CN101182531B - Recombination plasmid related with EB virus specificity TCR gene and method for constructing anti-EBV specific CTL - Google Patents
Recombination plasmid related with EB virus specificity TCR gene and method for constructing anti-EBV specific CTL Download PDFInfo
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Abstract
The invention discloses a recombinant plasmid relevant to the EB virus characteristic TCR gene and a method for reverting normal T cell to obtain the anti-EBV characteristic CTL. The recombinant plasmid consists of TCRV Alpha 15 gene, pIRES vector and TCR V Beta 1gene; the TCRV Alpha 15-p-IRES-TCR V V Alpha 1gene recombinant plasmid relevant to the clone of EB virus characteristic cell toxin T cell of the invention is delivered to the peripheral blood T cell to obtain a large number of high-efficiency cell toxin T cells of the antigen relevant to the characteristic EV virus; in this way, the cell of EBV<+> can be killed on purpose and the safety property is high; therefore, the invention has comparatively good prospects for curing diseases relevant to the EB virus.
Description
Technical field
The invention belongs to antiviral and antineoplastic immune technical field in the neoplastic hematologic disorder, be particularly related to a kind of EBV (Epstein-Barr virus, Epstein Barr virus) specificity TCR (TXi Baoshouti, T cell receptor) TCR V α 15-pIRES-TCR V β 1 recombinant plasmid of gene-correlation, and the normal T cell of transduceing obtains the method for anti-EBV specific CTL (cytotoxic T cells, cytotoxic T cell).
Background technology
Epstein-Barr virus (Epstein Barr virus, EBV) closely related with adenoid malignant tumour.Induce and produce anti-EBV
+(cytotoxic T cells CTL), brings into play its antitumor or ntiviral characteristic to the Epstein-Barr virus specific cytotoxic T cell of tumour cell, can remove EBV effectively
+Tumour cell has good prospects for application for the treatment of Epstein-Barr virus related neoplasms.But the specific cytotoxic T cell of the donor that increases is merely often originated limited or dose-effect is not enough; Abroad have utilize to import suicide gene or gene Modified Dendritic Cells and carry out immunotherapy of tumors behind the HSCT, obtained certain effect, but operation is comparatively consuming time loaded down with trivial details, be difficult to promote as the knurl seedling.
Induce the corresponding disease of specific cytotoxic T cell therapy and carry out to some extent abroad from normal human T-cell, small-scale I phase clinical study has been carried out in the application of antigen-specific cytotoxic T cell such as EBV-CTL; And external tcr gene transduction patient T cell produces the feasibility of antitumor cell poison T cell and has been confirmed in the melanoma treatment, the tcr gene transfection that studies confirm that identification Epstein-Barr virus antigen LMP is abroad also arranged after, can form the anti-EBV of specificity
+The cytotoxic T cell of cell, but owing to the use of retroviral vector has limited applicability.
Induce and produce anti-EBV
+The Epstein-Barr virus specific cytotoxic T cell of tumour cell can be removed EBV effectively
+Tumour cell has good prospects for application for the treatment of Epstein-Barr virus related neoplasms.But the specific cytotoxic T cell of the simple amplification of the past dependence donor is often originated limited or dose-effect is not enough; Abroad have utilize to import suicide gene or gene Modified Dendritic Cells and carry out immunotherapy of tumors behind the HSCT, obtained certain effect, but operation is comparatively consuming time loaded down with trivial details, be difficult to promote as the knurl seedling.
Summary of the invention
In order to overcome the deficiency that above-mentioned prior art exists, primary and foremost purpose of the present invention is to provide a kind of TCR V α 15-pIRES-TCR V β 1 recombinant plasmid of Epstein-Barr virus specificity TCR gene-correlation.
Another object of the present invention is to provide the method for utilizing above-mentioned TCR V α 15-pIRES-TCR V β 1 construction of recombinant plasmid anti EB virus specific cytotoxic T cell.
The present invention utilizes the Epstein-Barr virus antigen peptide to obtain virus-specific cytotoxic T cell clone, after carrying out the analysis of T cell clone, change the TCRV α 15 and V β 1 gene of predominant expression in the Epstein-Barr virus specific cytotoxic T cell clone over to carrier for expression of eukaryon, make up TCR V α 15-pIRES-TCR V β 1 recombinant plasmid, again by transgenic technology with specificity TCR V α 15-pIRES-TCR V β 1 plasmid transduction to the T cell, thereby obtain a large amount of Epstein-Barr virus specific cytotoxic T cells.
The present invention is with identification Epstein-Barr virus related antigen BMLF (crack protein BMLF, BamHfragment M leftward reading frame) TCR V α 15 and TCR V β 1 dual-gene transduction simultaneously obtain the antigenic efficient cytotoxic T cell of specific recognition Epstein-Barr virus, adopt safer and rotaring transfecting mode efficiently, have higher feasibility.By transgenic technology TCR V α 15-pIRES-TCR V β 1 plasmid transduction that Epstein-Barr virus specific cytotoxic T cell clone is relevant to periphery blood T cell, can obtain the efficient cytotoxic T cell of specific recognition Epstein-Barr virus related antigen after the conventional cultivation amplification, this specific cytotoxic T cell has more operability and versatility.
The objective of the invention is to be achieved through the following technical solutions: a kind of TCR V α 15-pIRES-TCR V β 1 recombinant plasmid of Epstein-Barr virus specificity TCR gene-correlation, it is characterized in that: described TCR V α 15-pIRES-TCR V β 1 recombinant plasmid is by TCR V α 15 genes, pIRES carrier and TCR V β 1 genomic constitution, and described TCR V α 15 gene orders are as follows:
ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCT
GGACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGT
GTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCT
CCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAG
TTGCTGACGTATATTTTTTCAAATATGGACATGAAACAAGACCAAAGACT
CACTGTTCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGCATTGCAG
ACACCCAGACTGGGGACTCAGCTATCTACTTCTGTGCAGAGAacctATACA
GCACCCTCACCTTTGGGAAGGGGACTATGCTTCTAGTCTCTCCAGATATC
CAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTG
ACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCA
CAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACAT
GAGGTCTATGGACTTCAAGAGCAACAGTGCCGCGGCCTGGAGCAACAA
ATCTGACTTTGCTGTGCAAACGCCTTCACACAGCATTATTCCAAAAGACA
CCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAA
AAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATT
GGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGAC
GCTGCGGTTGTGGTCCAGC；
Described TCR V α 15 gene orders are 810bp altogether.Wherein:
The V district is: ATGAAGACATTT..........................TGTGCAGAGA;
The J district is: ATACAGCACCCTC............................CTTCTAGTCTCTCCA G;
The C district is: ATATCCAGAACC.........................GGTTGTGGTCCAGC;
Wherein the CDR3 district is:
Aminoacid sequence C A E N L Y S T L T F
Nucleotide sequence tgt gca gag aac cta tac agc acc ctc acc ttt
Rearrangement mode: V α 15 (called after again: V α 5*01)-N-J α 11*01-C α.
