CN107827958A - Canine parvovirus synthetic peptide vaccine and its preparation method and application - Google Patents
Canine parvovirus synthetic peptide vaccine and its preparation method and application Download PDFInfo
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- CN107827958A CN107827958A CN201711109724.XA CN201711109724A CN107827958A CN 107827958 A CN107827958 A CN 107827958A CN 201711109724 A CN201711109724 A CN 201711109724A CN 107827958 A CN107827958 A CN 107827958A
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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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- 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
- C12N2750/14011—Parvoviridae
- C12N2750/14311—Parvovirus, e.g. minute virus of mice
- C12N2750/14322—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
- C12N2750/14011—Parvoviridae
- C12N2750/14311—Parvovirus, e.g. minute virus of mice
- C12N2750/14334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
The invention discloses a kind of antigen polypeptide, peptide composition and vaccine for being used to prepare the tiny synthetic peptide vaccine of dog.Antigen polypeptide is the polypeptide shown in sequence 1 or sequence 2.The vaccine of the present invention includes polypeptide or peptide composition.Vaccine can successfully manage the antigenic variation of canine parvovirus made of the polypeptide, also in the absence of safety issue, be easy to synthesize on a large scale, have a good application prospect.
Description
Technical field
The present invention relates to a kind of polypeptide or its polypeptide polymer for canine parvovirus synthetic peptide vaccine, and contain this
The vaccine and their preparation method of polypeptide or its polypeptide polymer.
Background technology
Canine parvovirus disease is high degree in contact caused by being infected by canine parvovirus (Canine Parvovirus, CPV)
Canine infectious disease, clinical manifestation are hemorrhagic enteritis and acute myocarditis, have greatly threat to the health of canine.In source of parents
Under the protection of antibody, the typically seldom infection morbidity of newborn pup, but the young dog being born 2 months is relatively easy to pathogenic infection.And with
Bitch Immunization coverage rate to increase, in recent years more rare, the currently a popular canine parvovirus of the myocarditis symptom of new cub dog
The clinical symptoms of infection show as hemorrhagic enteritis more.Canine parvovirus separated from the sick dog excrement for suffering from enteritis first in 1978
Obtain, be named as CPV-2；Its viral nucleic acid is single-stranded DNA, full length viral genome 5200nt, 3 structural proteins of main code
(VP1, VP2 and VP3) and 2 non-structural proteins (NS1 and NS2).VP1 albumen is 82KDa, and VP2 albumen is 65KDa, due to
The gene of VP2 albumen is contained among the gene of VP1 albumen, so VP2 albumen is also the product of VP1 albumen excision aminoterminal,
Meanwhile the two together constitutes the shell (wherein VP2 albumen accounts for 90%) of CPV maturation particles again, research shows, for shell egg
White antibody can effectively suppress CPV infection effect.
CPV-2 VP2 albumen determines the antigenicity of the cause of disease, preferendum and other biological natures inside and outside host.
The cause of disease separated first from 1978, had occurred many variants so far, and its variant sites is located at VP2 protein domains more.1985
Year, Parrish carries out antigens genotyping to the 49 plants of CPV-2 viruses for being isolated from country variant, wherein 26 plants (be 1980 with
Separate afterwards) made a variation, it is named as " CPV-2a ".Variant compared to CPV-2, its VP2 albumen there occurs Met87Leu,
Ile101Tyr, Ala300Gly, Asp305Tyr and Ile555Val are mutated (in the 555th separation strains after nineteen ninety
There occurs the mutation of Val555Ile recoveries).Due to the variation in other sites on VP2 albumen, also identification occurs successively afterwards
New variant CPV-2b, CPV-2c, NewCPV-2a and NewCPV-2b.Wherein NewCPV-2a and NewCPV-2b are compared
In CPV-2a and CPV-2b, 297 of its VP2 albumen have been mutated into Ala by Ser.The site is closed on space three-dimensional structure
The surface of capsid protein is fine prominent, and after mutation and the antigenicity of virus is not significantly changed, and is more that possible influence virus and place
The affinity of main animal recipient.
There are the VP2 genes that scholar analyzes 22 plants of canine parvovirus strains of domestic separation during 1983-2008, knot in the country
Fruit shows that isolated strain can be divided into CPV-2, CPV-2a (VP2 the 297th sports Ala, i.e. NewCPV-2a) and CPV-2b tri-
Kind genotype.There are many scholars to do the VP2 gene sequencing analysis work that a large amount of canine parvovirus are clinically separated strain afterwards.
