CN1284854C - Mosaic type DNA vaccine in use for preventing tuberculosis and immunological therapy - Google Patents

Abstract

The invention relates to a mosaic type DNA vaccine in use for preventing tuberculosis and immunological therapy, including as a full-length open read code frame such as showed in SEQ ID NO.1, or its conservative variant. A mycobacterium hsp70 gene and human B7 -1 gene are included in the SEQ ID NO.1, and a joint sequence is designed between the two genes to connect them together. The vaccine of the invention may be used into the tuberculosis induced by the tubercle bacillus; or it may express the corresponding protein in eukaryotic cells outside the body, for the prevention and treatment of tuberculosis. The vaccine may also be used an immunopotentiator, which is used in the immunity treatment of corresponding virus disease as well as tumorous.

Description

Be used to prevent the mosaic type dna vaccination of tuberculosis and immunotherapy

Technical field

The present invention relates to a kind of novel Vaccinum Calmette-Guerini, relate in particular to a kind of chimeric dna molecule that can induce and strengthen protective immune response (humoral immunization and cellular immunization) in the human or animal body.This dna molecular is used for prevention lungy and treatment as vaccine; As immunostimulant, can improve the intravital immunologic function of human or animal simultaneously, reach the effect of control virus disease and tumour.

Background technology

After the first time and twice tuberculosis peak in rebound of World War II whole world appearance, the mid-80, owing to tuberculosis epidemic situation in the world wide again rapid deterioration form peak in rebound for the third time, according to " World Health Organization's bulletin ", global institute has age death toll in 1996, tuberculosis is arranged in after the dead routine number of ischemic heart disease, cerebrovascular disease and acute lower respiratory infection, occupy the 4th, account for single sick kind the in the whole world and cause first of the dead routine number tidemark of wound one-hundred-year history.Die from number lungy and reach 3,000,000 global every year, has every day 8000 people to die from tuberculosis, and kind just had 1 people to die from tuberculosis in per 10 seconds.Do poverty, floating population, anti-multiple medicines tuberculosis increase and the popular tuberculosis that makes of AIDS becomes global serious public health problem (Cole ST.Why sequence the genome of Mycobacterium tuberculosis more? Tubercle Lung.Dis.1996; 77:486-490.), thereby cause the very big concern of international community.The secret of using modern molecular biology method announcement mycobacterium is the central issue of present research strategy.By analyzing tubercle bacillus gene group dna sequence dna, use these information and can understand generation lungy, evolution, and can strengthen research diagnosis lungy, treatment and the new measure of prevention with obtaining the bioinformations of a large amount of relevant tubercule bacillus.

Bacille Calmette-Guerin vaccine (BCG) is to be to the phthisical vaccine of unique prevention that goes through to use so far, and use above 100,000,000 doses in annual market.But bacille Calmette-Guerin vaccine but is not the ideal vaccine.Though bacille Calmette-Guerin vaccine has the certain protection effect to children, it does not have effectiveness to the adult.

The bacille Calmette-Guerin vaccine that uses is overbated at present.Bacille Calmette-Guerin vaccine is a live bacterial vaccines.Since bacille Calmette-Guerin vaccine by the Frenchman after 1908-1921 succeeds in developing, bacterial strain is by the continuous passage in nutrient solution of laboratory, various countries, until the appearance of low temperature (subzero 80 degree) refrigerator in 1961 is just by cryopreservation.Nearest gene pool (Genome) studies show that; tens kinds of bacille Calmette-Guerin vaccines that use in the whole world no longer have been same bacterial strains at present; many gene fragments are lost. and why early stage this is to cause one of reason that the bacille Calmette-Guerin vaccine protective effect weakens, also explained clinical experiment, and bacille Calmette-Guerin vaccine shows excellent protective (nineteen thirty; Britain; 80% protection ratio), and later stage bacille Calmette-Guerin vaccine result far unsatisfactory (1970, India; 0% protection ratio) (Liu Jun, Canadian national health institute.National defence consumptive disease magazine in the diagnosis of pulmonary tuberculosis (TB) and the prevention new technology.2003,25, supplementary issue: 20-22); Nineteen ninety-five WHO also issues a statement and points out, the BCG multiple cropping does not have scientific basis, so do not advocate multiple cropping.Also relevant (the Baldwin SL of BCG immuning failure with following factors; D ' Souza C; Roberts A D; et al.Evaluation of new vaccines in themouse and guinea pig model of tuberculosis.Infect.Immun.1998; 66 (6): 2951-2959.): (1) BCG can not improve because other factors have had immunizing power crowd's protection level; (2) individuality with age; might continue to touch the mycobacterium antigen of surrounding environment; thereby push this immune response to Th2 type (Here it is BCG prevention childhood tuberculosis is respond well, and to the major cause of adult's weak effect) from the Th1 type.In light of this situation, a kind of qualified novel vaccine should return to the protectiveness and the memory immunological status of Th1 type; Therefore, safe, effective, the inexpensive vaccine of new generation of development becomes a urgent and important job.

(heat shock protein is the protein that a class has the high conservative of important biomolecule and immunologic function hsp) to heat shock protein(HSP), is present in protokaryon and the eukaryotic cell.Mycobacterium hsp70 comprises a plurality of T cells and B cell epitope, immunogenicity is very strong, characteristics with immunodominance antigen (immunodominant antigen), can induce the cell response of specificity T h1 type, and generation r-Interferon, rabbit (r-IFN) and interleukin 12, attack to tubercule bacillus can produce stronger protective immunity effect (Mckenzie.KR, Adams E, BrittonWJ, et al.Sequence and immunogenicity of the 70-kDa heat shock protein ofMycobacterium leprae.J.Immune.1991; 147:312-319.).

B7-1 (B7-1) is one of important costimulatory molecules with immune activation function (costimulatorymolecule).Studies show that immunoregulation in recent years, during the T cell activation, essential two signals, be that TXi Baoshouti (TCR) combines first signal that produces with the MHC-Ag on antigen presenting cell (APCs) surface, with the second signal that provides by costimulatory molecules (costimulatory signal), if shortage costimulatory signal, T cell then can not be activated fully and present state of anergy (Anergy), apoptosis (Apoptosis) perhaps appears.(costimulatory receptor CD28 common with it and/or CTLA-4 interphase interaction can provide the T cell activation necessary costimulatory signal to B7-1, inducing T cell produces r-IFN, humoral immunization and cellular immunization had comprehensive activation, systemic autoimmune encephalomyelitis (EAE) model proves that B7-1 can promote Th1 type cell response (Orme I M Progress in the development of new vaccinesagainst tuberculosis.Int.J.Tuberc.Lung.Dis.1997 by experiment; 1 (2): 95-100 and Lenschow D J, Walunas T L and Bluestone J A.CD28/B7system of T cell costimulation.Annu.Rev.Immunol.1996; 14:233.).

R-IFN is mainly produced by the T cell; Main activity is to participate in immunomodulatory, is important immune-regulating factor in the body, realizes its antiviral, antitumor action by enhancing body immunologic mechanism, booster immunization supervisory role.The immunoregulation effect of r-IFN shows the influence to the host immune cell activity, as can make the expression of its surperficial mhc class ii molecule to scavenger cell, strengthens its antigen presentation ability; Can also strengthen scavenger cell surface expression Fc acceptor in addition, promote the macrophage phagocytic immunity to bring back to life the pathogenic agent and the tumour cell of thing, antibody sandwich.Simultaneously, differentiation to B cell and CD8+T cell has promoter action, can strengthen the Th1 cell activity, strengthen cellular immune function (Hathcock KS, Laszlo G, Pucillo C, et al.Comparitive analysis ofB7-1 and B7-2 costimulatory ligands:Expression and function.J.Exp.Med.1994; 180:631-640.)

Summary of the invention

Because BCG can not turn to the TH1 type from the TH2 type with immunological status, caused BCG as the existing defective of vaccine prevention tuberculosis, the objective of the invention is by selecting mycobacterium hsp70 gene and people B7-1 gene to carry out chimericly, be built into the novel tuberculosis dna vaccination at molecular level; Wherein hsp70 provides the immunologic opsonin signal, induces the cell response of specificity T h1 type, produces r-IFN and il-1 2; It is costimulatory signal that B7-1 provides second signal, and indirect induction T cell produces r-IFN, comprehensively active cells and humoral immunization.By both actings in conjunction, push the immunological status of humans and animals to the Th1 type by the Th2 type, reach the effect of prevention and treatment tuberculosis and other infectious diseases.

According to an aspect of the present invention, Novel DNA vaccine of the present invention comprises the total length open reading frame of nucleotide sequence shown in SEQ ID NO.1, and the conservative property varient of this total length open reading frame, comprised mycobacterium hsp70 gene and people B7-1 gene among the SEQID NO.1, passed through design one joint sequence (SEQ ID NO.3) between these two genes the two connection.

In the present invention, term " total length open reading frame " is meant and is not subjected to termination codon interferential reading frame, its source generally from DNA sequence inference draw.

Term " conservative property varient " is meant same worker's gene of coding same products in the identical or different organism, as tubercule bacillus hsp60, hsp65, and leprosy bacillus hsp70 etc.

