CN102532269B

CN102532269B - Dominant sequence of delta 1 chain complementary determining region (CDR) 3 in gamma delta T lymphocytes, and T cell receptor (TCR) transfected cells and application thereof

Info

Publication number: CN102532269B
Application number: CN201110126369.3A
Authority: CN
Inventors: 何维; 姜燕; 崔莲仙
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of AMMS; Institute of Basic Medical Sciences of CAMS
Priority date: 2011-05-16
Filing date: 2011-05-16
Publication date: 2014-07-09
Anticipated expiration: 2031-05-16
Also published as: CN102532269A

Abstract

The invention discloses a dominant sequence GTM of a delta 1 chain complementary determining region (CDR) 3 in human gastric cancer tissue-derived tumor-infiltrating gamma delta T lymphocytes, a T cell receptor (TCR) containing the sequence, and TCR transfected cells. The amino acid residue sequence of the GTM is shown as SEQ ID NO.1 in a sequence table. Experiments prove that GTM peptide can be specifically combined with tumor cells, tumor tissue and V delta 1 T cell ligand MICA protein; and a J.RT3-T3.5 cell platform for expressing the TCR containing the dominant sequence GTM of the delta 1 chain CDR3 in the human gastric cancer tissue-derived tumor-infiltrating gamma delta T lymphocytes on a J.RT3-T3.5 surface is established by a lentivirus expression system, gamma delta 1 tumor-infiltrating lymphocytes (TIL) and a tumor identifying and killing mechanism thereof are simulated in the cell level, the killing activity of the transfected cells TCR gamma 4 delta 1 on the tumor cells is obviously increased, and the killing effect is TCR-dependent. The invention plays an important role in the development of medicines for treating malignant tumors and the adoptive treatment of malignant tumors, and has wide application prospects.

Description

Complementary master sequence and TCR acceptor transfectional cell and the application that determines territory 3 of δ 1 chain in gamma delta T lymphocytes

Technical field

The invention belongs to polypeptide and encoding gene and application in biological technical field, particularly relate to complementary master sequence GTM and TCR acceptor transfectional cell and the application in preparation treatment malignant tumor medicine that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of Human Stomach Tissue source.

Background technology

The immunity system of body has produced four kinds of main antigen recognition mechanism during evolution, be divided into the pattern recognition of scavenger cell and dendritic cell (DC), the specific recognition of α β T cell and B cell, NK cell is for identification and identification (the Morita CTHH of gamma delta T cells to antigen of the self-abnormal change in inside, HHMariuzza RAHH, HHBrenner MBHH.Antigen recognition by human gamma delta T cells:pattern recognition by the adaptive immune system.HHSpringer Semin Immunopathol.HH2000, 22 (3): 191-217, Zocchi MRHH, HHPoggi AHH.Role of gammadelta T lymphocytesin tumor defense.HHFront Biosci.HH 2004Sep 1, 9:2588-604, Hochstenbach FHH, HHBrenner MBHH.Newly identified gamma delta and beta delta T-cell receptors.HHJ Clin Immunol.HH 1990Jan, 10 (1): 1-18.).At present, basic research is clear for first three kind antigen recognition mechanism.Scavenger cell and dendritic cell (DC) are by pattern recognition receptors (pattern recognition receptor, PRR) if marker molecule between TLR (toll-like receptor) identification kind is as pathogen-associated molecular (pathogen associated molecular pattern, PAMPs), according to the mutual identification between two standpatters, the pathogenic micro-organism of invading host is removed, be characterized in simple and quick (Takeda KHH, HHKaisho THH, HHAkira SHH.Toll-like receptors.HHAnnu Rev Immunol.HH2003, 21:335-76.Epub 2001 Dec 19.).α β T cell and B cell be main antigen recognition taking meticulous and accurately RM as feature, antigen of differentiation that they can be meticulous according to height diversity and many signals synergy in its antigen receptor storehouse and exotic antigen (epitope) fine distinction of other One's name is legion.NK cell can Direct Recognition act on without antigen with killing and wounding some tumour cells in advance.The activity of NK cell is by its activated receptor (killer activatory receptor, or suppress acceptor (killer cell inhibitory receptor KAR), KIR) some part and on target cell or the interaction of MHC-I quasi-molecule are controlled, its recognition point is by the acceptor with conserved sequence, constant part to be identified, for inside self-abnormal change (Allison TJ, Winter CC, Fournie JJ, Bonneville M, Garboczi DN:Structure of a human gammadelta T-cell antigen receptor.Nature 2001, 411:820-824, Kabelitz D, Wesch D, Hinz T:gamma delta T cells, their T cell receptor usage and role in human diseases.Springer Semin Immunopathol 1999,21:55-75.).But not clear at present as the recognition mechanism that the gamma delta T cells of bridge between the natural immunity and acquired immunity is concrete.

Human gamma delta t cells accounts for human peripheral CD3 ⁺the 2-5% of T cell, but in the local organization such as small intestine or skin, be main T cell subsets.Gamma delta T cells is according to the difference of δ chain V gene, mainly be divided into two hypotypes, peripheral blood is mainly γ 9 δ 2T (V δ 2) cell subsets, and in epithelium taking δ 1 type (V δ 1) as main, be furnished with different γ chains (Hayday AC. gamma delta T cells:a right time and a right place for a conserved third way of protection.Annu Rev Immunol 2000 simultaneously; 18:975-1026.).The gamma delta T cells of activation has very strong cytotoxic effector cell activity, and can produce various cytokines (generally including TNF-α and IFN-γ).But, due to the feature of the non-limiting identification of MHCI to antigen, gamma delta T may be identified the antigen part different from α β T cell, and this makes First Line (the Hayday AC. gamma delta T cells:a right time and a right place for a conserved third way of protection.Annu Rev Immunol 2000 of gamma delta T cells in body defence and immunosurveillance; 18:975-1026.Girardi M, Oppenheim DE, Steele CR, et al.Regulation of cutaneous mal ignancy by gy T cells.Science 2001; 294:605-9.).

The antigen that human gamma delta t cells identification tumor surface is expressed.V δ 1 identifies MICA/B and ULBP family.V δ 1 gamma delta T cells of separating from colorectal cancer patients tumor infiltrating T lymphocyte (TIL) can not only cracking from autologous, also can various allogeneic epithelial tumor cell (the Maeurer MJ of cracking, Martin D, Walter W, et al.Human intestinal Vy1+T lymphocytes recognize tumor cells of epithelial origin.J Exp Med 1996; 183:1681-96.).Under heat stress or oxidative pressure condition, can induce MICA/B and ULBP1-3 on many epithelial tumor cells and some lymphoma cell surfaces with various horizontal expressions.Expressing tumour and the lymphoma cell of MICA/B and ULBP1-3 can be identified and kill and wound by V δ 1T cell, and this killing and wounding by MICA Direct Recognition gamma delta T CR (gamma delta T cells acceptor) realized (Wu J., Groh V., Spies T.T cell antigen receptor engagement and specificity in the recognition of stress-inducible MIC by human epithelial gamma delta T cells.J.Immumol2002; 169:1236-1240.Zhao J, Huang J, Chen H, Cui L, He W.V δ 1 T cell receptor binds specifically to MHC I chain related A:molecular and biochemical evidences.Biochem Biophys Res Commun 2006; 339:232-40.).The V δ 2T cell recognition non-peptide antigen of phosphoric acid class and F1-ATPase.This class antigen is normally from the product of isoprenoid (isoprenoid) biosynthetic pathway.Wherein to gamma delta T stimulating activity the strongest be hydroxyl first isopentenyl diphosphate ((E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate, HMBPP).