It can be drawn from their analysis result, domestic canine parvovirus advantage cloth strain is NewCPV-2a types at present.
Because VP2 albumen constitutes the overwhelming majority (90%) of capsid protein, many relevant CPV epitopes identifications
Work also focuses mostly in the protein domain.LANGEVELD etc. is had found possible anti-at least there is 10 on VP2 by research
It is in situ, wherein at least there is the surface that 6 sites are exposed to capsid, and have three N-terminals for being located at VP2 among this 6 sites,
The other three is located at (Loop1 and Loop3) on two Loop near triad.
Nineteen ninety, G.F.Rimmelzwaan et al. has done correlative study work to the t cell epitope of canine parvovirus.He
Find that three regions in VP1 sequences be present can activate two kinds of CPV specific dog T cells clone respectively, be that effective T is thin
Born of the same parents' epitope.2001, SOURAVI GHOSH etc. were identified that they recognize to the helper T lymphocyte epitope on CDV
It is suitable candidate's helper T lymphocyte epitope for polypeptide P25-P37.
The vaccine that the country is used to prevent canine parvovirus has two major classes：Homologous or heterologous inactivated vaccine and Attenuate vaccine.
Due to canine parvovirus and Feline Panleukopenia Virus (FPV) nucleic acid and amino acid homology more than 98%, should
The immunoprophylaxis of canine parvovirus disease can be also applied to well by preparing heterologous inactivation or Attenuate vaccine with FPV.It is domestic at present general
What it is all over application is attenuated live vaccines, and majority introduces strain to be external, certain bio-safety be present, it is clear that be unfavorable for disease
Purification remove.Synthetic peptide vaccine is the vaccine prepared using antigen epitope polypeptide, compared to traditional inactivated vaccine and weak poison
Seedling, synthetic peptide vaccine is safer, because it, which is avoided, dissipates the risk that poison is propagated in traditional vaccine production process, while also effectively keeps away
Having exempted from Attenuate vaccine, virulence returns security risk that is strong, making a variation in use.In addition, the epitope that synthetic peptide vaccine is applied is not by
With amino acid residue permutation and combination, then chemical synthesis linear polypeptide, its cost is low, stably, is readily produced, and quality control
It is easy.Synthetic peptide vaccine preparation technology is more ripe, and suitable B cell epitope and t cell epitope are combined into coupling, coordinates
Adjuvant co-immunization animal can produce ideal protecting effect.Develop effective synthetic peptide vaccine it is the most key be sieve
The antigen polypeptide epitope of immundominance is selected, this is those skilled in the art's urgent problem to be solved.
The content of the invention
The present invention studies domestic canine parvovirus prevalence strain major antigen position by the sequencing to the tiny strain of dog
The variation situation of point, the analysis that the tiny antigen site of dog is carried out in combination with computer-aid method is predicted, develops a kind of effect
The synthetic peptide vaccine that power is good, safe, stable processing technique and cost are low.
Therefore, it is an object of the invention to provide a kind of polypeptide for the tiny synthetic peptide vaccine of dog or its polypeptide to polymerize
Thing, and the vaccine containing the polypeptide.
It is a further object to provide a kind of aforementioned polypeptides and the preparation method of above-mentioned vaccine.
To realize objectives stated above, this invention takes following technical scheme：
The present invention provides a kind of antigen polypeptide for being used to prepare canine parvovirus synthetic peptide vaccine, for sequence 1 or the institute of sequence 2
Show polypeptide, or, the polypeptide polymer of polypeptide shown in the polypeptide polymer of polypeptide shown in sequence 1 or sequence 2, or, by sequence 1
The polypeptide polymer of the composition of polypeptide shown in the polypeptide and sequence 2.
The mixtures of polypeptides for being used to prepare canine parvovirus synthetic peptide vaccine that this discovery provides, is as shown in sequence 1
The polypeptide polymer of polypeptide shown in polypeptide, sequence 1 shown in polypeptide, sequence 2, polypeptide shown in sequence 2 polypeptide polymer and by
One or both of polypeptide polymer of the composition of polypeptide shown in polypeptide described in sequence 1 and sequence 2 any of the above combination composition.
Heretofore described polypeptide and polypeptide polymer can be the peptide materials obtained by Solid-phase organic synthesis.