Six primers of total P3～P8 that are used for synthetic SEQ ID NO.1 sequence of the present invention's design, its nucleotide sequence is shown in SEQ ID NO.6～11.The combination of primers of mycobacterium part hsp70 gene and joint wherein is used to increase, upstream primer P3 (SEQ ID NO.6) is the single stranded DNA of long 30 Nucleotide, and downstream primer P4 (SEQ IDNO.7) is the single stranded DNA of long 76 Nucleotide (containing joint).The base sequence of upstream primer P3 is identical with SEQ ID NO:1 open reading frame 1593-1623 position Nucleotide, and at its 5 ' end design restriction enzyme site; 1844～1920 Nucleotide of the base sequence of downstream primer P4 and SEQ ID NO:1 open reading frame are identical, the minus strand sequence that contains SEQ ID NO:3 is increase mycobacterium part hsp70 gene and a joint of template with plasmid pcDNA3.1 (+)-hsp70.

Be used to the to increase combination of primers of people B7-1 gene and part joint, upstream primer P5 (SEQ ID NO.8) is the single stranded DNA of long 53 Nucleotide, downstream primer P6 (SEQ ID NO.9) is the single stranded DNA of long 39 Nucleotide.The base sequence of upstream primer P5 is identical with SEQ ID NO:1 open reading frame 1896-1948 position Nucleotide, at 3 ' end 24 Nucleotide (part joint sequence) of its 5 ' end for SEQ ID NO:3; 2745～2784 Nucleotide of the base sequence of downstream primer P6 and SEQ ID NO:1 open reading frame are identical, and design has restriction enzyme site behind termination codon TAA, is template amplification people B7-1 gene and part joint with plasmid pcDNA3.1 (+)-B7-1.Above two sections goal gene are increased with P3 and P6 for the third time as template, the directed insertion of gained goal gene is advanced to cut among the eukaryotic expression vector pcDNA3.1 (+) through enzyme, be built into pcDNA3.1 (+) and contain part hsp70-B7-1 plasmid.

The goal gene that pcDNA3.1 (+)-hsp70 is obtained after cutting with the KpaI enzyme is connected into pcDNA3.1 (+) and contains in the part hsp70-B7-1 plasmid, is built into pcDNA3.1 (+) Hsp70/B7-1 chimeric plasmid mosaic gene.

In the amplification below, the base sequence of upstream primer P7 (SEQ ID NO.10) is identical with SEQ ID NO:1 open reading frame 1-21 position Nucleotide, and at its 5 ' end design restriction enzyme site; 2764～2784 Nucleotide of the base sequence of downstream primer P8 (SEQID NO.11) and SEQ ID NO:1 open reading frame are identical, and design has restriction enzyme site behind termination codon TAA.Plate is touched in conduct with above constructed pcDNA3.1 (+) Hsp70/B7-1 chimeric plasmid, amplifies the chimeric goal gene of hsp70/B7-1.

With the hsp70/B7-1DNA transfecting eukaryotic cells, extract mRNA and carry out reverse transcription PCR, amplified production shows hsp70/B7-1 genetic expression.

Hsp70/B7-1 chimeric DNA vaccine inoculation mouse is attacked with the tubercule bacillus virulent strain, observes the prophylactic effect of this vaccine to mycobacterium tuberculosis infection; And carry out elder generation and attack with the tubercule bacillus virulent strain, behind the formation mouse tuberculosis model, treating with hsp70/B7-1 chimeric DNA vaccine, the serum I FN-γ that observes mouse produces level and liver, the bacterial count of spleen tissue smear and tissue culture enumeration.With hsp70/B7-1 chimeric DNA vaccine immunity and treatment macaque tuberculosis model,, observe its immunoprotection and therapeutic action by the CT pulmonary scanning.

The mycobacterium hsp70 gene that is comprised among the SEQ ID No:1 of the present invention can obtain by several different methods.Enzyme is cut, polymerase chain (PCR) amplification as the 1. cloned gene (plasmid that contains the hsp70 gene) known in this area, or 2. extracts pcr amplification behind the mycobacterium genome.In one embodiment of the invention, the hsp70 gene source is in tubercule bacillus virulent strain H37RV genomic dna, through pcr amplification, is connected into pcDNA3.1 (+) after the amplified production enzyme that obtains cut, and obtains eukaryon expression plasmid pcDNA3.1 (+)-hsp70.

The people B7-1 gene that SEQ ID No:1 of the present invention is comprised also can obtain by several different methods.Enzyme is cut, polymerase chain (PCR) amplification as 1. cloned gene (containing people B7-1 gene), or 2. extracts the total RNA of peripheral blood mononuclear cell, and reverse transcription cDNA carries out pcr amplification then, or 3. uses specific probe, obtains from corresponding cDNA library, or is purchased.In one embodiment of the invention, adopting the PCR primer of synthetic, is template with above-mentioned pcDNA3.1 (+)-hsp70, synthetic hsp70/B7-1 mosaic gene.

In the chimeric gene sequence of the present invention, by pcr amplification, inserted SEQID No:3 sequence between hsp70 and B7-1, the coded amino acid residue sequence of this segment DNA sequence is (GGGGS) ₃Because the physicochemical property that it is unique, can be between the mycobacterium hsp70 and people B7-1 protein of whole SEQ ID No:1 coding, form one section rigidity at interval, hsp70 and people B7-1 are separated, they are not disturbed on space structure mutually, can form natural structure picture separately, bring into play biological function separately.

According to a further aspect in the invention, provide the expression vector that contains nucleotide sequence shown in the SEQ ID NO.1 of the present invention.In one embodiment of the invention, adopting plasmid pcDNA3.1 (+) is carrier, by digestion with restriction enzyme, the mosaic gene that obtains is loaded in the plasmid, is built into expression vector.Except that plasmid pcDNA3.1 (+), other multiple expression vector system can be used for the expression of SEQ ID No:1, these carriers comprise its eukaryon expression plasmid, adenovirus and adeno-associated virus, retrovirus etc., in a single day these carriers have cloned SEQ ID No:1 total length open reading frame, can play identical or similar effect in body.The method that adopts those skilled in that art to know can be used for structure and contains SEQ ID No:1 total length open reading frame expression vector.These methods comprise reorganization/gene recombination in extracorporeal recombinant DNA technology, synthetic technology and the body.See as by Maniatis etc., the molecular cloning laboratory manual, cold spring harbor laboratory, N.Y., the 12nd chapter (1982) is described.

The present invention also provides the host cell that comprises expression vector of the present invention, multiple host expresses carrier system can be used for vivoexpression DNA, and these systems include but not limited to the recombinant phage dna that contains SEQ ID No:1 sequence, plasmid DNA or cosmid DNA expression vector microorganism transformed such as bacterium; Recombination microzyme with the expression vector that contains SEQ ID No:1 sequence; The insect cell system that infects with the recombinant expressed virus expression carrier (as baculovirus) of the expression vector that contains SEQ ID No:1 sequence; Recombinant expressed virus expression carrier (as adenovirus, adeno-associated virus or retrovirus) infected animals cell system with the expression vector that contains SEQ ID No:1 sequence.Its experimental technique: as the method that enzyme is cut, connected, conversion, PCR etc. are also known for this molecule field of biology.

Vaccine of the present invention can be with the form direct immunization animal of naked DNA, absorb through host myocyte, utilize the host cell expression system to express in vivo except directly expressing the respective egg white matter in vivo, also can in external eukaryotic cell, express the respective egg white matter, adopt the protein of proper method separation and purification, behind the injection body, also can produce identical or similar effect, therefore the present invention has also comprised the coded aminoacid sequence of SEQ ID NO.1, and this sequence is shown in SEQ ID NO.2.

According to another aspect of the invention, provide a kind of pharmaceutical composition, it contains DNA chimeric of the present invention and pharmaceutically acceptable carrier and vehicle.

The present invention also provides the application of the chimeric DNA vaccine that contains SEQ ID NO.1 sequence, and vaccine of the present invention can be used for prevention lungy and treatment, also can be used as immunostimulant, is used to improve immunologic function.

The invention provides design philosophy, the technological line of a kind of novel Vaccinum Calmette-Guerini of preparation, particularly be used for inducing in the humans and animals body and strengthening the DNA of protective immune response (humoral immunization and cellular immunization).Selection mycobacterium hsp70 and people's B7-1 gene carries out chimeric at molecular level, make up novel Vaccinum Calmette-Guerini, and wherein hsp70 provides the immunologic opsonin signal, induces the cell response of specificity T h1 type and produces r-IFN and interleukin 12; It is costimulatory signal that B7-1 provides second signal, and indirect induction T cell produces r-IFN, comprehensively active cells and humoral immunization.By both actings in conjunction, push the immunological status of humans and animals to the Th1 type by the Th2 type.Vaccine of the present invention can be used for the tuberculosis due to the tubercule bacillus; Perhaps in external eukaryotic cell, express corresponding proteins matter, as prevention and treatment tuberculosis, particularly to the treatment of anti-multiple medicines tuberculosis (MDR-TB); Vaccine of the present invention also can be used as a kind of immune promotor, is used for the immunotherapy of corresponding virus disease and tumour.

Brief description of drawings

Fig. 1 is a hsp/B7-1 mosaic type plasmid construction route map.

Fig. 2 is pcDNA3.1 (+) carrier collection of illustrative plates.Its structure is:

Pcmv is the CMV promotor, is positioned at 232-819bp; T7 promotor/primer sites is positioned at 863-882bp; Multiple clone site is positioned at 895-1010bp; PcDNA3.1/BGH reverse primer site is positioned at 1022-1039bp; BGHpA is BGHpoly (A) sequence, is positioned at 1028-1052bp; F1 ori is the F1 replication origin, is positioned at 1298-1726bp; SV40 ori is SV40 early promoter and replication origin, is positioned at 1731-2074bp; PUC ori is the PUC replication origin, is positioned at 3617-4287bp (complementary strand); Ampicllin is ampicillin resistance gene (bla), is positioned at 4432-5428bp (complementary strand); ORF (open reading frame) is positioned at 4432-5292bp (complementary strand); Ribosome bind site is positioned at 5300-5304bp (complementary strand); Bla promotor (P3): 5327-5333bp (complementary strand).