The γ chain that TCR (human T cell receptor) mates by δ chain with it forms, its antigen-binding site is formed by six antigenic determinants (CDR), these six CDR effect in the time of identification antigen is different, and wherein CDR3 district, in the time of antigen recognition, occupies dominant position.In view of the CDR3 district of TCR δ CDR3 district (CDR3 δ) and the heavy chain of antibody similarity on structure and function, therefore, the primary structure that researchist has proposed CDR3 δ is the academic hypothesis that determines TCR γ δ identification antigen-specific key position.According to the gene order (OT3) of a CDR3 δ of the gamma delta T cells of epithelial ovarian cancer (OEC) tissue infiltration (gamma delta T IL) TCR, synthetic OT3 peptide, and by external a series of OT3 peptides and target cell, target tissue or derive from the combination experiment of the albumen of target cell, the binding specificity of checking OT3 peptide; Meanwhile, in the corresponding experiment of carrying out with the OT3 grafted antibody (OT3Ab) that OT3 sequence replacing heavy chain of antibody CDR3 region sequence builds, further verified.Prove that CDR3 δ plays a crucial role really in TCR γ delta antigen identification specificity.Utilize OT3 peptide in human ovarian cancer SKOV3 total protein of cell, to search out the molecule hMSH2 part (Novel Ligands that TCR γ δ identifies) of combination with it as probe by the method for affinity chromatography.Confirm that by methods such as RT-PCR, Western Blot, flow cytometry, immunohistochemical methodss hMSH2 expresses in tumour cell and tissue; And experiment in vitro confirms hMSH2 albumen energy specificity and V δ 2T Cell binding, and stimulates its activation and proliferation, secretion IFN-γ, promotes the killing activity of V δ 2T cell to target cell.

The above results is the research to tumour identification from γ 9 δ 2T cells.And the molecular mechanism of the tumour antigen of the V δ 1T cell of another kind of epithelial origin and the evident characteristics of tumour, CDR3 region sequence feature, identification and identification tumour antigen have not been reported.V δ 1 and V δ 2T cell are in very large differences of Fang Douyou such as sequence and structure, distribution, identification antigen part, functions, and therefore, γ δ 2TCR structure C DR3 sequence can not represent other CDR3 sequence of other type TCR and the recognition mechanism of tumour.

Summary of the invention

The invention provides the complementary master sequence GTM that determines territory 3 (CDR3 δ 1) of δ 1 chain in a Human Stomach Tissue source tumor infiltrating gamma delta T lymphocytes (tumor-infiltrating gamma delta T cell, gamma delta T IL).

The complementary master sequence that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of Human Stomach Tissue provided by the present invention source, called after GTM is one of following amino acid residue sequences:

1) the SEQ ID NO:1 in sequence table;

2) by the amino acid residue sequence of SEQ ID NO:1 in sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and there is the protein of antigen recognition effect.

SEQ ID NO:1 in sequence table is made up of 15 amino-acid residues.

δ 1 catenin that contains the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source also belongs to protection scope of the present invention.

Specifically, δ 1 chain that contains the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source, called after TRD1G is one of following amino acid residue sequences:

1) the SEQ ID NO:2 in sequence table;

2) by the amino acid residue sequence of SEQ ID NO:2 in sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and there is the protein of antigen recognition effect.

SEQ ID NO:2 in sequence table is made up of 286 amino-acid residues, is GTM sequence from aminoterminal 111-125 amino acids residue.

With γ 4 chains that the above-mentioned δ 1 chain TRD1G that contains the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source matches, called after TRG4G is one of following amino acid residue sequences:

1) the SEQ ID NO:3 in sequence table;

2) by the amino acid residue sequence of SEQ ID NO:3 in sequence table through replacement, disappearance or the interpolation of one to ten amino-acid residue and there is the protein of antigen recognition effect.

SEQ ID NO:3 in sequence table is made up of 324 amino-acid residues.

The TCR of a kind of stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL), the amino acid residue sequence of its δ 1 chain is as shown in SEQ ID NO:2 in sequence table (TRD1G), and the amino acid residue sequence of its γ 4 chains is as shown in SEQ ID NO:3 in sequence table (TRG4G).

The encode gene (GTM) of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source is one of following nucleotide sequence:

1) DNA sequence dna of SEQ ID NO:4 in sequence table;

2) DNA sequence dna of SEQ ID NO:1 in code sequence list;

3) with sequence table in the nucleotide sequence that limits of SEQ ID NO:4 there is 90% above homology and there is the nucleotide sequence of antigen recognition effect;

4) under the rigorous condition of height can with sequence table in the nucleotide sequence of the DNA sequence dna hybridization that limits of SEQ ID NO:4.

The rigorous condition of described height is used containing the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1% SDS and wash film at 65 DEG C for hybridization is rear.

SEQ ID NO:4 in sequence table is by 45 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID NO:1 in sequence table from 5 ' end 1-45 bit base.

The gene that coding contains δ 1 catenin of the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source also belongs to protection scope of the present invention.

Specifically, the gene (TRD1G) that coding contains the δ 1 chain TRD1G of the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source is one of following nucleotide sequence:

1) DNA sequence dna of SEQ ID NO:5 in sequence table;

2) DNA sequence dna of SEQ ID NO:2 in code sequence list;

3) with sequence table in the nucleotide sequence that limits of SEQ ID NO:5 there is 90% above homology and there is the nucleotide sequence of antigen recognition effect;

4) under the rigorous condition of height can with sequence table in the nucleotide sequence of the DNA sequence dna hybridization that limits of SEQ ID NO:5.

SEQ ID NO:5 in sequence table is by 858 based compositions, its encoding sequence is from 5 ' end 1-858 bit base, be GTM encoding sequence from 5 ' end 331-375 bit base, coding has the protein of the amino acid residue sequence of SEQ ID NO:2 in sequence table.

The gene (TRG4G) of the γ 4 chain TRG4G that the δ 1 chain TRD1G that coding contains the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source matches is one of following nucleotide sequence:

1) DNA sequence dna of SEQ ID NO:6 in sequence table;

2) DNA sequence dna of SEQ ID NO:3 in code sequence list;

3) with sequence table in the nucleotide sequence that limits of SEQ ID NO:6 there is 90% above homology and there is the nucleotide sequence of antigen recognition effect;

4) under the rigorous condition of height can with sequence table in the nucleotide sequence of the DNA sequence dna hybridization that limits of SEQ ID NO:6.

SEQ ID NO:6 in sequence table is by 972 based compositions, and its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID NO:3 in sequence table from 5 ' end 1-972 bit base.

The encode gene of TCR of described stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL), the nucleotide sequence of its δ 1 chain encoding gene is as shown in SEQ ID NO:5 in sequence table (TRD1G), and the nucleotide sequence of its γ 4 chain encoding genes is as shown in SEQ ID NO:6 in sequence table (TRG4G).

