CN1318592C - O type foot and mouth disease virus DNA vaccine and its preparing method - Google Patents
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- CN1318592C CN1318592C CNB2003101156023A CN200310115602A CN1318592C CN 1318592 C CN1318592 C CN 1318592C CN B2003101156023 A CNB2003101156023 A CN B2003101156023A CN 200310115602 A CN200310115602 A CN 200310115602A CN 1318592 C CN1318592 C CN 1318592C
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Abstract
The present invention discloses an O type foot-and-mouth disease virus DNA vaccine and a development method thereof, more specifically a DNA vaccine containing a foot-and-mouth disease virus (preferably an O type foot-and-mouth disease virus, particularly a China99 strain O type foot-and-mouth disease virus) structural protein P1 gene, non-structural protein 2A, 2B and 3C genes and a non-essential 3D gene, and a preparation method thereof.
Description
Technical field
The present invention relates generally to O type foot and mouth disease virus dna vaccination and method of production thereof, particularly, relate to and utilize gene recombination technology, make up method of production of two kinds of dna vaccinations that comprise O type foot and mouth disease virus structural protein gene and a part of nonstructural protein gene and products thereof, purposes.
Background technology
Dna vaccination can be induced protectiveness cellular immunization and humoral immune reaction, is used to develop the vaccine of the multiple Different Kinds of Pathogens of opposing in recent years.Because used antiviral vaccine is mainly induced humoral immunization at present, so dna immunization may be very useful for the protection that long lasting antiviral property disease is provided.The article in the dna vaccination field of delivering in a large number is main relevant with virus disease, and this has reflected at anti-virus aspect for strong needs novel, safe and effective immunization strategy.(Foot-and-Mouth disease virus FMDV) is a kind of virus that causes the artiodactyl serious disease to foot and mouth disease virus, because it often causes serious economy loss, thereby is classified as the category-A transmissible disease by International Office of Epizootics (OIE).At present, immunoprophylaxis from the traditional vaccine of live virus is mainly used in the control of foot and mouth disease, although this traditional vaccine is effectively, in recent years, especially may and escape relevant with the not complete inactivation of virus from production of vaccine ground in the foot and mouth disease several times of Europe outburst.Therefore must develop a kind of safer, effective aftosa vaccine.
Data shows, the natural hollow capsid that lacks nucleic acid and comprise VP1, VP3 and VP0 can be induced the antibody response similar to complete virus particle (Rowlands et al in the cavy body, 1975), and structural protein precursor P1-2A is VP0 by the 3C protease cracking under the 2A mediation at first, VP3 and VP1, wherein VP0 continues to be cracked into VP2, VP4.These four kinds of structural protein constitute 20 body capsid structures of virus particle with the form of each 60 copy, and antigenic determinant is all arranged on these structural protein, and are especially important with the epitope on the VP1.Nonstructural Protein 2B, 3D also are the potential stimulus factors of cellular immunization and humoral immunization.(Foster-cuevas，1996，Foster etal，1998，Collen et al，1998)。
Summary of the invention
The invention provides DNA sequences encoding, its (preferred sequence ground) comprises foot and mouth disease virus (preferred O type foot and mouth disease virus, China99 strain O type foot and mouth disease virus particularly) structural protein P1 gene and Nonstructural Protein 2A, 2B, 3C gene and dispensable 3D gene, preferably it has the sequence of SEQ ID NO:1 or 2.
The present invention also provides transcription unit, its contain DNA sequences encoding of the present invention and be operably connected with this dna sequence dna, in eukaryotic cell (particularly mammalian cell), express the necessary expression regulation element of this dna sequence dna (promotor especially, as viral promotors or mammalian cell promotor, and transcription termination sequence), preferably, it is the transcription unit that sequence plasmid pcDNA3.1/P12X3C or pcDNA3.1/P12X3C3D are comprised.
The present invention further provides the carrier for expression of eukaryon as the FMDV dna vaccination, it contains transcription unit of the present invention.
In a preferred implementation, expression vector of the present invention is a plasmid, for example contains the eukaryon expression plasmid of transcription unit of the present invention, and preferably, it is plasmid pcDNA3.1/P12X3C or pcDNA3.1/P12X3C3D.
The present invention also provides the method that makes up expression vector of the present invention, and this method comprises:
Obtain the dna sequence dna of coding foot and mouth disease virus (preferred O type foot and mouth disease virus, particularly China99 strain O type foot and mouth disease virus) structural protein P1 gene and Nonstructural Protein 2A, 2B, 3C gene and dispensable 3D gene,
Adopt will the encode dna sequence dna segmentation of foot and mouth disease virus structural protein P1 gene and Nonstructural Protein 2A, 2B, 3C gene and dispensable 3D gene of known technology to be cloned in the carrier for expression of eukaryon (particularly plasmid) of sky, formation can expression structure albumen P1 gene and the recombinant expression vector of Nonstructural Protein 2A, 2B, 3C gene and dispensable 3D gene.
The present invention also provides dna vaccination, and it comprises one or more expression vectors of the present invention and one or more pharmaceutically acceptable carrier, for example thinner.
The present invention also relates to prepare the method for dna vaccination, described method comprises
Adopt known technology to make up one or more expression vectors of the present invention,
The expression vector that obtains is mixed with one or more pharmaceutically acceptable carrier.
The invention still further relates to foot and mouth disease virus structural protein P1 gene and Nonstructural Protein 2A, 2B, 3C gene and the dispensable 3D gene purposes in the preparation dna vaccination.
Also relate to transcription unit of the present invention, the purposes of expression vector of the present invention in the preparation dna vaccination.
Of the present invention is the method that prevents and/or treats non-human animal's foot and mouth disease more on the one hand, comprises the vaccine of the present invention of using significant quantity to described animal.
The present invention also provides the host cell that contains expression vector of the present invention, prokaryotic cell prokaryocyte for example, and as Bacillus coli cells, and eukaryotic cell, as mammalian cell.
Behind reference as detailed below and accompanying drawing, these and other aspect of the present invention will be obvious.All reference disclosed herein are this all complete quoting as a reference.
Description of drawings
Fig. 1 be the present invention obtain 3 ' sudden change fragment I, 5 ' sudden change fragment II and the agarose gel electrophoretogram of total length P12X3C.
Fig. 2 is an agarose gel electrophoretogram, shows the qualification result of positive plasmid pcDNA3.1/P12X3C.
Fig. 3 is 3D fragment, P12X3C fragment and the segmental agarose gel electrophoretogram of P12X3C3D that the present invention obtains.
Fig. 4 is an agarose gel electrophoretogram, shows the qualification result of positive plasmid pcDNA3.1/P12X3C3D.
Fig. 5 is the BHK-21 cellmediated immunity fluorescent dye photo of transfection.
Fig. 6 is the ELISA broken line graph, shows the OD492 value of sample under the different extent of dilution.
Fig. 7 is an antibody horizontal dynamic change broken line graph in immunity back in the body.
Fig. 8 shows immune guinea pig T lymphproliferation response.
Fig. 9 illustrates to obtain the process of purpose segment III.
Figure 10 illustrates the building process of plasmid pcDNA3.1/P12X3C.
Figure 11 illustrates the building process of plasmid pcDNA3.1/P12X3C3D.
Embodiment
Unless specialize, the term that this paper adopted has the common implication of the technical field of the invention.
The virus that the present invention uses can be any foot and mouth disease virus (belonging to aphthovirus genus (Aphtovirus)), preferred O type foot and mouth disease virus, particularly China99 strain O type foot and mouth disease virus.Foot-and-mouth disease virus gene group coding sequence can be divided into three part: 5 '-P1-P2-P3-3 ' by function, becomes various final products through protease hydrolysis again.For example the final product of P2 is 2A, 2B and 2C, and the final product of P3 can be 3A, 3B, 3C and 3D.Characteristics of the present invention are P1 gene, 2A, 2B, 3C and dispensable 3D gene are used to make up the foot and mouth disease virus dna vaccination.
Therefore, at first, the invention provides the DNA sequences encoding that comprises FMDV P1,2A, 2B, 3C and dispensable 3D gene.Preferably, this dna sequence dna sequentially comprises foot and mouth disease virus (preferred O type foot and mouth disease virus successively, China99 strain O type foot and mouth disease virus particularly) structural protein P1 gene and Nonstructural Protein 2A, 2B, 3C gene and dispensable 3D gene, or sequentially form by them successively.
In DNA sequences encoding, the corresponding sequence of FMDV P1,2A, 2B, 3C and 3D gene virus preferably natural or wild-type also can be the sequence of modifying.The preferably less change of character of described modification (comprise alternative, lack, insert, add), promptly conserved amino acid substitutes and folding or active the substituting of other not remarkably influenced protein or polypeptide; Little disappearance is typically 1 to about 30 amino acid whose disappearances; Extend with little amino or carboxyl terminal, for example N-terminal methionine residues, be no more than the little joint peptide or the affinity tag of about 20-25 residue.In a preferred implementation, described modification does not change or does not change substantially coded aminoacid sequence.
In a concrete embodiment, above-mentioned DNA sequences encoding has the sequence of SEQ ID NO:1 or 2.
The segmental dna sequence dna of coding FMDV genome purpose can adopt known Protocols in Molecular Biology to obtain from FMDV.For example can obtain the first chain cDNA from viral RNA, utilize suitable primer then, randomly be cloned in one or more carriers then through splicing from the double-stranded DNA of this first chain by the synthetic expectation in polymerase chain reaction by reverse transcription.
The present invention also is provided for expressing the transcription unit of above-mentioned DNA sequences encoding.This transcription unit except containing above-mentioned DNA sequences encoding, also contain can be operatively connected with this dna sequence dna, in eukaryotic cell, express the necessary expression regulation element of (transcribe and translate) this dna sequence dna.The most basic expression regulation element comprises promotor and transcription terminator, and the latter can stop effectively transcribing and add Poly (A) tail effectively to transcription product.Other controlling element can have enhanser etc.These controlling elements are known in the art, referring to for example Sambrook etc., molecular cloning: laboratory manual (Molecular Cloning:A Laboratory Manual), second edition, ColdSpring Harbor, NY, 1989.
Usually, this transcription unit is present in the expression vector.Expression vector is linearity or ring-shaped DNA molecule, and it contains the fragment of the desired polypeptides of encoding and other fragment that can be operatively connected with it in order to express this purpose fragment.These other fragments can comprise promotor and terminator sequence and optional one or more replication origin, one or more selective marker, enhanser, polyadenylation signal or the like.Expression vector generally derives from plasmid or viral DNA, or can contain both elements simultaneously.
The intensity of these elements may be different with specificity.According to used carrier system and host, can use any amount of element of suitably transcribing and translate, comprise composing type and inducible promoter, preferred constitutive promoter.In the mammal cell line system, preferred usually promotor from mammalian genes or mammalian virus.
