CN100381170C - Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use - Google Patents
Foot and mouth disease bivalent polypeptide vaccine and its preparation method and use Download PDFInfo
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- CN100381170C CN100381170C CNB031507514A CN03150751A CN100381170C CN 100381170 C CN100381170 C CN 100381170C CN B031507514 A CNB031507514 A CN B031507514A CN 03150751 A CN03150751 A CN 03150751A CN 100381170 C CN100381170 C CN 100381170C
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Abstract
The present invention relates to a bivalent polypeptide vaccine generated with a genetic engineering method, a preparation method thereof and purposes thereof. The vaccine contains 2<n-1> nucleotide sequence encoded polypeptides connected in series and disclosed in SEQ ID NO: 1 or SEQ ID NO: 2, wherein n is an integral number between 1 and 5. The vaccine of the present invention has the advantages of high safety and high immunizing titer. The vaccine suitable for large-scale production, which can effectively prevent and treat the foot-and-mouth disease, can also be used for distinguishing infected animals from immunized animals.
Description
Invention field
The present invention relates to a kind of novel foot-and-mouth disease vaccine, particularly a kind of two valency polypeptide vaccines that produce by gene engineering method and its production and use.
Background technology
Foot and mouth disease (FMD) is the very strong a kind of disease of infectiousness in the animal husbandry, and artiodactylous animals such as cattle, pig, sheep, camel etc. are easily infected.Up to now, countries in the world are except that North America, Australia, all once broke out the popular on a large scale of foot and mouth disease, the popular of this disease once caused enormous economic loss to Germany (1937-38), Europe (1951-52), Turkey (1964-65), Britain (1967-68), Austria (1973), France (1974), Korea S countries such as (2002).
The cause of disease of foot and mouth disease is a kind of Pironavirus, i.e. foot and mouth disease virus (FMDV).Contain normal chain single stranded RNA polar, that form by 8500 nucleotide in the virion.The cyst membrane of virion comprises four kinds of structural protein VP1 (molecular weight 34,000), the VP that holds single stranded RNA
2(30,000), VP
3(26,000), VP
4(13500).Every kind of 60 molecule of four kinds of structural protein constitutes an icosahedral virion.Icosahedral summit is by structural protein VP
1Form.VP
1With infection viral, that stop foot and mouth disease, viral ability is lower than the antibody of complete virus particle generation but it neutralizes in the proteic antibody capable.Further studies show that structural protein VP
1The immunologic determinants zone be 141-160 zone and 200-213 zone.In the polypeptide fragment in these two zones and the antibody capable that carrier protein produces after chemistry is connected and virion (Nature, l982,298 30-33).
After the infection by respiratory tract, the first level replication of FMDV is pharyngeal, infects the lymph node that closes on then and enters blood flow, and then be diffused in various organs and the tissue.Clinical symptoms appearred in most cases, zoogenetic infection 2-14 days.Seldom occur death behind the more old zoogenetic infection FMDV, but animal reproduction ability, growth and health are subjected to very big influence.And though behind some zoogenetic infection FMDV, occur higher antibody titer in the body, these healthy animal might be secreted FMDV and be infected other animal.
FMDV has significant antigen multiformity, and the FMDV that has now found that has 7 kinds of serotypes, and each serotype can also continue to be divided into multiple hypotype, sees following table for details:
Because foot and mouth disease virus is easy to propagate, have the ability that infects multiple animal and have multiple antigen form, make foot and mouth disease be difficult to control.
Develop vaccine animal is carried out one of effective ways that immunity is control FMDV.Traditional deactivation FMDV vaccine is by a large amount of cultivation zooblasts, infects foot and mouth disease virus then, through cultivation, isolated viral, the reuse chemical reagent makes to be made after virally inactivated.France Merieux company is maximum deactivation FMDV production of vaccine producer, and countries such as Brazil, Argentina, Russia also use same technology and produce this vaccine, and the protection effect is fine.But the shortcoming of preparation deactivation FMDV vaccine is to set up the environment of a strict sealing, in case stopping leak reveals and pollutes surrounding, this potential risk limits the use of this vaccine.And the change of tiring of deactivation FMDV vaccine is bigger, and particularly remaining vestige live virus can cause the popular and large-scale outbreak of disease in the vaccine.In addition, vaccine need be preserved under refrigerated condition and carry, and has increased cost.At last, though use the FMDV vaccine of deactivation that anaphylactic shock or stupor seldom take place, in case serious consequence takes place just to be enough to cause.
Foot-and-mouth disease gene engineering polypeptide vaccine is through the research of more than ten years course, obtained good effect, use escherichia coli only to need 20 hours as the host cell fermented incubation time, period ratio is with zooblast production deactivation FMDV vaccine much shorter, recombinant vaccine does not pollute environment, does not have the danger of leakage, and is as safe as a house, and the vaccine of producing do not need cold preservation in storage, transportation and use, and it is stable to tire.But the protection effect of polypeptide vaccine is lower than whole virus vaccine, for example, the aminoacid of synthetic VP1 protein 14 1-160, the immunogenicity of the polypeptide vaccine of preparation be lower than respective amount deactivation FMDV vaccine 100-1000 doubly.And present FMDV vaccine no matter be deactivation FMDV vaccine or polypeptide vaccine, all can not be differentiated immunized animal and infection animal by serology experiment, and immunized animal is obscured and can be caused very big influence to the market import and export with infection animal.
Therefore, preparing immunizing potency and improve and can be used for differentiating the immunized animal and the genetic engineering foot-and-mouth disease vaccine of infection animal, is constantly the target of pursuit.
Summary of the invention
The inventor is according to the VP of O, two types of FMDV of A
1The aminoacid sequence of immunologic determinants, the design oligonucleotides fragment at expression in escherichia coli, obtains having ideal immunogenic polypeptide with gene polyphone back.And, with VP among the FMDV
1After immunologic determinants amino acid sequence of polypeptide counter-rotating (holding the end to C from N) is expressed, carry out immunity test, obtain good effect, the time that produces neutralizing antibody greatly prolongs, and the polypeptide half-life in vivo also obtains prolonging.Be prepared into vaccine with polypeptide of the present invention, can prevent and treat the foot and mouth disease of two types of O, A, and can distinguish easily and measure immune animal and infection animal.
An object of the present invention is to provide a kind of novel genetic engineering foot and mouth disease polypeptide vaccine.
Another purpose of the present invention provides the construction method of described foot-and-mouth disease vaccine.
Fork one purpose of the present invention provides the engineering strain that can express described foot and mouth disease polypeptide vaccine.
Another purpose of the present invention provides the purposes of described foot-and-mouth disease vaccine in preventing and treating the foot and mouth disease disease.
A further object of the present invention provides the purposes of described foot-and-mouth disease vaccine in distinguishing infection animal and immunized animal.
