CN102875666B - Tuberculosis antigen specificity TCR (T cell receptor), recombinant retroviral vector thereof and application - Google Patents

Abstract

The invention discloses tuberculosis antigen specificity TCR (T cell receptor), a recombinant retroviral vector of the tuberculosis antigen specificity TCR and application. According to the tuberculosis antigen specificity TCR and the recombinant retroviral vector, the tuberculosis specific 38kDa antigen specific TCR is sleeved successfully, and is subject to transfection to iNKT cell through a retroviral vector, so that the iNKT cell for expressing the tuberculosis specificity TCR can be obtained; and the exogenous TCR gene can be successfully expressed through the iNKT cell; and the tuberculosis specific 38kDa antigen is distinguished based on the specificity, and the IFN-gamma (interferon-gamma) and TNF-alpha (tumor necrosis factor-alpha) cytokines secretion and cytotoxic activity can be mediated; and the application value on treatment of tuberculosis gene can be achieved; and a new way is provided for the adoptive cellular immunotherapy of the tuberculosis.

Description

A kind of tuberculosis antigen specificity TCR, its recombinant retroviral vector and application

Technical field

The invention belongs to bioengineering field, be specifically related to a kind of tuberculosis antigen specific t-cell receptor (TCR), and utilize a kind of retroviral vector for tuberculosis treatment that this TCR prepares, the iNKT cell that retroviral vector transfection obtains and in the application of preparing in anti-tuberculosis drugs.

Background technology

Tuberculosis is the highest transmissible disease of infection rate in global range, and case fatality rate is only second to acquired immune deficiency syndrome (AIDS).In recent years, occur together and infect and resistance and multiple antibiotic resistant strain popular along with increase, HIV and the tubercule bacillus of movement of population, make global tuberculosis revivable, present the gesture of staging a comeback.According to WHO statistical report, the whole world has 1/3 population to be subject to tubercle bacillus affection, and tuberculosis patient reaches 2,000 ten thousand, annual neopathy number 800-1000 ten thousand, because of tuberculosis death toll 3,000,000.China is that 22 tuberculosis height are born one of country in the world, tuberculosis number is in the whole world second, tubercle bacillus affection number has exceeded 600,000,000 at present, tuberculosis patient reaches 6,000,000 more than, annual neopathy number 1,500,000, because of tuberculosis death toll 250,000, national pulmonary tuberculosis report number of the infected and death toll are in first of various transmissible diseases always.

Tuberculotherapy is still taking chemotherapy as main at present, but there is long, the shortcoming such as toxic side effect the is large course for the treatment of, reduce patient's compliance of taking medicine, patient is irregular to take medicine, drug withdrawal voluntarily, selectivity are taken medicine etc. all may improve the probability of Mycobacterium tuberculosis drug-resistant, the generation of resistant organism is the basic reason that causes tuberculosis refractory, and single adjustment chemotherapy regimen effect is limited; And chemotherapy cannot solve Endogenous relapse and Exogenous reinfection low because of immunity of organisms or that defect causes.Therefore, starting new effective methods for the treatment of has become the task of top priority!

Tubercule bacillus belongs to born of the same parents' endophyte, and in body, the removing of tubercule bacillus needs inherent immunity and adaptive immunity acting in conjunction.INKT cell is one of subgroup of T cell, is the bridge of innate immunity and adaptive immune response, in the early immune reaction of tuberculosis infection, plays an important role.INKT cell can regulate the effect of scavenger cell performance tuberculosis by secrete cytokines IFN-γ, also can be by TNF secretion-α and GrB performance cytotoxicity.INKT cell also participates in granulomatous formation, suppresses the diffusion of tubercule bacillus.But, in tuberculosis mouse model, studies confirm that the multiplication capacity of iNKT cell weakens, immunocompetence declines.In active tuberculosis patient body, also find that iNKT cell quantity declines, function is suppressed.Therefore, improve in early days the function of iNKT cell at tuberculosis infection, by regulating the natural immunity and adaptive immune response, be conducive to control tuberculosis infection, may become a kind of promising alternative Immunotherapy Strategy.

The research that utilizes at present iNKT cell to carry out tuberculosis immunity treatment is carried out.INKT cell adoptive immunity is infused in tuberculosis mouse, finds in mouse lung tissue that tubercule bacillus quantity significantly reduces, pulmonary lesion obviously alleviates.Find tuberculosis mouse lung IFN-γ secretion increasing, mouse survival time significant prolongation to the activator KRN7000 that injects iNKT cell in tuberculosis mouse model.But the iNKT cell function of tubercular's body endogenous origin is impaired, amplification in vitro difficulty, has limited it in the clinical application in cellular immunization treatment field of adopting.

T cell antigen receptor (T cell receptor, TCR) is the characteristic mark of all T cell surfaces, in the identification of T cell antigen, plays a crucial role.TCR is the heterodimer being made up of α, two peptide chains of β, and every peptide chain can be divided into again variable region (V district), constant region (C district), several parts such as cross-film district and cytoplasmic region; Its cytoplasmic region is very short, and signal transmission is mainly undertaken by the CD3 molecule of being combined with non covalent bond with it.TCR molecule contactin, its antigen-specific is present in V district; Respectively there are again three hypervariable region CDR1, CDR2, CDR3 in V district (V α, V β), wherein maximum with CDR3 variation, has directly determined the antigen-binding specificity of TCR.In the time of TCR identification MHC-antigen peptide complex body, CDR1, CDR2 identification and the sidewall in conjunction with MHC molecular antigen engagement groove, and CDR3 directly combines with antigen peptide.

According to the homology of TCR V α, V β gene, more than 80 TCR V α genes can be divided into 32 families, more than 60 TCR V β gene is divided into 24 families.Utilize each T cell clone all to have the feature of its unique CDR3 sequence, adopt CDR3 spectral pattern analytical technology, can measure the frequency that each TCR each CDR3 of family occurs, reflect thus clone's property of T cell.Do not accept, in the T cell of antigenic stimulation, to be evenly distributed for the T cell clone of various antigens, show as many families and polyclone, particularly, show as approximately 8 CDR3 peaks that Gaussian distribution all appears being in each family; Antigenic stimulation causes the some of this antigen of identification or several specific TCR family t cell responses hyperplasia, show as the CDR3 member of this family and occur that the widow clone or the mono-clonal that are less than 4 peaks distribute, the TCR family wherein with mono-clonal CDR3 distribution (showing as unimodal) is the TCR family of antigen-specific mono-clonal hyperplasia.The PCR of this family product is checked order, can obtain antigen-specific TCR CDR3 sequence.

Summary of the invention

The object of the invention is to: the TCR that filters out tuberculosis 38kDa antigen-specific, utilize retroviral vector to be transfected in iNKT cell, obtain the iNKT cell of expressing tuberculosis specific TCR, and the iNKT cell that this process tcr gene is modified is in the application of preparing in anti-tuberculosis drugs.

