CN106380509B - One boar annulus synthetic peptide vaccine and its preparation method and application - Google Patents
One boar annulus synthetic peptide vaccine and its preparation method and application Download PDFInfo
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- CN106380509B CN106380509B CN201610754866.0A CN201610754866A CN106380509B CN 106380509 B CN106380509 B CN 106380509B CN 201610754866 A CN201610754866 A CN 201610754866A CN 106380509 B CN106380509 B CN 106380509B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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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
- 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
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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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The present invention provides a kind of antigen polypeptide for being used to prepare pig annulus synthetic peptide vaccine, peptide composition and vaccine.Antigen polypeptide is sequence 1, sequence 2, polypeptide shown in sequence 3 or sequence 4.Vaccine of the invention includes polypeptide or peptide composition.Pig annulus synthetic peptide vaccine provided by the invention has good immune efficacy, and the problems such as existing fever of traditional vaccine, redness will not be caused, therefore vaccine of the invention can successfully manage the antigenic variation of current pig circular ring virus, biological safety is not present, it is easy to synthesize on a large scale, has a good application prospect.The present invention also provides the preparation methods and its pharmaceutical applications of the polypeptide and polypeptide vaccine.
Description
Technical field
The present invention relates to the invention belongs to pharmaceutical technology fields, specifically, the present invention relates to a boar annulus synthetic peptides
Vaccine, with and preparation method thereof.
Background technique
Porcine circovirus desease (porcine circovirus-associated disease, PCVAD) refers to and is drawn by PCV2
A series of comprehensive syndromes or the related disease with PCV2 infection risen.It is different according to PCV antigenicity and gene composition, by its point
For two kinds of genotype (serotype), pig circular ring virus relevant to clinical disease is named as porcine circovirus 2 type (PCV2), and nothing is faced
The pathogenic pig circular ring virus of bed is named as 1 type of pig circular ring virus (PCV1).Pig circular ring virus is the one kind found so far
The smallest animal virus, diameter 17nm, icosahedral symmetry, no cyst membrane.Viral genome is by sub-thread minus strand closed hoop DNA group
At wherein PCV1 contains 1759 nucleotide, and PCV2 contains 1767 or 1768 nucleotide.
Its monocyte and macrophage are encroached on after PCV2 infection body, leads to the immunosupress and Abwehrkraft des Koepers drop of pig
Low, the generation and maintenance of interference and destruction pig to other diseases immune antiboidy cause the concurrent or secondary infection of a variety of diseases.Pig
After infection PCV2 virus, also horizontal transmission can be carried out by excrement etc. by placenta vertical transmission to newborn piglet.
The resistance to environmental factor ability of PCV2 virus is strong, has very strong repellence to various disinfectants, even if under high temperature and acidic environment
Survival a period of time is remained to, pig farm hardly results in purification.The virus can be present in multiple organs of body, especially lungs and
In lymph node, corresponding organ is caused the symptoms such as dysfunction occur.Infection pig not only will appear respiratory dysfunction, but will be because of
Inhibit immunity of organism and resistance is caused to decline, raising Other diseases illness is possible, including postweaning multisystemic failure is comprehensive
Levy (PMWS), pigskin inflammation nephrotic syndrome (PDNS), respiratory diseases in pigs mixing disease (PRDC), pig breeding dysfunction disease, granuloma
The illnesss such as property enteritis, acute pulmonary edema (APE), Hypertrophic necrotizing pneumonia (PNP).
Porcine circovirus desease was proved in 1997 in Canada West for the first time, successive North America, Europe and Asia majority state
There is the report of this disease prevalence in family.For the disease incidence of the disease up to 50%, the death rate may be due to pig farm condition, secondary infection situation
Difference, generally 5%~7%.In Porcine circovirus desease, endangered caused by pig breeding industry with PMWS the most seriously, it, should in Europe
Disease can cause about 600,000,000 Euros of pig breeding industry of loss every year.
The effective means that a large amount of industry aquiculture status and experimental study show to control PCV2 infection is to carry out epidemic disease to piglet
Seedling is immune.Presently commercially available PCV2 commercialized vaccine is mainly the vector-viral vaccine and domestic inactivated vaccine of import.Both
Vaccine has played important function in China's prevention and control PCV2 infection, but simultaneously is difficult to cultivate, produce there is also inactivated vaccine
The more difficult unification of quality, production technology such as require further improvement at the difficulties between batch, so that inactivated vaccine preparation yield is not high, while also depositing
In the danger for dissipating poison.And although baculovirus vector vaccine can guarantee the stability and consistency of vaccine, be easy immune animal
It is distinguished with infection animal, but expensive, many pig farms cannot largely be purchased, so the control to China's Porcine circovirus desease
Effect processed is limited.Since PCV2 vaccine still has defect in tradition in preparation method or price, cost effective is developed
New generation vaccine gradually by the concern of scholars.In recent years, attention is transferred to subunit and more by more and more scholars
The research of peptide vaccine, a part of polypeptide antigen as intact virus albumen do not have the risk for infecting disease, and can
To be largely synthetically produced, therefore have become one of the important content of vaccine research exploitation.
Summary of the invention
The purpose of the present invention is to provide a kind of antigen polypeptide for being used to prepare pig circular ring virus synthetic peptide vaccine, polypeptide groups
Close object, and the vaccine containing the peptide composition.