One aspect of the present invention provides a kind of novel foot-and-mouth disease gene engineering polypeptide vaccine, and it contains 2
N-1Nucleotide sequence encoded polypeptide shown in the SEQ ID NO.1 of individual polyphone or the SEQ ID NO.2, wherein, n is the integer of 1-5.SEQ ID NO.1 contains O type and A type foot and mouth disease virus VP
1The coded sequence of protein immunization determinant, A type that its forward of can encoding is expressed and the antigenic determinant of O type foot and mouth disease virus, its expression product can stimulate the antibody that produces anti-A type and O type foot and mouth disease; SEQ ID NO.2 then can encode the A type oppositely expressed and the antigenic determinant of O type foot and mouth disease virus, its expression product also can stimulate the antibody that produces anti-A type and O type foot and mouth disease.
Preferably, vaccine of the present invention contains 2
N-1The SEQ ID NO:1 of individual polyphone or SEQ ID NO:2 encoded polypeptide, wherein, n is the integer of 2-4.
Preferred, vaccine of the present invention contains 2
N-1The SEQ ID NO:1 of individual polyphone or SEQ IDNO:2 encoded polypeptide, wherein, n is 3.
Most preferred, vaccine of the present invention contains the SEQ ID NO:2 encoded polypeptide of 4 polyphones.
In a preferred embodiment, vaccine of the present invention contains the expressed albumen of plasmid that carries among the bacterial strain CGMCC No.0893.Described plasmid contains the nucleotide sequence shown in the SEQ ID NO.1 of 4 polyphones.
In a further preferred embodiment, vaccine of the present invention contains the expressed albumen of plasmid that carries among the bacterial strain CGMCC No.0894.Described plasmid contains the nucleotide sequence shown in the SEQ ID NO.2 of 4 polyphones.
In order to improve purposes such as bioavailability, enhance immunity effect, vaccine of the present invention can also contain materials such as pharmaceutically acceptable carrier, adjuvant and/or immunological adjuvant.
Immunological adjuvant be a kind of can enhance immunity the material of reaction, it can mix use with antigen, not only help the deposition of injection mass or compile, and can strengthen antibody response.
The material that can be used as immunological adjuvant has: 1. microorganism and product thereof, microorganism commonly used such as mycobacteria, coryne bacterium parvum, bordetella pertussis and the extract lipopolysaccharide of locating left Lan Shi negative bacillus are from the extraction of substance muramyldipeptide of mycobacteria etc.2. polynucleotide is as poly: cytidylic acid (poly I:C), polyadenylic acid (poly I:A:u) etc.3. freund adjuvant (Freund ' s adjuvant), comprise that incomplete freund adjuvant is (with antigen aqueous solution and oil preparation (paraffin oil or vegetable oil) mixed in equal amounts, add the Water-In-Oil antigen Emulsion that emulsifying agent (lanoline or Tween 80) is made again) and complete freund adjuvant (in Freund, adding mycobacteria, as bacillus calmette-guerin vaccine).4. inorganic matter is as Alumen and aluminium hydroxide etc.
In recent years, find that following material also can be used as immunological adjuvant, comprising: 1. bacteriotoxin, for example cholera toxin (CT) and escherichia coli thermal instability enterotoxin (LT); 2. attenuation derivant or the variant of CT and LT; 3. the endogenous immunoregulatory factor of people, as IL-2, IL-12, GM-GSF; 4. hormone; 5. lipopeptid; 6. saponin, saponin derivant QS-21; 7. the synthetic oligonucleotide fragment (CpG ODN) that contains CpG motif; 8. the derivant of lipoid A, for example lipopolysaccharide derivant monophosphoryl lipid A (MPL); 9. the derivant of muramyldipeptide (MDP).
In addition, some delivery system with intrinsic immunostimulatory activity also can be used as immunological adjuvant and is used for vaccine construction, and these delivery systems include but not limited to: liposome, Emulsion, spiral zooid (cochleate), virion, micropartical and immunostimulating complex (ISCOMs).
Various types of immunological adjuvant mentioned above all can be used for the present invention.Adjuvant can proper dosage and the present invention have immunogenic polypeptide and be used, form vaccine of the present invention.
Preferably, vaccine of the present invention contains incomplete freund adjuvant or Al (OH)
3, preferred, vaccine of the present invention contains incomplete freund adjuvant as immunostimulant.Not exclusively the ratio of lanoline and paraffin oil is slightly different with changes of seasons in the freund adjuvant, and for example, spring, summer, autumn, the ratio of lanoline and paraffin oil was about 3: 7; Winter, the ratio of the two was about 1.5: 8.5.
Another aspect of the invention provides a kind of method for preparing described gene engineering polypeptide vaccine, comprises 2
N-1The step that nucleotide sequence shown in the SEQ ID NO.1 of individual polyphone or the SEQ ID NO.2 is cloned into carrier and expresses in suitable expression system, wherein n is the integer of 1-5.
Preferably, with 2
N-1Nucleotide sequence shown in the SEQ ID NO.1 of individual polyphone or the SEQ ID NO.2 is expressed, and wherein, n is the integer of 2-4.
Preferred, the SEQ ID NO.1 of 4 polyphones or the nucleotide sequence shown in the SEQ ID NO.2 are expressed, prepare vaccine of the present invention.
Most preferred, the nucleotide sequence shown in the SEQ ID NO.2 of 4 polyphones is expressed, prepare vaccine of the present invention.
Nucleotide sequence shown in SEQ ID NO:1 of the present invention or the SEQ ID NO:2 can directly synthesize, and also can synthesize several oligonucleotide fragments earlier, connects the nucleotide sequence of back generation shown in SEQ ID NO:1 or SEQ ID NO:2.For the convenience of subsequent operation, when the design oligonucleotides fragment, can be with proper restriction site calling sequence two ends, so that target sequence is cloned into suitable carriers.
In a preferred embodiment of the invention, the inventor designs synthetic 4 oligonucleotide fragments, and the sequence F1 (+) that connects the back generation contains nucleotide sequence shown in the SEQ ID NO:1 and suitable restricted enzyme point of contact.Utilize engineered means that F1 (+) is cloned into carrier, then, by having the restricted enzyme of the sticking end that matches each other behind the enzyme action, F2 (+) (F1 (+) sequence that contains 2 polyphones) is cloned into carrier, and the polypeptide after the expression contains the O type of 2 forwards connections and the immunologic determinants of A type FMDV.According to similar method, can successively F4 (+), F8 (+), F16 (+) be cloned into carrier, they contain F1 (+) sequence of 4,8,16 polyphones successively, after expressing in suitable expression system, the polypeptide that obtains contains the O type of 4,8,16 forwards connections and the immunologic determinants of A type FMDV respectively.
