CN101293098A - Recombined cattle O type foot and mouth disease virus amalgamation protein vaccine - Google Patents
Recombined cattle O type foot and mouth disease virus amalgamation protein vaccine Download PDFInfo
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Abstract
The invention provides a recombinant foot-and-mouth disease virus fusion protein vaccine designed on the basis of O type foot-and-mouth disease virus. Experiments prove that the vaccine of the invention which is produced by applying the genetic engineering technology has good effects for the prevention and treatment of foot-and-mouth disease virus infection. The invention provides an amino acid sequence of the recombinant foot-and-mouth disease virus fusion protein vaccine, a nucleotide sequence thereof and a research on the prevention of foot-and-mouth disease virus infection.
Description
Technical field
The present invention relates to the genetic engineering field, relate to the genetic engineering recombinant protein vaccine particularly, particularly relate to a kind of recombined foot and mouth disease virus amalgamation protein vaccine that prevents the infection of animal foot and mouth disease virus and preparation method thereof.
Background technology
Foot and mouth disease (Foot and mouth disease, FMD) be a kind of wild and domestic cattle that mainly betides, sheep, the disease of the hyperinfection of the acute general of cloven-hoofed animal such as pig, (Foot-and-mouth disease virus FMDV) is the pathogen that causes that this is sick to foot and mouth disease virus.The FMDV circulation way mainly comprises digestive tract, respiratory tract, skin and mucosa.The infection rate of animal almost reaches 100%, and fervescence to 40 behind the zoogenetic infection～41 ℃ are sluggish.After 1～2 day, blister appears in the oral cavity, simultaneously or after a while, and between toe, on the soft skin of coronet blister takes place also.Animal stops in the production of meat and milk during one's sickness, meat and milk yield are long-term after being ill reduces and plants with being worth to lose and also can cause bigger loss.Because FMDV has bigger economic impact to relevant farming, animal husbandry, prevention and treatment foot and mouth disease are the problems that the whole world is paid close attention to always.
Foot and mouth disease virus belongs to Picornaviridae (Picornaviridae) aphthovirus genus (Aphthovirus), is the pathogen of cloven-hoofed animal hyperinfection disease foot and mouth disease.Foot and mouth disease virus comprises seven serotypes: A type, O type, C type, Asia 1, SAT1, SAT2, SAT3, variation of genome height and no cross reaction (Margarita S á iz, et al.2002) between each serotype.
Behind the FMDV infection animal, mainly excite specific humoral immunoresponse(HI).Humoral immunization protection body avoids similar antigenic infect again (J.S.Sallt, 1993).The level of neutralizing antibody and protective effect are closely related in the serum.Can detect the neutralizing antibody in the serum behind infection or the immune FMDV soon.Initial infection (3-4 days) IgM at first improves, and IgG begins to improve subsequently, is to infect interior main neutralizing antibody (MargaritaS á iz, et al.2002) of 2 weeks of back.The infection of FMDV or immunity also can activated t cell immunoreation.Experiment all proves in the body of pig and cattle: B cell-stimulating and production of antibodies usually are accompanied by the cell-mediated lymphocytic hyperplasia of CD4+T; these complementary T cells can be discerned the immune epitope on FMDV structural protein or the non-structural protein; also be at the requisite composition of the protective immunity of FMDV, play the secretion that promotes neutralizing antibody, the effect of adjusting the immunoreation microenvironment.Simultaneously, the infection of FMDV has also mediated the expression of infection cell surface MHCI quasi-molecule, influence the surperficial virus antigen peptide of FMDV infection cell to the offering of CTLs, thereby makes the virus escape host's CTL cell killing (F.Sobrino, 2001).
The FMDV vaccine of using mainly is inactivated vaccine and attenuated vaccine at present.Attenuated vaccine adopts the mode of attenuated virus, and mother's poison is passed through Cavia porcellus, and Embryo Gallus domesticus or cell kind make virus producer sudden change gradually, poison strain a little less than producing through polybasic cultivation reduction.Through after a large amount of amplifications, adding adjuvant etc. is made vaccine to weak poison strain in tissue culture.The FMD attenuated vaccine can produce the protection of certain level in the process of carrying out immunoprophylaxis, FMD popular had certain control action.But because attenuated vaccine is live virus, in premunitive process, may cause animal to carry the state of virus, in the impressibility object in the process of long-term surviving, the virulence that the FMDV strain of weak poison is very possible have been recovered to weaken.Simultaneously, weak viral disease poison also can have the effect of inhibition to the protection immunity that produces in the intravital propagation of susceptible animal, can not reach best immune effect.Therefore, the application of attenuated vaccine is restricted.After inactivated vaccine is a large amount of amplicon virus, handle, add the vaccine product for preparing behind the adjuvant through chemical ablation.The immunogenicity of FMDV inactivated vaccine is better, and owing to the use and the incomplete probability of inactivation of virus of strong poison in the preparation process, potential insecurity influences its use.So,, urgently seek a kind of new safely and effectively FMDV vaccine in order to overcome attenuated vaccine and the inactivated vaccine hidden danger on safety.
The recombined foot-and-mouth disease virus protein vaccine is to adopt the means of gene clone that the recombiant protein of preparing is cloned, recombinates, expressed to the antigen encoding gene of foot and mouth disease virus.Because recombinant protein vaccine only contains the part of pathogen, can not cause the animal morbidity, aspect safety, improve a lot.In addition, can prepare a plurality of compositions of same virus or multivalent virus vaccine as required.Therefore, the recombined foot-and-mouth disease virus protein vaccine has broad application prospects.
Summary of the invention
A kind of recombined foot and mouth disease virus amalgamation protein vaccine that using gene engineering technique provided by the invention is produced in escherichia coli has preventive and therapeutic effect to the infection of O type foot and mouth disease virus.
Among the present invention, use recombined foot and mouth disease virus amalgamation protein vaccine immune guinea pig and cattle, the serum that the immunity back obtains is in neonatal rat and in the experiment, have good in and the effect of O type foot and mouth disease virus.
Recombined foot and mouth disease virus amalgamation protein vaccine provided by the invention is different from traditional deactivation foot and mouth disease virus vaccine on the mode of production, and has the incomparable advantage of traditional deactivation foot and mouth disease virus vaccine in safety.
The invention still further relates to this recombined foot and mouth disease virus amalgamation protein vaccine and be used for preventing and treating the purposes of the biological product that the animal foot and mouth disease virus infects and the vaccine product that contains this genetic engineering recombiant protein in preparation.
The invention still further relates to subject, application process, pharmaceutical carriers and the application dosage of this recombined foot and mouth disease virus amalgamation protein vaccine.