To achieve the above object, this laboratory devises four amino acid compositions according to PCV2-Cap gene antigen epitope altogether
Polypeptide sequence, respectively SEQNO.1, SEQNO.2, SEQNO.3 and SEQNO.4 polypeptide sequence, using Solid phase synthesis, with
ISA50V adjuvant is mixed and made into oil emulsion vaccine.Pass through animal safety and effect Journal of Sex Research, it was demonstrated that single in minimum age in days piglet
It is all safe to pig in the case of dosage, piglet repeated inoculation, overdose of piglet and farrowing sow doubling dosage, and
Head exempts from the ELISA antibody titer of rear 35 days vaccine immunity groups up to 1:3520-1:5760, and IFA antibody titer is up to 1:3328-1:
4068, reach protecting effect identical with commercialized vaccine.
Synthetic peptide vaccine greatly improves safety and specificity, and its preparation process is simpler, production cost more
It is cheap, vaccine quality is relatively reliable, be conducive to large-scale production.As a kind of novel annulus vaccine, advantage can for enterprise and
New competitiveness and point of economic increase are brought in market, and the market space is huge.
The invention adopts the following technical scheme:
The present invention provides a kind of antigen polypeptide for being used to prepare pig circular ring virus synthetic peptide vaccine, is sequence 1, sequence 2, sequence
Polypeptide described in column 3 or sequence 4.
Provided by the present invention for preparing the peptide composition of pig circular ring virus synthetic peptide vaccine, including shown in sequence 1
Any group of one or more of polypeptide shown in polypeptide, sequence 2, polypeptide shown in polypeptide and sequence 4 shown in sequence 3
It closes.
Wherein, from the 33rd amino acids (X of aminoterminal of the sequence 1 in sequence table1It aa is) Ser or Arg, from sequence table
The 53rd amino acids (X of 1 aminoterminal of sequence2It aa is) Thr or Ser, from the 54th amino acids of 1 aminoterminal of sequence in sequence table
(X2It aa) is Arg or Gly；
It is Thr or Asn from the 44th amino acids (Xaa) of aminoterminal of the sequence 2 in sequence table；
From the 31st amino acids (X of aminoterminal of the sequence 3 in sequence table1It aa is) Ile or Phe, from the sequence in sequence table
The 37th amino acids (X of aminoterminal of column 32It aa is) Lys or Arg, from the 41st amino acids of aminoterminal of the sequence 3 in sequence table
(X3It aa is) Arg or Lys, from the 55th amino acids (X of aminoterminal of the sequence 3 in sequence table4It aa is) Asn or Asp, from sequence
The 58th amino acids (X of aminoterminal of sequence 3 in table5It aa is) Leu or Val, from the aminoterminal the 64th of the sequence 3 in sequence table
Amino acids (X6It aa) is Ser or Thr；
From the 34th amino acids (X of aminoterminal of the sequence 4 in sequence table1It aa is) Ile or Lys, from the sequence in sequence table
The 38th amino acids (X of aminoterminal of column 42It aa is) Asp or Glu, from the 43rd amino acids of aminoterminal of the sequence 3 in sequence table
(X3It aa is) Ile or Val, from the 60th amino acids (X of aminoterminal of the sequence 3 in sequence table4It aa) is Asn or Lys.
Wherein, as described above, including polypeptide shown in polypeptide shown in sequence 1, sequence 2, polypeptide and sequence shown in sequence 3
In polypeptide shown in 4 have polymorphic site (i.e. there are two kinds of optional amino acid for an amino acid sites) when, these due to
The not homotactic polypeptide of loci polymorphism bring, can individually or be mixed in peptide composition.Of the invention one
In a embodiment, in the synthesis of polypeptide, two kinds of amino acid are loaded simultaneously there are two types of optional amino acid sites in tool, at random
Synthesis.
Heretofore described polypeptide and polypeptide fragment can be the peptide material obtained by Solid-phase organic synthesis.
The sequence of specific above-mentioned each polypeptide is as follows:
Polypeptide 1 (polypeptide shown in sequence 1 in sequence table):
ISITEIGKVIVRHIATIFLεKHSRYFTPKPVLDX1TIDYFQPNNKRN QLWLRLQ X2X3GNV
(X1=R/S, X2=T/S, X3=R/G)
Polypeptide 2 (polypeptide shown in sequence 2 in sequence table):
ISITEIGKVIVRHIATIFL ε KITQGDRGVGSTAVILDDNFVTKAXALTYDPYVNYSSR (X=T/N)
Polypeptide 3 (polypeptide shown in sequence 3 in sequence table):
ISITEIGKVIVRHIATIFLεKGIFNTRLSRTX1GYTVKX2TTVX3TPS
WAVDMMRFNIX4DFX5PPGGGX6N
(X1=I/F, X2=K/R, X3=R/K, X4=N/D, X5=L/V, X6=S/T)
Polypeptide 4 (polypeptide shown in sequence 4 in sequence table):
ISITEIGKVIVRHIATIFLεKVDHVGLGTAFENSX1YDQX2YNIRX3T MYVQFREFNLKDPPL X4PK
(X1=K/I, X2=D/E, X3=I/V, X4=N/K)
Sequence is aminoterminal to c-terminus from left to right in above-mentioned each polypeptide sequence, from the carboxylic of the 19th amino acids of aminoterminal
Base and the ε bit amino of the 20th amino acids form peptide key connection.
Wherein, ε K is that the ε bit amino of lysine forms peptide bond, and concrete principle is as follows:
Each amino acid contains at least one "-COOH " (carboxyl) and "-a NH3" (amino), it is connected to alpha-carbon atom
On carboxyl and amino be known as " α carboxyl " and " α amino ".The reaction of general peptide synthesis be a upper amino acid " α amino " and
" α carboxyl " reaction of next amino acid forms amido bond (peptide bond) and forms.