The technical solution adopted in the present invention is:

A kind of tuberculosis antigen specific t-cell receptor (TCR), comprises α chain and β chain, and wherein, the sequence described in SEQ ID NO:3 is contained in the CDR3 district of α chain; The sequence shown in SEQ ID NO:4 is contained in the CDR3 district of β chain.

Preferably, the α chain of above-mentioned tuberculosis antigen specificity TCR is be substituted, lack and/or increased one or more amino acid and/or the special and exogenous β chain of energy that obtains is assembled into the aminoacid sequence of TCR protein molecular after end modified by the aminoacid sequence shown in SEQ ID NO:8; β chain is be substituted, lack and/or increased one or more amino acid and/or the special and exogenous α chain of energy that obtains is assembled into the aminoacid sequence of TCR protein molecular after end modified by the aminoacid sequence shown in SEQ ID NO:6.

Preferably, the aminoacid sequence of the α chain of above-mentioned tuberculosis antigen specificity TCR is as shown in SEQ ID NO:12, and the aminoacid sequence of β chain is as shown in SEQ ID NO:10.

The encode gene of above-mentioned TCR.

A fusion gene for tuberculosis antigen specificity TCR, its sequence is as shown in SEQ ID NO:13.

A kind of recombinant retroviral vector, the gene that contains aminoacid sequence shown in coding SEQ ID NO:10 and SEQ ID NO:12.

A kind of recombinant retroviral vector, contains the gene shown in SEQ ID NO:13.

Preferably, the carrier that sets out of above-mentioned recombinant retroviral vector is pMX-IRES-GFP, pMCs-IRES-GFP or pMYx-IRES-GFP.

The retrovirus that above-mentioned recombinant retroviral vector obtains after packaging.

The iNKT cell of above-mentioned Retroviral Transfer.

Tuberculosis antigen specificity TCR, its encoding gene, the iNKT cell of the recombinant retroviral vector that contains this gene, retrovirus, described Retroviral Transfer is in the application of preparing in anti-tuberculosis drugs.

The concrete steps flow process that realizes technique scheme is as follows:

1, filter out the special TCR of tuberculosis antigen

1. lymphocyte separation medium separating health volunteer peripheral blood mononuclear cell (PBMC);

2. adherent method obtains dendritic cell (dendritic cells, DC), IL-4 and GM-CSF induction DC maturation;

3. magnetic bead sorting goes out CD8 ⁺t cell;

4. 38kDa antigen load DCs is to CD8 ⁺t cell carries out three-wheel stimulation, the special CD8 of inducing antigen ⁺t cell generation clonal expansion;

5. extract CD8 ⁺t cell mRNA, reverse transcription is cDNA;

6. utilizing genescan (GeneScan) to monitor stimulates front and back CDR3 spectral pattern to change, and finds out the CD8 of the antigen-specific that stimulates rear mono-clonal amplification ⁺t cell tcr gene family.

2, build recombinant retroviral vector

1. according to people α, β gene family variable region (the variable region of GeneBank report, and constant region (constant region V), C) gene order, design α, β chain gene full length sequence primer, specific amplification TCR α, β full-length gene;

2. (minimum murinized C region, referred to as mC to adopt the mouse Yuan Hua C district that this laboratory built.Comprise mC α and mC β, wherein totally 9 amino acid by the amino acid substitution in corresponding site, mouse C district) replace respectively α, β full-length gene C district (reference literature: Luo W. et al. Development of genetically engineered CD4 ⁺and CD8 ⁺t-cells expressing TCRs specific for a 38 kDa M. tuberculosis antigen. J Mol Med. 2011,89 (9): 903-13).The object of mouse sourceization sudden change is to reduce the mispairing of interior exogenous TCR α, β chain, because there is the expression of endogenous TCR α, β gene in iNKT cell, can impel the albumen of exogenous α and beta gene expression to be correctly assembled into TCR protein molecular stably express at iNKT cell surface by sudden change, and be beneficial to its competition in conjunction with the CD3 molecule of iNKT cell surface, enhancing signal conduction function, improves the tuberculosis activity of modifying rear iNKT cell.Certainly, can also take other strategies to reduce the mispairing of interior exogenous TCR α, β chain herein, as: α, β full-length gene part C district (Sebestyen, Z. replaced with CD3 ζ chain et al.(2008) Human TCR that incorporate CD3{zeta} induce highly preferred pairing between TCR{alpha} and beta} chains following gene transfer. J. Immunol. 180,7736 – 7746); Introduce disulfide linkage (Boulter, J.M. in exogenous α, β gene C district et al. (2003) Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng. 16,707 – 711); Suddenly change exogenous α, β gene C district key amino acid to change static charge (Voss, the R.H. between α, β chain et al.(2008) Molecular design of the Cab interface favors specific pairing of introduced TCRab in human T cells. J. Immunol. 180,391 – 401); Exogenous α, β gene V district are merged into a strand TCR and merge (Willemsen, R.A. with CD3 ζ chain et al. (2000) Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR. Gene Ther. 7,1369 – 1377); Utilizing 2A to connect exogenous α, β gene realizes balance and expresses (Leisegang M, Engels B, Meyerhuber P, Kieback E, Sommermeyer D, Xue SA, Reuss S, Stauss H, Uckert W. Enhanced functionality of T cell receptor-redirected T cells is defined by the transgene cassette. J Mol Med. 2008,86:573-583.) etc.

3. utilize recombinant PCR technology, minimum mutation T CR α, β gene, by connecting from shearing polypeptide 2A, and are cloned into pGEM-T carrier order-checking qualification.

4. α correct order-checking, β full-length gene are inserted to retroviral vector pMX-IRES-GFP, enzyme is cut qualification;

5. adopt calcium phosphate transfection method, packaging retrovirus, hypothermal differential centrifugation concentrating virus;

6. recombinant retrovirus transfection NIH-3T3 cell, utilizes the expression amount of Flow Cytometry Assay virus infection NIH3T3 cell GFP, calculates recombinant retrovirus titre.Calculation formula: virus infection titre (IU/ml)=NIH3T3 total cellular score × GFP positive rate/virus concentrates liquid measure (ml).

3, the tuberculosis activity of the iNKT cell of qualification recombinant retrovirus transfection

1. adopt Ficoll density gradient centrifugation, separating health volunteer peripheral blood PBMC, IL-2 and KRN7000 amplification iNKT cell;

2. magnetic bead sorting iNKT cell;

3. retrovirus empty carrier pMX-IRES-GFP transfection iNKT cell, Flow cytometry transfection efficiency, determines optimal multiplicity of infection (multiplicity of infection, MOI);

4. with best MOI by recombinant retrovirus pMX-β-2A-α-IRES-GFP transfection iNKT cell;

5. utilize the fluorescently-labeled mouse anti human TCR monoclonal antibody of APC, Flow cytometry tuberculosis antigen specific TCR positive cell percentage;

6. carry out immunofluorescence dyeing with the fluorescently-labeled mouse anti human TCR monoclonal antibody of APC, simultaneously with PI fluorescence dye transfect cell core, under laser confocal microscope, observe the expression of iNKT cell surface tuberculosis specific TCR;

7. imitate targets than (effector:target by difference, E:T), tcr gene is modified to the DC mixed culture of iNKT cell and load 38kDa, with the negative contrast of iNKT cell of untransfected and empty carrier transfection, with chicken ovalbumin (ovalbumin, OVA) and ESAT-6(early secreted antigenic target, 6kDa) be heterogenetic antigen contrast;

8. enzyme linked immunosorbent assay analysis method (ELISA) detects the secretion level of IFN-γ, TNF-α, GrB in different time points iNKT cells and supernatant;

9. utilize time resolved fluoro-immunoassay (TRFIA) technology, detect tcr gene and modify the killing activity of iNKT cell to load 38kDa antigen DC.

