CN110256539A - O-shaped foot and mouth disease virus novel gene engineering subunit vaccine - Google Patents

O-shaped foot and mouth disease virus novel gene engineering subunit vaccine Download PDF

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Publication number
CN110256539A
CN110256539A CN201910620895.1A CN201910620895A CN110256539A CN 110256539 A CN110256539 A CN 110256539A CN 201910620895 A CN201910620895 A CN 201910620895A CN 110256539 A CN110256539 A CN 110256539A
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mouth disease
seq
sequence
amino acid
albumen
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CN110256539B (en
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曹文龙
孔迪
滕小锘
易小萍
张大鹤
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Suzhou Womei Biology Co ltd
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Suzhou Shino Biotechnology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

Present invention discloses a kind of immune composition, it includes: O-shaped Foot-and-mouth disease VP3 and VP1 albumen, and, O-shaped Foot-and-mouth disease VP2 and/or VP4 albumen.Further, the immune composition may also include O-shaped Foot-and-mouth disease VP0.The immune composition can be used for preparing O-shaped foot and mouth disease virus novel gene engineering subunit vaccine, the antigenicity of the vaccine, immunogenicity and function are similar to native protein, expression is higher, immunogenicity is strong, it is not pathogenic to animal, and vaccine of the invention can be prepared by the extensive serum free suspension culture of bioreactor, greatly reduce production of vaccine cost.

Description

O-shaped foot and mouth disease virus novel gene engineering subunit vaccine
Technical field
The present invention relates to animal immune technical field of pharmaceuticals, in particular to a kind of O-shaped foot and mouth disease virus novel gene Engineering subunit vaccine.
Background technique
Aftosa (foot-and-mouth disease, FMD) is one kind by foot and mouth disease virus (foot and mouth Disease virus, FMDV) disease caused by artiodactyls (such as pig, ox, sheep, deer etc.) is infected, it is characterized in that infected The area skins such as mouth, the foot of artiodactyls will appear bubble, to cause Some Animals dead.
FMDV belongs to Picornaviridae (Picornaviridae) aphthovirus genus (Aphthovirus), is cloven-hoofed dynamic The cause of disease of object highly contagious disease (aftosa).It is a single-stranded positive chain RNA at the center of virus, by about 8000 alkali Base composition is the basis of infection and heredity；Surrounding is wrapped in protein and determines that the antigenicity, immunity and serology of virus are anti- It should be able to power；Virus coat is symmetrical 20 face body.The immune of FMDV is the B cell response for relying on T cell, and vaccine inoculation mainly lures Lead the generation of neutralizing antibody.FMDV includes the blood of the not no Cross immunogenicity of A, O, C, Asia1 and SAT1, SAT2, SAT3 etc. 7 Clear type forms a hypotype more than 80 again in long-term mutual propagation infection, brings severe challenge for the prevention and control of aftosa.
The animal to catch a foot and mouth disease will appear fever, limping and occur the symptoms such as blister macula on skin and skin and mucosa. For domestic animals, the symptom of aftosa is mainly the high fever of rapid decrease after two to three days；Blister in mouthful leads to viscosity or foam The excessive secretion of shape saliva and trickling exports outer；The bubble of foot is possible to rupture and leads to maimed person.It can after adults infection Energy can be with the testis swelling for the weight loss and bull animal that the several months can not restore, for cow, milk crop meeting It significantly reduces.Although most of animal obtains can be self-healing after being ill, myocarditis (heart flesh also may cause when the disease is serious Voluptuousness dye) or it is dead, it is especially such for new born animal.Some infected animals may not have symptom, they are not in hair It burns or any disease symptoms；But these animals are also aftosa carrier, they can equally infect the disease.
The infection of aftosa is generally region, and virus is dynamic to easy infection by directly contacting infected zoochory Object, or pass through contaminated animal house or the lorry of Transport Animal.Ill domestic animal and with poison poultry be the main infection sources, they can lead to Immediate contagion is crossed, and can be infected by mediate contact (such as secretion, excreta, livestock products, the air of pollution, feed Deng) it is transmitted to susceptible animal.Water that the coat or skin, animal contact of the care of animal people (such as farm hand) is crossed and uncooked Food debris and feed addictive comprising infection animal product all may be the carrying source of virus.
Past, aftosa once occur in many areas in the whole world (including Europe, Africa, Asia and South Americas), the disease it is big Scope spreading and rapidly propagation cause the extensive concern of international community.World Organization for Animal Health is listed in A class animal First of disease list, it is the first that the Chinese government is also come a kind of zoonosis.Pork production and marketing of the China as the first in the world State, Pork Security problem substantial connection the benign sustainable development of China's animal husbandry.Now with molecular biology and it is immunized Make constant progress, and the new generation vaccine that cost performance is high, safety is good is also imperative.
Having some researchs at present is to express tri- albumen of VP1, VP3 and VP0 respectively using Escherichia coli, then external group Dress, the method protein purification difficulty is big, and the outer packaging efficiency of proteosome is very low；Separately having some researchs is using multiple baculovirals VP1, VP0 and VP3 albumen are expressed respectively, then with multiple viral coinfection Sf9 cells to co-express these three albumen, this side Method needs virus infection plural number very big, and the same cell needs while infection 2 or more viral, and it is very low that there are efficiency The problem of.
Summary of the invention
The present invention is intended to provide a kind of immune composition, to solve the problems of the prior art.
To achieve the goals above, according to an aspect of the invention, there is provided a kind of immune composition, it includes:
Using sequence nucleic acid molecules as shown in SEQ ID NO:1 or the nucleotide sequence 95% with SEQ ID NO:1 The O-shaped Foot-and-mouth disease VP3 albumen of the above identical nucleic acid molecule encoding；
Using sequence nucleic acid molecules as shown in SEQ ID NO:12 or the nucleotide sequence with SEQ ID NO:12 The O-shaped Foot-and-mouth disease VP1 albumen of 95% or more identical nucleic acid molecule encoding；
And selected from O-shaped Foot-and-mouth disease VP2 albumen or O-shaped Foot-and-mouth disease VP4 albumen One or two kinds of any combination.
The further technical solution of the present invention is: the O-shaped Foot-and-mouth disease VP3 albumen includes SEQ ID The amino acid sequence of NO:19 or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:19；
The O-shaped Foot-and-mouth disease VP1 albumen include SEQ ID NO:22 amino acid sequence or with The identical amino acid sequence of 95% or more full length amino acid sequence of SEQ ID NO:22.
The further technical solution of the present invention is: the immune composition also includes O-shaped Foot-and-mouth disease One of VP2, VP4 or two or more any combination；Wherein,
The O-shaped Foot-and-mouth disease VP2 albumen include SEQ ID NO:21 amino acid sequence or with The identical amino acid sequence of 95% or more full length amino acid sequence of SEQ ID NO:21；
The O-shaped Foot-and-mouth disease VP4 albumen include SEQ ID NO:23 amino acid sequence or with The identical amino acid sequence of 95% or more full length amino acid sequence of SEQ ID NO:23.
The further technical solution of the present invention is: where
The O-shaped Foot-and-mouth disease VP2 albumen is by sequence nucleic acid as shown in SEQ ID NO:8 point Son is obtained with the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:8；
The O-shaped Foot-and-mouth disease VP4 albumen is by sequence nucleic acid as shown in SEQ ID NO:16 Molecule is obtained with the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:16.
The further technical solution of the present invention is: the immune composition further includes O-shaped foot and mouth disease virus structure egg White VP0, the O-shaped Foot-and-mouth disease VP0 albumen are by sequence nucleic acid molecules as shown in SEQ ID NO:4 Or it is obtained with the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:4.
The further technical solution of the present invention is: the O-shaped Foot-and-mouth disease VP0 albumen includes SEQ ID The amino acid sequence of NO:20 or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:20.
The further technical solution of the present invention is: the immune composition is to include the O-shaped foot and mouth disease virus structure egg The composition of white VP3, VP1, VP2 albumen.The further technical solution of the present invention is: the immune composition is comprising the O The composition of type Foot-and-mouth disease VP3, VP1, VP4 albumen.The further technical solution of the present invention is: described immune Composition is the composition comprising O-shaped Foot-and-mouth disease VP3, VP1, VP2 and VP0 albumen.The present invention is into one The technical solution of step is: the immune composition is to include O-shaped Foot-and-mouth disease VP3, VP1, VP2, VP4 egg White composition.
The further technical solution of the present invention is: the immune composition is to include the O-shaped foot and mouth disease virus structure egg The composition of white VP3, VP1, VP4 and VP0 albumen.
The further technical solution of the present invention is: the immune composition is to include the O-shaped foot and mouth disease virus structure egg The composition of white VP3, VP1, VP2, VP4 and VP0 albumen.
Another object of the present invention is to provide a kind of preparation methods of immune composition, and the method includes following Step:
S1, recombination shuttle load will in the gene cloning of O-shaped Foot-and-mouth disease to corresponding shuttle vector, be obtained Body；
S2, recombinant shuttle vector is transformed into the DH10Bac bacterium containing Baculovirus Gene group, in recombinant shuttle vector Destination gene expression is confined to being inserted into rod-shaped viral genome plasmid, and the rod-shaped disease of the recombination containing destination gene expression frame is obtained Virus gene group plasmid；
S3, recombinant baculovirus geneome plasmid transfection insect cell is obtained into recombinant baculovirus；
S4, the recombinant baculovirus of acquisition is inoculated with insect cell, O-shaped foot and mouth disease virus is mass produced in the reactor Structural proteins；
S5, the O-shaped Foot-and-mouth disease obtained in S4 step is added in adjuvant to get the immune combination Object.
The further technical solution of the present invention is that step S1 includes: by O-shaped Foot-and-mouth disease VP3 and VP1 Encoding gene and in VP2, VP4, VP0 any one, the expression cassettes of two kinds or three kinds be cloned into same shuttle On carrier to get arrive the recombinant shuttle vector.
Preferably, step S1 includes: by the encoding gene of O-shaped Foot-and-mouth disease VP3 and VP1 and selected from O The expression cassette of any one or two kinds in type Foot-and-mouth disease VP2, VP4 is cloned on same shuttle vector, Obtain the recombinant shuttle vector.
It is furthermore preferred that step S1 includes: by the encoding gene of O-shaped Foot-and-mouth disease VP3 and VP1, selected from O-shaped The expression cassette and O-shaped Foot-and-mouth disease of any one or two kinds in Foot-and-mouth disease VP2, VP4 The expression cassette of VP0 is cloned on same shuttle vector to arrive the recombinant shuttle vector.
Especially preferred, the step S1 includes: by VP1 albumen, VP3 albumen, VP0 albumen, VP2 albumen, VP4 albumen Gene is cloned on same shuttle vector, obtains the recombinant shuttle vector.
The further technical solution of the present invention is: the shuttle vector is pFastBac Dual.
The further technical solution of the present invention is: it is thin that the insect cell is selected from Sf9, High Five, S2 or Sf21 cell Any one in born of the same parents, but not limited to this.
In previously-described implementation of the present invention, using a virus simultaneously co-express VP1 and VP3 albumen and selected from VP2, In VP4, VP0 any one, two or three albumen.More preferably, by using a virus simultaneously co-express VP1, Five albumen of VP3, VP2, VP4 and VP0.Wherein VP2 and VP4 can be from VP0 protein cleavage.VP1, VP3 and VP0 albumen It can be assembled into VLP automatically, VP1, VP3, VP2 and VP4 albumen can also be assembled into VLP automatically, therefore use a baculoviral Multiple albumen are co-expressed, so that the packaging efficiency of VLP greatly improves.
Another object of the present invention is to provide the immune compositions described in one kind for producing in animal subject Purposes of the induction for the medicament of the immune response of O-shaped foot-and-mouth disease virus antigen.
Another object of the present invention is to provide the immune compositions described in one kind for producing for preventing animal by O-shaped The purposes of the medicament of mouth disease virus infection.
Another object of the present invention is to provide a kind of nucleic acid molecules composition comprising: for encoding O-shaped hoof-and-mouth disease Malicious structural proteins VP3 albumen, the sequential nucleotide sequence comprising SEQ ID NO:1 or the nucleotides sequence with SEQ ID NO:1 The identical sequential nucleotide sequence of 95% or more column；
For encoding O-shaped Foot-and-mouth disease VP1 albumen, the sequential nucleotide sequence comprising SEQ ID NO:12 Or the identical sequential nucleotide sequence of 95% or more nucleotide sequence with SEQ ID NO:12.
Another object of the present invention is to provide nucleic acid molecules composition described in one kind for produce for it is tested move Purposes of the induction for the medicament of the immune response of O-shaped foot-and-mouth disease virus antigen in object.
Another object of the present invention is to provide nucleic acid molecules composition described in one kind for produce for preventing animal By the purposes of the medicament of O-shaped mouth disease virus infection.For example, the medicament can be O-shaped foot and mouth disease virus novel gene work Journey subunit vaccine.
Another object of the present invention is to provide a kind of protein composition comprising:
The identical albumen of 95% or more full length amino acid sequence of SEQ ID NO:19 and SEQ ID NO:19；SEQ ID The identical albumen of 95% or more full length amino acid sequence of NO:22 and SEQ ID NO:22.
In preferred technical solution, in step S3, culture medium used in fermented and cultured is secondary culture base.