The implication of term among the present invention
In the context of the present invention, employed term generally has the implication of those of ordinary skill in the art's common sense unless otherwise indicated.Especially, following term has following implication:
Foot and mouth disease virus infects the relevant disease that causes: be the contagious disease of being suffered from altogether by the cloven-hoofed animal that foot and mouth disease virus causes.Foot and mouth disease virus is a kind of RNA viruses, and its genome comprises 75008000 bases approximately.Foot and mouth disease virus is mainly propagated in the mode of saliva, and the speed of propagation is very fast, easily causes large-area popular.Cattle, pig, sheep, deer etc. are the infection animal of foot and mouth disease virus.
Recombinant protein vaccine: be to utilize molecule clone technology, the gene clone that coding is had immunogenic albumen or polypeptide is on protokaryon or eukaryotic expression vector, have immunogenic protein or polypeptide with this carrier what antibacterial or eukaryotic cell (yeast cells or mammalian cell) were produced, this protein or polypeptide can stimulate body (human or animal) that specific humoral immunoresponse(HI) and/or cellullar immunologic response take place after being applied to body.If on protokaryon or eukaryotic expression vector, is recombinant protein vaccine with antivirus action in antibacterial or eukaryotic cell (yeast cells or mammalian cell) production by viral gene encoded protein matter or polypeptide with it with the gene clone with immunogenic albumen or polypeptide of virus as foot-and-mouth disease virus gene coding.This vaccine can induce body that specific humoral immunoresponse(HI) and/or cellullar immunologic response take place after being applied to body (human or animal), prevents the infection that virus causes as foot and mouth disease virus.
Nucleic acid vaccine: being also referred to as dna vaccination, is the recombinant mammalian expressing vector that carries the antigen gene that can cause protective immunological reaction.After the injection, dna vaccination can be by the picked-up of muscle cell and dendritic cell, stops the time of growing with the form of exchromosomal DNA in cell, and gives expression to corresponding proteins matter, and the latter stimulates the immune system generation immunne response of body.
Adjuvant: adjuvant (adjuvant) is can the immunogenic material of enhancement antigen after using together with antigen.Include but not limited to: the heat shock protein of the composition of aluminium adjuvant, oily adjuvant, colloidal solid adjuvant, various bacteria or bacteria cell wall (the pertussis bacillus venenosus that kills, the muramyldipeptide of mycobacteria, bacteria lipopolysaccharide), Fu Shi Freund's complete adjuvant, antibacterial, the DNA of antibacterial, the oligonucleotide that contains CpG of synthetic, the activator of Toll sample receptor.
Nonspecific immunity strengthening agent: the preparation that is meant the non-special enhancing individual immunity reaction of energy, these preparations include but not limited to bacillus calmette-guerin vaccine (Bacillis Calmette-Gruen, BCG), coryne bacterium parvum (Corynabacterium parvum, staphylococcus aureus, the CD40 part, bacterial ribosome extract (Ribosomal fractions), bacterial membrane extract (membranar bacterial fractions), bacterial membrane proteoglycanes, polyarginine, HSP-60, HSP-70, HSP-90 and CTLA-4 inhibitor etc.
Pathogen carrier bacterin: be that gene with a certain proteantigen of coding changes the virus of attenuation or antibacterial over to and the vaccine made.The pathogen carrier bacterin after being applied to human body, can be in vivo propagation and the gene expression of proteantigen become corresponding proteins matter, the latter stimulates human body generation immunne response.The proteantigen encoding gene kind of a kind of encoding gene of two or more proteantigens of pathogen, two kinds or a plurality of pathogen can be changed over to a kind of pathogen and make the pathogen carrier bacterin.Vaccinia virus (vacciniavirus) is the carrier of using always, has been used to the development of recombinant vector vaccines such as hepatitis A virus, hepatitis B virus, Measles virus, herpes simplex virus.In addition, adenovirus, canary pox virus (canarypox virus), the poliovirus of attenuation, attenuated typhoid bacillus, bacillus calmette-guerin vaccine also can be used as the carrier of pathogen carrier bacterin.Some pathogen carrier bacterin is that the cholera of carrier and dysentery oral vaccine can be through natural pathogenic infection approach inoculations as adopting attenuated typhoid bacillus, and can secretion inducing type IgA, shows tangible advantage.
The materia medica acceptable carrier: materia medica acceptable carrier (pharmaceutically acceptable carrier) is meant filler, diluent or the encapsulating substance of one or more solids or liquid, and this carrier is fit to recombined foot and mouth disease virus amalgamation protein vaccine provided by the invention is applied to individuality.This carrier can be organic, inorganic, natural or synthetic.This carrier comprises various solution, diluent, solvent, dispersant, liposome, emulsifying agent, antibacterial, antifungal, isoosmotic and the reagent of delayed absorption and the recombined foot and mouth disease virus amalgamation protein vaccine application carrier among other suitable the present invention.The selection of materia medica acceptable carrier is decided by the application mode of recombined foot and mouth disease virus amalgamation protein vaccine provided by the invention among the present invention.The carrier that injectable is used comprises water, normal saline, PBS buffer (phosphate buffered saline solution), balanced salt solution, glucose solution, glycerol and emulsifying agent and wetting agent etc.Emulsifying agent can comprise oil-water emulsifiers (oil/wateremulsion) and water-in-oil emulsifier (o water/oil emulsion).
Treatment effective dose: be meant the individual dosage that produces the recombined foot and mouth disease virus amalgamation protein vaccine of desirable NAT after application.This " dosage " how much be decided by the standard technique that those skilled in the art should be known, also to comprise being not limited to individual size, the order of severity of health condition and disease etc. with reference to other factors.For stiffening effect, can carry out 1 time booster immunization in 14 or 21 days in the immunity back first time.Those skilled in the art can be done 10 times to 1000 times adjustment to dosage.
Route of administration: when using, the recombined foot and mouth disease virus amalgamation protein vaccine among the present invention can reach ideal effect via various suitable route of administration (Route).Recombined foot and mouth disease virus amalgamation protein vaccine among the present invention can be used through intestinal external administration approach, and these approach comprise subcutaneous, in the muscle, abdominal cavity, sheath, injection in Intradermal and the lymph node.
Sample before the last nickel affinity column: " the going up the preceding sample of nickel affinity column " among the present invention is meant the antibacterial cracking supernatant that contains reorganization O type foot and mouth disease virus amalgamation protein vaccine prokaryotic expression carrier of embodiment 3.
Stream is worn sample: " stream is worn sample " among the present invention is meant in embodiment 3, the antibacterial cracking supernatant application of sample that contains reorganization O type foot and mouth disease virus amalgamation protein vaccine prokaryotic expression carrier is during to the nickel metal chelating column, begins to finish the sample of flow through the nickel metal chelating and the post of collection to application of sample from application of sample.
Cleaning mixture is washed the post sample: when " cleaning mixture is washed the post sample " among the present invention is meant in embodiment 3 and washes nickel metal chelating and post with lavation buffer solution, and the lavation buffer solution of flow through the nickel metal chelating and the post of collection.