Wherein, iNKT cell is a kind of cell subsets in human body, can be obtained by separation in human peripheral, and carry out amplification in vitro cultivation, and the experimental installation that separates and cultivate requires low, technology maturation.

Retroviral vector is the gene delivery vehicle being built by certain retroviral sequence, can foreign gene-carrying or DNA enter host cell, and be incorporated in chromogene group, become at present commercially produced product, easily buy and obtain.

The construction process of recombinant retroviral vector is the conventional molecule clone technology in this area, recombinant retrovirus transfection method is current conventional animal nutrition, the calcium phosphate method using in the present invention, can also use other chemical infection protocol, comprise: DEAE-dextran method, artificial liposome method; And physical method, comprising: microinjection, electroporation, particle gun etc.

beneficial effect of the present invention is:

The present invention successfully filters out the TCR of tuberculosis 38kDa antigen-specific, the iNKT cell that carries the Retroviral Transfer of this tuberculosis antigen specific TCR gene can the exogenous tcr gene of successful expression, specific recognition tuberculosis 38kDa antigen also mediates IFN-γ, TNF-α cytokine secretion and cytotoxic activity, there is the using value of tuberculosis gene therapy, can be the cellular immunization treatment of adopting lungy and open up new footpath.

Brief description of the drawings

CD8 before and after Fig. 1 tuberculosis 38kDa antigenic stimulation ⁺t cell TCR α and β chain CDR3 spectral pattern are analyzed;

Fig. 2 recombinant retroviral vector pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP builds schematic diagram;

The enzyme of Fig. 3 pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP is cut qualification (M.DL15000 marker; 1. pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP; The HindIII enzyme of 2.pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP is cut product; The EcoRI+XhoI double digestion product of 3.pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP; The XhoI+HindIII double digestion product of 4.pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP);

Expression (× 20) (a. fluorescence of NIH3T3 cell GFP after the transfection of Fig. 4 fluorescence microscope recombinant virus; B. light field; C. stacking diagram)

GFP the positive expression rate (a. untransfected of NIH3T3 cell after the transfection of Fig. 5 Flow cytometry recombinant virus; B. pMX-IRES-GFP; C. pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP)

Fig. 6 Flow cytometry iNKT cell (left figure: homotype control antibodies IgG1-PE mark; Right figure: anti-human V α 24-PE antibody labeling);

The expression of iNKT cell GFP after the transfection of Fig. 7 Flow cytometry recombinant virus;

Under Fig. 8 laser confocal microscope, observe expression (the left figure: homotype control antibodies IgG1-APC mark of iNKT cell surface tuberculosis specific TCR; Right figure: anti-human TCR-V β 5-APC monoclonal antibody mark).

Fig. 9 Flow cytometry is expressed positive cell rate (the left figure: homotype control antibodies IgG1-APC mark of tuberculosis antigen specific TCR; Right figure: anti-human TCR-V β 5-APC antibody labeling);

The different effect of Figure 10 ELISA detection targets compare the secretion level with time point iNKT cell IFN-γ;

The different effect of Figure 11 ELISA detection targets compare the secretion level with time point iNKT cell TNF-α;

The different effect of Figure 12 ELISA detection targets compare the secretion level with time point iNKT cell GrB;

Figure 13 TRFIA detect different effect targets than time iNKT cell killing activity.

Embodiment

Below in conjunction with embodiment, the present invention is further illustrated, but be not limited to this.

The experimental technique of unreceipted actual conditions in following examples, operate according to normal condition, " molecular cloning experiment guide " (third edition) (Sambrook J that such as Sambrook etc. writes, Russell DW, Janssen K, the yellow training hall of Argentine J. waits to be translated, and 2002, Beijing: Science Press) described in condition, or the condition of advising according to manufacturer.

In following examples, all measurement data results represent with ± s, adopt the difference of cytokine IFN-γ, TNF-α secretion level between one-way analysis of variance (One-Way ANOVA) more each group, when heterogeneity of variance, proofread and correct with Welch, adopt LSD method to carry out comparing between two between each group, when heterogeneity of variance, adopt Dunnett ' s T3 method to proofread and correct.Inspection level α=0.05, two-tailed test.Adopt SPSS17.0 for windows statistical package to carry out data analysis.

Embodiment

1. filter out the TCR of tuberculosis 38kDa antigen-specific

1.1 density gradient centrifugation separation and purification PBMC

(1) add appropriate Ficoll lymphocyte separation medium at the aseptic centrifuge tube of 15ml scale;

(2) peripheric venous blood of taking heparin anti-freezing and equivalent RPMI 1640 liquid fully mix dilution, and the anticoagulation of drawing 2 times of volumes with pasteur dropper is slowly superimposed on lymphocyte separation medium along tube wall, note keeping interface complete.18 ~ 20 ° of C, 1800 ~ 2000rpm/min horizontal centrifugal, 20 ~ 30min;

(3) centrifugal rear liquid in pipe is divided into four layers, and upper strata is blood plasma and diluent, and the pipe end is mainly red corpuscle and GCL.Middle level is lymphocyte separation medium, has one taking mononuclearcell as main canescence cloud and mist layer in upper, interface, middle level;

(4) be inserted into the canescence cellular layer on lymphocyte separation medium interface with suction pipe, draw mononuclearcell, be placed in another centrifuge tube, add 5 times of RPMI 1640 liquid with upper volume, 18 ~ 20 ° of C, the centrifugal 10min of 1500rpm/min, washed cell is PBMC after removing the thrombocyte that major part mixes for twice;

(5) cell counting and cell viability detect: PBMC suspension mixes with the blue dye liquor of 0.4 % platform phenol of 1/10 volume, and four total cell count of large grid on angle on tally on blood counting chamber always 1/4th of cell count are severally multiplied by 10 ⁴be every ml concn; Dead cell pigmentable trypan blue, that lives is not painted, counts 200 lymphocytes, calculating viable cell percentage living cell rate %=(viable count/total cell count) × 100%.