Aforementioned polypeptides particular sequence is as follows：
Polypeptide 1 (polypeptide in sequence table shown in sequence 1)：
KKAGSVIX1X2CSRAEVGDYERLVNSDLVεKEQDIFNLX3VX4 TASDSVTQPPTRVYNNE
(X1=E/D, X2=N/I, X3=V/A, X4=K/R)
Polypeptide 2 (polypeptide in sequence table shown in sequence 2)：
IKSITTAVQITGX1IAVHQTX2LNVFATIεKQPALX3NEX4LTAS GQGSGAGGTG
(X1=G/A, X2=N/I, X3=R/K, X4=R/K)
Polypeptide fragment wherein described in sequence 1, from the carboxyl and the ε of the 28th amino acids by the amino acids of aminoterminal the 27th
Bit amino forms peptide key connection；Polypeptide fragment shown in sequence 2, from the carboxyl and the 28th bit amino of the amino acids of aminoterminal the 27th
The ε bit aminos (ε K) of sour (lysine) form peptide key connection.(sequence is from left to right aminoterminal to c-terminus)
Wherein, ε K are that the ε bit aminos of lysine form peptide bond, and concrete principle is as follows：
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 ".In 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.
Wherein, as described above, including the polypeptide shown in sequence 1, there is pleomorphism site (i.e. in the polypeptide shown in sequence 2
Two kinds of optional amino acid be present 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.
When choosing the composition vaccine combination of the polypeptide described in above two sequence, their mol ratio is (0.5-1.5):
(0.5-1.5)；Most preferably, their mol ratio is 1:1.
Application of the described peptide composition in canine parvovirus synthetic peptide vaccine is prepared falls within the protection of the present invention
Scope.
A kind of canine parvovirus synthetic peptide vaccine provided by the invention, includes above-mentioned peptide composition.
The canine parvovirus synthetic peptide vaccine also includes adjuvant.The invention provides described canine parvovirus synthetic peptide
The preparation method of vaccine, the preparation method comprise the following steps：
(1) peptide composition is diluted to 10-100 μ g/ml (preferably 100 μ g/ml) concentration with water for injection,
So as to obtain polypeptide antigen aqueous phase；
(2) adjuvant is sterilized (sterilize 30min 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, and 1.5~3min is stirred under 90~150rpm, polypeptide antigen aqueous phase is then slowly added into, then stirs
20~30min, then 15-30min is stirred under 8000~10000rpm, stand 3-10min (preferably 5min), packing.
Preferably, the adjuvant is selected from white oil, 50V, 50VII (one in MONTANIDE ISA 50V, 50VII adjuvants
Kind is a variety of.
Present invention also offers the preparation method of the polypeptide in aforementioned polypeptides or polypeptide polymer, the preparation method includes
Following steps：
(1) using amino resins as initiation material, using by the amino acid that 9-fluorenylmethyloxycarbonyl is protected as monomer, according to the ammonia
Base acid sequence is condensed successively connects amino acid to synthesize the polypeptide, unreacted with acetyl imidazole closing after often step condensation reaction
Aminoterminal；
(2) lytic reagent is added after synthesizing, so as to which 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, 25~40min is reacted 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 dries up, and then washs amino resins with 1-METHYLPYRROLIDONE；
(1-c) condensation reaction：Add 1- hydroxyls azimidobenzene (HOBT), N, N- DICs (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) is washed：Nitrogen dries up, and then washs amino resins with 1-METHYLPYRROLIDONE；
(1-e) capping：Add the N- crassitudes for the acetyl imidazole that percent weight in volume is 1.5%~4%
Ketone (NMP) solution, 20~40min is reacted 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：H2O；Also, the pyrolysis time of the step (2) is 1-4h.
In above-mentioned preparation method, the step (3) specifically includes：
(3-a) precipitates the polypeptide using ether, is then washed with dimethylformamide；
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) uses 0.2 μm of degerming preservation of filter.
Further aspect, the invention provides aforementioned polypeptides or polypeptide polymer or peptide composition to be used for preventing canine in preparation
Purposes in the biological products of parvovirus synthetic peptide vaccine.