The invention discloses a kind of Sf9 cell expression O-shaped foot and mouth disease virus recombinant subunit vaccine preparation method and Using, and prove that the vaccine can generate stronger humoral immunity in pig, ox body, the mammals energy such as pig, ox after being immunized The infection for enough resisting O-shaped foot and mouth disease virus belongs to animal vaccine and veterinary biologics technical field, and its purpose is to provide one Kind can large-scale industrial production O-shaped foot and mouth disease virus recombinant subunit vaccine preparation method.
After adopting the above scheme, the present invention has the advantages that following prominent and effect compared with prior art:
Antigenicity, immunogenicity and the function and day of the O-shaped foot and mouth disease virus novel gene engineering subunit vaccine of the present invention Right protein is similar, and expression is higher, and immunogenicity is strong, does not have pathogenic and of the invention vaccine that can lead to animal The extensive serum free suspension culture preparation of bioreactor is crossed, while greatly reducing production of vaccine cost.
Detailed description of the invention
The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:
Fig. 1 is that PCR product after VP3 protein gene PCR amplification is carried out gel electrophoresis result, it can be seen that near 0.7kbp There is a band；Wherein 1 is FMDV-VP3 gene, and 2 be negative control, and M is molecular weight marker；
Fig. 2 carries out the knot of gel electrophoresis for PCR product after the PCR amplification of the bacterium colony sample of multiple VP3 protein gene conversion Nearby there is positive sample in fruit, 0.7kbp band.Wherein 1~5 be VP3 protein gene conversion bacterium colony sample P CR amplification after Product, 6 be non-positive sample, M is molecular weight marker；
Fig. 3 is that PCR product after VP0 protein gene PCR amplification is carried out gel electrophoresis result, it can be seen that near 0.9kbp There is a band；Wherein 1 is FMDV-VP0 gene, and 2 be negative control, and M is molecular weight marker；
Fig. 4 carries out the knot of gel electrophoresis for PCR product after the PCR amplification of the bacterium colony sample of multiple VP0 protein gene conversion Nearby there is positive sample in fruit, 0.9kbp band.Wherein 1~5 be VP0 protein gene conversion bacterium colony sample P CR amplification after Product, 6 be non-positive sample, M is molecular weight marker；
Fig. 5 is that PCR product after VP2 protein expression frame PCR amplification is carried out gel electrophoresis result, it can be seen that 1.4kbp is attached Closely there is a band；Wherein 1 is FMDV-VP2 protein expression frame gene, and 2 be negative control, and M is molecular weight marker；
Fig. 6 carries out gel electrophoresis for PCR product after the PCR amplification of the bacterium colony sample of multiple VP2 protein expression frames conversion As a result, nearby there is positive sample in 1.4kbp band.Wherein 1~5 be VP2 protein expression frame conversion bacterium colony sample P CR expand Product after increasing, 6 be non-positive sample, and M is molecular weight marker；
Fig. 7 is that PCR product after VP1 protein expression frame PCR amplification is carried out gel electrophoresis result, it can be seen that 1.3kbp is attached Closely there is a band；Wherein 1 is FMDV-VP1 protein expression frame gene, and 2 be negative control, and M is molecular weight marker；
Fig. 8 carries out gel electrophoresis for PCR product after the PCR amplification of the bacterium colony sample of multiple VP1 protein expression frames conversion As a result, nearby there is positive sample in 1.3kbp band.Wherein 1~5 be VP1 protein expression frame conversion bacterium colony sample P CR expand Product after increasing, 6 be non-positive sample, and M is molecular weight marker；
Fig. 9 is that PCR product after VP4 protein expression frame PCR amplification is carried out gel electrophoresis result, it can be seen that 1.0kbp is attached Closely there is a band；Wherein 1 is FMDV-VP4 protein expression frame gene, and 2 be negative control, and M is molecular weight marker；
Figure 10 carries out gel electrophoresis for PCR product after the PCR amplification of the bacterium colony sample of multiple VP4 protein expression frames conversion As a result, nearby there is positive sample in 1.0kbp band.Wherein 1~5 be VP4 protein expression frame conversion bacterium colony sample P CR expand Product after increasing, 6 be non-positive sample, and M is molecular weight marker；
Figure 11 is the transfer vector Dual-VP3-VP0-VP2-VP1-VP4 map containing target gene of building；
Figure 12 is the cell culture supernatant progress PAGE gel electrophoresis harvested in embodiment 3 as a result, VP3- The cell culture of VP0-VP2-VP1-VP4 recombinant protein is respectively in molecular weight about 21kDa, 33kDa, 26kDa, 24kDa, 9kDa Nearby there is purpose band；Wherein 1 is negative control, and 2 cell culture to harvest in embodiment 3, M is molecular weight marker；
Figure 13 is the product WesternBlot testing result in embodiment 4 after SDS-PAGE electrophoresis；Wherein 1 is recombination bar Shape expressing viral sample, 2 be negative control, and M is molecular weight marker；
Figure 14 is Immunofluorescence test result；
Figure 15 is that Electronic Speculum observes result；
Figure 16 is the result after protein purification.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
The present invention provides a kind of immune compositions, include:
It is identical using the nucleic acid molecules of SEQ ID NO:1 or with 95% or more the nucleotide sequence of SEQ ID NO:1 The O-shaped Foot-and-mouth disease VP3 albumen of nucleic acid molecule encoding；
Using the nucleic acid molecules of SEQ ID NO:12 or identical as 95% or more the nucleotide sequence of SEQ ID NO:12 Nucleic acid molecule encoding O-shaped Foot-and-mouth disease VP1 albumen；
And selected from O-shaped Foot-and-mouth disease VP2 albumen or O-shaped Foot-and-mouth disease VP4 albumen One or two kinds of any combination.
A kind of method the present invention also relates to induction for the immune response of O-shaped foot-and-mouth disease virus antigen, the method packet It includes and applies vaccine of the invention to animal subject.
The present invention also relates to a kind of method for protecting animal subject from O-shaped mouth disease virus infection, the method includes Vaccine of the invention is applied to the animal subject.
The invention also includes the vaccines for being suitable for inducing the immune response for O-shaped foot and mouth disease virus.Vaccine of the invention It can be the plasmid comprising above-mentioned nucleic acid molecules.Nucleic acid molecules can be incorporated into that in virion.Vaccine can also include adjuvant molecules.Assistant Agent can for IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNF α, TNF β, GM-CSF, Epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33 or its Combination；It and in some embodiments, can be IL-12, IL-15, IL-28 or RANTES.
Vaccine of the invention includes protein molecule composition.Provided herein is the albumen selected from the group being made up of: including The albumen of SEQ ID NO:19 or 22 or 21 or 23 or 20；In the amino acid sequence of SEQ ID NO:19 or 22 or 21 or 23 or 20 Whole length on 95% identical albumen；The segment of SEQ ID NO:19 or 22 or 21 or 23 or 20；With SEQ ID NO:19 Or 22 or 21 or 23 or 20 the identical albumen of segment 95%.
A kind of protein composition selected from the group being made up of: (a) SEQ ID NO:19 or 22 or 21 is also provided herein Or 23 or 20；(b) long in the entire amino acid sequence of the full length sequence as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 Spend upper 95% identical albumen；(c) that SEQ ID NO:19 or 22 or 21 or 23 or 20 includes SEQ ID NO:19 or 22 or 21 Or 23 or 20 20 or more amino acid immunogenic fragments；And (d) in SEQ ID NO:19 or 22 or 21 or 23 Or 20 amino acid sequence whole length on 95% identical albumen the immunogenicity comprising 20 or more amino acid Segment.Certainly, a kind of protein composition is also provided herein, further includes selected from one of the group being made up of protein molecular Or the combination of multiple protein molecule: (a) SEQ ID NO:20 or 21 or 23；(b) in such as institute of SEQ ID NO:20 or 21 or 23 95% identical albumen on the entire length amino acid sequence for the full length sequence stated；(c) packet of SEQ ID NO:20 or 21 or 23 The immunogenic fragments of 20 or more the amino acid of the NO:20 or 21 or 23 of ID containing SEQ；And (d) in SEQ ID NO: 95% identical albumen includes exempting from for 20 or more amino acid in the whole length of 20 or 21 or 23 amino acid sequence Epidemic disease immunogenic fragment.
The present invention provides the nucleic acid molecules composition of the sequence comprising encoding one or more protein moleculars described above. In some embodiments, the nucleic acid molecules include the sequence selected from the group being made up of: SEQ ID NO:1 or 12 or 8 Or 16 or 4；The 95% identical nucleic acid sequence in the whole length of the nucleotide sequence of SEQ ID NO:1 or 12 or 8 or 16 or 4 Column；The segment of SEQ ID NO:1 or 12 or 8 or 16 or 4；It is identical as the segment 95% of SEQ ID NO:1 or 12 or 8 or 16 or 4 Nucleotide sequence.The nucleic acid molecules composition further includes selected from one of the group being made up of nucleic acid molecules Or the combination of multiple nucleic acid molecules: SEQ ID NO:4 or 8 or 16；In the nucleotide sequence of SEQ ID NO:4 or 8 or 16 95% identical nucleic acid sequence in whole length；The segment of SEQ ID NO:4 or 8 or 16；With SEQ ID NO:4's or 8 or 16 The identical nucleotide sequence of segment 95%.
Some aspects of the present invention provide the method for immune response of the induction for O-shaped foot and mouth disease virus, the method includes Following steps: O-shaped foot-and-mouth disease virus antigen and/or combination thereof object is applied to individual.
The other aspect of the present invention provides the method for protecting individuals from O-shaped mouth disease virus infection.The method includes Following steps: to the nucleic acid molecules or composition comprising such nucleic acid sequence of the individual application prevention effective dose；Wherein institute It states nucleic acid sequence to express in the cell of the individual, and is directed to and is exempted from by the protein induced protectiveness of the nucleic acid sequence encoding Epidemic disease reaction.
Some aspects of the invention provide a kind of method for inducing the immune response for O-shaped foot-and-mouth disease virus antigen, institute The method of stating includes that nucleic acid molecules of the invention are applied to animal subject.
Some aspects of the invention provide a kind of method for protecting animal subject from O-shaped mouth disease virus infection, described Method includes that nucleic acid molecules of the invention are applied to the animal subject.
Some aspects of the invention provide a kind of suitable for generating animal subject for the immune of O-shaped foot and mouth disease virus The vaccine of reaction, the vaccine includes: nucleic acid molecules and adjuvant molecules of the invention.The adjuvant can for IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNF α, TNF β, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-21, IL-31, IL-33 or combinations thereof；And in some implementations It can be IL-12, IL-15, IL-28 or RANTES in scheme.
Vaccine of the invention further includes one or more nucleic acid molecules as described above and one or more by the nucleic acid The albumen of molecule encoding.
1. defining
The term as used herein is not intended to limit merely for the sake of the purpose for describing specific embodiment.Such as illustrating Used in book and the claim, in addition to the other clear stipulaties of context, singular "one", "an" and " institute State " it include plural form.
For numberical range cited herein, clearly cover in the number for having each insertion between identical precision Word.For example, also covering number 7 and 8 other than 6 and 9 for the range of 6-9, and for the range of 6.0-7.0, clearly cover Number 6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9 and 7.0.
" adjuvant ", which means to be added in vaccine as described herein, as used herein enhances by coding core described below Any molecule of the immunogenicity of the encoded antigen of acid sequence.
As used herein " antibody " mean type IgG, IgM, IgA, IgD or IgE antibody or segment, its segment or Derivative, including Fab, F (ab') 2, Fd and single-chain antibody, double-chain antibody, bispecific antibody, bifunctional antibody and its spread out Biology.The antibody can be the antibody isolated from the serum sample of animal, polyclonal antibody, affinity purification antibody or its Mixture, to required epitope or derived from it, sequence shows enough binding specificities to the mixture.
" coded sequence " or " code nucleic acid " means the nucleic acid of the nucleotide sequence comprising coding albumen as used herein (RNA or DNA molecular).The coded sequence may further include the initial signal for being operably coupled to controlling element and end Stop signal, the controlling element include the promoter and more that expression can be instructed in the individual of administration of nucleic acid or the cell of animal Polyadenylation signal.
" complement " or " complementation " means that nucleic acid can refer to the nucleotide in nucleotide or nucleic acid molecules as used herein Watson-Crick (for example, A-T/U and C-G) or Hoogsteen base pairing between analog.
" shared " or " consensus sequence " means based on the team for analyzing specific O-shaped foot-and-mouth disease virus antigen as used herein The polypeptide sequence of multiple hypotypes of column.The nucleic acid sequence for encoding shared polypeptide sequence can be prepared.Wrapping protein-contg vaccine can be by For inducing a variety of hypotypes for being directed to specific O-shaped foot-and-mouth disease virus antigen or the extensive of serotype to be immunized, the vaccine includes to compile The consensus sequence and/or nucleic acid molecules of these albumen of code.
As " electroporation " used interchangeably herein, " electricity-permeabilization " or " electronic enhancing " (" EP ") mean using across Film electric field pulse induces the microcosmic approach (hole) in biomembrane；Their presence allows biomolecule such as plasmid, few core Thuja acid, siRNA, drug, ion and water flow to the other side from the side of cell membrane.
Mean that coding can be O-shaped with overall length wild-type strain relative to " segment " of nucleic acid sequence as used herein The nucleic acid sequence or part of it of the polypeptide triggered an immune response in the animal of foot-and-mouth disease virus antigen cross reaction.The segment It can be at least one DNA fragmentation selected from the various nucleotide sequences for encoding protein fragments described below.