1.2 DC induction and load mycobacterium tuberculosis 38kDa antigens

(1) PBMC cultivates based on 37 ° of C, 5% CO with 10% FBS-RPMI 1640 ₂in incubator, cultivate 1.5h;

(2) sucking-off supernatant PBMC changes hole cultivation, adds the 10% FBS-1640 substratum containing IL-2 100U/ml, anti-CD3 monoclonal antibody 30ng/ml, for subsequent use;

(3) pre-temperature substratum rinsing twice gently for DC, adds the 10% FBS-1640 substratum containing GM-CSF 100ng/ml, IL-4 100ng/ml;

(4) the 2nd, 4,6 days, DC culture is carried out to half amount and change liquid;

(5) the 7th days, abandon supernatant, change with the 10% FBS-1640 substratum 5ml containing TNF-α 20ng/ml, 38kDa 10 μ g/ml;

1. 3 stimulate and mycobacterium tuberculosis 38kDa antigen-specific T cell clone

(1) DC cultivates the 8th day, counts respectively DC and the PBMC of load 38kDa, by both mixed culture, adds 10ng/ml IL-7 by 1:10;

(2) after mixed culture the 3rd, 6 days, half amount was changed liquid, and adds 50U/ml IL-2, continued to cultivate;

(3) take turns taking 5 days as one, repeat above-mentioned steps, carry out 3 and take turns stimulation.

1.4 immunomagnetic beadses (German Mei Tian Ni biotech firm) sorting CD4 ⁺, CD8 ⁺t cell.

1. 5 total RNA extraction reagent boxes (OMEGA) extract total RNA of the above-mentioned cell precipitation of collecting.

1.6 reverse transcriptions (RT) test kits (Fermentas) synthesize cDNA.

1.7 34 of pcr amplifications TCR V α gene family CDR3 fragment (reference literatures: XIN-SHENG etc., 2006, Clinical & Laboratory Haematology, 28:405 – 415. doi:10.1111/j.1365-2257.2006.00827.x)

Utilize that 34 TCR V α family specificity upstream primers and shared downstream C α are outer, inner side primer does heminested PCR:

First round PCR: every sample does 34 PCR reaction tubess, 1st ~ 34 pipes add respectively TCR V α 1 to V α 34 family's upstream primers, every pipe to add downstream to share primer 1 μ l outside C α, and each primer concentration is 10 μ M.Every PCR reaction tubes volume is 25 μ l, containing cDNA template 1.0 μ l, and 10mmol/L dNTP 0.5 μ l, 10 × Buffer, 2.5 μ l, 25mmol/L MgCl ₂1.5 μ l, Taq archaeal dna polymerase 0.625U.PCR reaction conditions: 95 ° of C denaturation 3min; 95 ° of C 30s, 60 ° of C 30s, 72 ° of C 1min, 35 circulations; 72 ° of C extend 10min.

Second takes turns PCR: reaction cumulative volume is 25 μ l, containing first round PCR product 2 μ l, and 10mmol/L dNTP 0.5 μ l, 10 × Buffer, 2.5 μ l, 25mmol/L MgCl ₂1.5 μ l, Taq archaeal dna polymerase 0.625U, 34 family's upstream primers of TCR V α, 1 μ l, C α primer 1 μ l inside the FAM mark of downstream, each primer concentration is 10 μ M.PCR reaction conditions: 95 ° of C 2min; 60 ° of C 2min, 72 ° of C 10min, 4 circulations.

1. 24 TCR V β gene family CDR3 fragment (reference literatures: XIN-SHENG etc. of 8 pcr amplification, 2006, Clinical & Laboratory Haematology, 28:405 – 415. doi:10.1111/j.1365-2257.2006.00827.x):

Every sample does 24 PCR reaction tubess, and every pipe adds TCR C β-FAM downstream primer 0.8 μ l, and the 1st to the 24th pipe adds respectively TCR V β 1 to TCR V β 24 upstream primer 0.8 μ l, and each primer concentration is 10 μ M.PCR reaction volume is 25 μ l, containing cDNA template 1 μ l, and 10mmol/L dNTP 0.5 μ l, 10 × Buffer, 2.5 μ l, 25mmol/L MgCl ₂1.5 μ l, Taq archaeal dna polymerase 0.625U.PCR reaction conditions: 94 ° of C sex change 3min; 94 ° of C 1min, 55 ° of C 1min, 72 ° of C 1min, 35 circulations; 72 ° of C extend 10min.

1. 9 agarose gel electrophoresis

Get 34 TCR V α and 24 each 8 μ l of TCR V β gene family PCR product, 2% agarose gel electrophoresis, 100V, 20min, adopts gel imaging system to take a picture.A residue PCR product-20 ° C saves backup.

1. 10 CDR3 spectral patterns are analyzed

Get 34 V α, 24 V β gene family FAM fluorescent mark PCR product 2 μ l, at 373DNA sequenator (ABI, Perkin Elmer) on carry out 6% polyacrylamide gel electrophoresis, collect the fluorescent signal of the varying strength that in electrophoresis process, different time occurs, the data that GeneScan 672 software automatic analysis are collected, be converted to the peak of different positions, height and form, represent the frequency that each TCR CDR3 member of family occurs, reflect thus clone's property of each TCR family.Wherein, the TCR family that has a unimodal distribution is the TCR family of antigen-specific mono-clonal hyperplasia.

CDR3 spectral pattern analytical results shows, mycobacterium tuberculosis 38kDa antigenic stimulation CD8 ⁺after T cell, part tcr gene family spectral pattern changes, and from original 8 or become more than the Gaussian distribution of 8 peak types the few peak of list that is less than 8 peaks and distribute, shows that these families are because 38kDa antigen continues to stimulate the widow clone or the mono-clonal hyperplasia that cause.The variation of CDR3 spectral pattern before and after comparison stimulus, finding out stimulation front is polyclone, is V α 9, the V β 5 gene family (see figure 1)s of mono-clonal amplification after stimulation.

Sequencing result demonstration, the CDR3 sequence of TCR α 9, β 5 genes is respectively as shown in SEQ ID NO:1 and SEQ ID NO:2, and the aminoacid sequence of these two CDR3 sequence encodings is respectively as shown in SEQ ID NO:3 and SEQ ID NO:4.

2. build recombinant retroviral vector (build flow process and see Fig. 2)

2.1 synthetic primer

According to V α 9, the V β 5 gene family V region sequence features of GeneBank report, design full-length gene upstream and downstream primer, design respectively upstream and downstream primer according to the sequence (being mC α and mC β) after the key amino acid sudden change of 9, people C district, according to 2A peptide catenation sequence design primer, all primer is synthetic by Invitrogen Shanghai Ying Jun Bioisystech Co., Ltd, and (5 ' to 3 ') is as follows for primer title and sequence:

2.2 recombinant PCR amplification hV β 5mC β-2A-hV α 9mC α merge full-length gene

(1) cDNA preparing taking step 1.6 is template, utilize primer P1 and P9, Pfu archaeal dna polymerase, pcr amplification hV β 5hC β full-length gene order (nucleotide sequence is as shown in SEQ ID NO:5, and its coded aminoacid sequence is as shown in SEQ ID NO:6).

(2) as template, utilize primer P1 and P2 taking hV β 5hC β full-length gene order (SEQ ID NO:5), Pfu archaeal dna polymerase, the sequence (S1:hV β 5th district) on pcr amplification hV β 5th district and mC β before mutational site.