Fig. 3 is mycobacterium hsp70-joint-people B7-1 complete genome sequence open reading frame (SEQ ID NO:1), and initiator codon and terminator codon all are expressed as the black matrix underscore.

Fig. 4 is that the PCR that pcDNA3.1 (+)-hsp70/B7-1 chimeric plasmid makes up identifies figure.Wherein: M:Lambda-pUC hybrid dna molecular weight marker; 1 for being template with the pcDNA3-hsp70/B7 chimeric plasmid, and P5, P6 are the PCR product of primer; 2 for being template with the pcDNA3 plasmid, is the PCR product of primer with P1, P4; 3 for being template with the pcDNA3-hap70/B7 chimeric plasmid, and P3, P4 are the PCR product of primer; 4 for being template with the pcDNA3-hsp70/B7 chimeric plasmid, is the PCR product of primer with P1, P2; 5 are sterilization distilled water blank

Fig. 5 is that the enzyme of pcDNA3.1 (+)-hsp70/B7-1 chimeric plasmid is cut evaluation figure.Wherein:

M is a Lambda-pUC hybrid dna molecular weight marker; 1 pcDNA3.1 (+) hsp70/B7-1 plasmid for structure; 2 is with pcDNA3.1 (+) carrier and goal gene hsp70/B7-1 behind HindIII and the XbaI enzyme cutting; 3 are sterilization distilled water blank.

Fig. 6 is the expression product agarose gel electrophoresis figure of pcDNA3.1 (+) hsp70/B7-1 chimeric DNA in the COS.7 cell.Wherein:

1,2,3 is respectively pcDNA3.1 (+) hsp70/B7-1 transfection COS-7 cell after 24,48,72 hours, the RT-PCR result of P5, P2 primer; 4,5,6 be respectively pcDNA3.1 (+) hsp70/137-1 transfection COS-7 cell after 24,48,72 hours, the RT-PCR result of P3, P4 primer; 7,8,9 be respectively pcDNA3.1 (+) transfection COS-7 cell after 24,48,72 hours, the RT-PCR result of P3, P4 primer; M is a Lambda-pUC hybrid dna molecular weight marker.

Embodiment

Below, in conjunction with the accompanying drawings,, describe in detail but do not limit the present invention by description to better embodiment of the present invention.

The structure of [embodiment 1] pcDNA3.1 (+)-hsp70/B7-1 chimeric plasmid.

Referring to Fig. 1 and Fig. 2:

Main experiment material

One, plasmid and bacterial strain:; PcDNA3.1 (+)-B7-1 (containing people B7-1 encoding gene and both sides regulatory gene) and plasmid pcDNA3.1 (+) purchase in Invitrogen, and engineering bacteria Dh5 α purchases Shanghai and gives birth to worker bio-engineering corporation.

Two, Oligonucleolide primers design: according to the gene order (Mckenzie.KR of the bacillus tuberculosis typus humanus H37Rv strain hsp70 that delivers, Adams E, Britton WJ, et al.Sequence and immunogenicity of the 70-kDa heatshock protein of Mycobacterium leprae.J.Immune.1991; 147.:312-319.) and people B7-1 sequence (GenBank accession number NM005191) design.

The reorganization pcDNA3.1 (+)-hsp70 plasmid primer:

P1 (SEQ ID NO.4): 5 ' ggagccATGGCTCGTGCGGTCGGGATC 3 ' (containing BamHI site and codon ATG)

P2 (SEQ ID NO.5): 5 ' gaattcATGGCTCGTGCGGTCGGGATC 3 ' (containing the EcoRI site).

2.hsp70-linker-B7-1 primer:

P3:GGTGGTTCGAAggtaccTGAAGACACGCTG is positioned at the 1604bp place (containing the KapI site) of hsp70 sequence;

P4: ACTCCCTCCGCCACCGCTCCCTCCGCCACCACTCCCTCCGCCACCCTThe TGGCCTCCCGGCCGTCGTCGACCACCTCC setting-out partly is linker

P5: GGAGGGAGCGGTGGCGGAGGGAGTThe GGCCACACACGGAGGCAGGGAACATCACC setting-out partly is the linker complementary sequence

P6:GACACtctagaTTATACAGGGCGTACACTTTCCCTTCTC (containing XbaI site and termination codon TTA)

3. reorganization pcDNA3.1 (+) hsp70/B7-1 plasmid primers designed

P7:5 ' aagcttATGGCTCGTGCGGTCGGGATC 3 ' (containing HindIII site and codon ATG)

P8:5 ' tctagaTTATACAGGGCGTACACTTTC 3 ' (containing XbaI site and termination codon TTA)

Above primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.

Three, relevant enzyme: restriction enzyme, Taq+Pfu enzyme are given birth to worker bio-engineering corporation available from Shanghai; T4DNA connects test kit available from the biological Dalian of treasured company.

Four, plasmid extraction kit is used for plasmid and extracts available from Shanghai Hua Shun company.

Five, the DNA sepharose reclaims test kit available from Shanghai Hua Shun company, is used for the recovery of DNA electrophoresis product.

Six, PCR product purification test kit is a Shanghai Hua Shun company product, is used for the purifying of PCR product.

Seven. it is that chemical product is given birth in Shanghai that eukaryotic cell mRNA extracts reagent and box and RT-PCR test kit.

Eight .COS-7 cells are purchased the Shanghai cell bank in the Chinese Academy of Sciences.

Nine, tubercule bacillus virulent strain H37Rv, institute is so kind as to give by Sichuan Province's tuberculosis prevention and treatment.

Ten. cationic-liposome transfection reagent box is purchased and Shenzhen brilliant U.S. company.

11. single stage method RT-PCR reagent is that worker bio-engineering corporation product is given birth in Shanghai

12. laboratory animal: C57BL mouse, cleaning level; Purchase in Chongqing medical courses in general large animal institute.Macaque, 2 grades; Purchase Suzhou Western Hills Experimental Animal Center in the Chinese Academy of Sciences.

Experimental technique

1.Hsp70 the preparation of goal gene:

Extract tubercule bacillus virulent strain H37RV genomic dna, carry out pcr amplification, its reaction system is:

H37RV genomic dna 2 μ l

5 ' primer solution (P1), 3 μ l

3 ' primer solution (P2), 3 μ l

dNTP 2.5μl

Taq+Pfu enzyme 1 μ l

10 * PCR damping fluid, 5 μ l

Sterilization distilled water 33.5 μ l2.

Be 50 μ l systems, the PCR conditional parameter is 94 ℃ of pre-sex change 5 minutes, transfers 94 ℃ of sex change 1 minute after the warm start to, 56 ℃ of annealing 1 minute, 72 ℃ were extended 1.5 minutes, carry out 25 circulations after, 72 ℃ were extended 10 minutes.The about 1875bp of reaction gained clip size gets 5 μ l electrophoresis.And serve the sea and give birth to the confirmation of checking order respectively of worker biotech firm.The purifying of PCR product: the specification sheets that provides by a small amount of PCR of Shanghai Hua Shun company product purification test kit carries out.

The extraction of (2.pcDNA3.1+) plasmid:

A small amount of plasmid extraction kit operation by Shanghai Hua Shun company; BamH I and EcoR I double digestion vector plasmid pcDNA3.1 (+); BamH I and EcoR I double digestion hsp70 goal gene; be connected into pcDNA3.1 (+); adopt the connection test kit of the precious biotech firm in Dalian, obtain eukaryon expression plasmid pcDNA3.1 (+)-hsp70.

3.hsp70/B7-1 the preparation of goal gene:

Three PCR reactions.

PCR reaction (obtaining part Hsp70-linker) for the first time, reaction system is:

PcDNA3.1 (+)-hsp70 plasmid DNA 6.01 μ M 2 μ l

5 ' primer solution (P1), 17.5 μ M, 3 μ l

3 ' primer solution (P2) 10mM, 3 μ l

dNTP 2.5μl

Taq+Pfu enzyme 1 μ l

10 * PCR damping fluid, 5 μ l

Sterilization distilled water 33.5 μ l

PCR reaction (obtaining linker-B7-1) for the second time, reaction system is:

6.01 μ M plasmid pcDNA3.1 (+)-B7-1 2 μ l

Solution 3 μ l

16.6 μ M 5 ' primer solution (P3) 3 μ l

16.0 μ M 3 ' primer solution (P4) 2.5 μ l

10mM?dNTP 1μl

Taq+Pfu enzyme 5 μ l

10 * PCR damping fluid, 33.5 μ l

The sterilization distilled water

Be 50 μ l systems, the PCR conditional parameter is 94 ℃ of pre-sex change 5 minutes, transfers 94 ℃ of sex change 1 minute after the warm start to, 55 ℃ of annealing 1 minute, 72 ℃ were extended 2 minutes, carry out 25 circulations after, 72 ℃ were extended 10 minutes.PCR reacts the about 327bp of gained clip size for the first time, and the about 915bp of PCR reaction gained clip size gets 5 μ l electrophoresis for the second time.Make blank with the sterilization distilled water simultaneously.And serve the sea and give birth to the confirmation of checking order respectively of worker biotech firm.The purifying of PCR product: the specification sheets that provides by a small amount of PCR of Shanghai Hua Shun company product purification test kit carries out.

