CN102887951A - Tuberculosis peptide Ag85B199-207 specific TCR and recombinant retroviral vector and use thereof - Google Patents

Abstract

The invention discloses a tuberculosis peptide Ag85B199-207 specific TCR (T cell receptor), and a recombinant retroviral vector and use thereof. A tuberculosis peptide Ag 85B199-207 (KLVANNTRL) TCR is screened out; the TCR is carried by a retroviral vector to transfect CD8+T cells and thus CD8+T cells in which tuberculosis peptide Ag85B199-207 specific TCR is expressed are obtained. The CD8+T cells modified by the TCR gene can express exogenous TCR genes, specifically identify tuberculosis peptide Ag85B199-207, and mediate secretion of IFN-gamma and TNF-alpha cytokines and the cytotoxicity of cells tuberculosis. The TCR has gene therapy value of tuberculosis, so a new way for adoptive cellular immunotherapy of tuberculosis is provided.

Description

A kind of tuberculosis peptide Ag85B 199-207Specificity TCR, its recombinant retroviral vector and application

Technical field

The invention belongs to bioengineering field, be specifically related to a kind of tuberculosis antigen peptide specific φt cell receptor (TCR), and a kind of retroviral vector for the tuberculosis treatment that utilizes this TCR to prepare, the CD8 that the retroviral vector transfection obtains ⁺T cell 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!

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.

CD8 ⁺The T cell is that T is lymphocytic a kind of, by antigen recognition receptor (TCR α β) Recognition polypeptide antigen and the mixture that forms from body MHC I quasi-molecule, the target cell of can specific killing expressing the epitope that its TCR identifies, antibacterium and virus infection, acute be important effector cell in special-shaped transplant rejection and the lethal effect to tumour cell, be called as cytotoxic T lymphocyte (CTL).

In recent years, in the mouse model that infects tubercule bacillus, find functional CD8 ⁺The shortage of T cell significantly increases mouse to the susceptibility of tubercule bacillus, shows CD8 ⁺T cell and CD4 ⁺The T cell is the same, has important provide protection in resisting tuberculosis infection.Clinical study is also found, CD8 ⁺The T cell can be identified the cell of mycobacterium tuberculosis infection, by discharging bacterium in IFN-γ, the target cell of dissolving infection and the direct killing cell, plays a significant role in the anti-mycobacterium tuberculosis infection immune response.Identify the MHC I restricted CTL epitope of tuberculosis antigen, for illustrating CD8 ⁺The protective effect of T cell in tuberculosis, understand tuberculosis immune response and research and development Antitubercular vaccine etc. in depth and have vital effect.

The Ag85 complex body is that tubercule bacillus is expressed a kind ofly has an immunodominant antigen protein, albumen by 85A, 85B and 3 kinds of height of 85C homology forms, relevant with the transferase active of mycobacterium, participate in the cell walls of synthetic mycobacterium, can inducing T cell propagation and IFN-γ release in majority contacts the healthy human body of tubercule bacillus, be the comparatively ideal candidate antigens of Vaccinum Calmette-Guerini.Ag85B _199-207Peptide is the tuberculosis antigen peptide of a high conservative, and it is restricted to have a HLA-A*0201, and the frequency of this equipotential gene in the crowd surpasses 40%.Ag85B _199-207Peptide can be induced the Th1 type protective immunological reaction of anti-mycobacterium tuberculosis, makes T effector cell express the cytokine performance anti-microbial effects such as IFN-γ and TNF-α.Experiment shows in the body, with Ag85B _199-207Immunity HLA-A2/Kb transgenic mice can improve T cell proliferation level and cytotoxicity level.

Summary of the invention

The object of the invention is to: filter out tuberculosis peptide Ag85B _{199 – 207}(KLVANNTRL) special TCR utilizes retroviral vector to be transfected into CD8 ⁺In the T cell, obtain to express tuberculosis peptide Ag85B _{199 – 207}The CD8 of specific TCR ⁺The T cell, and should be through the CD8 of tcr gene modification ⁺The application of T cell in the preparation anti-tuberculosis drugs.

The technical solution adopted in the present invention is:

Tuberculosis peptide Ag85B _{199 – 207}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, described tuberculosis peptide Ag85B _{199 – 207}The α chain of specificity TCR is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous β chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:10; The β chain is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous α chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:6.

Preferably, described tuberculosis peptide Ag85B _{199 – 207}The aminoacid sequence of the α chain of specificity TCR is shown in SEQ ID NO:12, and the aminoacid sequence of β chain is shown in SEQ ID NO:8.

Coding tuberculosis peptide Ag85B _{199 – 207}The gene of specificity TCR.

A kind of tuberculosis peptide Ag85B _{199 – 207}The fusion gene of specificity TCR, its sequence is shown in SEQ ID NO:13.

A kind of recombinant retroviral vector contains coding tuberculosis peptide Ag85B _{199 – 207}The gene of specificity TCR.

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 upper recombinant retroviral vector obtains after packing.

The CD8 of above-mentioned Retroviral Transfer ⁺The T cell.

Tuberculosis peptide Ag85B specificity TCR, the gene of this TCR that encodes contains recombinant retroviral vector, the retrovirus of this gene, the CD8 of this Retroviral Transfer ⁺The application of T cell in the preparation anti-tuberculosis drugs.

