CN103880932B - Swine fever synthesized peptide vaccine and preparation method thereof - Google Patents
Swine fever synthesized peptide vaccine and preparation method thereof Download PDFInfo
- Publication number
- CN103880932B CN103880932B CN201410160524.7A CN201410160524A CN103880932B CN 103880932 B CN103880932 B CN 103880932B CN 201410160524 A CN201410160524 A CN 201410160524A CN 103880932 B CN103880932 B CN 103880932B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- vaccine
- seq
- resin
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24311—Pestivirus, e.g. bovine viral diarrhea virus
- C12N2770/24334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides a kind of swine fever synthesized peptide vaccine and preparation method thereof, be specifically related to the polypeptide for swine fever synthesized peptide vaccine, and contain vaccine and their preparation method of this polypeptide.The aminoacid sequence of described polypeptide is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or the aminoacid sequence shown in SEQ ID NO.4.The vaccine be made up of this polypeptide can successfully manage the antigenic variation of Pestivirus suis, also there is not biosafety issues, is easy to extensive synthesis, has a good application prospect.
Description
The divisional application that the application is the applying date is on October 9th, 2009, application number is 200910235802.X, denomination of invention is the application for a patent for invention of " swine fever synthesized peptide vaccine and preparation method thereof ".
Technical field
The present invention relates to a kind of polypeptide for swine fever synthesized peptide vaccine, and contain vaccine and their preparation method of this polypeptide.
Background technology
Swine fever (Classical Swine Fever, CSF) be the important viral infectious of serious harm pig industry, its main manifestations is characteristic pathological change, visceral hemorrhage, infraction and necrosis, mortality ratio is up to 80% ~ 90%, almost (except North America and Oceania) all over the world, great financial loss is caused to pig industry.International Animal Health tissue lists swine fever in category-A deadly infectious disease.Swine fever is an infected pigs under field conditions (factors), and the pig of different ages, sex, kind and wild boar all susceptibles, all can occur throughout the year.In addition the sow that ill and low virulent strain infects also through placenta vertical infection fetus, can produce weak piglet, stillborn foetus etc.The latent period of this disease is generally 5 ~ 7 days, can be divided into the most acute, acute, chronic and mild Four types according to clinical symptom.
Pestivirus suis (CSFV) belongs to flaviviridae (Flavividae) pestivirus (Pestivirus), genome is single strand plus RNA virus, size is about 12.3kb, containing an open reading frame (ORF), and a poly precursor protein containing 3898aa of encoding.CSFV genome is by 11 genomic constitutions, oriented albumen has 5 kinds: Npro, C, E0, E1 and E2, non-expliciting the position have 6 kinds: NS2, NS3, NS4A, NS4B, NS5A and NS5B, wherein C is nucleocapsid protein, E0, E1 and E2 are structural glycoprotein, and all the other are Nonstructural Protein.In structural protein, what have immune protection researching value most is E0 and raq gene.
E2 membrane glycoprotein (gp55) is that induction body produces neutralizing antibody and excites the major antigen albumen of protective immune response, has species specificity.The variation of raq gene will affect immunogenicity and the antigenicity of CSFV.There is A, B, C and D4 relatively independent antigenic domains in E2, and A is further divided into A1, A2 and A3 tri-subprovinces.A1, A2 subprovince is conservative between the different isolated strain of CSFV, and A3, B, C, D are all in variability in various degree.A, B and C district is Neutralization and crystallization district, can the neutralization reaction of induced animal, and the neutralization reaction of inducing between A1 and B, A1 and C exists synergy.In all antigenic regions or subprovince, only have A1 subprovince to induce the monoclonal antibody produced not only to have neutralizing effect to virus infection, and all CSFV isolated strains can be identified, and with correlated virus BVDV and BDV no cross reaction.Therefore, the accurate location of A1 antigenic region and clone, expression, have important value to the diagnosis of CSFV, immune Research.The epitope number in each region of A, B, C and D is respectively: 8,6,3 and 1, and the epitope number of A1, A2 and A3 is respectively: 4,3 and 1.Research is had to further determined that on E2 albumen the position of the epitope of 4 relatively independent antigenic domains: all antigen sites are all on the 176aa of the N end 690 ~ 866 of E2, wherein all epi-positions of B and an epi-position of C are between 1st ~ 83aa residue, residing for other epi-position in C region, region is wider, between 1st ~ 110aa residue, A1 is between 86th ~ 176aa residue, A2, A3 are between 86th ~ 123aa residue, only there is an epitope in D region, between 86th ~ 110aa residue.