PCR reaction (obtaining part hsp70-linker-B7-1 goal gene) for the third time, reaction system is as follows:

PCR product (purifying) 2 μ l for the first time

PCR product (purifying) 2 μ l for the second time

16.0 μ M 5 ' primer solution (P1) 3 μ l

16.0 μ M 3 ' primer solution (P4) 3 μ l

10mM?dNTP 2.5μl

Taq+Pfu enzyme 1 μ l

10 * PCR damping fluid, 5 μ l

Sterilization distilled water 31.5 μ l

50 μ l reaction systems, PCR conditional parameter be 94 ℃ 5 minutes, 45 ℃ 2 minutes, 70 ℃ 10 minutes, carry out 1 circulation after, transfer to 94 ℃ 2 minutes, 50 ℃ 2 minutes, 70 ℃ 2 minutes, carry out 25 circulations after, 72 ℃ were extended 10 minutes.Get whole PCR product electrophoresis, find to comprise in the gained fragment fragment of the about 1197bp of size.Make blank with the sterilization distilled water simultaneously.The glue of PCR product reclaims: carry out glue and reclaim by the fragment that preceding method will about 1197bp, get the visible corresponding electrophoresis band of 5 μ l electrophoresis, mosaic gene is behind KpaI and XbaI double digestion, and 65 ℃ of deactivations have obtained part hsp70 joint B7-1, and goal gene cDNA is ready to complete.

4.KpaI and Xba I double digestion vector plasmid pcDNA3.1 (+), is connected with PCR product for the third time, adopt the connection test kit of the precious biotech firm in Dalian, by specification is operated; Be built into pcDNA3.1 (+) part hsp70 (327bp)-Linker-B7-1 plasmid.

5.KpaI single endonuclease digestion pcDNA3.1 (+)-hsp70 obtains the 1.6KbHsp70 gene; KpaI single endonuclease digestion pcDNA3.1 (+) part hsp70 (327bp)-Linker-B7-1 plasmid is connected into the 1.6KbHsp70 gene wherein.Adopt the connection test kit of the precious biotech firm in Dalian, the by specification operation; Be built into pcDNA3.1 (+) hsp70-Lenker-B7-1 plasmid.With above-mentioned connection product transformed into escherichia coli DH5 α, undertaken by the method for " molecular cloning ".And serve the sea and give birth to worker biotech firm order-checking (sequence such as Fig. 3) and confirm that chimeric eukaryon expression plasmid pcDNA3.1 (+) hsp70/B7-1 successfully constructs.

The agarose electrophoresis figure that identifies in the building process as shown in Figure 4 and Figure 5.

The expression of [embodiment 2] pcDNA3.1 (+) hsp70/B7-1 chimeric DNA in eukaryotic cell

With pcDNA3.1 (+) hsp70/B7-1 chimeric DNA liposome transfecting eukaryotic cells (COS.7), the extraction cell mRNA was carried out RT-PCR in 24.48.72 hour, got product and carried out agarose electrophoresis, saw purpose band (Fig. 6).

[embodiment 3] pcDNA3.1 (+) hsp70/B7-1 chimeric DNA vaccine is to the immunoprophylaxis effect research of mouse

Zooprophylazis experiment: the inoculation of dna vaccination: the healthy mice C57BL/6N (available from Medical University Of Chongqing experimentation on animals center) of laboratory animal 6-8 age in week, cleaning level.Every group 8, male and female half and half are divided into experimental group and control group, and experimental group is injected pcDNA.3.1 (+), pcDNA.3.1 (+)-hsp70, pcDNA.3.1 (+)-hsp70/B7-1 100 μ g (0.5ml) respectively, and BCG is by the body weight injection; Control group injecting normal saline 0.5ml.The injection site is the both sides tibialis anterior, every side dosage half and half.3 weeks of inoculation and 6 weeks are separately by former scheme booster shot once.Immunity back three weeks every mouse is used H37RV tail vein injection 10 for the third time ⁴-10 ⁵CFU.

The experiment of [embodiment 4] treatment of animals

Mice group, vaccine inoculation and H37RV attack method, all identical with " experimentation on animals of immune postoperative infection earlier ".Only in this experiment, attack the transfection tubercule bacillus preceding, corresponding D NA vaccine be seeded in back (control group then only injecting normal saline), the vaccine inoculation time be after H37RV attacks the 7th day and the 14th day respectively once.

The vitro detection of IL-4, γ-INF: the prevention group is put to death animal after 14 days in two weeks of H37RV tail vein injection after the vaccine inoculation for the second time of treatment group, gets serum and detects, and detection method is by the mouse ELISA of brilliant U.S. company detection kit specification sheets.

Pathological change is observed: observe Mouse Liver, spleen tissue smear, section pathological change and and cultivation tubercule bacillus numeration.

Statistical procedures method: adopt SPSS for windows10.0 statistical package to carry out data processing.

Result of study

1. the vitro detection of γ-IFN, IL-4: detect with the ELISA method and to accept inoculation pcDNA3, pcDNA3-hsp70, pcDNA3-hsp70/B7-1, BCG, physiological saline is respectively organized γ-IFN in the mice serum, IL-4.

The vitro detection of table 1: γ-IFN, IL-4 (X ± SD)

Group	γ-IFN(pg/ml)		IL-4(pg/ml)
					Prevention	Treatment	Prevention	Treatment
	Physiological saline (n=8) BCG (n=8) pcDNA 3 (n＝8) hsp 70 (n＝8) hsp 70/B 7-1? (n＝8)	146.58±62.41 ? ? 134.74±55.32 ? 516.94±58.76 △? 542.82±92.34 △? 3230.80±114.35 ▲△?	132.66±74.68 ? ? 121.54±56.39 ? 192.00±64.38 ? 542.33±99.77 △? 1336.98±129.6 ? 4	141.86±81.23 ? ? 175.70±72.35 ? 171.25±63.24 ? 1224.38±98.64 △? ? 1423.70±86.19 ?					20.61±7.65 ? ? 45.32±9.45 ? 106.67±34.12 △? 215.44±61.25 △? ? 843.57±97.34 ▲△?

Annotate: ▲: with physiological saline, BCG, pcDNA ₃And HSP ₇₀Relatively, p＜0.001; △: compare p＜0.01. with physiological saline, BCG

1. pathological observation: the prevention group is at H ₃₇Two weeks of RV tail vein injection are after the vaccine inoculation for the second time of treatment group after 14 days

Put to death animal and get liver, spleen tissue grinding back cultivation, and the tissue that takes a morsel carries out smear.

Table 2: liver, spleen tissue smear (Z-N dyeing)

Group	The hepatic tissue smear		The spleen tissue smear
					Prevention	Treatment	Prevention	Treatment
	Physiological saline (n=8) BCG (n=8) pcDNA 3? ?(n＝8) ?hsp 70?(n＝8) ?hsp 70/B 7-1?(n＝8)	? ++++ ? ? ++ ? ++ ? ? + △? - ▲?	? ++++ ? ? ++++ ? ++ △? ? ++ △? + ▲?	? ++++ ? ? ++ ? ++ ? ? + △? - ▲?					? ++++ ? ? ++ ? +++ ? ? + △? - ▲?

Annotate ,+: be 50 below the bacterium colony, ++: be 500 below the bacterium colony, +++: be 1000 below the bacterium colony, ++ ++: be 5000 more than the bacterium colony.▲: compare p＜0.01 with other groups; △: compare p＜0.05 with other groups (unmarked)

Table 3. liver, tuberculosis of spleen bacterium are cultivated colony count

Group	Hepatic tissue		The spleen tissue
					Prevention	Treatment	Prevention	Treatment
	Physiological saline (n=8) BCG (n=8) pcDNA 3? ?(n＝8) ?hsp 70? (n＝8) ?hsp 70/B 7-1? (n＝8)	? +++ ? ? +++ ? ? +++ ? ? ++ ? ? + ?	? ++++ ? ? ++++ ? ? ++ ? ? ++ ? ? + ?	? ++++ ? ? ++ ? ? +++ ? ? + ? ? + ?					? ++++ ? ? ++ ? ? +++ ? ? + ? ? + ?

Annotate ,+: be 50 below the bacterium colony, ++: be 500 below the bacterium colony, +++: be 1000 below the bacterium colony, ++ ++: be 5000 more than the bacterium colony.

The immune macaque preventive effect research of [embodiment 5] hsp70/B7-1 chimeric DNA vaccine

Zooprophylazis experimental group: the inoculation of dna vaccination: laboratory animal macaque, 2 grades, (available from Chinese Academy of Sciences's Suzhou Western Hills Experimental Animal Center).Every group 2, male and female half and half; Be divided into hsp70/B7-1 chimeric DNA vaccine prevention group, BCG prevention group, infected group and normal control group; Experimental group injection pcDNA.3.1 (+)-hsp70/B7-1 500 μ g (1ml), BCG is by the body weight injection; Control group injecting normal saline 1ml.The injection site is the right arm deltoid muscle.3 weeks of experimental group inoculation and 6 weeks are separately by former scheme booster shot once.Immunity back three weeks every macaque is used tubercule bacillus virulent strain H37RV ulnar vein injection 50CFU for the third time.Attack the back and use CT scan January, see that the two lungs of BCG prevention group are dispersed in tubercle shadow size for about 1-5mm, infected group occurred number 1-10mm empty, hsp70/B7-1 chimeric DNA vaccine prevention group is a little tubercle of lung field as follows; Reduce gradually with the two lung tubercles of CT scan second and third month; Per then two months one time two lungs of CT scan see after 6 months that tubercle obviously reduces and absorption; The physiological saline group is that normal chest changes.