Realize that technique scheme concrete steps flow process is as follows:

1, screening tuberculosis peptide Ag85B _{199 – 207}(KLVANNTRL) special TCR

1. adopt lymphocyte separation medium to separate HLA-A*0201 type healthy volunteer's peripheral blood mononuclear cell (peripheral blood mononuclear cell, PBMC);

2. count PBMC, adjusting the PBMC number is 1 * 10 ⁶/ hole, every porocyte add respectively and contain tuberculosis peptide Ag85B _{199 – 207}(KLVANNTRL) 10% FBS-1640 substratum 2ml;

3. behind the adherent culture 2h, add 25U/ml IL-2, added IL-2 to 50U/ml, added IL-2 100U/ml, and continued to cultivate 11 days with this concentration afterwards in the 5th day in the 3rd day;

4. magnetic bead sorting goes out CD8 ⁺The T cell extracts its mRNA, and reverse transcription is cDNA;

5. CDR3 spectral pattern before and after complementary determining region 3(complementarity determining region3, CDR3) the spectral pattern analyzing and testing stimulates is found out the tuberculosis peptide Ag85B that is the mono-clonal hyperplasia after the stimulation _{199 – 207}Special TCR α, β gene family.

2, make up recombinant retroviral vector

1. the people TCR α, variable region, β gene family upstream (the variable region that report according to GeneBank, V) and downstream constant region (constant region, C) gene order, design TCR α, β chain full-length gene upstream and downstream primer amplify the special TCR α of tuberculosis peptide, β full-length gene.

2. design upstream and downstream, C region mutation site primer, adopt the recombinant PCR method with 9 key amino acids in TCR α, β chain C district (reference literature: Luo W. et al. Development of genetically engineered CD4 that suddenlys change ⁺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 this step is to reduce the mispairing of interior exogenous TCR α, β gene, because CD8 ⁺There is the expression of endogenous TCR α, β gene in the T cell, can impel the albumen of exogenous α and beta gene expression correctly to be assembled into TCR protein molecular and stably express at CD8 by sudden change ⁺The T cell surface is beneficial to its competition simultaneously in conjunction with CD8 ⁺The CD3 molecule of T cell surface, the enhancing signal conduction function improves and modifies rear CD8 ⁺The tuberculosis of T cell is active.Certainly, can also take other strategies to reduce the mispairing of interior exogenous TCR α, β chain herein, as: replace α, β full-length gene part C district (Sebestyen with CD3 ζ chain, Z. 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 in exogenous α, β gene C district, J.M. 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 the static charge (Voss between α, the β chain, R.H. 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 with CD3 ζ chain, R.A. 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, with tuberculosis peptide Ag85B _{199 – 207}Special TCR α, beta gene fragment connect the minimum mutation T CR gene of acquisition by certainly shearing polypeptide P2A;

The hV β 16mC β that 4. will check order correct-P2A-hV α 13mC alpha fusion gene inserts retroviral vector pMX-IRES-GFP, and enzyme is cut evaluation;

5. adopt the liposome transfection method, with recombinant retrovirus expression plasmid pMX-hV β mC β-P2A-hV α mC α-IRES-GFP and envelope protein plasmid VSV-G cotransfection GP2-293;

6. collect 48h-72h virus supernatant, the concentrated and purified virus of low temperature ultracentrifugation;

7. recombinant virus infection NIH3T3 cell, the Flow cytometry virus titer, calculation formula: virus titer (IU/ml)=NIH3T3 cell count * GFP positive rate/virus concentrates liquid measure (ml).

3, identify the CD8 of recombinant retrovirus transfection ⁺The tuberculosis of T cell is active

1. adopt the Ficoll density gradient centrifugation, separate HLA-A*0201 type donor peripheral blood PBMC;

2. magnetic bead sorting CD8 ⁺The T cell;

3. IL-2 and anti-CD3 monoclonal antibody activate the T cell that sub-elects;

4. by infection multiplicity (multiplicity of infection, MOI)=13 above-mentioned recombinant retrovirus is infected CD8 ⁺The T cell;

5. use IL-2 and anti-CD3 monoclonal antibody to stimulate metainfective CD8 ⁺The T cell;

6. the per-cent of positive cell behind the Flow cytometry virus infection;

7. measure the CD8 of virus transfection ⁺The anti-TB of T cell is active:

Experiment arranges: negative control group (tuberculosis peptide Ag85B _{199 – 207}The CD8 of specific TCR genetic modification ⁺T cell+dendritic cell (dendritic cells, DC)), untransfected group (untransfected CD8 ⁺T cell+load tuberculosis peptide Ag85B _{199 – 207}DC), empty carrier transfection group (empty carrier transfection CD8 ⁺T cell+load tuberculosis peptide Ag85B _{199 – 207}DC), irrelevant peptide group (tuberculosis peptide Ag85B _{199 – 207}The CD8 of specific TCR genetic modification ⁺The DC of T cell+load C MVpp65), TBTd+TB-DC group (tuberculosis peptide Ag85B _{199 – 207}The CD8 of specific TCR genetic modification ⁺T cell+load tuberculosis peptide Ag85B _{199 – 207}DC).

Detect the above-mentioned secretion level of respectively organizing IFN-γ in the cells and supernatant, TNF-α with enzyme linked immunosorbent assay analysis method (enzyme-linked immunosorbent assay, ELISA); Detect CD8 with time resolved fluoro-immunoassay technology (time-resolved fluoroimmuno-assay, TRFIA) ⁺The T cell is to the killing activity of DC.

