospect.
Embodiment
Illustrate the present invention referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, it does not limit the scope of the present invention in any way.
Experimental method in following embodiments, is conventional method unless otherwise specified.Medicine used in following embodiments
Material raw material, reagent material etc., unless otherwise specified, are commercially available products.
Embodiment 1, ox aftosa are O-shaped, the polypeptide antigen synthesis in solid state of A type divalence synthetic peptide vaccines
The present invention by the sequencing of popular strain and combining aftosa vaccine strain sequence recently to domestic aftosa,
The variation situation of aftosa major antigenic sites is studied, the frequency of its variation is counted for the amino acid sites mainly made a variation, together
When the analysis prediction in foot-and-mouth disease antigen site is carried out with reference to area of computer aided, chemical conjunction is carried out to possible antigen site peptide fragment
Into that is, for variation frequency of the easy variant sites according to statistics, different amino acid is used in these sites, obtains covering current
A variety of candidate polypeptide antigens of be possible to variant sites.And then, by substantial amounts of animal experiment to these candidate polypeptide antigens
Screened, obtain causing the immune response of animal, and immune response level is high, can be good at protecting animal from
The polypeptide antigen of aftosa prevalence strain attack.In addition, the present inventor according to screening experiment result to foot-and-mouth disease virus antigen position
Point is optimized, and efficient combination t cell epitope and B cell epitope, enhances the immune effect of polypeptide antigen.
U.S.'s 433A full-automatic polypeptide synthetic instruments can be used by the antigenic synthetic peptide of the invention for screening and identifying, profit
Prepared with Merrifield solid-phase synthesis, wherein employing the amino acid modified by 9-fluorenylmethyloxycarbonyl (Fmoc), solid phase is carried
Body is U.S. sigma Rink Amide mbha resins.Production process generally includes the synthesis in solid state of polypeptide antigen, polypeptide
Cracking, antigen purification are preserved with degerming.
The synthesis in solid state of 1.1 antigenic synthetic peptides
1.1.1 synthesis material prepares
The sequence of synthetic polypeptide antigen is as follows：
Polypeptide 1 (polypeptide in sequence table shown in sequence 1)：
RMEAX1KTGIEIAX2DERR-εK-VYNGNCKYTGGSAPNVRGDLQVLAPKA ARCLPTSFNYGAIK
X1=A/K, X2=Y/G
Polypeptide 2 (polypeptide in sequence table shown in sequence 2)：
KKGAGLX1GVMX2TESVX3FRK-εK-VYSGTCKYSAPQNRRGDLGPLAAR LAACLPASFNFGAIR
X1=A/T X2=V/Y X3=G/R
Wherein, ε K are the ε bit aminos formation peptide bond of the linking arm between polypeptide fragment, i.e. lysine.That is, shown in sequence 1
Polypeptide fragment, be connected (sequence from the carboxyls of the amino acids of aminoterminal the 17th and the ε bit aminos of the 18th amino acids formation peptide bond
It is from left to right aminoterminal to c-terminus).Polypeptide fragment shown in sequence 2, from the carboxyl of the amino acids of aminoterminal the 19th and
The ε bit aminos formation peptide bond connection of 20 amino acids (sequence is from left to right aminoterminal to c-terminus).(in following building-up processes
In, reaction solution is alkalescence, and its ε bit amino of the finished product lysine of purchase is protected by alkali sensitive group, and its α bit amino is by acid
Sensitive group is protected.In course of reaction, the blocking group of ε bit aminos can only be removed, its amino of exposure, with next amino acid
Carboxyl reacted.)
According to the sequence and 1mmol synthesis scale of antigen, the amino acid for preparing suitable Fmoc modifications [is purchased from Shanghai
Gill is biochemical], add in corresponding amino acid bottle.Rink Amide mbha resins are equally weighed on request, are put into reaction chamber
In, it will tighten lid, label up and down, and record the weight of title, lot number, the tare weight of reaction chamber and the alleged resin of synthesized peptide
Amount.Reaction chamber is loaded into synthesizer.Prepare synthetic agent, including 1-METHYLPYRROLIDONE (NMP), acetyl imidazole (AIM), piperidines
(PIP), methanol etc. is placed into corresponding reagent bottle.