The immune macaque result of treatment research of [embodiment 6] hsp70/B7-1 chimeric DNA vaccine

The treatment of animals experimental group: 4 macaques, male and female half and half are with tubercule bacillus virulent strain H37RV ulnar vein injection 50CFU.Attack the back and use CT scan January, see that two lungs fill the air the tubercle that distributes and differ in size.2 macaques, male and female half and half usefulness hsp70/B7-1 chimeric DNA vaccine 500ug/ml treats, and per three weeks injection is once; 2 macaques, male and female do not have obvious change with the CT scan result with vazadrine 50mg, sharp secondary flat 75mg, the acyl ammonia 125mg of the pyrrole Qin, Streptomycin sulphate 100mg treatment after January half and half every day, and reduce gradually with the two lung tubercles of CT scan second and third month; Per then two months one time the two lung tubercles of CT scan obviously reduce and absorption gradually; See after 6 each months that two lung hsp70/B7-1 chimeric DNA vaccine group tubercles have certain absorption; Certain therapeutic action of having compared with antituberculosis drugs thing group.

Sequence table

＜110〉clock, gloomy

Fourth, intelligent loyalty

History, Xiao Ling

Clock, woods

＜120〉be used to prevent the mosaic type dna vaccination of tuberculosis and immunotherapy

<130>A2095

<160>13

<170>PatentIn?version?3.1

<210>1

<211>2784

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>CDS

<222>(4)..(2781)