Elution samples (containing destination protein): " elution samples (containing destination protein) " among the present invention is meant in embodiment 3, when elution buffer is flowed through nickel metal chelating after the cleaning mixture washing and post, and the sample of collection (containing destination protein).
Description of drawings
Swimming lane one: the visible target DNA fragment in about 189bp place
Swimming lane two: DNA Marker 2000
Accompanying drawing 2 explanations: reorganization O type foot and mouth disease virus amalgamation protein N fragment coding gene electrophoretogram
Swimming lane one: the visible target DNA fragment in about 174bp place
Swimming lane two: DNA Marker 2000
Accompanying drawing 3 explanations: reorganization O type foot and mouth disease virus amalgamation protein A fragment coding gene dimer electrophoretogram
Swimming lane one: DNA Marker 2000
Swimming lane two: the visible target DNA fragment in about 348bp place
Accompanying drawing 4 explanations: reorganization O type foot and mouth disease virus amalgamation protein A fragment coding gene dimer (AA) and N fragment coding gene Fusion gene electrophoretogram
Swimming lane one: DNA Marker 2000
Swimming lane two: the visible target DNA fragment in about 504bp place
Accompanying drawing 5 explanations: reorganization O type foot and mouth disease virus amalgamation protein encoding gene electrophoretogram
Swimming lane one: DNA Marker 2000
Swimming lane two: the visible target DNA fragment in about 1002bp place
Accompanying drawing 6 explanations: reorganization O type foot and mouth disease virus amalgamation protein encoding gene sub-clone is gone into expression vector restriction enzyme digestion and electrophoresis figure
Swimming lane one: DNA Marker 2000
Swimming lane two: the visible endonuclease bamhi in about 1014bp place
Accompanying drawing 7 explanations: the reorganization O type foot and mouth disease virus amalgamation protein SDS-PAGE electrophoretogram that contains reorganization pET-28a (+) plasmid escherichia coli expression
Swimming lane one: albumen Marker
Swimming lane two: before inducing
Swimming lane three: IPTG induces 3h.At the 41KD place is destination protein
Accompanying drawing 8 explanations: the Western hybridization that anti-His tag antibody is identified reorganization O type foot and mouth disease virus amalgamation protein
Swimming lane one: albumen Marker
Swimming lane two: before inducing
Swimming lane three: IPTG induces 3h.The visible positive hybridization band of Western hybridization.
Accompanying drawing 9 explanations: the SDS-PAGE electrophoresis that the recombined foot and mouth disease virus amalgamation protein purification is identified
Swimming lane one: albumen Marker
Swimming lane two: go up the preceding sample of nickel affinity column
Swimming lane three: stream is worn sample
Swimming lane four: cleaning mixture is washed the post sample
Swimming lane five: elution samples (containing destination protein)
Swimming lane six: sample 1 (containing destination protein) behind the desalination
Swimming lane seven: sample 2 (containing destination protein) behind the desalination
Accompanying drawing 10 explanations: the ELISA of Cavia porcellus immune serum foot and mouth disease virus specific antibody detects
Abscissa is represented the time after the immunity, and vertical coordinate is represented the OD value of chromogenic reaction in the ELISA detection.
Meander line M represents meansigma methods.On behalf of this Cavia porcellus, PBS inject PBS merely as negative control.
Below in conjunction with concrete preparation embodiment and biology effect embodiment, and the present invention is described in further detail with reference to accompanying drawing.These embodiment are in order to demonstrate the invention, but not limit the scope of the invention by any way.
The specific embodiment
In following embodiment, various processes of Xiang Ximiaoshuing and method are not conventional methods as known in the art, for example Molecular Cloning one book (J.Sambrook, Cold Spring Harbor Laboratory Press, Molecularcloning, 1989) described method.
The source of agents useful for same, strain, virus, animal in the embodiment of the invention:
Cobra venom endonuclease EcoRI, HindIII, BglII, BamHI and T4DNA ligase, alkali phosphatase are available from Dalian TAKARA bio-engineering corporation; DNA reclaims test kit available from Beijing ancient cooking vessel state biotech company; IPTG, anti-histidine monoclonal antibody are available from Sigma company; The goat anti-rabbit antibody of horseradish peroxidase-labeled, the anti-Cavia porcellus antibody of the rabbit of horseradish peroxidase-labeled are available from Beijing ancient cooking vessel state Bioisystech Co., Ltd; Nitrocellulose filter (NC) is available from U.S. Schleicher﹠amp; Schuell company; Kanamycin, ampicillin are purchased in Beijing Huamei Bio-Engrg Co..Metal is integrated chromatography media (Sepharose-6B), exclusion chromatography medium (polydextran gel Sephadex-G-25) etc. all available from Pharmacia company.
The pMD-18T plasmid is purchased the bio-engineering corporation in Dalian TAKARA; The PET28a plasmid is purchased the company in Novagen.
Cavia porcellus: available from the clean laboratory animal of the high-new Experimental Animal Center in Changchun.
Experiment is provided by Inner Mongol Jin Yu biotech firm experimental center with neonatal rat, cattle.
Cattle Asia I type, O type foot and mouth disease virus bivalence inactivated vaccine, O type foot and mouth disease virus (Jiamusi strain) are provided by Inner Mongol Jin Yu biotech firm experimental center.
The structure of embodiment 1 reorganization O type foot and mouth disease virus amalgamation protein vaccine encoding gene
1.1 the structure of reorganization O type foot and mouth disease virus amalgamation protein vaccine A fragment coding gene
Adopt two to take turns the synthetic reorganization of PCR method O type foot and mouth disease virus amalgamation protein vaccine encoding gene dna fragmentation A.Designed and synthesized PCR primer (it is synthetic that worker's biological engineering company limited is given birth in Shanghai) with following sequence:
Primer 1:5 ' TCTTCAGGTTCTGGCTCAGAAAGCTGCTCGTACTCTGCCAGGTGGCGAAGAAAACT ACGGTGGT 3 '
Primer 2: 5 ' GAAGGATACGTCGGTGTGCTGACGACGCTGAACCTGAGCTTCACCACCGTAGTTTT CTT 3 '
Primer 3:5 ' GAATTCAGATCTGGTGGCCCGGTAACCAACGTTCGTGGTGATCTTCAGGTTCTGGC TC 3 '
Primer 4:5 ' AAGCTTGGATCCCGGAGTTACTTTAACGAAACGGTCCAGGATGAAGGATACGTCGG TGT 3 '
With primer 1, primer 2 each other template be first round PCR.First round PCR reaction condition: 94 ℃, 45s; 58 ℃, 30S; 74 ℃, 10min.
As template, primer 3, primer 4 are primer, do second and take turns PCR with the PCR product that obtains.
Second takes turns the PCR reaction condition: 94 ℃, and 45s; 58 ℃, 30S; 74 ℃, 1min; 25 circulations, 25 circulations are extended 10min for back 72 ℃.
