CN102875666A - 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 a kind of retroviral vector for the tuberculosis treatment that utilizes this TCR to prepare, the iNKT cell that the retroviral vector transfection obtains and the application in the preparation anti-tuberculosis drugs thereof.

Background technology

Tuberculosis is the highest transmissible disease of infection rate in the 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 the 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, the tuberculosis number is in the whole world second, the tubercle bacillus affection number surpasses 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 the various transmissible diseases always.

Tuberculotherapy is still take chemotherapy as main at present, but there is long, the shortcoming such as toxic side effect the is large course for the treatment of, reduced patient's compliance of taking medicine, patient is irregular to take medicine, voluntarily drug withdrawal, 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 the tuberculosis refractory, and single adjustment chemotherapy regimen effect is limited; And chemotherapy can't solve Endogenous relapse and Exogenous reinfection low because of immunity of organisms or that defective 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 the removing of tubercule bacillus needs inherent immunity and adaptive immunity acting in conjunction in the body.The iNKT cell is one of subgroup of T cell, is the bridge of innate immunity and adaptive immune response, plays an important role in the early immune reaction of tuberculosis infection.The iNKT cell can be regulated the effect of scavenger cell performance tuberculosis by secrete cytokines IFN-γ, also can be by TNF secretion-α and GrB performance cytotoxicity.The iNKT cell also participates in granulomatous formation, suppresses the diffusion of tubercule bacillus.Yet, studies confirm that in the tuberculosis mouse model multiplication capacity of iNKT cell weakens, immunocompetence descends.Find also that in the active tuberculosis patient body iNKT cell quantity descends, 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 the iNKT cell to carry out the tuberculosis immunity treatment is carried out.INKT cell adoptive immunity is infused in the tuberculosis mouse, finds that tubercule bacillus quantity significantly reduces in the mouse lung tissue, pulmonary lesion obviously alleviates.The activator KRN7000 of injection iNKT cell finds tuberculosis mouse lung IFN-γ secretion increasing, mouse survival time significant prolongation in the tuberculosis mouse model.Yet the iNKT cell function of tubercular's body endogenous origin is impaired, and the 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 sign of all T cell surfaces, plays a crucial role in the identification of T cell antigen.TCR is the heterodimer that is made of two peptide chains of α, 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 the signal transmission is mainly by carrying out with its CD3 molecule of being combined with non covalent bond.TCR molecule contactin, its antigen-specific is present in the V district; Respectively there are again three hypervariable region CDR1, CDR2, CDR3 in V district (V α, V β), and is wherein maximum with the CDR3 variation, directly determined the antigen-binding specificity of TCR.When TCR identification MHC-antigen peptide complex body, CDR1, CDR2 identification and in conjunction with the sidewall of MHC molecular antigen engagement groove, and CDR3 directly combines with antigen peptide.

According to TCR V α, V β gene homology, 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 that the characteristics of its unique CDR3 sequence are all arranged, adopt CDR3 spectral pattern analytical technology, can measure the frequency that each CDR3 of each TCR family occurs, reflect thus clone's property of T cell.Do not accept in the T cell of antigenic stimulation, be evenly distributed for the T cell clone of various antigens, show as many families and polyclone, particularly, show as about 8 CDR3 peaks that Gaussian distribution all appears being in each family; Antigenic stimulation then causes the some or several specific TCR family t cell responses hyperplasia of this antigen of identification, show as widow clone or mono-clonal distribution that 4 peaks appear being less than in the CDR3 member of this family, the TCR family that wherein has mono-clonal CDR3 distribution (showing as unimodal) namely 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 the iNKT cell, obtain to express the iNKT cell of tuberculosis specific TCR, and should be through the application of iNKT cell in the preparation anti-tuberculosis drugs of tcr gene modification.

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 described sequence of SEQ ID NO:3 is contained in the CDR3 district of α chain; The sequence shown in the 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 to be substituted, to lack and/or increased one or more amino acid and/or the special and exogenous β chain of resulting energy is assembled into the TCR protein molecular after end modified aminoacid sequence by the aminoacid sequence shown in the SEQ ID NO:8; The β chain is to be substituted, to lack and/or increased one or more amino acid and/or the special and exogenous α chain of resulting energy is assembled into the TCR protein molecular after end modified aminoacid sequence by the aminoacid sequence shown in the SEQ ID NO:6.

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

The encode gene of above-mentioned TCR.

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

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

A kind of recombinant retroviral vector contains the gene shown in the 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 packing.

The iNKT cell of above-mentioned Retroviral Transfer.