<223>

<400>1

atg?gct?cgt?gcg?gtc?ggg?atc?gac?ctc?ggg?acc?acc?aac?tcc?gtc?gtc 48

Ala?Arg?Ala?Val?Gly?Ile?Asp?Leu?Gly?Thr?Thr?Asn?Ser?Val?Val

1 5 10 15

tcg?gtt?ctg?gaa?ggt?ggc?gac?ccg?gtc?gtc?gtc?gcc?aac?tcc?gag?ggc 96

Ser?Val?Leu?Glu?Gly?Gly?Asp?Pro?Val?Val?Val?Ala?Asn?Ser?Glu?Gly

20 25 30

tcc?agg?acc?acc?ccg?tca?att?gtc?gcg?ttc?gcc?cgc?aac?ggt?gag?gtg 144

Ser?Arg?Thr?Thr?Pro?Ser?Ile?Val?Ala?Phe?Ala?Arg?Asn?Gly?Glu?Val

35 40 45

ctg?gtc?ggc?cag?ccc?gcc?aag?aac?cag?gca?gtg?acc?aac?gtc?gat?cgc 192

Leu?Val?Gly?Gln?Pro?Ala?Lys?Asn?Gln?Ala?Val?Thr?Asn?Val?Asp?Arg

50 55 60

acc?gtg?cgc?tcg?gtc?aag?cga?cac?atg?ggc?agc?gac?tgg?tcc?ata?gag 240

Thr?Val?Arg?Ser?Val?Lys?Arg?His?Met?Gly?Ser?Asp?Trp?Ser?Ile?Glu

65 70 75

att?gac?ggc?aag?aaa?tac?acc?gcg?ccg?gag?atc?agc?gcc?cgc?att?ctg 288

Ile?Asp?Gly?Lys?Lys?Tyr?Thr?Ala?Pro?Glu?Ile?Ser?Ala?Arg?Ile?Leu

80 85 90 95

atg?aag?ctg?aag?cgc?gac?gcc?gag?gcc?tac?ctc?ggt?gag?gac?att?acc 336

Met?Lys?Leu?Lys?Arg?Asp?Ala?Glu?Ala?Tyr?Leu?Gly?Glu?Asp?Ile?Thr

100 105 110

gac?gcg?gtt?atc?acg?acg?ccc?gcc?tac?ttc?aat?gac?gcc?cag?cgt?cag 384

Asp?Ala?Val?Ile?Thr?Thr?Pro?Ala?Tyr?Phe?Asn?Asp?Ala?Gln?Arg?Gln

115 120 125

gcc?acc?aag?gac?gcc?ggc?cag?atc?gcc?ggc?ctc?aac?gtg?ctg?cgg?atc 432

Ala?Thr?Lys?Asp?Ala?Gly?Gln?Ile?Ala?Gly?Leu?Asn?Val?Leu?Arg?Ile

130 135 140

gtc?aac?gag?ccg?acc?gcg?gcc?gcg?ctg?gcc?tac?ggc?ctc?gac?aag?ggc 480

Val?Asn?Glu?Pro?Thr?Ala?Ala?Ala?Leu?Ala?Tyr?Gly?Leu?Asp?Lys?Gly

145 150 155

gag?aag?gag?cag?cga?atc?ctg?gtc?ttc?gac?ttg?ggt?ggt?ggc?act?ttc 528

Glu?Lys?Glu?Gln?Arg?Ile?Leu?Val?Phe?Asp?Leu?Gly?Gly?Gly?Thr?Phe

160 165 170 175

gac?gtt?tcc?ctg?ctg?gag?atc?ggc?gag?ggt?gtg?gtt?gag?gtc?cgt?gcc 576

Asp?Val?Ser?Leu?Leu?Glu?Ile?Gly?Glu?Gly?Val?Val?Glu?Val?Arg?Ala

180 185 190

act?tcg?ggt?gac?aac?cac?ctc?ggc?ggc?gac?gac?tgg?gac?cag?cgg?gtc 624

Thr?Ser?Gly?Asp?Asn?His?Leu?Gly?Gly?Asp?Asp?Trp?Asp?Gln?Arg?Val

195 200 205

gtc?gat?tgg?ctg?gtg?gac?aag?ttc?aag?ggc?acc?agc?ggc?atc?gat?ctg 672

Val?Asp?Trp?Leu?Val?Asp?Lys?Phe?Lys?Gly?Thr?Ser?Gly?Ile?Asp?Leu

210 215 220

acc?aag?gac?aag?atg?gcg?atg?cag?cgg?ctg?cgg?gaa?gcc?gcc?gag?aag 720

Thr?Lys?Asp?Lys?Met?Ala?Met?Gln?Arg?Leu?Arg?Glu?Ala?Ala?Glu?Lys

225 230 235

gca?aag?atc?gag?ctg?agt?tcg?agt?cag?tcc?acc?tcg?atc?aac?ctg?ccc 768

Ala?Lys?Ile?Glu?Leu?Ser?Ser?Ser?Gln?Ser?Thr?Ser?Ile?Asn?Leu?Pro

240 245 250 255

tac?atc?acc?gtc?gac?gcc?gac?aag?aac?ccg?ttg?ttc?tta?gac?gag?cag 816

Tyr?Ile?Thr?Val?Asp?Ala?Asp?Lys?Asn?Pro?Leu?Phe?Leu?Asp?Glu?Gln

260 265 270

ctg?acc?cgc?gcg?gag?ttc?caa?cgg?atc?act?cag?gac?ctg?ctg?gac?cgc 864

Leu?Thr?Arg?Ala?Glu?Phe?Gln?Arg?Ile?Thr?Gln?Asp?Leu?Leu?Asp?Arg

275 280 285

act?cgc?aag?ccg?ttc?cag?tcg?gtg?atc?gct?gac?acc?ggc?att?tcg?gtg 912

Thr?Arg?Lys?Pro?Phe?Gln?Ser?Val?Ile?Ala?Asp?Thr?Gly?Ile?Ser?Val

290 295 300

tcg?gag?atc?gat?cac?gtt?gtg?ctc?gtg?ggt?ggt?tcg?acc?cgg?atg?ccc 960

Ser?Glu?Ile?Asp?His?Val?Val?Leu?Val?Gly?Gly?Ser?Thr?Arg?Met?Pro

305 310 315

gcg?gtg?acc?gat?ctg?gtc?aag?gaa?ctc?acc?ggc?ggc?aag?gaa?ccc?aac 1008

Ala?Val?Thr?Asp?Leu?Val?Lys?Glu?Leu?Thr?Gly?Gly?Lys?Glu?Pro?Asn

320 325 330 335

aag?ggc?gtc?aac?ccc?gat?gag?gtt?gtc?gcg?gtg?gga?gcc?gct?ctg?cag 1056

Lys?Gly?Val?Asn?Pro?Asp?Glu?Val?Val?Ala?Val?Gly?Ala?Ala?Leu?Gln

340 345 350

gcc?ggc?gtc?ctc?aag?ggc?gag?gtg?aaa?gac?gtt?ctg?ctg?ctt?gat?gtt 1104

Ala?Gly?Val?Leu?Lys?Gly?Glu?Val?Lys?Asp?Val?Leu?Leu?Leu?Asp?Val

355 360 365

acc?ccg?ctg?agc?ctg?ggt?atc?gag?acc?aag?ggc?ggg?gtg?atg?acc?agg 1152

Thr?Pro?Leu?Ser?Leu?Gly?Ile?Glu?Thr?Lys?Gly?Gly?Val?Met?Thr?Arg

370 375 380

ctc?atc?gag?cgc?aac?acc?acg?atc?ccc?acc?aag?cgg?tcg?gag?act?ttc 1200

Leu?Ile?Glu?Arg?Asn?Thr?Thr?Ile?Pro?Thr?Lys?Arg?Ser?Glu?Thr?Phe

385 390 395

acc?acc?gcc?gac?gac?aac?caa?ccg?tcg?gtg?cag?atc?cag?gtc?tat?cag 1248

Thr?Thr?Ala?Asp?Asp?Asn?Gln?Pro?Ser?Val?Gln?Ile?Gln?Val?Tyr?Gln

400 405 410 415

ggg?gag?cgt?gag?atc?gcc?gcg?cac?aac?aag?ttg?ctc?ggg?tcc?ttc?gag 1296

Gly?Glu?Arg?Glu?Ile?Ala?Ala?His?Asn?Lys?Leu?Leu?Gly?Ser?Phe?Glu

420 425 430

ctg?acc?ggc?atc?ccg?ccg?gcg?ccg?cgg?ggg?att?ccg?cag?atc?gag?gtc 1344

Leu?Thr?Gly?Ile?Pro?Pro?Ala?Pro?Arg?Gly?Ile?Pro?Gln?Ile?Glu?Val

435 440 445

act?ttc?gac?atc?gac?gcc?aac?ggc?att?gtg?cac?gtc?acc?gcc?aag?gac 1392

Thr?Phe?Asp?Ile?Asp?Ala?Asn?Gly?Ile?Val?His?Val?Thr?Ala?Lys?Asp

450 455 460

aag?ggc?acc?ggc?aag?gag?aac?acg?atc?cga?atc?cag?gaa?ggc?tcg?ggc 1440

Lys?Gly?Thr?Gly?Lys?Glu?Asn?Thr?Ile?Arg?Ile?Gln?Glu?Gly?Ser?Gly

465 470 475

ctg?tcc?aag?gaa?gac?att?gac?cgc?atg?atc?aag?gac?gcc?gaa?gcg?cac 1488

Leu?Ser?Lys?Glu?Asp?Ile?Asp?Arg?Met?Ile?Lys?Asp?Ala?Glu?Ala?His

480 485 490 495

gcc?gag?gag?gat?cgc?aag?cgt?cgc?gag?gag?gcc?gat?gtt?cgt?aat?caa 1536

Ala?Glu?Glu?Asp?Arg?Lys?Arg?Arg?Glu?Glu?Ala?Asp?Val?Arg?Asn?Gln

500 505 510

gcc?gag?aca?ttg?gtc?tac?cag?acg?gag?aag?ttc?gtc?aaa?gaa?cag?cgt 1584

Ala?Glu?Thr?Leu?Val?Tyr?Gln?Thr?Glu?Lys?Phe?Val?Lys?Glu?Gln?Arg

515 520 525

gag?gcc?gag?ggt?ggt?tcg?aag?gta?cct?gaa?gac?acg?ctg?aac?aag?gtt 1632

Glu?Ala?Glu?Gly?Gly?Ser?Lys?Val?Pro?Glu?Asp?Thr?Leu?Asn?Lys?Val

530 535 540

gat?gcc?gcg?gtg?gcg?gaa?gcg?aag?gcg?gca?ctt?ggc?gga?tcg?gat?att 1680

Asp?Ala?Ala?Val?Ala?Glu?Ala?Lys?Ala?Ala?Leu?Gly?Gly?Ser?Asp?Ile

545 550 555

tcg?gcc?atc?aag?tcg?gcg?atg?gag?aag?ctg?ggc?cag?gag?tcg?cag?gct 1728

Ser?Ala?Ile?Lys?Ser?Ala?Met?Glu?Lys?Leu?Gly?Gln?Glu?Ser?Gln?Ala

560 565 570 575

ctg?ggg?caa?gcg?atc?tac?gaa?gca?gct?cag?gct?gcg?tca?cag?gcc?act 1776

Leu?Gly?Gln?Ala?Ile?Tyr?Glu?Ala?Ala?Gln?Ala?Ala?Ser?Gln?Ala?Thr

580 585 590

ggc?gct?gcc?cac?ccc?ggc?ggc?gag?ccg?ggc?ggt?gcc?cac?ccc?ggc?tcg 1824

Gly?Ala?Ala?His?Pro?Gly?Gly?Glu?Pro?Gly?Gly?Ala?His?Pro?Gly?Ser

595 600 605

gct?gat?gac?gtt?gtg?gac?gcg?gag?gtg?gtc?gac?gac?ggc?cgg?gag?gcc 1872

Ala?Asp?Asp?Val?Val?Asp?Ala?Glu?Val?Val?Asp?Asp?Gly?Arg?Glu?Ala

610 615 620

aag?ggt?ggc?gga?ggg?agt?ggt?ggc?gga?ggg?agc?ggt?ggc?gga?ggg?agt 1920

Lys?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser

625 630 635

ggc?cac?aca?cgg?agg?cag?gga?aca?tca?cca?tcc?aag?tgt?cca?tac?ctc 1968

Gly?His?Thr?Arg?Arg?Gln?Gly?Thr?Ser?Pro?Ser?Lys?Cys?Pro?Tyr?Leu

640 645 650 655

aat?ttc?ttt?cag?ctc?ttg?gtg?ctg?gct?ggt?ctt?tct?cac?ttc?tgt?tca 2016

Asn?Phe?Phe?Gln?Leu?Leu?Val?Leu?Ala?Gly?Leu?Ser?His?Phe?Cys?Ser

660 665 670

ggt?gtt?atc?cac?gtg?acc?aag?gaa?gtg?aaa?gaa?gtg?gca?acg?ctg?tcc 2064

Gly?Val?Ile?His?Val?Thr?Lys?Glu?Val?Lys?Glu?Val?Ala?Thr?Leu?Ser

675 680 685

tgt?ggt?cac?aat?gtt?tct?gtt?gaa?gag?ctg?gca?caa?act?cgc?atc?tac 2112

Cys?Gly?His?Asn?Val?Ser?Val?Glu?Glu?Leu?Ala?Gln?Thr?Arg?Ile?Tyr

690 695 700

tgg?caa?aag?gag?aag?aaa?atg?gtg?ctg?act?atg?atg?tct?ggg?gac?atg 2160

Trp?Gln?Lys?Glu?Lys?Lys?Met?Val?Leu?Thr?Met?Met?Ser?Gly?Asp?Met

705 710 715

aat?ata?tgg?ccc?gag?tac?aag?aac?cgg?acc?atc?ttt?gat?atc?act?aat 2208

Asn?Ile?Trp?Pro?Glu?Tyr?Lys?Asn?Arg?Thr?Ile?Phe?Asp?Ile?Thr?Asn

720 725 730 735

aac?ctc?tcc?att?gtg?atc?ctg?gct?ctg?cgc?cca?tct?gac?gag?ggc?aca 2256

Asn?Leu?Ser?Ile?Val?Ile?Leu?Ala?Leu?Arg?Pro?Ser?Asp?Glu?Gly?Thr

740 745 750

tac?gag?tgt?gtt?gtt?ctg?aag?tat?gaa?aaa?gac?gct?ttc?aag?cgg?gaa 2304

Tyr?Glu?Cys?Val?Val?Leu?Lys?Tyr?Glu?Lys?Asp?Ala?Phe?Lys?Arg?Glu

755 760 765

cac?ctg?gct?gaa?gtg?acg?tta?tca?gtc?aaa?gct?gac?ttc?cct?aca?cct 2352

His?Leu?Ala?Glu?Val?Thr?Leu?Ser?Val?Lys?Ala?Asp?Phe?Pro?Thr?Pro

770 775 780

agt?ata?tct?gac?ttt?gaa?att?cca?act?tct?aat?att?aga?agg?ata?att 2400

Ser?Ile?Ser?Asp?Phe?Glu?Ile?Pro?Thr?Ser?Asn?Ile?Arg?Arg?Ile?Ile

785 790 795

tgc?tca?acc?tct?gga?ggt?ttt?cca?gag?cct?cac?ctc?tcc?tgg?ttg?gaa 2448

Cys?Ser?Thr?Ser?Gly?Gly?Phe?Pro?Glu?Pro?His?Leu?Ser?Trp?Leu?Glu

800 805 810 815

aat?gga?gaa?gaa?tta?aat?gcc?atc?aac?aca?aca?gtt?tcc?caa?gat?cct 2496

Asn?Gly?Glu?Glu?Leu?Asn?Ala?Ile?Asn?Thr?Thr?Val?Ser?Gln?Asp?Pro

820 825 830

gaa?act?gag?ctc?tat?gct?gtt?agc?agc?aaa?ctg?gat?ttc?aat?atg?aca 2544

Glu?Thr?Glu?Leu?Tyr?Ala?Val?Ser?Ser?Lys?Leu?Asp?Phe?Asn?Met?Thr

835 840 845

acc?aac?cac?agc?ttc?atg?tgt?ctc?atc?aag?tat?gga?cat?tta?aga?gtg 2592

Thr?Asn?His?Ser?Phe?Met?Cys?Leu?Ile?Lys?Tyr?Gly?His?Leu?Arg?Val

850 855 860

aat?cag?acc?ttc?aac?tgg?aat?aca?acc?aag?caa?gag?cat?ttt?cct?gat 2640

Asn?Gln?Thr?Phe?Asn?Trp?Asn?Thr?Thr?Lys?Gln?Glu?His?Phe?Pro?Asp

865 870 875

aac?ctg?ctc?cca?tcc?tgg?gcc?att?acc?tta?atc?tca?gta?aat?gga?att 2688

Asn?Leu?Leu?Pro?Ser?Trp?Ala?Ile?Thr?Leu?Ile?Ser?Val?Asn?Gly?Ile

880 885 890 895

ttt?gtg?ata?tgc?tgc?ctg?acc?tac?tgc?ttt?gcc?cca?aga?tgc?aga?gag 2736

Phe?Val?Ile?Cys?Cys?Leu?Thr?Tyr?Cys?Phe?Ala?Pro?Arg?Cys?Arg?Glu

900 905 910

aga?agg?agg?aat?gag?aga?ttg?aga?agg?gaa?agt?gta?cgc?cct?gta?taa 2784

Arg?Arg?Arg?Asn?Glu?Arg?Leu?Arg?Arg?Glu?Ser?Val?Arg?Pro?Val

915 920 925

<210>2

<211>926

<212>PRT

＜213〉artificial sequence (Artificial)