Described TCRV β 1 gene order is as follows:
ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGC
AGGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACA
GCAACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACC
TCTCTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCT
CATTCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAA
CGATTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGA
GCTCTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTA
GCGGGAGGGgACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTC
ACAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGC
CATCAGAAGCAGAGATATCCCACACCCAAAAGGCCACACTGGTATGCCT
GGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAAT
GGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAG
GAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGA
GGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCA
AGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGG
GCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCA
GACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCA
CCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTG
GTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAG
GC；
Described TCR V β 1 gene order is 930bp altogether.Wherein:
The V district is: ATGGGCTTCAGGC...................GCCAGCAGCGTAG;
The D district is: CGGGAGGG;
The J district is: ACGAGCAGTAC........................GGGCCGGGCAC;
The C district is: AGGACCTGAAAA........................ATTCCAGAGGC;
Wherein the CDR3 district is:
Aminoacid sequence C A S S V A G G D E Q Y F
Nucleotide sequence tgt gcc agc agc gta gcg gga ggg gac gag cag tac ttc resets mode: V β 1 (called after V β 9*01 again)-D β 2*02-N-J β 2-7*01-C β 2.
Utilize the method for above-mentioned TCR V α 15-pIRES-TCR V β 1 construction of recombinant plasmid anti EB virus specific cytotoxic T cell, comprise the steps:
(1) make up the relevant TCR V α-pIRES-TCR V β recombinant plasmid of EBV specific CTL clone:
(a) TCR V α 15 and TCR V β 1 subfamily gene order (comprising complete CDR3 district) the design primer according to clone's property propagation carries out the PCR reaction, have on the upstream primer of amplification TCR V α 15 gene orders and have EcoR I restriction enzyme site on Xho I restriction enzyme site, the downstream primer, used upstream primer is: CGCTCGAGAAGCAGTGGTATTAACGCAGAGTC; Used downstream primer is: TCGAATTCTCAGCTGGACCACAACCCGCAGCG, have on the upstream primer of amplification TCR V β 1 gene order and have Sal I restriction enzyme site on Xba I restriction enzyme site, the downstream primer, upstream primer is: CGTCTAGAAAGCAGTGGTATCAACGCAGAGTA; Downstream primer is: CGGTCGACCTAGCCTCTGGAATCCTTTCTCTT; Obtain the TCR V α 15 and the TCR V β 1 subfamily gene PCR product of total length respectively.
(b) the TCR V α 15PCR product behind carrier for expression of eukaryon pIRES and the purifying is cut 37 ℃ of water-bath 3h respectively with Xho I and EcoR I enzyme; PIRES carrier behind the double digestion and TCR V α 15PCR product carry out ligation: transform then; Select positive bacterium colony, extracting plasmid DNA after the amplification cultivation, identify with Xho I and EcoR I double digestion, design simultaneously a downstream primer I RES-R (5 '-TATAGACAAACGCACACCGG-3 ') in the IRES district of pIRES carrier, carrying out PCR in conjunction with the T7 consensus primer of pIRES carrier upstream (5 '-TACGACTCACTATAGGCTAG-3 ') identifies, and it is carried out two-way order-checking identify, confirm sequence correct back amplification positive colony and extract plasmid, obtain the relevant TCR V α 15-pIRES recombinant plasmid of Epstein-Barr virus specific cytotoxic T cell.
(c) TCR V β 1PCR product behind TCR V α 15-pIRES plasmid and the purifying is cut 37 ℃ of water-bath 3h respectively with Xba I and Sal I enzyme; TCR V α 15-pIRES plasmid behind the double digestion carries out ligation with TCR V β 1PCR product: will be connected product (10 μ L) again and change the competence bacillus coli DH 5 alpha over to; Select positive bacterium colony, extracting plasmid DNA after the amplification cultivation is respectively with Xba I and Sal I and Xho I and two groups of double digestions evaluations of EcoR I; The one upstream primer IRES-F of IRES district design simultaneously (5 '-TAAAAAAACGTCTAGGCCCC-3 '), carrying out PCR in conjunction with the T3 consensus primer of pIRES carrier downstream (5 '-TAACCCTCACTAAAGGGAAG-3 ') identifies, again it being carried out segment of double identifies to order-checking, after confirming in the plasmid that contained TCR V α 15 and TCR V β 1 sequence are all correct, large quantity extracting plasmid obtains relevant TCRV α 15-pIRES-TCR V β 1 recombinant plasmid of Epstein-Barr virus specific cytotoxic T cell.
(2) the relevant TCR V α-pIRES-TCR V β recombinant plasmid transfection T cell of EBV specific CTL clone, obtain the efficient CTL of specific recognition EBV related antigen: utilize liposome transfection or electrotransfection or consideration convey dyeing technique that TCR V α 15-pIRES-TCR V β 1 plasmid of EBV specificity TCR gene-correlation is changed in the T cell, cultivate the amplification back and obtain anti-EBV specific CTL.
The present invention compared with prior art has following advantage and beneficial effect:
Because the TCR pedigree use of advantage that neoplastic hematologic disorder is relevant and clone's property have certain specificity and proneness, utilize the Epstein-Barr virus antigen peptide to obtain virus-specific cytotoxic T cell clone, after carrying out the analysis of T cell clone, change the TCR V α 15 and V β 1 gene of specific cytotoxic T cell clone predominant expression over to carrier for expression of eukaryon, make up TCR V α 15-pIRES-TCR V β 1 recombinant plasmid, again by transgenic technology with the specificity TCR V α 15-pIRES-TCR V β 1 recombinant plasmid T cell of transduceing, thereby obtain a large amount of Epstein-Barr virus specific cytotoxic T cells.Carry out genetic modification T cell by chimeric TCR, vitro culture and amplification back obtain the antigenic a large amount of specific efficient cytotoxic T cells of specific recognition Epstein-Barr virus, be possible effectively treat the relevant tumour of Epstein-Barr virus or the adoptive immunotherapy strategy of post-transplantation complication, the specific active immunotherapy that can be the Epstein-Barr virus relative disease provides new treatment approach, has application promise in clinical practice.TCRV α 15-pIRES-TCR V β 1 recombinant plasmid that Epstein-Barr virus specific cytotoxic T cell clone of the present invention is relevant is transduceed to periphery blood T cell, can obtain a large amount of efficient cytotoxic T cell of specific recognition Epstein-Barr virus related antigen, can kill and wound the cell of EBV+ specifically, safe, the treatment of Epstein-Barr virus relative disease there is application promise in clinical practice.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited thereto.