Lys (lysine) is a kind of basic amino acid, other than normal " α amino " and " α carboxyl ", on the position ε also
There are an amino, such as following formula.
ε K refers in particular to participate in the amino of peptide formation to be " ε amino " rather than " α amino ", because of its longer arm structure, because
This, be used to connect the epitope that needs to be used in conjunction with but not interact.
Preferably, when choosing the composition vaccine composition of polypeptide described in two of them sequence, their molar ratio is
(0.5-1.5):(0.5-1.5)；Most preferably, their molar ratio is 1:1.When choosing wherein polypeptide group described in three kinds of sequences
When at vaccine composition, their molar ratio is (0.5-1.5): (0.5-1.5): (0.5-1.5)；Most preferably, they rub
You are than being 1:1:1.When choosing the composition vaccine composition of polypeptide described in wherein four kinds of sequences, (0.5-1.5): (0.5-1.5):
(0.5-1.5):(0.5-1.5)；Most preferably, their molar ratio is 1:1:1:1.
The peptide composition also belongs to protection of the invention in the application prepared in pig circular ring virus synthetic peptide vaccine
Range.
A kind of pig circular ring virus synthetic peptide vaccine provided by the invention, includes above-mentioned peptide composition.
The peptide vaccine also includes adjuvant.
The present invention provides the preparation method of the pig circular ring virus synthetic peptide vaccine, the preparation method includes following
Step:
(1) peptide composition is diluted to the concentration of 10-100 μ g/ml (preferably 50 μ g/ml) with water for injection, from
And obtain polypeptide antigen water phase；
(2) adjuvant is sterilized into (sterilizing 30 minutes under the conditions of 121 DEG C)；
(3) under the conditions of 20~28 DEG C, according to the volume ratio of the polypeptide antigen water phase and the adjuvant 1:1, first high-ranking officer
Agent is added in emulsion tank, stirs 1.5~3 minutes at 90~150rpm, is then slowly added into polypeptide antigen water phase, then stirs
20~30 minutes, then stirred 15-30 minutes at 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 company)) one of or it is a variety of.
The present invention also provides the preparation method of the polypeptide in aforementioned polypeptides polymer, the preparation method includes following step
It is rapid:
(1) using amino resins as starting material, using the amino acid protected by 9-fluorenylmethyloxycarbonyl as monomer, according to the ammonia
Base acid sequence, which is successively condensed, connects amino acid to synthesize the polypeptide, is closed after every step condensation reaction with acetyl imidazole unreacted
Aminoterminal；
(2) lytic reagent is added after synthesizing, so that the polypeptide is 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 the above preparation method, the step (1) specifically includes the 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, 25~40min is reacted under the conditions of 20~28 DEG C, to remove the 9- fluorenes on amino resins
Methoxycarbonyl group blocking group；
(1-b) washing: it is dried with nitrogen, then washs amino resins with N-Methyl pyrrolidone；
(1-c) condensation reaction: be added 1- hydroxyl azimidobenzene (HOBT), N, N- diisopropylcarbodiimide (DIC) with by
The amino acid of 9-fluorenylmethyloxycarbonyl protection, then reacts 0.5~2.5h under the conditions of 20~28 DEG C；
(1-d) washing: it is dried with nitrogen, then washs amino resins with N-Methyl pyrrolidone；
(1-e) capping: the N- crassitude for the acetyl imidazole that percent weight in volume is 1.5%~4% is added
Ketone (NMP) solution reacts 20~40min under the conditions of 20~28 DEG C.
In the above preparation method, the group of lytic reagent is divided into the trifluoro that volume ratio is 85:8:6:1 in the step (2)
Acetic acid: tri isopropyl silane: phenol: H2O；Also, the pyrolysis time of the step (2) is 1-4h.
In the above preparation method, the step (3) specifically includes:
(3-a) precipitates the polypeptide using ether, is then washed with dimethylformamide；
In the above preparation method, the step (4) specifically includes the following steps:
(4-a) uses tangential flow filtration film packet polypeptide described in ultrafiltration under the conditions of 20~28 DEG C, to remove small molecular weight impurity；
(4-b) is saved using 0.2 μm of filter degerming.
Also on the one hand, the present invention provides aforementioned polypeptides or polypeptide vaccine to prepare for preventing pig circular ring virus synthetic peptide
Purposes in the biological products of vaccine.
Specifically, the present invention passes through the sequencing to domestic porcine circovirus 2 type prevalence strain and combines pig annulus
Viral vaccine strain sequence studies the variation situation of the major antigenic sites of porcine circovirus type 2 strain, for what is mainly made a variation
Amino acid sites count the frequency of its variation, and the analysis of porcine circovirus 2 type antigen site is carried out in combination with area of computer aided
Prediction carries out chemical synthesis to possible antigen site peptide fragment, i.e., for easy variant sites according to the variation frequency of statistics, at this
A little sites use different amino acid, obtain a variety of candidate polypeptide antigens for covering current all possible variant sites.In turn, lead to
It crosses a large amount of animal experiment to screen these candidate polypeptide antigens, obtains the immune response that can cause animal, and exempt from
Epidemic disease reaction level is high, can be good at the polypeptide antigen for protecting animal to attack from porcine circovirus 2 type prevalence strain.In addition,
The present inventor is optimized porcine circovirus 2 type antigen site according to screening experiment result, and efficient combination T cell table
Position and B cell epitope, enhance the immune effect of polypeptide antigen.