For polypeptide sequence, " segment " or " immunogenic fragments " means can be O-shaped with overall length wild-type strain The polypeptide triggered an immune response in the animal of foot-and-mouth disease virus antigen cross reaction.The segment of albumen can wrap it is protein-contg at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% Or at least 95%.In some embodiments, the segment of albumen can wrap protein-contg at least 20 amino acid or more, at least 30 amino acid or more, at least 40 amino acid or more, at least 50 amino acid or more, at least 60 amino acid or more More, at least 70 amino acid or more, at least 80 amino acid or more, at least 90 amino acid or more, at least 100 ammonia Base acid or more, at least 110 amino acid or more, at least 120 amino acid or more, at least 130 amino acid or more, At least 140 amino acid or more, at least 150 amino acid or more, at least 160 amino acid or more, at least 170 ammonia Base acid or more, at least 180 amino acid or more, at least 190 amino acid or more, at least 200 amino acid or more, At least 210 amino acid or more, at least 220 amino acid or more, at least 230 amino acid or more or at least 240 Amino acid or more.
Term " genetic constructs " as used herein refers to DNA or RNA points of the nucleotide sequence comprising coding albumen Son.The coded sequence includes the initial signal and termination signal for being operably coupled to controlling element, the controlling element packet Include the promoter and polyadenylation signal that expression can be instructed in the cell of the individual of administration of nucleic acid molecule.Such as this paper institute Term " expression-form " refers to that gene construct, the gene construct contain the volume for being operably coupled to coding albumen The necessary controlling element of code sequence, so that coded sequence will express when in the cell for being present in the individual.
Term " homology " as used herein refers to complementary degree.Homoeology or complete homology may be present (that is, identity).At least partly inhibiting fully-complementary sequence to hybridize in the partial complementarity sequence of target nucleic acids is using function art Language " substantially homologous " refers to.It is when the double-strandednucleic acid sequence about such as cDNA or genomic clone in use, as used herein Term " substantially homologous " refer to that probe can hybridize under the conditions of property low strict in the chain of the double-strandednucleic acid sequence.When about single-stranded Nucleic acid sequence is in use, term " substantially homologous " as used herein refers to that probe can hybridize under the conditions of property low strict in list Chain sequence of template of nucleic acid (that is, being the complementary series of single stranded nucleic acid template sequence).
In the case where two or more nucleic acid or polypeptide sequence, " identical " or " identity " as used herein means Sequence has the prescribed percentage of identical residue in specified region.The percentage can be calculated by following: most preferably Compare two sequences, specified region compare two sequences, the quantity for the position for determining residue identical in the two sequences with Generate the quantity of matching position, the total quantity with the quantity of matching position divided by the position in specified region, and by result The percentage of sequence identity is generated multiplied by 100.There is different length in two sequences or compare generation one or more In the case that a staggered end and the specified region compared only include unique sequence, the residue of unique sequence is included in meter In the denominator of calculation rather than in molecule.When comparison dna and RNA, thymidine (T) and uracil (U) are considered With.Identity can be executed manually or by computer sequence algorithm such as BLAST or BLAST2.0 is used.
It " is immunoreacted " introducing for meaning that antigen is shared in response to for example O-shaped foot and mouth disease virus of antigen, place as used herein The activation of main immune system (such as immune system of animal).It is described immune response can be cell effect or humoral response or The form of the two.
" nucleic acid " or " oligonucleotides " or " polynucleotides " mean at least two be covalently joined together as used herein A nucleotide.Single-stranded description also defines the sequence of complementary strand.Therefore, nucleic acid also covers described single-stranded complementation Chain.Many variants of nucleic acid can be used for purpose identical with given nucleic acid.Therefore, nucleic acid also covers substantially the same Nucleic acid and its complement.The probe that single-stranded offer can hybridize under stringent hybridization conditions with target sequence.Therefore, nucleic acid is also Cover the probe hybridized under stringent hybridization conditions.
Nucleic acid can be single-stranded or double-strand or can be containing the part of both double-strand or single stranded sequence.The core Acid can be both DNA, genome and cDNA, RNA or heterozygote, wherein the nucleic acid can containing deoxyribonucleotide and The combination of ribonucleotide, and it is yellow including uracil, adenine, thymidine, cytimidine, the fast quinoline of bird, inosine, xanthine time The combination of the base of purine, iso-cytosine and isoguanine.Nucleic acid can by chemical synthesis process or pass through recombination side Method obtains.
The expression of gene be the promoter being spatially attached thereto control under carry out.At the control, promoter The upstream 5'(of gene can be positioned in) or the downstream 3'().The distance between the promoter and gene can about with institute It states promoter and its distance between the gene controlled in the promoter therefrom gene of derivation is identical.Such as this field institute Know, the variation of this distance can be adjusted in the case where not losing promoter function." promoter " means synthesis Or the molecule of natural source, the molecule can assign, activate or enhance the expression of the nucleic acid in cell.Promoter may include one A or multiple specific transcription regulating nucleotide sequences are to further enhance expression and/or to change expression and/or the time in its space Expression.Promoter can also include Distal enhancer or repressor elements, they can be located at the difference since the starting point of transcription At few thousands of pairs of base-pairs.Promoter can be from including obtaining in virus, bacterium, fungi, plant, insect and the source of animal ?.Promoter can relative to cell, tissue or the organ wherein expressed or relative to the stage of development expressed or The basically or distinctively controlling gene component in response to outside stimulus such as physiological stress, pathogen, metal ion or inducer Expression.The representative example of promoter includes phage t7 promoter, bacteriophage T3 promoter, SP6 promoter, lactose manipulation Son-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMVIE promoter, SV40 early promoter or SV40 late promoter and CMVIE promoter.
" signal peptide " and " leader sequence " refers to the amino that can be connected O-shaped hoof-and-mouth disease toxalbumin as described herein The amino acid sequence of end.Signal peptide/leader sequence is indicated generally at the position of albumen.Signal peptide/leader sequence used herein Albumen is preferably facilitated to secrete from the cell for generating it.Signal peptide/leader sequence is usually cracked from the remainder of albumen, institute Albumen is stated after secreting from cell through being commonly referred to as maturation protein.Signal peptide/leader sequence is connected to the N-terminal of the albumen.
" stringent hybridization conditions " mean the first nucleic acid sequence (example such as in the complex mixture of nucleic acid under the described conditions Such as, probe) it will hybridize with second nucleotide sequence (for example, target).Stringent condition is sequence dependent and not It will be different in same environment.Stringent condition can be selected as the heat than particular sequence at the ionic strength pH of restriction Mechanics fusing point (Tm) is about 5-10 DEG C low.The Tm can be such temperature (in the ionic strength of restriction, pH and nucleic acid concentration Under), hybridized in the state of the equilibrium with the 50% of target-complementary probe with target sequence at said temperatures (because of target sequence It is present in excess, at Tm, 50% probe is occupied in the state of the equilibrium).Stringent condition can be those conditions, i.e. wherein salt Sodium ion of the concentration less than about 1.0M, the Na ion concentration (or other salt) of about 0.01-1.0M such as at pH7.0 to 8.3, and Temperature is at least about 30 DEG C and for long probe (for example, greater than about for short probe (for example, about 10-50 nucleotide) 50 nucleotide) it is at least about 60 DEG C.Stringent condition can also be realized by addition destabilizing agent such as formamide.For choosing Hybridization select or specific, positive signal can be at least 2 to 10 times of background hybridization.Illustrative stringent hybridization conditions packet It includes following: 50% formamide, 5x SSC and 1%SDS, being incubated for or 5xSSC, 1%SDS, incubate at 65 DEG C at 42 DEG C It educates, washed at 65 DEG C with 0.2xSSC and 0.1%SDS.
" be substantially complementary " as used herein mean First ray 8,9,10,11,12,13,14,15,16,17,18, 19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、180、270、 360, in the region of 450,540 or more nucleotide or amino acid with the complement at least 60% of the second sequence, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% are identical, or mean two sequences stringent miscellaneous Hybridized under the conditions of friendship.
As used herein " substantially the same " mean First ray and the second sequence 8,9,10,11,12,13,14, 15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、 100, be at least 60% in 180,270,360,450,540 or more nucleotide or amino acid region, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% are identical, or for nucleic acid, if First ray and the second sequence The complement of column is substantially complementary, then First ray and the second sequence are also identical in this way.
" hypotype " or " serotype ": as be used interchangeably herein and about O-shaped foot and mouth disease virus, it is intended that O-shaped aftosa The genetic mutation of virus a, so that hypotype is identified and separated from different hypotypes by immune system.
" variant " used for nucleic acid means a part or segment of (i) reference nucleotide sequence herein；(ii) join Examine the complement of nucleotide sequence or part thereof；(iii) nucleic acid substantially the same with reference nucleic acid or its complement；Or (iv) nucleic acid hybridized under strict conditions with reference nucleic acid, its complement or the sequence substantially the same with its.
" variant " for peptide or polypeptide is by the insertion of amino acid, missing or conservative replaces in amino acid sequence Upper difference, but retain at least one bioactivity.Variant, which is still meant that, has the amino acid sequence substantially the same with reference protein The albumen of column, the reference protein have the amino acid sequence for retaining at least one bioactivity.The conservative replaces of amino acid, Amino acid is replaced with the different aminoacids of similar characteristic (for example, hydrophily, the degree of charging zone and distribution), in ability It is considered being usually directed to minor change in domain.As understood in the art, these minor changes can be partially by considering amino The hydrophilic and hydrophobic index of acid identifies.Kate (Kyte) etc., J. Mol. BioL (J.Mol.Biol.) 157:105-132 (1982).The hydrophilic and hydrophobic index of the amino acid is based on the considerations of its hydrophobicity and charge.It is known in the art that similar Hydrophilic and hydrophobic index amino acid can be substituted and still retain protein function.In one aspect, hydrophilic and hydrophobic index is ± 2 amino acid is substituted.The hydrophily of amino acid, which can be utilized to disclose, can generate taking for the albumen for retaining biological function Generation.Consider that the hydrophily of amino acid allows to calculate the peptide maximum local average hydrophilicity in the case of peptide, this is It is reported and antigenicity and the good associated useful measurement of immunogenicity.As this field is understood, there is similar hydropathic The substitution of the amino acid of value can produce the peptide for retaining bioactivity (such as immunogenicity).Can use has each other in ± 2 The amino acid of hydrophilicity value is replaced.The hydrophilic and hydrophobic index and hydrophilicity value of amino acid are both by the spy of the amino acid Determine side chain influence.Consistent with the observation to be, the amino acid substitution compatible with biological function is understood to depend on these ammonia The opposite similitude of base acid, and especially those amino acid side chain, such as by hydrophobicity, hydrophily, charge, size and Other characteristics are revealed.
" carrier " means the nucleic acid sequence containing replication orgin as used herein.Carrier can be viral vectors, phagocytosis Body, bacterial artificial chromosome or yeast artificial chromosome.Carrier can be DNA or RNA carrier.Carrier can be self-replacation The outer carrier of chromosome, and preferably DNA vector.
2. vaccine
Vaccine of the invention can be designed in control animal subject for one or more O-shaped hoof-and-mouth disease serotypes Immune response degree or intensity.Vaccine may include that it is inhibited to be integrated into element or reagent in chromosome.Vaccine can be volume The RNA of the O-shaped Foot-and-mouth disease of code.RNA vaccine can be introduced into cell.Vaccine of the invention may include O-shaped mouth hoof Epidemic disease virus structural protein.O-shaped Foot-and-mouth disease is by inducing 1) cytotoxic T lymphocyte (CTL) reaction, 2) T helper cell reaction and/or 3) B cell reaction, or preferably all above-mentioned reactions, are carried out with reaching intersection submission Immune-mediated virus sweep target.
The antigen may include the protein epitope for making them particularly effectively be used as immunogene, can be directed to the immunogene Induce resisting O-type foot and mouth disease virus immune response.O-shaped foot-and-mouth disease virus antigen may include overall length translation product, its variant, its piece Section or combinations thereof.
What some embodiments were related to encoding immunogenic albumen has 95% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 96% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 97% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 98% homology with this paper nucleic acid coding sequence Nucleic acid molecules.What some embodiments were related to encoding immunogenic albumen has 99% homology with this paper nucleic acid coding sequence Nucleic acid molecules.In some embodiments, have and homologous disclosed herein of the coded sequence of albumen disclosed herein The nucleic acid molecules of coded sequence are connected to the volume for encoding homologous protein sequence disclosed herein comprising coding IgE leader sequence The sequence of 5 ' ends of code sequence.
In some embodiments, the nucleic acid sequence is free of the coded sequence of encoding leader sequence.In some embodiment party In case, coded sequence of the nucleic acid sequence without coding IgE lead.
Some embodiments are related to the segment of SEQ ID NO:1 or 12 or 8 or 16 or 4.Segment can be at least 10%, extremely Few 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, At least 96%, at least 97%, at least 98% or at least 99% SEQ ID NO:1 or 12 or 8 or 16 or 4.Segment can be with SEQ The segment of ID NO:1 or 12 or 8 or 16 or 4 at least 95%, at least 96%, at least 97%, at least 98% or at least 99% are identical. Segment can with the segment at least 80% of SEQ ID NO:1 or 12 or 8 or 16 or 4, at least 85%, at least 90% at least 91%, extremely Few 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% are identical.? In some embodiments, segment includes the sequence of encoding leader sequence, such as immunoglobulin leader object, such as IgE lead.? In some embodiments, segment is free of the coded sequence of encoding leader sequence.In some embodiments, before segment is without coding Lead sequence, the such as coded sequence of IgE lead.