(3) with the plasmid pMX-mmTCR β 8-P2A-mmTCR α 3-IRES-GFP in the mouse Yuan Hua C district (mC) that built containing this laboratory, (concrete sequence and construction process are referring to document: Luo W.et al. J Mol Med. 2011,89 (9): 903-13) be template, utilize primer P3 and P4, Pfu archaeal dna polymerase, pcr amplification mCβ district and 5 ' end P2A sequence (S2:mC β-5 ' DuanP2A district).

(4) cDNA preparing taking step 1.6 is template, utilize primer P5 and P10, Pfu archaeal dna polymerase, pcr amplification hV α 9hC α full-length gene order (nucleotide sequence is as shown in SEQ ID NO:7, and its coded aminoacid sequence is as shown in SEQ ID NO:8).

(5) as template, utilize primer P5 and P6 taking hV α 9hC α full-length gene order (SEQ ID NO:7), Pfu archaeal dna polymerase, pcr amplification is containing the sequence of 3 ' end P2A, hV α 9th district and 5 ' end mC α (S3:3 ' end P2A-hV α 9th district).

(6) taking the plasmid pMX-mmTCR β 8-P2A-mmTCR α 3-IRES-GFP in the mouse Yuan Hua C district (mC) that built containing this laboratory as template, utilize primer P7 and P8, Pfu archaeal dna polymerase, pcr amplification mCα district (S4:mC α district).

(7) S2 that the S1 obtaining using step (2) and step (3) obtain is as template, utilize primer P1 and P4, Pfu archaeal dna polymerase, wherein the sequence of hV β 5mC β section is as shown in SEQ ID NO:9 for recombinant PCR amplification hV β 5mC β-5 ' end P2A sequence S5(, and its coded aminoacid sequence is as shown in SEQ ID NO:10).

(8) S4 that the S3 obtaining using step (5) and step (6) obtain is as template, utilize primer P5 and P8, Pfu archaeal dna polymerase, wherein the sequence of hV α 9mC α section is as shown in SEQ ID NO:11 for recombinant PCR amplification 3 ' end 2A-hV α 9mC α sequence S6(, and the aminoacid sequence of its coding is as shown in SEQ ID NO:12).

(9) S6 that the S5 obtaining using step (7) and step (8) obtain is as template, utilize primer P1 and P8, Pfu archaeal dna polymerase, pcr amplification hV β 5mC β-2A-hV α 9mC alpha fusion gene full length sequence (SEQ ID NO:13).

Above conventional PCR reaction system 25 μ l, containing 10 × Buffer, 2.5 μ l, 10mmol/L dNTP 0.5 μ l, Pfu archaeal dna polymerase 0.2U, the each 0.8 μ l of 10 μ M primer, template DNA 1 μ l.PCR reaction conditions: 95 ° of C sex change 2min; 95 ° of C 1min, 72 ° of C 2min, 35 circulations; 72 ° of C extend 10min.

Recombinant PCR reaction system 25 μ l, containing 10 × Buffer, 2.5 μ l, 10mmol/L dNTP 0.5 μ l, Pfu archaeal dna polymerase 0.2U, 10 μ M primer P5, the each 0.8 μ l of P8, two kinds of each 1.5 μ l of PCR product template.PCR reaction conditions: 94 ° of C sex change 2min; 94 ° of C 30s, 52 ° of C 45s, 72 ° of C 1min, 3 circulations; 94 ° of C 1min, 72 ° of C 2min, 32 circulations; 72 ° of C extend 10min.

2.3 build the cloning vector containing hV β 5mC β-2A-hV α 9mC alpha fusion gene

(1) reclaim test kit (Omega) with glue and reclaim hV β 5mC β-2A-hV α 9mC alpha fusion gene fragment;

(2) add A with DNA A-Tailing test kit (TaKaRa) at said gene fragment end;

(3) by hV β 5mC β-2A-hV α 9mC alpha fusion gene fragment access pGEM-T carrier, ligation system 10ml:pGEM-T carrier 1ml, 10 ' ligation Buffer 1ml, T4 DNA ligase enzyme 1ml, 0.2pmol have added the PCR product of A purifying, and 16 ° of C connections are spent the night.

(4) ordinary method will connect correct plasmid Transformed E .coli DH5 α competence bacteria.Then bacterium is coated on the penbritin flat board of 4ml 200mg/ml IPTG, 40ml 20 mg/ml X-gal; Overnight incubation, the bacterium colony of recombinant plasmid transformed is for white, and the bacterium colony that empty plasmid transforms is blue.Select dull and stereotyped upper white colony, forward to 3ml Amp is housed ⁺in the test tube of LB nutrient solution, 37 ° of C, 160rpm jolting 12-16h.

(5) extract plasmid with plasmid extraction test kit (Omega), the positive recombinant plasmid of primary dcreening operation is identified with corresponding restriction enzyme, and taking recombinant plasmid as template, carried out pcr amplification, agarose gel electrophoresis is identified clip size.Finally send Invitrogen Shanghai Ying Jun Bioisystech Co., Ltd to check order primary dcreening operation positive plasmid.Sequencing result shows, the contained exogenous gene sequence of this recombinant plasmid and forecasting sequence are in full accord.

The structure of 2.4 recombinant retroviral vectors

(1) cut T carrier and the pMX-IRES-GFP empty plasmid containing hV β 5mC β-2A-hV α 9mC alpha fusion gene with restriction enzyme EcoR I, Xho I enzyme, glue reclaims hV β 5mC β-2A-hV α 9mC alpha fusion gene fragment and vector gene fragment (method is with step 2.3) respectively;

(2) hV β 5mC β-2A-hV α 9mC alpha fusion gene is connected into after pMX-IRES-GFP carrier (method is with step 2.3), transformed competence colibacillus bacterium XL-10, coat containing the solid medium of penbritin and cultivate after 12-16h, select single bacterium colony, shake bacterium and spend the night;

(3) extracting plasmid, enzyme is cut qualification result and is shown, and gene fragment is inserted correct (see figure 3);

(4) select positive bacteria to drop into row amplification cultivation, a large amount of extracting plasmid DNA.

The packaging of 2.5 retrovirus recombinant vectorss

Recombinant plasmid mixes with the ratio of 1:1 with envelope protein plasmid VSV-G, adopts calcium phosphate transfection method transfection GP2-293 cell with packaging retrovirus, and calcium phosphate method cell transfecting test kit (the green skies) description operation is pressed in experiment.