Take turns the PCR product with second and do 2% agarose gel electrophoresis (Fig. 1), the purpose fragment length is 189bp.Adopt the DNA of Beijing ancient cooking vessel state company to reclaim the test kit recovery synthetic recombined foot and mouth disease virus amalgamation protein encoding gene of PCR dna fragmentation A, and it is cloned into pMD18-T Vect plasmid (TakaRa company).There is the encode pMD18-T Vect plasmid of basic dna fragmentation A of recombined foot and mouth disease virus amalgamation protein to be transformed into e. coli jm109 (Novagen company) clone.Select positive colony, extract plasmid, enzyme action is identified direction, measures and inserts segmental DNA sequence (worker's biological engineering company limited is given birth in Shanghai), and sequencing result is correct.
1.2 the structure of reorganization O type foot and mouth disease virus amalgamation protein vaccine N fragment coding gene
Adopt two to take turns the synthetic reorganization of PCR method O type foot and mouth disease virus amalgamation protein vaccine encoding gene dna fragmentation N.Designed and synthesized PCR primer (it is synthetic that worker's biological engineering company limited is given birth in Shanghai) with following sequence:
Primer 1:5 ' GTTACTAACGTTCGTGGCGATCTGCAGGTTCTGGCTCAGAAGGCAGCACGTACTCT G 3 '
Primer 2: 5 ' TTTCTGTTTATGACGAGCTTCAGACGGTTCACCACCCGGCAGAGTACGTGCTGCCT T 3 '
Primer 3:5 ' GAATTCAGATCTGGTTCCTCTAAATACGGCGAGTCTCCGGTTACTAACGTTCGTGG C 3 '
Primer 4:5 ' AAGCTTGGATCCCAGCAGTTGCTTAACTGGAGCAACGATTTTCTGTTTATGACGAG C 3 '
With primer 1, primer 2 each other template be first round PCR.First round PCR reaction condition: 94 ℃, 45s; 60 ℃, 30S; 74 ℃, 10min.
As template, primer 3, primer 4 are primer, do second and take turns PCR with the PCR product that obtains.
Second takes turns the PCR reaction condition: 94 ℃, and 45s; 55 ℃, 30S; 74 ℃, 1min; 25 circulations, 25 circulations are extended 10min for back 72 ℃.
Take turns the PCR product with second and do 2% agarose gel electrophoresis (Fig. 2), the purpose fragment length is 174bp.Adopt the DNA of Beijing ancient cooking vessel state company to reclaim the test kit recovery synthetic recombined foot and mouth disease virus amalgamation protein encoding gene of PCR dna fragmentation N, and it is cloned into pMD18-T Vect plasmid (TakaRa company).There is the encode pMD18-T Vect plasmid of basic dna fragmentation N of recombined foot and mouth disease virus amalgamation protein to be transformed into e. coli jm109 (Novagen company) clone.Select positive colony, extract plasmid, enzyme action is identified direction, measures and inserts segmental DNA sequence (worker's biological engineering company limited is given birth in Shanghai), and sequencing result is correct.
1.3 the structure of reorganization O type foot and mouth disease virus amalgamation protein vaccine A fragment coding gene dimer (called after AA)
With being loaded with pMD18-T carrier BglII, the BamHI double digestion of A fragment coding gene, obtain the segmental encoding gene of A; To be loaded with the pMD18-T carrier BglII single endonuclease digestion of A fragment coding gene, obtain containing the pMD18-T linear carrier of A fragment coding gene; Then the above-mentioned pMD18-T linear carrier that contains A fragment coding gene is connected with the T4DNA ligase with the A fragment, has both obtained to contain the pMD18-T carrier of AA fragment coding gene.Change the host cell amplification over to, BglII, BamHI double digestion identify that the purpose fragment length is 348bp (Fig. 3) behind the extraction plasmid.
1.4 the structure of reorganization O type foot and mouth disease virus amalgamation protein vaccine A fragment coding gene dimer and N fragment coding gene Fusion gene (called after AAN)
With being loaded with pMD18-T carrier BglII, the BamHI double digestion of AA fragment coding gene, obtain the segmental encoding gene of AA; To be loaded with the pMD18-T carrier BglII single endonuclease digestion of N fragment coding gene, obtain containing the pMD18-T linear carrier of N fragment coding gene; Then the above-mentioned pMD18-T linear carrier that contains N fragment coding gene is connected with the T4DNA ligase with the AA fragment, has both obtained to insert the pMD18-T carrier of AAN fragment coding gene.Change the host cell amplification over to, BglII, BamHI double digestion identify that the purpose fragment length should be 504bp (Fig. 4) behind the extraction plasmid.
1.5 the structure of reorganization O type foot and mouth disease virus amalgamation protein vaccine encoding gene (dimer of AAN)
With being loaded with pMD18-T carrier BglII, the BamHI double digestion of AAN fragment coding gene, obtain the segmental encoding gene of AAN; To be loaded with the pMD18-T carrier BglII single endonuclease digestion of AAN fragment coding gene, obtain containing the pMD18-T linear carrier of AAN fragment coding gene; Then the above-mentioned pMD18-T linear carrier that contains AAN fragment coding gene is connected with the T4DNA ligase with the AAN fragment, has both obtained to contain the pMD18-T carrier of AAN dimer encoding gene.Change the host cell amplification over to, BglII, BamHI double digestion identify that the purpose fragment length should be 1002bp (Fig. 5) behind the extraction plasmid.
To insert the pMD18-T carrier EcoR I of AAN dimer encoding gene, the HindIII double digestion obtains the dimeric encoding gene of AAN; With pET28a plasmid EcoR I, the HindIII double digestion had both obtained the pET28a linear carrier.The fragment that above-mentioned two steps are obtained is connected with the T4DNA enzyme with carrier, has both obtained to contain the pET28a carrier [called after pET28a-(AAN) 2] of AAN dimer encoding gene.Change the host cell amplification over to, EcoRI, HindIII double digestion enzyme action identify that the purpose fragment length should be 1014bp (Fig. 6) behind the extraction plasmid.
Expression, evaluation and the purification of embodiment 3 reorganization O type foot and mouth disease virus amalgamation protein vaccines
PET28a-(AAN) 2 recombinant expression plasmids are changed in the BL21 recipient bacterium, after colony screening, evaluation, the expression of higher level is arranged with IPTG induced protein J8.(Fig. 7)
Plasmid pET28a has one section histidine tail (His tag) at the segmental rear end of insertion purpose, termination codon front end, selects anti-His tag antibody that corresponding protein is identified.The result shows that expressing protein can be illustrated that we induce and the albumen of successful expression is corresponding target albumen (Fig. 8) by anti-His tag antibody recognition.