Some embodiments are related to the albumen homologous with SEQ ID NO:19 or 22 or 21 or 23 or 20.Some embodiments It is related to the immunogenicity that there is 95% homology with the protein sequence as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 Albumen.Some embodiments are related to having 96% with the protein sequence as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of homology.Some embodiments are related to and as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 Protein sequence has the immunogenic protein of 97% homology.Some embodiments are related to and such as SEQ ID NO:19 or 22 or 21 Or protein sequence described in 23 or 20 has the immunogenic protein of 98% homology.Some embodiments are related to and such as SEQ Protein sequence described in ID NO:19 or 22 or 21 or 23 or 20 has the immunogenic protein of 99% homology.
Some embodiments are related to albumen identical with SEQ ID NO:19 or 22 or 21 or 23 or 20.Some embodiments It is related to the entire amino in the overall length consensus amino acid sequences as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of 80% identical amino acid sequence in acid sequence length.Some embodiments are related to having in such as SEQ ID 85% is identical on the entire length amino acid sequence of overall length consensus amino acid sequences described in NO:19 or 22 or 21 or 23 or 20 Amino acid sequence immunogenic protein.Some embodiments be related to having in such as SEQ ID NO:19 or 22 or 21 or 23 or 90% identical amino acid sequence is immune on the entire length amino acid sequence of overall length consensus amino acid sequences described in 20 Immunogenic peptide.Some embodiments are related to having shared in the overall length as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of 91% identical amino acid sequence on the entire length amino acid sequence of amino acid sequence.Some embodiment party Case is related to the entire ammonia in the overall length consensus amino acid sequences as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of 92% identical amino acid sequence in base acid sequence length.Some embodiments are related to having in such as SEQ 93% on the entire length amino acid sequence of overall length consensus amino acid sequences described in ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of identical amino acid sequence.Some embodiments be related to having in such as SEQ ID NO:19 or 22 or 21 or 94% identical amino acid sequence on the entire length amino acid sequence of overall length consensus amino acid sequences described in 23 or 20 Immunogenic protein.Some embodiments are related to having in the overall length as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of 95% identical amino acid sequence on the entire length amino acid sequence of consensus amino acid sequences.Some realities The scheme of applying is related to having in the whole of the overall length consensus amino acid sequences as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of 96% identical amino acid sequence on a length amino acid sequence.Some embodiments are related to having such as On the entire length amino acid sequence of overall length consensus amino acid sequences described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of 97% identical amino acid sequence.Some embodiments be related to having in such as SEQ ID NO:19 or 22 or 98% identical amino acid sequence on the entire length amino acid sequence of overall length consensus amino acid sequences described in 21 or 23 or 20 The immunogenic protein of column.Some embodiments are related to having as described in SEQ ID NO:19 or 22 or 21 or 23 or 20 The immunogenic protein of 99% identical amino acid sequence on the entire length amino acid sequence of overall length consensus amino acid sequences.
In some embodiments, albumen is free of leader sequence.In some embodiments, albumen is free of IgE lead. The segment of albumen may include at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% albumen.It can The immunogenic fragments of SEQ ID NO:19 or 22 or 21 or 23 or 20 are provided.Immunogenic fragments may include at least 10%, extremely Few 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, At least 96%, at least 97%, at least 98% or at least 99% SEQ ID NO:19 or 22 or 21 or 23 or 20.In some implementations In scheme, segment includes leader sequence, such as immunoglobulin leader object, such as IgE lead.In some embodiments, Segment is free of leader sequence.In some embodiments, segment is free of leader sequence, such as IgE lead.
It can provide the amino acid sequence egg homologous with the immunogenic fragments of SEQ ID NO:19 or 22 or 21 or 23 or 20 White immunogenic fragments.The immunogenic fragments may include at least 10%, at least 15%, at least 20%, at least 25%, extremely Few 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% Or at least 99% the albumen homologous with SEQ ID NO:19 or 22 or 21 or 23 or 2095%.Some embodiments are related to and this The immunogenic fragments of literary protein sequence have the immunogenic fragments of 96% homology.Some embodiments are related to and this paper egg The immunogenic fragments of Bai Xulie have the immunogenic fragments of 97% homology.Some embodiments are related to and this paper albumen sequence The immunogenic fragments of column have the immunogenic fragments of 98% homology.Some embodiments are related to and this paper protein sequence Immunogenic fragments have the immunogenic fragments of 99% homology.In some embodiments, segment includes leader sequence, example As immunoglobulin leader sequence, such as IgE lead.In some embodiments, segment is free of leader sequence.In some realities It applies in scheme, segment is free of leader sequence, such as IgE lead.
It can provide amino acid sequence egg identical with the immunogenic fragments of SEQ ID NO:19 or 22 or 21 or 23 or 20 White immunogenic fragments.The immunogenic fragments may include at least 10%, at least 15%, at least 20%, at least 25%, extremely Few 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% Or at least 99% the amino acid sequence described in SEQ ID NO:19 or 22 or 21 or 23 or 20 whole length on 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical albumen.In some embodiment party In case, segment includes leader sequence, such as immunoglobulin leader object, such as IgE lead.In some embodiments, piece Duan Buhan leader sequence.In some embodiments, segment is free of leader sequence, such as IgE lead.
3. vaccine constructs and plasmid
Vaccine may include encoding O-shaped Foot-and-mouth disease, O-shaped foot-and-mouth disease virus antigen and O-shaped hoof-and-mouth disease The combined nucleic acid construct or plasmid of malicious structural proteins/antigen.Provided herein is may include coding O-shaped mouth disclosed herein The genetic constructs of the nucleic acid sequence of aphtovirus antigen, the core antigen include that protein sequence and protein sequence are homologous Sequence, the segment of protein sequence and the sequence homologous with the segment of protein sequence.In addition, provided herein is may include coding this paper Disclosed O-shaped foot and mouth disease virus surface antigen is (including protein sequence and the homologous sequence of protein sequence, the segment of protein sequence And the homologous sequence with the segment of protein sequence) nucleic acid sequence genetic constructs.The genetic constructs can be used as Molecule outside functional genomics and exist.The genetic constructs can be including centromere, telomere or plasmid or clay Linear minichromosome.
The genetic constructs can also be a part of the genome of recombinant viral vector, the recombinant viral vector packet Include recombined adhenovirus, recombinant adeno-associated virus and recombinant vaccinia.Genetic constructs can be the viable microbial in attenuation Or the part of the inhereditary material in the recombinant microorganism carrier living in cell.
Genetic constructs may include the controlling element of the gene expression of the coded sequence for nucleic acid.Controlling element can be with It is promoter, enhancer, initiation codon, terminator codon or polyadenylation signal.
Nucleic acid sequence can be the genetic constructs of carrier.The carrier can be exempted from effectively causing in animal The amount of epidemic disease reaction expresses antigen in the cell of animal.Institute+state carrier can be recombinant.It is anti-that the carrier may include coding Former heterologous nucleic acids.The carrier can be plasmid.The carrier can be adapted for transfecting cell with the nucleic acid of coding for antigens, The host cell of the conversion is cultivated and is maintained under conditions of antigen presentation wherein occurs.
Coded sequence can be optimized to be used in the stability and high level of expression.In some cases, codon is selected To reduce the formation of RNA secondary structure, the secondary structure such as formed due to intramolecular bond.
The carrier may include the heterologous nucleic acids of coding for antigens, and can further include can be in antigen encoding sequence The initiation codon of the upstream of column and can be in the terminator codon in the downstream of antigen encoding sequences.Initiation codon and termination are close Numeral can be with antigen encoding sequences in frame.The carrier also includes the starting for being operably coupled to antigen encoding sequences Son.The promoter for being operably coupled to antigen encoding sequences can be promoter from simian virus 40 (SV40), mouse The long end weight of mammary tumour virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter such as bovine immunodeficiency virus (BIV) Multiple (LTR) promoter, Moloney (Moloney) viral promotors, avian leukosis virus (ALV) promoter, cytomegalovirus (CMV) viral (EBV) promoter of promoter such as CMV immediate early promoter, love bar Er Shi (EpsteinBarr) or rous sarcoma Viral (RSV) promoter.The promoter can also be the promoter from people's gene, the people's gene such as human actin, People's myosin, people's ferroheme, people's muscle creatin or human metal thioalbumen.The promoter can also be that tissue specificity starts Son, such as natural or synthetic muscle or skin-specific promoter.
The carrier can also include polyadenylation signal, and the polyadenylation signal can be in O-shaped mouth hoof The downstream of epidemic disease viral core protein coded sequence.The polyadenylation signal can be SV40 polyadenylation signal, LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone (HGH) (hGH) polyadenylic acid Change signal or people's beta-globin polyadenylation signal.The SV40 polyadenylation signal can be to be carried from pCEP4 The polyadenylation signal of body (Invitrogen, San Diego, CA).
Carrier, which can also reside in, to be shared O-shaped foot and mouth disease virus core protein coded sequence or shares O-shaped foot and mouth disease virus table The enhancer of the upstream of face antigen protein coded sequence.The enhancer is necessary for DNA expression.The enhancer can be with Human actin, people's myosin, people's ferroheme, people's muscle creatin or virus enhancer, such as from CMV, HA, RSV or A kind of enhancer of EBV.
Carrier can also include the replication orgin of animal, to maintain carrier outside chromosome and to generate load in cell Multiple copies of body.The carrier can be pVAX1, pCEP4 from Invitrogen (San Diego, CA) or PREP4 may include the replication orgin and nuclear antigen EBNA-1 coding region of love bar Er Shi virus, this can not integrated In the case where generate high copy episomal replication.The carrier can be modified pVAX1 pVax1 variant, such as originally Variant plasmid described in text.The variant pVax1 plasmid is Backbone Vector plasmid pVAX1 (Invitrogen, Carlsbad CA) 2998 base-pair variants.The CMV promoter is located at base 137-724.T7 promoter/initiation site is located at base 664- At 683.Multiple cloning sites are located at base 696-811.Ox GH polyadenylation signal is at base 829-1053.Block that Mycin (Kanamycin) resistant gene is at base 1226-2020.PUC starting point is at base 2320-2993.
The carrier can be pSE420 (Invitrogen, San Diego, Calif.), can be used in Escherichia coli (E.coli) albumen is generated in.The carrier can be pYES2 (Invitrogen, San Diego, Calif.), can be used for Albumen is generated in the Wine brewing yeast strain (Saccharomyces cerevisiae strain) of yeast.The carrier may be used also Can be used for the complete baculovirus expression system of MAXBACTM (Invitrogen, San Diego, Calif.) Albumen is generated in insect cell.The carrier can also be pcDNAI or pcDNA3 (Invitrogen, SanDiego, Calif.), can be used for generating albumen in zooblast such as Sf9 cell line.The carrier can be through routine techniques and Available initial substance is easy to generate the expression vector or system of albumen, the technology and substance include Sambrook etc., Molecular Cloning and Laboratory Manual, second edition, ColdSpring Harbor (1989).
VP1 in the present invention, VP3, VP0, VP2, VP4 protein sequence can be original series, increase and truncated sequence. The virus expression carrier expresses any three, four or five albumen in this five albumen simultaneously, and wherein VP1 and VP3 are The structural proteins of indispensability expression.Baculovirus expression system transfer vector in recombination bacillary viral vector is pFastBac Dual.The promoter of five albumen can be P10 promoter, PH promoter, prawn β-actin gene promoter, OpIE starting Any of son, but it is not limited to this four promoters, it is also possible to it is other bacilliform virus promoters.The transcription of five albumen Termination signal is that SV40polyA transfection termination signal, HSV tk polyA transfection termination signal or OpIE poly A terminate letter Any of number, it should be not limited to using these three transcription stop signals, it is also possible to other transcription stop signals.Insect cell System can be as Sf9, High Five, S2 or Sf21 cell, preferably use Sf9.Animal of the present invention, especially farming animals Animal, such as ox, sheep, pig, caprine species.
The principle of the invention lies in by one recombinant baculovirus shuttle plasmid of building, on the plasmid comprising expression VP3, Expressing gene (the wherein VP1 of any three or four or five albumen in five albumen of VP0, VP2, VP1 and VP4 It is indispensable with VP3 albumen).VP3 and VP0 albumen and VP2, tri- protein expression frames of VP1 and VP4 can be used P10 and open Any of mover, PH promoter, prawn β-actin gene promoter, OpIE promoter (should be not limited to start using this four Son, it is also possible to other promoters), VP3 and VP0 albumen and VP2, the tanscription termination of tri- protein expression frames of VP1 and VP4 Signal can be SV40 polyA transfection termination signal, HSV tk polyA transfection termination signal, OpIE poly A termination signal Any of (should be not limited to using these three transcription stop signals, it is also possible to other transcription stop signals).In this way, one This five albumen or three or four therein are expressed on carrier simultaneously, but must include VP1 and VP3 albumen, expression It is greatly improved with packaging efficiency.It constructs shuttle plasmid conversion DH10Bac bacterium and obtains recombination bacmid The recombination bacmid of (Baculovirus plasmid, baculovirus plasmid), acquisition transfects Sf9 cell, obtains and recombinates rod-shaped disease Poison, the recombinant baculovirus are inoculated with Sf9 cell, can express VP3, VP0 and VP2 simultaneously, five albumen of VP1, VP4, VP1, VP3, VP2, VP4 and VP0 albumen can be assembled into VLP automatically, and VP1, VP3 and VP2, VP4 albumen can also be assembled into VLP automatically.
