CN101225388A - MR gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 6 subfamily and uses thereof - Google Patents
MR gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 6 subfamily and uses thereof Download PDFInfo
- Publication number
- CN101225388A CN101225388A CNA2008100259947A CN200810025994A CN101225388A CN 101225388 A CN101225388 A CN 101225388A CN A2008100259947 A CNA2008100259947 A CN A2008100259947A CN 200810025994 A CN200810025994 A CN 200810025994A CN 101225388 A CN101225388 A CN 101225388A
- Authority
- CN
- China
- Prior art keywords
- tcr
- cell
- pcr
- specific
- cell lymphoma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a nucleotide sequence and the application of an antigen-specific T cell receptor of the diffusive large B cell lymphoma. According to the RT-PCR genescan analysis, the TCR V Alpha 6 subfamilies in the peripheral blood of patients shows an obviously monoclonal state, and the V-N-J region, namely, the complementary determining region 3, where the PCR products conduct the sequence analysis of the specific TCR sequence, is a part recognizably bound to the antigenic specificity; the genescan analysis also shows that the TCR of the clonal T cell is a specific TCR produced due to the stimulus of the DLBL associated antigens, and is the basis and key for performing DLBL molecular targeted adoptive immunotherapy by means of the TCR transgenic technique. The nucleotide sequence of the specific TCR provides a significant basis for further carrying out lymphoma-specific cellular immunotherapy.
Description
Technical field
The present invention relates to the nucleotide sequence of a TXi Baoshouti (TCR), particularly the MR gene order and the application of diffuse large B cell lymphoma (DLBL) correlation antigen specific TCR V α 6 subfamilies.
Background technology
In recent years, non-Hodgkin lymphoma (NHL) sickness rate is lasting ascendant trend.According to estimates, at the beginning of 21 century, NHL will account for 4.4% of aggressive malignant tumour, and account for 4.8% of all cancer mortality numbers.Wherein diffuse large B cell lymphoma (DLBL) is modal NHL, accounts for 30%～40%, and the gerontal patient who wherein surpasses 60 years old accounts for 70%.The main chemotherapy that adopts based on CHOP scheme (endoxan, Dx, vincristine(VCR), prednisone) over more than 20 year.But to the toxic effect of part old man, long-term survival rate is low.Target immunotherapy of carrying out in the recent period such as Rituximab (CD20 monoclonal antibody) though appearance obtained new curative effect, the palindromia that the existence of microresidual disease causes remains the thorny problem of present clinical treatment.For many years, the various countries scholar is making great efforts to seek more effective methods of treatment always.The clinical tumor scholar places high hopes to specific cellular immunization treatment.Xue equals to report that utilization suffers from the CTL that isolates WT1 epitope that can specific recognition HLA-A2 restriction from leukemia, with the tcr gene transfection patient's of CTL T lymphocyte, demonstrates the specific killing of transfectional cell in mouse test.Subsequently Tsuji etc. will from further proved the Th1 that utilizes the special tcr gene of transduction WT1 and Tc1 demonstrate the HLA restriction lethal effect, the Th1 cell of transfection simultaneously has the function of a large amount of IFN-γ of secretion and IL-2, because IFN-γ and IL-2 play an important role in the containment tumour, thereby method that can be by the corresponding tcr gene of this transfection plays an important role to the immunotherapy of tumour.
Utilize at present the tcr gene of tumour cell correlation antigen specific modify normal T cell (from body or donor source T cell), thereby obtain to have the cytotoxic T cell (CTL) of killing and wounding corresponding tumour cell, this is an important discovery of antineoplastic specificity cellular immunization treatment in recent years.Antigen-specific TCR modifies normal T cell and the research that obtains specificity anti-tumor T cell clone at first launches in melanoma, there are a plurality of researchs to expand this The Application of Technology subsequently, as the CTL of specificity anti EB virus positive cell, the CTL of anti-WT1, formed the TCL product individually and be used for clinical treatment.The key of preparation CTL product is clear and definite tumour/leukemia cell's a related antigen, thereby obtains the TCR of antigen-specific, and then induces the T cell clone that produces special leukemia/tumour.Lymphadenomatous research is mainly concentrated on T cellular type lymphoma both at home and abroad at present, and less to the research data of B lymphocytic lymphoma, major cause may be to the lymphadenomatous related antigen of bone-marrow-derived lymphocyte type solve less relevant.
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 MR gene order of filling the air large B cell lymphoma correlation antigen specific TCR V α 6 subfamilies.