Also available special start signal realizes more effective translation of the sequence of coding desired polypeptides.These signals comprise ATG initiator codon and flanking sequence.When sequence, its initiator codon and the upstream sequence of coded polypeptide insert in the suitable expression vector, can not need other to transcribe or translate control signal.Yet, when only inserting encoding sequence or its part, should use external source translation control signal, comprise the ATG initiator codon.In addition, initiator codon should be in correct reading frame, to guarantee the segmental translation of whole insertion.External source translation element and initiator codon can be different sourcess, be nature and synthetic.
The expression vector that is used to express DNA sequences encoding of the present invention can be any carrier (as plasmid or virus) that can be advantageously used in the recombinant DNA program and dna sequence dna is expressed.Typically, the consistency between the host cell that carrier and carrier will import is depended in the selection of carrier.This carrier also can be a virus vector, for example, and adenovirus, adeno-associated virus, retrovirus or vaccinia virus or other poxvirus (for example bird poxvirus).This carrier is linearity or closed hoop plasmid preferably.
This carrier can be a self-replicating type carrier, promptly exists as the outer entity of karyomit(e), it duplicates the carrier that is independent of chromosome duplication, as plasmid, extrachromosomal element, minichromosome or artificial chromosome.This carrier can contain the sequence of any assurance self-replacation.Perhaps, this carrier can be to be incorporated into after importing host cell in the genome and and the carrier that duplicates together of its karyomit(e) of integrating.And, single carrier or plasmid be can use, or the two or more carriers or the plasmid of all DNA in the host cell gene group to be imported contained together.
Carrier of the present invention can contain one or more selective markers that allow to select easily transformant.Selective marker is that its product provides biocide or virus resistance, heavy metal resistance, the gene of auxotrophic prototroph etc. relatively.The example of bacterium selective marker is the dal gene of subtilis or Bacillus licheniformis, or gives the mark of antibiotics resistance such as penbritin, kantlex, paraxin or tetracyclin resistance.The suitable mark that is used for yeast host cell is ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3.Carrier of the present invention can comprise and allows this carrier stable integration to the host cell gene group or allow this carrier to be independent of genome and one or more elements of self-replicating in this cell.
In order to be integrated in the host cell gene group, this carrier can depend on the nucleotide sequence of coding said polypeptide or be used for making this carrier by homology or non-homogeneous reorganization stable integration any other carrier element to genome.Perhaps, this carrier can contain to be useful on by homologous recombination instruct the extra nucleotide sequence integrate in the host cell gene group.
For self-replicating, this carrier can further comprise make carrier can be in bacterial cell the replication orgin of self-replicating.The example of bacterium replication orgin is to allow the replication orgin of pBR322, pUC19, pACYC177 and the pACYC184 plasmid duplicate and the replication orgin of pUB110, the pE194, pTA1060 and pAM β 1 plasmid that allow to duplicate in bacillus in intestinal bacteria.
Can in host cell, insert the nucleotide sequence of the present invention of copy more than, to increase the output of gene product.The increase of this nucleotide sequence copy number can realize by the following method: this sequence of integrating an additional copy in the host cell gene group at least again; Or in cell, comprise the increased selectable marker gene that has this nucleotide sequence, and when suitably selective agent exists, cultivate these cells, can select the selectable marker gene that containing the amplification copy, therefore also contain the cell of this nucleotide sequence of additional copy.
Being used to connect said elements is well known to those skilled in the artly (for example to see with the program that makes up recombinant expression vector of the present invention, Sambrook etc., molecular cloning: laboratory manual (MolecularCloning:A Laboratory Manual), second edition, Cold Spring Harbor, NY, 1989; With volumes such as Ausubel, contemporary molecular biology experiment guide (Current Protocolsin Molecular Biology), John Wiley and Sons, Inc., NY, 1987).
The present invention also provides the host cell of carrier conversion of the present invention or transfection.Host cell can be a prokaryotic cell prokaryocyte, as Bacillus coli cells, or eukaryotic cell, as mammalian cell.The technology that foreign DNA is imported in the various host cells is disclosed in for example Sambrook etc., and the same and Ausubel etc. is the same.
The host cell that contains and express the polynucleotide sequence of wishing can be identified with the known several different methods of those skilled in the art.These methods include but not limited to: DNA-DNA or DNA-RNA hybridization and protein biological assay or immunoassay comprise as the technology based on film, solution or chip, PCR or the like.
Recombinant host cell of the present invention can be as the source that produces dna sequence dna of the present invention, transcription unit, expression vector.
The present invention also provides the dna vaccination that comprises expression vector of the present invention, is used for preventing and/or treating foot and mouth disease animal (particularly Mammals).Dna vaccination of the present invention can also contain pharmaceutically acceptable carrier except containing expression vector of the present invention, for example thinner, adjuvant etc., and these pharmaceutically acceptable carrier can be any auxiliary substances that is adopted in the common dna vaccination.
About preparation, route of administration, mode and dosage, dna vaccination of the present invention can with common vaccine, what especially common dna vaccination adopted is similar.And, can easily determine by those skilled in the art.
In dna vaccination, the dosage of plasmid of the present invention can be 0.01-0.1mg/kg, preferred 0.02-0.05mg/kg.
In specific embodiments, the invention provides and carry FMDV structural protein P1 gene and part Nonstructural Protein 2A, 2B, 3C gene or carry FMDV structural protein P1 gene and two kinds of plasmid DNA of part Nonstructural Protein 2A, 2B, 3C, 3D gene, and make dna vaccination effective control livestock foot-and-mouth disease, safety and stability respectively with it, its method of production is provided simultaneously.
For realizing above purpose, the invention provides following scheme: obtain the various objectives gene fragment from the T carrier that carries different fragments, obtain total length P12X3C gene, cut connection by enzyme again and make up the plasmid that has the P12X3C gene by site-directed mutagenesis technique.And on plasmid vector that has the P12X3C gene that builds and the T carrier that has the 3D gene, obtain gene fragment P12X3C and 3D respectively, cut connection by enzyme and make up the plasmid DNA that has the P12X3C3D gene.
The present invention includes two kinds of O type FMDV dna vaccinations; comprise the gene of coding FMDV China99 strain structural protein P1, Nonstructural Protein 2A, 2B, 3C and the gene of encode FMDV China99 strain structural protein P1, Nonstructural Protein 2A, 2B, 3C, 3D respectively; the plasmid DNA of its structure is purified, dilution directly can stimulate animal body generation immune response and immunoprotection is provided as dna vaccination inoculation domestic animal.
The manufacture method of domestic animal FMDV dna vaccination of the present invention comprises the preparation of gene recombination, cell transfecting, Protein Detection, dna vaccination, and its concrete manufacturing processed is:
(1) to be connected with the T support template of China99 strain FMDV corresponding gene fragment, amplify fragment I that is partly with the P1-2A gene and the fragment II that has another part 2B, 3C gene, connect the fragment III that becomes to have total length P1-2A2B3C gene by the pcr amplification method according to the rite-directed mutagenesis principle, and KpnI and XbaI site are introduced in the downstream thereon, cut the plasmid vector that the same enzyme of itself and warp is handled by enzyme and link to each other, thereby make up the plasmid DNA pcDNA3.1/P12X3C that has P1-2A, 2B, 3C gene.
(2) be template with plasmid pcDNA3.1/P12X3C with the T carrier that has the 3D gene fragment respectively, amplify total length P1-2A2B3C and 3D gene, respectively AflII, KpnI and KpnI, XbaI site are introduced in the downstream thereon, cut method of attachment by enzyme, it is connected with the pcDNA3.1 (+) that cuts processing through same enzyme, thereby makes up the plasmid pcDNA3.1/P12X3C3D that has P1-2A, 2B, 3C, 3D gene.
(3) with behind the plasmid DNA purifying that makes up, transfection BHK-21 cell adopts indirect fluorescent staining and sandwich ELISA method, the target protein that the testing goal gene is expressed in external environment under the control of the CMV promotor on the carrier.
Two kinds of plasmid DNA that (4) will make up are extracted and purifying with alkaline lysis is a large amount of respectively, be diluted to finite concentration with damping fluid DPBS after, with it as dna vaccination direct injection animal.
In specific implementation process of the present invention, the manufacturing of dna vaccination comprises with special primer and obtains goal gene, construction of recombinant plasmid, recombinant plasmid in external transfection, preparation dna vaccination and with its immune animal.With the carrier of eukaryon expression plasmid pcDNA3.1 (+) (available from Invitrogen company, Cat No:V860-20) as the constructed dna vaccine.Utilize the special primer of design to amplify purpose fragment I, II from the T carrier that has the various objectives gene, and use a pair of expression primer according to the principle of rite-directed mutagenesis fragment I and II are obtained fragment III with the pcr amplification method, at 5 of fragment III ' end and 3 ' hold and introduce KpnI and XbaI site respectively.After simultaneously fragment III enzyme being cut with restriction endonuclease KpnI and XbaI, with the pcDNA3.1 (+) that handles with same two kinds of enzymes thus be connected and make up first kind of plasmid pcDNA3.1/P12X3C.Wherein, the upstream and downstream primer of fragment I is respectively:
Upstream primer: 5 '-ACCTCCRACGGGTGGTACGC-3 '
Downstream primer: 5 '-CACTGCCACTTCAAGTTCTGCGAAG-3 '
The upstream and downstream primer of fragment II is respectively:
Upstream primer: 5 '-CTTCGCAGAACTTGAAGTGCCAGTG-3 '
Downstream primer: 5 '-CAGCTATGACCATGATTACGCCAAG-3 '
The expression primer of amplified fragments III is respectively:
Upstream primer: 5 '-GG
ATGGGAGCCGGACAATCCAGCCCGGCGAC-3 '
Downstream primer: 5 '-GC
CCTCGTGGTGTGGTTCGGGATCGATGTGTG-3 '
Be template with the T carrier that has the 3D gene fragment and first kind of carrier pcDNA3.1/P12X3C of structure respectively again, use two pairs of special primers and obtain 3D gene fragment and P12X3C gene fragment by pcr amplification, and introduce KpnI and XbaI site at 5 of 3D gene fragment ' end and 3 ' end respectively, introduce AflII and KpnI site at 5 of P12X3C gene fragment ' end and 3 ' end.Handle the 3D gene fragment with restriction endonuclease KpnI and XbaI enzyme cutting, cut processing P12X3C gene fragment with restriction endonuclease AflII and KpnI enzyme, successively respectively enzyme is cut 3D gene fragment and the P12X3C gene fragment and the pcDNA3.1 (+) that successively handles of processing, thereby made up second kind of plasmid pcDNA3.1/P12X3C3D with restriction endonuclease KpnI, XbaI, AflII.Wherein, the amplimer of 3D gene is respectively:
Upstream primer: C (+) 5 '-GG
CACTCCGCAGGCGGCAACGGAGTTGG-3 '
Downstream primer: Dg (-) 5 '-GG
ATGCGTCACCGCACACGGCGTTACAA-3 '
The primer of P12X3C gene amplification is respectively:
Upstream primer: A (+) 5 '-GG
ATGGGAGCCGGACAATCCAGCCCGGCGAC-3 '
Downstream primer: B (-) 5 '-GG
AACGATTGAAAGTCTCGGCTCCGTCC-3 '
Two kinds of recombinant plasmids are adopted the liposome transfection method respectively, on the 35mm plate, change seven BHK-21 cell, after transfection 24-48 hour, the harvested cell individual layer is also used cell pyrolysis liquid (100mMTris-HCl, pH 8.3-8.6,2%Triton X-100,150nm NaCl, 0.6M KCl, 5mMEDTA, 1% aprotinin, 3mMPMSF, 1 μ g/ml leupeptin, 5 μ g/ml trypsininhibitor) handle with certain proportion.With this lysate PBST doubling dilution, on 96 hole enzyme plates, adopt double-antibodies sandwich ELISA to measure the expression of FMDV capsid protein.And will cover with the cover glass of the BHK-21 cell after the transfection, detect the expression of FMDV differential protein in the BHK-21 cell with the indirect fluorescent staining.Two kinds of methods all detect under the effect of CMV promotor on the carrier for expression of eukaryon, and the FMDV capsid protein gene can be expressed in the cultured cell in vitro system, and have proved that these albumen have immunogenicity.