2.6 recombinant retrovirus concentrated and purified

(1) collect viral supernatant, 10000g, 4 ° of centrifugal 10min of C;

(2) reclaim viral supernatant, 50000g, 4 ° of centrifugal 2h of C;

(3) TNE of 1%-3% original volume is resuspended, after virus is dissolved completely, and packing ,-80 ° of C storages; ;

2.7 Flow Cytometry Assay virus titers

(1) in advance by NIH3T3 cell (2 × 10 ⁵/ hole) inoculation culture 24h;

(2) add polybrene (PB) to final concentration 8mg/L, add the viral supernatant of 10 μ l titre to be measured;

(3) infect after 24h, change fresh medium, remove pseudovirion;

(4) 37 ° of C continue to cultivate after 3d, observe the expression of green fluorescent protein (GFP) under inverted fluorescence microscope;

(5) 37 ° of C continue to cultivate after 5d, and trysinization, PBS wash after 3 times, resuspended with 200 ~ 300 μ l PBS, and preparation density is 1 ~ 5 × 10 ⁶the single cell suspension of/ml, flow cytometer detects its GFP the positive expression rate, calculates virus titer by following formula: virus titer (GFU/ml)=NIH3T3 cell count × positive rate/transfection virus supernatant amount (ml).

Fluorescence microscopy Microscopic observation, the NIH3T3 cell expressing green fluorescence of pMX-hV β 5mC β-2A-hV α 9mC α-IRES-GFP retroviral infection, shows the expression (Fig. 4) of GFP in cell.Through Flow cytometry, the titre that calculates recombinant virus is 1.97 × 10 ⁷iU/ml(Fig. 5).

3. the tuberculosis activity of the iNKT cell of qualification recombinant retrovirus transfection

The cultivation of 3.1 iNKT cells and sorting

(1) separate with counting PBMC(method with step 1.1);

(2) (taking 24 orifice plates as example) every hole paving 2 × 10 ⁶individual PBMC, containing RPMI 1640 complete culture solutions of 10% FBS at 37 ° of C, 5% CO ₂under condition, cultivate;

(3) added respectively IL-2 10U/ml, KRN7000 100ng/ml at 1,3,5 day;

(4) by frozen remaining PBMC, for the amplification of 7-14 days iNKT;

(5) the 8th days, frozen PBMC is taken out, in the iNKT cell hole of having bred, add 2 × 10 ⁶individual PBMC, continues to cultivate 7 days;

(6) the 15th days, with V α 24 ⁺tCR immunological magnetic bead sorting iNKT cell (method is with step 1.4);

(7) purity >95%(Fig. 6 of Flow cytometry iNKT cell).

3.2 measure the suitableeest MOI of recombinant virus infection iNKT cell

(1) by iNKT cell infection the day before yesterday with 5 × 10 ⁵individual/hole is inoculated in 6 orifice plates;

(2) the old liquid of cell cultures was abandoned in transfection the same day, was 2,4,5,6,8,10,12 to add viral stock solution by MOI, and adding PB is 8mg/L to final concentration, and 37 ° of C cultivate 4h;

(3) add substratum, dilution PB to 2mg/L, continues to cultivate 5 days;

(4) centrifugal collecting cell, PBS washing 2 times, 2% paraformaldehyde is fixed;

(5) flow cytometry analysis iNKT cell GFP the positive expression rate is 36.7%, determines the suitableeest MOI=8(Fig. 7).

3.3 immunofluorescences detect the expression of genetic modification iNKT cell external source TCR

(1) recombinant virus is pressed to the conventional iNKT of infection of MOI=8 cell;

Centrifugal collecting cell after (2) 5 days, PBS washing 2 times;

(3) cell is resuspended with PBS, adds fluorescein-labeled mouse anti human TCR-V β 5 monoclonal antibodies of APC, and 4 DEG C of lucifuges are hatched 30 min;

(4) centrifugal collecting cell, PBS washing 2 times, 2% paraformaldehyde is fixed;

(5) carry out immunofluorescence dyeing with the homotype control antibodies IgG1-APC of APC mark and mouse anti human TCR-V β 5 monoclonal antibodies of APC mark respectively, with PI fluorescence dye transfect cell core, under laser confocal microscope, observe the expression (Fig. 8) of iNKT cell surface tuberculosis specific TCR simultaneously.INKT cell surface after visible modification is significantly expressed external source TCR albumen;

(6) respectively with homotype control antibodies IgG1-APC and people TCR-V β 5-APC antibody labeling, Flow cytometry, result shows, the positive cell rate of expressing tuberculosis antigen specific TCR is 60.9%(Fig. 9).

3.4 ELISA test kits (Wuhan Boster Biological Technology Co., Ltd.) are measured IFN-γ, TNF-α, the GrB secretion level of iNKT cell

Experiment contrast group arranges: 1. untransfected group (DC that untransfected iNKT cell+38kDa antigen impacts); 2. empty carrier transfection group (DC that empty carrier transfection iNKT cell+38kDa antigen impacts); 3. irrelevant antigen group (tcr gene is modified the DC that iNKT cell+OVA impacts); 4. related antigen group (tcr gene is modified the DC that iNKT cell+ESAT-6 impacts) and 5. tcr gene modification group (tcr gene is modified the DC that iNKT cell+tuberculosis 38kDa antigen impacts).Following experiment contrast arranges all herewith.Experiment repeats 3 times, and method is as follows:

(1) with 5 × 10 ³the DC of individual/hole inoculation load 38kDa antigen is in 96 orifice plates, respectively according to certain E:T value (E:T=1,3,5,7,10; 15,20,25,30) add antigen-specific tcr gene to modify iNKT cell, carry out common cultivation with DC, establish two multiple holes for every group;

(2) cultivate altogether 6,12,18,24,30, after 36h, collect each hole culture supernatant, operate by test kit specification sheets.

ELISA result shows:

1. imitating target than (E:T)=7, during with the DC mixed culture 18h of load tuberculosis 38kDa antigen, the iNKT cell IFN-γ secretion level that 38kDa antigen-specific tcr gene is modified reaches maximum 2225.954 ± 53.655pg/ml, the iNKT cell that is significantly higher than untransfected, the iNKT cell that dyes of idle running and modifies with the tcr gene that DC of load tuberculosis antigen or other non-specific antigen of load does not cultivate altogether ( p<0.001), see Figure 10;

2. at E:T=20, during with the DC mixed culture 24h of load tuberculosis 38kDa antigen, the iNKT cell TNF-α secretion level that 38kDa antigen-specific tcr gene is modified reaches maximum 1299.701 ± 13.183pg/ml, the iNKT cell that is significantly higher than untransfected, the iNKT cell that dyes of idle running and modifies with the tcr gene that DC of load tuberculosis antigen or other non-specific antigen of load does not cultivate altogether ( p<0.001), see Figure 11.

3. at E:T=20, during with the DC mixed culture 24h of load tuberculosis 38kDa antigen, 38kDa antigen-specific tcr gene modification group GrB secretory volume (11.364 ± 0.031pg/ml) is significantly higher than untransfected, iNKT cell that idle running is dyed and the iNKT cell modified with the tcr gene that DC of load tuberculosis antigen or other non-specific antigen of load does not cultivate altogether ( p<0.001), see Figure 12.

3.5 time resolved fluoro-immunoassay test kits (PerkinElmer) are measured the killing activity of iNKT cell.