1.1.2 synthesizer state-detection
Check whether Peptide synthesizer normally runs.After start, Run Self Test programs are run, instrument self checking is every
Whether index is normal.N is checked in addition2Whether sufficient, whether system gauge pressure is normal.The performance of instrument has been tackled before synthesis
Solution, so to be measured to the flow velocity of every kind of synthetic agent.Flow Rate1-18 are sent to synthesizer, Main is selected
Menu-Module Test-looking for ModuleA, ModuleD, ModuleI, ModuleI, ModuleA by Prer or next-are pressed
Start-measured or observed by more, if flow is improper, adjusts lower valve pressure, until reaching requirement.
1.1.3 the synthesis of antigenic synthetic peptide starts
The Sol DIC90 of method Std Fmoc 1.0 for synthesizing needs are sent on synthesizer in the program of synthesizer.
The sequence of File-New-Sequence- Edit and Compose peptides, is preserved.File-New-Run, checks Chemistry；Sequence is
It is no to be deposited name；Set Cycles；Preserve.It is finally sent on synthesizer.
Main Menu-Cycle Monitor-begin, bring into operation.
1.1.4 the synthesis of antigenic synthetic peptide
Peptide sequence described above, is to N-terminal, according to given order, successively constantly when synthesis since C-terminal
It is repeated below synthesis step：
(1) deprotection reaction：Above-mentioned amino resins is placed in the NMP for the hexahydropyridine that percent by volume is 15%~30%
In solution, in the Fmoc blocking groups under the conditions of 20~28 DEG C on 25~40 minutes removing amino resins of reaction；
(2) wash：Nitrogen is dried up, NMP washing amino resins；
(3) condensation reaction：The amino acid for adding HOBT, DIC and Fmoc protection reacts 0.5-2.5 under the conditions of 20~28 DEG C
Hour；
(4) wash：Nitrogen is dried up, NMP washing amino resins；
(5) capping：Nmp solution of the percent weight in volume for 1.5%~4% acetyl imidazole is added, 20~
Reacted 20~40 minutes under the conditions of 28 DEG C.
1.1.5 antigenic synthetic peptide end of synthesis
Synthesizer will be automatically stopped after antigen end of synthesis.Then reactor is removed from Peptide synthesizer, then with 100%
Methanol washing polypeptide resin 3 times, then dries up in fume hood, polypeptide resin is transferred in brown bottle, -20 DEG C of refrigerators are put into
Interior, sealed membrane sealing is standby.
The cracking and identification of 1.2 antigenic synthetic peptides
1.2.1 the cracking of antigenic synthetic peptide
Prepare and split according to volume ratio (trifluoroacetic acid (TFA)/tri isopropyl silane (TIS)/phenol/water=85/8/6/1)
Liquid is solved, the polypeptide resin of synthesis is then taken out out of refrigerator, is put into round-bottomed flask, adds and prepares into flask in fume hood
Good lysate and magnetic stick, is then stably placed on magnetic stirring apparatus, persistently stirs 1h until having reacted at room temperature
Entirely.After reaction terminates, the TFA removed in crude product for 30~120 minutes is persistently evaporated using the Rotary Evaporators with cold-trap.Then
Collected using ether, precipitated polypeptide, the crude product of polypeptide antigen is then cleaned multiple times with dimethylformamide (DMF), finally will be mixed
The resin being combined is filtered out with sand core funnel, that is, obtains polypeptide antigen.
1.2.2 the identification of antigenic synthetic peptide
Antigen synthesis finish after with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF) and reversed phase high-pressure liquid
Phase chromatography (RP-HPLC) carries out qualitative and quantitative analysis.