His tag can combine with metal ion nickel specificity, so select the method purification destination protein of metal ion Ni2+ sequestration affinity chromatograph.Adorn post (20mM phosphate, pH 7.2 1M NaCl) with phosphate buffer, chromatography media is Sepharose4B-Ni2+ (Pharmacia) and balance.The antibacterial cracking supernatant that will contain recombined foot and mouth disease virus amalgamation protein vaccine J8 prokaryotic expression carrier is added in the nickel metal chelating column, and collects stream and wear sample.With lavation buffer solution (20mM Tris, pH 7.9,0.5M NaCl, the 20mM imidazoles, 3M carbamide is washed post, collects the lavation buffer solution wash behind the post.With elution buffer (20mM Tris, pH7.9,0.5M NaCl, 1M imidazoles, 2M carbamide) eluting, collect the albumen of eluting.Till absorption value no longer descends, access the effluent of different periods with container.
Desalination
Also wash post with Sephadex 625 (Pharmacia) dress post with the distilled water of 2 bed volumes.
With the 10mM PBS of 2 bed volumes, after pH7.2 washes post, behind the albumen upper prop with nickel affinity column eluting, reuse 10mM PBS, the pH7.2 upper prop, the albumen of Fractional Collections eluting is preserved in-70 ℃ of lyophilizing after the packing.
With the SDS-PAGE electrophoresis reorganization O type foot and mouth disease virus amalgamation protein of purification is identified that behind the prokaryotic expression carrier expressed proteins purification of nucleotide sequence shown in wherein containing, its purity reaches 95% (Fig. 9).
The ELISA of embodiment 4 guinea pig serum O type foot and mouth disease virus specific antibodies detects
With PBS dissolving reorganization O type foot and mouth disease virus amalgamation protein vaccine, concentration is 800 μ g/mL, with equal-volume oil adjuvant (MONTANIDE ISA 206 oily adjuvants, Seppic, France) uniform mixing.Get the healthy male guinea pig about 5 body weight 250g, vastus medialis meat injection, 4 Cavia porcellus immunity reorganization O type foot and mouth disease virus amalgamation protein vaccines wherein, dosage be 200 μ g/500 μ L/ only, 1 injection equal-volume PBS is as negative control.After immunity the 3rd day respectively gathered the guinea pig serum sample in 7 days, 14 days, 21 days, 29 days, 36 days, 43 days, 71 days, 85 days.
Detect the level of O type foot and mouth disease virus specific antibody in the serum with the indirect ELISA method of Jiamusi strain foot and mouth disease virus of deactivation bag quilt.Method is as follows: with coating buffer (PBS:8g/L NaCl, 0.2g/L KCl, 3.58g/L Na2HPO412H2O, 0.24g/L KH2PO4; 0.8% glutaraldehyde) Jiamusi strain hoof-and-mouth disease venom of dilution deactivation (dilution in 1: 4), 100 μ L/ holes add ELISA Plate, seal plate, 4 ℃ of bags were by 14-16 hour; Cleaning mixture (PBS; 0.05%Tween-20) washing is dull and stereotyped three times, 300 μ L/ holes, 3 minutes/time; Add confining liquid (PBS; 5%FBS), 2h is placed for 37 ℃ in 200 μ L/ holes; Washing is dull and stereotyped once more; With sample diluting liquid (PBS; 5%FBS) dilution animal serum adds serum 100 μ L/ holes, dilution back, places 1h for 37 ℃; The dull and stereotyped back of washing resists (dilution in 1: 5000), 100 μ L/ holes with the anti-Cavia porcellus two of rabbit of sample diluting liquid dilution horseradish peroxidase labelling once more.Lucifuge is placed 1h for 37 ℃; Behind the washing flat board, add the substrate solution (citric acid 0.01M among the 10ml, Na2HPO40.02M, ultra-pure water 9ml, 3,0%H,2O2 15 μ L, OPD4mg) every hole, 100 μ L/ holes, the room temperature lucifuge 20min that now join; Every hole adds 50 μ L stop buffers (20% sulphuric acid).Detect OD value (A492) at once.
The indirect ELISA result shows: the serum of reorganization O type foot and mouth disease virus amalgamation protein vaccine immune guinea pig contains at the specific antibody of O type foot and mouth disease virus (Figure 10).
For the serum that detects reorganization O type foot and mouth disease virus amalgamation protein vaccine immune guinea pig under the condition that O type foot and mouth disease virus is attacked to the protective effect of animal, carried out the neonatal rat virus neutralization tests.
After exempting from 21 days guinea pig serum and two and exempt from 21 days guinea pig serum and carry out serial dilution and heat inactivation complement one, mix with equal-volume O type foot and mouth disease virus (200 ID50), after carrying out the neutralization reaction of 1h, inject in the neonatal rat body, observe 24 hours, 48 hours, 72 hours neonatal rat survival condition, with the negative contrast of the guinea pig serum of PBS immunity, with O type foot and mouth disease virus 2 immunity positive contrasts of 21 days guinea pig serum of inactivated vaccine (table 1).In the table 1,1-A table is to exempt from 21 days guinea pig serum neonatal rats and result of experiment; 1-B table is two to exempt from 21 days guinea pig serum neonatal rats and result of experiment.
In the neonatal rat and experimental result show: it is specific at O type foot and mouth disease virus antibody that reorganization O type foot and mouth disease virus amalgamation protein vaccine induces Cavia porcellus to produce, can in and O type foot and mouth disease virus, the protection neonatal rat avoids the attack of O type foot and mouth disease virus.
Table 1: the guinea pig serum of reorganization O type foot and mouth disease virus protein vaccine immunity is to the protective effect of the neonatal rat of O type foot and mouth disease virus attack
1-A
In the table: OJ3-1, OJ3-2, OJ3-3, OJ3-4 representative are through once 21 days 4 Cavia porcelluss of immunity of O type foot and mouth disease virus protein vaccine of recombinating; 2 Cavia porcelluss of equal-volume PBS immunity as negative control are used in PBS-1, PBS-2 representative.Inactivated vaccine-4 is represented 21 days Cavia porcellus of 2 immunity of O type foot and mouth disease virus inactivated vaccine.
1--B
In the table: OJ3-1, OJ3-2, OJ3-3, OJ3-4 representative are through 4 Cavia porcelluss of 21 days of reorganization O type foot and mouth disease virus protein vaccine secondary immunity; Inactivated vaccine-4 is represented 21 days Cavia porcellus of 2 immunity of O type foot and mouth disease virus inactivated vaccine.