Embodiment 1
1 makes up the relevant TCR V α-pIRES-TCR V β recombinant plasmid of Epstein-Barr virus specific cytotoxic T cell clone;
Obtain Epstein-Barr virus specific cytotoxic T cell clone after at first inducing normal human T-cell (HLA-A02 is restricted) and carry out vitro culture by the nonapeptide fragment GLCTLVAML of Epstein-Barr virus related antigen BMLF, in the IMDM substratum that contains 10% (v/v) foetal calf serum, 20U/mL recombinant human il-2,100U/mL green grass or young crops-Streptomycin sulphate, 37 ℃, 5% (v/v) CO
2In the saturated humidity incubator, conventional cultured and amplified in vitro; Collect Epstein-Barr virus specific cytotoxic T cell clone 1 * 10 then
7Individual cell, routine is carried out RNA extraction, cDNA synthetic (operating by corresponding reagent box specification sheets), then utilize TCRV α and TCRV β subfamily primer to carry out pcr amplification, analyze the TCR V α and the TCR V β subfamily of predominant expression in the Epstein-Barr virus specific cytotoxic T cell clone, respectively with 29 TCR V α subfamilies and 24 corresponding specificity upstream primers of TCR V β subfamily, and, carry out V α 1～29 and C α and V β 1～24 and C β totally 53 PCR reactions respectively in conjunction with corresponding C α and C β downstream primer.PCR total reaction system 20 μ L, it is arbitrary to V α and V β primer, 0.1mmol/L dNTP, 1.25U Taq polysaccharase, 10 * PCR damping fluid, 1.5mmol/L MgCl wherein to contain 0.5 μ mol/L
2, ultrapure water and 1 μ L cDNA template, the positive and negative control are set simultaneously.Be reflected in the pcr amplification instrument and carry out reaction conditions: 94 ℃ of 1min (being 3min first), 60 ℃ of 1min, 72 ℃ of 1min (last is 10min), totally 40 circulations, the PCR product is stored in 4 ℃.Get 8 μ LPCR products in 2% sepharose behind the electrophoresis with Gene genius gel imaging analysis systems analysis result, positive band person occurs and further carry out asymmetric amplification (reaction system of 10 μ L contains PCR product, 0.15 μ mol/L C α-Fam primer or C β-fam primer, the 1.5mmol/L MgCl of 2.5 μ LTCR subfamily genes with fluorescein-labeled C α-Fam primer or C β-Fam primer
2, 0.1mmol/L dNTP, 0.75U Taq polysaccharase, 10 * PCR damping fluid and ultrapure water.PCR reaction conditions: 94 ℃ of 1min (3min first), 66 ℃ of 1min, 72 ℃ of 1min (last 6min), totally 35 circulations), obtain fluorescently-labeled strand PCR product, carry out the genescan analysis again and (high-quality deionized formamide and Genescan-500LIZ molecular weight standard product are prepared the mixed solution of 10 μ L in 20: 1 ratios, the PCR product that adds 2 μ L fluorescein Fam marks, through 94 ℃ of sex change 4min, place cooled on ice 2min immediately, add 96 orifice plates then, directly be loaded on the 3100DNA sequenator (ABI PRISM 3100-Avant Genetic Analyzer) and carry out capillary electrophoresis, carry out the genescan analysis), determine its T cell clone (it is as shown in table 1 to relate to relevant primer sequence).
The primer sequence that table 1 is relevant
Carry out nucleotide sequence analysis by subfamily (TCR V α 15 and TCR V β 1 subfamily), determine the TCR V α 15 and the TCRV β 1 subfamily gene order of clone's property propagation in the Epstein-Barr virus specific cytotoxic T cell clone mono-clonal propagation; TCR V α 15 and TCR V β 1 subfamily gene order (comprising complete CDR3 district) design primer according to clone's property propagation, have on the upstream primer of amplification TCR V α 15 gene orders and have EcoR I restriction enzyme site on Xho I restriction enzyme site, the downstream primer (corresponding with the restriction enzyme site of insertion site A among the carrier for expression of eukaryon pIRES, used upstream primer is: CGCTCGAGAAGCAGTGGTATTAACGCAGAGTC; Used downstream primer is: TCGAATTCTCAGCTGGACCACAACCCGCAGCG), have on the upstream primer of amplification TCR V β 1 gene order and have Sal I restriction enzyme site on Xba I restriction enzyme site, the downstream primer (corresponding with the restriction enzyme site of insertion site B among the carrier for expression of eukaryon pIRES, upstream primer is: CGTCTAGAAAGCAGTGGTATCAACGCAGAGTA; Downstream primer is: CGGTCGACCTAGCCTCTGGAATCCTTTCTCTT).Press BD Advantage
TM2PCR test kit specification sheets carries out the PCR reaction: PCR total reaction system 50 μ L, wherein contain 10 * BDAdvantage2PCR Buffer, upstream and downstream primer each 0.5 μ mol/L, dNTP Mix 0.2mmol/L, 50 * BDAdvantage, 2 Polymerase Mix, ultrapure water and 1 μ LcDNA template, the positive and negative control are set simultaneously.Reaction conditions: 94 ℃ of 1min (being 3min first), 60 ℃ of 1min, 72 ℃ of 2min (last is 7min), totally 35 circulations.