Specifically, VP3 the and VP0 protein coding gene for the FMDV for optimizing and synthesizing is cloned into pFastBac respectively first Below the PH promoter and P10 promoter of Dual carrier.Construct Dual-VP3-VP0 carrier.Again in Dual-VP3-VP0 carrier I restriction enzyme site of SnaB at be inserted into VP2 protein expression frame, which includes prawn β-actin gene promoter, optimization VP2 gene and SV40polyA transcription stop signals, to construct Dual-VP3-VP0-VP2 carrier.Again in Dual-VP3- VP1 protein expression frame is inserted at II restriction enzyme site of Avr of VP0-VP2 carrier, which includes OpIE promoter, it is Second in Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) virus A early stage regulatory factor promoter, VP1 gene and OpIE poly A transcription stop signals after optimization, to construct Dual- VP3-VP0-VP2-VP1 carrier.VP4 albumen table is inserted at the BsrGI restriction enzyme site of Dual-VP3-VP0-VP2-VP1 carrier Up to frame, which includes prawn β-actin gene promoter sequence (Litopenaeus vannamei beta-actin Gene promoter), the VP4 gene and SV40 polyA transcription stop signals of optimization, to construct Dual-VP3-VP0- VP2-VP1-VP4 carrier.Carrier after building is converted into DH10Bac bacterium, selects recombinant bacterium, extracting genome transfects Sf9 cell, Obtain recombinant baculovirus.
Express five albumen simultaneously using a baculoviral and Sf9 cell in this way, that is, the VP1 optimized, VP0 and VP3, VP2, VP4 protein sequence, what this five albumen of expression can be spontaneous be assembled into VLP, and because the inside include VP0 with And VP2 and VP4, it can preferably be assembled with VP1, VP3, packaging efficiency is high.The present invention expresses VP1, VP3, VP2 simultaneously, Five albumen of VP4 and VP0, VP1, VP3 and VP0 albumen can be assembled into VLP, VP1, VP3 and VP2, and VP4 albumen can also assemble At VLP, this five albumen are co-expressed, so that the packaging efficiency of VLP greatly improves.The VLP's of the FMDV of formation is antigenic, immune Originality and function are similar to native protein, and expression is higher, and immunogenicity is strong, do not have to pig pathogenic and of the invention Vaccine bioreactor extensive serum free suspension culture preparation can be used, be greatly reduced production of vaccine cost, can With large-scale serial production, Quality Control is easy, and highly-safe, immunogenicity is good, is stablized between batch.
1 embodiment of embodiment, 1 transfer vector Dual-VP3-VP0-VP2-VP1-VP4 building and identification
1. transfer vector Dual-VP3 building and identification
1.1VP3 gene magnification has synthesized the VP3 gene (SEQ after codon optimization in Nanjing Jin Sirui company with purifying ID NO:1) and be cloned on pUC17 carrier and obtain pUC-VP3 plasmid vector.Using pUC-VP3 plasmid as template, VP3-F, VP3-R carries out PCR amplification (gene order of VP3-F, VP3-R are as shown in SEQ ID NO.2,3) as upstream and downstream primer, amplification System is shown in Table 1.
1 VP3 gene magnification system of table
Reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；94 DEG C be denaturalized 45 seconds, 54 DEG C anneal 45 seconds, 72 DEG C extend 1 minute, 35 A circulation；72 DEG C extend 10 minutes.
PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in Figure 1, the position near 0.7kbp goes out Existing purpose band, target gene expand successfully, carry out recovery purifying with gel purification kit.
PFastBacDual plasmid and VP3 gene PCR amplified production are used BamH I, Hind III 37 with purifying by 1.2 digestions DEG C double digestion 3 hours, specific endonuclease reaction system was shown in Table 2, table 3.
Digestion products are subjected to gel electrophoresis, use the pFastBac of gel purification kits digestion respectively Dual plasmid and VP3 genetic fragment.
2 VP3 gene endonuclease reaction system of table
3 pFastbacDual plasmid enzyme restriction reaction system of table
The pFastBac Dual plasmid of double digestion and VP3 gene digestion products are used T4 DNA ligase 16 by 1.3 connections DEG C water-bath connection is overnight.Specific coupled reaction system is shown in Table 4.
4 VP3 gene of table and pFastBac Dual plasmid linked system
10 μ l connection products are added in the DH5 α competent cell of 100 μ l and mix by 1.4 conversions, and ice bath 30 minutes, 42 DEG C Water-bath heat shock 90 seconds, then ice bath 2 minutes, the LB liquid medium that 900 μ l are free of Amp is added, 37 DEG C are cultivated 1 hour.It takes 1.0ml bacterium solution is condensed into 100 μ l and is coated on the LB solid medium containing Amp, and 37 DEG C are cultivated 16 hours.
Single colonie on 1.5 bacterium colony PCR and sequencing identification picking plate is inoculated with LB liquid medium, 37 DEG C of cultures 2 respectively Hour, using bacterium solution as template, PCR product is carried out gel electrophoresis as primer progress bacterium colony PCR identification by VP3-F and VP3-R The size of verifying purpose gene, as shown in Fig. 2, the sample for 0.7kbp band occur is positive sample.By the positive bacterium solution of identification Sequencing company is sent to be sequenced, selection is sequenced correct bacterium solution and saves.
2. transfer vector Dual-VP3-VP0 building and identification
2.1VP0 gene magnification has synthesized the VP0 gene (SEQ after codon optimization in Nanjing Jin Sirui company with purifying ID NO:4) and be cloned on pUC17 carrier and obtain pUC-VP0 plasmid vector.Using pUC-VP0 plasmid as template, VP0-F, VP0-R carries out PCR amplification (gene order of VP0-F, VP0-R are as shown in SEQ ID NO.5,6) as upstream and downstream primer, amplification System is shown in Table 5.
5 VP0 gene magnification system of table
Reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；94 DEG C be denaturalized 45 seconds, 54 DEG C anneal 45 seconds, 72 DEG C extend 1 minute, 35 A circulation；72 DEG C extend 10 minutes.
PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in figure 3, the position near 0.9kbp goes out Existing purpose band, target gene expand successfully, carry out recovery purifying with gel purification kit.
Dual-VP3 plasmid and VP0 gene PCR amplified production are used Xho I, Kpn I 37 DEG C pairs with purifying by 2.2 digestions Digestion 3 hours, specific endonuclease reaction system was shown in Table 6, table 7.
Digestion products are subjected to gel electrophoresis, use the pFastBac of gel purification kits digestion respectively Dual plasmid and VP0 genetic fragment.
6 VP0 gene endonuclease reaction system of table
7 Dual-VP3 plasmid enzyme restriction reaction system of table
The Dual-VP3 plasmid of double digestion and VP0 gene digestion products are used the 16 DEG C of water-baths of T4DNA ligase by 2.3 connections Connection is overnight.Specific coupled reaction system is shown in Table 8.
8 VP0 gene of table and Dual-VP3 plasmid linked system
10 μ l connection products are added in the DH5 α competent cell of 100 μ l and mix by 2.4 conversions, and ice bath 30 minutes, 42 DEG C Water-bath heat shock 90 seconds, then ice bath 2 minutes, the LB liquid medium that 900 μ l are free of Amp is added, 37 DEG C are cultivated 1 hour.It takes 1.0ml bacterium solution is condensed into 100 μ l and is coated on the LB solid medium containing Amp, and 37 DEG C are cultivated 16 hours.
Single colonie on 2.5 bacterium colony PCR and sequencing identification picking plate is inoculated with LB liquid medium, 37 DEG C of cultures 2 respectively Hour, using bacterium solution as template, PCR product is carried out gel electrophoresis as primer progress bacterium colony PCR identification by VP0-F and VP0-R The size of verifying purpose gene, as shown in figure 4, the sample for 0.9kbp band occur is positive sample.By the positive bacterium solution of identification Sequencing company is sent to be sequenced, selection is sequenced correct bacterium solution and saves.
3. transfer vector Dual-VP3-VP0-VP2 building and identification
The amplification of 3.1 VP2 gene expression frames has synthesized VP2 gene expression frame (SEQ ID in Nanjing Jin Sirui company with purifying NO:7 it) and is cloned on pUC17 carrier and obtains pUC-VP2 plasmid vector, which includes that prawn β-actin gene opens Mover, the FMDV VP2 gene and SV40 poly A termination signal sequence of codon optimization.Using pUC-VP2 plasmid as mould Plate, VP2-F, VP2-R carry out PCR amplification (gene order of VP2-F, VP2-R such as SEQ ID NO.9,10 as upstream and downstream primer It is shown), amplification system is shown in Table 9.
9 VP2 gene magnification system of table
Reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；94 DEG C be denaturalized 45 seconds, 54 DEG C anneal 45 seconds, 72 DEG C extend 1 minute, 35 A circulation；72 DEG C extend 10 minutes.
PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in figure 5, the position near 1.4kbp goes out Existing purpose band, target gene expand successfully, carry out recovery purifying with gel purification kit.
Dual-VP3-VP0 plasmid and VP2 gene expression frame pcr amplification product are used SnaB I 37 with purifying by 3.2 digestions DEG C single endonuclease digestion 3 hours, specific endonuclease reaction system was shown in Table 10, table 11.
Digestion products are subjected to gel electrophoresis, use the Dual-VP3- of gel purification kits digestion respectively VP0 plasmid and VP2 gene expression frame pcr amplification product.
10 VP2 gene endonuclease reaction system of table
11 Dual-VP3-VP0 plasmid enzyme restriction reaction system of table
3.3 connections connect the Dual-VP3-VP0 plasmid of double digestion with VP2 gene expression frame digestion products using T4DNA 16 DEG C of water-bath connections of enzyme are overnight.Specific coupled reaction system is shown in Table 12.
12 VP2 gene expression frame PCR product of table and Dual-VP3-VP0 plasmid linked system
10 μ l connection products are added in the DH5 α competent cell of 100 μ l and mix by 3.4 conversions, and ice bath 30 minutes, 42 DEG C Water-bath heat shock 90 seconds, then ice bath 2 minutes, the LB liquid medium that 900 μ l are free of Amp is added, 37 DEG C are cultivated 1 hour.It takes 1.0ml bacterium solution is condensed into 100 μ l and is coated on the LB solid medium containing Amp, and 37 DEG C are cultivated 16 hours.
Single colonie on 3.5 bacterium colony PCR and sequencing identification picking plate is inoculated with LB liquid medium, 37 DEG C of cultures 2 respectively Hour, using bacterium solution as template, PCR product is carried out gel electrophoresis as primer progress bacterium colony PCR identification by VP2-F and VP2-R The size of verifying purpose gene, as shown in fig. 6, the sample for 1.4kbp band occur is positive sample.By the positive bacterium solution of identification Sequencing company is sent to be sequenced, selection is sequenced correct bacterium solution and saves.
4. transfer vector Dual-VP3-VP0-VP2-VP1 building and identification
The amplification of 4.1VP1 gene expression frame has synthesized VP1 gene expression frame (SEQ ID in Nanjing Jin Sirui company with purifying NO:11 it) and is cloned on pUC17 carrier and obtains pUC-VP1 plasmid vector, which includes OpIE promoter, close The FMDVVP1 gene and OpIEpolyA transcription stop signals sequence of numeral optimization.Using pUC-VP1 plasmid as template, VP1- F, VP1-R carries out PCR amplification (gene order of VP1-F, VP1-R are as shown in SEQ ID NO.13,14) as upstream and downstream primer, Amplification system is shown in Table 13.
13 VP1 gene expression frame amplification system of table
Reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；94 DEG C be denaturalized 45 seconds, 54 DEG C anneal 45 seconds, 72 DEG C extend 1 minute, 35 A circulation；72 DEG C extend 10 minutes.
PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in fig. 7, the position near 1.3kbp goes out Existing purpose band, target gene expand successfully, carry out recovery purifying with gel purification kit.
Dual-VP3-VP0-VP2 plasmid and VP1 gene expression frame pcr amplification product are used Avr with purifying by 4.2 digestions II 37 DEG C of restriction endonuclease double digestion 3 hours, specific endonuclease reaction system is shown in Table 14, table 15.
Digestion products are subjected to gel electrophoresis, use the Dual-VP3- of gel purification kits digestion respectively VP0-VP2 plasmid and VP1 gene expression frame pcr amplification product.
14 VP1 gene expression frame PCR product endonuclease reaction system of table
15 Dual-VP3-VP0-VP2 plasmid enzyme restriction reaction system of table
The Dual-VP3-VP0-VP2 plasmid of double digestion and VP1 gene expression frame digestion products are used T4 by 4.3 connections 16 DEG C of water-bath connections of DNA ligase are overnight.Specific coupled reaction system is shown in Table 16.
16 VP1 gene expression frame of table and Dual-VP3-VP0-VP2 plasmid linked system
10 μ l connection products are added in the DH5 α competent cell of 100 μ l and mix by 4.4 conversions, and ice bath 30 minutes, 42 DEG C Water-bath heat shock 90 seconds, then ice bath 2 minutes, the LB liquid medium that 900 μ l are free of Amp is added, 37 DEG C are cultivated 1 hour.It takes 1.0ml bacterium solution is condensed into 100 μ l and is coated on the LB solid medium containing Amp, and 37 DEG C are cultivated 16 hours.