The ELISA that embodiment 6 reorganization O type foot and mouth disease virus amalgamation protein vaccine immune cattles produce O type foot and mouth disease virus specific antibody detects
With PBS dissolving reorganization O type foot and mouth disease virus amalgamation protein vaccine, making it concentration is 1.6mg/0.92ml, gets 5.52ml and oily adjuvant (MONTANIDE ISA 206 oily adjuvants, Seppic, France) 6.48ml uniform mixing.Get the young cattle of 5 healthy 8 months big or small anti-FMDV negative antibodies in pure pastoral area, the mode immunity of intragluteal injection, the dosage of every cattle immunizing antigen solution is the 1.6mg/2mL/ head.Cattle Asia I/O FMDV bivalent inactivated vaccine is made positive control.Get dilution with 0.04M PBS buffer solution, carry out emulsifying with 206 adjuvants and do the solvent contrast.Respectively at 0 day and 28 days immune 2 times.Exempt to gather in back 14 days the solcoseryl sample two.Jugular vein blood sampling, the blood of collection place 37 ℃ to place 15min, place 2h, fully separate out serum for 4 ℃; The centrifugal 10min of 4000r/min, the centrifugal 4min of 11000r/min, careful sucking-off serum ,-70 ℃ of preservations are standby.
The indirect ELISA method that wraps quilt with Jiamusi strain O type foot and mouth disease virus of deactivation detects O type foot and mouth disease virus specific antibody level (concrete grammar is with reference to embodiment 4) in the serum.The indirect ELISA result shows: produced in the serum of the young cattle of reorganization O type foot and mouth disease virus amalgamation protein vaccine immunity high titre at the specific antibody of O type foot and mouth disease virus.(table 2).
Table 2: the ELISA that reorganization O type foot and mouth disease virus amalgamation protein vaccine immune cattle produces O type foot and mouth disease virus specific antibody detects
In the table: OJ3 representative reorganization O type foot and mouth disease virus amalgamation protein vaccine; Inactivated vaccine is represented cattle Asia I/O FMDV bivalent inactivated vaccine.Each digitized representation a head of cattle in the cattle hurdle.
For the serum that detects the young cattle of reorganization O type foot and mouth disease virus amalgamation protein vaccine immunity under the condition that homotype O type foot and mouth disease virus is attacked to the protective effect of animal, carried out the neonatal rat virus neutralization tests.Back 14 days young cattle (6-18 month) of back 21 days of immunity for the first time of O type foot and mouth disease virus amalgamation protein vaccine and inactivated vaccine and the immunity for the second time serum heat inactivation complement of will recombinating respectively carries out serial dilution, with equal-volume O type foot and mouth disease virus (200 ID
50) mix, carry out the neutralization reaction of 1h after, inject in the neonatal rat body, carry out cultivation and the observation (table 3) of 72h.Show with experimental result in the neonatal rat: produced at the specific antibody of O type foot and mouth disease virus in the serum of the young cattle of reorganization O type foot and mouth disease virus amalgamation protein vaccine immunity.
Table 3: the Ox blood serum of reorganization O type foot and mouth disease virus amalgamation protein vaccine immunity is to the protective effect of the neonatal rat of foot and mouth disease virus attack
A
B
In the table: OJ3 representative reorganization O type foot and mouth disease virus amalgamation protein vaccine; Inactivated vaccine is represented cattle Asia I/O FMDV bivalent inactivated vaccine.Each digitized representation a head of cattle in the cattle hurdle.The PBS representative group of equal-volume PBS immunity as negative control.Other neonatal rat of serum matched group is the neonatal rat that the relevant serum of only injection does not give virus.Normal healthy controls group neonatal rat is not give any experiment to handle the neonatal rat that the raising condition is identical with experimental group.
The research that embodiment 8 reorganization O type foot and mouth disease virus amalgamation protein vaccine immune cattle prevention cattle O type foot and mouth disease virus infect
Laboratory animal
6-18 month in same breed, source healthy cattle, Asia I type, O type foot and mouth disease virus antibody are all negative in check serum.
The experiment grouping
Reorganization O type foot and mouth disease virus amalgamation protein vaccine group: mix water with 206 adjuvants through the dissolved reorganization of PBS O type foot and mouth disease virus amalgamation protein vaccine: oil phase=46: 54, reorganization O type foot and mouth disease virus amalgamation protein vaccine final concentration is 800 mcg/ml.2 milliliters/only/time, intragluteal injection.
Cattle foot and mouth disease bivalence inactivated vaccine positive controls: commercialization Niu YazhouI type, O type foot and mouth disease virus bivalence inactivated vaccine, 2 milliliters/only/time, intragluteal injection.
PBS negative control group: PBS, 2 milliliters/only/time, intragluteal injection.
The counteracting toxic substances experiment
In each group the 28th day after immunity, injection O type foot and mouth disease virus Jiamusi strain under the cow tongue dough cover, 10000ID50/0.2ml.The 10th day judgement incidence behind injecting virus.Except that the lingual surface of injecting virus, blister appears in gingiva, oral cavity, any position of four hoof, can be judged as morbidity.Blister appears in few three hoofs of the Adeps Bovis seu Bubali of negative control group, can judge counteracting toxic substances experiment establishment.The results are shown in Table 4.Experimental result shows, has the effect that the prevention cattle O type foot and mouth disease virus infects behind the reorganization O type foot and mouth disease virus amalgamation protein vaccine immune cattle, and its preventive effect and commercialization Niu YazhouI type, O type foot and mouth disease virus bivalence inactivated vaccine are suitable.