The expression vector, transgenic cell line and the Host Strains that contain gene of the present invention all belong to protection scope of the present invention.

The primer pair of the arbitrary fragment in gene of the present invention of increasing is also within protection scope of the present invention.

Described transgenic cell is transgenosis J.RT3-T3.5 cell, and this is a kind of redundant organism of people Jurkat Leukemia Cell Lines, and it can not produce the TCR acceptor of function, but the TCR that can be used for other external source of transfection is to study its antigen-specific; Can be according to will the encode gene transfection J.RT3-T3.5 cell of TCR of described stomach organization source tumor infiltrating lymphocyte (gamma delta T IL) cell of ordinary method, as the recombinant eukaryon expression vector of the tcr gene by containing the described stomach organization source tumor infiltrating gamma delta T lymphocytes of coding (gamma delta T IL) will encode as described in the originate tcr gene importing J.RT3-T3.5 cell of tumor infiltrating gamma delta T lymphocytes (gamma delta T IL) of stomach organization.

The carrier that sets out of the recombinant eukaryon expression vector of the tcr gene that contains the described stomach organization source tumor infiltrating gamma delta T lymphocytes of coding (gamma delta T IL) for structure can be the Lentivirals such as pRSV-rev, pLentilox 3.7, pMDLg, pRRE, pWPXL or pLVX-DsRed, is preferably pWPXL; With pWPXL for the carrier that sets out, the recombinant eukaryon expression vector of the δ 1 chain encoding gene (TRD1G) of the TCR that carries described stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL) building is pWPXL-δ 1, and the recombinant eukaryon expression vector of the γ 4 chain encoding genes (TRG4G) of the TCR that carries described stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL) of structure is pWPXL-γ 4.

The conventional biological methods such as the recombinant eukaryon expression vector of the described tcr gene that carries coding described stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL) can turn by liposome mediated-method, electricity, adenovirus, adeno-associated virus, retrovirus or slow virus transform J.RT3-T3.5 cell.

It is TCR γ 4 δ 1 transfectional cells that described transfection has the J.RT3-T3.5 cell of the tcr gene of the described stomach organization source tumor infiltrating gamma delta T lymphocytes of coding (gamma delta T IL).

The complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of described Human Stomach Tissue source, δ 1 catenin that contains the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in above-mentioned Human Stomach Tissue source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL), δ 1 chain is TRD1G, TCR and the coding said polypeptide of the stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL) that γ 4 chains are TRG4G, the gene of albumen can be used for preparing medicine or the preparation of killing off tumor cells, thereby the present invention provides foundation for further developing a kind of antitumor drug.

The antitumor drug of indication of the present invention, its activeconstituents is the complementary master sequence GTM that determines territory 3 of δ 1 chain in the above-mentioned Human Stomach Tissue source tumor infiltrating gamma delta T lymphocytes with antitumor action, or δ 1 catenin that contains the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source, or δ 1 chain is the gene of the TCR of TRD1G, the γ 4 chains stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL) that is TRG4G or coding said polypeptide, albumen.

The gene of coding said polypeptide, albumen can be present in J.RT3-T3.5 cell, and wherein to have the J.RT3-T3.5 cell of the gene of the TCR of coding described stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL) be TCR γ 4 δ 1 in transfection.

When needs, in said medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises thinner, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent and the absorption carrier etc. of pharmaceutical field routine.

Medicine of the present invention can be made the various ways such as injection liquid or lyophilisate.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.

The consumption of above-mentioned protein drug and genomic medicine can adopt the routine dose of protein drug and genomic medicine in pharmaceutical field, and can be according to practical situation adjustment.Generally, TCR γ 4 δ 1 cells of the present invention are 1.0 × 10 in injected dose ⁵-2.0 × 10 ⁵individual/when kg body weight, can to delay the growth of tumour.

The invention provides complementary master sequence GTM, the TCR that contains this sequence and the TCR acceptor transfectional cell that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of Human Stomach Tissue source.Experiment showed, that GTM peptide can be combined with tumour cell, tumor tissues and V δ 1T cell ligand MICA protein-specific.Set up the J.RT3-T3.5 cell platform that contains the TCR of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of Human Stomach Tissue source at J.RT3-T3.5 surface expression by Lentivirus expression system, on cell levels, simulated γ δ 1TIL and identification killing tumor cells mechanism (wherein an approach is: TCR identifies tumour antigen, and then release cells factor TNF α carrys out killing tumor cell), result shows that transfectional cell TCR γ 4 δ 1 obviously increase the killing activity of tumour cell, and this lethal effect is that TCR is dependent.The present invention, by playing a significant role in the exploitation for the treatment of malignant tumor medicine and the treatment of adopting thereof, has a extensive future.

Below in conjunction with specific embodiment, the present invention is described in further details.

Brief description of the drawings

Fig. 1 is the ratio of gamma delta T IL in 4 routine cancer of the stomach source TIL

Fig. 2 is the Subtype of δ chain in a routine cancer of the stomach source gamma delta T IL (W2-gamma delta T IL)

Fig. 3 is the agarose gel electrophoresis detected result of the V district full length sequence of the δ of pcr amplification W2-gamma delta T IL

Fig. 4 is W2-gamma delta T IL (A) and the lethal effect of S2-gamma delta T IL (B) to different tumour cells

Fig. 5 is the phenotype of Flow cytometry W2-gamma delta T IL

Fig. 6 is GTM peptide and the combination experimental result of different tumour cells, tumor tissues and ligandin MICA

Fig. 7 is the agarose gel electrophoresis detected result of δ 1 chain full-length gene and the γ 4 chain full-length genes of pcr amplification

Fig. 8 is the Flow cytometry result (pWPXL transfectional cell is contrast) of TCR γ 4 δ 1 transfectional cell surface gamma δ TCR expressions

Fig. 9 is the lethal effects of TCR γ 4 δ 1 transfectional cells to Daudi cell

Figure 10 is Flow cytometry TCR γ 4 δ 1 transfectional cells (pWPXL transfectional cell is contrast) are hatched respectively rear TNF α expression with anti-gamma delta T CR (A) and MICA albumen (B)

Embodiment

The present invention has optimized the condition that amplification in vitro is cultivated tumor infiltrating gamma delta T cells (gamma delta T IL), and the gamma delta T IL of V δ 1 phenotype is turned out in success from the tumor tissues in 4 routine cancer of the stomach sources.External have killing activity to tumour cell.

CDR3 δ 1 sequence of the tumour γ δ 1TIL that the present invention cultivates amplification in vitro is analyzed, and found that: CDR3 δ 1 sequence of every routine γ δ 1TIL has comparative advantage feature; Wherein in 4 routine cancer of the stomach source γ δ 1TIL, the CDR3 district master sequence of 3 examples is identical and total master sequence is short compared with other sequence.Choose the basis of the total CDR3 δ 1 district master sequence GTM of 3 routine cancer of the stomach source gamma delta T IL as follow-up structural research.