In another preferred embodiment of the present invention, the inventor designs synthetic 4 oligonucleotide fragments, and the sequence F1 (-) that connects the back generation contains nucleotide sequence shown in the SEQ ID NO:2 and suitable restricted enzyme point of contact.Utilize engineered means that F1 (-) is cloned into carrier, then, by having the restricted enzyme of the sticking end that matches each other behind the enzyme action, F2 (-) (F1 (-) sequence that contains 2 polyphones) is cloned into carrier, and the polypeptide after the expression contains 2 O types that oppositely connect and the immunologic determinants of A type FMDV.According to similar method, can successively F4 (-), F8 (-), F16 (-) be cloned into carrier, they contain F1 (-) sequence of 4,8,16 polyphones successively, after expressing in suitable expression system, the polypeptide that obtains contains 4,8,16 O types that oppositely connect and the immunologic determinants of A type FMDV respectively.
In preparation method of the present invention, the expression system that is used for expression of polypeptides can be a prokaryotic expression system, can also be eukaryotic expression system.Expression system comprises proper host cell, and can duplicate the also plasmid or the carrier of stable existence in host cell.
The example that can be used as host cell includes but not limited to: bacterial cell, as escherichia coli, streptomycete, Salmonella typhimurium etc.; Fungal cell: as yeast etc.
That operable carrier can comprise is chromosome source, the non-chromosome source, and synthetic DNA sequence.For example: phage DNA, baculovirus, bacterial plasmid, yeast plasmid and make up deutero-carrier by plasmid, phage and viral DNA.
Preferably, in prokaryotic expression system, express polypeptide of the present invention.Preferred, polypeptide of the present invention can be cloned into high efficiency expression vector (for example commercially available expression vector), carry out amalgamation and expression with carrier protein.
The invention still further relates to a kind of engineering strain, its plasmid that carries contains 2
N-1Nucleotide sequence shown in the SEQ ID NO.1 of individual polyphone or the SEQ ID NO.2, wherein n is the integer of 1-5.
Preferably, the plasmid that carries of described engineering bacteria contains 2
N-1Nucleotide sequence shown in the SEQ ID NO.1 of individual polyphone or the SEQ ID NO.2, wherein n is the integer of 2-4.
Preferred, the plasmid that described engineering bacteria carries contains 2
N-1Nucleotide sequence shown in the SEQ ID NO.1 of individual polyphone or the SEQ ID NO.2, wherein n is 3.
In a preferred embodiment of the invention, contain the regulation of the engineering strain of nucleotide sequence shown in the SEQ ID NO.1 of 4 polyphones and the SEQID NO.2, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) according to " the microbial preservation budapest treaty that is used for the international recognition of proprietary program ".Preservation date is on February 25th, 2003, and preserving number is respectively CGMCC No.0893 and CGMCC No.0894.
Preservation thing CGMCC No.0893 prepares after changing escherichia coli HB101 over to by plasmid pEZZ18/F4 (+).PEZZ18/F4 (+) is with behind EcoRI and the HindIII enzyme action pUC18/F4 (+), reclaims F4 (+) fragment, is cloned into that the pEZZ18 carrier (GenBankAccession No.M74186) handled through same enzyme action makes again.Contain the coding A type of 4 forwards polyphone and the nucleotide sequence of O type FMDV antigenic determinant among the pEZZ18/F4 (+), i.e. the SEQ IDNO.I of 4 polyphones, its expression product contains the A type and the O type FMDV antigenic determinant of 4 forwards polyphones.
Preservation thing CGMCC No.0894 prepares after changing escherichia coli HB101 over to by plasmid pEZZ18/F4 (-).PEZZ18/F4 (-) is with behind EcoRI and the HindIII enzyme action pUC18/F4 (-), reclaim F4 (-) fragment, is cloned into to make through the pEZZ18 of same enzyme action carrier again.Contain 4 oppositely the coding A types of polyphone and the nucleotide sequences of O type FMDV antigenic determinant among the pEZZ18/F4 (-), i.e. the SEQ ID NO.II of 4 polyphones, its expression product contains 4 oppositely the A type and the O type FMDV antigenic determinants of polyphone.
The preservation thing is for fully openly the present invention, for those skilled in the art provides convenience.The behavior of any manufacturing, use or sale preservation thing need not give such permission at this through inventor's permission.
The present invention also provides the purposes of described gene engineering polypeptide vaccine in prevention and treatment foot and mouth disease.Can give animal with vaccine injection of the present invention, stimulate the antibody that produces specific resisting O-type and A type foot and mouth disease in the animal body, thereby reach the purpose of prevention and treatment.
The invention provides the purposes of described gene engineering polypeptide vaccine in distinguishing infection animal and immunized animal.Because the animal of natural infection foot and mouth disease only produces monospecific antibody (a kind of serotype) usually, and animal is given in vaccine injection of the present invention, vaccine of the present invention stimulates the antibody that produces specific resisting O-type and A type foot and mouth disease in the animal body, so can be used to distinguish immunity inoculation animal and infection animal.
Use vaccine of the present invention to have the following advantages: 1. safety is good, and vaccine of the present invention is a gene engineering product, is not inactivated vaccine, does not have the danger because of trace live virus leakage causing illness outbreak.In zoopery, with the vaccine of higher dosage mice and Cavia porcellus are carried out subcutaneous injection, in the long observation phase, the healthy survival of laboratory animal, and produced specific antibody in the body.2. vaccine of the present invention is suitable for having reduced cost with engineered method large-scale production.3. utilize vaccine of the present invention to be easy to distinguish infection animal and immune animal.At occurring in nature, infect the animal of A type foot and mouth disease, can produce A type foot-and-mouth disease antibody in its body, the animal that infects O type foot and mouth disease then produces the antibody of O type foot and mouth disease.The existing A type of vaccine of the present invention antigen has the antigenic immunologic determinants of O type again, and after the inoculation, producing the existing A type of antibody in the animal body has the O type again, can distinguish infection animal and immune animal by this, avoids causing confusion to the animal import and export.4. vaccine of the present invention contains VP among A type and the O type FMDV
1Immunologic determinants polyphone back counter-rotating (holding end) polypeptide expressed to C from N, it obtains good effect in immunity test, and the polypeptide half-life in vivo prolongs, and its time that produces neutralizing antibody also greatly prolongs.
In order more to be expressly understood the present invention, referring now to the following drawings and embodiment the present invention is made an explanation, but drawings and Examples to the present invention without any limited significance.
Brief description of drawings
Fig. 1 is that synthetic 4 oligonucleotide fragment P1+P2 are connected the fragment F1 (+) that the back produces with P3+P4, O type that the generation forward of can encoding connects and the immunologic determinants of A type foot and mouth disease virus.