Table 4: reorganization O type foot and mouth disease virus amalgamation protein vaccine immune cattle prevention cattle O type foot and mouth disease virus infects
| The experiment grouping | Cattle (only) does not fall ill | Morbidity cattle (only) | Protective rate |
| Reorganization O type foot and mouth disease virus amalgamation |
4 | 1 | 80％ |
| Commercialization Niu YazhouI type, O type foot and mouth disease virus bivalence inactivated vaccine |
4 | 1 | 80％ |
| The PBS |
0 | 3 | 0 |
Sequence table
＜110〉Beijing DiWeiHuaYu Biological Technology Co., Ltd
＜120〉a kind of recombined cattle O type foot and mouth disease virus amalgamation protein vaccine
<160>2
<210>1
<211>1152
<212>DNA
＜213〉artificial sequence
<400>1
ATGGGCAGCA GCCATCATCA TCATCATCAC AGCAGCGGCC TGGTGCCGCG CGGCAGCCAT 60
ATGGCTAGCA TGACTGGTGG ACAGCAAATG GGTCGCGGAT CCGAATTCAG ATCTGGTGGC 120
CCGGTAACCA ACGTTCGTGG TGATCTTCAG GTTCTGGCTC AGAAAGCTGC TCGTACTCTG 180
CCAGGTGGCG AAGAAAACTA CGGTGGTGAA GCTCAGGTTC AGCGTCGTCA GCACACCGAC 240
GTATCCTTCA TCCTGGACCG TTTCGTTAAA GTAACTCCGG GATCTGGTGG CCCGGTAACC 300
AACGTTCGTG GTGATCTTCA GGTTCTGGCT CAGAAAGCTG CTCGTACTCT GCCAGGTGGC 360
GAAGAAAACT ACGGTGGTGA AGCTCAGGTT CAGCGTCGTC AGCACACCGA CGTATCCTTC 420
ATCCTGGACC GTTTCGTTAA AGTAACTCCG GGATCTGGTT CCTCTAAATA CGGCGAGTCT 480
CCGGTTACTA ACGTTCGTGG CGATCTGCAG GTTCTGGCTC AGAAGGCAGC ACGTACTCTG 540
CCGGGTGGTG AACCGTCTGA AGCTCGTCAT AAACAGAAAA TCGTTGCTCC AGTTAAGCAA 600
CTGCTGGGAT CTGGTGGCCC GGTAACCAAC GTTCGTGGTG ATCTTCAGGT TCTGGCTCAG 660
AAAGCTGCTC GTACTCTGCC AGGTGGCGAA GAAAACTACG GTGGTGAAGC TCAGGTTCAG 720
CGTCGTCAGC ACACCGACGT ATCCTTCATC CTGGACCGTT TCGTTAAAGT AACTCCGGGA 780
TCTGGTGGCC CGGTAACCAA CGTTCGTGGT GATCTTCAGG TTCTGGCTCA GAAAGCTGCT 840
CGTACTCTGC CAGGTGGCGA AGAAAACTAC GGTGGTGAAG CTCAGGTTCA GCGTCGTCAG 900
CACACCGACG TATCCTTCAT CCTGGACCGT TTCGTTAAAG TAACTCCGGG ATCTGGTTCC 960
TCTAAATACG GCGAGTCTCC GGTTACTAAC GTTCGTGGCG ATCTGCAGGT TCTGGCTCAG 1020
AAGGCAGCAC GTACTCTGCC GGGTGGTGAA CCGTCTGAAG CTCGTCATAA ACAGAAAATC 1080
GTTGCTCCAG TTAAGCAACT GCTGGGATCC AAGCTTGCGG CCGCACTCGA GCACCACCAC 1140
CACCACCACT GA 1152
<210>2
<211>383
<212>PRT
＜213〉artificial sequence
<400>2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val 15
Pro Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met 30
Gly Arg Gly Ser Glu Phe Arg Ser Gly Gly Pro Val Thr Asn Val 45
Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Thr Leu 60
Pro Gly Gly Glu Glu Asn Tyr Gly Gly Glu Ala Gln Val Gln Arg 75
Arg Gln His Thr Asp Val Ser Phe Ile Leu Asp Arg Phe Val Lys 90
Val Thr Pro Gly Ser Gly Gly Pro Val Thr Asn Val Arg Gly Asp 105
Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Thr Leu Pro Gly Gly 120
Glu Glu Asn Tyr Gly Gly Glu Ala Gln Val Gln Arg Arg Gln His 135
Thr Asp Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro 150
Gly Ser Gly Ser Ser Lys Tyr Gly Glu Ser Pro Val Thr Asn Val 165
Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Thr Leu 180
Pro Gly Gly Glu Pro Ser Glu Ala Arg His Lys Gln Lys Ile Val 195
Ala Pro Val Lys Gln Leu Leu Gly Ser Gly Gly Pro Val Thr Asn 210
Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Thr 225
Leu Pro Gly Gly Glu Glu Asn Tyr Gly Gly Glu Ala Gln Val Gln 240
Arg Arg Gln His Thr Asp Val Ser Phe Ile Leu Asp Arg Phe Val 255
Lys Val Thr Pro Gly Ser Gly Gly Pro Val Thr Asn Val Arg Gly 270
Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Thr Leu Pro Gly 285
Gly Glu Glu Asn Tyr Gly Gly Glu Ala Gln Val Gln Arg Arg Gln 300
His Thr Asp Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr 315
Pro Gly Ser Gly Ser Ser Lys Tyr Gly Glu Ser Pro Val Thr Asn 330
Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Thr 345
Leu Pro Gly Gly Glu Pro Ser Glu Ala Arg His Lys Gln Lys Ile 360
Val Ala Pro Val Lys Gln Leu Leu Gly Ser Lys Leu Ala Ala Ala 375
Leu Glu His His His His His His 383
Claims (16)
1. the recombined foot and mouth disease virus amalgamation protein vaccine produced of a using gene engineering method, the nucleotide sequence of this recombiant vaccine of encoding is shown in SEQ NO.1, and its aminoacid sequence is shown in SEQ NO.2.
2. be the protide vaccine of core texture with the described aminoacid sequence of claim 1, the protide vaccine here is meant the protide vaccine of adopting brave following a kind of method or several method Joint Production:
A) recombinant protein vaccine that suddenlys change with reference to the described aminoacid sequence of claim 1.The sudden change here is meant that N-terminal, middle part, the C-terminal at recombinant protein vaccine as claimed in claim 1 carries out amino acid whose point mutation or a plurality of amino acid whose sudden change.
B) make amendment with reference to the nucleotide sequence of the described recombined foot and mouth disease virus amalgamation protein vaccine of claim 1 and the recombinant protein vaccine for preparing.Here the 5` that " modification " is meant at the nucleotide of sequence shown in the SEQ NO.1 holds the deletion of the nucleotide of mid portion or/and 3` holds or/and add the nucleotide of other sequence, or/and the nucleotide of sequence shown in the SEQNO.1 is carried out change single or that the part base is done.
C) with the recombinant protein vaccine of the described aminoacid sequence of claim 1 a) or b) described in recombinant protein vaccine be core texture, at its N-terminal or/and the recombinant protein vaccine that C-terminal adds or the deletion aminoacid sequence is produced.
D) with the recombinant protein vaccine of the described aminoacid sequence of a plurality of claim 1 a) or b) or c) described in the recombinant protein vaccine recombiant protein class vaccine that is connected in series and produced.
3. the nucleic acid vaccine of nucleotide sequence that contains this protein vaccine of aminoacid sequence corresponding codes of arbitrary described recombinant protein vaccine in claim 1 or the claim 2.
4. be loaded with claim 1 or claim 2 in the pathogen carrier bacterin of nucleotide sequence of this protein vaccine of aminoacid sequence corresponding codes of arbitrary described recombinant protein vaccine.
5. arbitrary described vaccine can stimulate Cavia porcellus to produce specificity resisting O-type foot and mouth disease virus antibody after immunity in the application rights requirement 1,2,3,4.
6. arbitrary described vaccine can stimulate cattle to produce specificity resisting O-type foot and mouth disease virus antibody after immunity in the application rights requirement 1,2,3,4.
7. the arbitrary specificity foot-and-mouth disease virus resistant antibody described in claim 5 or the claim 6 is in the neonatal rat neutralization test of foot and mouth disease virus, can in and O type foot and mouth disease virus, neonatal rat is had protective effect.
8. arbitrary described vaccine can protect cattle to avoid infection and the attack of O type foot and mouth disease virus behind immune cattle in the application rights requirement 1,2,3,4.
9. arbitrary described vaccine is used to prepare and is used to prevent and treat the purposes that O type foot and mouth disease virus or O type hoof-and-mouth disease virus mutants infect the vaccine of the relevant disease that causes in the claim 1,2,3,4.