Peptide vaccine potency test, safety experiment the result shows that, pig circular ring virus synthetic peptide vaccine provided by the invention tool
The problems such as having good immune efficacy, and the existing fever of traditional vaccine, redness will not be caused, therefore vaccine of the invention can
To successfully manage the antigenic variation of current pig circular ring virus, biological safety is not present, it is easy to synthesize on a large scale, has good
Application prospect.
Detailed description of the invention
1-8 synthetic peptide vaccine antigen polypeptide the selection result in Fig. 1 embodiment 2
9-14 synthetic peptide vaccine antigen polypeptide the selection result in Fig. 2 embodiment 2
15-26 synthetic peptide vaccine antigen polypeptide the selection result in Fig. 3 embodiment 2
27-36 synthetic peptide vaccine antigen polypeptide the selection result in Fig. 4 embodiment 2
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples
Material raw material, reagent material etc. are commercially available products unless otherwise specified.
The synthesis in solid state of embodiment 1, pig circular ring virus synthetic peptide vaccine polypeptide
The present invention is by the sequencing of popular strain and combining pig annulus vaccine virus recently to domestic porcine circovirus 2 type
Strain sequence, studies the variation situation of porcine circovirus 2 type major antigenic sites, counts it for the amino acid sites mainly to make a variation
The frequency of variation, the analysis for carrying out foot-and-mouth disease antigen site in combination with area of computer aided is predicted, to possible antigen site peptide
Duan Jinhang chemical synthesis uses different amino acid in these sites that is, for easy variant sites according to the variation frequency of statistics,
Obtain a variety of candidate polypeptide antigens for covering current all possible variant sites.In turn, by a large amount of animal experiment to these
Candidate polypeptide antigen is screened, and the immune response that can cause animal is obtained, and is immunoreacted horizontal height, can be good at
The polypeptide antigen for protecting animal to attack from porcine circovirus 2 type prevalence strain.In addition, the present inventor is according to screening experiment result
Porcine circovirus 2 type antigen site is optimized, and efficient combination t cell epitope and B cell epitope, enhances polypeptide
The immune effect of antigen.
U.S.'s 433A full-automatic polypeptide synthetic instrument can be used by the antigenic synthetic peptide of the invention for screening and identifying, benefit
It is prepared with Merrifield solid-phase synthesis, wherein using the amino acid modified by 9-fluorenylmethyloxycarbonyl (Fmoc), solid phase is carried
Body is the Rink Amide MBHA resin of Tianjin Nankai.Production process generally includes the synthesis in solid state of polypeptide antigen, polypeptide is split
Solution, antigen purification and degerming save.
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 (in sequence table shown in sequence 1):
ISITEIGKVIVRHIATIFLεKHSRYFTPKPVLDX1TIDYFQPNNKRN QLWLRLQ X2X3GNV
In above-mentioned sequence, X1=R/S, X2=T/S, X3=R/G, two different aminoacids shown in these sites simultaneously plus
Sample, random synthesis.
Polypeptide 2 (in sequence table shown in sequence 2):
ISITEIGKVIVRHIATIFLεKITQGDRGVGSTAVILDDNFVTKAXALTYDPYVNYSSR
In the sequence, X=T/N；Two different aminoacids shown in this site are loaded simultaneously, random synthesis.Polypeptide 3
(in sequence table shown in sequence 3):
ISITEIGKVIVRHIATIFLεKGIFNTRLSRTX1GYTVKX2TTVX3TPS
WAVDMMRFNIX4DFX5PPGGGX6N
In the sequence, X1=I/F, X2=K/R, X3=R/K, X4=N/D, X5=L/V, X6=S/T, shown in these sites
Two different aminoacids are loaded simultaneously, random synthesis.
Polypeptide 4 (in sequence table shown in sequence 4):
ISITEIGKVIVRHIATIFLεKVDHVGLGTAFENSX1YDQX2YNIRX3T MYVQFREFNLKDPPLX4PK
In the sequence, X1=K/I, X2=D/E, X3=I/V, X4=N/K) two different aminoacids shown in these sites
It is loaded simultaneously, random synthesis.
Wherein, ε K is that the ε bit amino of lysine and the carboxyl of the 19th amino acids form peptide bond.
In following synthesis processes, reaction solution is alkalinity, its ε bit amino of the finished product lysine of purchase is by alkali sensitivity base
Group's protection, and its α bit amino is protected by acid sensitive group.In reaction process, the blocking group of ε bit amino can only be removed, cruelly
Reveal its amino, is reacted with the carboxyl of next amino acid.
According to the sequence of antigen and the synthesis scale of 1mmol, prepare the amino acid of suitable Fmoc modification [purchased from Shanghai
Gill is biochemical], it is added in corresponding amino-acid reagent bottle.Rink Amide MBHA resin is equally weighed as required, is put into reaction
In chamber, upper and lower lid is tightened, is labelled, the weight of the title of synthesized peptide, lot number, the tare weight of reaction chamber and alleged resin is recorded
Amount.Reaction chamber is packed into synthesizer.Prepare synthetic agent, including N-Methyl pyrrolidone (NMP), acetyl imidazole (AIM), piperidines
(PIP), methanol etc. is placed into corresponding reagent bottle.
1.1.2 synthesizer state-detection
Check PSI300 Peptide synthesizer whether normal operation.In addition N is checked2Whether sufficient, whether system gauge pressure is normal.
The performance of reply instrument is had gained some understanding before synthesis, so to be measured to the flow velocity of every kind of synthetic agent.It is separately operable A to
RV,B to RV,C to RV,D to RV,E to RV,F to RV,A to AA,C to AA.It measures or observes, if
Flow is improper, then lower valve pressure or each solution parameter of modification is adjusted, until reaching requirement.