Specifically, the present invention by the sequencing to domestic canine parvovirus prevalence strain and combines canine parvovirus
Vaccine strain sequence, the variation situation of the major antigenic sites of canine parvovirus poison strain is studied, to main variant amino acid position
Line frequency statistics is clicked through, the analysis that Canine Parvovirus antigen site is carried out in combination with area of computer aided is predicted, to possible anti-
Original position point polypeptide fragment carries out chemical synthesis, the i.e. variation frequency for easy variant sites, and different ammonia is used in these sites
Base acid.And then these candidate polypeptide antigens are screened by substantial amounts of animal experiment, obtain that the immune of animal can be caused
Reaction, and immune response is horizontal high.In addition, the present inventor is carried out according to screening experiment result to Canine Parvovirus antigen site
Optimization, and efficient combination B, t cell epitope, enhance the immune effect of polypeptide antigen.
Polypeptide vaccine potency test and safety experiment result show, canine parvovirus synthetic peptide vaccine provided by the invention
With good immune efficacy, and will not trigger heating, it is red and swollen the problems such as, therefore the present invention vaccine can successfully manage mesh
The antigenic variation of preceding canine parvovirus, in the absence of biological safety risk, be easy to synthesize on a large scale, before there is good application
Scape.
Brief description of the drawings
Fig. 1 is 1-8 antigen polypeptide the selection results in embodiment 2；
Fig. 2 is 9-16 antigen polypeptide the selection results in embodiment 2；
Fig. 3 is the HI antibody titer results after 3 groups of polypeptide immunes.
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, its scope not limiting the invention in any way.
Experimental method in following embodiments, it is conventional method unless otherwise specified.Medicine used in following embodiments
Material raw material, reagent material etc., unless otherwise specified, it is commercially available products.
The synthesis in solid state of embodiment 1, canine parvovirus synthetic peptide vaccine polypeptide
The present embodiment is the synthesis in solid state of the polypeptide antigen of synthetic peptide vaccine provided by the present invention.The synthetic peptide of the present invention
Antigen can use full-automatic polypeptide synthetic instrument, be prepared using Merrifield solid-phase synthesis, wherein employing by 9- fluorenes first
The amino acid of oxygen carbonyl (Fmoc) modification, solid phase carrier are the Rink Amide mbha resins of Tianjin Nankai.Production process is usual
Cracking, antigen purification and the degerming preservation of synthesis in solid state, polypeptide including polypeptide antigen.
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)：
KKAGSVIX1X2CSRAEVGDYERLVNSDLVεKEQDIFNLX3VX4 TASDSVTQPPTRVYNNE
In above-mentioned sequence, X1=E/D, X2=N/I, X3=V/A, X4=K/R, two different amino shown in these sites
Acid is loaded simultaneously, random synthesis.
Polypeptide 2 (polypeptide in sequence table shown in sequence 2)：
IKSITTAVQITGX1IAVHQTX2LNVFATIεKQPALX3NEX4LTAS GQGSGAGGTG
In above-mentioned sequence, X1=G/A, X2=N/I, X3=R/K, X4=R/K, two different amino shown in these sites
Acid is loaded simultaneously, random synthesis.
Polypeptide fragment wherein described in polypeptide 1, from the carboxyl and the ε of the 28th amino acids by the amino acids of aminoterminal the 27th
Bit amino forms peptide key connection；Polypeptide fragment shown in sequence 2, from the carboxyl and the 28th bit amino of the amino acids of aminoterminal the 27th
The ε bit aminos (ε K) of sour (lysine) form peptide key connection.(in following building-up processes, reaction solution be alkalescence, purchase into
Its ε bit amino of product lysine is protected by alkali sensitive group, and its α bit amino is protected by acid sensitive group.In course of reaction, only
The blocking group of ε bit aminos can be removed, its amino of exposure, be reacted with the carboxyl of next amino acid.)
According to the sequence of antigen and 1mmol synthesis scale, the amino acid for preparing suitable Fmoc modifications [is purchased from Shanghai
Gill is biochemical], add in corresponding amino-acid reagent bottle.Rink Amide mbha resins are equally weighed on request, are put into reaction
In chamber, upper and lower lid is tightened, labelled, the title of peptide synthesized by record, lot number, the weight of the tare weight of reaction chamber and alleged resin
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 Peptide synthesizer whether normal operation.Check whether N2 is sufficient, and whether system gauge pressure is normal in addition.Before synthesis
The performance of reply instrument is had gained some understanding, 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.Measure or observe, if flow
It is improper, then lower valve pressure or each solution parameter of modification are 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 preserved.Opened again in operating file, set amino acid
Bottle prepares quantity, brings into operation.