Temporal resolution immunofluorescence detected result shows: at E:T=30, during with the DC mixed culture 4h of load tuberculosis 38kDa antigen, the iNKT cell killing activity that tcr gene is modified is the highest, reach 84.20%, be significantly higher than untransfected, the iNKT cell that dyes of idle running and the level of killing and wounding of the iNKT cell modified with the tcr gene that DC of load tuberculosis antigen or other non-specific antigen of load does not cultivate altogether ( p<0.001), see Figure 13.

Above-mentioned experimental result shows: the iNKT cell that carries the Retroviral Transfer of tuberculosis antigen specific TCR gene can the exogenous tcr gene of successful expression, specific recognition tuberculosis 38kDa antigen also mediates IFN-γ, TNF-α cytokine secretion and cytotoxic activity, there is the using value of tuberculosis gene therapy, can be the cellular immunization treatment of adopting lungy and open up new footpath.

<110> Nanfang Medical Univ

<120> tuberculosis antigen specificity TCR, its recombinant retroviral vector and application

<160> 23

<170> PatentIn version 3

<210> 1

<211> 63

<212> DNA

<213> Human

<400> 1

gcccgaggag gaaacacacc tcttgtcttt ggaaagggca caagactttc tgtgattgca 60

aat 63

<210> 2

<211> 72

<212> DNA

<213> Human

<400> 2

gcgcctgaca ccggctcagg agctttcttt ggacaaggca ccagactcac agttgtagag 60

gacctgaaca ag 72

<210> 3

<211> 21

<212> PRT

<213> Human

<400> 3

Ala Arg Gly Gly Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu

1 5 10 15

Ser Val Ile Ala Asn

20

<210> 4

<211> 24

<212> PRT

<213> Human

<400> 4

Ala Pro Asp Thr Gly Ser Gly Ala Phe Phe Gly Gln Gly Thr Arg Leu

1 5 10 15

Thr Val Val Glu Asp Leu Asn Lys

20

<210> 5

<211> 927

<212> DNA

<213> Human

<400> 5

atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60

gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120

agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180

ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240

ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300

gagctggggg actcggccct ttatctttgc gccagcagcg cgcctgacac cggctcagga 360

gctttctttg gacaaggcac cagactcaca gttgtagagg acctgaacaa ggtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttcttcccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gctttacctc ggtgtcctac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 900

atggccatgg tcaagagaaa ggatttc 927

<210> 6

<211> 308

<212> PRT

<213> Human

<400> 6

Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala Gly

1 5 10 15

Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys Thr

20 25 30

Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His Arg

35 40 45

Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe Leu

50 55 60

Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro Gly

65 70 75 80

Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn Val

85 90 95

Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser

100 105 110

Ala Pro Asp Thr Gly Ser Gly Ala Phe Phe Gly Gln Gly Thr Arg Leu

115 120 125

Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val

130 135 140

Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu

145 150 155 160

Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp

165 170 175

Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln

180 185 190

Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser

195 200 205

Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His

210 215 220

Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp

225 230 235 240

Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala

245 250 255

Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly

260 265 270

Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr

275 280 285

Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys

290 295 300

Arg Lys Asp Phe

305

<210> 7

<211> 810

<212> DNA

<213> Human

<400> 7

atgtggggag ctttccttct ctatgtttcc atgaagatgg gaggcactgc aggacaaagc 60

cttgagcagc cctctgaagt gacagctgtg gaaggagcca ttgtccagat aaactgcacg 120

taccagacat ctgggtttta tgggctgtcc tggtaccagc aacatgatgg cggagcaccc 180

acatttcttt cttacaatgc tctggatggt ttggaggaga caggtcgttt ttcttcattc 240

cttagtcgct ctgatagtta tggttacctc cttctacagg agctccagat gaaagactct 300

gcctcttact tctgcgctgt gagagcccga ggaggaaaca cacctcttgt ctttggaaag 360

ggcacaagac tttctgtgat tgcaaatatc cagaaccctg accctgccgt gtaccagctg 420

agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca 480

aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg 540

aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca 600

tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagcccagaa 660

agttcctgtg atgtcaagct ggtcgagaaa agctttgaaa cagatacgaa cctaaacttt 720

caaaacctgt cagtgattgg gttccgaatc ctcctcctga aagtggccgg gtttaatctg 780

ctcatgacgc tgcggctgtg gtccagctga 810

<210> 8

<211> 268

<212> PRT

<213> Human

<400> 8

Trp Gly Ala Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr Ala

1 5 10 15

Gly Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly Ala

20 25 30

Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly Leu

35 40 45

Ser Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser Tyr

50 55 60

Asn Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe Leu

65 70 75 80

Ser Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln Met

85 90 95

Lys Asp Ser Ala Ser Tyr Phe Cys Ala Val Arg Ala Arg Gly Gly Asn

100 105 110

Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala Asn

115 120 125

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser

130 135 140

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn

145 150 155 160

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val

165 170 175

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp

180 185 190

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

195 200 205

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val

210 215 220

Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln

225 230 235 240

Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly

245 250 255

Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 9

<211> 927

<212> DNA

<213> artificial sequence

<400> 9

atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60

gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120

agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180

ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240

ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300

gagctggggg actcggccct ttatctttgc gccagcagcg cgcctgacac cggctcagga 360

gctttctttg gacaaggcac cagactcaca gttgtagagg acctgaacaa ggtgttccca 420

cccgaggtcg ctgtgtttga gccatcaaaa gcagagatcg cacacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttcttcccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gcattacctc ggcatcctac caccaagggg tcctgtctgc caccatcctc 840

tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 900

atggccatgg tcaagagaaa ggatttc 927

<210> 10

<211> 308

<212> PRT

<213> artificial sequence

<400> 10

Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala Gly

1 5 10 15

Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys Thr

20 25 30

Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His Arg

35 40 45

Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe Leu

50 55 60

Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro Gly

65 70 75 80

Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn Val

85 90 95

Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser

100 105 110

Ala Pro Asp Thr Gly Ser Gly Ala Phe Phe Gly Gln Gly Thr Arg Leu

115 120 125

Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val

130 135 140

Phe Glu Pro Ser Lys Ala Glu Ile Ala His Thr Gln Lys Ala Thr Leu

145 150 155 160

Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp

165 170 175

Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln

180 185 190

Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser

195 200 205

Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His

210 215 220

Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp

225 230 235 240

Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala

245 250 255

Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr His Gln Gly

260 265 270

Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr

275 280 285

Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys

290 295 300

Arg Lys Asp Phe

305

<210> 11

<211> 810

<212> DNA

<213> artificial sequence

<400> 11

atgtggggag ctttccttct ctatgtttcc atgaagatgg gaggcactgc aggacaaagc 60

cttgagcagc cctctgaagt gacagctgtg gaaggagcca ttgtccagat aaactgcacg 120

taccagacat ctgggtttta tgggctgtcc tggtaccagc aacatgatgg cggagcaccc 180

acatttcttt cttacaatgc tctggatggt ttggaggaga caggtcgttt ttcttcattc 240

cttagtcgct ctgatagtta tggttacctc cttctacagg agctccagat gaaagactct 300

gcctcttact tctgcgctgt gagagcccga ggaggaaaca cacctcttgt ctttggaaag 360

ggcacaagac tttctgtgat tgcaaatatc cagaaccctg accctgccgt gtaccagctg 420

agagactcta aatccagtga caagtctgtc tgcctattca ccgattttga ttctcaaaca 480

aatgtgtcac aaagtaagga ttctgatgtg tatatcacag acaaaactgt gctagacatg 540

aggtctatgg acttcaagag caacagtgct gtggcctgga gcaacaaatc tgactttgca 600

tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca ccttcttccc cagctcagac 660