Synthetic GTM peptide, utilizes flow cytometry, laser confocal scanning microscope technology, ELISA, SPR, ImmunohistochemistryMethods Methods prove GTM Toplink and tumour cell or organize district's specific binding in γ δ 1T-CDR3 peptide level.Affinity chromatography method confirms that disappearance exists the albumen of being combined with GTM peptide specific, i.e. tumour antigen part in tumour cell total protein.

Success amplifies complete γ 4 chains and δ 1 chain total length from the gamma delta T IL in a routine cancer of the stomach source, and set up the platform at complete TCR γ 4 δ 1 of J.RT3-T3.5 cell surface stably express by Lentivirus expression system, to simulating γ δ 1TIL and then study it molecular mechanism of identifying tumour on cell levels.Transfectional cell can killing tumor cell and this lethal effect be that TCR is dependent, brought wide possibility to functional genome and following gene therapy application.

Embodiment implements under taking technical solution of the present invention as prerequisite, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.

In following embodiment, method therefor is ordinary method if no special instructions.

The acquisition of the complementary master sequence GTM that determines territory 3 (CDR3 δ 1) of δ 1 chain in embodiment 1, Human Stomach Tissue source tumor infiltrating gamma delta T lymphocytes (tumor-infiltrating gamma delta T cell, gamma delta T IL)

One, cultivation and the qualification of gamma delta T IL cell in epithelial origin tumour

1, the cultivation of gamma delta T IL cell

With coated 24 orifice plates of anti-pan-TCR γ δ mAb (purchased from Beckman Coulter), 5 μ g/ holes, hatch 2h for 37 DEG C.The solid tumor such as cancer of the stomach, the kidney sample (epithelial origin) that aseptic operation is gathered embathes 2 times in RPMI-1640 substratum (containing penicillin 200 μ g/mL, Streptomycin sulphate 100 μ g/mL, gentamicin 100 μ g/mL), 10min/ time; Remove necrotic tissue, surrounding blood vessel and healthy tissues; Washed tissue is shredded, wash 2 times with RPMI-1640 substratum (containing penicillin 200 μ g/mL, Streptomycin sulphate 100 μ g/mL), 10min/ time, be then resuspended in RPMI-1640 perfect medium.By the resuspended fragment upper plate of organizing, add IL-2 (interleukin II, purchased from Ruide, Beijing He Tong pharmaceutcal corporation, Ltd, 200U/mL); 37 DEG C, 5%CO ₂cultivate two weeks, within every 3 days, change liquid once.

2, flow cytometer detects the TCR phenotype of cultivating til cell:

Collecting cell, the centrifugal 5min of 1000rpm, adjusts every tube cell number to 1 × 10 ⁶individual.Add 1mL PBS washing lotion and blow and beat gently, re-suspended cell, the centrifugal 5min of 1000rpm, washes 2 times.Add antibody (FITC-anti-TCR γ δ/PE-anti-TCR α β or FITC-anti-V δ 1, PE-anti-V δ 1, FITC-anti-V δ 3, purchased from Beckman Coulter or Pierce) and homotype contrast (FITC-mouse IgG/PE-mouse IgG, purchased from Beckman Coulter or Pierce), do not add antibody pipe and make negative control, 4 DEG C of lucifuges are hatched 30min.Add 1mL PBS washing lotion washed cell, the centrifugal 5min of 3000rpm, washes 3 times.Every tube cell adds the PBS stationary liquid 0.5mL of 4 DEG C of precoolings, and piping and druming, has hanged cell gently, and 400 mesh filter screens filter, and 4 DEG C keep in Dark Place to be measured.

As Fig. 1, (X-coordinate represents TCR γ δ to partial detection, and ordinate zou represents TCR α β; W2 represents a routine stomach organization source gamma delta T IL (called after W2), W3 represents a routine stomach organization source gamma delta T IL (called after W3), W4 represents a routine stomach organization source gamma delta T IL (called after W4), W5 represents that (X-coordinate represents V δ 1 or V δ 2 or V δ 3 to routine stomach organization source gamma delta T IL (called after W5) and a Fig. 2, ordinate zou represents cell count) shown in, the gamma delta T IL that successfully cultivates altogether the different tumor tissues of many cases source, in Flow cytometry epithelial origin tumour, gamma delta T IL cell majority is δ 1 type.Meet document (Bennett WTHH, HHPandolfi FHH, HHGrove BHHH, HHHawes GEHH, HHBoyle LAHH, HHKradin RLHH, HHKurnick JTHH.Dominant rearrangements among human tumor infiltrating lymphocytes.Analysis of T cells derived from 32 patients with melanoma, lung, and renal cell carcinoma.HHCancer.HH 1992 May 1; 69 (9): 2379-84.) the gamma delta T IL cell of the epithelial origin of report is mainly V δ 1 type, and the gamma delta T IL cell of derived from peripheral blood is mainly V δ 2 types.

Two, γ δ 1TIL CDR3 δ 1 region sequence analysis

1, the extraction of cell total rna

The gamma delta T IL cell of collecting the different sources (deriving from the solid tumor such as cancer of the stomach, kidney tissue) of cultivating, PBS washes once; By every 5 × 10 ⁶the ratio of cells/mL adds Trizol reagent, and piping and druming makes the complete cracking of cell, incubated at room 5min repeatedly; Move in the EP pipe of processing through DEPC, add the chloroform of 1/5 volume, thermal agitation 15sec, room temperature leaves standstill 3min; 4 DEG C, 10, the centrifugal 15min of 000rpm, carefully draws in upper strata water to new pipe; Add the Virahol of 1/2 volume, ice bath 10min; 4 DEG C, 12, the centrifugal 10min of 000rpm, abandons supernatant, adds 1mL 75% ethanol washing and precipitating, washes twice.4 DEG C, 12, the centrifugal 10min of 000rpm, inhales and abandons supernatant, drying at room temperature precipitation.By DEPC water dissolution precipitation, save backup for reverse transcription or-80 DEG C immediately.

2, synthetic cDNA the first chain of reverse transcription

Use the MMLV reverse transcription test kit purchased from Promega, the cell total rna extracting taking step 1 is template, and its cDNA first chain is synthesized in reverse transcription, and method is: get RNA sample 2 μ g and Oligo (dT) ₁₅(500 μ g/ml) 1 μ l, adds DEPC water to 13 μ l, 70 DEG C of 10min; After taking out, be placed in ice bath, add successively 5 × Buffer5 μ l, dNTP (10mmol/L) 5 μ l, RNA enzyme inhibitors 1 μ l, MMLV reversed transcriptive enzyme 1 μ l, add DEPC water to 25 μ l, 42 DEG C, 60min carries out reverse transcription reaction at once; 70 DEG C of 15min inactivator activity.