Two kinds of plasmid DNA are extracted in a large number with alkaline lysis, and with film absorb-elute method purifying, being diluted to concentration with the DPBS damping fluid is 1 μ g/ μ l, with its as dna vaccination direct injection cavy after the shank intramuscular, the 1st week after the immunity first time, 2 weeks, 3 weeks, the 1st week after 4 weeks and the immunity for the second time, 2 weeks, 3 weeks took a blood sample respectively, survey the FMDV specific antibody in its serum, and before immunity, the 28th day of immunity for the first time, the 21st day of immunity for the second time, use the microneutralization experiment and detect serum serum NAT, and use the T lymphocyte proliferation assay and detect the propagation situation of animal splenic T lymphocyte under phytohaemagglutinin (PHA) stimulates.The result shows, in the cavy body of two kinds of plasmid immune group, produces FMDV specific antibody and neutralizing antibody, and stronger proliferative response appears in the spleen lymphocyte of plasmid immune guinea pig, and control group guinea pig serum and T lymphocyte do not have similar variation.The 3rd week after the immunity second time attacks with 100ID
50FMDV China99 strain, the protection ratio of immune guinea pig is 100%, and the control group cavy is all dead.
Two kinds of plasmids that make up among the present invention have following outstanding feature as the FMDV dna vaccination: can express the FMDV capsid protein in animal body, thereby stimulate special antibody and neutralizing antibody; Can simulate the process of FMDV natural infection cell, experience is transcribed in cell, expression, submission etc. as endogenous antigen albumen, stimulate the FMDV capsid protein differentiation of body T lymphopoiesis, thereby induce stronger cell immune response.Therefore two kinds of dna vaccinations among the present invention can significantly improve the immunological competence of immune domestic animal, can make the infection of domestic animal opposing FMDV.
Below by non-restrictive example the present invention is described in more detail:
Embodiment
The structure of 1 first kind of plasmid pcDNA3.1/P12X3C of embodiment
For when obtaining total length P12X3C gene, in downstream primer, introduce the restriction enzyme site of XbaI site as clone's usefulness, utilize the terminator codon in the XbaI site simultaneously, on the P1 gene fragment must be fallen by existing XbaI enzyme cutting site mutation.Therefore, utilize upstream primer A:5 '-ACCTCCRACGGGTGGTACGC-3 ' and downstream primer A1:5 '-CACTGCCACTTCAAG
TTCTGCGAAG-3 ', wherein in downstream primer, replace (inefficacy) XbaI site of having introduced change by base, make up with this laboratory of T carrier pT/P1-2A[that has the P1-2A gene fragment, for the P1-2A gene is connected to pGEM-T Easy VectorSystem (available from Promega company, Cat No:A3610) product in] be template, pcr amplification obtains the part P1 gene (fragment I) that 3 ' XbaI site is suddenlyd change.The reaction system of amplified fragments I (50 μ l) is: 10 * damping fluid, 5 μ l, dNTPs4 μ l, MgCl
23 μ l, pT/P1-2A0.5 μ l, upstream primer A0.5 μ l, downstream primer A10.5 μ l, Taq archaeal dna polymerase (5U/ μ l; Available from Promega company, Cat No:M1661) 0.5 μ l, H
2O 36 μ l.Reaction conditions is: 94 ℃ of 3min, a circulation; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min30sec circulate 35 times; Be 72 ℃ of 5min at last.The PCR product electrophoretic separation on 1% sepharose that takes a morsel, EB dyeing.The results are shown in Figure 1 swimming lane 1.As seen from the figure, obtained the fragment of expection size.
In addition, utilize upstream primer B:5 '-CTTCGCAGAACTTGAAGTGGCAGTG-3 ' and downstream primer B1:5 '-CAGCTATGACCATGATTACGCCAAG-3 ', wherein replace (inefficacy) XbaI site of introducing change by base among the upstream primer B, to have part P1 gene, this laboratory of the T carrier pT/P12X3C[of 2B gene and total length 3C gene makes up, for the P12X3C gene is connected to pGEM-T Easy Vector System (available from Promega company, Cat No:A3610) product in] be template, pcr amplification obtains the part P1-2A that 5 ' XbaI site is suddenlyd change, 2B and total length 3C gene (fragment II).The reaction system of amplified fragments II (50 μ l) is: 10 * damping fluid, 5 μ l, dNTPs 4 μ l, MgCl
23 μ l, pT/P12X3C 0.5 μ l, upstream primer B 0.5 μ l, downstream primer B1 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l, H
2O 36 μ l.Reaction conditions is: 94 ℃ of 5min, a circulation; 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 3min circulate 35 times; Be 72 ℃ of 8min at last.The PCR product electrophoretic separation on 1% sepharose that takes a morsel, EB dyeing.The results are shown in Figure 1 swimming lane 2.As seen from the figure, obtained the fragment of expection size.
With fragment I (not purifying) and fragment II (not purifying) as template, with upstream primer C:5 '-GG
ATGGGAGCCGGACAATCCACCCCGGCGAC-3 ' and downstream primer C1:5 '-GC
CCTCGTGGTGTGGTTCGGGATCGATGTGTG-3 ' is by the gene fragment P12A283C (fragment III) of pcr amplification acquisition total length.Wherein introduce the KpnI restriction enzyme site in the upstream primer, introduce the XbaI site in the downstream primer.The reaction system of amplified fragments III (50 μ l) is: 10 * damping fluid, 5 μ l, dNTPs 4 μ l, MgCl
23 μ l, fragment I (not purifying) 0.5 μ l, fragment II (not purifying) 0.5 μ l, upstream primer C 0.5 μ l, downstream primer C1 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l, H
2O 35.5 μ l.Reaction conditions is: 94 ℃ of 5min, a circulation; 94 ℃ of 1min20sec, 60 ℃ of 1min, 72 ℃ of 3min30sec circulate 35 times; Be 72 ℃ of 10min at last.The PCR product electrophoretic separation on 1% sepharose that takes a morsel, EB dyeing.The results are shown in Figure 1 swimming lane 3.As seen from the figure, obtained the fragment of expection size.Fig. 9 has illustrated to obtain the process of purpose segment III.
Fragment III is remained PCR product electrophoretic separation purifying (according to the AgaroseGel DNA Purification Kit Ver of Takara company 2.0, Cat No:DV805A operation instructions is carried out, concentration is 0.5 μ g/ μ l) use restriction endonuclease KpnI (available from Takara company, Cat No:D1068A) and XbaI (available from Takara company, Cat No:D1093A) simultaneously fragment III enzyme is cut also purifying, use the T4 dna ligase (available from Takara company then, Cat No:D2011A) according to supplier's explanation enzyme is cut fragment III and pcDNA3.1 (+) (available from Invitrogen company, the Cat No:V860-20) connection of handling with same two kinds of enzymes behind the purifying.To connect product according to calcium chloride method transformed into escherichia coli JM109 bacterial strain.The positive bacterium colony that picking is possible, amplification cultivation is extracted plasmid in a small amount with alkaline lysis.Use pcr amplification method (adopting primer C and C1) and restriction enzyme complete digestion method (adopting the XbaI/KpnI enzyme of independent EcoRI and associating to cut respectively) to identify positive plasmid respectively.Fig. 2 is the qualification result (1% agarose gel electrophoresis) of a positive plasmid, and wherein, swimming lane 5 is PCR qualification results, shows the product that has obtained the expection size; Swimming lane 3 is EcoRI complete degestion results, and swimming lane 4 is XbaI/KpnI complete degestion results, and the result meets the restriction enzyme mapping of expection.This positive plasmid is called first kind of plasmid pcDNA3.1/P12X3C (building process and plasmid map are referring to Figure 10).
With plasmid pcDNA3.1/P12X3C order-checking (by the order-checking of Takara company), obtain the segmental complete sequence of being cloned, see SEQ ID NO:1, as follows.