The conformation of 1.3 antigenic synthetic peptides is formed
Polypeptide antigen is configured to the polypeptide solution that concentration is 2mg/ml with 15%DMSO, then with 0.1N NaOH or
The pH value of 0.1N HCl adjustment initial gross separation polypeptide solutions is 5.5~7.5, being shaken in rotating speed for 110rpm in the environment of 25 DEG C
Placed 48 hours on bed, make to form disulfide bond, form cyclic structure.Or, carry out following head and the tail and be cyclized：
"-the COOH " of head and the tail amino acid and "-NH2" cyclization method is referring to the Peptide such as Mengfen Protein
Reserch 1996.48:229-239；"-the COOH " and "-OH " of head and the tail amino acid is set to be reacted and form cyclic structure
Method is referring to the Chem.Soc such as Mmenhofer 1970.92:3771-3777.Thus obtaining being capable of the natural structure of simulated virus particle
The polypeptide cyclisation structure of elephant.
The purifying of 1.4 antigenic synthetic peptides is degerming
Antigenic synthetic peptide carries out ultrafiltration (Tangential using circulating tangential flow filtration film bag under the conditions of 20~28 DEG C
The circulating tangential flow filtration film bags of Flow Device and the peristaltic pump supporting with it), polypeptide antigen is that macromolecular can not be by one
The filter membrane of set aperture, and early stage building-up process and the small molecular weight impurity of the formation of later stage cyclization or introduction can then pass through filter
Film.Then it is degerming for 0.2 μm of filter by aperture again, the solution finally obtained is dispensed into aseptic plastic bottle, mark is sticked
Label.Title, numbering, product batch number, concentration, date of manufacture, pot-life and the preservation condition of polypeptide, packing are indicated on label
Afterwards, be stored in -20 DEG C or -40 DEG C it is standby.
The need for for the ease of transport and long-term preservation, polypeptide antigen is freeze-dried to obtain many of solid state
Peptide.The polypeptide antigen frozen in advance is taken out, is dried on Labconco freeze driers, obtains the polypeptide of solid state
Antigen.It is simultaneously labelled.Indicated on label the title of polypeptide, numbering, product batch number, concentration, the date of manufacture, the pot-life and
Preservation condition.
Embodiment 2, ox aftosa are O-shaped, the preparation of A type divalence synthetic peptide vaccines
The preparation of 1.1 antigen aqueous phases
Weigh respectively according to the antigenic synthetic peptide shown in the polypeptide 1 or polypeptide 2 of the preparation of embodiment 1, be according to molar ratio
1:1 mixing, is then diluted to 50 μ g/ml with sterilized water for injection by antigenic synthetic peptide total concentration.By gained antigenic solution through hole
Footpath is filtered for 0.2 μm of filter, degerming.
It is prepared by 1.2 oil phase adjuvants
It is standby by oil phase adjuvant 50V through 121 DEG C of 30min that sterilize.
The emulsification of 1.3 synthetic peptide vaccines
IKA emulsifying devices are cleaned with the distilled water 2000ml of sterilizing 3 times, and oil phase adjuvant is then pressed under the conditions of 20~28 DEG C
It is 1 with antigen aqueous phase:1 volume ratio, first adds oil phase in emulsion tank, starts motor and is stirred with 90~150rpm slow rotations
After 2min, while being slowly added to aqueous phase antigen, 30min is stirred after adding, then with 10000rpm high-speed stirred 20min, stand
5min, makes vaccine be emulsified into the single-phase vaccine of Water-In-Oil, and it is 1 to obtain containing mol ratio:1 polypeptide 1, the polyvalent vaccine of polypeptide 2.
The vaccine for comprising only one-component is prepared in the same manner, and antigen concentration is 50 μ g/ml.