TCR V α 15PCR product behind carrier for expression of eukaryon pIRES and the purifying is cut 37 ℃ of water-bath 3h respectively with Xho I and EcoR I enzyme; PIRES carrier behind the double digestion and TCR V α 15PCR product carry out ligation: system is: the pIRES carrier 2 μ L behind 10 * T4 buffer1 μ L, T4DNA enzyme 1 μ L, the double digestion, the TCRV α 15PCR product 6 μ L behind the double digestion, and mixing is placed on 16 ℃ and spends the night; Transform then: 1. will connect product (10 μ L) and add the competence bacillus coli DH 5 alpha, and put 30min in the ice; 2. behind 42 ℃ of heat-shocked 90s, put 3min in the ice rapidly; 3. add SOC substratum 800 μ L, put 37 ℃ of 180～200rpm jolting 60min in the dry constant temperature vibration case; 4. bacterium liquid is napped on 1.5%LB solid medium (containing penbritin 100 μ g/mL) with glass, puts 37 ℃ of incubator incubated overnight; Select positive bacterium colony, extracting plasmid DNA after the amplification cultivation, identify with Xho I and EcoR I double digestion, design simultaneously a downstream primer I RES-R (5 '-TATAGACAAACGCACACCGG-3 ') in the IRES district of pIRES carrier, carry out PCR in conjunction with the T7 consensus primer of pIRES carrier upstream (5 '-TACGACTCACTATAGGCTAG-3 ') and identify that the PCR reaction system comprises 0.1mmol/L dNTP, 1.5U Taq polysaccharase, 10 * PCR damping fluid, 1.5mmol/L MgCl
2And ultrapure water, primer concentration is 0.4 μ mol/L, with 1: 500 the dilution plasmid DNA 1 μ L as template.The pcr amplification condition is 94 ℃ of 3min, (94 ℃ of 30s, 60 ℃ of 1min, 72 ℃ of 2min, 35 circulations), 72 ℃ of 5min; And it is carried out two-way order-checking identify, confirm sequence correct back amplification positive colony and extract plasmid, obtain the relevant TCR V α 15-pIRES recombinant plasmid of Epstein-Barr virus specific cytotoxic T cell earlier.
TCR V β 1PCR product behind TCR V α 15-pIRES recombinant plasmid and the purifying is cut 37 ℃ of water-bath 3h respectively with Xba I and SalI enzyme; TCR V α 15-pIRES recombinant plasmid behind the double digestion and TCR V β 1PCR product carry out ligation: system is: the TCR V α 15-pIRES recombinant plasmid 2 μ L behind 10 * T4buffer1 μ L, T4DNA enzyme 1 μ L, the double digestion, the TCR V β 1PCR product 6 μ L behind the double digestion, and mixing is placed on 16 ℃ and spends the night; To connect product (10 μ L) again and change the competence bacillus coli DH 5 alpha over to, method is the same; Select positive bacterium colony, extracting plasmid DNA after the amplification cultivation identifies that with Xba I and SalI and Xho I and two groups of double digestions of EcoR I system is respectively: 1. TCR V α 15-pIRES-TCR V β 1 recombinant plasmid 3 μ L, Tango
TMBuffer 4 μ L, Xba I and Sal I restriction endonuclease each 1.5 μ L, ultrapure water 10 μ L; 2. TCR V α 15-pIRES-TCR V β 1 plasmid 3 μ L, Tango
TMBuffer 4 μ L, Xho I and EcoR I restriction endonuclease each 1.5 μ L, ultrapure water 10 μ L; Mixing is placed on 37 ℃ of water-bath 3h; The one upstream primer IRES-F of IRES district design simultaneously (5 '-TAAAAAAACGTCTAGGCCCC-3 '), carrying out PCR in conjunction with the T3 consensus primer of pIRES carrier downstream (5 '-TAACCCTCACTAAAGGGAAG-3 ') identifies, PCR reaction system and condition are the same, again it being carried out segment of double identifies to order-checking, after confirming in the plasmid that contained TCR V α 15 and TCR V β 1 sequence are all correct, large quantity extracting plasmid obtains relevant TCR V α 15-pIRES-TCR V β 1 recombinant plasmid of Epstein-Barr virus specific cytotoxic T cell.
The long 939bp of fragment sequence (wherein containing TCRV α 15 gene order total length 810bp and the non-coding leader sequence of part) in the constructed plasmid in the insertion site A of pIRES carrier, sequence is as follows:
CTCGAGAAGCAGTGGTATTAACGCAGAGTCAGGGGGACCACAGTGCTTT
GGAGGAAAGGAAGAGATACTTGATAATATAGCTCTCTTGGCTGGAGATTG
CAGGTCCCAGTGGGGAGAACAATGAAGACATTTGCTGGATTTTCGTTCC
TGTTTTTGTGGCTGCAGCTGGACTGTATGAGTAGAGGAGAGGATGTGGA
GCAGAGTCTTTTCCTGAGTGTCCGAGAGGGAGACAGCTCCGTTATAAAC
TGCACTTACACAGACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGA
ACCTGGAGCAGGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACA
TGAAACAAGACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACA
TCTGTCTCTGCGCATTGCAGACACCCAGACTGGGGACTCAGCTATCTACT
TCTGTGCAGAGAacctATACAGCACCCTCACCTTTGGGAAGGGGACTATGC
TTCTAGTCTCTCCAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTG
AGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGA
TTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAG
ACAAAACTGTGCTAGACATGAGGTCTATAATGGACTTCAAGAGCAACAGTGC
CGCGGCCTGGAGCAACAAATCTGACTTTGCTGTGCAAACGCCTTCACAC
AGCATTATTCCAAAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGA
TGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTT
CAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCG
GGTTTAATCTGCTCATGACGCTGCGGTTGTGGTCCAGCTGAGAATTC。
Wherein, initiator codon: ATG, terminator codon: TGA; Xho I restriction enzyme site: CTCGAG;
EcoR I restriction enzyme site: GAATTC.
The V district is:
ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGGA
CTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTC
CGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCT
CCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTG
CTGACGTATATTTTTTCAAATATGGACATGAAACAAGACCAAAGACTCAC
TGTTCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACA
CCCAGACTGGGGACTCAGCTATCTACTTCTGTGCAGAGA。
The J district is:
ATACAGCACCCTCACCTTTGGGAAGGGGACTATGCTTCTAGTCTCTCCAG。
The C district is:
ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCC
AGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGT
GTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAG
ACATGAGGTCTATGGACTTCAAGAGCAACAGTGCCGCGGCCTGGAGCAA
CAAATCTGACTTTGCTGTGCAAACGCCTTCACACAGCATTATTCCAAAAG
ACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGA
GAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTG
ATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCAT
GACGCTGCGGTTGTGGTCCAGC。
Wherein the CDR3 district is:
Aminoacid sequence C A E N L Y S T L T F
Nucleotide sequence tgt gca gag aac cta tac agc acc ctc acc ttt
Rearrangement mode: V α 15 (called after is again: V α 5*01)-and N-J α 11*01-C α (comprising exons 1,2,3)
The aminoacid sequence of TCR V α 15 genes of being translated is as follows:
MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDSSST
YLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQ
TGDSAIYFCAENLYSTLTFGKGTMLLVSPDIQNPDPAVYQLRDSKSSDKSVC
LFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAAAWSNKSDFAV
QTPSHSIIPKDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVA
GFNLLMTLRLWSS。
Wherein, molecular weight is: MW (mono)=30485.01226; MW (avg)=30504.6118.