GGTACCATGGGAGCCGGACAATCCAGCCCGGCGCGACTGGGGTCACAGAACCAGTCAGGCAACACTGGAAGCATTATCAACAA
TTACTACATGCAGCAGTACCAGAACTCCATGGACACGCAACTTGGTGATAACGCTATTAGCGGAGGCTCCAACGAGGGGT
CCACGGACACCACCTCCACCCACACAACCAACACTCAGAACAATGACTGGTTTTCAAAGCTGGCCAGTTCCGCTTTTAGC
GGTCTTTTCGGCGCTCTTCTCGCCGACAAGAAAACCGAGGAGACCACTCTTCTCGAGGACCGCATCCTCACTACCCGCAA
CGGACACACGACCTCGACAACCCAGTCGAGCGTTGGAGTCACTTACGGGTACGCAACAGCTGAGGACTTTGTGAGCGGAC
CAAACACATCTGGGCTTGAGACCAGGGTTGTGCAGGCAGAGCGGTTCTTCAAAACCCACTTGTTCGACTGGGTCACCGGT
GACCCGTTCGGACGGTGCTACCTGCTGGAACTCCCAACTGACCACAAAGGTGTCTACGGCGGCCTGACTGACTCTTATGC
TTACATGAGAAACGGTTGGGATGTTGAGGTCACTGCAGTGGGAAATCAGTTCAACGGAGGATGTCTGTTGGTGGCCATGG
TGCCAGAACTTTGCTCTATTGACAAGAGAGAGCTGTACCAGCTCACGCTCTTTCCCCACCAGTTCATCAACCCCCGGACG
AACATGACGGCGCACATCACTGTGCCCTTTGTTGGTGTCAACCGCTACGACCAGTACAAGGTACACAAACCTTGGACCCT
CGTGGTTATGGTTGTGGCCCCGCTGACTGTCAACACCGAAGGTGCCCCACAGATCAAGGTCTATGCCAACATCGCCCCTA
CCAACGTGCACGTTGCGGGTGAGTTCCCTTCTAAGGAAGGGATCTTCCCCGTGGCATGTAGCGACGGTTACGGTGGTCTG
GTGACCACTGACCCAAAGACGGCTGACCCCGCCTACGGGAAAGTGTTCAATCCACCTCGCAACATGTTGCCGGGGCGGTT
CACCAACTTCCTTGATGTGGCTGAGGCGTGCCCTACGTTTCTGCACTTTGAGGGTGACGTGCCGTACGTGACCACAAAGA
CGGACTCAGACAGGGTGCTCGCCCAGTTTGACTTGTCTCTGGCAGCAAAGCACATGTCAAACACCTTCCTGGCAGGTCTC
GCCCAGTACTACACACAGTACAGCGGCACCATCAACCTGCACTTCATGTTCACAGGACCCACTGACGCGAAAGCGCGTTA
CATGATTGCATACGCCCCCCCTGGCATGGAGCCGCCCAAAACACCTGAGGCGGCCGCTCACTGCATTCATGCGGAGTGGG
ACACAGGGTTGAATTCAAAATTCACATTTTCAATCCCTTACCTTTCGGCGGCTGATTACGCGTACACCGCGTCTGACGCT
GCGGAGACCACAAATGTACAGGGATGGGTCTGCCTGTTTCAAATTACACACGGGAAGGCTGACGGCGACGCACTGGTCGT
TCTAGCTAGCGCCGGTAAGGACTTTGAGCTGCGTCTGCCAGTTGACGCTCGCACGCAGACCACCTCCACAGGTGAGTCGG
CTGACCCCGTGACTGCCACTGTTGAGAACTACGGTGGTGAGACACAGGTCCAGAGACGCCAACACACGGATGTCTCGTTC
ATATTAGACAGATTTGTGAAAGTAACACCAAAAGACCAAATTAATGTGTTGGACCTGATGCAAACCCCTGCACACACTTT
GGTAGGCGCGCTCCTCCGTACTGCCACCTACTACTTCGCAGATCTAGAAGTGGCAGTGAAACACGAGGGGAACCTTACCT
GGGTCCCGAATGGGGCGCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCGCTCACCCGG
CTTGCCTCTTAGCACACGGCACCACACCATGTCTTGGCTACTGTTTACAACGGGAACTGCAAGTATGACGAGAGCCCCGT
GACCAATGTGAGAGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGCTGCCTACCTCCTTCAATTACGGTGCCG
TCAAAGCCACTCGGGTGACTGAACTGCTTTACCGCATGAAGAGGGCCGAAACATACTGCCCCCGGCCTCTTTTGGCTATT
CACCCGAGCGAAGCTAGACACAAACAAAAGATCGTGGCGCCTGTGAAACAGCTTTTGAACTTTGACCTGCTCAAGTTGGC
AGGAGACGTCGAGTCCAACCCTGGGCCCTTCTTCTCTGACGTCAGGTCAAATCTTTCCAAGTTGGTTGAAACCATCAACC
AGATGCAGGAGGACATGTCGGATCCAGGACCGGTGAAGAAACCTGTCGCTTTGAAAGTGAAAGCAAAGAATTTGATTGTC
ACTGAGAGTGGTGCTCCCCCGACTGACTTGCAAAAGATGGTCATGGGTAACACCAAGCCTGTTGAGCTCATCCTCGACGG
GAAGACGGTGGCCATCTGCTGCGCCACCGGAGTGTTTGGTACTGCCTACCTTGTTCCTCGTCATCTTTTCGCAGAGAAGT
ATGACAAGATCATGTTGGACGGCAGAGCCATGACAGACAGTGACTACAGAGTGTTTGAGTTTGAGATTAAAGTGAAAGGA
CAGGACATGCTCTCAGACGCCGCGCTCATGGTGCTTCACCGTGGGAATCGCGTGCGGGACATCACGAAGcACTTCCGTGA
TGTGGCAAGAATGAAGAAAGGCACCCCCGTCGTCGGCGTGATCAACAACGCTGATGTTGGGAGACTGATCTTCTCTGGTG
AGGCCCTTACCTACAAGGACATCGTAGTGTGCATGGACGGAGACACCATGCCCGGTCTCTTCGCCTACAAAGCCGCCACC
AAGGCGGGTTACTGTGGAGGAGCCGTTCTTGCAAAGGACGGAGCCGAGACTTTCATCGTCGGCACTCACTCCGCAGGCGG
CAATGGAGTTGGATACTGCTCATGCGTTTCCAGGTCTATGCTGCTTAAAATGAAGGCACACATCGATCCCGAACCACACC
ACGAGGTCTAGA
The structure of 2 second kinds of plasmid pcDNA3.1/P12X3C3D of embodiment
With the T carrier that has the 3D gene fragment [pGEM/3D is the product that 3D gene and pGEM-T EasyVector System (available from Promega company, Cat No:A3610) connect] be template, application upstream primer C (+) 5 '-GG
CACTCCGCAGGCGGCAACGGAGTTGG-3 ' and downstream primer Dg (-) 5 '-GG
ATGCGTCACCGCACACGGCGTTACAA-3 ' obtains the 3D gene fragment by pcr amplification, introduces KpnI and XbaI site at 5 of 3D gene fragment ' end and 3 ' end respectively.The reaction system of amplified fragments 3D gene (50 μ l) is: 10 * damping fluid, 5 μ l, dNTPs 4 μ l, MgCl
23 μ l, primer C (+) 0.5 μ l, primer Dg (-) 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l, H
2O 36.5 μ l.Reaction conditions is: 94 ℃ of 5min, a circulation; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 1min30sec circulate 35 times; Be 72 ℃ of 10min at last.The PCR product electrophoretic separation on 1% sepharose that takes a morsel, EB dyeing.The results are shown in Figure 3 swimming lanes 1.As seen from the figure, obtained the fragment of expection size, to remain 3D amplified fragments purifying (according to the Agarose Gel DNA Purification Kit Ver2.0 of Takara company, Cat No:DV805A operation instructions is carried out, concentration is 0.5 μ g/ μ l) afterwards use restriction endonuclease KpnI (available from Takara company, Cat No:D1068A) and the XbaI enzyme (available from Takara company, CatNo:D1093A) cut and handle the 3D gene fragment, and once more purifying in order to make up pcDNA3.1/P12X3C3D.
First kind of carrier pcDNA3.1/P12X3C with structure is template, uses upstream primer A (+): 5 '-GG
ATGGGAGCCGGACAATCCAGCCCGGCGAC-3 ' and downstream primer B (-): 5 '-GG
AACGATTGAAAGTCTCGGCTCCGTCC-3 ' obtains the P12X3C gene fragment by pcr amplification, introduce AflII and KpnI site at 5 of P12X3C gene fragment ' end and 3 ' end respectively. the reaction system of amplified fragments P12X3C gene (50 μ l) is: 10 * damping fluid, 5 μ l, dNTPs 4 μ l, MgCl
23 μ l, primer A (+) 0.5 μ l, primer B (-) 0.5 μ l, Taq archaeal dna polymerase 0.5 μ l, H
2O 36.5 μ l.Reaction conditions is: 94 ℃ of 5min, a circulation; 94 ℃ of 1min30sec, 60 ℃ of 1min20sec, 72 ℃ of 4min30sec circulate 35 times; Be 72 ℃ of 10min at last.The PCR product electrophoretic separation on 1% sepharose that takes a morsel, EB dyeing. the results are shown in Figure 3 swimming lanes 2.As seen from the figure, obtained the fragment of expection size.With gained P12X3C fragment purification (according to the Agarose Gel DNAPurification Kit Ver of Takara company 2.0, Cat No:DV805A operation instructions is carried out, concentration is 0.5 μ g/ μ l) afterwards use restriction endonuclease AflII (available from Takara company, Cat No:D1003A) and KpnI (available from Takara company, Cat No:D1068A) enzyme is cut and purifying, the P12X3C gene fragment of handling is connected with the pcDNA3.1 (+) that handles with restriction endonuclease KpnI, AflII, obtains interstitial granules in.The middle interstitial granules that will obtain then is with KpnI, XbaI enzyme cutting, is connected with the 3D fragment with same double digestion in advance afterwards, thereby makes up second kind of plasmid pcDNA3.1/P12X3C3D (building process and plasmid map are referring to Figure 11).To connect product according to calcium chloride method transformed into escherichia coli JM109 bacterial strain.The positive bacterium colony that picking is possible, amplification cultivation is extracted plasmid in a small amount with alkaline lysis.Use pcr amplification method (adopting primer A (+) and Dg (-)) and restriction enzyme complete digestion method (adopting AflII/KpnI and KpnI/ respectively) XbaI enzyme cutting respectively) the evaluation positive plasmid.Fig. 4 is the qualification result (1% agarose gel electrophoresis) of a positive plasmid, wherein, swimming lane 3,4,5th, the PCR qualification result shows the product that has obtained the expection size; Swimming lane 6,7th, complete degestion result, result meet the restriction enzyme mapping of expection.
With plasmid pcDNA3.1/P12X3C3D order-checking (by the order-checking of Takara company), obtain the segmental complete sequence of being cloned, see SEQ ID NO:2, as follows.