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

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

1, filters 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), and IL-4 and GM-CSF induce DC ripe;

3. magnetic bead sorting goes out CD8 ⁺The T cell;

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

5. extract CD8 ⁺T cell mRNA, reverse transcription are 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, make up recombinant retroviral vector

1. the people α, β gene family variable region (the variable region that report according to GeneBank, V) and constant region (constant region, 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 is referred to as mC to adopt the C district, mouse source 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 purpose of mouse source 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 the iNKT cell, can impel the albumen of exogenous α and beta gene expression correctly to be assembled into TCR protein molecular and stably express at the iNKT cell surface by sudden change, and be beneficial to its competition in conjunction with the CD3 molecule of iNKT cell surface, the enhancing signal conduction function, it is active to improve the tuberculosis 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, the 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); Utilize 2A to connect exogenous α, β gene realization balance expression (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 the recombinant PCR technology, minimum mutation T CR α, β gene connect by certainly shearing polypeptide 2A, and are cloned into pGEM-T carrier order-checking evaluation.

4. will check order correct α, β full-length gene inserts retroviral vector pMX-IRES-GFP, and enzyme is cut evaluation;

5. adopt calcium phosphate transfection method, packing 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 the recombinant retrovirus titre.Calculation formula: virus infection titre (IU/ml)=NIH3T3 total cellular score * GFP positive rate/virus concentrates liquid measure (ml).

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

1. adopt the 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, the Flow cytometry transfection efficiency is determined optimal multiplicity of infection (multiplicity of infection, MOI);

4. with best MOI with 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 nuclear, observe the expression of iNKT cell surface tuberculosis specific TCR under the laser confocal microscope;

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

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

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

Wherein, the iNKT cell is a kind of cell subsets in the human body, can obtain by separating in the human peripheral, and carry out amplification in vitro and cultivate, and separation and the experimental installation of cultivating require low, technology maturation.

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

The construction process of recombinant retroviral vector is this area molecule clone technology commonly used, the recombinant retrovirus transfection method is animal nutrition commonly used at present, the calcium phosphate method that in the present invention, uses, 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, but carry the exogenous tcr gene of iNKT cell successful expression of the Retroviral Transfer of this tuberculosis antigen specific TCR gene, specific recognition tuberculosis 38kDa antigen also mediates IFN-γ, TNF-α cytokine secretion and cytotoxic activity, using value with tuberculosis gene therapy can be the cellular immunization treatment of adopting lungy and opens up new footpath.

Description of 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 makes up synoptic diagram;

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

Observe expression (the left figure: homotype control antibodies IgG1-APC mark of iNKT cell surface tuberculosis specific TCR under Fig. 8 laser confocal microscope; 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 the time iNKT cell killing activity.

Embodiment

The present invention is further illustrated below in conjunction with embodiment, 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) (the Sambrook J that writes such as Sambrook etc., Russell DW, Janssen K, the yellow training hall of Argentine J. waits translates 2002, Beijing: the condition Science Press), or the condition of advising according to manufacturer.

In following examples, all measurement data results represent with ± s, adopt the relatively difference of cytokine IFN-γ, TNF-α secretion level between each group of one-way analysis of variance (One-Way ANOVA), proofread and correct with Welch during heterogeneity of variance, adopt the LSD method to carry out comparing in twos between each group, adopt Dunnett ' s T3 method to proofread and correct during heterogeneity of variance.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) adds an amount of Ficoll lymphocyte separation medium at the aseptic centrifuge tube of 15ml scale;

(2) the abundant mixing dilution of the peripheric venous blood of taking heparin anti-freezing and equivalent RPMI 1640 liquid, the anticoagulation of drawing 2 times of volumes with the pasteur dropper slowly is superimposed on the lymphocyte separation medium along tube wall, notes keeping the 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 the upper strata is blood plasma and diluent, and the pipe end is mainly red corpuscle and GCL.The middle level is lymphocyte separation medium, has at the interface one in upper, middle level take mononuclearcell as main canescence cloud and mist layer;

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

(5) cell counting and cell viability detect: the PBMC suspension mixes with the blue dye liquor of 0.4 % platform phenol of 1/10 volume, four total cell count of large grid on angle on the tally on the blood counting chamber, and 1/4th numbers of total cell count multiply 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 induces and load mycobacterium tuberculosis 38kDa antigen

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

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

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

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

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

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

(1) DC cultivated the 8th day, counted respectively DC and the PBMC of load 38kDa, pressed 1:10 with both mixed culture, added 10ng/ml IL-7;

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

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

1.4 immunomagnetic beads (beautiful day Ni biotech firm of Germany) sorting CD4 ⁺, CD8 ⁺The T cell.