The swine fever B cell linear epitope reported at present has: the TAVSPTTLR (aa829-837) on E2, SPTTL(aa289-293), CTAVSPTTLRTEVVK (aa828-842), LFDGTNP(aa772-778) and Erns on DKN(aa117-119), the determination of these epitopes is by the development of the diagnositc analysis and Epitope tag vaccine that help lend some impetus to Pestivirus suis.
Pathogen inactivated or attenuation are made by traditional vaccine, there is biohazardous and heritable variation causes the problems such as former vaccine potency forfeiture.Epiposition vaccine is the vaccine prepared with epitope, is the direction of developing infectious diseases vaccine at present.Relative to traditional Attenuate vaccine and deactivation vaccine and other several novel vaccines, peptide vaccine is safe, nontoxic, stable, simple because its molecular structure is little, seldom there will be serious complication and nosocomial infection problem.Peptide vaccine can accurately be located, and synthesis T, B cell antigen epi-position and can carry out different combinations, make the vaccine for multiple pathogens.Particularly can select in microorganism and prepare synthetic peptide vaccine without the small segment of sudden change.Some cause of disease (as influenza virus) escapes host immune system by the quick and sudden change of high frequency, if select the little peptide fragment without sudden change just can overcome the limitation of himself albumen as vaccine.The most important step of development improvement on synthesis vaccine finds epitope.
The people such as the Liu Siguo of the military veterinary institute of military supplies university of PLA, on the basis of CSFV raq gene Genetic Variation Analysis, according to the aminoacid sequence of deriving, by the epitope of Computer Analysis, prediction E2 albumen, synthesize small peptide.React with the rabbit anteserum (or monoclonal antibody) of anti-mE2 albumen, qualification small peptide reactionogenicity, and with carrier proteins (BSA) coupling after immunization experiment animal, measure immunogenicity.Result shows, and only wherein a polypeptide can produce antibody.Dong etc. report synthesis 5 sections of swine fever Strain Shimen B/C district 693-777 amino acids, and coupling BSA makes synthetic peptide seedling, and result immune swine can produce the antibody of high titre, and can protect the attack of strong poison completely.With this polypeptide preparation hyper-immune serum, this serum 1:4 and 1:6 can suppress the rabbit body heat of C strain to be reacted after diluting, and after carrying out affinity chromatography process with improvement on synthesis to hyper-immune serum, then the rabbit body heat of C strain can not be suppressed to react.
Summary of the invention
Therefore, the object of the present invention is to provide a kind of polypeptide for swine fever synthesized peptide vaccine, and the vaccine containing this polypeptide.
Another object of the present invention is to provide the preparation method of a kind of aforementioned polypeptides and above-mentioned vaccine.
For achieving the above object, present invention employs following technical scheme:
On the one hand, the invention provides a kind of polypeptide for swine fever synthesized peptide vaccine, the aminoacid sequence of wherein said polypeptide be SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or the aminoacid sequence shown in SEQ ID NO.4.
On the other hand, the invention provides a kind of swine fever synthesized peptide vaccine, is the polypeptide of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4 comprising one or more aminoacid sequences; Preferably, described vaccine comprises adjuvant.