1.1.3 the synthesis of antigenic synthetic peptide starts
Composition sequence editor Sequence is pressed in the program of synthesizer, then edits specific synthesis Program, finally handle
" Sequence " and " Program " is combined into one " runfile ", is saved.It is opened in operating file again, sets amino acid
Bottle prepares quantity, brings into operation.
1.1.4 the synthesis of antigenic synthetic peptide
Such as above-mentioned polypeptide sequence, synthesis when is since C-terminal to N-terminal, according to given sequence, successively constantly
Repeat following synthesis step:
(1) deprotection reaction: it is 15%~30% hexahydropyridine that above-mentioned amino resins, which is placed in containing percent by volume,
In nmp solution, the Fmoc blocking group on 25~40min removing amino resins is reacted under the conditions of 20~28 DEG C；
(2) it washs: being dried with nitrogen, NMP washs amino resins；
(3) condensation reaction: HOBT, DIC is added with fmoc-protected amino acid and reacts 0.5- under the conditions of 20~28 DEG C
2.5h；
(4) it washs: being dried with nitrogen, NMP washs amino resins；
(5) capping: being added the nmp solution for the acetyl imidazole that percent weight in volume is 1.5%~4%, 20~
20~40min is reacted under the conditions of 28 DEG C.
1.1.5 antigenic synthetic peptide synthesis terminates
Synthesizer will be automatically stopped after antigen synthesizes.Then reactor is removed from Peptide synthesizer, then with 100%
Methanol washs polypeptide resin 3 times, then dries up in draught cupboard, polypeptide resin is transferred in brown bottle, be put into -20 DEG C of refrigerators
Interior, sealed membrane sealing is spare.
The cracking and identification of 1.2 antigenic synthetic peptides
1.2.1 the cracking of antigenic synthetic peptide
According to volume ratio (trifluoroacetic acid (TFA)/tri isopropyl silane (TIS)/phenol/H2O=85/8/6/1 it) prepares
Then lysate takes out the polypeptide resin of synthesis out of refrigerator, is put into round-bottomed flask, be added and match into flask in draught cupboard
The lysate and magnetic stick made, is then stably placed on magnetic stirring apparatus, persistently stirs 1h at room temperature until reaction
Completely.After reaction, the TFA in 30~120min removing crude product is persistently evaporated using the Rotary Evaporators with cold-trap.It connects
Using ether collect, precipitated polypeptide, the crude product of polypeptide antigen is then cleaned multiple times with dimethylformamide (DMF), finally general
The resin mixed is filtered out to arrive polypeptide antigen with sand core funnel.
1.2.2 the identification of antigenic synthetic peptide
Polypeptide antigen is high with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF) and reverse phase after synthesizing
Pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis.
The purifying degerming of 1.3 antigenic synthetic peptides
Antigenic synthetic peptide carries out ultrafiltration (Tangential using circulating tangential flow filtration film packet under the conditions of 20~28 DEG C
The circulating tangential flow filtration film packet of Flow Device and peristaltic pump matched with its), polypeptide antigen is that macromolecular cannot be by one
The filter membrane of set aperture, and the small molecular weight impurity that synthesis process early period and later period cyclization are formed or introduced can then pass through filter
Film.Then passing through aperture again is 0.2 μm of filter degerming, and the solution finally obtained is dispensed into aseptic plastic bottle, mark is sticked
Label.Title, number, 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 spare.
For the ease of transporting the needs with long-term preservation, polypeptide antigen is freeze-dried to obtain the more of solid state
Peptide.The polypeptide antigen frozen in advance is taken out, is dried on Labconco freeze drier, obtains the polypeptide of solid state
Antigen.It is labelled simultaneously.Indicated on label the title of polypeptide, number, product batch number, concentration, the date of manufacture, the pot-life and
Preservation condition.
The screening of embodiment 2, pig circular ring virus synthetic peptide vaccine antigen polypeptide
The synthesis of 1.1 candidate antigen polypeptides
Synthetic method is the same as embodiment 1.
The screening of 1.2 antigen polypeptides
The present inventor passes through a large amount of Literature Consult, is designed antigen polypeptide and has been screened, at up to ten thousand
Pass through multiple screening in polypeptide, and modify and optimize some sites repeatedly, finally obtains 36 polypeptides and waited as polypeptide vaccine
Choosing.
36 candidate antigen polypeptides are coated with ELISA reaction plate respectively with the concentration of 1 μ g/ml, are stood overnight at 4~8 DEG C,
Combine polypeptide antigen sufficiently with enzyme-linked reaction plate.Then 37 DEG C of 2~3h of Seal treatment of confining liquid are added according to 300 holes μ l/.With
It is dried after washing buffer washing.According to 100 holes μ l/ be added 60 times of diluted porcine circovirus 2 type antibody positive serums, 37 DEG C
In incubator or water-bath, 30min is reacted.After washing, 10000 times of diluted horseradish peroxidase-labeleds are added in 100 holes μ l/
Rabbit-anti pig IgG antibody (Sigma, article No. A5670) in 37 DEG C of incubators or water-bath, reacts 30min.It is aobvious that substrate is added after washing
Color.Read OD450Value.Negative control antigen (BSA, 2.5 μ g/ml) coating reaction plate, positive control antigen (StarNet, pig are set simultaneously
Circovirus 2-dCap-ELISA antibody assay kit) coating reaction plate.