Fig. 2 is that synthetic 4 oligonucleotide fragment P1 '+P2 ' are connected the fragment F1 (-) that the back produces with P3 '+P4 ', can encode to produce reverse O type that connects and the immunologic determinants of A type foot and mouth disease virus.
The building process sketch map of Fig. 3 plasmid pUC8/F1.
Fig. 4 is the sketch map that is made up plasmid pUC8/F8 by plasmid pUC8/F1.
Fig. 5 is with the structure sketch map of the gene among plasmid pUC8/F4 series connection fragment cloning in the expression vector pGEX-6P-3.
Fig. 6 is after pGEX-6P-3/F4 (-) changes escherichia coli over to, the SDS-PAGE collection of illustrative plates of fusion rotein.Wherein, 1 is pGEX-6p-3 carrier protein (contrast), and 2-6 is the fusion expressed product of pGEX-6p-3/F4 (-), i.e. the carrier protein that carries of pGEX-6p-3 and F4 (-) product that carries out amalgamation and expression, and 7 is molecular weight standard.
Fig. 7 is the engineering bacteria growth collection of illustrative plates during the fermentation that contains pGEX-6P-3/F4 (-).
Fig. 8 is the testing result of two valency polypeptide vaccines (O type).
1 is "+" reagent (positive control), and 8 is "-" reagent (negative control);
2-5 respectively is * 1, * 2, * 4, * 8 times of dilutions for the TE buffer pGEX-6P-3/F4 (-) expression product being made the sample of doubling dilution;
9-12 is a sample of making doubling dilution with the supernatant that the TE buffer is handled guanidine, is followed successively by * 1, * 2, * 4, * 8 dilutions;
6-7,13-14 is the diluent that detectable provided, and pGEX-6P-3/F4 (-) expression product is made the sample of doubling dilution, is followed successively by * 1, * 2, * 4, * 8 dilutions.
Fig. 9 is the measurement result of two-way immunodiffusion test (antigen antibody reaction).
Among Fig. 9 A:
Antigen is 1 and 2, and wherein, 1 is the polypeptide that obtains behind pGEX-6P-3/F4 (-) the expression product purification, and 2 is the polypeptide that obtains behind pGEX-6P-3/F4 (+) the expression product purification;
Antibody is A, is to expand positive serum available from the A type FMD fine jade that the Lanzhou veterinary is provided, and * 1 is stock solution, and * 5 is 5 times of diluents;
Among Fig. 9 B:
Antigen be the Lanzhou veterinary A type FMD agp antigen, * 1 is stock solution, * 5 is 5 times of diluents;
Antibody is A and F, and wherein, A is the serum that the polypeptide immune Cavia porcellus behind pGEX-6P-3/F4 (-) the expression product purification obtains, and F is the serum that the polypeptide immune Cavia porcellus behind pGEX-6P-3/F4 (+) the expression product purification obtains.
Among Fig. 9 C:
Antigen is 1 and 2, and wherein, 1 is the polypeptide for obtaining behind pGEX-6P-3/F4 (-) the expression product purification, and 2 is the polypeptide that obtains behind pGEX-6P-3/F4 (+) expression and purification;
Antibody is A and D, and wherein, A is the serum that the polypeptide immune Cavia porcellus behind pGEX-6P-3/F4 (-) the expression product purification obtains, and D is the serum that the polypeptide immune Cavia porcellus behind pGEX-6P-3/F4 (+) the expression product purification obtains;
Figure 10 is the testing result with immuno-electrophoresis.
Among Figure 10 A:
Antibody B is the antiserum that makes behind the protein immunization Cavia porcellus behind pGEX-6P-3/F4 (-) the expression product purification, O type "+" reagent that antigen is provided for the Lanzhou veterinary.
Among Figure 10 B:
Antibody A expands positive serum (Lanzhou the veterinary provided) by the FMDV fine jade, and antigen is the polypeptide that obtains behind pGEX-6P-3/F4 (+) the expression product purification.
Among Figure 10 C:
Antibody A is that A type FMD fine jade expands positive serum (Lanzhou veterinary institute); Antigen is the polypeptide that obtains behind pGEX-6P-3/F4 (-) the expression product purification.
Among Figure 10 D:
Antibody E is the serum that the polypeptide immune Cavia porcellus behind pGEX-6P-3/F4 (+) the expression product purification obtains, O type "+" reagent that antigen is provided for the Lanzhou veterinary.
The specific embodiment
The structure of O type that the coding forward is arranged and the plasmid pUC8/F1 (+) of A type antigenic determinant
According to O type and A type foot and mouth disease virus VP
1Proteic 141-160 amino acids sequence, synthetic four oligonucleotide fragments are gone into carrier through connecting rear clone, and formation contains 0 type of forward arrangement and the recombiant plasmid pUC8/F1 (+) of A type antigenic determinant.
1. the preparation of oligonucleotide fragment
According to O type and A type foot and mouth disease virus VP
1Proteic 141-160 amino acids sequence designs following oligonucleotide fragment, and company is synthetic by the match Parkson.
P1：5′-AAT TCC ATG AGA TCT GGT TCT GGT GTT CGT CGT
GAT TTC GGT TCT CTG GCG CCG CGT GTT GCG CGT CAG CTG A-3′
P2：5′-T GTT GGT CAG CTG ACG CGC AAC ACG CGG CGC
CAG AGA ACC GAA ATC ACG ACG AAC ACC AGA ACC AGA TCT CAT GG-3′
P3：5′-CC AAC AAC GTT CGT GGT GAT CTG CAG GTT CTG
GCG CAG AAA GCG GAA CGT ACC CTG CCG GGA TCC TAG A-3′
P4：5′-AG CTT CTA GGA TCC CGG CAG GGT ACG TTC CGC
TTT CTG CGC CAG AAC CTG CAG ATC ACC ACG AAC GT-3′
With concentration is A
260nm4 oligonucleotide fragments of=20D are dissolved in 49 μ l sterile deionized water respectively.P1 after the dissolving and P2 respectively get 2.5 μ l, place in 1 0.5ml centrifugal; P3 and P4 respectively get 2.5 μ l, place in the centrifuge tube of other 1 0.5ml.The T4 polynucleotide kinase (Promega company) that in 2 centrifuge tubes, adds 1 μ l respectively, 10 * T4 polynucleotide kinase buffer of 1 μ l, 1 μ l concentration is the ATP (BoehringerMannheim company) of 0.1mol/L, 2 μ l deionized waters.37 ℃ of insulations placed 92 ℃ of water-bath annealing after 1 hour, naturally cooled to room temperature then.