CTTAAGATGGGAGCCGGACAATCCAGCCCGGCGACTGGGTCACAGAACCAGTCAGGCAACACTGGAAGCATTATCAACAA
TTACTACATGCAGCAGTACAGCAACTCCATGGACACGCAACTTGGTGATAACGCTATTAGCGGAGGCTCCAACGAGGGGT
CCACGGACACCACCTCCACCCACACAACCAACACTCAGAACAATGACTGGTTTTCAAAGCTGGCCAGTTCCGCTTTTAGC
GGTCTTTTCGGCGCTCTTCTCGCCGACAAGAAAACCGAGGAGACCACTCTTCTCGAGGACCGCATCCTCACTACCCGCAA
CGGACACACGACCTCGACAACCCAGTCGAGCGTTGGAGTCACTTACGGGTACGCAACAGCTGAGGACTTTGTGAGCGGAC
CAAACACATCTGGGCTTGAGACCAGGGTTGTGCAGGCAGAGCGGTTCTTCAAAACCCACTTGTTCGACTGGGTCACCGGT
GACCCGTTCGGACGGTGCTACCTGCTGGAACTCCCAACTGACCACAAAGGTGTCTACGGCGGCCTGACTGACTCTTATGC
TTACATGAGAAACGGTTGGGATGTTGAGGTCACTGCAGTGGGAAATCAGTTCAACGGAGGATGTCTGTTGGTGGCCATGG
TGCCAGAACTTTGCTCTATTGACAAGAGAGAGCTGTACCAGCTCACGCTCTTTCCCCACCAGTTCATCAACCCCCGGACG
AACATGACGGCGCACATCACTGTGCCCTTTGTTGGTGTCAACCGCTACGACCAGTACAAGGTACACAAACCTTGGACCCT
CGTGGTTATGGTTGTGGCCCCGCTGACTGTCAACACCGAAGGTGCCCCACAGATCAAGGTCTATGCCAACATCGCCCCTA
CCAACGTGCACGTTGCGGGTGAGTTCCCTTCTAAGGAAGGGATCTTCCCCGTGGCATGTAGCGACGGTTACGGTGGTCTG
GTGACCACTGACCCAAAGACGGCTGACCCCGCCTACGGGAAAGTGTTCAATCCACCTCGCAACATGTTGCCGGGGCGGTT
CACCAACTTCCTTGATGTGGCTGAGGCGTGCCCTACGTTTCTGCACTTTGAGGGTGACGTGCCGTACGTGACCACAAAGA
CGGACTCAGACAGGGTGCTCGCCCAGTTTGACTTGTCTCTGGCAGCAAAGCACATGTCAAACACCTTCCTGGCAGGTCTC
GCCCAGTACTACACACAGTACAGCGGCACCATCAACCTGCACTTCATGTTCACAGGACCCACTGACGCGAAAGCGCGTTA
CATGATTGCATACGCCCCCCCTGGCATGGAGCCGCCCAAAACACCTGAGGCGGCCGCTCACTGCATTCATGCGGAGTGGG
ACACAGGGTTGAATTCAAAATTCACATTTTCAATCCCTTACCTTTCGGCGGCTGATTACGCGTACACCGCGTCTGACGCT
GCGGAGACCACAAATGTACAGGGATGGGTCTGCCTGTTTCAAATTACACACGGGAAGGCTGACGGCGACGCACTGGTCGT
TCTAGCTAGCGCCGGTAAGGACTTTGAGCTGCGTCTGCCAGTTGACGCTCGCACGCAGACCACCTCCACAGGTGAGTCGG
CTGACCCCGTGACTGCCACTGTTGAGAACTACGGTGGTGAGACACAGGTCCAGAGACGCCAACACACGGATGTCTCGTTC
ATATTAGACAGATTTGTGAAAGTAACACCAAAAGACCAAATTAATGTGTTGGACCTGATGCAAACCCCTGCACACACTTT
GGTAGGCGCGCTCCTCCGTACTGCCACCTACTACTTCGCAGATCTAGAAGTGGCAGTGAAACACGAGGGGAACCTTACCT
GGGTCCCGAATGGGGCGCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCGCTCACCCGG
CTTGCACTGCCTTACACGGCACCACACCATGTCTTGGCTACTGTTTACAACGGGAACTGCAAGTATGACGAGAGCCCCGT
GACCAATGTGAGAGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGCTGCCTACCTCCTTCAATTACGGTGCCG
TCAAAGCCACTCGGGTGACTGAACTGCTTTACCGCATGAAGAGGGCCGAAACATACTGCCCCCGGCCTCTTTTGGCTATT
CACCCGAGCGAAGCTAGACACAAACAAAAGATCGTGGCGCCTGTGAAACAGCTTTTGAACTTTGACCTGCTCAAGTTGGC
AGGAGACGTCGAGTCCAACCCTGGGCCCTTCTTCTCTGACGTCAGGTCAAATCTTTCCAAGTTGGTTGAAACCATCAACC
AGATGCAGGAGGACATGTCGGATCCAGGACCGGTGAAGAAACCTGTCGCTTTGAAAGTGAAAGCAAAGAATTTGATTGTC
ACTGAGAGTGGTGGTCCCCCGACTGACTTGCAAAAGATGGTCATGGGTAACACCAAGCCTGTTGAGCTCATCCTCGACGG
GAAGACGGTGGCCATCTGCTGCGCCACCGGAGTGTTTGGTACTGCCTACCTTGTTCCTCGTCATCTTTTCGCAGAGAAGT
ATGACAAGATCATGTTGGACGGCAGAGCCATGACAGACAGTGACTACAGAGTGTTTGAGTTTGAGATTAAAGTGAAAGGA
CAGGACATGCTCTCAGACGCCGCGCTCATGGTGCTTCACCGTGGGAATCGCGTGCGGGACATCACGAAGCACTTCCGTGA
TGTGGCAAGAATGAAGAAAGGCACCCCCGTCGTCGGCGTGATCAACAACGCTGATGTTGGGAGACTGATCTTCTCTGGTG
AGGCCCTTACCTACAAGGACATCGTAGTGTGCATGGACGGAGACACCATGCCCGGTCTCTTCGCCTACAAAGCCGCCACC
AAGGCGGGTTACTGTGGAGGAGCCGTTCTTGCAAAGGACGGAGCCGAGACTTTCATCGTTGGTACCCACTCCGCAGGCGG
CAACGGAGTTGGATACTGCTCATGCGTTTCCAGGTCTATGCTGCTTAAAATGAAGGCACACATCGATCCCGAACCACACC
ACGAGGGATTGATAGTTGACACCAGAGATGTTGAGGAGCGCGTACATGTCATGCGCAAAACCAAGCTCGCACCCACCGTG
GCACACGGTGTGTTTAACCCCGAATTTGGGCCTGCCGCCTTGTCCAACAAGGGCCCGCGCCTGAATGAGGGGGTTGTCCT
CGATGAAGCCATCTTCTCCAAACACAAAGGAAACACAAAGATGTCTGAGGAGGACAAAGCGCTGTTCCGCCGCTGTGCTG
CTGACTACGCGTCGCGTCTACATAGCGTGCTGGGTACGGCAAATGCCCCACTGAGCACTTACGTGGCAATCAAGGGCGTC
GACGGACTTGACGCCATGGAACCAGACACCGCGCCTGGTCTCCCCTGGGCTCTCCAGGGGAAACGCCGTGGTGCGCTCAT
TGATTTCGAGAACGGCACTGTCGGACCCGAGGTTGAAGCTGCCTTGAAGCTCATGGAGAAAAGAGAGTACAAGTTTGTAT
GCCAGACCTTCCTGAAGGACGAGATTCGCCCGATGGAGAAGGTACGTGCCGGCAAGACTCGCATTGTCGACGTCCTGCCT
GTTGAACACATTCTTTACACCAGGATGATGATTGGCAGATTTTGTGCTCAAATGCACTCAAACAACGGACCGCAAATTGG
CTCGGCGGTTGGTTGTAATCCTGATGTTGATTGGCAAAGATTTGGCACGCATTTTGCTCAGTACAGAAACGTGTGGGATG
TGGACTATTCGGCCTTTGATGCCAACCACTGCAGTGACGCAATGAACATCATGTTTGAGGAGGTGTTCAACACGGATTTC
GGGTTCCACCCAAACGCTGAGTGGATCCTGAAAACTCTCGTGAACACTGAACACGCCTATGAGAACAAACGCATCACTGT
TGAAGGCGGGATGCCGTCTGGTTGTTCCGCAACAAGCATCATCAACACAATTTTGAACAACATCTACGTGCTCTACGCCT
TGCGTAGACACTATGAGGGAGTTGAGCTGGACTCTTACACCATGATCTCCTACGGAGACGACATCGTGGTTGCAAGTGAT
TACGATCTGGACTTTGAGGCCCTCAAGCCTCACTTCAAATCCCTTGGTCAAACCATTACTCCAGCTGACAAAAGCGACAA
AGGTTTTGTTCTTGGTCACTCCATTACCGAGTTCACTTTCCTCAAAAGACACTTCCACATGGACTATGGAACTGGGTTTT
ACAAACCTGTGATGGCTTCGAAGACCCTCGAGGCTATCCTCTCCTTTGCACGCCGTGGGACCATACAGGAGAAGTTGATC
TCCGTGGCAGGACTCGCCGTCCACTCTGGACCTGACGAGTACCGGCGTCTCTTTGAGCCTTTCCAGGGCCTCTTTGAGAT
TCCAAGTTACAGATCACTTTACCTGCGTTGGGTGAACGCCGTGTGCGGTGACGCATTCTAGA
The expression of embodiment 3 recombinant plasmid transfecting animal cells and goal gene
The JM109 intestinal bacteria that will have two kinds of plasmid DNA breed in the less salt LB nutritive medium (NaCl 5g/L) that contains Zeomycin (25 μ g/ml), extract plasmid DNA (concentration 300 μ g/ml) according to the S N A PMidiPrep Kit of Invitrogen company (Cat No:K1910-01) operation instructions, measure its OD value to determine transfection BHK-21 cell and used transfection reagent ratio.Transfection BHK-21 cell on the 35mm plate is dissolved in 4 μ g plasmid DNA in the 250 Ke DMEM nutrient solutions (no foetal calf serum and microbiotic), adds 8 μ L lipofectamine LipofectaminePlus again
TMReagent (available from Invitrogen company, Cat No:10964-013) mixes the back room temperature and places 20min, in addition with 12 μ L lipofectamine Lipofectamine Plus
TMReagent is (available from Invitrogen company, Cat No:10964-013) is dissolved in the 250 μ L DMEM nutrient solutions (no foetal calf serum and microbiotic), room temperature is placed 20min, both mix the back and place 20min in room temperature, mixed solution are slowly added in the cell to 37 ℃ of hatching 5h, remove the transfection mixed solution, add the nutritive medium that 5mL contains 10% foetal calf serum, behind 37 ℃ of cultivation 24-48h, the results transfectional cell.Adopt indirect immunofluorescence and double-antibodies sandwich ELISA to detect the expression of FMDV specific gene fragment under the effect of CMV promotor in the plasmid DNA.
Indirect immunofluorescence is collected transfectional cell stand density in the culture dish and is reached cover glass more than 90% when detecting, 0.01%PBS liquid rinsing 1-2 time, cold acetone-20 is 30min ℃ fixedly, blot liquid after the 0.01%PBS rinsing, drip 1: 800 foot and mouth disease rabbit positive serum (preserve in this laboratory), hatch 30min in 37 ℃ of wet boxes, 0.01%PBS liquid rinsing 5 times, the FITC-goat anti-rabbit igg that adds 1: 160 is (available from Sigma company, Cat No:F0382), the 30min that dyes in 37 ℃ of wet boxes, glycerine mounting after the 0.01%PBS liquid rinsing 5 times is observed down in the Olympus fluorescent microscope.The result shows, there is specific fluorescence to produce in the BHK-21 cell of transfection pcDNA3.1/P12X3C, and specific fluorescence does not appear in the blank cell of the negative cells of transfection pcDNA3.1 (+) and untransfected, illustration purpose albumen is the correct (see figure 5) of expressing in the BHK-21 cell.