3, pcr amplification TCR δ gene

The primer that this step is used is synthesized by Qing Ke Bioisystech Co., Ltd, primer concentration is 10pmol/ μ l, uses internal reference primer P-UP, P-DN and gamma delta T IL δ chain V district (being called for short V δ) total length sequencing primer V δ 1, V δ 2, V δ 3, C δ.Wherein V δ is 5 ' terminal specific primer, uses with 3 ' end primer C δ pairing, can amplify CDR3 gene fragment, and primer sequence is as shown in table 1:

Table 1 primer sequence

primer title	primer sequence
		v δ
1	5 '-GCCCAGAAGGTTACTCAAGCCCAG-3 ';
		v δ 2	5 '-GCCATTGAGTTGGTGCCTGAACAC-3 ';
v δ 3	5 '-TGTGACAAAGTAACCCAGAGTTCCCC-3 ';
		c δ (reverse)	5 '-AAACGGATGGTTTGGTATGAGGC-3 ';
p-UP (β-actin-UP)	5 '-TACCACTGGCATCGTGATGGACTC-3 ';
		p-DN (β-actin-DN)	5 '-TCCTGCTTGCTGATCCACATCTGC-3 '.

Taking synthetic cDNA the first chain of step 2 reverse transcription as template, pcr amplification gamma delta T IL δ chain V region sequence under the guiding of above-mentioned primer (primer pair V δ 1 and C δ amplification V δ 1 gene, primer pair V δ 2 and C δ amplification V δ 2 genes, primer pair V δ 3 and C δ amplification V δ 3 genes), PCR reaction system is as follows:

cDNA 1μl，

The each 1 μ l of primer,

10 × PCR reaction buffer, 5 μ l,

MgClRR ₂RR(25mM) 3μl，

dNTP(2.5mM) 4μl，

Taq plusDNA polysaccharase (1u/ μ is 1 μ l l),

dd HRR ₂RRO 34μl，

Cumulative volume 50 μ l;

PCR parameter is: 94 DEG C of denaturation 5min, and 94 DEG C of 30sec, 58 DEG C of annealing 45sec, 72 DEG C are extended 1min, 30 circulations, last 72 DEG C are extended 10min again.After reaction finishes, get 5 μ l PCR products and carry out 1% agarose gel electrophoresis analysis, as Fig. 3, (DNA molecular amount Marker is DL-2000 to result, swimming lane P represents internal reference β-actin, swimming lane 1 represents V δ 1/C δ amplified production, swimming lane 2 represents V δ 2/C δ amplified production, swimming lane 3 represents V δ 3/C δ amplified production) shown in, obtain the DNA fragmentation of the V δ 1 of 367bp through amplification under the guiding of primer pair V δ 1 and C δ, primer pair V δ 2 and C δ, V δ 3 and the C δ goal gene that do not increase, conforms to expected results.

4, δ 1 gene clone of gamma delta T IL

(1) recovery of target DNA fragment: reclaim the object fragment that test kit (AXYGEN) recycling step 3 increases with DNA gel;

(2) ligation: the object fragment of recovery is connected in pGEM-T Easy carrier (purchased from Promega), and reaction system is as follows:

Above-mentioned reactant fully mix and centrifugal make its be sunken to pipe the end after, 22 DEG C connect 2h.

(3) conversion of recombinant plasmid

Get E.coli DH5 α competent cell 50 μ l, add 10 μ l to connect product, mix gently, place 30min on ice; 42 DEG C of heat-shocked 90sec, insert rapidly in ice ice bath 5min; Add 200 μ l LB substratum, 37 DEG C, 200rpm shaking culture 1h; Now can be by LB flat board (containing 50 μ g/mL Amp) coating IPTG (40 μ g/mL) and X-gal (24 μ g/mL), to carry out blue hickie screening.After coated plate, 37 DEG C of overnight incubation to colony diameters are 1～2mm.Picking white colony is inoculated in 5mL LB substratum as positive colony, 37 DEG C of overnight incubation.

5, positive colony sequential analysis

Send positive colony bacterium liquid to the order-checking of Nuo Sai biotech company, sequencing primer uses the universal primer of T carrier.DNAMAN software analysis sequence results.Result is as shown in table 2, and CDR3 δ 1 sequence of every routine γ δ 1TIL has comparative advantage feature; Wherein in 4 examples (W2, W3, W4, W5) cancer of the stomach source γ δ 1TIL, CDR3 δ 1 master sequence of 3 examples is identical, called after GTM sequence ( cA FLPHA DKLIFGKG, i.e. SEQ ID NO:1 in sequence table), and this total master sequence is short compared with other sequence.

CDR3 δ 1 sequence and frequency brief summary thereof in the routine cancer of the stomach of table 24 source γ δ 1TIL

The killing ability to tumour and the phenotype analytical of embodiment 2, γ δ 1TIL

1, the killing ability of gamma delta T IL to tumour cell

Use cytotox 96 non-radioactive cytotoxicity assay reagents kit (Promega, USA) reference reagent box specification sheets to carry out fragmentation test.Taking W2-gamma delta T IL (deriving from stomach organization) and S2-gamma delta T IL (deriving from renal carcinoma tissue) as effector cell, detect respectively its killing-efficiency to different tumour cell-gastric adenocarcinoma cells BGC-823 (deriving from people's cancer of the stomach), kidney cancer cell G401 (deriving from people's kidney) and colon cancer cell HT-29 (deriving from human colon carcinoma).Target cell 1 × 10 ⁴/ hole adds 96 orifice plates, makes to imitate target ratio and is respectively 2.5: 1,5: 1,10: 1.(figure A is the W2-gamma delta T IL in the cancer of the stomach source kill rate to tumour cell to result as shown in Figure 4, figure B is the S2-gamma delta T IL in the kidney source kill rate to tumour cell), this two routine gamma delta T IL all has certain lethal effect to 3 kinds of tumour cells, and kill and wound and there is source specificity, the W2-gamma delta T IL that is cancer of the stomach source is the highest to gastric carcinoma cell lines BGC-823 killing-efficiency, and the S2-gamma delta T IL in kidney source is the highest to renal carcinoma cell line G401 killing-efficiency.

2, the phenotype analytical of W2-gamma delta T IL

Fluidic cell staining procedure is with embodiment 1.Detect the cell surface molecule expression (NKG2D, CD4, CD8, CD80, CD86, CD56, CD69) of W2-gamma delta T IL.

(X-coordinate represents TCR γ δ to result as shown in Figure 5, ordinate zou represents various surface moleculars), the phenotype analytical of W2-gamma delta T IL shows W2-gamma delta T IL surface NKG2D, CD8, CD86, CD56, the equal high expression level of CD69, and CD4, CD80 be without expression, shows to be mainly to kill and wound phenotype.

δ 1 chain complementary decision master sequence-CDR3 δ 1 (GTM) peptide in territory 3 and the combination of tumour cell, tumor tissues and V δ 1 ligandin MICA in embodiment 3, Human Stomach Tissue source tumor infiltrating gamma delta T lymphocytes

1, the synthetic and biotinylation of GTM peptide

Complementary master sequence-CDR3 δ 1 (GTM) peptide that determines territory 3 of δ 1 chain in Human Stomach Tissue source tumor infiltrating gamma delta T lymphocytes shown in SEQ ID NO:1 in employing chemical synthesis composition sequence table, direct biotin molecule of mark endways when synthetic, taking PEPC H3 (WPHNWWPHFKVK) as contrast.