In addition, present invention also offers the preparation method of aforementioned polypeptides, comprising following steps:
(1) protective reaction: be in the N-Methyl pyrrolidone solution of the hexahydropyridine of 15%-30% in volume ratio, 25-40 minute is reacted under 20-28 DEG C of condition, 9-fluorenylmethyloxycarbonyl blocking group on deresinate amino, nitrogen dries up, and N-Methyl pyrrolidone washs;
(2) amino acid whose activation: by each amino acid with 9-fluorenylmethyloxycarbonyl blocking group of synthesis and 1-hydroxyl azimidobenzene Reactive Synthesis amino acid-1-hydroxyl azimidobenzene ester;
(3) condensation reaction: use Peptide synthesizer above-mentioned each seed amino acid and resin and DIC automatically to be added in reactor, react 0.5-2.5 hour under 20-28 DEG C of condition, nitrogen dries up, N-Methyl pyrrolidone washing resin;
(4) acetylization reaction: the N-Methyl pyrrolidone solution of the acetyl imidazole that operating weight volume ratio (g/ml) is 1.5%-4% and the middle resin obtained of step (3) react 20-40 minute under 20-28 DEG C of condition, and nitrogen dries up, methanol wash resin;
(5) building-up process is held by C and is held to N, according to the continuous repeating step of aminoacid sequence (1) ~ (4), after having reacted, with N-Methyl pyrrolidone cleaning, obtains dry polypeptide resin;
(6) being separated of polypeptide and resin: add lytic reagent in the polypeptide resin of drying, at the uniform velocity to stir within 1-4 hour, being placed on 0 DEG C reaction 10 minutes, returns to room temperature, fling to trifluoroacetic acid, t-butyl methyl ether and diethyl ether are added polypeptide solution, agitator treating, filter to obtain polypeptide solution;
(7) ultrafiltration purification polypeptide and aseptically process: use film to wrap in ultrafiltration polypeptide under 20-28 DEG C of condition, use 0.22 micron of degerming preservation of online filter.
In the preparation method of aforementioned polypeptides, the volume components of described lytic reagent is than being trifluoroacetic acid: tri isopropyl silane: dithioglycol: phenol: water=85:8:3:3:1.
Present invention also offers the preparation method of above-mentioned vaccine, comprising following steps:
(1) with water for injection, polypeptide is diluted to 200 μ g/ml and obtains antigen aqueous phase;
(2) under 20-28 DEG C of condition, according to antigen aqueous phase: the volume ratio of adjuvant=1:1, first adjuvant is added in emulsion tank, 90-150 rev/min of low rate mixing 1.5-3 minute;
(3) slowly add antigen aqueous phase, stir 20-30 minute;
(4) 5 minutes are left standstill after high-speed stirring 15-30 minute, after packing and get final product.
In the preparation method of above-mentioned vaccine, described high-speed stirring is 8000-10000 rev/min.
Invention further provides above-mentioned polypeptide or vaccine and prepare the purposes treated and/or prevented in the medicine of swine fever.
In sum, the present invention is by the sequencing to swine fever strain, the variation situation of research swine fever major antigenic sites, carry out the analyses and prediction in swine fever antigen site in conjunction with computer-aid method simultaneously, chemosynthesis is carried out to possible antigen site peptide section, and then through a large amount of animal experiments, candidate polypeptide antigen is screened, obtain immune response level high, can watch for animals well from the polypeptide antigen of the attack of swine fever strain, and it is good to develop a kind of effect based on this, security is high, stable processing technique and the synthetic peptide vaccine of the low swine fever virus resistant of cost.The vaccine safety carry out these polypeptide and efficacy test results show, vaccine provided by the invention can successfully manage the antigenic variation of Pestivirus suis, also there is not biosafety issues, are easy to extensive synthesis, have a good application prospect.
Accompanying drawing explanation
Fig. 1 is the Virus monitory result in the embodiment of the present invention 3.
Embodiment
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiments are only for illustration of the present invention, its scope do not limited the present invention in any way.
embodiment 1: the solid phase synthesis of synthetic peptide vaccine polypeptide antigen
The present embodiment is the solid phase synthesis of the polypeptide antigen of synthetic peptide vaccine provided by the present invention.Polypeptide antigen of the present invention can pass through ABI433A full-automatic polypeptide synthetic instrument, utilizes Merrifield solid-phase synthesis to prepare, wherein have employed the amino acid that 9-fluorenylmethyloxycarbonyl (Fmoc) is modified, and solid phase carrier is Rink Amide mbha resin.Production process generally includes the solid phase synthesis of polypeptide antigen, the cracking of polypeptide, antigen purification and degerming preservation.
1, the preparation of synthesis material
Synthetic peptide vaccine polypeptide antigen sequence is respectively SEQ ID NO.1, SEQ ID NO.2, the aminoacid sequence shown in SEQ INNO.3 and SEQ ID NO.4.