1.1.4 the synthesis of antigenic synthetic peptide
Peptide sequence described above, it is to N-terminal, according to given order, successively constantly when synthesis since C-terminal
It is repeated below 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 groups on 25~40min removing amino resins are reacted under the conditions of 20~28 DEG C；
(2) wash：Nitrogen dries up, NMP washing amino resins；
(3) condensation reaction：The amino acid for adding HOBT, DIC and Fmoc protection reacts 0.5- under the conditions of 20~28 DEG C
2.5h；
(4) wash：Nitrogen dries up, NMP washing amino resins；
(5) capping：The nmp solution for the acetyl imidazole that percent weight in volume is 1.5%~4% is added, 20~
20~40min is reacted 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, be put into -20 DEG C of refrigerators
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
Prepared according to volume ratio (trifluoroacetic acid (TFA)/tri isopropyl silane (TIS)/phenol/H2O=85/8/6/1)
Lysate, the polypeptide resin of synthesis is then taken out out of refrigerator, is put into round-bottomed flask, added and match somebody with somebody into flask in fume hood
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 terminates, the TFA in 30~120min removing crude products is persistently evaporated using the Rotary Evaporators with cold-trap.Connect
The crude product that polypeptide antigen using ether collection, precipitated polypeptide, is then cleaned multiple times with dimethylformamide (DMF), finally will
The resin mixed is filtered out with sand core funnel, that is, obtains polypeptide antigen.
1.2.2 the identification of antigenic synthetic peptide
Polypeptide antigen synthesize after with substance assistant laser desorpted time-of-flight mass spectrometry (TOFMS) (MALDL-TOF) and anti-phase
High pressure lipuid chromatography (HPLC) (RP-HPLC) carries out qualitative and quantitative analysis.
The purifying of 1.3 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 the small molecular weight impurity that building-up process early stage and later stage cyclization are formed or introduced 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, sticks mark
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.
For the ease of the needs for transporting and preserving for a long time, 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 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.
The screening of embodiment 2, canine parvovirus synthetic peptide vaccine antigen polypeptide
The synthesis of 1.1 candidate antigen polypeptides
Method is the same as embodiment 1.
The screening of 1.2 polypeptide antigens
32 candidate polypeptides are coated with ELISA reaction plates with 1 μ g/ml concentration respectively, add 100 μ l per hole, 4~8 DEG C
Left overnight, polypeptide antigen is set fully to be combined with enzyme-linked reaction plate.Then 37 DEG C of closings of confining liquid are added according to 300 μ l/ holes
Handle 3h.Dried after being washed with lavation buffer solution.The canine parvovirus antibody positive blood of 50 times of dilutions is added according to 100 μ l/ holes
(offer of Zhongmu Industry Co., Ltd Jiangxi biology pharmaceutical factory) clearly, 45min is reacted in 37 DEG C of incubators.After washing, 100 μ l/ holes
The rabbit-anti dog IgG antibody of the horseradish peroxidase-labeled of 25000 times of dilutions is added, reacts 45min in 37 DEG C of incubators.After washing
Add substrate colour developing.Read OD450Value.Negative control (BSA, 2.5 μ g/ml) coating reaction plate is set simultaneously, and (dog is thin for positive control
Small virus inactivation antigen, Zhongmu Industry Co., Ltd Jiangxi biology pharmaceutical factory provide) coating reaction plate.
The selection result is shown in Fig. 1, Fig. 2.Compared with positive control antigen, the antigen polypeptide of 8 excellent effects is filtered out.This
The sequence of 8 polypeptides is shown in Table 1.Wherein, shown in table 1, the sequence in sequence table shown in sequence 1, four amino acid positions therein
Point (8,9,36,38) has two kinds of optional amino acid, and sequence 1 (is represented) from the amino acids of aminoterminal the 8th in table 1 with X1aa
=Glu or Asp, sequence 1 (represent)=Asn or Ile from the amino acids of aminoterminal the 9th with X2aa in table 1, sequence 1 from ammonia
The amino acids of cardinal extremity the 36th (represent)=Val or Ala with X3aa in table 1, sequence 1 from the amino acids (table 1 of aminoterminal the 38th
It is middle to be represented with X4aa)=Lys or Arg any combination, its OD of the polypeptide antigen of synthesis450Value is 1.0 or so.Sequence in sequence table
Sequence shown in row 2, amino acid sites (13,20,33,36) therein have two kinds of optional amino acid, sequence 2 from amino
The 13rd amino acids are held (to represent)=Gly or Ala in table 1 with X1aa, sequence 2 (is used from the amino acids of aminoterminal the 20th in table 1
X2aa is represented)=Asn or Ile, sequence 2 (represent)=Arg or Lys from the amino acids of aminoterminal the 33rd with X3aa in table 1,
Sequence 2 (represents)=Arg or Lys, its OD of the polypeptide antigen of synthesis from the amino acids of aminoterminal the 36th with X4aa in table 1450
Value is 1.0 or so.