<400>2

Ala?Arg?Ala?Val?Gly?Ile?Asp?Leu?Gly?Thr?Thr?Asn?Ser?Val?Val?Ser

1 5 10 15

Val?Leu?Glu?Gly?Gly?Asp?Pro?Val?Val?Val?Ala?Asn?Ser?Glu?Gly?Ser

20 25 30

Arg?Thr?Thr?Pro?Ser?Ile?Val?Ala?Phe?Ala?Arg?Asn?Gly?Glu?Val?Leu

35 40 45

Val?Gly?Gln?Pro?Ala?Lys?Asn?Gln?Ala?Val?Thr?Asn?Val?Asp?Arg?Thr

50 55 60

Val?Arg?Ser?Val?Lys?Arg?His?Met?Gly?Ser?Asp?Trp?Ser?Ile?Glu?Ile

65 70 75 80

Asp?Gly?Lys?Lys?Tyr?Thr?Ala?Pro?Glu?Ile?Ser?Ala?Arg?Ile?Leu?Met

85 90 95

Lys?Leu?Lys?Arg?Asp?Ala?Glu?Ala?Tyr?Leu?Gly?Glu?Asp?Ile?Thr?Asp

100 105 110

Ala?Val?Ile?Thr?Thr?Pro?Ala?Tyr?Phe?Asn?Asp?Ala?Gln?Arg?Gln?Ala

115 120 125

Thr?Lys?Asp?Ala?Gly?Gln?Ile?Ala?Gly?Leu?Asn?Val?Leu?Arg?Ile?Val

130 135 140

Asn?Glu?Pro?Thr?Ala?Ala?Ala?Leu?Ala?Tyr?Gly?Leu?Asp?Lys?Gly?Glu

145 150 155 160

Lys?Glu?Gln?Arg?Ile?Leu?Val?Phe?Asp?Leu?Gly?Gly?Gly?Thr?Phe?Asp

165 170 175

Val?Ser?Leu?Leu?Glu?Ile?Gly?Glu?Gly?Val?Val?Glu?Val?Arg?Ala?Thr

180 185 190

Ser?Gly?Asp?Asn?His?Leu?Gly?Gly?Asp?Asp?Trp?Asp?Gln?Arg?Val?Val

195 200 205

Asp?Trp?Leu?Val?Asp?Lys?Phe?Lys?Gly?Thr?Ser?Gly?Ile?Asp?Leu?Thr

210 215 220

Lys?Asp?Lys?Met?Ala?Met?Gln?Arg?Leu?Arg?Glu?Ala?Ala?Glu?Lys?Ala

225 230 235 240

Lys?Ile?Glu?Leu?Ser?Ser?Ser?Gln?Ser?Thr?Ser?Ile?Asn?Leu?Pro?Tyr

245 250 255

Ile?Thr?Val?Asp?Ala?Asp?Lys?Asn?Pro?Leu?Phe?Leu?Asp?Glu?Gln?Leu

260 265 270

Thr?Arg?Ala?Glu?Phe?Gln?Arg?Ile?Thr?Gln?Asp?Leu?Leu?Asp?Arg?Thr

275 280 285

Arg?Lys?Pro?Phe?Gln?Ser?Val?Ile?Ala?Asp?Thr?Gly?Ile?Ser?Val?Ser

290 295 300

Glu?Ile?Asp?His?Val?Val?Leu?Val?Gly?Gly?Ser?Thr?Arg?Met?Pro?Ala

305 310 315 320

Val?Thr?Asp?Leu?Val?Lys?Glu?Leu?Thr?Gly?Gly?Lys?Glu?Pro?Asn?Lys

325 330 335

Gly?Val?Asn?Pro?Asp?Glu?Val?Val?Ala?Val?Gly?Ala?Ala?Leu?Gln?Ala

340 345 350

Gly?Val?Leu?Lys?Gly?Glu?Val?Lys?Asp?Val?Leu?Leu?Leu?Asp?Val?Thr

355 360 365

Pro?Leu?Ser?Leu?Gly?Ile?Glu?Thr?Lys?Gly?Gly?Val?Met?Thr?Arg?Leu

370 375 380

Ile?Glu?Arg?Asn?Thr?Thr?Ile?Pro?Thr?Lys?Arg?Ser?Glu?Thr?Phe?Thr

385 390 395 400

Thr?Ala?Asp?Asp?Asn?Gln?Pro?Ser?Val?Gln?Ile?Gln?Val?Tyr?Gln?Gly

405 410 415

Glu?Arg?Glu?Ile?Ala?Ala?His?Asn?Lys?Leu?Leu?Gly?Ser?Phe?Glu?Leu

420 425 430

Thr?Gly?Ile?Pro?Pro?Ala?Pro?Arg?Gly?Ile?Pro?Gln?Ile?Glu?Val?Thr

435 440 445

Phe?Asp?Ile?Asp?Ala?Asn?Gly?Ile?Val?His?Val?Thr?Ala?Lys?Asp?Lys

450 455 460

Gly?Thr?Gly?Lys?Glu?Asn?Thr?Ile?Arg?Ile?Gln?Glu?Gly?Ser?Gly?Leu

465 470 475 480

Ser?Lys?Glu?Asp?Ile?Asp?Arg?Met?Ile?Lys?Asp?Ala?Glu?Ala?His?Ala

485 490 495

Glu?Glu?Asp?Arg?Lys?Arg?Arg?Glu?Glu?Ala?Asp?Val?Arg?Asn?Gln?Ala

500 505 510

Glu?Thr?Leu?Val?Tyr?Gln?Thr?Glu?Lys?Phe?Val?Lys?Glu?Gln?Arg?Glu

515 520 525

Ala?Glu?Gly?Gly?Ser?Lys?Val?Pro?Glu?Asp?Thr?Leu?Asn?Lys?Val?Asp

530 535 540

Ala?Ala?Val?Ala?Glu?Ala?Lys?Ala?Ala?Leu?Gly?Gly?Ser?Asp?Ile?Ser

545 550 555 560

Ala?Ile?Lys?Ser?Ala?Met?Glu?Lys?Leu?Gly?Gln?Glu?Ser?Gln?Ala?Leu

565 570 575

Gly?Gln?Ala?Ile?Tyr?Glu?Ala?Ala?Gln?Ala?Ala?Ser?Gln?Ala?Thr?Gly

580 585 590

Ala?Ala?His?Pro?Gly?Gly?Glu?Pro?Gly?Gly?Ala?His?Pro?Gly?Ser?Ala

595 600 605

Asp?Asp?Val?Val?Asp?Ala?Glu?Val?Val?Asp?Asp?Gly?Arg?Glu?Ala?Lys

610 615 620

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly

625 630 635 640

His?Thr?Arg?Arg?Gln?Gly?Thr?Ser?Pro?Ser?Lys?Cys?Pro?Tyr?Leu?Asn

645 650 655

Phe?Phe?Gln?Leu?Leu?Val?Leu?Ala?Gly?Leu?Ser?His?Phe?Cys?Ser?Gly

660 665 670

Val?Ile?His?Val?Thr?Lys?Glu?Val?Lys?Glu?Val?Ala?Thr?Leu?Ser?Cys

675 680 685

Gly?His?Asn?Val?Ser?Val?Glu?Glu?Leu?Ala?Gln?Thr?Arg?Ile?Tyr?Trp

690 695 700

Gln?Lys?Glu?Lys?Lys?Met?Val?Leu?Thr?Met?Met?Ser?Gly?Asp?Met?Asn

705 710 715 720

Ile?Trp?Pro?Glu?Tyr?Lys?Asn?Arg?Thr?Ile?Phe?Asp?Ile?Thr?Asn?Asn

725 730 735

Leu?Ser?Ile?Val?Ile?Leu?Ala?Leu?Arg?Pro?Ser?Asp?Glu?Gly?Thr?Tyr

740 745 750

Glu?Cys?Val?Val?Leu?Lys?Tyr?Glu?Lys?Asp?Ala?Phe?Lys?Arg?Glu?His

755 760 765

Leu?Ala?Glu?Val?Thr?Leu?Ser?Val?Lys?Ala?Asp?Phe?Pro?Thr?Pro?Ser

770 775 780

Ile?Ser?Asp?Phe?Glu?Ile?Pro?Thr?Ser?Asn?Ile?Arg?Arg?Ile?Ile?Cys

785 790 795 800

Ser?Thr?Ser?Gly?Gly?Phe?Pro?Glu?Pro?His?Leu?Ser?Trp?Leu?Glu?Asn

805 810 815

Gly?Glu?Glu?Leu?Asn?Ala?Ile?Asn?Thr?Thr?Val?Ser?Gln?Asp?Pro?Glu

820 825 830

Thr?Glu?Leu?Tyr?Ala?Val?Ser?Ser?Lys?Leu?Asp?Phe?Asn?Met?Thr?Thr

835 840 845

Asn?His?Ser?Phe?Met?Cys?Leu?Ile?Lys?Tyr?Gly?His?Leu?Arg?Val?Asn

850 855 860

Gln?Thr?Phe?Asn?Trp?Asn?Thr?Thr?Lys?Gln?Glu?His?Phe?Pro?Asp?Asn

865 870 875 880

Leu?Leu?Pro?Ser?Trp?Ala?Ile?Thr?Leu?Ile?Ser?Val?Asn?Gly?Ile?Phe

885 890 895

Val?Ile?Cys?Cys?Leu?Thr?Tyr?Cys?Phe?Ala?Pro?Arg?Cys?Arg?Glu?Arg

900 905 910

Arg?Arg?Asn?Glu?Arg?Leu?Arg?Arg?Glu?Ser?Val?Arg?Pro?Val

915 920 925

<210>3

<211>45

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(45)