Embodiment 3, ox aftosa are O-shaped, the experiment of the antigen selection of A type divalence synthetic peptide vaccines
1. materials and methods
1.1 material
1.1.1 synthetic peptide vaccine
The screening antigen of divalence synthetic peptide vaccine is prepared according to the method for embodiment 1, is then prepared respectively according to embodiment 2
One-component vaccine containing screening antigen.For the vaccine of the alternative antigen preparation of O-shaped foot and mouth disease virus, correspondence lot number is：
NO1301、NO1302、NO1303、NO1304.For the vaccine of the alternative antigen preparation of A type foot and mouth disease viruses, correspondence lot number is：
NA1401、NA1402、NA1403、NA1404、NA1405。
1.1.2 experimental animal
Select negative (suckling mouse neutralizing antibody titers≤1 of foot-and-mouth disease antibody:4) 6 monthly ages, healthy ox 45 [was purchased from Lanzhou
Regional cattle farm].
1.1.3 foot and mouth disease virus seed culture of viruses AF/72 plants, OS/99 plants of seed culture of viruses [Zhongmu Industry Co., Ltd]
Determined with 3~4 age in days suckling mouses [from numerous] and adjust malicious valency, be placed in -70 DEG C of freezen protectives standby.
1.2 test method
By healthy susceptible ox 45, be randomly divided into 9 groups, every group 5, be immunized respectively NO1301, NO1302, NO1303,
NO1304、NA1401、NA1402、NA1403、NA1404、NA1405.Immunizing dose is every part 1ml, and neck is used when being immunized
Intramuscular injection.The 14th after inoculation, 21 days, gather venous blood respectively, serum separated, with " the O-shaped antibody liquid of aftosa is mutually blocked
ELISA detection kit " determines O-shaped and A type mouthful hoof respectively with " foot-and-mouth disease a type antibody liquid mutually blocks ELISA detection kit "
Epidemic disease antibody titer.
By the 1st, 2,3,4 groups of ox attack OS/99 it is malicious by force, the 5th, 6,7,8,9 groups of ox attack AF/72 it is malicious by force, compare ox 4
Head, wherein 2 cow tongue upper surfaces both sides are divided to 2 intracutaneous injection AF/72 malicious by force, another 2 beef injection OS/99 are malicious by force.Every is
0.1ml (common 0.2ml, containing 104ID50), Continuous Observation 10 days.
1.3 result judgement
Control ox answers more than 3 hoof lesion (bubble or ulcer) occur, and bubble or ulcer occurs in immune cattle only lingual surface, and its
Protection is judged to when his position is without lesion, sentencing during typical aftosa lesion (bubble or ulcer) occurs in any position in addition to lingual surface
Not protect.6 groups of vaccines to be checked carry out protest test according to the method described above.
2. result of the test is with discussing
2.1 result of the test
Animal is immunized according to 1.2 methods in above-mentioned 6 batches of laboratory vaccines, respectively at 14, detection serum antibody titer on the 21st, 21
After day detection antibody titer, the strong poison of AF/72 is attacked immediately and OS/99 is malicious by force, result of determination after 10 days.Antibody level and attack malicious protection
It the results are shown in Table 1.
As a result show, O-shaped vaccine antigen OP1, OP2 resist immune latter 14 days and 21 days antibody titers higher than other two
Original, attacks malicious protective rate for 5/5 full guard, therefore selects the antigen as the O-shaped antigen of divalence synthetic peptide vaccine (as X1aa (oneself
The amino acids of amino acid the 5th of sequence 1)=Ala or Lys, X2aa (from the amino acids of amino acid the 13rd of sequence 1)=Tyr or
During Gly, antibody level highest is above OP3 and OP4, and 5/5) attack malicious protecting effect rate is.A type vaccine antigens AP2, AP3,
AP4 was higher than other two antigens immune latter 14 days and 21 days antibody titers, attacked malicious protective rate for 5/5 full guard, therefore selection
The antigen as divalence synthetic peptide vaccine A types antigen (as X1aa (from the amino acids of amino acid the 7th of sequence 2)=Ala or
Thr, X2aa (from the amino acids of amino acid the 11st of sequence 2)=Val or Tyr, X3aa are (from the 16th ammonia of amino acid of sequence 2
Base acid)=Gly or Arg when, be immunized after 14 days and 21 days antibody titer highests, be above AP1 and AP5, attacking malicious protective rate is
5/5 full guard).
The alternative antigen selection result of the test of the vaccine of table 1.