The long 1023bp of fragment sequence (wherein containing TCR V β 1 gene order total length 930bp and the non-coding leader sequence of part) in the constructed plasmid in the insertion site B of pIRES carrier, sequence is as follows:
TCTAGAAAGCAGTGGTATCAACGCAGAGTACGCGGGGAGAATGCTTACT
ACAGAGACACCAGCCCCAAGCTAGGAGATCCTGCCATGGGCTTCAGGCT
CCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGGCCCAGTGGATTCTG
GAGTCACACAAACCCCAAAGCACCTGATCACAGCAACTGGACAGCGAG
TGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCTCTGTGTACTGGTAC
CAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTCAGTATTATAATGG
AGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACA
GTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTGGAGCTGGGG
GACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAGCGGGAGGGgACGAGC
AGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAA
ACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGAT
ATCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTAC
CCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCAC
AGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTC
AATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCT
TCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGG
GCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCAC
CCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACC
TCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGAT
CTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGC
TGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAGGTCGAC。
Wherein, initiator codon: ATG, terminator codon: TAG; Xba I restriction enzyme site: TCTAGA; SalI restriction enzyme site: GTCGAC.
The V district is:
ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCAGG
CCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGCA
ACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCT
CTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCAT
TCAGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGA
TTCTCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCT
CTCTGGAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAG。
The D district is: CGGGAGGG.
The J district is: ACGAGCAGTACTTCGGGCCGGGCAC.
The C district is:
AGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATC
AGAAGCAGAGATATCCCACACCCAAAAGGCCACACTGGTATGCCTGGCC
ACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGA
AGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGC
AGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGT
CTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTC
CAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCA
AACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACT
GTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCAT
CCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCA
GTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC。
Wherein the CDR3 district is:
Aminoacid sequence C A S S V A G G D E Q Y F
Nucleotide sequence tgt gcc agc agc gta gcg gga ggg gac gag cag tac ttc
Rearrangement mode: V β 1 (called after V β 9*01 again)-D β 2*02-N-J β 2-7*01-C β 2
The aminoacid sequence of TCRV β 1 gene of being translated is as follows:
MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTLRCSPRSGD
LSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSS
LELGDSALYFCASSVAGGDEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEI
SHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALND
SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS
AEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVK
RKDSRG。
Molecular weight is: MW (mono)=34408.08665; MW (avg)=34429.9602.
(2) TCR V α-pIRES-TCR V β recombinant plasmid transfection T cell;
With liposome Lipofectamine
TM2000 transfections are example:
1. normal people T cell cultures:
Count after separating normal people's peripheral blood mononuclear cell, get 1 * 10
6Individual cell inoculation is in 50mL 25cm
2Culturing bottle in, cultivate total liquid measure 5mL, contain the IMDM substratum of 15% (v/v) foetal calf serum, 500U/mL recombinant human il-2, the anti-people CD3 of 1 μ g/mL monoclonal antibody, the anti-people CD28 of 0.6 μ g/mL monoclonal antibody, 100U/mL green grass or young crops-Streptomycin sulphate, 37 ℃, 5%CO
2The conventional cultivation in the saturated humidity incubator; Can go down to posterity in 3～4 days, culture system becomes: the IMDM substratum of 15% foetal calf serum, 250U/mL recombinant human il-2,100U/mL green grass or young crops-Streptomycin sulphate, 37 ℃, 5%CO
2Keep cultivation in the saturated humidity incubator.
2. TCR V α-normal human T-cell of pIRES-TCR V β recombinant plasmid transfection
The normal human T-cell that will be in logarithmic phase is with 2 * 10
6The density of individual cells/well is inoculated 6 orifice plates, gets an amount of TCR V α-pIRES-TCR V β recombinant plasmid and liposome Lipofectamine
TM2000 mix the normal human T-cell of back transfection, working method is pressed transfection reagent box specification sheets, cultivate the T cell that obtains expressing the relevant TCR V α β gene of Epstein-Barr virus specific cytotoxic T cell after the amplification, utilize corresponding TCRV α/V β subfamily primer to carry out real-time quantitative PCR again and carry out streaming with corresponding TCR V α/V β subfamily monoclonal antibody and detect and verify recombinant plasmid expression in the cell after transfection.
(3) utilize TCR V α-pIRES-TCR V β recombinant plasmid transfection, obtain the efficient cytotoxic T cell of specific recognition Epstein-Barr virus related antigen.
Behind TCR V α-normal human T-cell of pIRES-TCR V β recombinant plasmid transfection, (lactate dehydrogenase, LDH) detection kit the 1st week, the 3rd week is carried out cell toxicity test respectively after transfection, operates and is undertaken by explanation with serum lactic dehydrogenase.Be the effector cell with the normal T cell of transfection recombinant plasmid, the normal T cell of transfection empty carrier respectively, with Raji cell (EBV
+), Molt4 cell (EBV-) is target cell, the normal T cell that other establishes the untransfected plasmid is blank group.Imitating the target ratio is 10: 1, target cell concentration 1 * 10
4Individual cells/well, 3 every group multiple holes; Put in U type 96 well culture plates, centrifugal slightly 2500rpm30s is to contact between promotion effect, the target cell, in 37 ℃, 5%CO
2Hatch 4h in the saturated humidity incubator; With the centrifugal 5min of 1000rpm, draw supernatant 100 μ L and add in the flat enzyme plate, add 100 μ LLDH substrate reactions liquid, lucifuge effect 30min under the room temperature, every hole adds 50 μ L 1mol/L hydrochloric acid and stops enzymatic reaction; The Elx-800 microplate reader is measured optical density(OD), and (optical density, OD) value is measured wavelength 490nm, reference wavelength 670nm.Cytotoxic T cell cytotoxic activity calculation formula: cytotoxic T cell activity (%)=(experimental group OD value-effector cell's nature release group OD value-target cell nature release group OD value)/(maximum release group OD value-target cell nature release group OD value).