The synthesis scale of foundation aforementioned polypeptides antigen sequence and 1mmol prepares the amino acid of suitable Fmoc modification, adds corresponding Cartridge(and fills amino acid whose bottle) in.Weigh RinkAmide mbha resin equally on request, put into reaction chamber, upper and lower lid is tightened, label, the title of the peptide synthesized by record, lot number, the tare weight of reaction chamber and take the weight of resin.Reaction chamber is loaded synthesizer.Preparation synthetic agent comprises N-Methyl pyrrolidone (NMP), acyl imidazoles (AIM), piperidines (PIP), methyl alcohol etc. and is placed in corresponding reagent bottle.
2, the detection of synthesizer state
Check whether 433A Peptide systhesis instrument normally runs, after start, run Run Self Test program, whether instrument self checking indices is normal.Check that whether N2 is sufficient in addition, whether system gauge pressure is normal.Before synthesis, the performance of reply instrument is had gained some understanding, so will measure the flow velocity of often kind of synthetic agent.433A synthesizer: send Flow Rate1-18 to synthesizer, select Main Menu-ModuleTest-look for Module A, ModuleD, ModuleI, ModuleI, Module A-by Start-undertaken measuring or observing by more by Prer or next, if flow is improper, then regulate lower valve pressure, until reach requirement.
3, the preparation of synthetic peptide vaccine polypeptide antigen
(1) protective reaction: in 15%-30%(volume/volume) hexahydropyridine N-Methyl pyrrolidone solution in, 25-40 minute is reacted under 20-28 DEG C of condition, 9-fluorenylmethyloxycarbonyl blocking group on deresinate amino, nitrogen dries up, and N-Methyl pyrrolidone washs;
(2) amino acid whose activation: by each amino acid with 9-fluorenylmethyloxycarbonyl blocking group of synthesis and 1-hydroxyl azimidobenzene Reactive Synthesis amino acid-1-hydroxyl azimidobenzene ester;
(3) condensation reaction: use Peptide synthesizer above-mentioned each seed amino acid and resin and DIC automatically to be added in reactor, react 0.5-2.5 hour under 20-28 DEG C of condition, nitrogen dries up, N-Methyl pyrrolidone washing resin;
(4) acetylization reaction: use 1.5%-4%(weight/volume) acetyl imidazole N-Methyl pyrrolidone solution under 20-28 DEG C of condition, react 20-40 minute with the middle resin obtained of step (3), nitrogen dries up, methanol wash resin;
(5) building-up process is held by C and is held to N, according to the continuous repeating step of aminoacid sequence (1) ~ (4), after having reacted, with N-Methyl pyrrolidone cleaning, obtains dry polypeptide resin;
(6) being separated of polypeptide and resin: add lytic reagent (volume ratio of each component is trifluoroacetic acid: tri isopropyl silane: dithioglycol: phenol: water=85:8:3:3:1) in the polypeptide resin of drying, at the uniform velocity stir within 1-4 hour, being placed on 0 DEG C and react 10 minutes, return to room temperature, fling to trifluoroacetic acid, t-butyl methyl ether and diethyl ether are added polypeptide solution, agitator treating, filters to obtain polypeptide solution;
(7) ultrafiltration purification polypeptide and aseptically process: use film to wrap in ultrafiltration polypeptide under 20-28 DEG C of condition, use 0.22 micron of degerming preservation of online filter.
embodiment 2: the preparation of synthetic peptide vaccine
The present embodiment is the preparation of synthetic peptide vaccine provided by the present invention.
With water for injection, polypeptide solution is diluted to 50 μ g/ml respectively, makes antigen aqueous phase; By SEPPICMONTANIDE ISA50V oil adjuvant through 121 DEG C, sterilizing in 30 minutes, for subsequent use as oil phase.Under 22 DEG C of conditions, be the volume ratio of 1:1 according to the oil phase 50V of antigen aqueous phase and sterilizing, first oil phase is added in emulsion tank, 100 revs/min of low rate mixings 2 minutes, slowly add aqueous phase, add rear stirring 30 minutes, high speed 9000 revs/min stirs 20 minutes again, then 5 minutes are left standstill, namely the synthetic peptide vaccine of anti-avian influenza virus is obtained after packing, the i.e. synthetic peptide vaccine of aminoacid sequence shown in SEQ ID NO.1, the synthetic peptide vaccine of aminoacid sequence shown in SEQID NO.2, the synthetic peptide vaccine of aminoacid sequence shown in SEQ ID NO.3, the synthetic peptide vaccine of aminoacid sequence shown in SEQ ID NO.4.
embodiment 3: the potency test of synthetic peptide vaccine
Present embodiments provide the potency test of synthetic peptide vaccine of the present invention, specifically details are as follows.