The T4DNA ligase (Promega company) that in the centrifuge tube that contains annealed nucleotide fragments mixture P1 and P2, P3 and P4, adds 2 μ l, the T4DNA ligase buffer of 2.5 μ l, 0.5 μ l deionized water.16 ℃ are incubated 1-2 hour, and 4-8 ℃ is incubated 12-16 hour.Through the 15%PAGE electrophoretic separation, be contrast with φ χ 174/HaeIII enzyme action molecular weight standard (Promega company), extract the gel between the 118-194bp, soak, with phenol, chloroform extracting, the dehydrated alcohol precipitation, standby after reuse 70% washing with alcohol, the drying.
The sequence that the connection back produces represents with F1 (+) that as shown in Figure 1 it contains the O type FMDV of 1 forward arrangement and the antigenic determinant of A type FMDV.
2. clone
The building process of recombiant plasmid pUC8/F1 (+) as shown in Figure 3.Operating procedure is as follows:
Get pUC8 carrier 1 μ l (1ug/ μ l), carry out double digestion with restricted enzyme EcoRI and HindIII, big fragment is wherein reclaimed in dialysis.With phenol, chloroform extracting, reclaim liquid phase, with dehydrated alcohol precipitation, reuse 70% washing with alcohol, drying for standby.
F1 (+) genetic fragment is connected with carrier segments behind the enzyme action.Dissolve target gene fragment and carrier segments fully with 4 μ l sterile deionized water respectively, the dna fragmentation after the dissolving is mixed, add T4DNA ligase 1 μ l, T4DNA ligase buffer 1 μ l.16 ℃ are incubated 1-2 hour, then 4-8 ℃ of insulation 12-16 hour.
Picking is stored in the escherichia coli JM103 bacterial strain in the glycerol pipe, rules 37 ℃ of overnight incubation on the LB culture dish.Second day picking list colony inoculation treats that in 100ml LB fluid medium bacterial growth is to A
600nm=0.6 o'clock, stop cultivating, in 4 ℃, centrifugal 4 minutes of 6000rpm.Collect thalline, with the calcium chloride solution of the 0.1mol/L of the 20ml pre-cooling thalline that suspends again.Placed on ice 30 minutes.4 ℃, centrifugal 4 minutes of 6000rpm.With the 0.1mol/L calcium chloride solution of the 2ml pre-cooling thalline that suspends again, divide to be filled to aseptic tubule then, every pipe 100 μ l, adding glycerol adding, to make ultimate density be that 15% back is standby in-84 ℃ of preservations.
To connect product and join competent cell, mixing was placed 30 minutes on ice, placed 2 minutes, placed cooled on ice again 2 minutes for 42 ℃.Add the fresh LB culture medium of 120 μ l, 37 ℃ vibrated 1 hour.Conversional solution is coated LB culture dish (containing the 100ug/ml ampicillin), 37 ℃ of overnight incubation.Second day, the single bacterium colony on the picking plate placed 2ml LB culture fluid (containing the 100ug/ml ampicillin).Method extracting plasmid according to molecular cloning is described obtains pUC8/F1 (+).
3. enzyme action is identified
Get 6 μ l DNA extracts and add 2 μ l M μ lti-core 10x buffer 0.5 μ l restricted enzyme EcoRI and 0.5 μ l restricted enzyme HindIII, 0.2 μ l BSA, 10.8 μ l sterile deionized water, 37 ℃ of insulation enzyme action were used the 15%PAGE electrophoresis detection after 1 hour.With φ X174/HaeIII is that molecular weight standard detects.The result detects the dna fragmentation of about 141bp.
Make single endonuclease digestion with Bg1II, BamHI, EcoRI, HindIII restricted enzyme, as seen it is single point of contact.
4, dna sequence analysis
With PE company 377 type nucleotide sequence analysis instrument, adopt four color fluorometric methods to carry out nucleotide sequencing.Sequencing result conforms to expection.
The structure of the O type of coding reversed arrangement and the plasmid pUC8/F1 (-) of A type antigenic determinant
According to O type and A type foot and mouth disease virus VP
1Proteic 141-160 amino acids sequence, synthetic four oligonucleotide fragments are gone into carrier through connecting rear clone, form and contain the O type of reversed arrangement and the recombiant plasmid pUC8/F1 (-) of A type antigenic determinant.
According to O type and A type foot and mouth disease virus VP
1Proteic 141-160 amino acids sequence designs following oligonucleotide fragment, and company is synthetic by match Parkson, Beijing.
P1’：5′-AAT TCC AGA TCT CCG CTG ACC CGT GAA GCG AAA CAG GC G
CTG GTT CAG CTG GAT GGT CGT GTT AAC AAC A-3′
P2’：5′-G CAG GGT GTT GTT AAC ACG ACC ATC CAG GTG AAC CAG CGC
CTG TTT CGC TTC ACG GGT CAG CGG AGA TCT GG-3′
P3’：5′-CC CTG CAG CGT GCG GTT CGT CCG GCG CTG TCT GGT TTC
GAT CGT CGT GTT GGT TCT GGT GGA TCC TAG A-3′
P4’：5′-AG CTT CTA GGA TCC ACC AGA ACC AAC ACG ACG ATC GAA ACC
AGA CAG CGC CGG ACG AAC CGC ACG CT-3′
After 4 synthetic oligonucleotide fragment 5 ' phosphorylations, connect, the sequence of generation represents that with F1 (-) it contains the antigenic determinant of the O type FMDV and the A type FMDV of 1 reversed arrangement as shown in Figure 2.
With reference to the operation of embodiment 1, the nucleic acid fragment after connecting is cloned into the pUC8 carrier, make up pUC8/F1 (-) recombiant plasmid.Building process as shown in Figure 3.
The series connection of embodiment 3, gene and clone
As shown in Figure 4, pUC8/F1 (+) or pUC8/F1 (-) (hereinafter to be referred as pUC8/F1) are carried out double digestion, reclaim big fragment with restricted enzyme BamHI and HindIII; With restricted enzyme Bg1II and HindIII gene monomer pUC8/F1 is made double digestion again, reclaim small fragment.
Because the restriction enzyme site otch of Bg1II is A ↓ G A T C ↑ T, obtains a sticky end A or G A T C T behind the Bg1II enzyme action; And the restriction enzyme site of Bam HI is G ↓ G AT C ↑ C, obtains sticky end G or G A T C C after with Bam HI enzyme action.Behind two enzyme enzyme action, the lucky complementary pairing of the otch of generation.
Two fragments that will reclaim with the T4 ligase connect.Connect product and transform the JM103 competent cell, and identify the plasmid DNA of gained transformant, can obtain 2 monomer series-connected junctional complex pUC8/F2, it contains 2 O type FMDV that arrange forward or backwards and the antigenic determinant of A type FMDV.
Repeat above-mentioned steps, can be prepared into pUC8/F4, pUC8/F8 and pUC8/F16, it contains 4,8 and 16 O type FMDV that arrange forward or backwards and the antigenic determinant of A type FMDV respectively.