Embodiment 4, ox aftosa are O-shaped, the potency test of A type divalence synthetic peptide vaccines
1. materials and methods
1.1 material
1.1.1 synthetic peptide vaccine
Prepared according to embodiment 1 selected in embodiment 3 such as polypeptide 1 and the polypeptide antigen of polypeptide 2, then according to embodiment
2 ox aftosas of the preparation containing polypeptide 1 and the blending ingredients of polypeptide 2 are O-shaped, A type divalence synthetic peptide vaccines, and correspondence lot number is：
NOA1601、NOA1602、NOA1603。
1.1.2 experimental animal
Select negative (suckling mouse neutralizing antibody titers≤1 of foot-and-mouth disease antibody:4) 6 monthly ages, healthy ox 124 was [purchased from orchid
State area cattle farm].
1.1.3 foot and mouth disease virus seed culture of viruses AF/72 plants, OS/99 plants of seed culture of viruses [Zhongmu Industry Co., Ltd]
Determined with 3~4 age in days suckling mouses [from numerous] and adjust malicious valency, be placed in -70 DEG C of freezen protectives standby.
1.2 test method
By healthy susceptible ox 120,8 groups are randomly divided into, every group 15,1-2 groups are immunized NOA1601,3-4 groups and exempted from
NOA1603 is immunized in epidemic disease NOA1602,5-6 groups, and O-shaped comparable product control seedling aftosa, A types, Type Asia 1 three is immunized in 7-8 groups
Bivalent inactivated vaccine (O/HB/HK/99 plants+AF/72 plants+Asia-1/XJ/KLMY/04 plants).It is specific immune per 3 dosage groups of component
Dosage is：1 part (5), 1/3 part (5), 1/9 part (5), wherein, the every head of NOA1601, NOA1602, NOA1603
Part it is 1ml, comparable product control seedling 1607002 (commercialization ox aftosa is O-shaped, A types, Type Asia 1 tervalence inactivated vaccine) every head
Part is 3ml.Using musculi colli injection when immune, injection dosage and specific packet situation refer to table 1., will after inoculation after 21 days
1st, 3,5,7 groups of ox attack AF/72 it is malicious by force, the 2nd, 4,6,8 groups of ox attack OS/99 it is malicious by force.Ox 4 is compareed, wherein 2 oxen
Tongue upper surface both sides are divided to 2 intracutaneous injection AF/72 malicious by force, and another 2 beef injection OS/99 are malicious by force, and every is 0.1ml (common
0.2ml, containing 104ID50), Continuous Observation 10 days.
1.3 result judgement
Control ox answers more than 3 hoof lesion (bubble or ulcer) occur, and bubble or ulcer occurs in immune cattle only lingual surface, and its
Protection is judged to when his position is without lesion, sentencing during typical aftosa lesion (bubble or ulcer) occurs in any position in addition to lingual surface
Not protect.According to the protection number of immune cattle, the PD of tested vaccine is calculated by Read-Muench methods50.8 groups of vaccines to be checked are pressed
Potency test is carried out according to the above method.
2. result of the test is with discussing
2.1 result of the test
Animal is immunized according to 1.2 methods in above-mentioned 3 batches of laboratory vaccines, control vaccine, and the strong poison of AF/72 and OS/ are attacked after 21 days
The last 99 poison, result of determination after 10 days the results are shown in Table 2.
2. Ns of aftosas of table are O-shaped, A type divalence synthetic peptide vaccine efficacy test results
2.2 discussion of results
By the above results it can be seen that：The ox aftosas of three batches is O-shaped, A type divalence synthetic peptide vaccines NOA1601,
Animal is immunized in NOA1602, NOA1603 respectively, and the PD after AF/72 strong virus attacks is used after 21 days50Value is respectively：9.00、9.00、
9.00, the PD that comparable product control vaccine 1607002 is attacked after poison using AF/7250It is worth for 7.49.Three batch polypeptide vaccines are used
PD after OS/99 strong virus attacks50Value is respectively：10.81st, 10.32,9.00, comparable product control vaccine 1607002 uses OS/
PD after 99 strong virus attacks50It is worth for 7.05.