＜223〉manual splice

<400>3

ggtggcggag?ggagtggtgg?cggagggagc?ggtggcggag?ggagt 45

<210>4

<211>27

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉reorganization pcDNA3.1 (+)-hsp70 plasmid primer

<400>4

ggagccatgg?ctcgtgcggt?cgggatc 27

<210>5

<211>27

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉reorganization pcDNA3.1 (+)-hsp70 plasmid primer

<400>5

gaattcatgg?ctcgtgcggt?cgggatc 27

<210>6

<211>30

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(30)

＜223〉hsp70-linker-B7-1 primer

<400>6

ggtggttcga?aggtacctga?agacacgctg 30

<210>7

<211>76

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(76)

＜223〉hsp70-linker-B7-1 primer

<400>7

actccctccg?ccaccgctcc?ctccgccacc?actccctccg?ccacccttgg?cctcccggcc 60

gtcgtcgacc?acctcc 76

<210>8

<211>53

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(53)

<223>hsp70-linker-B7-1

<220>

<221>misc_feature

<222>(1)..(53)

＜223〉hsp70-linker-B7-1 primer

<400>8

ggagggagcg?gtggcggagg?gagtggccac?acacggaggc?agggaacatc?acc 53

<210>9

<211>39

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(39)

＜223〉hsp70-linker-B7-1 primer

<400>9

gacactctag?attatacagg?gcgtacactt?tcccttctc 39

<210>10

<211>27

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉reorganization pcDNA3.1 (+) hsp70/B7-1 plasmid primers designed

<400>10

aagcttatgg?ctcgtgcggt?cgggatc 27

<210>11

<211>27

<212>DNA

＜213〉artificial sequence (Artificial)

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉reorganization pcDNA3.1 (+) hsp70/B7-1 plasmid primers designed

<400>11

tctagattat?acagggcgta?cactttc 27

<210>12

<211>1875

<212>DNA

＜213〉tubercule bacillus (H37Rv) strain

<400>12

atggctcgtg?cggtcgggat?cgacctcggg?accaccaact?ccgtcgtctc?ggttctggaa 60

ggtggcgacc?cggtcgtcgt?cgccaactcc?gagggctcca?ggaccacccc?gtcaattgtc 120

gcgttcgccc?gcaacggtga?ggtgctggtc?ggccagcccg?ccaagaacca?ggcagtgacc 180

aacgtcgatc?gcaccgtgcg?ctcggtcaag?cgacacatgg?gcagcgactg?gtccatagag 240

attgacggca?agaaatacac?cgcgccggag?atcagcgccc?gcattctgat?gaagctgaag 300

cgcgacgccg?aggcctacct?cggtgaggac?attaccgacg?cggttatcac?gacgcccgcc 360

tacttcaatg?acgcccagcg?tcaggccacc?aaggacgccg?gccagatcgc?cggcctcaac 420

gtgctgcgga?tcgtcaacga?gccgaccgcg?gccgcgctgg?cctacggcct?cgacaagggc 480

gagaaggagc?agcgaatcct?ggtcttcgac?ttgggtggtg?gcactttcga?cgtttccctg 540

ctggagatcg?gcgagggtgt?ggttgaggtc?cgtgccactt?cgggtgacaa?ccacctcggc 600

ggcgacgact?gggaccagcg?ggtcgtcgat?tggctggtgg?acaagttcaa?gggcaccagc 660

ggcatcgatc?tgaccaagga?caagatggcg?atgcagcggc?tgcgggaagc?cgccgagaag 720

gcaaagatcg?agctgagttc?gagtcagtcc?acctcgatca?acctgcccta?catcaccgtc 780

gacgccgaca?agaacccgtt?gttcttagac?gagcagctga?cccgcgcgga?gttccaacgg 840

atcactcagg?acctgctgga?ccgcactcgc?aagccgttcc?agtcggtgat?cgctgacacc 900

ggcatttcgg?tgtcggagat?cgatcacgtt?gtgctcgtgg?gtggttcgac?ccggatgccc 960

gcggtgaccg?atctggtcaa?ggaactcacc?ggcggcaagg?aacccaacaa?gggcgtcaac 1020

cccgatgagg?ttgtcgcggt?gggagccgct?ctgcaggccg?gcgtcctcaa?gggcgaggtg 1080

aaagacgttc?tgctgcttga?tgttaccccg?ctgagcctgg?gtatcgagac?caagggcggg 1140

gtgatgacca?ggctcatcga?gcgcaacacc?acgatcccca?ccaagcggtc?ggagactttc 1200

accaccgccg?acgacaacca?accgtcggtg?cagatccagg?tctatcaggg?ggagcgtgag 1260

atcgccgcgc?acaacaagtt?gctcgggtcc?ttcgagctga?ccggcatccc?gccggcgccg 1320

cgggggattc?cgcagatcga?ggtcactttc?gacatcgacg?ccaacggcat?tgtgcacgtc 1380

accgccaagg?acaagggcac?cggcaaggag?aacacgatcc?gaatccagga?aggctcgggc 1440

ctgtccaagg?aagacattga?ccgcatgatc?aaggacgccg?aagcgcacgc?cgaggaggat 1500

cgcaagcgtc?gcgaggaggc?cgatgttcgt?aatcaagccg?agacattggt?ctaccagacg 1560

gagaagttcg?tcaaagaaca?gcgtgaggcc?gagggtggtt?cgaaggtacc?tgaagacacg 1620

ctgaacaagg?ttgatgccgc?ggtggcggaa?gcgaaggcgg?cacttggcgg?atcggatatt 1680

tcggccatca?agtcggcgat?ggagaagctg?ggccaggagt?cgcaggctct?ggggcaagcg 1740

atctacgaag?cagctcaggc?tgcgtcacag?gccactggcg?ctgcccaccc?cggcggcgag 1800

ccgggcggtg?cccaccccgg?ctcggctgat?gacgttgtgg?acgcggaggt?ggtcgacgac 1860

ggccgggagg?ccaag 1875

<210>13

<211>864

<212>DNA

＜213〉people (Homo sapiens)

<400>13

ggccacacac?ggaggcaggg?aacatcacca?tccaagtgtc?catacctcaa?tttctttcag 60

ctcttggtgc?tggctggtct?ttctcacttc?tgttcaggtg?ttatccacgt?gaccaaggaa 120

gtgaaagaag?tggcaacgct?gtcctgtggt?cacaatgttt?ctgttgaaga?gctggcacaa 180

actcgcatct?actggcaaaa?ggagaagaaa?atggtgctga?ctatgatgtc?tggggacatg 240

aatatatggc?ccgagtacaa?gaaccggacc?atctttgata?tcactaataa?cctctccatt 300

gtgatcctgg?ctctgcgccc?atctgacgag?ggcacatacg?agtgtgttgt?tctgaagtat 360

gaaaaagacg?ctttcaagcg?ggaacacctg?gctgaagtga?cgttatcagt?caaagctgac 420

ttccctacac?ctagtatatc?tgactttgaa?attccaactt?ctaatattag?aaggataatt 480

tgctcaacct?ctggaggttt?tccagagcct?cacctctcct?ggttggaaaa?tggagaagaa 540

ttaaatgcca?tcaacacaac?agtttcccaa?gatcctgaaa?ctgagctcta?tgctgttagc 600

agcaaactgg?atttcaatat?gacaaccaac?cacagcttca?tgtgtctcat?caagtatgga 660

catttaagag?tgaatcagac?cttcaactgg?aatacaacca?agcaagagca?ttttcctgat 720

aacctgctcc?catcctgggc?cattacctta?atctcagtaa?atggaatttt?tgtgatatgc 780

tgcctgacct?actgctttgc?cccaagatgc?agagagagaa?ggaggaatga?gagattgaga 840

agggaaagtg?tacgccctgt?ataa 864

Claims (14)

1, the Nucleotide that has SEQ ID NO.1 sequence.

2, by the coded aminoacid sequence of SEQ ID NO.1 nucleotide sequence, it has the sequence of SEQ ID NO.2.

3, the expression vector that contains the nucleotide sequence of SEQ ID NO.1.

4, expression vector according to claim 3 is characterized in that described carrier is a plasmid vector.

5, expression vector according to claim 4 is characterized in that described plasmid vector is pcDNA3.1 (+).

6, the host cell that contains the described expression vector of claim 3.

7, a kind of mosaic type dna vaccination has the total length open reading frame of nucleotide sequence shown in SEQ ID NO.1.

8, mosaic type dna vaccination according to claim 7 is characterized in that it further comprises an expression vector.

9, mosaic type dna vaccination according to claim 8 is characterized in that described expression vector is a plasmid vector.

10, mosaic type dna vaccination according to claim 9 is characterized in that described plasmid vector is pcDNA3.1 (+).

11, a kind of pharmaceutical composition contains the described mosaic type dna vaccination of claim 8 and pharmaceutically acceptable carrier and vehicle.

12, a kind of pharmaceutical composition contains protein and pharmaceutically acceptable carrier and vehicle with SEQ ID NO.2.

13, the application of the described mosaic type dna vaccination of claim 8 in preparation prevention and treatment tuberculosis medicine.

14, the application of the described mosaic type dna vaccination of claim 8 in the preparation immunotherapy medicaments.

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