Find transfection recombinant plasmid group, the cytotoxic T cell activity of transfection empty carrier group and blank group all strengthens with the prolongation of incubation time, the T cell in the 1st week and the 3rd week to the killing activity of Raji cell (EBV+) is behind the transfection recombinant plasmid: 4.64 ± 0.07% and 59.44 ± 2.27%, transfection empty carrier group T cell the 1st week and the 3rd week, the killing activity to the Raji cell was: 3.73 ± 0.16% and 28.23 ± 0.23%, and normal the 1st week of T cell of blank group and the 3rd week to the killing activity of Raji cell are: 1.39 ± 0.47% and 11.29 ± 3.19%, explanation is the 1st week and the 3rd week after transfection, and the T cell of transfection recombinant plasmid all is higher than the T cell of transfection empty carrier and the normal T cell of blank group (the P value all＜0.05) to the killing activity of Raji cell.And the T cell of transfection recombinant plasmid group, transfection empty carrier group and blank group by formula calculates the cytotoxic activity of Molt4 cell (EBV-) and is negative value, so think that transfection recombinant plasmid group, transfection empty carrier group and the blank T cell of organizing all do not have obviously lethal to the Molt4 cell; Above result shows: the normal T cell of transfection TCR V α-pIRES-TCR V β recombinant plasmid has specific killing to the EBV+ cell strain, has successfully made up anti-EBV specific cytotoxic T cell.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Ji'nan University
＜120〉recombinant plasmid of Epstein-Barr virus specificity TCR gene-correlation and make up the method for anti-EBV specific CTL
<130>31
<160>4
<170>PatentntIn?version?3.2
<210>1
<211>810
<212>DNA
<213>Artificial?sequence
<220>
＜223〉TCR V α 15 gene orders
<400>1
atgaagacat?ttgctggatt?ttcgttcctg?tttttgtggc?tgcagctgga?ctgtatgagt 60
agaggagagg?atgtggagca?gagtcttttc?ctgagtgtcc?gagagggaga?cagctccgtt 120
ataaactgca?cttacacaga?cagctcctcc?acctacttat?actggtataa?gcaagaacct 180
ggagcaggtc?tccagttgct?gacgtatatt?ttttcaaata?tggacatgaa?acaagaccaa 240
agactcactg?ttctattgaa?taaaaaggat?aaacatctgt?ctctgcgcat?tgcagacacc 300
cagactgggg?actcagctat?ctacttctgt?gcagagaacc?tatacagcac?cctcaccttt 360
gggaagggga?ctatgcttct?agtctctcca?gatatccaga?accctgaccc?tgccgtgtac 420
cagctgagag?actctaaatc?cagtgacaag?tctgtctgcc?tattcaccga?ttttgattct 480
caaacaaatg?tgtcacaaag?taaggattct?gatgtgtata?tcacagacaa?aactgtgcta 540
gacatgaggt?ctatggactt?caagagcaac?agtgccgcgg?cctggagcaa?caaatctgac 600
tttgctgtgc?aaacgccttc?acacagcatt?attccaaaag?acaccttctt?ccccagccca 660
gaaagttcct?gtgatgtcaa?gctggtcgag?aaaagctttg?aaacagatac?gaacctaaac 720
tttcaaaacc?tgtcagtgat?tgggttccga?atcctcctcc?tgaaagtggc?cgggtttaat 780
ctgctcatga?cgctgcggtt?gtggtccagc 810
<210>2
<211>930
<212>DNA
<213>Artificial?sequence
<220>
＜223〉TCRV β 1 gene order
<400>2
atgggcttca?ggctcctctg?ctgtgtggcc?ttttgtctcc?tgggagcagg?cccagtggat 60
tctggagtca?cacaaacccc?aaagcacctg?atcacagcaa?ctggacagcg?agtgacgctg 120
agatgctccc?ctaggtctgg?agacctctct?gtgtactggt?accaacagag?cctggaccag 180
ggcctccagt?tcctcattca?gtattataat?ggagaagaga?gagcaaaagg?aaacattctt 240
gaacgattct?ccgcacaaca?gttccctgac?ttgcactctg?aactaaacct?gagctctctg 300
gagctggggg?actcagcttt?gtatttctgt?gccagcagcg?tagcgggagg?ggacgagcag 360
tacttcgggc?cgggcaccag?gctcacggtc?acagaggacc?tgaaaaacgt?gttcccaccc 420
gaggtcgctg?tgtttgagcc?atcagaagca?gagatatccc?acacccaaaa?ggccacactg 480
gtatgcctgg?ccacaggctt?ctaccccgac?cacgtggagc?tgagctggtg?ggtgaatggg 540
aaggaggtgc?acagtggggt?cagcacagac?ccgcagcccc?tcaaggagca?gcccgccctc 600
aatgactcca?gatactgcct?gagcagccgc?ctgagggtct?cggccacctt?ctggcagaac 660
ccccgcaacc?acttccgctg?tcaagtccag?ttctacgggc?tctcggagaa?tgacgagtgg 720
acccaggata?gggccaaacc?cgtcacccag?atcgtcagcg?ccgaggcctg?gggtagagca 780
gactgtggct?tcacctccga?gtcttaccag?caaggggtcc?tgtctgccac?catcctctat 840
gagatcttgc?tagggaaggc?caccttgtat?gccgtgctgg?tcagtgccct?cgtgctgatg 900
gccatggtca?agagaaagga?ttccagaggc 930
<210>3
<211>270
<212>PRT
<213>Artificial?sequence
<220>
The aminoacid sequence of TCR V α 15 genes of＜223〉being translated
<400>3
Met?Lys?Thr?Phe?Ala?Gly?Phe?Ser?Phe?Leu?Phe?Leu?Trp?Leu?Gln?Leu
1 5 10 15
Asp?Cys?Met?Ser?Arg?Gly?Glu?Asp?Val?Glu?Gln?Ser?Leu?Phe?Leu?Ser
20 25 30
Val?Arg?Glu?Gly?Asp?Ser?Ser?Val?Ile?Asn?Cys?Thr?Tyr?Thr?Asp?Ser
35 40 45
Ser?Ser?Thr?Tyr?Leu?Tyr?Trp?Tyr?Lys?Gln?Glu?Pro?Gly?Ala?Gly?Leu
50 55 60
Gln?Leu?Leu?Thr?Tyr?Ile?Phe?Ser?Asn?Met?Asp?Met?Lys?Gln?Asp?Gln
65 70 75 80
Arg?Leu?Thr?Val?Leu?Leu?Asn?Lys?Lys?Asp?Lys?His?Leu?Ser?Leu?Arg
85 90 95
Ile?Ala?Asp?Thr?Gln?Thr?Gly?Asp?Ser?Ala?Ile?Tyr?Phe?Cys?Ala?Glu
100 105 110