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

1.6 reverse transcription (RT) test kit (Fermentas) synthesizes cDNA.

1.7 34 TCR V of pcr amplification α gene family CDR3 fragment (reference literature: XIN-SHENG etc., 2006, Clinical ﹠amp; 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, inboard primer is done heminested PCR:

First round PCR: every sample is done 34 PCR reaction tubess, and the 1st ~ 34 pipe adds respectively TCR V α 1 to V α 34 family's upstream primers, and every pipe adds the downstream and shares C α outside primer 1 μ l, and each primer concentration is 10 μ M.Every PCR reaction tubes volume is 25 μ l, contains cDNA template 1.0 μ l, 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: the reaction cumulative volume is 25 μ l, contains first round PCR product 2 μ l, 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, the inboard C α of downstream FAM mark primer 1 μ l, 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 of 8 pcr amplifications β gene family CDR3 fragment (reference literature: XIN-SHENG etc., 2006, Clinical ﹠amp; Laboratory Haematology, 28:405 – 415. doi:10.1111/j.1365-2257.2006.00827.x):

Every sample is done 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 primers 0.8 μ l, and each primer concentration is 10 μ M.The PCR reaction volume is 25 μ l, contains cDNA template 1 μ l, 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.Residue PCR product-20 a ° 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) carries out 6% polyacrylamide gel electrophoresis on, collect the fluorescent signal of the varying strength that different time occurs in the electrophoresis process, 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 CDR3 member of TCR family occurs, reflect thus clone's property of each TCR family.Wherein, the TCR family that has a unimodal distribution namely is the TCR family of antigen-specific mono-clonal hyperplasia.

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

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

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

2.1 synthetic primer

V α 9, V β 5 gene family V region sequence characteristics according to the GeneBank report, design full-length gene upstream and downstream primer, design respectively the upstream and downstream primer according to the sequence (being mC α and mC β) after 9 the key amino acid sudden changes in 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 merges full-length gene

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

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

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

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

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

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

(7) S2 that the S1 that obtains with step (2) and step (3) obtain is as template, utilize primer P1 and P4, the Pfu archaeal dna polymerase, wherein the sequence of hV β 5mC β section is 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 shown in SEQ ID NO:10).

(8) S4 that the S3 that obtains with step (5) and step (6) obtain is as template, utilize primer P5 and P8, the Pfu archaeal dna polymerase, wherein the sequence of hV α 9mC α section is 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 shown in SEQ ID NO:12).

(9) S6 that the S5 that obtains with step (7) and step (8) obtain utilizes 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) as template.

Above conventional PCR reaction system 25 μ l contain 10 * Buffer, 2.5 μ l, 10mmol/L dNTP 0.5 μ l, Pfu archaeal dna polymerase 0.2U, each 0.8 μ l of 10 μ M primers, 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 contain 10 * Buffer, 2.5 μ l, 10mmol/L dNTP 0.5 μ l, Pfu archaeal dna polymerase 0.2U, each 0.8 μ l of 10 μ M primer P5, 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 make up the cloning vector that contains hV β 5mC β-2A-hV α 9mC alpha fusion gene

(1) reclaims 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) with in 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 Transformation 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 are and are 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), with corresponding restriction enzyme the positive recombinant plasmid of primary dcreening operation is identified, and take recombinant plasmid as template, carried out pcr amplification, agarose gel electrophoresis is identified clip size.Send Invitrogen Shanghai Ying Jun Bioisystech Co., Ltd to check order the primary dcreening operation positive plasmid at last.Sequencing result shows that the contained exogenous gene sequence of this recombinant plasmid and forecasting sequence are in full accord.

2.4 the structure of recombinant retroviral vector

(1) cut T carrier and the pMX-IRES-GFP empty plasmid that contains 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) with after hV β 5mC β-2A-hV α 9mC alpha fusion gene connects into pMX-IRES-GFP carrier (method is with step 2.3), transformed competence colibacillus bacterium XL-10, after coating the solid medium cultivation 12-16h that contains penbritin, select single bacterium colony, shake bacterium and spend the night;

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

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

2.5 the packing of retrovirus recombinant vectors

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 the packing retrovirus, and calcium phosphate method cell transfecting test kit (the green skies) description operation is pressed in experiment.