The above results show：Ox aftosa is O-shaped, A type divalence synthetic peptide vaccine is attacked for the strong poison of AF/72 or the strong poison of OS/99
The PD hit50Value reaches that protection is required more than 6.Illustrate the energy that polypeptide vaccine has protection test animal ox from virus attack
Power.And ox aftosa is O-shaped, A type divalence synthetic peptide vaccine is for the strong poison of AF/72, the PD of OS/99 strong virus attacks50Value is higher than ox mouthful
The PD of fever aphthous inactivated vaccine50Value, illustrates that polypeptide vaccine is compared than conventional inactivated vaccine, with more preferable protecting effect.This experiment is proved
Synthetic peptide seedling had both met the requirement of prevention from suffering from the diseases, and the requirement of food security is met again, and the market space is very wide.
Embodiment 5, ox aftosa are O-shaped, the safety testing of A type divalence synthetic peptide vaccines
1. materials and methods
1.1 synthetic peptide vaccine
Be the same as Example 3.
1.2 experimental animals [from numerous]
350~450g cavy；18~22g mouse；The healthy susceptible ox at least 6 monthly ages.
1.3 test method
1.3.1 350~450g of body weight cavy 15 is used, every is subcutaneously injected vaccine 2ml；It is small with 18~22g of body weight
Mouse 15, every is subcutaneously injected vaccine 0.5ml.Continuous Observation 7 days, must not occur dead or obvious caused by vaccinating
Local adverse reaction or general reaction.
1.3.2 with the healthy susceptible ox at least 6 monthly ages, (cell neutralize antibody titers are not higher than 1:8) 9, in every cow tongue
20 points of back side intracutaneous injection vaccine, every 0.1ml, after observing 4 day by day, every ox intramuscular injection vaccine 6ml continues day by day
Observation 6 days.Aftosa symptom or the obvious toxic reaction caused by vaccinating must not occur.
2. result of the test
2.1 vaccine in guinea pigs and the security of mouse
Cavy 15, every is subcutaneously injected vaccine 2ml；Mouse 15, every hypodermic injection 0.5ml.Continuous Observation 7 days,
Do not occur dead caused by vaccinating or obvious local adverse reaction or general reaction, concrete outcome such as table 3 below.
The cavy of table 3. and the vaccine safety result of the test of mouse
2.2. security of the vaccine to healthy susceptible ox
Synthetic peptide vaccine is taken out and equilibrated to after room temperature, 2ml, every point are vaccinated in intracutaneous point of 20 points of every cow tongue
0.1ml, is observed at least 4 days day by day.Afterwards, every ox intramuscular injection vaccine 9ml, continues to observe 6 day by day.Concrete outcome is shown in Table
4。
The vaccine safety result of the test of the healthy susceptible ox of table 4.
The above results illustrate that ox aftosa is O-shaped, A type divalence synthetic peptide vaccines are safe to cavy, mouse and ox, without dissipating
It is malicious dangerous, so with good promotion prospect and market value.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deformed, and without departing from the spirit of the present invention, all should belong to the model of appended claims of the present invention
Enclose.