The canine parvovirus synthetic peptide vaccine antigen polypeptide of table 1.
The preparation of embodiment 3, canine parvovirus synthetic peptide vaccine
The preparation of 1.1 antigen aqueous phases
The polypeptide 1 prepared according to embodiment 1, the antigenic synthetic peptide shown in polypeptide 2 are weighed respectively, according to antigen ratio mole
For 1:1 mixing, is then diluted to 100 μ g/ml with sterilized water for injection by antigenic synthetic peptide total concentration.Gained antigenic solution is passed through
The filter that aperture is 0.2 μm filters, degerming.
It is prepared by 1.2 oil phase adjuvants
It is standby by oil phase adjuvant 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 oil phase is added in emulsion tank, start motor and stirred with 90~150rpm slow rotations
After 2min, while aqueous phase antigen is slowly added to, 30min is stirred after adding, then with 10000rpm high-speed stirred 20min, stand
5min, vaccine is set to be emulsified into the single-phase vaccine of Water-In-Oil, it is 1 to obtain containing mol ratio:1 polypeptide 1, the polyvalent vaccine of polypeptide 2.
By above-mentioned identical method, the vaccine for antigen with polypeptide 1, polypeptide 2 is prepared respectively, ensures that polypeptide antigen concentration is
100μg/ml。
The safety testing of embodiment 4, canine parvovirus synthetic peptide vaccine
1. materials and methods
1.1 synthetic peptide vaccines are prepared
With embodiment 3.Mol ratio will be contained for 1:1 polypeptide 1, the polyvalent vaccine of polypeptide 2 are named as ZMCPV2M2；Polypeptide resists
Original content is 100 μ g/ml.
By above-mentioned identical method, prepare with polypeptide 1, polypeptide 2 as the vaccine of antigen, be respectively designated as respectively
ZMCPV2001、ZMCPV2002；Polypeptide antigen concentration is respectively 100 μ g/ml.
1.2 experimental animal
CPV2 blood clottings suppress (Hemagglutination Inhibition, HI) negative antibody and CPV2 antigen negatives 45
Age in days pup.
1.3 test method
1.3.1 pup single dose repeated inoculation
45 age in days pup 20 is chosen, is weighed respectively, it is subcutaneous to be randomly divided into 4 groups, every group 5, the 1st group and the 2nd group difference
Inject the synthetic peptide vaccine that lot number is ZMCPV2001, ZMCPV2002, the synthetic peptide that the 3rd group of hypodermic injection lot number is ZMCPV2M2
Vaccine, continuous 2 times, inoculation every time is spaced 2 weeks, and dosage of inoculation 1mL//times, the 4th group is blank control group.Mark overbit simultaneously claims
After body weight, isolated rearing 45 days, observation pup whether there is clinical symptoms.Two exempt from latter 1st~7 day measurement body temperature (rectum temperature daily
Degree).Weighed during off-test.
1.3.2 an overdose inoculation
45 age in days piglet 20 is taken, is weighed respectively, is randomly divided into 4 groups, every group 5, the 1st group and the 2nd group subcutaneous notes respectively
Penetrate the synthetic peptide vaccine that lot number is ZMCPV2001, ZMCPV2002, the synthetic peptide epidemic disease that the 3rd group of hypodermic injection lot number is ZMCPV2M2
Seedling, dosage of inoculation 4mL/ is only；4th group is blank control group, and isolated rearing 30 days after marking overbit and weighing, observation pup has
Without clinical symptoms.Body temperature (rectal temperature) is measured daily within 1~7 day after inoculation.Weighed during off-test.