2, the binding characteristic of Flow cytometry GTM peptide and different tumour cells

BGC-823 good growth conditions, G401, lung adenocarcinoma cell GLC-82 (deriving from human lung adenocarcinoma), HT-29, Proliferation of Human Ovarian Cell SKOV3 (deriving from human ovarian cancer) are digested to collection, and centrifugal rear to be adjusted to concentration with PBS be 1 × 10 ⁶the cell suspension of cells/mL, respectively asks for 0.5mL in each EP pipe, and washed cell adds biotinylated GTM peptide or control peptide CH340 μ g/ pipe; Blank pipe and two anti-control tube only add PBS washing lotion; Hatch 30min for 4 DEG C, wash three times.Add FITC-Streptavidin (purchased from eBioscience) 5 μ l, blank pipe only adds PBS washing lotion, 4 DEG C of lucifuges are hatched 30min, wash three times, in every tube cell, add the PBS stationary liquid 0.5mL of 4 DEG C of precoolings, gently piping and druming, hang cell, cell suspension is filtered in new EP pipe, and 4 DEG C keep in Dark Place, and flow cytometer detects.

As shown in Figure 6A, compared with the control, GTM peptide all has combination in various degree from different tumour cells to Flow cytometry result, and combination rate is higher.

3, laser confocal scanning microscope detects the combination of GTM peptide and different tumour cells

Process cover glass: in acid (potassium bichromate 120g: vitriol oil 200mL: distilled water 1000mL), soak 48h, clean, in 95% ethanol, soak, dry, in poly-lysine (concentration 0.1mg/mL), soak 1h, dry high-temperature sterilization.Paving cell: get aseptic 24 orifice plates, the rear also dry cover glass of a slice sterilization is put in every hole.It is 1 × 10 that BGC-823 good growth conditions, G401, GLC-82, HT-29, SKOV3 cell are adjusted to concentration ⁵the cell suspension of cells/mL, every hole 1mL, 37 DEG C of hold over night.Washed cell: every hole adds 1mL PBS damping fluid, 3min × 4 time, in whole operating process, action softly, avoids touching the cell of having grown on slide.Fixed cell: every hole adds 0.5mL 4% paraformaldehyde, and room temperature leaves standstill 20min; Stationary liquid is abandoned in suction, washed cell 1 time.Closing cell: every hole adds 0.5mL to contain the PBS confining liquid of 10% lowlenthal serum, does not have cover glass, incubated at room 45min; Confining liquid is abandoned in suction.The liquid in hole that exhausts completely, it is moistening that slide keeps, and every hole adds 0.25mL peptide (PBS confining liquid is joined, 10 μ g/mL), incubated at room 45min.Primary antibodie is abandoned in suction, washed cell.Then every hole adds 0.25ml FITC-Streptavidin (dilution in 1: 500), and room temperature lucifuge is hatched 45min; Suction is abandoned two and is resisted, washed cell 3 times.In green skies kit, Hochest dyes core 1min.Washed cell.Mountant mounting in green skies kit, laser confocal scanning microscope detects.

As shown in Figure 6B, compared with the control, GTM peptide all has combination in various degree from different tumour cells to laser confocal scanning microscope detected result, and combination rate is higher.

4, utilize the combination of BIA technology for detection GTM peptide and MICA

(1) the coated CM5 chip of V δ 1 ligandin MICA

First to fixation reaction damping fluid (10mM acetic acid (acetate), purchased from GE) pH value and the concentration of coating protein be optimized screening, the acetate buffer of choosing the pH value (pH5.0) that maximum response is corresponding is as the immobilised buffer system of experiment.The concentration of the coated CM5 chip (purchased from GE) of MICA albumen (this group expression and purification) is decided to be to 8000RU.

(2) grope elution requirement, this elution requirement (20mM glycine-HCl) is lower can be washed down GTM peptide and mutant peptide thereof completely and can not make again the albumen being coated on chip be washed down, gets back to baseline.

(3) select protein-bonded maximum concentration, gradient dilution sample according to Rmax value.

(4) utilize auto-programming to carry out avidity mensuration according to the specification sheets of Biacore3000.Each circulation is divided into balance baseline-combination-dissociate-regenerate-reply baseline, can carry out next circulation after completing, and each concentration is at least done 2 circulations.

(5) BIACORE 3000 Evaluation softwares carry out data analysis.

Biacore detected result as shown in Figure 6 C, compared with the control, can be combined with tumour antigen part-V δ 1 ligandin MICA by GTM peptide.

5, immunohistochemical method detects the combination of GTM peptide and various cancerous tissues

As shown in Figure 6 D, tissue slice is the paraffin section of cancer of the stomach (G), kidney (R), lung cancer (L), colorectal carcinoma (C) and ovarian cancer (O) tissue.Roasting sheet: 60 DEG C of 2h.Paraffin section routine dewaxes to water (dimethylbenzene-alcohol-water).Citric Acid is repaired, and exposes antigen, 93 DEG C of 10min, ddH ₂o washed twice.3% H ₂o ₂process 10min to remove the activity of endogenous peroxydase.Aquation, PBS rinses, and 3min/ time, totally 3 times.Sealing, 10% normal sheep serum (PBS dilution), hatches 10min for 37 DEG C.On slide, drip GTM peptide or CH3 peptide (final concentration is 50 μ g/mL), make it just cover cell, hatch 1h for 37 DEG C.Separately establish antibody diluent in contrast.PBS rinses, and 3min/ time, totally 3 times.Drip HRP-Streptavidin (horseradish peroxidase-labeled Streptavidin, purchased from Pierce, dilutes at 1: 100), incubated at room 30min.PBS rinses, and 3min/ time, totally 3 times.Drip DAB nitrite ion, rinse termination reaction with tap water in good time.Hematorylin is redyed, and routine is dewatered to dimethylbenzene (water-ethanol-dimethylbenzene).Gelatin mounting, takes a picture.

As shown in Figure 6 D, compared with the control, GTM peptide all has combination in various degree from different tumor tissues (cancer of the stomach, kidney, lung cancer, colorectal carcinoma, ovarian cancer) to immunohistochemical methods detected result.

Embodiment 4, transfection have the acquisition of J.RT3-T3.5 gamma delta T lymphocytes TCR γ 4 δ 1 of the gene of the TCR of the described stomach organization source tumor infiltrating gamma delta T lymphocytes of coding (gamma delta T IL)

One, carry respectively the structure of the recombinant slow virus expression vector of total length δ 1 (TRD1G, containing GTM), γ 4 (TRG4G) gene

The δ 1 chain called after TRD1G of the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of above-mentioned Human Stomach Tissue source will be contained, by the γ 4 chain called after TRG4G that match with TRD1G, build and carry respectively total length δ 1 (TRD1G, containing GTM), the recombinant slow virus expression vector of γ 4 (TRG4G) gene, concrete grammar comprises the following steps:

1, the extraction of cell total rna

The RNA that extracts the gamma delta T IL (W2) in cancer of the stomach source with Trizol reagent, method is with embodiment 1.

2, synthetic cDNA the first chain of reverse transcription

Method is with embodiment 1.

3, pcr amplification TCR δ 1 and γ 4 full-length genes

δ 1 chain gene total length amplimer is TR δ 1-UP-BamHI and TR δ-DN-MluI, and γ 4 chain gene total length amplimers are TR γ 4-UP-PmeI and TR γ-DN-MluI, and primer sequence is as shown in table 3.