1, test method: adopt stop band restrain to detect the effect of the swine fever synthesized peptide vaccine immune swine serum that embodiment 2 is prepared.
Four batches of synthetic peptide vaccines are set to altogether four groups, experimental animal be 40 ages in days after testing CSF antigen, antibody be negative health pig 16, concrete grouping situation is in table 1.Front and 2 immune 21d ear edge vein exploitating blood separation of serum respectively at immunity, as detection serum.
Table 1 vaccine grouping situation
| Grouping | Vaccinate | Experimental animal number (head) | Immunizing dose (ml/ head) |
| 1 group | SEQ ID NO.1 polypeptide vaccine | 4 | 2 |
| 2 groups | SEQ ID NO.2 polypeptide vaccine | 4 | 2 |
| 3 groups | SEQ ID NO.3 polypeptide vaccine | 4 | 2 |
| 4 groups | SEQ ID NO.4 polypeptide vaccine | 4 | 2 |
Application IDEXX CSFV antibody assay kit (purchased from Ai get Shi Yuan Heng bio tech ltd, Beijing) detects, concrete grammar is as follows: add in each detect aperture and control wells by 50 μ l sample diluting liquids respectively, add the serum 50 μ l of detection, feminine gender and positive control serum 50 μ l, after mixing, work is felt 2 hours by micro-reaction plate at ambient temperature wet box, discard the liquid in reacting hole, application PBST(pH7.4, 0.01mol/L phosphoric acid salt (PBS) damping fluid, containing 0.05%Tween-20) wash plate 3 times, add anti-CSFV ELIAS secondary antibody, 100 μ l/ holes, room temperature sense does 30 minutes, application PBST washes plate 3 times, add substrate solution TMB (TMB), 100 μ l/ holes, the sense of room temperature lucifuge does 10 minutes, add the stop buffer 0.5M H in 100 μ l/ holes
2sO
4, adopt the full-automatic microplate reader ELx800 of U.S. Bio-Tek to detect OD
450nm.
According to blocking-up rate=(OD
neg-OD
test)/OD
neg* 100% calculates blocking-up rate, wherein OD
negfor the OD of negative serum
450nmvalue, OD
testfor the OD of tested serum
450nmvalue.Positive serum controls blocking-up rate is greater than 50%, the average OD of negative control
450nmshould be greater than 0.5, blocking-up rate is less than 30% for negative, and blocking-up rate is suspicious between 30% ~ 40%, is greater than 40% for positive.
2, test-results:
According to the criterion of IDEXX test kit, positive serum controls blocking-up rate is greater than 50%, the average OD of negative control
450nmbe greater than 0.5, the blocking-up rate of this touchstone positive serum is 86%, the OD of negative serum
450nmbe 1.115, show this test and set up.
Serum before immunization is feminine gender (blocking-up rate is less than 30% for negative, and blocking-up rate is suspicious between 30% ~ 40%, is greater than 40% for positive); Serum after immunity is the positive, and as shown in Figure 1, wherein numbering 1-4 represents the aminoacid sequence prepared in example 2 is the synthetic peptide vaccine shown in SEQID NO.1-4 to Virus monitory result.Shown in Fig. 1, result shows, swine fever synthesized peptide vaccine can induce pig to produce immunne response.
embodiment 4: the proof test of synthetic peptide vaccine
Present embodiments provide the proof test of synthetic peptide vaccine of the present invention.The swine fever synthesized peptide vaccine injection laboratory animal adopting embodiment 2 to prepare respectively, comprises cavy, mouse and piglet, Continuous Observation 15 days, judges whether laboratory animal occurs death because vaccinate causes or significantly local untoward reaction or systemic reaction.Details are as follows for detailed process:
With the cavy 20 of body weight 350 ~ 450g, often criticize vaccination 5, every subcutaneous injection 2ml;
With the mouse 20 of body weight 18 ~ 22g, often criticize vaccination 5, vaccine every subcutaneous injection 0.5ml;
With the healthy susceptible piglet 20 of 27 ages in days, often criticize vaccination 5, every subcutaneous injection 2ml.Observations is in table 2.