2. result of the test
The vaccine safety result of the test of the age in days pup of table 1.45
As a result show (table 1), vaccine (ZMCPV2001, ZMCPV2002) and immune mixing that independent sequence is prepared is immunized
The vaccine (ZMCPV2M2) that sequence is prepared, 45 age in days pups do not have adverse reaction generation, and body temperature and weightening are normal.Illustrate containing orderly
The security that pup is immunized for row 1 and the synthetic peptide vaccine of sequence 2 is good.
The pup immunity test of embodiment 5, canine parvovirus synthetic peptide vaccine
1. materials and methods
1.1 synthetic peptide vaccines are prepared
With embodiment 3.
1.2 experimental animal
Canine parvovirus HI negative antibodies and Canine Parvovirus antigen feminine gender 2-3 monthly ages pup 20.
1.3 test method
1.3.1 immunity inoculation
Suppress (Hemagglutination Inhibition, HI) negative antibody with 2 monthly age CPV-2 blood clottings and (be less than 1:
4) and CPV-2 antigen negative health susceptible canine 20, it is divided into 4 groups, every group 5, the 1st group every is subcutaneously injected lot number and is
ZMCPV2001 synthetic peptide vaccines 1ml, the 2nd inoculation is carried out by identical approach and dosage after 2 weeks.2nd group is immunized
ZMCPV2002 synthetic peptide vaccines 1ml, the 2nd inoculation is carried out by identical approach and dosage after 2 weeks.3rd group of immune ZMCPV2M2 is closed
Into peptide vaccine 1ml, the 2nd inoculation is carried out by identical approach and dosage after 2 weeks.4th group is made blank control (nonimmune), is isolated
Breeding observing.Head takes a blood sample for the 0th day, 7 days, 14 days, 21 days, 28 days, 35 days, 42 days respectively after exempting from, and separates serum, carries out HI antibody
Detection.Vaccine immunity and packet situation are shown in Table 3.
The vaccine immunity of table 2. and packet situation
1.3.2 blood clotting suppresses the detection of (Hemagglutination Inhibition, HI) antibody
(given birth in Zhongmu Industry Co., Ltd Jiangxi with 1% swine erythrocyte suspension (self-control) measure canine parvovirus venom
Thing pharmaceutical factory provide) agglutination titer (according to national standard GB/T14926.53-2001 carry out), it is appropriate with PBS according to agglutination titer
4 unit virus liquids are prepared in dilution；Gather after each immune group and blank control group animal immune the 0th day, 7 days, 14 days, 21 days,
28 days, 35 days, the serum of 42 days, after 56 DEG C inactivate 30min, handled with the kaolinite soil suspension and swine erythrocyte mud of 25% acidifying
Serum removes the plain and nonspecific aggegation inhibiting factor of nonspecific agglutination, is carried out with reference to national standard GB/T14926.54-2001
HI is tested, and detects the HI antibody titers of serum to be checked.
As a result such as Fig. 3, from result, it is immunized the 7th day afterwards, each immune group detects the tiny HI antibody of dog, the 1st group
Vaccine immunity group HI potency average out to 1:27.6, the 2nd group of vaccine immunity group HI potency average out to 1:27.8, the 3rd group of vaccine immunity group
HI potency average out to 1:28.2.Over time, each group HI potency slightly declines.14th day after immune, each group HI potency
Average out to 1:27.2-1:27.4Between.HI antibody increases rapidly after booster immunization, after head exempts from the 21st day (i.e. 7 days after booster immunization),
Its HI potency average out to 1：29.8-1:211.0.Elapse over time, the HI potency after each group booster immunization is also on a declining curve.The
The HI potency average out to 1 of 28 days (after booster immunization 14 days):29.8-1:210.6, the HI potency of the 35th day is 1:29.6-1:210.2；
42nd day is 1:29.2-1:29.8.Control group does not detect the tiny HI antibody of dog.