Wherein, CD8 ⁺The T 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 a kind of 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 artificial liposome method of in the present invention, using, can also use other chemical infection protocol, comprise: DEAE-dextran method, calcium phosphate method; And physical method, comprising: microinjection, electroporation, particle gun etc.

Beneficial effect of the present invention is:

The present invention successfully filters out tuberculosis peptide Ag85B _{199 – 207}Special TCR carries the CD8 of the Retroviral Transfer of this peptide specific TCR gene ⁺But the exogenous tcr gene of T cell successful expression, specific recognition tuberculosis peptide Ag85B _{199 – 207}And mediation IFN-γ, TNF-α cytokine secretion and cytotoxic activity, have the using value of tuberculosis gene therapy, can be the cellular immunization treatment of adopting lungy and open up new footpath.

Description of drawings

Fig. 1 tuberculosis peptide Ag85B _{199 – 207}CD8 before and after stimulating ⁺T cell TCR α and β chain CDR3 spectral pattern are analyzed;

Fig. 2 recombinant retroviral vector pMX-hV β 16mC β-P2A-hV α 13mC α-IRES-GFP makes up synoptic diagram;

The enzyme of Fig. 3 pMX-hV β 16mC β-P2A-hV α 13mC α-IRES-GFP is cut evaluation (M.DL15000 marker; 1. pMX-IRES-GFP empty carrier; 2. the XhoI enzyme of pMX-IRES-GFP empty carrier is cut product; 3. pMX-hV β 16mC β-P2A-hV α 13mC α-IRES-GFP; 4. the XhoI enzyme of pMX-hV β 16mC β-P2A-hV α 13mC α-IRES-GFP is cut product; 5. the XhoI+NotI double digestion product of pMX-hV β 16mC β-P2A-hV α 13mC α-IRES-GFP);

Expression (* 10) (a. light field of NIH3T3 cell GFP after the transfection of Fig. 4 fluorescence microscope recombinant virus; B. fluorescence; 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-hV β 16mC β-P2A-hV α 13mC α-IRES-GFP);

CD8 after the transfection of Fig. 6 fluorescence microscope recombinant virus ⁺The expression (* 10 of T cell GFP; EmTd: empty carrier transfection group; TBTd: the recombinant virus transfection group);

CD8 after the transfection of Fig. 7 Flow cytometry recombinant virus ⁺The expression of T cell GFP (UnTd: untransfected group; EmTd: empty carrier transfection group; TBTd: the recombinant virus transfection group);

Fig. 8 ELISA detects CD8 ⁺The secretion level of T cell IFN-γ;

Fig. 9 ELISA detects CD8 ⁺The secretion level of T cell TNF-α;

Figure 10 TRFIA detects CD8 ⁺The killing activity of T cell.

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. screen tuberculosis peptide Ag85B _{199 – 207} (KLVANNTRL) special TCR

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 buffy coat 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 cell 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 preparation tuberculosis peptide, the special T cell clone of HIV peptide:

(1) counting PBMC, adjusting the PBMC number is 1 * 10 ⁶/ hole adds respectively and contains 50ng/ml tuberculosis peptide Ag85B _{199 – 207}(KLVANNTRL) 10% FBS-1640 substratum 2ml;

(2) 37 ° of C, 5% CO ₂After cultivating 2h under the condition, add IL-2 25U/ml;

Added IL-2 to 50U/ml in (3) the 3rd days, added IL-2 100U/ml, and continued to cultivate 11 days with this concentration afterwards in the 5th day.

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

1. 4 total RNA extraction reagent boxes (OMEGA) extract total RNA of the above-mentioned cell precipitation of collecting.

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

1.6 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 shared downstream 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/LMgCl 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.

1.7 24 TCR V of pcr amplification 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 1 μ l, and the 1st to the 24th pipe adds respectively TCR V β 1 to TCR V β 24 upstream primers 1 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.

1.8 agarose gel electrophoresis:

Get 34 TCR V α and 24 each 5 μ l of TCR V β gene family PCR product, 2% agarose gel electrophoresis, 110V, 20min adopts gel imaging system to take a picture.Residue PCR product-20 a ° C saves backup.

1.9 the CDR3 spectral pattern is 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 that antigen peptide stimulates 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 antigen peptide 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 respectively V α 13, V β 16 gene families (Fig. 1) of mono-clonal amplification after the tuberculosis peptide stimulates.

Order-checking shows, tuberculosis peptide Ag85B _{199 – 207}(KLVANNTRL) nucleotide sequence in the CDR3 district of special TCR V α 13, V β 16 is shown in SEQ ID NO:1 and SEQ ID NO:2, and the aminoacid sequence of two CDR3 sequence encodings is respectively shown in SEQ ID NO:3 and SEQ ID NO:4.

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

2.1 synthetic primer

V α 13, V β 16 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 P2A peptide catenation sequence design primer, all primer is synthetic by Invitrogen Shanghai Ying Jun Bioisystech Co., Ltd, and primer title and sequence are as follows:

2.2 recombinant PCR amplification hV β 16mC β-P2A-hV α 13mC α merges full-length gene

(1) take the cDNA of step 1.5 preparation as template, utilize primer P1 and P2, the Pfu archaeal dna polymerase, pcr amplification hV β 16hC β full-length gene upstream is to sequence B 1(hV β 16mC β-1 district between the upper first group of mutational site of hC β).Wherein, the nucleotide sequence of hV β 16hC β full-length gene is shown in SEQ ID NO:5, and its coded aminoacid sequence is shown in SEQ ID NO:6.