The selection result is shown in Fig. 1, Fig. 2, Fig. 3 and Fig. 4.Compared with positive control antigen, 16 antigen polypeptides are filtered out, it is more
The sequence of peptide is shown in Table 1.Wherein, sequence shown in sequence 1 in sequence table, three amino acid sites (33,53,54) therein,
X1Aa=Ser or Arg, X2Aa=Thr or Ser, X3Aa=Arg or Gly, any combination, its OD of the polypeptide antigen of synthesis450Value is equal
1.5 or so.Sequence shown in sequence 2 in sequence table, amino acid sites (44) therein, Xaa=Thr or Asn, synthesis it is more
Its OD of peptide antigen450Value is 1.5 or so.Sequence shown in sequence 3 in sequence table, six amino acid sites therein (31,37,
41,55,58,64), X1Aa=Ile or Phe, X2Aa=Lys or Arg, X3Aa=Arg or Lys, X4Aa=Asn or Asp, X5Aa=
Leu or Val, X6Aa=Ser or Thr, any combination, its OD of the polypeptide antigen of synthesis450Value is 1.5 or so.Sequence in sequence table
Sequence shown in column 4, four amino acid sites (34,38,44,60) therein, X1Aa=Ile or Lys, X2Aa=Asp or Glu,
X3Aa=Ile or Val, X4Aa=Asn or Lys, any combination, its OD of the polypeptide antigen of synthesis450Value is 1.5 or so.
1. porcine circovirus 2 type synthetic peptide vaccine antigen polypeptide of table
The preparation of embodiment 3, pig circular ring virus synthetic peptide vaccine
The preparation of 1.1 antigen water phases
Antigenic synthetic peptide shown in polypeptide 1, polypeptide 2, polypeptide 3 or the polypeptide 4 prepared according to embodiment 1 is weighed respectively, is pressed
It is 1:1:1:1 mixing according to antigen ratio mole, antigenic synthetic peptide total concentration is then diluted to 100 μ g/ with sterilized water for injection
ml.The filter that gained antigenic solution via hole diameter is 0.2 μm is filtered, degerming.
1.2 oily phase adjuvant preparations
It is spare by oily phase adjuvant 50V through 121 DEG C of sterilizing 30min.
The emulsification of 1.3 synthetic peptide vaccines
It is cleaned IKA emulsifying device 3 times with the distilled water 2000ml of sterilizing, then by oily phase adjuvant under the conditions of 20~28 DEG C
Oil, is first added in emulsion tank by the volume ratio for being 1:1 with antigen water phase, starts motor with the stirring of 90~150rpm slow rotation
After 2min, while it being slowly added to water 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, obtains polypeptide 1, polypeptide 2,3 and of polypeptide for being 1:1:1:1 containing molar ratio
4 polyvalent vaccine of polypeptide.
According to above-mentioned identical method, prepares respectively and four kinds of polypeptide mixing are replaced with polypeptide 1, polypeptide 2, polypeptide 3 or polypeptide 4
Object is the mixture of the vaccine of antigen or polypeptide that any two kinds of antigen molar ratios are 1:1, any three kinds of polypeptide antigen molar ratios
Mixture for 1:1:1 is the vaccine of antigen.Guarantee polypeptide antigen total concentration is 100 μ g/ml.
The safety testing of embodiment 4, pig circular ring virus synthetic peptide vaccine
1. materials and methods
1.1 synthetic peptide vaccines are prepared
With embodiment 3.
1.2 experimental animal
PCV2ELISA negative antibody and PCV2 antigen negative 14,21 age in days piglets, the sow being pregnant 65~95 days.
1.3 test method
1.3.1 single dose inoculation of minimum the age of immune piglet
14 age in days suckling pig 30 is taken, is weighed respectively, is divided into 6 groups, every group 5, the 1st~4 group of difference intramuscular injection
The synthetic peptide vaccine of ZMPCV001, ZMPCV002, ZMPCV003, ZMPCV004, the 5th group of intramuscular injection lot number are ZMPCVH4's
Synthetic peptide vaccine, dosage of inoculation 1mL/ head；6th group is blank control group, isolated rearing 30 days after cutting overbit and weighing, observation
Whether there is or not clinical symptoms for piglet.Measurement body temperature (rectal temperature) daily in 1~7 day after inoculation.At the end of weigh in.
1.3.2 piglet single dose repeated inoculation
21 age in days piglet 30 is taken, is weighed respectively, is divided into 6 groups, every group 5, the 1st~4 group of difference intramuscular injection lot number be
The synthetic peptide vaccine of ZMPCV001, ZMPCV002, ZMPCV003, ZMPCV004, the 5th group of intramuscular injection lot number are ZMPCVH4's
Synthetic peptide vaccine, continuous 2 times, every minor tick 2 weeks, dosage of inoculation 1mL/ head/time, the 6th group is blank control group.It cuts overbit and claims
After weight, isolated rearing 45 days, whether there is or not clinical symptoms for observation piglet.Two exempt from latter 1st~7 day measurement body temperature (rectum temperature daily
Degree).It weighs when off-test.
1.3.3 overdose inoculation of piglet
21 age in days piglet 30 is taken, is weighed respectively, is divided into 6 groups, every group 5, the 1st~4 group of difference intramuscular injection lot number be
The synthetic peptide vaccine of ZMPCV001, ZMPCV002, ZMPCV003, ZMPCV004, the 5th group of intramuscular injection lot number are ZMPCVH4's
Synthetic peptide vaccine, dosage of inoculation 4mL/ head；6th group is blank control group, isolated rearing 30 days after cutting overbit and weighing, observation
Whether there is or not clinical symptoms for piglet.Measurement body temperature (rectal temperature) daily in 1~7 day after inoculation.It weighs when off-test.