2.6 recombinant retrovirus is concentrated and purified

(1) collects 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 fully, and packing ,-80 ° of C store;

2.7 Flow Cytometry Assay virus titer

(1) in advance with 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) behind the infection 24h, change fresh medium, remove pseudovirion;

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

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

The 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. identify the tuberculosis activity of the iNKT cell of recombinant retrovirus transfection

3.1 the cultivation of iNKT cell and sorting

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

(2) (take 24 orifice plates as example) every hole spreads 2 * 10 ⁶Individual PBMC, at RPMI 1640 complete culture solutions that contain 10% FBS at 37 ° of C, 5% CO ₂Cultivate under the condition;

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

(4) remaining PBMC is frozen, be used 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 of Flow cytometry iNKT cell〉95%(Fig. 6).

3.2 measure the suitableeest MOI of recombinant virus infection iNKT cell

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

(2) same day was abandoned the old liquid of cell cultures in transfection, was the viral stock solutions of 2,4,5,6,8,10,12 addings 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 96 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 immunofluorescence detects the expression of genetic modification iNKT cell external source TCR

(1) recombinant virus is pressed 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 ℃ of lucifuges are hatched 30 min;

(4) centrifugal collecting cell, PBS washing 2 times, 2% Paraformaldehyde 96 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 nuclear, observe the expression (Fig. 8) of iNKT cell surface tuberculosis specific TCR under the laser confocal microscope simultaneously.As seen the iNKT cell surface after modifying 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 show that the positive cell rate of expressing the tuberculosis antigen specific TCR is 60.9%(Fig. 9).

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

The 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. the antigen group that has nothing to do (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 the antigen-specific tcr gene and modify the iNKT cell, carry out common cultivation with DC, establish two multiple holes for every group;

(2) cultivate altogether 6,12,18,24,30, behind the 36h, collect each hole culture supernatant, operate by the 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 iNKT cell that untransfected, idle running dye and modifies with the tcr gene that the DC of load tuberculosis antigen or other non-specific antigen of load not cultivates altogether ( P＜0.001), sees 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 iNKT cell that untransfected, idle running dye and modifies with the tcr gene that the DC of load tuberculosis antigen or other non-specific antigen of load not cultivates altogether ( P＜0.001), sees 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) be significantly higher than iNKT cell that untransfected, idle running dye and the iNKT cell modified with the tcr gene that the DC of load tuberculosis antigen or other non-specific antigen of load not cultivates altogether ( P＜0.001), sees Figure 12.

3.5 time resolved fluoro-immunoassay test kit (PerkinElmer) is 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 the DC of load tuberculosis antigen or other non-specific antigen of load not cultivates altogether ( P＜0.001), sees Figure 13.

Above-mentioned experimental result shows: but carry the exogenous tcr gene of iNKT cell successful expression of the Retroviral Transfer of tuberculosis antigen specific TCR gene, specific recognition tuberculosis 38kDa antigen also mediates IFN-γ, TNF-α cytokine secretion and cytotoxic activity, using value with tuberculosis gene therapy can be the cellular immunization treatment of adopting lungy and opens 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 (10)

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

2. tuberculosis antigen specificity TCR according to claim 1, it is characterized in that the α chain is to be substituted, to lack and/or increased one or more amino acid and/or the special and exogenous β chain of resulting energy is assembled into the TCR protein molecular after end modified aminoacid sequence by the aminoacid sequence shown in the SEQ ID NO:8; The β chain is to be substituted, to lack and/or increased one or more amino acid and/or the special and exogenous α chain of resulting energy is assembled into the TCR protein molecular after end modified aminoacid sequence by the aminoacid sequence shown in the SEQ ID NO:6.

3. tuberculosis antigen specificity TCR according to claim 2 is characterized in that, the aminoacid sequence of described α chain is shown in SEQ ID NO:12, and the aminoacid sequence of β chain is shown in SEQ ID NO:10.

4. the gene of coding claim 1 ~ 3 each described TCR.

5. the fusion gene of a tuberculosis antigen specificity TCR, its sequence is shown in SEQ ID NO:13.

6. a recombinant retroviral vector contains claim 4 or 5 described genes.

7. recombinant retroviral vector according to claim 6 is characterized in that, the carrier that sets out comprises pMX-IRES-GFP, pMCs-IRES-GFP or pMYx-IRES-GFP.

8. claim 6 or the 7 described recombinant retroviral vectors retrovirus that obtains after packing.

9. the iNKT cell of Retroviral Transfer claimed in claim 8.

10. each described tuberculosis antigen specificity TCR of claim 1 ~ 3, claim 4 or 5 described genes, the application of the iNKT cell of claim 6 or 7 described recombinant retroviral vectors, retrovirus claimed in claim 8, Retroviral Transfer claimed in claim 9 in the preparation anti-tuberculosis drugs.

Images