(2) take the cDNA of step 1.5 preparation as template, utilize primer P3 and P4, Pfu archaeal dna polymerase, sequence B 2(mC β-2 district between group mutational site, the upper first group of mutational site to the second of pcr amplification hC β).

(3) take the cDNA of step 1.5 preparation as template, utilize primer P5 and P6, the Pfu archaeal dna polymerase, upper second group of mutational site, pcr amplification hC β district is to the sequence between the hC β downstream and 5 ' end P2A sequence B 3(mC β-3-5 ' end P2A district).

(4) B3 that the B2 that obtains with step (2) and step (3) obtain is as template, utilize primer P3 and P6, the Pfu archaeal dna polymerase, the upper first group of mutational site of recombinant PCR amplification hC β is to holding the P2A district to the sequence between the hC β downstream and 5 ' end P2A sequence B 4(mC β-2-5 ').

(5) B4 that the B1 that obtains with step (1) and step (4) obtain is as template, utilize primer P1 and P6, the Pfu archaeal dna polymerase, recombinant PCR amplification hV β 16 upstreams are to the sequence between the hC β downstream and 5 ' end P2A sequence, i.e. hV β 16mC β-5 ' end P2A region sequence B5.Wherein, the nucleotide sequence of hV β 16mC β section is shown in SEQ ID NO:7, and the aminoacid sequence of its coding is shown in SEQ ID NO:8.

(6) take the cDNA of step 1.5 preparation as template, utilize primer P7 and P8, the Pfu archaeal dna polymerase, the sequence A 1(3 ' between pcr amplification 3 ' end P2A and hV α 13hC α full-length gene upstream to the upper mutational site of hC α holds P2A-hV α 13mC α-1 district).Wherein, hV α 13hC α full-length gene order is shown in SEQ ID NO:9, and its coded aminoacid sequence is shown in SEQ ID NO:10.

(7) take the cDNA of step 1.5 preparation as template, utilize primer P9 and P10, Pfu archaeal dna polymerase, sequence A 2(hV α 13mC α-2 district on the pcr amplification hC α between mutational site to the hC α downstream).

(8) A2 that the A1 that obtains with step (6) and step (7) obtain is as template, utilize primer P7 and P10, the Pfu archaeal dna polymerase, the sequence between recombinant PCR amplification 3 ' end P2A and hV α 13 upstreams to the hC α downstream, i.e. 3 ' end P2A-hV α 13mC α region sequence A3.Wherein, the nucleotide sequence of hV α 13mC α section is shown in SEQ ID NO:11, and the aminoacid sequence of its coding is shown in SEQ ID NO:12.

(9) A3 that the B5 that obtains with step (5) and step (8) obtain utilizes primer P1 and P10 as template, Pfu archaeal dna polymerase, recombinant PCR amplification hV β 16mC β-P2A-hV α 13mC α (SEQ ID NO:13).

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 5min; 95 ° of C 1min, 72 ° of C 90s, 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 primers, two kinds of each 3 μ l of PCR product template.PCR reaction conditions: 95 ° of C sex change 5min; 95 ° of C 30s, 50 ° of C 45s, 72 ° of C 45s, 3 circulations; 95 ° of C 45s, 72 ° of C 90s, 32 circulations; 72 ° of C extend 10min.

2.3 make up the cloning vector that contains hV β 16mC β-P2A-hV α 13mC α

(1) reclaims test kit (Omega) Separation and Recovery hV β 16mC β-P2A-hV α 13mC alpha gene fragment with gel;

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

(3) with in hV β 16mC β-P2A-hV α 13mC alpha gene fragment access pGEM-T carrier, ligation system 10 μ l:pGEM-T carriers 1 μ l, 10 ' ligation Buffer, 1 μ l, T4 dna ligase 1 μ l, 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 is and is 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 exogenous gene sequence and forecasting sequence that this recombinant plasmid contains 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 16mC β-P2A-hV α 13mC alpha fusion gene with restriction enzyme Xho I, Not I enzyme, glue reclaims hV β 16mC β-P2A-hV α 13mC alpha fusion gene fragment and vector gene fragment (method is with step 2.3) respectively;

(2) with after hV β 16mC β-P2A-hV α 13mC 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, gene fragment is inserted correct (Fig. 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 liposome transfection method transfection GP2-293 cell, the packing retrovirus, and the Lipofectamine 2000 test kit description operation that Invitrogen is pressed in experiment are collected viral supernatant.

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;

2.14 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, after trysinization, PBS washed 3 times, resuspended with 200-300 μ l PBS, 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 the retroviral infection of packing through the GP2-293 cell shows that GFP is at cells (Fig. 4).The titre that calculates recombinant virus is 8 * 10 ⁶IU/mL(Fig. 5).