1.3.4 the safety (production performance) of farrowing sow
Sow 24 of pregnancy 65~95 days are taken, are divided into 6 groups, every group 4, the 1st~4 group of difference musculi colli injects lot number
For the synthetic peptide vaccine of ZMPCV001, ZMPCV002, ZMPCV003, ZMPCV004, the 5th group of intramuscular injection lot number is ZMPCVH4
Dosage of inoculation 8mL/ head；6th group is separately raised under the same conditions as control, each group sow, and observation to Farrowing is seen
Examine that whether there is or not the appearance of the breeding difficultys such as premature labor, stillborn foetus, the mummification of fetus and weak son.
2. test result
The vaccine safety test result of 2.14 age in days piglet of table, 21 age in days piglets and farrowing sow
The result shows that (table 2), be immunized vaccine that independent sequence prepares (ZMPCV001, ZMPCV002, ZMPCV003,
ZMPCV004) and immune mixed sequence prepare vaccine (ZMPCVH4), 14 age in days piglets, 21 age in days piglets do not have adverse reaction
It generates, body temperature and weight gain are normal.Farrowing sow has no adverse reaction generation, and appetite is normal, and production performance is not affected.Explanation
Synthetic peptide vaccine containing sequence 1 to sequence 5 good security immune to piglet and farrowing sow.
The potency test of embodiment 5, pig circular ring virus synthetic peptide vaccine
1. materials and methods
1.1 synthetic peptide vaccines are prepared
With embodiment 3.
1.2 experimental animal
PCV2 ELISA negative antibody and PCV2 antigen negative 14-21 age in days piglet 100.
1.3 test method
1.3.1 immunity inoculation
With 14~21 age in days PCV ELISA negative antibodies and PCV2 antigen negative health susceptible piglet 100, it is divided into 20
Group, every group 5, vaccine 1ml prepared by the every incidence intramuscular injection embodiment 2 of 1-15 group presses identical approach and dosage after 2 weeks
Carry out the 2nd inoculation.16-18 group is immune by market seedling explanation, the 19th group make it is nonimmune attack malicious control, the 20th group is made blank pair
According to (nonimmune, non-to attack poison), equal isolated rearing observation.Vaccine immunity and grouping situation are shown in Table 3.
3. vaccine immunity of table and grouping situation
It weighs within 5 weeks after head exempts from, 1-19 group is each, and with PCV2 type strain, (SH plants, Agricultural University Of Nanjing is saved, and contains
106.0TCID50/ ml) collunarium 1ml, intramuscular injection 2ml, isolated rearing.It is observed continuously 21 after attacking poison, body temperature is measured, in the 21st
It is slaughtered after day weighing, dissect.
1.3.2 criterion
Determined that three Testing index there are 2 positives to sentence according to body temperature, opposite daily gain, virus antigen detection result
For morbidity.Attack malicious control group should at least 4 hairs disease, immune group should at least 4 head protections.
Body temperature is greater than 40 DEG C and continues to be judged within 3 days or more body temperature raising.
Opposite daily gain=(attack the 21st daily weight after poison-attacks poison and work as daily weight) ÷ 21 ÷ attack and malicious work as daily weight.
Virus antigen detection: ImmunohistochemistryMethods Methods are used.
2. challenge test result
2.1 pathological examination
It visually observes: attacking the visible lungs of malicious control group and peripheral lymph nodes swelling is obvious, section is uniform white；Lung
There are taupe inflammation and swelling, is in diffusivity lesion；Liver obfuscation, in light yellow；Kidney is pale, oedema.The individual pigs of immune group
There are lungs and kidney oedema, but lesion situation is far below and attacks malicious control group.Each internal organ eye of blank control group is seen normal.
Histologic lesion: it takes lungs and lymph node tissue that slice is made, is checked.Attack the visible lungs of malicious control group and leaching
Lymph nodule missing, lymphocyte in tissue is fawned on to significantly reduce, and there are the lesions such as macrophages infiltration；Immune group has part pig
Show that lymphocyte is reduced, macrophages infiltration；Blank group is without lesion.
2.2 virus antigen detection
Using ImmunohistochemistryMethods Methods.It takes lymphoid tissue that slice is made, carries out immunohistochemistry with pig circular ring virus positive serum
Detection.Observe under high magnification microscope, attack in malicious control group lymph follicle occur obvious brown color dye cell, lymph follicle side
Edge is fuzzy, and boundary is unclear；Immune group also has a lymphocyte reduction, but cell brown color dye it is unobvious, illustrate immune antiboidy energy
Damage of the virus to body lymphocyte is reduced, is played a protective role.Attacking poison protection the results are shown in Table 4.
Pathological change and protective rate after 4. Immunization of table
* the identical expression difference of a letter is not significant (P > 0.05)
The results show that single peptide immune group protective rate is 60%~100%, two peptide mixed immunity group protective rates are 80%
~100%, three peptides and four peptide mixed immunity group protective rates are 100%, commercialized vaccine immune group protective rate is 80%~
100%.The above mixed immunity piglet of two peptides, protective rate is 80%~100%, similar to commercialized vaccine result.It is each immune
Group attacks after poison 14 days opposite daily gains and blank control group without significant difference, it is non-exempt to attack malicious control group be substantially less than with respect to daily gain
Blank control group.
Claims (13)
1. a kind of antigen polypeptide for being used to prepare pig circular ring virus synthetic peptide vaccine is polypeptide shown in sequence 1.