With the 0.01%PBS washing back trysinization of the cell behind the transfection 48h with sterilization, collecting cell Digestive system and the centrifugal supernatant of abandoning added cell pyrolysis liquid (100mM Tris-HCl in the cell precipitation when double-antibody sandwich elisa detected, pH 8.3-8.6,2%Triton X-100,150nm NaCl, 0.6MKCl, 5mM EDTA, 1%aprotinin, 3mM PMSF, 1 μ g/ml Leupeptin, 5 μ g/mlTrypsin Inhibitor) handle cell and centrifugal, supernatant is used for ELISA and detects.96 hole enzyme plates are with 1: 800 foot and mouth disease rabbit positive serum (preserve in this laboratory) embedding, 4 ℃ are spent the night, after the horse serum sealing, with FMDV antigen (preserve in this laboratory), the cell pyrolysis liquid of transfection pcDNA3.1/P12X3C, the negative control cell lysate of transfection pcDNA3.1 (+) and do not change seven blank cell pyrolysis liquid with 2 times of gradient dilutions, each extent of dilution is established two multiple holes, behind 37 ℃ of effect 1h, the PBST washing, the positive antiserum(antisera) of foot and mouth disease cavy (preserve in this laboratory) that adds 1: 1600,37 ℃ of effect 1h, the washing back adds 1: 2000 the anti-cavy IgG of HRP-rabbit (available from Sigma company, Cat No:A5545), 37 ℃ of effect 1h add substrate OPD-H
2O
2In 37 ℃ of effect 15min, with stop buffer (1.25M H
2SO
4) finish reaction, measure the OD value in λ 492nm place.The result shows that B group and C, D organize significant difference (P＜0.01), and different not remarkable (P＜0.05) (the seeing Table 1) of the OD value difference between C and D group.Along with the raising of doubling dilution degree, the OD value of the OD value of B group and A group reduces simultaneously gradually, but the OD value of the OD value of C group and D group changes little (see figure 6).
Table 1 sandwich ELISA method detects the target protein expression in the BHK-21 cell
| Group | | Extension rate | |||||||||||||
| 1∶2 | 1∶4 | 1∶8 | 1∶16 | 1∶32 | 1∶64 | 1∶128 | |||||||||
| A group B group C group D group | 1.73±0.01 0.5±0.02 0.18±0.00 0.18±0.09 | 1.60±0.02 0.50±0.04 0.14±0.01 0.14±0.02 | 1.51±0.01 0.37±0.01 0.15±0.03 0.15±0.02 | 1.24±0.01 0.34±0.02 0.13±0.02 0.12±0.00 | 0.92±0.03 0.22±0.02 0.13±0.02 0.13±0.03 | 0.73±0.01 0.20±0.00 0.22±0.03 0.17±0.01 | 0.58±0.04 0.16±0.00 0.16±0.03 0.13±0.00 | 0.45±0.00 0.13±0.03 0.15±0.05 0.16±0.04 | |||||||
A organizes (positive control): cell cultures is also used the FMDV of BEI deactivation; B organizes (sample to be checked): the BHK-21 cell pyrolysis liquid of pcDNA3.1/P12X3C transfection; C (negative control): the BHK-21 cell pyrolysis liquid of pcDNA3.1 transfection; D (blank): the BHK-21cell lysate of untransfected.
The detection of inoculation and immunne response in embodiment 4 bodies
With the DPBS damping fluid (Dulbecco ' s phosphate buffered saline is available from Sigma company, Cat No:D8537) to be diluted to concentration be 1 μ g/ μ l to two kinds of plasmid DNA will extracting purifying, with its as dna vaccination direct injection cavy after the shank intramuscular (inject 200 μ g plasmid DNA when exempting from for every one, two inject 400 μ g plasmid DNA when exempting from), the 1st week after the immunity first time, 2 weeks, 3 weeks, the 1st week after 4 weeks and the immunity for the second time, 2 weeks, 3 weeks took a blood sample respectively, and be contrast with the serum before the immunity, the FMDV that adopts indirect elisa method to survey in its serum is special anti-.Take a blood sample three at random for every group, guinea pig serum was diluted with 1: 32, on 96 hole enzyme plates, be envelope antigen (diluting at 1: 10) with FMDV vaccine strain purified virus (this laboratory preserve), tested guinea pig serum is one anti-, the anti-cavy IgG of the rabbit of peroxidase labelling in 1: 2000 is (available from Sigma company, CatNo:A5545) be two anti-, the OD value in the every hole of mensuration, λ 492nm place returns to zero with blank well.Get the mean value (seeing Table 2) of every group of indirect ELISA method measurement result, and with the dynamic change of antibody horizontal graphical representation (see figure 7).Table 2 and Fig. 7 show that all except that D group and E group, second week of the specific antibody level of each immune group cavy after the immunity first time obviously improves.
Table 2 indirect ELISA detects guinea pig serum FMDV specific antibody result
| Group | The A group | The B group | The C group | The D group | The E group | |
| Before the immunity | 0.2125± 0.001 | 0.1585± 0.019 | 0.2040± 0.033 | 0.2445± 0.034 | 0.2405± 0.060 | |
| The immunity for the second time of immunity for the first time | First all the 3rd all first all the 3rd weeks of second week of 4th week of second week | 0.3260± 0.044 0.5053± 0.046 0.5417± 0.092 0.9940± 0.222 1.4035± 0.204 1.7225± 0.161 0.9310± 0.121 | 0.3347± 0.062 0.7237± 0.422 0.4493± 0.234 0.3935± 0.157 0.4715± 0.135 0.4095± 0.022 0.2460± 0.04 | 0.3567± 0.057 0.5937± 0.168 0.2777± 0.051 0.3055± 0.025 0.2500± 0.044 0.4600± 0.100 0.2357± 0.049 | 0.2663± 0.031 0.3427± 0.025 0.2887± 0.045 0.2670± 0.003 0.2165± 0.003 0.3890± 0.059 0.2327± 0.011 | 0.2543± 0.017 0.3430± 0.010 0.2740± 0.010 0.2840± 0.087 0.2445± 0.029 0.4280± 0.026 0.2287± 0.024 |
A group: injection FMD inactivated vaccine; B group: injection plasmid pcDNA3.1/P12X3C; The C group: injection plasmid pcDNA3.1/P12X3C and Yeast system are expressed the also FMDV 3D albumen of purifying; D group: injection plasmid pcDNA3.1; E group: injection DPBS damping fluid.
Respectively with (the 0th day) before the immunity, for the first time (the 28th day) around the immunity back the, the guinea pig serum of immunity back the 3rd week (the 49th day) is used for microneutralization test for the second time, surveys two cavys for every group.In the microneutralization experiment, behind 56 ℃ of deactivation 30min of guinea pig serum with the physiological saline dilution, on the trace Tissue Culture Plate of 96 holes, (serum-free DMEM) makes a series of doubling dilutions with diluent, each extent of dilution is done 4 holes, and every hole adds 50 μ l virus liquid (100TCID
50), add 100 μ l cell suspensions with every hole after 1 hour in 37 ℃ of incubators, put 37 ℃ of cultivations of 5%CO2 incubator, observed to 144 hours from 48 hours and declare eventually.Be provided with positive and negative serum contrast, virus return experiment, the contrast of serum toxicity, normal cell and contrasts.Calculate and can protect 50% cell hole not produce cytopathic serum dilution, this extent of dilution is the NAT of this part serum.Result's (seeing Table 3) shows except that D group E group, the neutralizing antibody of each test group is in immunity back appearance for the first time, but have only the NAT of A group cavy behind second immunisation, further to improve, the slightly reduction after the NAT ratio immunity for the first time of B group and C group cavy.
Table 3. immune guinea pig neutralizing antibody
| The cavy numbering | The A group | The B group | The C group | The D group | The E group | |
| (the 0th day) immunity (the 28th day) for the first time immunity (the 49th day) for the second time before the |
1 2 1 2 1 2 | ＜0.6 ＜0.6 1.5 1.2 2.1 1.5 | ＜0.6 ＜0.6 0.75 0.9 0.75 0.75 | ＜0.6 ＜0.6 0.75 0.9 0.75 0.75 | ＜0.6 ＜0.6 ＜0.6 ＜0.6 ＜0.6 ＜0.6 | ＜0.6 ＜0.6 ＜0.6 ＜0.6 ＜0.6 ＜0.6 |
A group: injection FMD inactivated vaccine; B group: injection plasmid pcDNA3.1/P12X3C; The C group: injection plasmid pcDNA3.1/P12X3C and Yeast system are expressed the also FMDV 3D albumen of purifying; D group: injection plasmid pcDNA3.1; E group: injection DPBS damping fluid.
The T lymphocyte proliferation assay.Before immunity, the 28th day, immune the 21st day for the second time of immunity for the first time, the aseptic cavy spleen of adopting, use 100 order copper mesh aseptic condition separating spleen lymphocytes, detect the propagation situation (each group be provided with above-mentioned neutralization test identical) of animal splenic T lymphocyte under phytohaemagglutinin (PHA) stimulates.The cavy spleen cell of aseptic collection is diluted to 2 * 10 with RPMI1640 nutrient solution (available from Invitrogen company, Cat No:61870-036)
7Individual/milliliter, in 96 porocyte culture plates, every hole adds 50ul cell suspension and 50uLPHA solution (glad through company of section available from Beijing, Cat No:5030, concentration 50ug/ml), establishes three multiple holes, and other establishes control wells.Culture plate is put 37 ℃, 5%CO
2Cultivating after 45 hours every hole under the saturated humidity condition, to add MTT solution (glad through company of section available from Beijing, Cat No:15029,5mg/ml) 10ul, continue to cultivate 3 hours and with 10%SDS-0.01mol/L HCl solution termination reaction, treat to measure every hole OD value in λ 570nm place behind the resolution of precipitate, the result represents with three hole mean values.The result shows that (see figure 8) is except that D group and E group, proliferative response all appears in the splenic T lymphocyte of each immune group, wherein the T lymphocyte of B group cavy strengthens after the immunity second time to some extent, but the T lymphproliferation response that A organizes, C organizes behind the second immunisation weakens to some extent.Adopt the Anove analytical procedure that each group cavy T lymphopoiesis is changed and analyzes, the result shows between A group, B group, the C group does not have significant difference, does not have significant difference between C group, D group, the E group, the B group with the D group, there were significant differences for the E group.