3. recombinant retrovirus infects CD8 ⁺ The T cell

3.1 with CD8 ⁺T cell infection the day before yesterday is with 5 * 10 ⁵Individual/hole is inoculated in 24 orifice plates;

3.2 the same day was abandoned the old liquid of cell cultures in transfection, was the viral stock solutions of 13 addings by MOI, adding PB is 8mg/L to final concentration, and 37 ° of C cultivate 4h;

3.3 the adding substratum, dilution PB to 2mg/L continues to cultivate 5 days;

3.4 centrifugal collecting cell, PBS washing 2 times, 2% Paraformaldehyde 96 is fixed;

3.5 flow cytometry analysis CD8 ⁺T cell GFP the positive expression rate is 33.1%(Fig. 6,7).

4. identify the CD8 of recombinant retrovirus transfection ⁺ The tuberculosis of T cell is active

4.1 ELISA test kit (Wuhan Boster Biological Technology Co., Ltd.) is measured CD8 ⁺The IFN-γ of T cell, TNF-α secretion level

The experiment contrast group arranges: negative control group (TBTd+DC: tuberculosis peptide Ag85B _{199 – 207}The CD8 of specific TCR genetic modification ⁺T cell+DC), untransfected group (UnTd+DC-TB: untransfected CD8 ⁺T cell+load tuberculosis peptide Ag85B _{199 – 207}DC), empty carrier transfection group (EmTd+DC-TB: empty carrier transfection CD8 ⁺T cell+load tuberculosis peptide Ag85B _{199 – 207}DC), irrelevant peptide group (TBTd+DC-CMV: tuberculosis peptide Ag85B _{199 – 207}The CD8 of specific TCR genetic modification ⁺The DC of T cell+load C MVpp65), TBTd+DC-TB group (tuberculosis peptide Ag85B _{199 – 207}The CD8 of specific TCR genetic modification ⁺T cell+load tuberculosis peptide Ag85B _{199 – 207}DC); Following experiment contrast arranges all herewith.Experiment repeats 3 times, and method is as follows:

(1) DC is with 5 * 10 ³Individual/hole is inoculated in 96 orifice plates, E:T=7 when surveying the secretory volume of IFN-γ by the E:T(that groped, E:T=20 when surveying the secretory volume of TNF-α) add CD8 ⁺The T cell is established three multiple holes for every group;

(2) CD8 ⁺T cell and DC mixed culture certain hour (mixed culture 18h when surveying IFN-γ, mixed culture 24h when surveying TNF-α) are collected each hole culture supernatant, operate by the test kit specification sheets.

ELISA result shows:

1. imitating target than (E:T)=7, with load tuberculosis peptide Ag85B _{199 – 207}DC mixed culture 18h the time, the CD8 of tuberculosis peptide specific TCR genetic modification ⁺T cell IFN-γ secretion level reaches maximum 1427.930 ± 63.714pg/ml, the CD8 that be significantly higher than untransfected, dallies and dye ⁺T cell and the CD8 that modifies with the tcr gene that the DC of not load tuberculosis peptide or other irrelevant peptide of load cultivates altogether ⁺The T cell ( P＜0.001), sees Fig. 8;

2. at E:T=20, with load tuberculosis peptide Ag85B _{199 – 207}DC mixed culture 24h the time, the CD8 of tuberculosis peptide specific TCR genetic modification ⁺T cell TNF-α secretion level reaches maximum 1040.184 ± 31.769pg/ml, the CD8 that be significantly higher than untransfected, dallies and dye ⁺T cell and the CD8 that modifies with the tcr gene that the DC of not load tuberculosis peptide or other irrelevant peptide of load cultivates altogether ⁺The T cell ( P＜0.001), sees Fig. 9.

4. 2 time resolved fluoro-immunoassay test kits (PerkinElmer) are measured CD8 ⁺The T cell killing activity operates by the test kit specification sheets.

Temporal resolution immunofluorescence detected result shows: at E:T=30, with load tuberculosis peptide Ag85B _{199 – 207}DC mixed culture 4h the time, the CD8 that tcr gene is modified ⁺The T cell killing activity is the highest, reaches 57.50%, the CD8 that be significantly higher than untransfected, dallies and dye ⁺T cell and the CD8 that modifies with the tcr gene that the DC of not load tuberculosis peptide or other irrelevant peptide of load cultivates altogether ⁺The level of killing and wounding of T cell ( P＜0.001), sees Figure 10.

Above-mentioned experimental result shows: carry tuberculosis peptide Ag85B _{199 – 207}The CD8 of the Retroviral Transfer of specific TCR gene ⁺But the exogenous tcr gene of T cell successful expression, specific recognition tuberculosis peptide Ag85B _{199 – 207}And mediation IFN-γ, TNF-α cytokine secretion and cytotoxic activity, have 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〉a kind of tuberculosis peptide Ag85B _199-207Specificity TCR, its recombinant retroviral vector and application