Sequence table
<110>Zhongmu Industry Co., Ltd
<120>Canine parvovirus synthetic peptide vaccine and its preparation method and application
<130> WHOI170089
<170> Patent-In 3.5
<160> 2
<210> 1
<211> 55
<212> PRT
<213>Artificial sequence（Artificial sequence）
<220>
<221> UNSURE
<222> (8) ..(8)
<223>Xaa=Glu or Asp
<220>
<221> UNSURE
<222> (9) ..(9)
<223>Xaa=Asn or Ile
<220>
<221> UNSURE
<222> (36) ..(36)
<223>Xaa=Val or Ala
<220>
<221> UNSURE
<222> (38) ..(38)
<223>Xaa=Lys or Arg
<400> 1
Lys Lys Ala Gly Ser Val Ile Xaa Xaa Cys Ser Arg Ala Glu Val Gly
1 5 10 15
Asp Tyr Glu Arg Leu Val Asn Ser Asp Leu Val Lys Glu Gln Asp Ile
20 25 30
Phe Asn Leu Xaa Val Xaa Thr Ala Ser Asp Ser Val Thr Gln Pro Pro
35 40 45
Thr Arg Val Tyr Asn Asn Glu
50 55
<210> 2
<211> 50
<212> PRT
<213>Artificial sequence（Artificial sequence）
<220>
<221> UNSURE
<222> (13) ..(13)
<223>Xaa=Gly or Ala
<220>
<221> UNSURE
<222> (20) ..(20)
<223>Xaa=Asn or Ile
<220>
<221> UNSURE
<222> (33) ..(33)
<223>Xaa=Arg or Lys
<220>
<221> UNSURE
<222> (36) ..(36)
<223>Xaa=Arg or Lys
<400> 2
Ile Lys Ser Ile Thr Thr Ala Val Gln Ile Thr Gly Xaa Ile Ala Val
1 5 10 15
His Gln Thr Xaa Leu Asn Val Phe Ala Thr Ile Lys Gln Pro Ala Leu
20 25 30
Xaa Asn Glu Xaa Leu Thr Ala Ser Gly Gln Gly Ser Gly Ala Gly Gly
35 40 45
Thr Gly
50
Claims (8)
1. a kind of antigen polypeptide for being used to prepare canine parvovirus synthetic peptide vaccine, it is shown in polypeptide or the sequence 2 shown in sequence 1
Polypeptide, or, the polypeptide polymer of polypeptide shown in the polypeptide polymer of polypeptide shown in sequence 1 or sequence 2, or, by sequence 1
The polypeptide polymer of the composition of polypeptide shown in the polypeptide and sequence 2.
2. the antigen polypeptide according to claim 1 for being used to prepare canine parvovirus synthetic peptide vaccine, wherein described in sequence 1
Polypeptide fragment, form peptide key connection from the carboxyls of the amino acids of aminoterminal the 27th and the ε bit aminos of the 28th amino acids；Sequence
Polypeptide fragment shown in 2, the ε bit aminos of carboxyl and the 28th amino acids from the amino acids of aminoterminal the 27th form peptide bond and connected
Connect.
A kind of 3. peptide composition for being used to prepare canine parvovirus synthetic peptide vaccine, as the polypeptide shown in sequence 1, the institute of sequence 2
The polypeptide polymer of polypeptide shown in the polypeptide that shows, sequence 1, the polypeptide polymer of polypeptide shown in sequence 2 and the polypeptide as described in sequence 1
Formed with any of the above combination of one or both of the polypeptide polymer of the composition of polypeptide shown in sequence 2.
4. the peptide composition according to claim 3 for being used to prepare canine parvovirus synthetic peptide vaccine, the wherein institute of sequence 1
The polypeptide fragment stated, peptide key connection is formed from the carboxyl by the amino acids of aminoterminal the 27th and the ε bit aminos of the 28th amino acids；
Polypeptide fragment shown in sequence 2, the ε bit aminos of carboxyl and the 28th amino acids from the amino acids of aminoterminal the 27th form peptide bond
Connection.
5. peptide composition according to claim 3, when in the peptide composition, polypeptide, sequence 2 shown in sequence 1
The mol ratio of shown polypeptide is (0.5-1.5)：(0.5-1.5)；Preferably, their mol ratio is 1：1.
6. the peptide composition in the antigen polypeptide and claim 3-5 described in claim 1 or 2 described in any one is being made
Standby canine parvovirus synthetic peptide vaccine or prepare for prevent canine parvovirus biological products in application.
7. a kind of canine parvovirus synthetic peptide vaccine, include the peptide composition described in any one in claim 3-5.
8. the preparation method of canine parvovirus synthetic peptide vaccine, it is characterised in that the preparation method comprises the following steps：
(1) peptide composition described in any one in claim 3-5 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 1.5~3min, be then slowly added into polypeptide antigen aqueous phase, then stir 20~
30min, then 15~30min is stirred under 8000~10000rpm, stand 3-10min, packing.
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