The pGEX-6P-3 that selects pharmacia biotech company for use is as expression vector.PGEX-6P-3 contains derivable tac promoter, is used for the 1acIq gene of expressing at E.coli and the recognition site that is used for the PreScission enzyme of crack fusion protein (fusion expressed product of GST and destination protein).
As shown in Figure 5, pUC8/F4 (±) is cloned into pGEX-6P-3, is built into pGEX-6P-3/F4 (+) and pGEX-6P-3/F4 (-) recombiant plasmid (following represent) with pGEX-6p-3/F4.Wherein, M is artificial synthetic joint, has following sequence and multiple clone site:
PGEX-6p-3/F4 changes e. coli bl21 over to recombiant plasmid, 37 ℃ of cultivations, is that the IPTG of 0.2mmol/L-0.5mmol/L induces with the final concentration.
According to the method that " molecular cloning " laboratory manual (second edition) (Jin Dongyan, Li Mengfeng translate, and Science Press publishes) is described, carry out the SDS-PAGE electrophoresis of fusion rotein, the separation gel (H of preparation 12%
2O 3.3ml, 30% acrylamide 4.0ml, the Tris of 1.5mol/L (pH8.8) 2.5ml, 10% SDS 0.1ml, 10% Ammonium persulfate. 0.1ml, TEMED0.004m l, cumulative volume 10ml) and concentrate glue (H accordingly
2O 2.7ml, 30% acrylamide 0.67ml, the Tris of 1.0mol/L (pH6.8) 0.5ml, 10% SDS 0.004ml, 10% Ammonium persulfate. 0.004ml, TEMED 0.004ml, cumulative volume 4ml).The about 50 μ g of applied sample amount, electric current 10-15mA, voltage 100V, electrophoresis 1-1.5 hour, dyeing, observed result.
Fig. 6 is after pGEX-6p-3/F4 (-) changes escherichia coli over to, the Expression of Fusion Protein situation.1 is the pGEX-6p-3 contrast, and 2-6 is the fusion expressed product of pGEX-6p-3/F4 (-), i.e. the carrier protein that carries of pGEX-6p-3 and F4 (-) product that carries out amalgamation and expression, and 7 is molecular weight standard.
As seen from the figure, the molecular weight of pGEX-6p-3 carrier protein is about 26Kd, and destination protein and carrier protein merge back (pGEX-6p-3/F4 (-)), and the molecular weight of fusion rotein is about 43Kd.
The 250ml seed culture fluid (the 1000ml seed culture fluid contains peptone 10g, yeast extracting powder 5g, and the phosphate buffer 20ml of 0.02mol/L pH7.0) places the 1000ml triangular flask, sterilizes 20 minutes for 120 ℃, and the cooling back adds 20% glucose solution 5ml.The bacterial strain of lml cryopreservation in glycerol added above-mentioned solution, and adding ampicillin to final concentration again is 50 μ g/ml, and 37 ℃, 200rpm cultivates the 12-14 hour kind daughter bacteria as amplification culture.
The 1000ml seed culture fluid contains peptone 20g, yeast extracting powder 10g, the phosphate buffer 20ml of 0.2m0l/L, pH7.0, and trace elements of Ca Cl
2, NiNO
3, CoCl
3, MgSO
4, FeCl
3Each 1mg, 120 ℃ the sterilization 20 minutes, be cooled to 37 ℃ after, adding ampicillin to final concentration is 50mg.Add seed culture bacterium 20ml, add 20% glucose solution 5ml again, keep in the table listed condition and ferment.
| Fermentation tank | 35L |
| Temperature | 37℃ |
| Mixing speed | 700rpm |
| Ventilation | 35L/min |
| pH | 7-7.5 |
Certain hour is respectively got the 1ml fermentation liquid and is placed 2 plastic centrifuge tubes at interval, and centrifugal 10 minutes of 8000rpm removes supernatant, and taking by weighing thalline weight is weight in wet base (g/L).
As shown in Figure 7, after fermentation 12-15 hour, bacterial concentration reaches peak value.
After the fermentation, 4000rpm centrifugal (BeckmanJ6-HC centrifuge) collected thalline in 30 minutes.
Thalline is suspended in the solution of the potassium phosphate pH 7.0 that contains 1% sodium chloride, 1mmol/L EDTA, 20mmol/L with/3 liters ratios of 1000 grams.Add 1 gram lysozyme in suspension, stirring at room 1 hour is with smudge cells.Thallus suspension liquid is in 10, and centrifugal 30 minutes of 000rpm abandons supernatant.
The guanidine hydrochloride solution that above-mentioned precipitation is added 6mol/L with the ratio of 250 grams per liters.Stirring, extracting are spent the night.20, centrifugal 30 minutes of 000rpm gets supernatant, and the dress bag filter is dialysed with water.
Through dialysis has precipitation to produce to water.With 10, centrifugal 30 minutes of 000rpm gets precipitation, makes homogenate with sterilized water, and is standby.
The detection of embodiment 5, the two valency polypeptide vaccines (O type) of foot and mouth disease
1. reagent
O type swine pox lyophilizing forward blood clotting diagnosticum is available from Lanzhou Veterinary Inst., Chinese Academy of Agricultural Science.Diagnosticum comprises: 1. blood cell preparation, 2. positive reagent "+", 3. negative agents "-".
2. sample
The bacterial strain that contains pGEX-6P-3/F4 (-) after fermentation, centrifugal collection thalline, smudge cells, with the extracting of 6M guanidine hydrochloride, centrifugal back is collecting precipitation and supernatant respectively, does * 1, * 2, * 4, * 8 dilutions successively.
3. method
Blood cell reagent is shaken up, add 96 orifice plates (Denmark NUNC company), every hole 50ul.
According to design in advance, in the above-mentioned hole that contains the blood cell preparation, add 3ul O type foot and mouth disease "+" liquid respectively as positive control (sample 1), 3ul O type foot and mouth disease "-" liquid is as negative control (sample 8).And add 10ul successively and make the testing sample of * 1, * 2, * 4, * 8 times of dilutions, be i.e. fermentation, smudge cells, fusion rotein and the supernatant collected with the extracting of 6M guanidine hydrochloride, after centrifugal with the TE buffer.
The result: as shown in Figure 8, antigen antibody reaction all takes place in the fusion rotein of various extension rates and O type blood cell preparation, and does not contain fusion rotein in the supernatant, so O type blood cell preparation is not reacted.
The antigen-antibody reaction of embodiment 6, FMDV vaccine
Utilize protein when semisolid matrix (as gel) goes up diffusion, antigen and antibody can produce the principle of precipitation band or precipitation ring at the suitable position of concentration ratio, adopt double immunodiffusion to carry out the mensuration of vaccine immunogenicity of the present invention.