gttccctgtg atgtcaagct ggtcgagaaa agctttgaaa cagatacgaa cctaaacttt 720

caaaacctgt cagtgattgg gttccgaatc ctcctcctga aagtggccgg gtttaatctg 780

ctcatgacgc tgcggctgtg gtccagctga 810

<210> 12

<211> 268

<212> PRT

<213> artificial sequence

<400> 12

Trp Gly Ala Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr Ala

1 5 10 15

Gly Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly Ala

20 25 30

Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly Leu

35 40 45

Ser Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser Tyr

50 55 60

Asn Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe Leu

65 70 75 80

Ser Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln Met

85 90 95

Lys Asp Ser Ala Ser Tyr Phe Cys Ala Val Arg Ala Arg Gly Gly Asn

100 105 110

Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala Asn

115 120 125

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser

130 135 140

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn

145 150 155 160

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val

165 170 175

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp

180 185 190

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile

195 200 205

Ile Pro Glu Asp Thr Phe Phe Pro Ser Ser Asp Val Pro Cys Asp Val

210 215 220

Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln

225 230 235 240

Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly

245 250 255

Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 13

<211> 1803

<212> DNA

<213> artificial sequence

<400> 13

atgggctcca ggctgctctg ttgggtgctg ctttgtctcc tgggagcagg cccagtaaag 60

gctggagtca ctcaaactcc aagatatctg atcaaaacga gaggacagca agtgacactg 120

agctgctccc ctatctctgg gcataggagt gtatcctggt accaacagac cccaggacag 180

ggccttcagt tcctctttga atacttcagt gagacacaga gaaacaaagg aaacttccct 240

ggtcgattct cagggcgcca gttctctaac tctcgctctg agatgaatgt gagcaccttg 300

gagctggggg actcggccct ttatctttgc gccagcagcg cgcctgacac cggctcagga 360

gctttctttg gacaaggcac cagactcaca gttgtagagg acctgaacaa ggtgttccca 420

cccgaggtcg ctgtgtttga gccatcaaaa gcagagatcg cacacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttcttcccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gcattacctc ggcatcctac caccaagggg tcctgtctgc caccatcctc 840

tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 900

atggccatgg tcaagagaaa ggatttcggc tccggagcca cgaacttctc tctgttaaag 960

caagcaggag acgtggaaga aaaccccggt cccatgtggg gagctttcct tctctatgtt 1020

tccatgaaga tgggaggcac tgcaggacaa agccttgagc agccctctga agtgacagct 1080

gtggaaggag ccattgtcca gataaactgc acgtaccaga catctgggtt ttatgggctg 1140

tcctggtacc agcaacatga tggcggagca cccacatttc tttcttacaa tgctctggat 1200

ggtttggagg agacaggtcg tttttcttca ttccttagtc gctctgatag ttatggttac 1260

ctccttctac aggagctcca gatgaaagac tctgcctctt acttctgcgc tgtgagagcc 1320

cgaggaggaa acacacctct tgtctttgga aagggcacaa gactttctgt gattgcaaat 1380

atccagaacc ctgaccctgc cgtgtaccag ctgagagact ctaaatccag tgacaagtct 1440

gtctgcctat tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat 1500

gtgtatatca cagacaaaac tgtgctagac atgaggtcta tggacttcaa gagcaacagt 1560

gctgtggcct ggagcaacaa atctgacttt gcatgtgcaa acgccttcaa caacagcatt 1620

attccagaag acaccttctt ccccagctca gacgttccct gtgatgtcaa gctggtcgag 1680

aaaagctttg aaacagatac gaacctaaac tttcaaaacc tgtcagtgat tgggttccga 1740

atcctcctcc tgaaagtggc cgggtttaat ctgctcatga cgctgcggct gtggtccagc 1800

tga 1803

<210> 14

<211> 44

<212> DNA

<213> artificial sequence

<400> 14

ccggaattca tgggctccag gctgctctgt tgggtgctgc tttg 44

<210> 15

<211> 40

<212> DNA

<213> artificial sequence

<400> 15

cttgttcagg tcctctacaa ctgtgagtct ggtgccttgt 40

<210> 16

<211> 40

<212> DNA

<213> artificial sequence

<400> 16

agactcacag ttgtagagga cctgaacaag gtgttcccac 40

<210> 17

<211> 79

<212> DNA

<213> artificial sequence

<400> 17

cttccacgtc tcctgcttgc tttaacagag agaagttcgt ggctccggag ccgaaatcct 60

ttctcttgac catggccat 79

<210> 18

<211> 79

<212> DNA

<213> artificial sequence

<400> 18

cgaacttctc tctgttaaag caagcaggag acgtggaaga aaaccccggt cccatgtggg 60

gagctttcct tctctatgt 79

<210> 19

<211> 40

<212> DNA

<213> artificial sequence

<400> 19

gtcagggttc tggatatttg caatcacaga aagtcttgtg 40

<210> 20

<211> 40

<212> DNA

<213> artificial sequence

<400> 20

tctgtgattg caaatatcca gaaccctgac cctgccgtgt 40

<210> 21

<211> 42

<212> DNA

<213> artificial sequence

<400> 21

ccgctcgagt cagctggacc acagccgcag cgtcatgagc ag 42

<210> 22

<211> 45

<212> DNA

<213> artificial sequence

<400> 22

ccgctcgagt cagaaatcct ttctcttgac catggccatc aacac 45

<210> 23

<211> 43

<212> DNA

<213> artificial sequence

<400> 23

catgccatgg gctggaccac agccgcagcg tcatgagcag att 43

Claims (7)

1. a tuberculosis antigen specific t-cell receptor, comprises α chain and β chain, it is characterized in that, the aminoacid sequence of described α chain is as shown in SEQ ID NO:12, and the aminoacid sequence of β chain is as shown in SEQ ID NO:10.

2. the gene of tuberculosis antigen specific t-cell receptor described in coding claim 1.

3. a fusion gene for tuberculosis antigen specific t-cell receptor, its sequence is as shown in SEQ ID NO:13.

4. a recombinant retroviral vector, contains the gene described in claim 2 or 3.

5. recombinant retroviral vector according to claim 4, is characterized in that, the carrier that sets out comprises pMX-IRES-GFP, pMCs-IRES-GFP or pMYx-IRES-GFP.

6. the retrovirus that the recombinant retroviral vector described in claim 4 or 5 obtains after packaging.

7. the iNKT cell of Retroviral Transfer claimed in claim 6.

Images