2. the antigen polypeptide according to claim 1 for being used to prepare pig circular ring virus synthetic peptide vaccine, wherein described in sequence 1
Polypeptide fragment, form peptide key connection from the carboxyls of the 19th amino acids of aminoterminal and the ε bit amino of the 20th amino acids.
3. a kind of peptide composition for being used to prepare pig circular ring virus synthetic peptide vaccine, including polypeptide shown in sequence 1 and sequence
One or more of polypeptide shown in column 2, polypeptide shown in polypeptide and sequence 4 shown in sequence 3 any combination.
4. the peptide composition according to claim 3 for being used to prepare pig circular ring virus synthetic peptide vaccine, wherein sequence 1,
Polypeptide fragment described in sequence 2, sequence 3 or sequence 4, from the carboxyl of the 19th amino acids of aminoterminal and the ε of the 20th amino acids
Bit amino forms peptide key connection.
5. peptide composition according to claim 3, it is characterised in that: when the peptide composition includes shown in sequence 1
Polypeptide and sequence 2 shown in one of polypeptide, polypeptide shown in polypeptide and sequence 4 shown in sequence 3 when, two kinds of polypeptides
Molar ratio be (0.5-1.5): (0.5-1.5).
6. peptide composition according to claim 5, it is characterised in that: when the peptide composition includes shown in sequence 1
Polypeptide and sequence 2 shown in one of polypeptide, polypeptide shown in polypeptide and sequence 4 shown in sequence 3 when, two kinds of polypeptides
Molar ratio be 1:1.
7. peptide composition according to claim 3, it is characterised in that: when the peptide composition includes shown in sequence 1
Polypeptide and sequence 2 shown in polypeptide, two kinds in polypeptide shown in polypeptide and sequence 4 shown in sequence 3, three kinds of polypeptides
Molar ratio is (0.5-1.5): (0.5-1.5): (0.5-1.5).
8. peptide composition according to claim 7, it is characterised in that: when the peptide composition includes shown in sequence 1
Polypeptide and sequence 2 shown in polypeptide, two kinds in polypeptide shown in polypeptide and sequence 4 shown in sequence 3 when, any three kinds
The molar ratio of polypeptide is 1:1:1.
9. peptide composition according to claim 3, it is characterised in that: the peptide composition includes shown in sequence 1
Polypeptide shown in polypeptide, sequence 2, polypeptide shown in polypeptide and sequence 4 shown in sequence 3, their molar ratio are (0.5-
1.5): (0.5-1.5): (0.5-1.5): (0.5-1.5).
10. peptide composition according to claim 9, it is characterised in that: polypeptide shown in sequence 1, more shown in sequence 2
The molar ratio of polypeptide shown in polypeptide shown in peptide, sequence 3 and sequence 4 is 1:1:1:1.
11. peptide composition described in any one of antigen polypeptide of any of claims 1 or 2 or claim 3-10 is being made
Application in the biological products of standby pig circular ring virus synthetic peptide vaccine or preparation for preventing pig circular ring virus.
12. a kind of pig circular ring virus synthetic peptide vaccine, includes peptide composition described in any one of claim 3 to 10.
13. the preparation method of pig circular ring virus synthetic peptide vaccine, which is characterized in that the preparation method comprises the following steps:
(1) peptide composition described in any one of claim 3-7 is diluted to the concentration of 100 μ g/ml with water for injection, from
And obtain polypeptide antigen water phase；
(2) adjuvant is sterilized；
(3) under the conditions of 20~28 DEG C, according to the volume ratio of the polypeptide antigen water phase and the adjuvant 1:1, first adjuvant is added
Enter in emulsion tank, stirred 1.5~3 minutes at 90~150rpm, be then slowly added into polypeptide antigen water phase, then stir 20~
30 minutes, then stirred 15~30 minutes at 8000~10000rpm, 3-10 minutes are stood, packing.
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| CN101709080A (en) * | 2009-11-09 | 2010-05-19 | 中牧实业股份有限公司 | Synthetic peptide vaccine for resisting porcine circovirus and preparation method thereof |
| CN101920012A (en) * | 2010-07-22 | 2010-12-22 | 洛阳普莱柯生物工程有限公司 | Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof |
| CN102127533A (en) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | Preparation method of recombinant porcine circovirus type 2 Cap antigen |
| CN103108653A (en) * | 2010-07-08 | 2013-05-15 | 美国联合生物医学公司 | Designer peptide-based pcv2 vaccine |
| CN104098657A (en) * | 2014-07-07 | 2014-10-15 | 南京农业大学 | Porcine circovirus 2 immune protection polypeptide and vaccine comprising same |
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| CN101709080A (en) * | 2009-11-09 | 2010-05-19 | 中牧实业股份有限公司 | Synthetic peptide vaccine for resisting porcine circovirus and preparation method thereof |
| CN103108653A (en) * | 2010-07-08 | 2013-05-15 | 美国联合生物医学公司 | Designer peptide-based pcv2 vaccine |
| CN101920012A (en) * | 2010-07-22 | 2010-12-22 | 洛阳普莱柯生物工程有限公司 | Method for producing porcine circovirus type II recombinant capsid protein subunit vaccine by utilizing silkworm bioreactor and products thereof |
| CN102127533A (en) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | Preparation method of recombinant porcine circovirus type 2 Cap antigen |
| CN104098657A (en) * | 2014-07-07 | 2014-10-15 | 南京农业大学 | Porcine circovirus 2 immune protection polypeptide and vaccine comprising same |
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