The safety testing result of table 2 vaccine in guinea pigs, mouse and piglet
As can be seen from this test-results, all there is not the death because vaccinate causes or significantly local untoward reaction or systemic reaction in Continuous Observation 15 days after swine fever synthesized peptide vaccine immune guinea pig of the present invention, mouse and piglet.In whole process of the test, all not there are the phenomena of mortality in cavy, mouse and piglet, illustrates that swine fever synthesized peptide vaccine provided by the present invention is safe for cavy, mouse and piglet.
Claims (7)
1., for a polypeptide for swine fever synthesized peptide vaccine, the aminoacid sequence of wherein said polypeptide is the aminoacid sequence shown in SEQ ID NO.2 or SEQ ID NO.4.
2. a swine fever synthesized peptide vaccine, wherein said vaccine comprises aminoacid sequence for the polypeptide shown in SEQ IDNO.2 and/or SEQ ID NO.4.
3. vaccine according to claim 2, is characterized in that, described vaccine comprises adjuvant.
4. a preparation method for polypeptide described in claim 1, comprising following steps:
(1) protective reaction: be in the N-Methyl pyrrolidone solution of the hexahydropyridine of 15%-30% in volume ratio, 25-40 minute is reacted under 20-28 DEG C of condition, 9-fluorenylmethyloxycarbonyl blocking group on deresinate amino, nitrogen dries up, and N-Methyl pyrrolidone washs;
(2) amino acid whose activation: by each amino acid with 9-fluorenylmethyloxycarbonyl blocking group of synthesis and 1-hydroxyl azimidobenzene Reactive Synthesis amino acid-1-hydroxyl azimidobenzene ester;
(3) condensation reaction: use Peptide synthesizer above-mentioned each seed amino acid and resin and DIC automatically to be added in reactor, react 0.5-2.5 hour under 20-28 DEG C of condition, nitrogen dries up, N-Methyl pyrrolidone washing resin;
(4) acetylization reaction: operating weight volume ratio is that the resin obtained in the N-Methyl pyrrolidone solution of the acetyl imidazole of 1.5%-4% and step (3) reacts 20-40 minute under 20-28 DEG C of condition, and nitrogen dries up, methanol wash resin;
(5) building-up process is held by C and is held to N, according to the continuous repeating step of aminoacid sequence (1) ~ (4), after having reacted, with N-Methyl pyrrolidone cleaning, obtains dry polypeptide resin;
(6) being separated of polypeptide and resin: add lytic reagent in the polypeptide resin of drying, at the uniform velocity to stir within 1-4 hour, being placed on 0 DEG C reaction 10 minutes, returns to room temperature, fling to trifluoroacetic acid, t-butyl methyl ether and diethyl ether are added polypeptide solution, agitator treating, filter to obtain polypeptide solution;
(7) ultrafiltration purification polypeptide and aseptically process: use film to wrap in ultrafiltration polypeptide under 20-28 DEG C of condition, use 0.22 micron of degerming preservation of online filter;
Wherein, the volume components of described lytic reagent is than being trifluoroacetic acid: tri isopropyl silane: dithioglycol: phenol: water=85:8:3:3:1.
5. a preparation method for vaccine described in Claims 2 or 3, comprising following steps:
(1) with water for injection, polypeptide is diluted to 200 μ g/ml and obtains antigen aqueous phase;
(2) under 20-28 DEG C of condition, according to antigen aqueous phase: the volume ratio of adjuvant=1:1, first adjuvant is added in emulsion tank, 90-150 rev/min of low rate mixing 1.5-3 minute;
(3) slowly add antigen aqueous phase, stir 20-30 minute;
(4) 5 minutes are left standstill after high-speed stirring 15-30 minute, after packing and get final product.
6. the preparation method of vaccine according to claim 5, is characterized in that, described high-speed stirring is 8000-10000 rev/min.