Single colonie on 4.5 bacterium colony PCR and sequencing identification picking plate is inoculated with LB liquid medium, 37 DEG C of cultures 2 respectively Hour, using bacterium solution as template, PCR product is carried out gel electrophoresis as primer progress bacterium colony PCR identification by VP1-F and VP1-R The size of verifying purpose gene, as shown in figure 8, the sample for 1.3kbp band occur is positive sample.By the positive bacterium solution of identification Sequencing company is sent to be sequenced, selection is sequenced correct bacterium solution and saves.
The building of 5.Dual-VP3-VP0-VP2-VP1-VP4 carrier
The amplification of 5.1VP4 gene expression frame has synthesized VP4 gene expression frame (SEQ ID in Nanjing Jin Sirui company with purifying NO:15 it) and is cloned on pUC17 carrier and obtains pUC-VP4 plasmid vector, which includes prawn β-actin, The FMDV VP4 gene and SV40 poly A transcription stop signals sequence of codon optimization.Using pUC-VP4 plasmid as mould Plate, VP4-F, VP4-R as upstream and downstream primer carry out PCR amplification (gene order of VP4-F, VP4-R such as SEQ ID NO.17, Shown in 18), amplification system is shown in Table 17.
17 VP4 gene expression frame amplification system of table
Reaction condition are as follows: 95 DEG C initial denaturation 5 minutes；94 DEG C be denaturalized 45 seconds, 54 DEG C anneal 45 seconds, 72 DEG C extend 1 minute, 35 A circulation；72 DEG C extend 10 minutes.
PCR product is subjected to gel electrophoresis verifying purpose gene size, as shown in figure 9, the position near 1.0kbp goes out Existing purpose band, target gene expand successfully, carry out recovery purifying with gel purification kit.
5.2 digestions and purifying use Dual-VP3-VP0-VP2-VP1 plasmid and VP4 gene expression frame pcr amplification product BsrG 37 DEG C of I restriction endonuclease double digestion 3 hours, specific endonuclease reaction system is shown in Table 18, table 19.
Digestion products are subjected to gel electrophoresis, use the Dual-VP3- of gel purification kits digestion respectively VP0-VP2-VP1 plasmid and VP4 gene expression frame pcr amplification product.
18 VP4 gene expression frame PCR product endonuclease reaction system of table
19 Dual-VP3-VP0-VP2-VP1 plasmid enzyme restriction reaction system of table
5.3 connections use the Dual-VP3-VP0-VP2-VP1 plasmid of double digestion and VP4 gene expression frame digestion products The 16 DEG C of water-bath connections of T4DNA ligase are overnight.Specific coupled reaction system is shown in Table 20.
20 VP4 gene expression frame of table and Dual-VP3-VP0-VP2-VP1 plasmid linked system
10 μ l connection products are added in the DH5 α competent cell of 100 μ l and mix by 5.4 conversions, and ice bath 30 minutes, 42 DEG C Water-bath heat shock 90 seconds, then ice bath 2 minutes, the LB liquid medium that 900 μ l are free of Amp is added, 37 DEG C are cultivated 1 hour.It takes 1.0ml bacterium solution is condensed into 100 μ l and is coated on the LB solid medium containing Amp, and 37 DEG C are cultivated 16 hours.
Single colonie on 5.5 bacterium colony PCR and sequencing identification picking plate is inoculated with LB liquid medium, 37 DEG C of cultures 2 respectively Hour, using bacterium solution as template, PCR product is carried out gel electrophoresis as primer progress bacterium colony PCR identification by VP4-F and VP4-R The size of verifying purpose gene, as shown in Figure 10, the sample for 1.0kbp band occur is positive sample.By the positive bacterium solution of identification Sequencing company is sent to be sequenced, selection is sequenced correct bacterium solution and saves.The transfer vector Dual- containing target gene of building Its schematic diagram of VP3-VP0-VP2-VP1-VP4 such as Figure 11.
2 recombinant baculovirus genome Bac-VP3-VP0-VP2-VP1-VP4 of embodiment building
1.DH10Bac bacterium converts 1 μ l Dual-VP3-VP0-VP2-VP1-VP4 plasmid in Example 1 and is added 100 μ l's Mixed in DH10Bac competent cell, ice bath 30 minutes, 42 DEG C water-bath heat shock 90 seconds, it is then ice bath 2 minutes, each that 900 μ l are added LB liquid medium without Amp, 37 DEG C are cultivated 5 hours.After taking 100 μ l bacterium solutions to dilute 81 times, the diluted bacterium solution of 100 μ l is taken to apply On cloth to the LB solid medium containing gentamicin, kanamycins, tetracycline, X-gal and IPTG, 37 DEG C of cultures 48 are small When.
2. select the white colony that monoclonal uses transfer needle picking big respectively, then containing gentamicin, to block that mould Element, tetracycline, X-gal and IPTG LB solid medium on cross, 37 DEG C are cultivated 48 hours, and then picking single colonie is inoculated with LB liquid medium culture containing gentamicin, kanamycins, tetracycline saves strain, extracts plasmid.Obtain recombinant plasmid Bacmid-VP3-VP0-VP2-VP1-VP4。
The transfection of 3 recombinant baculovirus of embodiment
Each hole inoculation 0.8 × 10 in six orifice plates6A Sf9 cell, cell confluency degree are 50~70%.For each hole It prepares compound below: diluting the Cellfectin transfection reagent of 4 μ l, of short duration whirlpool shake with 100 μ l transfection media T1 It swings；The recombination Bacmid-VP3-VP0-VP2-VP1-VP4 plasmid in culture T1 base 3 μ g embodiments 2 of dilution is transfected with 100 μ l, it will Diluted transfection reagent and plasmid mixing, gently blow even, prepare transfection mixture.It is multiple that above-mentioned transfection is added after cell is adherent Object is closed, 27 DEG C are incubated for 5 hours, remove supernatant, add 2mlSF-SFM fresh culture, 27 DEG C of cultures harvest supernatant on the 4th~5.It obtains Recombinant baculovirus rBac-VP3-VP0-VP2-VP1-VP4 is obtained, the P1 of harvest is imitated for recombinant baculovirus using MTT is opposite Force method detects virus titer, and rBac-VP3-VP0-VP2-VP1-VP4P1 kind poison virus titer is 5.4 × 107pfu/mL.Amplification Recombinant baculovirus rBac-VP3-VP0-VP2-VP1-VP4 is spare as kind of poison.
4 SDS-PAGE of embodiment detection
The cell culture harvested in embodiment 3 is subjected to SDS-PAGE detection, while respectively using the empty rod-shaped disease of infection The Sf9 cell of poison is as negative control.Concrete operations are as follows: take 40 μ l harvest cell culture, be added 10 μ l 5 × Loading buffer, boiling water bath 5 minutes, 12000r/min was centrifuged 1 minute, and taking supernatant to carry out PAGE gel, (12% is dense Spend gel) electrophoresis, take gel to be dyed after electrophoresis, decolourize after observe purpose band.As shown in figure 12, molecular weight about 21kDa, Nearby there is purpose band in 33kDa, 26kDa, 24kDa and 9kDa, and negative control does not have band in corresponding position.
5 Western Blot of embodiment identification
Product after SDS-PAGE electrophoresis in embodiment 4 is transferred on NC (nitrocellulose) film, with 5% skim milk Closing 2 hours, the anti-FMDV positive serum in pig source are incubated for 2 hours, and the goat-anti pig polyclonal antibody secondary antibody of rinsing, HRP label is incubated for 2 Hour, then rinsing is added dropwise enhanced chemical luminous fluorescent substrate, is taken pictures using chemiluminescence imaging instrument.As shown in figure 13, weight Group baculovirus expression sample has purpose band, and negative control does not have purpose band, and illustration purpose antigen protein is in Sf9 cell Correctly expressed.
6 indirect immunofluorescene assay of embodiment
The Sf9 cell that addition transfected rBac-VP3-VP0-VP2-VP1-VP4 into 96 porocyte culture plates respectively hangs Liquid, (cell concentration is 2.5 × 10 in 100 holes μ l/5~4.0 × 105A/ml), 4 holes are inoculated with, 27 DEG C stand 15 minutes, keep Sf9 thin Born of the same parents are affixed on culture plate bottom wall, and then the seed culture of viruses that 10 μ l dilute 10 times is added in every hole.Blanc cell control is set simultaneously.Cell after inoculation It sets in 27 DEG C of constant incubators and cultivates 72-96 hours, abandon culture solution, cold methanol/acetone (1:1) is fixed.It is anti-with pig source first Then the reaction of FMDV polyvalent antibody is reacted with the goat-anti pig IgG of FITC label, inverted fluorescence microscope observes result.Such as Figure 14 Shown, inoculation sky baculoviral Sf9 cell is not it is observed that fluorescence, and being vaccinated with recombinant baculovirus Sf9 cell can observe To fluorescence, illustration purpose antigen protein is correctly expressed in Sf9 cell, and recombinant baculovirus building is correct.
The detection of 7 Electronic Speculum of embodiment
By recombinant baculovirus cell culture ultrasonication, 12000r/min is centrifuged 30 minutes, takes supernatant, 0.22 μm of filter Film filtering, removes impurity, is concentrated 10 times using the super filter tube that molecular cut off is 3kDa.It is 40% that concentration, which is added, in each centrifuge tube Sucrose solution 10ml, then be added 2.0ml be concentrated by ultrafiltration sample, 29000r/min ultracentrifugation 2 hours, abandon supernatant, precipitating It is resuspended with 2.0ml PBS.Then by suspension through over-richness be respectively 50%, 60%, 70%, 80% gradient concentration sucrose from The heart 29000r/min ultracentrifugation 2 hours, then collects the band for being located at 60%~70% concentration intersection.Use phosphotungstic acid Negative staining observes the product collected, and observes and virus-like similar in O-shaped FMDV virion sizableness, morphology under Electronic Speculum Grain, Electronic Speculum the result is shown in Figure 15.
The bioreactor serum free suspension culture of 8 insect cell of embodiment and VP3-VP0-VP2-VP1-VP4 expression are fixed Amount
Insect cell 3-4 days sterile culture Sf9 in 1000ml shaking flask, grow to 3-5 × 10 to concentration6Cell/mL, vigor It when greater than 95%, seeds cells into the bioreactor of 5L, inoculum density is 3-8 × 105cell/mL.Work as cell concentration Reach 3-55 × 106When cell/mL, seed cells into 50L bioreactor, to cell it is long to concentration be 3-55 × 106Cell/mL, is inoculated into 500L bioreactor, reaches 2-85 × 10 to cell concentration6When cell/mL, inoculation recombination bar Shape virus rBac-VP3-VP0-VP2-VP1-VP4, bioreactor culture condition are pH value 6.0-6.5,25-27 DEG C of temperature, dissolved oxygen 30-80%, mixing speed 100-180rpm.In view of the optimum condition of cell culture, preferred pH6.2, cell culture stage Temperature sets 27 DEG C, dissolved oxygen 50%, mixing speed 100-180rpm.After continuing culture after infection 5-9 days, addition thousand/ After one final concentration BEI, 37 DEG C of effect 48h, add 2/1000ths final concentration Na2S2O3Terminate inactivation.Pass through centrifugation or doughnut mistake The method of filter harvests cells and supernatant, sets 2-8 DEG C of preservation vaccinogen liquid.
9 protein purification of embodiment
The stoste of harvest is purified using cation exchange chromatography
Exchange of particles chromatography is carried out using strong sun particle chromatographic filler POROS50HS, filler uses 0.5M NaOH using preceding It carries out disinfection.Then with micro-filtration buffer in equilibrium at room temperature, then by vaccinogen liquid with the rate upper prop of 125mL/min, then 8 column volumes are eluted with rinsing bufferA (0.05M MOPS (sodium salt), pH=7.0,0.5M NaCl).Then linear gradient into Row elution, from 0% bufferA to 100% buffer B (0.05M MOPS (sodium salt), pH=7.0,1.5M NaCl), wherein One co-elute of linear elution, 10 column volumes, the then average eluate for harvesting this 10 column volumes respectively.Linear elution is complete After, then with bufferB 2 column volumes are eluted, and collect respectively.The sample of collection is placed in the sterile plastic bottle of 2L and is put It sets at 4 DEG C.Then the component collected under the last one eluting peak (A280) aseptic filtration is stored in 4 DEG C.
2.3 hydroxyapatite hydrophobic chromatographies
Use prepackage hydroxyapatite column (CHTTMCeramic Hydroxyapatite Type II Media), make first With 50mM MOPS (sodium salt), pH=7.0,1.25M NaCl are balanced, then by the sample of preliminary purification above with 90cm/ Hour carries out loading, elute using the equilibrium liquid of 8 volumes until UV value is zero after upper complete sample.Then eluent is used (0.2M phosphate, pH=7.0,1.25MNaCl) carries out gradient elution, and from 0% to 100%, speed is still eluate concentration 90cm/h, elution volume are 4 column volumes.Purifying obtains destination protein.
The unilateral use BCA total protein of the purpose of purifying is quantified, determines destination protein purity then in conjunction with gray scale scanning, it is pure Albumen after change is as shown in figure 16, and destination protein concentration is 312ug/mL, purity 92%.