10. in the described purposes of claim 9, O type foot and mouth disease virus infect to as if cattle, or sheep, or pig, perhaps deer, or the artiodactyl of other types, or people.
11. according to the described purposes of claim 9, the vaccine that wherein is used to prevent and treat the foot and mouth disease virus infection is when being applied to the described object of claim 10, its application dose is a medicine effective dose.
12. according to the described purposes of claim 9, the vaccine that wherein is used to prevent and treat the foot and mouth disease virus infection can be used with the carrying of materia medica acceptable carrier when being applied to the described object of claim 10.
13. according to the described purposes of claim 9, wherein be used to prevent and treat vaccine that foot and mouth disease virus infects when being applied to the described object of claim 10 and adjuvant or/and the nonspecific immunity strengthening agent use in conjunction.
14. according to the described purposes of claim 9, wherein be used to prevent and treat the vaccine of foot and mouth disease virus infection when being applied to the described object of claim 10, with one or more other foot-and-mouth disease vaccine use in conjunction, " other foot-and-mouth disease vaccine " here includes but not limited to recombined foot-and-mouth disease virus protein vaccine, nucleic acid vaccine, the pathogen carrier bacterin at foot-and-mouth disease virus Asia I and mutation thereof.
15., wherein be used to prevent and treat vaccine that foot and mouth disease virus infects when being applied to the described object of claim 10 according to the described purposes of claim 9, to use through intestinal external administration approach, that these approach comprise is subcutaneous, Intradermal, muscle, abdominal cavity.
16. arbitrary described vaccine is used to prepare and is used to prevent and treat and purposes that O type foot and mouth disease virus has the foot and mouth disease virus of other type of cross-reacting antigen to infect the vaccine of the relevant disease that causes in the claim 1,2,3,4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100981002A CN101293098A (en) | 2007-04-28 | 2007-04-28 | Recombined cattle O type foot and mouth disease virus amalgamation protein vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100981002A CN101293098A (en) | 2007-04-28 | 2007-04-28 | Recombined cattle O type foot and mouth disease virus amalgamation protein vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101293098A true CN101293098A (en) | 2008-10-29 |
Family
ID=40063807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100981002A Pending CN101293098A (en) | 2007-04-28 | 2007-04-28 | Recombined cattle O type foot and mouth disease virus amalgamation protein vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101293098A (en) |
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| CN101948510A (en) * | 2010-09-10 | 2011-01-19 | 中国人民解放军第二军医大学 | Molecular mimic peptide of type O foot and mouth disease virus VP2 epitope and applications thereof |
| CN102372766A (en) * | 2011-07-13 | 2012-03-14 | 青岛宝麦德生物医药科技有限公司 | O-type foot-and-mouth disease multi-epitope vaccine with cross immunity protective efficiency |
| CN103339140A (en) * | 2011-01-28 | 2013-10-02 | 米迪缪尼有限公司 | Expression of soluble viral fusion glycoproteins in mammalian cells |
| CN103849637A (en) * | 2012-11-29 | 2014-06-11 | 于力 | O-type foot and mouth disease virus acid-resistant mutant strain, capsid protein carried by O-type foot and mouth disease virus acid-resistant mutant strain, coding gene of capsid protein and use of O-type foot and mouth disease virus acid-resistant mutant strain and capsid protein |
| WO2017091418A1 (en) * | 2015-11-23 | 2017-06-01 | Merial, Inc. | Fmdv and e2 fusion proteins and uses thereof |
| CN109851663A (en) * | 2019-03-14 | 2019-06-07 | 天津威特生物医药有限责任公司 | Method for purifying His-tag marked foot-and-mouth disease epitope protein antigen by nickel-combined nano magnetic beads |
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2007
- 2007-04-28 CN CNA2007100981002A patent/CN101293098A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101948510A (en) * | 2010-09-10 | 2011-01-19 | 中国人民解放军第二军医大学 | Molecular mimic peptide of type O foot and mouth disease virus VP2 epitope and applications thereof |
| CN101948510B (en) * | 2010-09-10 | 2012-08-29 | 中国人民解放军第二军医大学 | Molecular mimic peptide of type O foot and mouth disease virus VP2 epitope and applications thereof |
| CN103339140A (en) * | 2011-01-28 | 2013-10-02 | 米迪缪尼有限公司 | Expression of soluble viral fusion glycoproteins in mammalian cells |
| CN102372766A (en) * | 2011-07-13 | 2012-03-14 | 青岛宝麦德生物医药科技有限公司 | O-type foot-and-mouth disease multi-epitope vaccine with cross immunity protective efficiency |
| CN103849637A (en) * | 2012-11-29 | 2014-06-11 | 于力 | O-type foot and mouth disease virus acid-resistant mutant strain, capsid protein carried by O-type foot and mouth disease virus acid-resistant mutant strain, coding gene of capsid protein and use of O-type foot and mouth disease virus acid-resistant mutant strain and capsid protein |
| CN103849637B (en) * | 2012-11-29 | 2015-12-23 | 于力 | The acidproof mutant strain of O type foot and mouth disease virus, its capsid protein carried and encoding gene thereof and application |
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| KR20180084995A (en) * | 2015-11-23 | 2018-07-25 | 메리얼 인코포레이티드 | FMDV and E2 fusion proteins and uses thereof |
| WO2017091418A1 (en) * | 2015-11-23 | 2017-06-01 | Merial, Inc. | Fmdv and e2 fusion proteins and uses thereof |
| EP3539566A1 (en) * | 2015-11-23 | 2019-09-18 | Boehringer Ingelheim Animal Health USA Inc. | Fmdv and e2 fusion proteins and uses thereof |
| AU2016361305B2 (en) * | 2015-11-23 | 2020-02-06 | Boehringer Ingelheim Animal Health USA Inc. | FMDV and E2 fusion proteins and uses thereof |
| RU2714428C2 (en) * | 2015-11-23 | 2020-02-14 | Мериал, Инк. | Fmdv-e2 fusion proteins and use thereof |
| KR102153303B1 (en) | 2015-11-23 | 2020-09-09 | 뵈링거 잉겔하임 애니멀 헬스 유에스에이 인코포레이티드 | FMDV and E2 fusion proteins and uses thereof |
| JP2020203890A (en) * | 2015-11-23 | 2020-12-24 | ベーリンガー インゲルハイム アニマル ヘルス ユーエスエイ インコーポレイテッド | Fmdv and e2 fusion proteins and uses thereof |
| CN108367067B (en) * | 2015-11-23 | 2022-05-03 | 勃林格殷格翰动物保健美国公司 | Fusion proteins of FMDV and E2 and their uses |
| CN109851663A (en) * | 2019-03-14 | 2019-06-07 | 天津威特生物医药有限责任公司 | Method for purifying His-tag marked foot-and-mouth disease epitope protein antigen by nickel-combined nano magnetic beads |
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