<130>

<160> 23

<170> PatentIn version 3.5

<210> 1

<211> 60

<212> DNA

<213> human

<400> 1

aatggtgcta caaacaagct catctttgga actggcactc tgattgttgt cctgcccaat 60

<210> 2

<211> 63

<212> DNA

<213> human

<400> 2

cgccttggac aggggatgaa cactgaagct ttctttggac aaggcaccag actcacagtt 60

gta 63

<210> 3

<211> 20

<212> PRT

<213> human

<400> 3

Asn Gly Ala Thr Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Ile Val

1 5 10 15

Val Leu Pro Asn

20

<210> 4

<211> 21

<212> PRT

<213> human

<400> 4

Arg Leu Gly Gln Gly Met Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr

1 5 10 15

Arg Leu Thr Val Val

20

<210> 5

<211> 951

<212> DNA

<213> human

<400> 5

ctcgaggcca ggatggtttc caggctcctc agtttagtgt ccctttgtct cctgggagca 60

aagcacatag aagctggagt tactcagttc cccagccaca gcgtaataga gaagggccag 120

actgtgactc tgagatgtga cccaatttct ggacatgata atctttattg gtatcgacgt 180

gttatgggaa aagaaataaa atttctgtta cattttgtga aagagtctaa acaggatgag 240

tccggtatgc ccaacaatcg attcttagct gaaaggactg gagggacgta ttctactctg 300

aaggtgcagc ctgcagaact ggaggattct ggagtttatt tctgtgccag cagccgcctt 360

ggacagggga tgaacactga agctttcttt ggacaaggca ccagactcac agttgtagag 420

gacctgaaca aggtgttccc acccgaggtc gctgtgtttg agccatcaga agcagagatc 480

tcccacaccc aaaaggccac actggtgtgc ctggccacag gcttcttccc tgaccacgtg 540

gagctgagct ggtgggtgaa tgggaaggag gtgcacagtg gggtcagcac ggacccgcag 600

cccctcaagg agcagcccgc cctcaatgac tccagatact gcctgagcag ccgcctgagg 660

gtctcggcca ccttctggca gaacccccgc aaccacttcc gctgtcaagt ccagttctac 720

gggctctcgg agaatgacga gtggacccag gatagggcca aacccgtcac ccagatcgtc 780

agcgccgagg cctggggtag agcagactgt ggctttacct cggtgtccta ccagcaaggg 840

gtcctgtctg ccaccatcct ctatgagatc ctgctaggga aggccaccct gtatgctgtg 900

ctggtcagcg cccttgtgtt gatggccatg gtcaagagaa aggatttctg a 951

<210> 6

<211> 311

<212> PRT

<213> human

<400> 6

Val Ser Arg Leu Leu Ser Leu Val Ser Leu Cys Leu Leu Gly Ala Lys

1 5 10 15

His Ile Glu Ala Gly Val Thr Gln Phe Pro Ser His Ser Val Ile Glu

20 25 30

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

35 40 45

Asn Leu Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe Leu

50 55 60

Leu His Phe Val Lys Glu Ser Lys Gln Asp Glu Ser Gly Met Pro Asn

65 70 75 80

Asn Arg Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys

85 90 95

Val Gln Pro Ala Glu Leu Glu Asp Ser Gly Val Tyr Phe Cys Ala Ser

100 105 110

Ser Arg Leu Gly Gln Gly Met Asn Thr Glu Ala Phe Phe Gly Gln Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