The preparation of 10 vaccine of embodiment
The O-shaped foot and mouth disease virus VP3-VP0-VP2-VP1-VP4 albumen purified in right amount is added to MONTANIDE ISA In 206 VG adjuvants (volume ratio 1:1), make final concentration of protein 100ug/mL, emulsify, quality inspection qualification is placed on 4 DEG C of preservations.
11 immunization experiment of embodiment
1 monthly age or so pig (antibody titer < 1:16), is randomly divided into 3 groups, every group 7, A group is injected embodiment 10 and made by 21 Standby recombinant vaccine；B group injecting normal saline；C group injects the commercially available O-shaped FMDV inactivated virus vaccine of certain brand.Respectively Musculi colli injects 2ml vaccine, 28 days booster immunizations after head exempts from, and is spaced 2 weeks and takes a blood sample 1 time, separates serum, detection antibody effect Valence.ELISA detection kit is mutually blocked to carry out antibody test using the O-shaped antibody liquid of aftosa purchased from Lanzhou veterinary institute. Experimental result is as follows
1, result judgement: antibody titer >=1:128 is judged to the O-shaped antibody positive of aftosa；When 1:64~1:128, being judged to can It doubts；< 1:64, is determined as feminine gender.Suspect serum specimens can be resurveyed, and resurvey antibody titer>=1:128 and be judged to the positive,<1:128 It is judged to feminine gender.
It can be seen that in negative control group whole experiment process according to upper result is schemed, antibody is all negative.Immune recombination Recombinant vaccine, pig all turn sun in immune 21d afterwards, and two exempt from seven days afterwards, and antibody level has a very high raising, and And continues to and exempt from rear 56d.Antibody arousal level and antibody duration are better than certain commercially available brand inactivated virus vaccine.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Suzhou Shi Nuo Bioisystech Co., Ltd
<120>foot and mouth disease virus novel gene engineering subunit vaccine
<160> 23
<170> SIPOSequenceListing 1.0
<210> 1
<211> 678
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 1
ggatccatgg gcatcttccc agtggcttgc tcggatggat acggaggact ggtgaccacc 60
gatccaaaga ccgccgatcc agtgtacggc aaggtgttca accccccacg caacatgctg 120
ccaggacgtt tcaccaacct gctggacgtg gccgaggcct gcccaacctt cctgcacttc 180
gacggagacg tgccatacgt gaccaccaag accgatagcg atcgcgtgct ggctcagttc 240
gatctgtcct tggccgccaa gcacatgagc aacaccttcc tggccggact ggctcagtac 300
tacacccagt acagcggcac cgtgaacctg cacttcatgt tcaccggccc aaccgacgct 360
aaggcccgtt acatgatcgc ctacgcccca ccaggcatgg aaccaccaaa gaccccagag 420
gccgccgctc attgtattca cgccgaatgg gacaccggcc tgaacagcaa gttcaccttc 480
agcatcccat acctgagcgc cgccgattac gcttacacag ctagcgacgc cgccgaaaca 540
accaacgtgc agggttgggt ctgcctgttc caaatcaccc acggaaaggc cgagggagac 600
gctttggtgg tgttggcttc ggccggaaag gatttcgagc tgcgactgcc agtggacgct 660
cgccagcagt aaaagctt 678
<210> 2
<211> 35
<212> DNA
<213>artificial primer (artificial sequence)
<400> 2
ataggatcca tgggcatctt cccagtggct tgctc 35
<210> 3
<211> 42
<212> DNA
<213>artificial primer (artificial sequence)
<400> 3
ataaagcttt tactgctggc gagcgtccac tggcagtcgc ag 42
<210> 4
<211> 927
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 4
ctcgagatgg gagctggaca aagtagccca gctaccggaa gccagaatca gagcggaaac 60
accggcagca tcatcaacaa ctactacatg cagcagtacc agaacagcat ggacacccag 120
ctgggcgata atgccatcag cggaggaagc aacgagggca gcacagatac caccagcacc 180
cacaccacca acacccagaa caacgattgg ttcagcaagc tggctagcag cgcctttagc 240
ggactgttcg gagctctgct ggccgataag aagaccgagg agaccaccct gctggaagat 300
cgcatcctga ccacccgcaa cggacataca accagcacca cccagagcag cgtgggaatt 360
acccacggat acgccacagc cgaggatttc gtgaacggcc caaataccag cggcctggag 420
acacgagtgg tgcaagccga gcgcttcttc aagacccacc tgttcgattg ggtgaccagc 480
gaccccttcg gacgttgcta tctgctggag ctgccaaccg accacaaggg agtgtacggc 540
tccctgaccg acagctacgc ctacatgcgc aacggttggg acgtggaagt gacagccgtg 600
ggaaaccagt tcaacggcgg ttgtctgctg gtggcgatgg tgcccgagtt gtgctccatt 660
gagcgtcgag agctgttcca actgaccctg ttcccacacc agttcatcaa cccccgcacc 720
aacatgaccg cccatatcaa ggtgccattc gtgggcgtga accgctacga ccagtacaag 780
gtgcacaagc cctggacatt ggtggtgatg gtggtggctc cactgacagt gaacaccgag 840
ggagctccac agatcaaggt gtacgccaac atcgccccaa ccaacgtgca cgtggccgga 900
gagttcccca gcaaggagta aggtacc 927
<210> 5
<211> 40
<212> DNA
<213>artificial primer (artificial sequence)
<400> 5
atactcgaga tgggagctgg acaaagtagc ccagctaccg 40
<210> 6
<211> 33
<212> DNA
<213>artificial primer (artificial sequence)
<400> 6
ataggtacct tactccttgc tggggaactc tcc 33
<210> 7
<211> 1386
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 7
tacgtaaaaa tgaggcggcg gcaatgattt acgggcatat attcggtcga ggaggacgaa 60
atattctgaa atgggacgaa aggggatgac gcggcgcggc tctcgtcttc ccgcctcgca 120
ttcaacgctc ggctcgacca atcagcggcc gagttttgcg ctatgaccat ataaggcgat 180
acgtttgtcc gggtggggtg ggacgagcca ttgcggctta tcgcgcgggg gagtaccctc 240
tcaaaatgca ctatgcactg ccgtaacact ctttcggaaa gaatataata catcagtaga 300
tacctcttga aaattaggat ccgatgcata ccataaatcc ccaaattaga gagaataaaa 360
ggggttaatt cgatcgagag taatgacact tggaacgacc tcccctctgg agaaagtcga 420
cgatccgaga ggtggagtaa gcgccctact cactctctca tggataagaa gaccgaggag 480
accaccctgc tggaagatcg catcctgacc acccgcaacg gacatacaac cagcaccacc 540
cagagcagcg tgggaattac ccacggatac gccacagccg aggatttcgt gaacggccca 600
aataccagcg gcctggagac acgagtggtg caagccgagc gcttcttcaa gacccacctg 660
ttcgattggg tgaccagcga ccccttcgga cgttgctatc tgctggagct gccaaccgac 720
cacaagggag tgtacggctc cctgaccgac agctacgcct acatgcgcaa cggttgggac 780
gtggaagtga cagccgtggg aaaccagttc aacggcggtt gtctgctggt ggcgatggtg 840
cccgagttgt gctccattga gcgtcgagag ctgttccaac tgaccctgtt cccacaccag 900
ttcatcaacc cccgcaccaa catgaccgcc catatcaagg tgccattcgt gggcgtgaac 960
cgctacgacc agtacaaggt gcacaagccc tggacattgg tggtgatggt ggtggctcca 1020
ctgacagtga acaccgaggg agctccacag atcaaggtgt acgccaacat cgccccaacc 1080
aacgtgcacg tggccggaga gttccccagc aaggagtaat gagtcgagaa gtactagagg 1140
atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 1200
ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 1260
gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 1320
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc 1380
tacgta 1386
<210> 8
<211> 663
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 8
atggataaga agaccgagga gaccaccctg ctggaagatc gcatcctgac cacccgcaac 60
ggacatacaa ccagcaccac ccagagcagc gtgggaatta cccacggata cgccacagcc 120
gaggatttcg tgaacggccc aaataccagc ggcctggaga cacgagtggt gcaagccgag 180
cgcttcttca agacccacct gttcgattgg gtgaccagcg accccttcgg acgttgctat 240
ctgctggagc tgccaaccga ccacaaggga gtgtacggct ccctgaccga cagctacgcc 300
tacatgcgca acggttggga cgtggaagtg acagccgtgg gaaaccagtt caacggcggt 360
tgtctgctgg tggcgatggt gcccgagttg tgctccattg agcgtcgaga gctgttccaa 420
ctgaccctgt tcccacacca gttcatcaac ccccgcacca acatgaccgc ccatatcaag 480
gtgccattcg tgggcgtgaa ccgctacgac cagtacaagg tgcacaagcc ctggacattg 540
gtggtgatgg tggtggctcc actgacagtg aacaccgagg gagctccaca gatcaaggtg 600
tacgccaaca tcgccccaac caacgtgcac gtggccggag agttccccag caaggagtaa 660
tga 663
<210> 9
<211> 36
<212> DNA
<213>artificial primer (artificial sequence)
<400> 9
atatacgtaa aaatgaggcg gcggcaatga tttacg 36
<210> 10
<211> 32
<212> DNA
<213>artificial primer (artificial sequence)
<400> 10
atatacgtag atccagacat gataagatac at 32
<210> 11
<211> 1330
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 11
cctaggtcat gatgataaac aatgtatggt gctaatgttg cttcaacaac aattctgttg 60
aactgtgttt tcatgtttgc caacaagcac ctttatactc ggtggcctcc ccaccaccaa 120
cttttttgca ctgcaaaaaa acacgctttt gcacgcgggc ccatacatag tacaaactct 180
acgtttcgta gactatttta cataaatagt ctacaccgtt gtatacgctc caaatacact 240
accacacatt gaaccttttt gcagtgcaaa aaagtacgtg tcggcagtca cgtaggccgg 300
ccttatcggg tcgcgtcctg tcacgtacga atcacattat cggaccggac gagtgttgtc 360
ttatcgtgac aggacgccag cttcctgtgt tgctaaccgc agccggacgc aactccttat 420
cggaacagga cgcgcctcca tatcagccgc gcgttatctc atgcgcgtga ccggacacga 480
ggcgcccgtc ccgcttatcg cgcctataaa tacagcccgc aacgatctgg taaacacagt 540
tgaacagcat ctgttatgac aacaagtacc ggagagagcg ccgatccagt gacagccaca 600
gtggagaact acggaggaga aacacaggtg cagcgtcgcc accataccga cgtgtccttc 660
atcctggacc gcttcgtgaa agtgaccccc aaggacagca tcaacgtgct ggacctgatg 720
cagaccccca gtcataccct ggtgggagct ctgctgcgta ccgccaccta ctacttcgcc 780
gatctggagg tcgccgtgaa gcacgaggga gatctgactt gggtgcccaa cggagctcca 840
gaagccgctc tggataacac caccaacccc accgcctacc ataaggctcc actgacacgc 900
ctggctctgc catataccgc tccacatcgc gtgctggcta ccgtgtacaa cggcaattgc 960
aagtacgccg gaggaagcct gccaaacgtg cgaggagatc tgcaagtgct ggcccaaaaa 1020
gccgctcgtc cactgccaac cagcttcaac tacggagcca tcaaggccac acgcgtgaca 1080
gagctgctgt accgcatgaa gcgcgccgag acctattgcc cacgtccatt gttggccgtg 1140
catccaagcg ccgcccgtca taagcagaag atcgtggctc ccgtgaagca gtaaatctta 1200
gtttgtattg tcatgtttta atacaatatg ttatgtttaa atatgttttt aataaatttt 1260
ataaaataat ttcaactttt attgtaacaa cattgtccat ttacacactc ctttcaagcg 1320
cgtgcctagg 1330
<210> 12
<211> 639
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 12
atgacaacaa gtaccggaga gagcgccgat ccagtgacag ccacagtgga gaactacgga 60
ggagaaacac aggtgcagcg tcgccaccat accgacgtgt ccttcatcct ggaccgcttc 120
gtgaaagtga cccccaagga cagcatcaac gtgctggacc tgatgcagac ccccagtcat 180
accctggtgg gagctctgct gcgtaccgcc acctactact tcgccgatct ggaggtcgcc 240
gtgaagcacg agggagatct gacttgggtg cccaacggag ctccagaagc cgctctggat 300
aacaccacca accccaccgc ctaccataag gctccactga cacgcctggc tctgccatat 360
accgctccac atcgcgtgct ggctaccgtg tacaacggca attgcaagta cgccggagga 420
agcctgccaa acgtgcgagg agatctgcaa gtgctggccc aaaaagccgc tcgtccactg 480
ccaaccagct tcaactacgg agccatcaag gccacacgcg tgacagagct gctgtaccgc 540
atgaagcgcg ccgagaccta ttgcccacgt ccattgttgg ccgtgcatcc aagcgccgcc 600
cgtcataagc agaagatcgt ggctcccgtg aagcagtaa 639
<210> 13
<211> 34
<212> DNA
<213>artificial primer (artificial sequence)
<400> 13
atacctaggt catgatgata aacaatgtat ggtg 34
<210> 14
<211> 33
<212> DNA
<213>artificial primer (artificial sequence)
<400> 14
atacctaggc acgcgcttga aaggagtgtg taa 33
<210> 15
<211> 987
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 15
tgtacaaaaa tgaggcggcg gcaatgattt acgggcatat attcggtcga ggaggacgaa 60
atattctgaa atgggacgaa aggggatgac gcggcgcggc tctcgtcttc ccgcctcgca 120
ttcaacgctc ggctcgacca atcagcggcc gagttttgcg ctatgaccat ataaggcgat 180
acgtttgtcc gggtggggtg ggacgagcca ttgcggctta tcgcgcgggg gagtaccctc 240
tcaaaatgca ctatgcactg ccgtaacact ctttcggaaa gaatataata catcagtaga 300
tacctcttga aaattaggat ccgatgcata ccataaatcc ccaaattaga gagaataaaa 360
ggggttaatt cgatcgagag taatgacact tggaacgacc tcccctctgg agaaagtcga 420
cgatccgaga ggtggagtaa gcgccctact cactctctca tgggagctgg acaaagtagc 480
ccagctaccg gaagccagaa tcagagcgga aacaccggca gcatcatcaa caactactac 540
atgcagcagt accagaacag catggacacc cagctgggcg ataatgccat cagcggagga 600
agcaacgagg gcagcacaga taccaccagc acccacacca ccaacaccca gaacaacgat 660
tggttcagca agctggctag cagcgccttt agcggactgt tcggagctct gctggcctaa 720
tgagtcgaga agtactagag gatcataatc agccatacca catttgtaga ggttttactt 780
gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt 840
gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 900
ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 960
gtatcttatc atgtctggat ctgtaca 987
<210> 16
<211> 264