Embodiment 5 inoculation animals are to the protection of virus attack
In the 3rd week behind the second immunisation, count infective dose (ID in every Guinea Pig Left hind paw subcutaneous puncture and intradermal vaccination 0.2ml100 sesquialter
50=10
5.5) O type FMDV China99 strain, incidence is observed in 1 week in inoculation back.The severity of pathology is expressed as " nothing " respectively, and promptly two hind paws all do not have pathology; " slightly ", promptly pathology is only sent out in single hind paw of injection; " seriously ", promptly pathology all appears in two hind paws.The severity that occurs with pathology is as the judgement of protection effect: protection is not for any pathology occurring fully, and the part protection is single the pin that pathology only betides injection.Cavy number/every group of cavy sum * 100 of protection ratio (%)=fully protection.The results are shown in following table 4.
Table 4 cavy anti-virus ability detects
| The cavy numbering | A organizes * | The B group | C group * * | The D group | The E group | F group * * * | |
| Protection is lesion degree protection ratio (%) as a |
1 2 3 4 1 2 3 4 | The protection of fully protection part of protection protection part does not have slight 50 (2/4) fully | Protection protects fully protection to protect the nothing nothing not have 100 (4/4) fully fully fully | The protection of fully protection part protection part protection part does not have serious slight 25 (1/4) | Unprotect unprotect unprotect unprotect serious 0 (0/4) | Unprotect unprotect unprotect unprotect serious 0 (0/4) | Unprotect unprotect unprotect unprotect serious 0 (0/4) |
* two cavy pregnancies; Two cavy pregnancies of *; * * is the normal healthy controls group.
A group: injection FMD inactivated vaccine; B group: injection plasmid pcDNA3.1/P12X3C; The C group: injection plasmid pcDNA3.1/P12X3C and Yeast system are expressed the also FMDV 3D albumen of purifying; D group: injection plasmid pcDNA3.1; E group: injection DPBS damping fluid.
Claims (16)
1. DNA sequences encoding, it comprises one of one of following sequence or following sequence sequence after modified, and described modification does not change coded aminoacid sequence,
ATGGGAGCCGGACAATCCAGCCCGGCGACTGGGTCACAGAACCAGTCAGGCAACACTGGAAGCATTATCAACAATTACTACATGCAGCAGTACCAGAACTCCATGGACACGCAACTTGGTGATAACGCTATTAGCGGAGGCTCCAACGAGGGGTCCACGGACACCACCTCCACCCACACAACCAACACTCAGAACAATGACTGGTTTTCAAAGCTGGCCAGTTCCGCTTTTAGCGGTCTTTTCGGCGCTCTTCTCGCCGACAAGAAAACCGAGGAGACCACTCTTCTCGAGGACCGCATCCTCACTACCCGCAACGGACACACGACCTCGACAACCCAGTCGAGCGTTGGAGTCACTTACGGGTACGCAACAGCTGAGGACTTTGTGAGCGGACCAAACACATCTGGGCTTGAGACCAGGGTTGTGCAGGCAGAGCGGTTCTTCAAAACCCACTTGTTCGACTGGGTCACCGGTGACCCGTTCGGACGGTGCTACCTGCTGGAACTCCCAACTGACCACAAAGGTGTCTACGGCGGCCTGACTGACTCTTATGCTTACATGAGAAACGGTTGGGATGTTGAGGTCACTGCAGTGGGAAATCAGTTCAACGGAGGATGTCTGTTGGTGGCCATGGTGCCAGAACTTTGCTCTATTGACAAGAGAGAGCTGTACCAGCTCACGCTCTTTCCCCACCAGTTCATCAACCCCCGGACGAACATGACGGCGCACATCACTGTGCCCTTTGTTGGTGTCAACCGCTACGACCAGTACAAGGTACACAAACCTTGGACCCTCGTGGTTATGGTTGTGGCCCCGCTGACTGTCAACACCGAAGGTGCCCCACAGATCAAGGTCTATGCCAACATCGCCCCTACCAACGTGCACGTTGCGGGTGAGTTCCCTTCTAAGGAAGGGATCTTCCCCGTGGCATGTAGCGACGGTTACGGTGGTCTGGTGACCACTGACCCAAAGACGGCTGACCCCGCCTACGGGAAAGTGTTCAATCCACCTCGCAACATGTTGCCGGGGCGGTTCACCAACTTCCTTGATGTGGCTGAGGCGTGCCCTACGTTTCTGCACTTTGAGGGTGACGTGCCGTACGTGACCACAAAGACGGACTCAGACAGGGTGCTCGCCCAGTTTGACTTGTCTCTGGCAGCAAAGCACATGTCAAACACCTTCCTGGCAGGTCTCGCCCAGTACTACACACAGTACAGCGGCACCATCAACCTGCACTTCATGTTCACAGGACCCACTGACGCGAAAGCGCGTTACATGATTGCATACGCCCCCCCTGGCATGGAGCCGCCCAAAACACCTGAGGCGGCCGCTCACTGCATTCATGCGGAGTGGGACACAGGGTTGAATTCAAAATTCACATTTTCAATCCCTTACCTTTCGGCGGCTGATTACGCGTACACCGCGTCTGACGCTGCGGAGACCACAAATGTACAGGGATGGGTCTGCCTGTTTCAAATTACACACGGGAAGGCTGACGGCGACGCACTGGTCGTTCTAGCTAGCGCCGGTAAGGACTTTGAGCTGCGTCTGCCAGTTGACGCTCGCACGCAGACCACCTCCACAGGTGAGTCGGCTGACCCCGTGACTGCCACTGTTGAGAACTACGGTGGTGAGACACAGGTCCAGAGACGCCAACACACGGATGTCTCGTTCATATTAGACAGATTTGTGAAAGTAACACCAAAAGACCAAATTAATGTGTTGGACCTGATGCAAACCCCTGCACACACTTTGGTAGGCGCGCTCCTCCGTACTGCCACCTACTACTTCGCAGATCTAGAAGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGCGCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCGCTCACCCGGCTTGCACTGCCTTACACGGCACCACACCATGTCTTGGCTACTGTTTACAACGGGAACTGCAAGTATGACGAGAGCCCCGTGACCAATGTGAGAGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGCTGCCTACCTCCTTCAATTACGGTGCCGTCAAAGCCACTCGGGTGACTGAACTGCTTTACCGCATGAAGAGGGCCGAAACATACTGCCCCCGGCCTCTTTTGGCTATTCACCCGAGCGAAGCTAGACACAAACAAAAGATCGTGGCGCCTGTGAAACAGCTTTTGAACTTTGACCTGCTCAAGTTGGCAGGAGACGTCGAGTCCAACCCTGGGCCCTTCTTCTCTGACGTCAGGTCAAATCTTTCCAAGTTGGTTGAAACCATCAACCAGATGCAGGAGGACATGTCGGATCCAGGACCGGTGAAGAAACCTGTCGCTTTGAAAGTGAAAGCAAAGAATTTGATTGTCACTGAGAGTGGTGCTCCCCCGACTGACTTGCAAAAGATGGTCATGGGTAACACCAAGCCTGTTGAGCTCATCCTCGACGGGAAGACGGTGGCCATCTGCTGCGCCACCGGAGTGTTTGGTACTGCCTACCTTGTTCCTCGTCATCTTTTCGCAGAGAAGTATGACAAGATCATGTTGGACGGCAGAGCCATGACAGACAGTGACTACAGAGTGTTTGAGTTTGAGATTAAAGTGAAAGGACAGGACATGCTCTCAGACGCCGCGCTCATGGTGCTTCACCGTGGGAATCGCGTGCGGGACATCACGAAGCACTTCCGTGATGTGGCAAGAATGAAGAAAGGCACCCCCGTCGTCGGCGTGATCAACAACGCTGATGTTGGGAGACTGATCTTCTCTGGTGAGGCCCTTACCTACAAGGACATCGTAGTGTGCATGGACGGAGACACCATGCCCGGTCTCTTCGCCTACAAAGCCGCCACCAAGGCGGGTTACTGTGGAGGAGCCGTTCTTGCAAAGGACGGAGCCGAGACTTTCATCGTCGGCACTCACTCCGCAGGCGGCAATGGAGTTGGATACTGCTCATGCGTTTCCAGGTCTATGCTGCTTAAAATGAAGGCACACATCGATCCCGAACCACACCACGAGG，
Perhaps
ATGGGAGCCGGACAATCCAGCCCGGCGACTGGGTCACAGAACCAGTCAGGCAACACTGGAAGCATTATCAACAATTACTACATGCAGCAGTACCAGAACTCCATGGACACGCAACTTGGTGATAACGCTATTAGCGGAGGCTCCAACGAGGGGTCCACGGACACCACCTCCACCCACACAACCAACACTCAGAACAATGACTGGTTTTCAAAGCTGGCCAGTTCCGCTTTTAGCGGTCTTTTCGGCGCTCTTCTCGCCGACAAGAAAACCGAGGAGACCACTCTTCTCGAGGACGCGATCCTCACTACCCGCAACGGACACACGACCTCGACAACCCAGTCGAGCGTTGGAGTCACTTACGGGTACGCAACAGCTGAGGACTTTGTGAGCGGACCAAACACATCTGGGCTTGAGACCAGGGTTGTGCAGGCAGAGCGGTTCTTCAAAACCCACTTGTTCGACTGGGTCACCGGTGACCCGTTCGGACGGTGCTACCTGCTGGAACTCCCAACTGACCACAAAGGTGTCTACGGCGGCCTGACTGACTCTTATGCTTACATGAGAAACGGTTGGGATGTTGAGGTCACTGCAGTGGGAAATCAGTTCAACGGAGGATGTCTGTTGGTGGCCATGGTGCCAGAACTTTGCTCTATTGACAAGAGAGAGCTGTACCAGCTCACGCTCTTTCCCCACCAGTTCATCAACCCCCGGACGAACATGACGGCGCACATCACTGTGCCCTTTGTTGGTGTCAACCGCTACGACCAGTACAAGGTACACAAACCTTGGACCCTCGTGGTTATGGTTGTGGCCCCGCTGACTGTCAACACCGAAGGTGCCCCACAGATCAAGGTCTATGCCAACATCGCCCCTACCAACGTGCACGTTGCGGGTGAGTTCCCTTCTAAGGAAGGGATCTTCCCCGTGGCATGTAGCGACGGTTACGGTGGTCTGGTGACCACTGACCCAAAGACGGCTGACCCCGCCTACGGGAAAGTGTTCAATCCACCTCGCAACATGTTGCCGGGGCGGTTCACCAACTTCCTTGATGTGGCTGAGGCGTGCCCTACGTTTCTGCACTTTGAGGGTGACGTGCCGTACGTGACCACAAAGACGGACTCAGACAGGGTGCTCGCCCAGTTTGACTTGTCTCTGGCAGCAAAGCACATGTCAAACACCTTCCTGGCAGGTCTCGCCCAGTACTACACACAGTACAGCGGCACCATCAACCTGCACTTCATGTTCACAGGACCCACTGACGCGAAAGCGCGTTACATGATTGCATACGCCCCCCCTGGCATGGAGCCGCCCAAAACACCTGAGGCGGCCGCTCACTGCATTCATGCGGAGTGGGACACAGGGTTGAATTCAAAATTCACATTTTCAATCCCTTACCTTTCGGCGGCTGATTACGCGTACACCGCGTCTGACGCTGCGGAGACCACAAATGTACAGGGATGGGTCTGCCTGTTTCAAATTACACACGGGAAGGCTGACGGCGACGCACTGGTCGTTCTAGCTAGCGCCGGTAAGGACTTTGAGCTGCGTCTGCCAGTTGACGCTCGCACGCAGACCACCTCCACAGGTGAGTCGGCTGACCCCGTGACTGCCACTGTTGAGAACTACGGTGGTGAGACACAGGTCCAGAGACGCCAACACACGGATGTCTCGTTCATATTAGACAGATTTGTGAAAGTAACACCAAAAGACCAAATTAATGTGTTGGACCTGATGCAAACCCCTGCACACACTTTGGTAGGCGCGCTCCTCCGTACTGCCACCTACTACTTCGCAGATCTAGAAGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGCGCCCGAGACAGCGTTGGACAACACCACCAATCCAACGGCTTACCACAAGGCACCGCTCACCCGGCTTGCACTGCCTTACACGGCACCACACCATGTCTTGGCTACTGTTTACAACGGGAACTGCAAGTATGACGAGAGCCCCGTGACCAATGTGAGAGGTGACCTGCAAGTGTTGGCCCAGAAGGCGGCAAGAACGCTGCCTACCTCCTTCAATTACGGTGCCGTCAAAGCCACTCGGGTGACTGAACTGCTTTACCGCATGAAGAGGGCCGAAACATACTGCCCCCGGCCTCTTTTGGCTATTCACCCGAGCGAAGCTAGACACAAACAAAAGATCGTGGCGCCTGTGAAACAGCTTTTGAACTTTGACCTGCTCAAGTTGGCAGGAGACGTCGAGTCCAACCCTGGGCCCTTCTTCTCTGACGTCAGGTCAAATCTTTCCAAGTTGGTTGAAACCATCAACCAGATGCAGGAGGACATGTCGGATCCAGGACCGGTGAAGAAACCTGTCGCTTTGAAAGTGAAAGCAAAGAATTTGATTGTCACTGAGAGTGGTGCTCCCCCGACTGACTTGCAAAAGATGGTCATGGGTAACACCAAGCCTGTTGAGCTCATCCTCGACGGGAAGACGGTGGCCATCTGCTGCGCCACCGGAGTGTTTGGTACTGCCTACCTTGTTCCTCGTCATCTTTTCGCAGAGAAGTATGACAAGATCATGTTGGACGGCAGAGCCATGACAGACAGTGACTACAGAGTGTTTGAGTTTGAGATTAAAGTGAAAGGACAGGACATGCTCTCAGACGCCGCGCTCATGGTGCTTCACCGTGGGAATCGCGTGCGGGACATCACGAAGCACTTCCGTGATGTGGCAAGAATGAAGAAAGGCACCCCCGTCGTCGGCGTGATCAACAACGCTGATGTTGGGAGACTGATCTTCTCTGGTGAGGCCCTTACCTACAAGGACATCGTAGTGTGCATGGACGGAGACACCATGCCCGGTCTCTTCGCCTACAAAGCCGCCACCAAGGCGGGTTACTGTGGAGGAGCCGTTCTTGCAAAGGACGGAGCCGAGACTTTCATCGTTGGTACCCACTCCGCAGGCGGCAACGGAGTTGGATACTGCTCATGCGTTTCCAGGTCTATGCTGCTTAAAATGAAGGCACACATCGATCCCGAACCACACCACGAGGGATTGATAGTTGACACCAGAGATGTTGAGGAGCGCGTACATGTCATGCGCAAAACCAAGCTCGCACCCACCGTGGCACACGGTGTGTTTAACCCCGAATTTGGGCCTGCCGCCTTGTCCAACAAGGGCCCGCGCCTGAATGAGGGGGTTGTCCTCGATGAAGCCATCTTCTCCAAACACAAAGGAAACACAAAGATGTCTGAGGAGGACAAAGCGCTGTTCCGCCGCTGTGCTGCTGACTACGCGTCGCGTCTACATAGCGTGCTGGGTACGGCAAATGCCCCACTGAGCACTTACGTGGCAATCAAGGGCGTCGACGGACTTGACGCCATGGAACCAGACACCGCGCCTGGTCTCCCCTGGGCTCTCCAGGGGAAACGCCGTGGTGCGCTCATTGATTTCGAGAACGGCACTGTCGGACCCGAGGTTGAAGCTGCCTTGAAGCTCATGGAGAAAAGAGAGTACAAGTTTGTATGCCAGACCTTCCTGAAGGACGAGATTCGCCCGATGGAGAAGGTACGTGCCGGCAAGACTCGCATTGTCGACGTCCTGCCTGTTGAACACATTCTTTACACCAGGATGATGATTGGCAGATTTTGTGCTCAAATGCACTCAAACAACGGACCGCAAATTGGCTCGGCGGTTGGTTGTAATCCTGATGTTGATTGGCAAAGATTTGGCACGCATTTTGCTCAGTACAGAAACGTGTGGGATGTGGACTATTCGGCCTTTGATGCCAACCACTGCAGTGACGCAATGAACATCATGTTTGAGGAGGTGTTCAACACGGATTTCGGGTTCCACCCAAACGCTGAGTGGATCCTGAAAACTCTCGTGAACACTGAACACGCCTATGAGAACAAACGCATCACTGTTGAAGGCGGGATGCCGTCTGGTTGTTCCGCAACAAGCATCATCAACACAATTTTGAACAACATCTACGTGCTCTACGCCTTGCGTAGACACTATGAGGGAGTTGAGCTGGACTCTTACACCATGATCTCCTACGGAGACGACATCGTGGTTGCAAGTGATTACGATCTGGACTTTGAGGCCCTCAAGCCTCACTTCAAATCCCTTGGTCAAACCATTACTCCAGCTGACAAAAGCGACAAAGGTTTTGTTCTTGGTCACTCCATTACCGATGTCACTTTCCTCAAAAGACACTTCCACATGGACTATGGAACTGGGTTTTACAAACCTGTGATGGCTTCGAAGACCCTCGAGGCTATCCTCTCCTTTGCACGCCGTGGGACCATACAGGAGAAGTTGATCTCCGTGGCAGGACTCGCCGTCCACTCTGGACCTGACGAGTACCGGCGTCTCTTTGAGCCTTTCCAGGGCCTCTTTGAGATTCCAAGTTACAGATCACTTTACCTGCGTTGGGTGAACGCCGTGTGCGGTGACGCATTCTAG。
2. transcription unit, its contain the DNA sequences encoding of claim 1 and be operably connected with this dna sequence dna, in eukaryotic cell, express the necessary expression regulation element of this dna sequence dna.
3. the transcription unit of claim 2, wherein said expression regulation element is a promotor.
4. the transcription unit of claim 3, wherein said promotor is viral promotors or mammalian cell promotor.
5. the transcription unit of claim 2, wherein said expression regulation element is a transcription termination sequence.
6. the transcription unit of claim 2, it is the transcription unit that plasmid pcDNA3.1/P12X3C or pcDNA3.1/P12X3C3D are comprised.
7. carrier for expression of eukaryon, it contains each transcription unit of claim 2-6.
8. the expression vector of claim 7, it is a plasmid.
9. the expression vector of claim 8, it is plasmid pcDNA3.1/P12X3C or pcDNA3.1/P12X3C3D.
10.DNA vaccine, its comprise one or more claims 7-9 each expression vector and one or more pharmaceutically acceptable carrier.
11. prepare the method for dna vaccination, comprise
Adopt known technology to make up each expression vector of one or more claims 7-9,
The expression vector that obtains is mixed with one or more pharmaceutically acceptable carrier.
12. the purposes of the dna sequence dna of claim 1 in the preparation dna vaccination.
13. claim 2-6 each transcription unit or each expression vector of claim 7-9 in the purposes of preparation in the dna vaccination.
14. contain each the host cell of expression vector of claim 7-9.
15. the host cell of claim 14, it is prokaryotic cell prokaryocyte or eukaryotic cell.
16. the host cell of claim 14, it is intestinal bacteria or mammalian cell.
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| CN100572541C (en) * | 2007-05-28 | 2009-12-23 | 中国农业科学院兰州兽医研究所 | Dual-purpose vaccine carrier for foot and mouth disease virus, its preparation method and application |
| CN101721698B (en) * | 2008-10-24 | 2014-04-02 | 法罗斯疫苗公司 | Foot-and-mouth disease resistant vaccine composition and preparation and application thereof |
| CN103933581A (en) * | 2014-05-04 | 2014-07-23 | 贵州大学 | Preparation method of engineered vaccine based on CSF-FMD duplex gene |
| CN113897392A (en) * | 2020-07-06 | 2022-01-07 | 嘉兴安宇生物科技有限公司 | Trivalent recombinant adenovirus vaccine for foot-and-mouth disease and construction method thereof |
| CN117210501A (en) * | 2023-09-13 | 2023-12-12 | 中国农业科学院兰州兽医研究所 | Construction of recombinant foot-and-mouth disease virus strain with foot-and-mouth disease virus 2B protein immunosuppression site mutation |
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| CN1090203A (en) * | 1993-10-21 | 1994-08-03 | 复旦大学 | Polypeptide vaccine for aftosa and preparation method thereof |
| US6107021A (en) * | 1998-06-20 | 2000-08-22 | United Biomedical, Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN1408349A (en) * | 2002-09-16 | 2003-04-09 | 复旦大学 | Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method |
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| CN1090203A (en) * | 1993-10-21 | 1994-08-03 | 复旦大学 | Polypeptide vaccine for aftosa and preparation method thereof |
| US6107021A (en) * | 1998-06-20 | 2000-08-22 | United Biomedical, Inc. | Synthetic peptide vaccines for foot-and-mouth disease |
| CN1408349A (en) * | 2002-09-16 | 2003-04-09 | 复旦大学 | Foot-and-mouth disease gene engineering polypeptide vaccine and its preparing method |
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| "口蹄疫病毒China/99基因组RNA序列测定及比较分析" 张显升等，中国科学(C辑)，第33卷第5期 2003;"口蹄疫病毒多基因重组质粒在BHK-21细胞中的表达" 郭慧琛等，生物工程学报，第19卷第3期 2003;"口蹄疫病毒真核表达质粒的构建及序列分析" 郭慧琛等，中国兽医科技，第33卷第4期 2003 * |
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