Asn?Leu?Tyr?Ser?Thr?Leu?Thr?Phe?Gly?Lys?Gly?Thr?Met?Leu?Leu?Val
115 120 125
Ser?Pro?Asp?Ile?Gln?Asn?Pro?Asp?Pro?Ala?Val?Tyr?Gln?Leu?Arg?Asp
130 135 140
Ser?Lys?Ser?Ser?Asp?Lys?Ser?Val?Cys?Leu?Phe?Thr?Asp?Phe?Asp?Ser
145 150 155 160
Gln?Thr?Asn?Val?Ser?Gln?Ser?Lys?Asp?Ser?Asp?Val?Tyr?Ile?Thr?Asp
165 170 175
Lys?Thr?Val?Leu?Asp?Met?Arg?Ser?Met?Asp?Phe?Lys?Ser?Asn?Ser?Ala
180 185 190
Ala?Ala?Trp?Ser?Asn?Lys?Ser?Asp?Phe?Ala?Val?Gln?Thr?Pro?Ser?His
195 200 205
Ser?Ile?Ile?Pro?Lys?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>4
<211>310
<212>PRT
<213>Artificial?sequence
<220>
The aminoacid sequence of TCR V β 1 gene of＜223〉being translated
<400>4
Met?Gly?Phe?Arg?Leu?Leu?Cys?Cys?Val?Ala?Phe?Cys?Leu?Leu?Gly?Ala
1 5 10 15
Gly?Pro?Val?Asp?Ser?Gly?Val?Thr?Gln?Thr?Pro?Lys?His?Leu?Ile?Thr
20 25 30
Ala?Thr?Gly?Gln?Arg?Val?Thr?Leu?Arg?Cys?Ser?Pro?Arg?Ser?Gly?Asp
35 40 45
Leu?Ser?Val?Tyr?Trp?Tyr?Gln?Gln?Ser?Leu?Asp?Gln?Gly?Leu?Gln?Phe
50 55 60
Leu?Ile?Gln?Tyr?Tyr?Asn?Gly?Glu?Glu?Arg?Ala?Lys?Gly?Asn?Ile?Leu
65 70 75 80
Glu?Arg?Phe?Ser?Ala?Gln?Gln?Phe?Pro?Asp?Leu?His?Ser?Glu?Leu?Asn
85 90 95
Leu?Ser?Ser?Leu?Glu?Leu?Gly?Asp?Ser?Ala?Leu?Tyr?Phe?Cys?Ala?Ser
100 105 110
Ser?Val?Ala?Gly?Gly?Asp?Glu?Gln?Tyr?Phe?Gly?Pro?Gly?Thr?Arg?Leu
115 120 125
Thr?Val?Thr?Glu?Asp?Leu?Lys?Asn?Val?Phe?Pro?Pro?Glu?Val?Ala?Val
130 135 140
Phe?Glu?Pro?Ser?Glu?Ala?Glu?Ile?Ser?His?Thr?Gln?Lys?Ala?Thr?Leu
145 150 155 160
Val?Cys?Leu?Ala?Thr?Gly?Phe?Tyr?Pro?Asp?His?Val?Glu?Leu?Ser?Trp
165 170 175
Trp?Val?Asn?Gly?Lys?Glu?Val?His?Ser?Gly?Val?Ser?Thr?Asp?Pro?Gln
180 185 190
Pro?Leu?Lys?Glu?Gln?Pro?Ala?Leu?Asn?Asp?Ser?Arp?Tyr?Cys?Leu?Ser
195 200 205
Ser?Arg?Leu?Arg?Val?Ser?Ala?Thr?Phe?Trp?Gln?Asn?Pro?Arg?Asn?His
210 215 220
Phe?Arg?Cys?Gln?Val?Gln?Phe?Tyr?Gly?Leu?Ser?Glu?Asn?Asp?Glu?trp
225 230 235 240
Thr?Gln?Asp?Arg?Ala?Lys?Pro?Val?Thr?Gln?Ile?Val?Ser?Ala?Glu?Ala
245 250 255
Trp?Gly?Arg?Ala?Asp?Cys?Gly?Phe?Thr?Ser?Glu?Ser?Tyr?Gln?Gln?Gly
260 265 270
Val?Leu?Ser?Ala?Thr?Ile?Leu?Tyr?Glu?Ile?Leu?Leu?Gly?Lys?Ala?Thr
275 280 285
Leu?Tyr?Ala?Val?Leu?Val?Ser?Ala?Leu?Val?Leu?Met?Ala?Met?val?Lys
290 295 300
Arg?Lys?Asp?Ser?Arg?Gly
305 310
Claims (2)
1. the recombinant plasmid of an Epstein-Barr virus specificity TCR gene-correlation, it is characterized in that: described recombinant plasmid is by TCR V α 15 genes, pIRES carrier and TCR V β 1 genomic constitution, be TCR V α 15-pIRES-TCR V β 1 recombinant plasmid, described TCR V α 15 gene orders are as follows:
ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCTGG
ACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTGAGTGTC
CGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAGACAGCTCCTC
CACCTACTTATACTGGTATAAGCAAGAACCTGGAGCAGGTCTCCAGTTGCT
GACGTATATTTTTTCAAATATGGACATGAAACAAGACCAAAGACTCACTGT
TCTATTGAATAAAAAGGATAAACATCTGTCTCTGCGCATTGCAGACACCCA
GACTGGGGACTCAGCTATCTACTTCTGTGCAGAGAacctATACAGCACCCTC
ACCTTTGGGAAGGGGACTATGCTTCTAGTCTCTCCAGATATCCAGAACCCT
GACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGT
CTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGA
TTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGA
CTTCAAGAGCAACAGTGCCGCGGCCTGGAGCAACAAATCTGACTTTGCTG
TGCAAACGCCTTCACACAGCATTATTCCAAAAGACACCTTCTTCCCCAGCC
CAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGAT
ACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTC
CTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGTTGTGGTCCAGC
；
Described TCR V β 1 gene order is as follows:
ATGGGCTTCAGGCTCCTCTGCTGTGTGGCCTTTTGTCTCCTGGGAGCA
GGCCCAGTGGATTCTGGAGTCACACAAACCCCAAAGCACCTGATCACAGC
AACTGGACAGCGAGTGACGCTGAGATGCTCCCCTAGGTCTGGAGACCTCT
CTGTGTACTGGTACCAACAGAGCCTGGACCAGGGCCTCCAGTTCCTCATTC
AGTATTATAATGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTC
TCCGCACAACAGTTCCCTGACTTGCACTCTGAACTAAACCTGAGCTCTCTG
GAGCTGGGGGACTCAGCTTTGTATTTCTGTGCCAGCAGCGTAGCGGGAGG
GgACGAGCAGTACTTCGGGCCGGGCACCAGGCTCACGGTCACAGAGGACC
TGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCA
GAGATATCCCACACCCAAA?GGCCACACTGGTATGCCTGGCCACAGGCTT
CTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGC
ACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCT
CAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCT
TCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGG
CTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCA
GATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCG
AGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGC
TAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGG
CCATGGTCAAGAGAAAGGATTCCAGAGGC。
2. utilize the method for the described TCR V of claim 1 α 15-pIRES-TCR V β 1 construction of recombinant plasmid anti EB virus specific cytotoxic T cell, it is characterized in that comprising the steps:
(1) make up the relevant TCR V α-pIRES-TCR V β recombinant plasmid of EBV specific CTL clone:
(a) TCR V α 15 and the TCR V β 1 subfamily gene order design primer according to clone's property propagation carries out the PCR reaction, have on the upstream primer of amplification TCR V α 15 gene orders and have EcoR I restriction enzyme site on Xho I restriction enzyme site, the downstream primer, used upstream primer is: CGCTCGAGAAGCAGTGGTATTAACGCAGAGTC; Used downstream primer is: TCGAATTCTCAGCTGGACCACAACCCGCAGCG, have on the upstream primer of amplification TCR V β 1 gene order and have Sal I restriction enzyme site on Xba I restriction enzyme site, the downstream primer, upstream primer is: CGTCTAGAAAGCAGTGGTATCAACGCAGAGTA; Downstream primer is: CGGTCGACCTAGCCTCTGGAATCCTTTCTCTT; Obtain the TCR V α 15 and the TCRV β 1 subfamily gene PCR product of total length respectively;
(b) the TCR V α 15 PCR products behind carrier for expression of eukaryon pIRES and the purifying are cut 37 ℃ of water-bath 3h respectively with Xho I and EcoR I enzyme; PIRES carrier behind the double digestion and TCR V α 15 PCR products carry out ligation: transform then; Select positive bacterium colony, extracting plasmid DNA after the amplification cultivation, identify with Xho I and EcoR I double digestion, be 5 '-TATAGACAAACGCACACCGG-3 ' at the IRES district of pIRES carrier design one downstream primer I RES-R simultaneously, T7 consensus primer in conjunction with pIRES carrier upstream is that 5 '-TACGACTCACTATAGGCTAG-3 ' carries out the PCR evaluation, and it is carried out two-way order-checking identify, confirm sequence correct back amplification positive colony and extract plasmid, obtain the relevant TCR V α 15-pIRES recombinant plasmid of Epstein-Barr virus specific cytotoxic T cell;
(c) TCR V β 1 PCR product behind TCR V α 15-pIRES plasmid and the purifying is cut 37 ℃ of water-bath 3h respectively with Xba I and Sal I enzyme; TCR V α 15-pIRES plasmid behind the double digestion carries out ligation with TCR V β 1 PCR product: will be connected product again and change the competence bacillus coli DH 5 alpha over to; Select positive bacterium colony, extracting plasmid DNA after the amplification cultivation is respectively with Xba I and Sal I and Xho I and two groups of double digestions evaluations of EcoR I; The one upstream primer IRES-F of IRES district design simultaneously i.e. 5 '-TAAAAAAACGTCTAGGCCCC-3 ', T3 consensus primer in conjunction with the pIRES carrier downstream is that 5 '-TAACCCTCACTAAAGGGAAG-3 ' carries out the PCR evaluation, again it being carried out segment of double identifies to order-checking, after confirming in the plasmid that contained TCR V α 15 and TCR V β 1 sequence are all correct, large quantity extracting plasmid obtains relevant TCR V α 15-pIRES-TCR V β 1 recombinant plasmid of Epstein-Barr virus specific cytotoxic T cell;
(2) the relevant TCRV α-pIRES-TCR V β recombinant plasmid transfection T cell of EBV specific CTL clone, obtain the efficient CTL of specific recognition EBV related antigen: utilize liposome transfection or electrotransfection or consideration convey dyeing technique that TCR V α 15-pIRES-TCR V β 1 plasmid of EBV specificity TCR gene-correlation is changed in the T cell, cultivate the amplification back and obtain anti-EBV specific CTL.
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| CN106632659A (en) * | 2016-08-05 | 2017-05-10 | 武汉赛云博生物科技有限公司 | EB (Epstein-Barr) virus specific TCR (T cell receptor) and recombinant lentiviral vector and application thereof |
| CN113234863A (en) * | 2021-06-18 | 2021-08-10 | 重庆天科雅生物科技有限公司 | TCR primer group for specifically identifying EBV virus peptide segment with HLAA11 immune typing and application thereof |
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Non-Patent Citations (2)
| Title |
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| Lisa A. Jurgens et. Al.（"Transduction of Primary Lymphocytes with Epstein-BarrVirus (EBV) Latent Membrane Protein-Specific T-CellReceptor Induces Lysis of Virus-Infected Cells: A NovelStrategy for the Treatment of Hodgkin’s Disease andNasopharyngeal Carcinoma.Journal of Clinical Immunology26 1.2006,26(1),22-32. |
| Lisa A.Jurgens et. Al.("Transduction of Primary Lymphocytes with Epstein-BarrVirus (EBV) Latent Membrane Protein-Specific T-CellReceptor Induces Lysis of Virus-Infected Cells: A NovelStrategy for the Treatment of Hodgkin’s Disease andNasopharyngeal Carcinoma.Journal of Clinical Immunology26 1.2006,26(1),22-32. * |
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