1. antigen:
Standard substance: 1. O type swine pox lyophilizing forward blood clotting diagnosticum, 2. A type FMD fine jade expands refining antigen, available from Lanzhou veterinary institute of the Chinese Academy of Agricultural Sciences.
The albumen that obtains behind test article: pGEX-6P-3/F4 of the present invention (-) and pGEX-6P-3/F4 (+) expression and purification.
2. antibody:
Standard substance: 1. O type swine pox lyophilizing forward blood clotting diagnostic agent, 2. A type FMD fine jade expands positive serum, provides by the Lanzhou veterinary of the Chinese Academy of Agricultural Sciences.
Test article: the albumen 1mg by pGEX-6P-3/F4 of the present invention (-) and pGEX-6P-3/F4 (+) expression and purification obtain, form water in oil emulsion with the 1ml freund adjuvant, subcutaneous injection is given Cavia porcellus, the serum that the back blood sampling of 3 weeks is produced.
3. two-way immunodiffusion test
Being mixed with concentration with the sodium phosphate buffer of 20mmol/L pH7.1 and agarose is 1.0% gel, after the heating and melting, is laid on (about 3ml/ piece) on the clean microscope slide.After the cooling, stamp aperture with card punch after, fill slit between microscope slide and the hole with a little hot agar solution again.Difference dilution (or not diluting) antigen, antibody are added in the aperture the about 20 μ l in every hole respectively.Naturally diffusion is 24 hours, observes its result.
The result: in this experiment, the precipitation band that observed antigen, antibody diffusion are taken place when meeting as shown in Figure 9.
In Fig. 9 A, with the albumen behind pGEX-6P-3/F4 of the present invention (-) and pGEX-6P-3/F4 (+) the expression product purification is antigen, A type FMDV antiserum (standard substance) is an antibody, and immunoprecipitation all takes place for F4 albumen and the A type FMDV antibody of observing forward expression and oppositely expression.
In Fig. 9 B, with A type FMD agp antigen (standard substance) is antigen, behind pGEX-6P-3/F4 of the present invention (-) and pGEX-6P-3/F4 (+) the expression product purification, the serum of producing behind the immune guinea pig is antibody, observes between antigen and the antibody immunoprecipitation takes place.
In Fig. 9 C, with O type swine pox lyophilizing forward blood clotting diagnosticum antigen is the antigenic positive control of O type, the hole of not adding reagent is a blank, with the albumen behind pGEX-6P-3/F4 of the present invention (-) and pGEX-6P-3/F4 (+) the expression product purification is antigen to be detected, and with behind pGEX-6P-3/F4 of the present invention (-) and pGEX-6P-3/F4 (+) the expression product purification, the serum of producing behind the immune guinea pig is antibody, between determined antigen and the antibody immunoprecipitation takes place.
Above result shows that FMDV vaccine of the present invention and O type and A type antibody all are immunological cross-reaction.
The gene engineering polypeptide vaccine of the present invention preparation has the antigenic determinant of A type FMDV and O type FMDV simultaneously, after the inoculation the antibody that produces not only anti-A type but also resisting O-type foot and mouth disease.Therefore, can be used for distinguishing infection animal and immune animal.
In the counter immunoelectrophoresis technology experiment, prepare 1.5% agarose solution with 0.05mol/L (pH8.6) barbital sodium-hydrochloride buffer, be laid on after the heating and melting on the microscope slide, the punching of cooling back is with hot agarose solution perforations adding.Add antibody at interstitial hole, bilateral adds antigen in the hole.Microscope slide is placed in the electrophoresis tank, indicate+,-utmost point, with 10mA electric current electrophoresis 1 hour.
After stopping electrophoresis, soak a few hours with 0.9% sodium chloride solution after, direct observation precipitation band is perhaps observed the precipitation band with amino black dyeing back.
Experimental result as shown in figure 10, this result shows that FMDV vaccine of the present invention can induce O type and two kinds of antibody of A type, and the animal of natural infection foot and mouth disease only produces monospecific antibody (a kind of serotype) usually, so vaccine of the present invention can be used to distinguish immunity inoculation animal and infection animal.The safety experiment of embodiment 8, vaccine
1. Cavia porcellus experiment
6 of male guinea pigs, every about 200g of body weight is provided by Chinese Academy of Sciences's Shanghai animal center.
The antigen protein of getting the 1mg purification suspends with the 1ml sterilized water, and add 0.1% SDS and promote protein dissolution, be added drop-wise to while grinding that (lanoline: paraffin oil=3: 7), be prepared into vaccine, it is subcutaneous to be injected into Cavia porcellus under the percutaneous in the water in oil freund 's incomplete adjuvant of 1ml.In 1 month, Cavia porcellus is continued to observe, and gather serum and detect.
Cavia porcellus does not go into a coma after the vaccinate.And in 1 month observation phase, 6 Cavia porcelluss all survive, and detect the antibody of not only resisting O-type but also anti-A type foot and mouth disease virus in the blood.
2. mouse experiment
3 of male mice in kunming, every about 20g of body weight is provided by Chinese Academy of Sciences's Shanghai animal center.
The antigen protein of getting the 1mg purification suspends with the 1ml sterilized water, and the SDS of adding 0.l% promotes that albumen fully dissolves.Get 100 μ l solution, mice is carried out subcutaneous injection.In 1 week, mice is observed, and gathered serum and detect.
In the observation phase in 1 week, 3 mices all survive.
Sequence table
＜110〉Shanghai Huayi Bio-Lab Co., Ltd
＜120〉two valency polypeptide vaccines of a kind of foot and mouth disease and its production and use
<160>2
<170>PatentIn version 3.1
<210>1
<211>117
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the design of O type and A type FMDV immunologic determinants, the forward polypeptide expressed
<400>1
ggttctggtg ttcgtcgtga tttcggttct ctggcgccgc gtgttgcgcg tcagctgacc 60
aacaacgttc gtggtgatct gcaggttctg gcgcagaaag cggaacgtac cctgccg 117
<210>2
<211>117
<212>DNA
＜213〉artificial sequence
<220>
<222>DNA
＜223〉according to O type and the design of A type FMDV immunologic determinants, reverse polypeptide expressed
<400>2
ccgctgaccc gtgaagcgaa acaggcgctg gttcagctgg atggtcgtgt taacaacacc 60
ctgcagcgtg cggttcgtcc ggcgctgtct ggtttcgatc gtcgtgttgg ttctggt 117
Claims (22)
1. two valency polypeptide vaccines of a foot and mouth disease, it contains 2
N-1The SEQ IDNO:1 of individual polyphone, or 2
N-1Nucleotide sequence encoded polypeptide shown in the SEQ ID NO:2 of individual polyphone, wherein n is the integer of 1-5.