Table 3 primer sequence

title	primer sequence
		tR δ 1-UP-BamHI	5 '-CGCGGATCCGCCACCATGCTGTTCTCCAGC CTGCTG-3 '
tR δ-DN-MluI	5 '-CGACGCGTTCATTACAAGAAAAATAACTTGGCAGTC-3 '
		tR γ 4-UP-PmeI	5 '-GGGTTTAAACGCCACCATGCAGTGGGCCCT AGCGG-3 '
tR γ-DN-MluI	5 '-CGACGCGTTCATCATGATTTCTCTCCATTGCAGCAG-3 '.

Taking synthetic cDNA the first chain of step 2 reverse transcription as template, pcr amplification δ 1 chain full-length gene under the guiding of TR δ 1-UP-BamHI and TR δ-DN-MluI, pcr amplification γ 4 chain full-length genes under the guiding of TR γ 4-UP-PmeI and TR γ-DN-MluI, the archaeal dna polymerase used that increases is Phusion archaeal dna polymerase (FINNZYMES), and PCR reaction system is as follows:

PCR parameter is: 98 DEG C of denaturation 30sec, and 98 DEG C of 10sec, 98 DEG C of annealing 30sec, 54 DEG C are extended 50sec, 30 circulations, last 72 DEG C are extended 5min again.After reaction finishes, get 5 μ l PCR products and carry out 1% agarose gel electrophoresis detection, as Fig. 7, (DNA molecular amount Marker is DL-2000 to result, δ 1 chain full-length gene (A) and γ 4 chain full-length genes (B)) shown in, obtain the δ 1DNA fragment of 858bp and the γ 4DNA fragment of 972bp through amplification, conformed to expected results.

4, being connected of TRD1G and TRG4G and carrier

(1) single endonuclease digestion of carrier pWPXL and PCR product reaction

Carrier pWPXL (purchased from Clontech) and TRD1G gene PCR amplified production carry out double digestion with BamHI and MluI respectively, and carrier pWPXL and TRG4G gene PCR amplified production carry out double digestion with PmeI and MluI respectively, and endonuclease reaction system is as follows:

37 DEG C of enzymes are cut 2 hours, reclaim test kit (AXYGEN) reclaim endonuclease bamhi with DNA gel.

(2) ligation

Endonuclease bamhi is connected in the carrier pWPXL cutting through same enzyme enzyme, and ligation system is as follows:

Above-mentioned reactant fully mixes and centrifugal it is sunken at pipe the end, and 4 DEG C of connections are spent the night, and obtain recombinant plasmid, respectively called after pWPXL-δ 1 and pWPXL-γ 4.

5, the conversion of recombinant plasmid and qualification

(1) conversion of recombinant plasmid

Method is with embodiment 1.

(2) enzyme of recombinant plasmid is cut qualification

Method is with embodiment 1, and pWPXL-δ 1 cuts through BamHI and MluI enzyme enzyme the DNA fragmentation that has obtained 858bp, and pWPXL-γ 4 cuts through PmeI and MluI enzyme enzyme the DNA fragmentation that has obtained 972bp, conforms to expected results.

(3) sequential analysis of DNA fragmentation

Identify that by cutting through enzyme the positive colony that contains target DNA Insert Fragment carries out sequencing, is completed by Nuo Sai biotech company.With DNAMAN software analysis sequencing result.Result has obtained that insertion sequence and position are all correct carries respectively the recombined lentivirus vector pWPXL-δ 1 that contains the δ 1 chain gene TRD1G of the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of Human Stomach Tissue source and carry the recombined lentivirus vector pWPXL-γ 4 of the γ 4 chain gene TRG4G that mate with δ 1 chain, wherein TRD1G has the nucleotide sequence of SEQ ID NO:5 in sequence table, by 858 based compositions, its encoding sequence is from 5 ' end 1-858 bit base, be GTM encoding sequence from 5 ' end 331-375 bit base, coding has the protein of the amino acid residue sequence of SEQ ID NO:2 in sequence table, TRG4G has the nucleotide sequence of SEQ ID NO:6 in sequence table, and by 972 based compositions, its encoding sequence is that coding has the protein of the amino acid residue sequence of SEQ ID NO:3 in sequence table from 5 ' end 1-972 bit base.Get the correct bacterium liquid of order-checking and add 10% glycerine as bacterial classification, save backup in-70 DEG C.

Two, transfection has the acquisition of the J.RT3-T3.5 cell (TCR γ 4 δ 1 transfectional cells) of the tcr gene of the described stomach organization source tumor infiltrating gamma delta T lymphocytes of coding (gamma delta T IL)

1, the transfection of J.RT3-T3.5 cell

With the little extraction reagent kit of Qiagen plasmid (purchased from Qiagen) extraction pWPXL (negative control), pWPXL-δ 1, pWPXL-γ 4 plasmids, psPAX2 plasmid (packaging plasmid, purchased from Clontech), pMD2.G plasmid (coating plasmid, purchased from Clontech), concentration and the purity of OD260/OD280 Detection and Extraction plasmid.Transfection the day before yesterday (D0) is by 293T cell (purchased from ATCC) bed board good growth conditions (the dull and stereotyped paving 2 × 10 of 6cm ⁶individual cell).Before transfection, 1h changes fresh culture (antibiotic-free) into (D1).With

i Reduced Serum Medium (purchased from Invitrogen) dilutes respectively plasmid, mixes gently.Mix gently before use Lipofectamine ^tM2000 (purchased from Invitrogen), use

i Reduced Serum Medium dilution.Incubated at room 5min.Then by the Lipofectamine after the plasmid after dilution and dilution ^tM2000 mix.Mix gently, incubated at room 20min, it is muddy that solution becomes.Added in the orifice plate containing cell and substratum, mixed.Hatch 24h for 37 DEG C.After transfection, second day (D2) changes substratum, and the 3rd day (D3) receives viral supernatant.The viral supernatant of cleer and peaceful pWPXL-γ 4, psPAX2 and pMD2.G transfection in the virus of pWPXL-δ 1, psPAX2 and pMD2.G transfection is added to (polybrene 8 μ g/mL in J.RT3-T3.5 cell (purchased from ATCC) simultaneously, Chinese 1,5-dimethyl-1,5-phenodiazine 11 methylene radical gather Methobromide), by transfectional cell called after pWPXL-δ 1/pWPXL-γ 4 transfectional cells.Using the viral supernatant of pWPXL transfection as negative control, called after pWPXL transfectional cell.After infecting, 48h changes fresh culture, then detects expression after hatching 48h.

2, the Flow cytometry of total length γ 4 and δ 1 chain transfectional cell surface gamma δ TCR expression

The expression of Flow cytometry transfectional cell surface gamma δ TCR: collect the J.RT3-T3.5 cell after transfection δ 1 and γ 4, carry out cell surface dyeing with PE-γ δ Ab antibody (purchased from Beckman Coulter), step is with embodiment 1.Flow cytometry result as shown in Figure 8, compared with negative control (pWPXL transfectional cell), in δ 1 and γ 4 transfectional cells (pWPXL-δ 1/pWPXL-γ 4 transfectional cells), detect that gamma delta T CR expresses, and has transfection J.RT3-T3.5 cell called after TCR γ 4 δ 1 transfectional cells of the tcr gene of the described stomach organization source tumor infiltrating gamma delta T lymphocytes of coding (gamma delta T IL).