7. the purposes of the vaccine described in polypeptide according to claim 1, Claims 2 or 3 in the medicine of preparation prevention swine fever.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910235802.XA CN102485749B (en) | 2009-10-09 | 2009-10-09 | Swine fever synthesized peptide vaccine and preparation method thereof |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910235802.XA Division CN102485749B (en) | 2009-10-09 | 2009-10-09 | Swine fever synthesized peptide vaccine and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103880932A CN103880932A (en) | 2014-06-25 |
| CN103880932B true CN103880932B (en) | 2015-09-23 |
Family
ID=46151324
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910235802.XA Expired - Fee Related CN102485749B (en) | 2009-10-09 | 2009-10-09 | Swine fever synthesized peptide vaccine and preparation method thereof |
| CN201410160524.7A Expired - Fee Related CN103880932B (en) | 2009-10-09 | 2009-10-09 | Swine fever synthesized peptide vaccine and preparation method thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910235802.XA Expired - Fee Related CN102485749B (en) | 2009-10-09 | 2009-10-09 | Swine fever synthesized peptide vaccine and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN102485749B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107217B (en) * | 2017-11-14 | 2020-07-24 | 中国农业科学院哈尔滨兽医研究所 | Classical swine fever virus truncated E2 protein and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338309A (en) * | 2000-08-10 | 2002-03-06 | 清华大学 | Synthetic peptide vaccine of hog cholera and its preparing process |
| US6887484B2 (en) * | 2001-07-31 | 2005-05-03 | Posco | Vaccine composition containing a promoter peptide |
| EP1983346A4 (en) * | 2006-02-08 | 2010-03-17 | Nec Corp | Method for cleavage of peptide bond at c-terminus of peptide, and method for determination of c-terminal amino acid sequence of peptide |
| CN101116749A (en) * | 2006-08-18 | 2008-02-06 | 中国农业科学院哈尔滨兽医研究所 | Swine fever replicon carrier vaccines, the method for preparing the same and application thereof |
-
2009
- 2009-10-09 CN CN200910235802.XA patent/CN102485749B/en not_active Expired - Fee Related
- 2009-10-09 CN CN201410160524.7A patent/CN103880932B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN103880932A (en) | 2014-06-25 |
| CN102485749B (en) | 2014-06-04 |
| CN102485749A (en) | 2012-06-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110618279B (en) | African swine fever virus epitope antigen polypeptide and application thereof | |
| CN102317449B (en) | Recombinant classical swine fever virus (CSFV) comprising a modified E2 protein and methods for generating said recombinant CSFV | |
| CN109485703B (en) | Foot-and-mouth disease A-type structural protein VP1 antigen epitope polypeptide and application thereof | |
| CN110642925B (en) | African swine fever virus synthetic peptide ELISA antibody detection kit | |
| US20160238601A1 (en) | Methods and compositions for coronavirus diagnostics and therapeutics | |
| CN103864906B (en) | Foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent | |
| CN102533668B (en) | Duck flavivirus, and vaccine and kit thereof | |
| KR20090025291A (en) | Novel virulence determinant within the e2 structural glycoprotein of classical swine fever virus | |
| CN111961120B (en) | African swine fever virus MGFs and CD2v ELISA antibody detection kit | |
| Kongsuwan et al. | Location of neutralizing epitopes on the G protein of bovine ephemeral fever rhabdovirus | |
| CN107973850A (en) | Recombinate CSFV E 2 protein pig source monoclonal antibody and its preparation method and application | |
| CN104987368B (en) | Foot-and-mouth disease antibody ELISA immunity detection reagent | |
| Vilček et al. | Genetic typing of pestiviruses from New Zealand | |
| Mavtchoutko et al. | Characterization of tick‐borne enchephalitis virus from Latvia | |
| CN104478998B (en) | Swine foot-and-mouth disease virus nonstructural protein 3A BC antibody ELISA immunity detection reagents | |
| CN101659695A (en) | O-type aftosa synthetic peptide vaccine | |
| Li et al. | Development of an immunochromatographic strip for rapid detection of antibodies against classical swine fever virus | |
| CN104961809B (en) | The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of ox aftosa | |
| CN103880932B (en) | Swine fever synthesized peptide vaccine and preparation method thereof | |
| CN107513101B (en) | Pig foot-and-mouth disease virus non-structural protein antibody enzyme-linked immunoassay kit | |
| CN103255111A (en) | Green fluorescent protein marked recombinant swine fever virus, its rescue method and application | |
| CN106380509B (en) | One boar annulus synthetic peptide vaccine and its preparation method and application | |
| CN109206492B (en) | Avian influenza synthetic peptide vaccine and preparation method thereof | |
| CN103936841A (en) | Peptide vaccine used for animal and its preparation | |
| CN101579522B (en) | Novel peptide-based vaccine used for domestic animal and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150923 Termination date: 20201009 |