2. according to claim 1 pair of valency polypeptide vaccine, it contains 2
N-1The SEQ ID NO:1 of individual polyphone, or 2
N-1Nucleotide sequence encoded polypeptide shown in the SEQ ID NO:2 of individual polyphone, wherein n is the integer of 2-4.
3. according to claim 2 pair of valency polypeptide vaccine, it contains: 2
N-1The SEQ I D NO:1 of individual polyphone, or 2
N-1Nucleotide sequence encoded polypeptide shown in the SEQ ID NO:2 of individual polyphone, wherein n is 3.
4. according to claim 2 pair of valency polypeptide vaccine, it contains the coded albumen of nucleotide sequence shown in the SEQ ID NO:1 of 4 polyphones.
5. according to claim 2 pair of valency polypeptide vaccine, it contains the coded albumen of nucleotide sequence shown in the SEQ ID NO:2 of 4 polyphones.
6. according to claim 4 pair of valency polypeptide vaccine, it contains the expressed albumen of plasmid that carries among the bacterial strain CGMCC No.0893.
7. according to claim 5 pair of valency polypeptide vaccine, it contains the expressed albumen of plasmid that carries among the bacterial strain CGMCC No.0894.
8. according to each described pair of valency polypeptide vaccine of claim 1-7, wherein said vaccine also comprises pharmaceutically acceptable carrier, adjuvant or immunological adjuvant.
9. according to claim 8 pair of valency polypeptide vaccine, wherein said immunological adjuvant is a freund 's incomplete adjuvant.
10. according to claim 8 pair of valency polypeptide vaccine, wherein said immunological adjuvant are Al (OH)
3
11. the preparation method of described pair of valency polypeptide vaccine of a claim 1 comprises 2
N-1The SEQ ID NO:1 of individual polyphone, or 2
N-1The step that nucleotide sequence shown in the SEQ ID NO:2 of individual polyphone is cloned into carrier and expresses in expression system, wherein n is the integer of 1-5.
12. preparation method according to claim 11, it comprises 2
N-1The SEQ ID NO:1 of individual polyphone, or 2
N-1The step that nucleotide sequence shown in the SEQ ID NO:2 of individual polyphone is cloned into carrier and expresses, wherein n is the integer of 2-4.
13. preparation method according to claim 12, it comprises 2
N-1The SEQ ID NO:1 of individual polyphone, or 2
N-1The step that nucleotide sequence shown in the SEQ ID NO:2 of individual polyphone is cloned into carrier and expresses, wherein n is 3.
14. according to each described method of claim 11-13, wherein said nucleotide sequence is expressed in prokaryotic expression system.
15. according to each described method of claim 11-13, wherein said nucleotide sequence is expressed in eukaryotic expression system.
16. an engineering strain, its plasmid that carries contains 2
N-1The SEQ IDNO:1 of individual polyphone, or 2
N-1Nucleotide sequence shown in the SEQ ID NO:2 of individual polyphone, wherein n is the integer of 1-5.
17. bacterial strain according to claim 16, its plasmid that carries contains 2
N-1The SEQ ID NO:1 of individual polyphone, or 2
N-1The SEQ ID NO:2 of individual polyphone, wherein n is the integer of 2-4.
18. bacterial strain according to claim 17, its plasmid that carries contains 2
N-1The SEQ ID NO:1 of individual polyphone, or
2n-1The SEQ ID NO:2 of individual polyphone, wherein n is 3.
19. bacterial strain according to claim 17, it is the genetic engineering bacterium of CGMCC No.0893 for preserving number.
20. bacterial strain according to claim 17, it is the genetic engineering bacterium of CGMCC No.0894 for preserving number.
21. the purposes of the described vaccine of claim 1 in preparation prevention and treatment A type and O type foot and mouth disease medicine.
22. the purposes of the described vaccine of claim 1 in preparation differentiation infection animal and immunized animal reagent.
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| PCT/CN2004/001016 WO2005021035A1 (en) | 2003-09-03 | 2004-09-03 | A bivalent vaccine against fmd, its preparation methods and applications |
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|---|---|---|---|---|
| CN100425291C (en) * | 2005-04-06 | 2008-10-15 | 中国农业科学院兰州兽医研究所 | O-type foot-and-mouth disease virus poly-gene duplication defect type adenovirus active carrier vaccine and process for preparing the same |
| CN101659695B (en) * | 2008-08-27 | 2012-08-29 | 中牧实业股份有限公司 | O-type aftosa synthetic peptide vaccine |
| CN101643500B (en) * | 2009-05-19 | 2012-06-06 | 中牧实业股份有限公司 | Asia I synthetic peptide vaccine of foot and mouth disease |
| CN102274496B (en) * | 2010-06-12 | 2015-05-06 | 吴晓琰 | O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose |
| CN102380095A (en) * | 2010-09-03 | 2012-03-21 | 吴晓琰 | FMD trivalence polypeptide vaccine and preparation method and application thereof |
| CN102772797B (en) * | 2011-05-13 | 2013-11-06 | 吴晓琰 | Genetic engineering polypeptide vaccine for 4 subtypes of O-type foot and mouth disease virus and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1319670A (en) * | 2001-03-17 | 2001-10-31 | 深圳市三方圆信息技术有限公司 | Foot-and-mouth disease virus gene engineered vaccine of domestic animal and preparation process thereof |
| GB2377017A (en) * | 2001-06-28 | 2002-12-31 | Animal Health Inst | Detection of foot and mouth disease virus |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1054544C (en) * | 1993-10-21 | 2000-07-19 | 复旦大学 | Polypeptide vaccine for aftosa and its preparation |
| FR2810888B1 (en) * | 2000-06-29 | 2004-07-30 | Merial Sas | VACCINE AGAINST FOOT AND MOUTH DISEASE |
| CN1287858C (en) * | 2002-09-16 | 2006-12-06 | 复旦大学 | Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method |
-
2003
- 2003-09-03 CN CNB031507514A patent/CN100381170C/en not_active Expired - Lifetime
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- 2004-09-03 WO PCT/CN2004/001016 patent/WO2005021035A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1319670A (en) * | 2001-03-17 | 2001-10-31 | 深圳市三方圆信息技术有限公司 | Foot-and-mouth disease virus gene engineered vaccine of domestic animal and preparation process thereof |
| GB2377017A (en) * | 2001-06-28 | 2002-12-31 | Animal Health Inst | Detection of foot and mouth disease virus |
Non-Patent Citations (1)
| Title |
|---|
| 牛O-A型口蹄疫双价灭活苗研究--小白鼠接种疫苗后的细胞免疫应答. 王永录等.中国兽医科技，第27卷第1期. 1997 * |
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| WO2005021035A1 (en) | 2005-03-10 |
| CN1589901A (en) | 2005-03-09 |
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