3, the transfectional cell of flow cytometry enrichment high expression level goal gene

The transfectional cell of flow cytometry enrichment high expression level goal gene, step is with embodiment 1, and just whole process is all wanted aseptic technique, and agents useful for same all needs aseptic.After sorting, as Fig. 8, (GFP represents green fluorescent protein to the expression of cell surface gamma delta T CR, be connected to the label protein of δ 1 gene 3 ' end) shown in, after airflow classification, TCR γ 4 δ 1 positive cell purity reach more than 90%, can carry out follow-up function experiment.

The Function Identification of embodiment 5, TCR γ 4 δ 1 transfectional cells

1, TCR γ 4 δ 1 transfectional cells are measured the killing activity of tumour cell

Use cytotox 96 non-radioactive cytotoxicity assay reagents kit (Promega, USA) to carry out tumor cytotoxicity test.Effector cell is TCR γ 4 δ 1 transfectional cells, and target cell behaviour B lymphoma cell Daudi (purchased from ATCC), adds in 96 orifice plates target cell 3 × 10 ⁴/ hole.Effector cell contrasts after 4 DEG C of IgG1 antibody are hatched 1h and kills and wounds with anti-gamma delta T CR antibody, homotype, and effect target ratio is respectively 1.25: 1, and 2.5: 1,5: 1.Also pWPXL and the killing activity of TCR γ 4 δ 1 transfectional cells to tumour cell have been detected by same procedure simultaneously.

As shown in Figure 9, compared with negative control (pWPXL transfectional cell), TCR γ 4 δ 1 transfectional cells improve the killing-efficiency of Daudi cell result, and this lethal effect is that TCR γ δ is dependent.

2, the cytokine assay of TCR γ 4 δ 1 transfectional cells

After transfection, 1 × 10 ⁶ individual TCR γ 4 δ 1 transfectional cells (pWPXL transfectional cell for contrast) are hatched 18h with 37 DEG C, anti-TCR γ δ (concentration 5 μ g/mL, purchased from Beckman Coulter) or MICA albumen (concentration 5 μ g/mL) respectively.Hatch last 6h and add BrefeldinA (Chinese Cyanein, BioLegend), 1ug/mL.Use the generation of staining examine transfectional cell TNF-a in Fixation/Permeabilization Wash Buffer (BioLegend) born of the same parents.4 DEG C of lucifuges keep to be measured.

(X-coordinate is TNF α intracellular expression situation to result, and ordinate zou is cell count as shown in figure 10; Figure A represents that transfectional cell and anti-TCR γ anti-δ hatch the interior TNF alpha expression situation of rear born of the same parents, figure B represents that transfectional cell and MICA albumen hatches the interior TNF alpha expression situation of rear born of the same parents), compared with negative control (pWPXL transfectional cell), after hatching with anti-TCR γ δ or MICA albumen, in TCR γ 4 δ 1 transfectional cells, TNF alpha expression improves.

In sum, after J.RT3-T3.5 cell expressing TCR γ δ, the lethal effect of tumour cell is strengthened, and this enhancing is that TCR γ δ is dependent, TCR γ 4 δ 1 transfectional cells of the present invention can be used as active fraction preparation and become antitumor drug.

Claims

1. the complementary master sequence that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of Human Stomach Tissue source, called after GTM, is the albumen that amino acid residue sequence is represented by the SEQ ID NO:1 in sequence table.

2. δ 1 catenin of the TCR acceptor of the complementary master sequence GTM that determines territory 3 of δ 1 chain in the tumor infiltrating gamma delta T lymphocytes of Human Stomach Tissue source, is characterized in that: be the albumen that amino acid residue sequence is represented by the SEQ ID NO:2 in sequence table.

3. γ 4 chains that match with δ 1 catenin claimed in claim 2 are the albumen that amino acid residue sequence is represented by the SEQ ID NO:3 in sequence table.

4. a TCR for stomach organization source tumor infiltrating gamma delta T lymphocytes (gamma delta T IL), the amino acid residue sequence of its δ 1 chain is by sequence table shown in SEQ ID NO:2, and the amino acid residue sequence of its γ 4 chains is by sequence table shown in SEQ ID NO:3.

5. the gene of master sequence GTM described in coding claim 1 is one of following nucleotide sequence:

1) DNA sequence dna of SEQ ID NO:4 in sequence table;

2) DNA sequence dna of SEQ ID NO:1 in code sequence list.

6. the gene of δ 1 catenin described in coding claim 2, is characterized in that: be one of following nucleotide sequence:

1) DNA sequence dna of SEQ ID NO:5 in sequence table;

2) DNA sequence dna of SEQ ID NO:2 in code sequence list.

7. the gene of γ 4 chains described in coding claim 3 is one of following nucleotide sequence:

1) DNA sequence dna of SEQ ID NO:6 in sequence table;

2) DNA sequence dna of SEQ ID NO:3 in code sequence list.

8. the gene of TCR described in coding claim 4, the nucleotide sequence of its δ 1 chain encoding gene is as shown in SEQ ID NO:5 in sequence table, and the nucleotide sequence of its γ 4 chain encoding genes is as shown in SEQ ID NO:6 in sequence table.

9. contain the expression vector of gene described in claim 5-8 any one.

10. contain the transgenic cell line of gene described in claim 5-8 any one.

11. transgenic cell lines according to claim 10, is characterized in that: described transgenic cell is transgenosis J.RT3-T3.5 cell; The tcr gene of described coding stomach organization source tumor infiltrating gamma delta T lymphocytes is imported J.RT3-T3.5 cell by the recombinant eukaryon expression vector of the tcr gene by containing the described stomach organization of coding source tumor infiltrating gamma delta T lymphocytes.

12. transgenic cell lines according to claim 11, is characterized in that: the carrier that sets out for the recombinant eukaryon expression vector that builds the tcr gene that contains the described stomach organization of coding source tumor infiltrating gamma delta T lymphocytes is pRSV-rev, pLentilox3.7, pMDLg, pRRE, pWPXL or pLVX-DsRed Lentiviral.

13. transgenic cell lines according to claim 12, is characterized in that: described in the carrier that sets out be pWPXL; With pWPXL for the carrier that sets out, the recombinant eukaryon expression vector called after pWPXL-δ 1 of the δ 1 chain encoding gene of the TCR that carries described stomach organization source tumor infiltrating gamma delta T lymphocytes building, the recombinant eukaryon expression vector called after pWPXL-γ 4 of the γ 4 chain encoding genes of the TCR that carries described stomach organization source tumor infiltrating gamma delta T lymphocytes of structure.

14. transgenic cell lines according to claim 13, is characterized in that: it is TCR γ 4 δ 1 transfectional cells that described transfection has the J.RT3-T3.5 cell of the tcr gene of the described stomach organization of coding source tumor infiltrating gamma delta T lymphocytes.

15. Host Strains that contain gene described in claim 5-8 any one.

The application of 16. TCR claimed in claim 4 in medicine or the preparation of preparing killing off tumor cells, described tumour cell behaviour B lymphoma cell Daudi.