Sequence table
<110>Zhongmu Industry Co., Ltd
<120>Ox aftosa is O-shaped, A type divalence synthetic peptide vaccines and its preparation method and application
<130> WHOI170023
<160> 2
<210> 1
<211> 59
<212> PRT
<213>Artificial sequence
<220>
<221> misc_feature
<222> (5) ..(5)
<223>X1aa=Ala or Lys
<220>
<221> misc_feature
<222> (13) ..(13)
<223>X2aa=Tyr or Gly
<400> 1
Arg Met Glu Ala X1aa Lys Thr Gly Ile Glu Ile Ala X2aa Asp Glu Arg
1 5 10 15
Arg Lys Val Tyr Asn Gly Asn Cys Lys Tyr Thr Gly Gly Ser Ala Pro
20 25 30
Asn Val Arg Gly Asp Leu Gln Val Leu Ala Pro Lys Ala Ala Arg Cys
35 40 45
Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys
50 55
<210> 2
<211> 59
<212> PRT
<213>Artificial sequence
<220>
<221> misc_feature
<222> (7) ..(7)
<223>X1aa=Ala or Thr
<220>
<221> misc_feature
<222> (11) ..(11)
<223>X2aa=Val or Tyr
<220>
<221> misc_feature
<222> (16) ..(16)
<223>X3aa=Gly or Arg
<400> 2
Lys Lys Gly Ala Gly Leu X1aa Gly Val Met X1aa Thr Glu Ser Val X1aa
1 5 10 15
Phe Arg Lys Lys Val Tyr Ser Gly Thr Cys Lys Tyr Ser Ala Pro Gln
20 25 30
Asn Arg Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Cys
35 40 45
Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile Arg
50 55
Claims (9)
1. it is used to preparing that ox aftosa to be O-shaped, A type divalence synthetic peptide vaccines antigen polypeptide composition a kind of, including in sequence table
Polypeptide in polypeptide and sequence table shown in sequence 1 shown in sequence 2.
2. it is used to preparing that ox aftosa to be O-shaped, the combination of antigen polypeptide of A type divalence synthetic peptide vaccines according to claim 1
Thing, the wherein polypeptide fragment shown in sequence 1, from the carboxyl and the ε bit aminos of the 18th amino acids of the amino acids of aminoterminal the 17th
Form peptide bond connection.
3. it is used to preparing that ox aftosa to be O-shaped, the combination of antigen polypeptide of A type divalence synthetic peptide vaccines according to claim 1
Thing, the wherein polypeptide fragment shown in sequence 2, from the carboxyl and the ε bit aminos of the 20th amino acids of the amino acids of aminoterminal the 19th
Form peptide bond connection.
4. it is used to preparing that ox aftosa to be O-shaped, the combination of antigen polypeptide of A type divalence synthetic peptide vaccines according to claim 1
Thing, it is described to be used to preparing that ox aftosa to be O-shaped, A type divalence synthetic peptide vaccines peptide composition, polypeptide and sequence as shown in sequence 1
Polypeptide shown in row 2 is constituted.
5. ox aftosa according to claim 4 is O-shaped, the antigen polypeptide composition of A type divalence synthetic peptide vaccines, wherein,
The mol ratio of the polypeptide shown in polypeptide, sequence 2 shown in sequence 1 is (0.5-1.5):(0.5-1.5).
6. ox aftosa according to claim 4 is O-shaped, the antigen polypeptide composition of A type divalence synthetic peptide vaccines, wherein,
The mol ratio of the polypeptide shown in polypeptide, sequence 2 shown in sequence 1 is 1:1.
7. for preparing, ox aftosa is O-shaped, A type divalence synthetic peptide vaccines antigen polypeptide, is many shown in sequence 1 in sequence table
Polypeptide in peptide or sequence table shown in sequence 2.
8. the antigen polypeptide composition in the antigen polypeptide and claim 1-6 described in claim 7 described in any one exists
Prepare O-shaped ox aftosa, A type divalence synthetic peptide vaccines or prepare for preventing in cattle O type, the biological products of A type aftosas
Using.
9. N aftosa is O-shaped, the preparation method of A type divalence synthetic peptide vaccines, it is characterised in that the preparation method includes following
Step：
(1) peptide composition described in any one in claim 1-6 is diluted to the dense of 10-100 μ g/ml with water for injection
Degree, so as to obtain polypeptide antigen aqueous phase；
(2) adjuvant is sterilized；
(3) under the conditions of 20~28 DEG C, according to the polypeptide antigen aqueous phase and the adjuvant 1:1 volume ratio, first adds adjuvant
Enter in emulsion tank, under 90~150rpm stir 1.5~3 minutes, be then slowly added into polypeptide antigen aqueous phase, then stir 20~
30 minutes, then stirred 15~30 minutes under 8000~10000rpm, 3~10 minutes are stood, packing.
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