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

195 200 205

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

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

Met Val Lys Arg Lys Asp Phe

305 310

<210> 7

<211> 939

<212> DNA

＜213〉artificial sequence

<400> 7

atggtttcca ggctcctcag tttagtgtcc ctttgtctcc tgggagcaaa gcacatagaa 60

gctggagtta ctcagttccc cagccacagc gtaatagaga agggccagac tgtgactctg 120

agatgtgacc caatttctgg acatgataat ctttattggt atcgacgtgt tatgggaaaa 180

gaaataaaat ttctgttaca ttttgtgaaa gagtctaaac aggatgagtc cggtatgccc 240

aacaatcgat tcttagctga aaggactgga gggacgtatt ctactctgaa ggtgcagcct 300

gcagaactgg aggattctgg agtttatttc tgtgccagca gccgccttgg acaggggatg 360

aacactgaag ctttctttgg acaaggcacc agactcacag ttgtagagga cctgaacaag 420

gtgttcccac ccgaggtcgc tgtgtttgag ccatcaaaag cagagatcgc acacacccaa 480

aaggccacac tggtgtgcct ggccacaggc ttcttccctg accacgtgga gctgagctgg 540

tgggtgaatg ggaaggaggt gcacagtggg gtcagcacgg acccgcagcc cctcaaggag 600

cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660

ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720

aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 780

tggggtagag cagactgtgg cattacctcg gcatcctacc accaaggggt cctgtctgcc 840

accatcctct atgagatcct gctagggaag gccaccctgt atgctgtgct ggtcagcgcc 900

cttgtgttga tggccatggt caagagaaag gatttctga 939

<210> 8

<211> 311

<212> PRT

＜213〉artificial sequence

<400> 8

Val Ser Arg Leu Leu Ser Leu Val Ser Leu Cys Leu Leu Gly Ala Lys

1 5 10 15

His Ile Glu Ala Gly Val Thr Gln Phe Pro Ser His Ser Val Ile Glu

20 25 30

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

35 40 45

Asn Leu Tyr Trp Tyr Arg Arg Val Met Gly Lys Glu Ile Lys Phe Leu

50 55 60

Leu His Phe Val Lys Glu Ser Lys Gln Asp Glu Ser Gly Met Pro Asn

65 70 75 80

Asn Arg Phe Leu Ala Glu Arg Thr Gly Gly Thr Tyr Ser Thr Leu Lys

85 90 95

Val Gln Pro Ala Glu Leu Glu Asp Ser Gly Val Tyr Phe Cys Ala Ser

100 105 110

Ser Arg Leu Gly Gln Gly Met Asn Thr Glu Ala Phe Phe Gly Gln Gly

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr

180 185 190

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

195 200 205

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

210 215 220

Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn

225 230 235 240

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

245 250 255

Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Ile Thr Ser Ala Ser Tyr

260 265 270

His Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly

275 280 285

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

290 295 300

Met Val Lys Arg Lys Asp Phe

305 310

<210> 9

<211> 816

<212> DNA

<213> human

<400> 9

atggctttgc agagcactct gggggcggtg tggctagggc ttctcctcaa ctctctctgg 60

aaggttgcag aaagcaagga ccaagtgttt cagccttcca cagtggcatc ttcagaggga 120

gctgtggtgg aaatcttctg taatcactct gtgtccaatg cttacaactt cttctggtac 180

cttcacttcc cgggatgtgc accaagactc cttgttaaag gctcaaagcc ttctcagcag 240

ggacgataca acatgaccta tgaacggttc tcttcatcgc tgctcatcct ccaggtgcgg 300

gaggcagatg ctgctgttta ctactgtgct gtgaatggtg ctacaaacaa gctcatcttt 360

ggaactggca ctctgattgt tgtcctgccc aatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

ccagaaagtt cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agctga 816

<210> 10

<211> 270

<212> PRT

<213> human

<400> 10

Ala Leu Gln Ser Thr Leu Gly Ala Val Trp Leu Gly Leu Leu Leu Asn

1 5 10 15

Ser Leu Trp Lys Val Ala Glu Ser Lys Asp Gln Val Phe Gln Pro Ser

20 25 30

Thr Val Ala Ser Ser Glu Gly Ala Val Val Glu Ile Phe Cys Asn His

35 40 45

Ser Val Ser Asn Ala Tyr Asn Phe Phe Trp Tyr Leu His Phe Pro Gly

50 55 60

Cys Ala Pro Arg Leu Leu Val Lys Gly Ser Lys Pro Ser Gln Gln Gly

65 70 75 80

Arg Tyr Asn Met Thr Tyr Glu Arg Phe Ser Ser Ser Leu Leu Ile Leu

85 90 95

Gln Val Arg Glu Ala Asp Ala Ala Val Tyr Tyr Cys Ala Val Asn Gly

100 105 110

Ala Thr Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Ile Val Val Leu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 11

<211> 816

<212> DNA

＜213〉artificial sequence

<400> 11

atggctttgc agagcactct gggggcggtg tggctagggc ttctcctcaa ctctctctgg 60

aaggttgcag aaagcaagga ccaagtgttt cagccttcca cagtggcatc ttcagaggga 120

gctgtggtgg aaatcttctg taatcactct gtgtccaatg cttacaactt cttctggtac 180

cttcacttcc cgggatgtgc accaagactc cttgttaaag gctcaaagcc ttctcagcag 240

ggacgataca acatgaccta tgaacggttc tcttcatcgc tgctcatcct ccaggtgcgg 300

gaggcagatg ctgctgttta ctactgtgct gtgaatggtg ctacaaacaa gctcatcttt 360

ggaactggca ctctgattgt tgtcctgccc aatatccaga accctgaccc tgccgtgtac 420

cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga ttttgattct 480

caaacaaatg tgtcacaaag taaggattct gatgtgtata tcacagacaa aactgtgcta 540

gacatgaggt ctatggactt caagagcaac agtgctgtgg cctggagcaa caaatctgac 600

tttgcatgtg caaacgcctt caacaacagc attattccag aagacacctt cttccccagc 660

tcagacgttc cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta 720

aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt ggccgggttt 780

aatctgctca tgacgctgcg gctgtggtcc agctga 816

<210> 12

<211> 270

<212> PRT

＜213〉artificial sequence

<400> 12

Ala Leu Gln Ser Thr Leu Gly Ala Val Trp Leu Gly Leu Leu Leu Asn

1 5 10 15

Ser Leu Trp Lys Val Ala Glu Ser Lys Asp Gln Val Phe Gln Pro Ser

20 25 30

Thr Val Ala Ser Ser Glu Gly Ala Val Val Glu Ile Phe Cys Asn His

35 40 45

Ser Val Ser Asn Ala Tyr Asn Phe Phe Trp Tyr Leu His Phe Pro Gly

50 55 60

Cys Ala Pro Arg Leu Leu Val Lys Gly Ser Lys Pro Ser Gln Gln Gly

65 70 75 80

Arg Tyr Asn Met Thr Tyr Glu Arg Phe Ser Ser Ser Leu Leu Ile Leu

85 90 95

Gln Val Arg Glu Ala Asp Ala Ala Val Tyr Tyr Cys Ala Val Asn Gly

100 105 110

Ala Thr Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Ile Val Val Leu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