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 16
atgggagctg gacaaagtag cccagctacc ggaagccaga atcagagcgg aaacaccggc 60
agcatcatca acaactacta catgcagcag taccagaaca gcatggacac ccagctgggc 120
gataatgcca tcagcggagg aagcaacgag ggcagcacag ataccaccag cacccacacc 180
accaacaccc agaacaacga ttggttcagc aagctggcta gcagcgcctt tagcggactg 240
ttcggagctc tgctggccta atga 264
<210> 17
<211> 36
<212> DNA
<213>artificial primer (artificial sequence)
<400> 17
atatgtacaa aaatgaggcg gcggcaatga tttacg 36
<210> 18
<211> 39
<212> DNA
<213>artificial primer (artificial sequence)
<400> 18
atatgtacag atccagacat gataagatac attgatgag 39
<210> 19
<211> 221
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 19
Met Gly Ile Phe Pro Val Ala Cys Ser Asp Gly Tyr Gly Gly Leu Val
1 5 10 15
Thr Thr Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Lys Val Phe Asn
20 25 30
Pro Pro Arg Asn Met Leu Pro Gly Arg Phe Thr Asn Leu Leu Asp Val
35 40 45
Ala Glu Ala Cys Pro Thr Phe Leu His Phe Asp Gly Asp Val Pro Tyr
50 55 60
Val Thr Thr Lys Thr Asp Ser Asp Arg Val Leu Ala Gln Phe Asp Leu
65 70 75 80
Ser Leu Ala Ala Lys His Met Ser Asn Thr Phe Leu Ala Gly Leu Ala
85 90 95
Gln Tyr Tyr Thr Gln Tyr Ser Gly Thr Val Asn Leu His Phe Met Phe
100 105 110
Thr Gly Pro Thr Asp Ala Lys Ala Arg Tyr Met Ile Ala Tyr Ala Pro
115 120 125
Pro Gly Met Glu Pro Pro Lys Thr Pro Glu Ala Ala Ala His Cys Ile
130 135 140
His Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile
145 150 155 160
Pro Tyr Leu Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Ala Ala
165 170 175
Glu Thr Thr Asn Val Gln Gly Trp Val Cys Leu Phe Gln Ile Thr His
180 185 190
Gly Lys Ala Glu Gly Asp Ala Leu Val Val Leu Ala Ser Ala Gly Lys
195 200 205
Asp Phe Glu Leu Arg Leu Pro Val Asp Ala Arg Gln Gln
210 215 220
<210> 20
<211> 304
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 20
Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser
1 5 10 15
Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln
20 25 30
Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser
35 40 45
Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln
50 55 60
Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Ser Gly Leu
65 70 75 80
Phe Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu
85 90 95
Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr
100 105 110
Gln Ser Ser Val Gly Ile Thr His Gly Tyr Ala Thr Ala Glu Asp Phe
115 120 125
Val Asn Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala
130 135 140
Glu Arg Phe Phe Lys Thr His Leu Phe Asp Trp Val Thr Ser Asp Pro
145 150 155 160
Phe Gly Arg Cys Tyr Leu Leu Glu Leu Pro Thr Asp His Lys Gly Val
165 170 175
Tyr Gly Ser Leu Thr Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp
180 185 190
Val Glu Val Thr Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu
195 200 205
Val Ala Met Val Pro Glu Leu Cys Ser Ile Glu Arg Arg Glu Leu Phe
210 215 220
Gln Leu Thr Leu Phe Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met
225 230 235 240
Thr Ala His Ile Lys Val Pro Phe Val Gly Val Asn Arg Tyr Asp Gln
245 250 255
Tyr Lys Val His Lys Pro Trp Thr Leu Val Val Met Val Val Ala Pro
260 265 270
Leu Thr Val Asn Thr Glu Gly Ala Pro Gln Ile Lys Val Tyr Ala Asn
275 280 285
Ile Ala Pro Thr Asn Val His Val Ala Gly Glu Phe Pro Ser Lys Glu
290 295 300
<210> 21
<211> 219
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 21
Met Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu
1 5 10 15
Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly
20 25 30
Ile Thr His Gly Tyr Ala Thr Ala Glu Asp Phe Val Asn Gly Pro Asn
35 40 45
Thr Ser Gly Leu Glu Thr Arg Val Val Gln Ala Glu Arg Phe Phe Lys
50 55 60
Thr His Leu Phe Asp Trp Val Thr Ser Asp Pro Phe Gly Arg Cys Tyr
65 70 75 80
Leu Leu Glu Leu Pro Thr Asp His Lys Gly Val Tyr Gly Ser Leu Thr
85 90 95
Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp Val Glu Val Thr Ala
100 105 110
Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu Val Ala Met Val Pro
115 120 125
Glu Leu Cys Ser Ile Glu Arg Arg Glu Leu Phe Gln Leu Thr Leu Phe
130 135 140
Pro His Gln Phe Ile Asn Pro Arg Thr Asn Met Thr Ala His Ile Lys
145 150 155 160
Val Pro Phe Val Gly Val Asn Arg Tyr Asp Gln Tyr Lys Val His Lys
165 170 175
Pro Trp Thr Leu Val Val Met Val Val Ala Pro Leu Thr Val Asn Thr
180 185 190
Glu Gly Ala Pro Gln Ile Lys Val Tyr Ala Asn Ile Ala Pro Thr Asn
195 200 205
Val His Val Ala Gly Glu Phe Pro Ser Lys Glu
210 215
<210> 22
<211> 212
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 22
Met Thr Thr Ser Thr Gly Glu Ser Ala Asp Pro Val Thr Ala Thr Val
1 5 10 15
Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp
20 25 30
Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro Lys Asp Ser
35 40 45
Ile Asn Val Leu Asp Leu Met Gln Thr Pro Ser His Thr Leu Val Gly
50 55 60
Ala Leu Leu Arg Thr Ala Thr Tyr Tyr Phe Ala Asp Leu Glu Val Ala
65 70 75 80
Val Lys His Glu Gly Asp Leu Thr Trp Val Pro Asn Gly Ala Pro Glu
85 90 95
Ala Ala Leu Asp Asn Thr Thr Asn Pro Thr Ala Tyr His Lys Ala Pro
100 105 110
Leu Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ala
115 120 125
Thr Val Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn
130 135 140
Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu
145 150 155 160
Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys Ala Thr Arg Val Thr Glu
165 170 175
Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu
180 185 190
Leu Ala Val His Pro Ser Ala Ala Arg His Lys Gln Lys Ile Val Ala
195 200 205
Pro Val Lys Gln
210
<210> 23
<211> 86
<212> PRT
<213>artificial sequence (artificial sequence)
<400> 23
Met Gly Ala Gly Gln Ser Ser Pro Ala Thr Gly Ser Gln Asn Gln Ser
1 5 10 15
Gly Asn Thr Gly Ser Ile Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln
20 25 30
Asn Ser Met Asp Thr Gln Leu Gly Asp Asn Ala Ile Ser Gly Gly Ser
35 40 45
Asn Glu Gly Ser Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln
50 55 60
Asn Asn Asp Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Ser Gly Leu
65 70 75 80
Phe Gly Ala Leu Leu Ala
85

Claims (12)

1. a kind of immune composition, it is characterised in that include: using sequence nucleic acid molecules as shown in SEQ ID NO:1 or with The O-shaped Foot-and-mouth disease VP3 egg of the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:1 It is white；Using sequence nucleic acid molecules as shown in SEQ ID NO:12 or with 95% or more the nucleotide sequence of SEQ ID NO:12 The O-shaped Foot-and-mouth disease VP1 albumen of identical nucleic acid molecule encoding；And it is selected from O-shaped foot and mouth disease virus structure egg One or two kinds of any combination of white VP2 albumen or O-shaped Foot-and-mouth disease VP4 albumen.
2. immune composition according to claim 1, which is characterized in that the O-shaped Foot-and-mouth disease VP3 Albumen includes the amino acid sequence of SEQ ID NO:19 or 95% or more the phase of full length amino acid sequence with SEQ ID NO:19 Same amino acid sequence；The O-shaped Foot-and-mouth disease VP1 albumen includes the amino acid sequence of SEQ ID NO:22 Or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:22.
3. immune composition according to claim 1, which is characterized in that the O-shaped Foot-and-mouth disease VP2 Albumen includes the amino acid sequence of SEQ ID NO:21 or 95% or more the phase of full length amino acid sequence with SEQ ID NO:21 Same amino acid sequence；The O-shaped Foot-and-mouth disease VP4 albumen includes the amino acid sequence of SEQ ID NO:23 Or the identical amino acid sequence of 95% or more full length amino acid sequence with SEQ ID NO:23.
4. immune composition according to claim 1, which is characterized in that the immune composition further includes O-shaped mouth Aphtovirus structural proteins VP0 albumen, the O-shaped Foot-and-mouth disease VP0 albumen are by sequence such as SEQ ID Nucleic acid molecules shown in NO:4 are obtained with the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:4 ?.
5. immune composition according to claim 4, which is characterized in that the O-shaped Foot-and-mouth disease VP0 Albumen includes the amino acid sequence of SEQ ID NO:20 or 95% or more the phase of full length amino acid sequence with SEQ ID NO:20 Same amino acid sequence.
6. immune composition according to claim 3, which is characterized in that wherein,
The O-shaped Foot-and-mouth disease VP2 albumen be by sequence nucleic acid molecules as shown in SEQ ID NO:8 or The identical nucleic acid molecule encoding of 95% or more nucleotide sequence of person and SEQ ID NO:8 obtain；
The O-shaped Foot-and-mouth disease VP4 albumen is by sequence nucleic acid molecules as shown in SEQ ID NO:16 Or it is obtained with the identical nucleic acid molecule encoding of 95% or more nucleotide sequence of SEQ ID NO:16.
7. the preparation method of immune composition described in claim 1~6 any one, it is characterised in that the following steps are included:
S1, by the gene cloning of O-shaped Foot-and-mouth disease to corresponding shuttle vector, obtain recombinant shuttle vector；
S2, recombinant shuttle vector is transformed into the DH10Bac bacterium containing Baculovirus Gene group plasmid, in recombinant shuttle vector Destination gene expression is confined to being inserted into rod-shaped viral genome plasmid, and the rod-shaped disease of the recombination containing destination gene expression frame is obtained Virus gene group plasmid；
S3, recombinant baculovirus geneome plasmid transfection insect cell is obtained into recombinant baculovirus；
S4, the recombinant baculovirus of acquisition is inoculated with insect cell, large-scale production recombinates O-shaped foot and mouth disease virus in the reactor Structural proteins；
S5, the O-shaped Foot-and-mouth disease of the recombination obtained in S4 step is added in adjuvant to get the immune combination Object.
8. preparation method according to claim 7, which is characterized in that step S1 includes: by O-shaped foot and mouth disease virus structure egg The encoding gene of white VP3 and VP1 and any one or two kinds in O-shaped Foot-and-mouth disease VP2, VP4 Expression cassette is cloned on same shuttle vector to arrive the recombinant shuttle vector.
9. preparation method according to claim 8, which is characterized in that step S1 includes: by O-shaped foot and mouth disease virus structure egg The encoding gene of white VP3 and VP1, the expression of any one or two kinds in O-shaped Foot-and-mouth disease VP2, VP4 The expression cassette of frame and O-shaped Foot-and-mouth disease VP0 are cloned on same shuttle vector to arrive the recombination Shuttle vector.
10. preparation method according to claim 9, which is characterized in that step S1 includes: by O-shaped foot and mouth disease virus structure The encoding gene of albumen VP3, VP1, VP2, VP4, VP0 are cloned on same shuttle vector to shuttle to get to the recombination Carrier.
11. immune composition described in claim 1~6 any one is directed to O for inducing in animal subject for producing The purposes of the medicament of the immune response of type foot-and-mouth disease virus antigen.
12. immune composition described in claim 1~6 any one is for producing for preventing animal by O-shaped hoof-and-mouth disease The purposes of the medicament of poison infection.
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