<210> 13

<211> 1818

<212> DNA

＜213〉artificial sequence

<400> 13

atggtttcca ggctcctcag tttagtgtcc ctttgtctcc tgggagcaaa gcacatagaa 60

gctggagtta ctcagttccc cagccacagc gtaatagaga agggccagac tgtgactctg 120

agatgtgacc caatttctgg acatgataat ctttattggt atcgacgtgt tatgggaaaa 180

gaaataaaat ttctgttaca ttttgtgaaa gagtctaaac aggatgagtc cggtatgccc 240

aacaatcgat tcttagctga aaggactgga gggacgtatt ctactctgaa ggtgcagcct 300

gcagaactgg aggattctgg agtttatttc tgtgccagca gccgccttgg acaggggatg 360

aacactgaag ctttctttgg acaaggcacc agactcacag ttgtagagga cctgaacaag 420

gtgttcccac ccgaggtcgc tgtgtttgag ccatcaaaag cagagatcgc acacacccaa 480

aaggccacac tggtgtgcct ggccacaggc ttcttccctg accacgtgga gctgagctgg 540

tgggtgaatg ggaaggaggt gcacagtggg gtcagcacgg acccgcagcc cctcaaggag 600

cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660

ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720

aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggcc 780

tggggtagag cagactgtgg cattacctcg gcatcctacc accaaggggt cctgtctgcc 840

accatcctct atgagatcct gctagggaag gccaccctgt atgctgtgct ggtcagcgcc 900

cttgtgttga tggccatggt caagagaaag gatttcggct ccggagccac gaacttctct 960

ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ccatggcttt gcagagcact 1020

ctgggggcgg tgtggctagg gcttctcctc aactctctct ggaaggttgc agaaagcaag 1080

gaccaagtgt ttcagccttc cacagtggca tcttcagagg gagctgtggt ggaaatcttc 1140

tgtaatcact ctgtgtccaa tgcttacaac ttcttctggt accttcactt cccgggatgt 1200

gcaccaagac tccttgttaa aggctcaaag ccttctcagc agggacgata caacatgacc 1260

tatgaacggt tctcttcatc gctgctcatc ctccaggtgc gggaggcaga tgctgctgtt 1320

tactactgtg ctgtgaatgg tgctacaaac aagctcatct ttggaactgg cactctgatt 1380

gttgtcctgc ccaatatcca gaaccctgac cctgccgtgt accagctgag agactctaaa 1440

tccagtgaca agtctgtctg cctattcacc gattttgatt ctcaaacaaa tgtgtcacaa 1500

agtaaggatt ctgatgtgta tatcacagac aaaactgtgc tagacatgag gtctatggac 1560

ttcaagagca acagtgctgt ggcctggagc aacaaatctg actttgcatg tgcaaacgcc 1620

ttcaacaaca gcattattcc agaagacacc ttcttcccca gctcagacgt tccctgtgat 1680

gtcaagctgg tcgagaaaag ctttgaaaca gatacgaacc taaactttca aaacctgtca 1740

gtgattgggt tccgaatcct cctcctgaaa gtggccgggt ttaatctgct catgacgctg 1800

cggctgtggt ccagctga 1818

<210> 14

<211> 42

<212> DNA

＜213〉artificial sequence

<400> 14

cgctcgaggc caggatggtt tccaggctcc tcagtttagt gt 42

<210> 15

<211> 37

<212> DNA

＜213〉artificial sequence

<400> 15

tgtgtgcgat ctctgctttt gatggctcaa acacagc 37

<210> 16

<211> 33

<212> DNA

＜213〉artificial sequence

<400> 16

atcaaaagca gagatcgcac acacccaaaa ggc 33

<210> 17

<211> 39

<212> DNA

＜213〉artificial sequence

<400> 17

ggtggtagga tgccgaggta atgccacagt ctgctctac 39

<210> 18

<211> 38

<212> DNA

＜213〉artificial sequence

<400> 18

gcattacctc ggcatcctac caccaagggg tcctgtct 38

<210> 19

<211> 79

<212> DNA

＜213〉artificial sequence

<400> 19

gttttcttcc acgtctcctg cttgctttaa cagagagaag ttcgtggctc cggagccgaa 60

atcctttctc ttgaccatg 79

<210> 20

<211> 71

<212> DNA

＜213〉artificial sequence

<400> 20

cttctctctg ttaaagcaag caggagacgt ggaagaaaac cccggtccca tggctttgca 60

gagcactctg g 71

<210> 21

<211> 37

<212> DNA

＜213〉artificial sequence

<400> 21

atcacaggga acgtctgagc tggggaagaa ggtgtct 37

<210> 22

<211> 33

<212> DNA

＜213〉artificial sequence

<400> 22

agctcagacg ttccctgtga tgtcaagctg gtc 33

<210> 23

<211> 42

<212> DNA

＜213〉artificial sequence

<400> 23

gataagaatg cggccgctca gctggaccac agccgcagcg tc 42

Claims (10)

1. tuberculosis peptide Ag85B _{199 – 207}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 peptide Ag85B according to claim 1 _{199 – 207}Specificity TCR, it is characterized in that the α chain of described TCR is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous β chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:10; The β chain is to be substituted, to lack and/or increased the aminoacid sequence that one or more amino acid and/or the special and exogenous α chain of end modified rear resulting energy are assembled into the TCR protein molecular by the aminoacid sequence shown in the SEQ ID NO:6.

3. tuberculosis peptide Ag85B according to claim 2 _{199 – 207}Specificity TCR 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:8.

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

5. tuberculosis peptide Ag85B _{199 – 207}The fusion gene of 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 CD8 of Retroviral Transfer claimed in claim 8 ⁺The T cell.

10. the CD8 of each described tuberculosis peptide Ag85B specificity TCR of claim 1～3, claim 4 or 5 described genes, claim 6 or 7 described recombinant retroviral vectors, retrovirus claimed in claim 8, Retroviral Transfer claimed in claim 9 ⁺The application of T cell in the preparation anti-tuberculosis drugs.

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