There is clone's property T cell of TXi Baoshouti (TCR) V α subfamily in the present invention from DLBL patient's peripheral blood (TCR α and TCR β chain are respectively the integral part of the expressed heteroduplex TCR of TCR α β+T cell surface, both have the status of no less important in T cellular immunization), the hypervariable region V-N-J district that shows its specificity TCR sequence through sequential analysis, be complementary determining region 3 (CDR3), with antigen-specific identification bonded position, show that the expressed TCR of this clone's property T cell for the DLBL related antigen stimulates the specificity TCR that is produced, has obtained the relevant tcr gene sequence of DLBL.
The invention discloses gene order at the specificity TCR of DLBL related antigen, especially the hypervariable region of TCR is CDR3, the latter is that the T cell surface receptor is discerned antigenic special zone, be directed to not synantigen, except the selected TCR V α subfamily T cell difference of body, more crucial is the difference of the CDR3 sequence of its tcr gene, and this position has determined the antigen that affiliated T cell can be discerned.The invention provides a kind of TCR V α 6 gene orders of the DLBL of identification related antigen, owing to the specificity of its CDR3, only come from the T cell of monospecific polyclonal, is the sequence of a high special.Simultaneously,, can be used to understand the associated antigen polypeptide of DLBL, for the biological characteristics of understanding DLBL is given information according to the gene order encoded protein matter of CDR3.
Another object of the present invention is to provide the application of said gene sequence.
Nucleotide sequence according to the specificity TCR of the DLBL related antigen that is obtained, can be used for (1) and understand the situation of the TCRV α 6 subfamily T cells of the existing anti-DLBL of patient DLBL, be used to estimate patient's immune functional state, (2) change in the normal T lymphocyte by the specificity TCR gene of transgenic technology the DLBL related antigen, make this species specific TCR of its forced expression, change T cell endogenous TCR and discern antigenic original pattern, possess secrete cytokines and (or) function of specific killing target cell, can produce the CTL of capacity through the amplification of external Short-term Culture, be fed back into again and make it that DLBL cell is exercised the targeted cells immunological effect in patient's body.
Purpose of the present invention realizes by following technical proposals: a kind of MR gene order of filling the air large B cell lymphoma correlation antigen specific TCR V α 6 subfamilies, and described MR gene order is as follows:
TCCGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTATTTCTGTGCAATGAGGGATTACTCTGGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCGGTCATACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCCGCGGCCTGGAGCAAC。
Wherein, described gene order is that 333 bp:1-65bp are the V district; 66-69bp is the N district; 70-133bp is the J district; 134-333bp is the C district.
The protein (111 amino acid) that above-mentioned MR gene order of filling the air large B cell lymphoma correlation antigen specific TCR V α 6 subfamilies is translated is as follows:
SANLVISASQLGDSAMYFCAMRDYSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN。
Wherein: the CDR3 district comprises the upstream extremity in downstream end, N district and the J district in V district.
The preparation method of the MR gene order of above-mentioned diffuse large B cell lymphoma related antigen specific TCR V α 6 subfamilies comprises the steps:
A. collect DLBL patient's peripheral blood, the Ficoll top and bottom process is separated peripheral blood mononuclear cell;
B. extract the RNA of peripheral blood mononuclear cell, RT-PCR synthesizes cDNA first chain;
C. in the Genebank of NCBI, search the gene order of TCR α chain, and mark V district and the C district position in genome of each subfamily;
D. design 29 V alpha specific upstream primers and C α downstream primer;
The V-N-JC district that 29 V α subfamily TCR of e.PCR amplification produce when resetting;
F.C α-Fam mark PCR product, genescan analyze expression and clone's property of T cell subfamily;
The subfamily PCR product that g. will be mono-clonal or few clone's scintigram is cut the glue purification sequential analysis.
The application of the MR gene order of above-mentioned diffuse large B cell lymphoma related antigen specific TCR V α 6 subfamilies in preparation diffuse large B cell lymphoma specific molecular target adoptive immunity medicine.
The present invention compared with prior art has following advantage and beneficial effect:
(1) the present invention has prepared a kind of antigenic relevant TCR V α 6 gene orders of DLBL of uniqueness, and this sequence can be used as a kind of new antigen recognition tcr gene (main variation zone is the CDR3 sequence) and replenishes human gene bank's data.
(2) this tcr gene can be used for studying specific immunity diagnosis and the treatment of DLBL.
The gene order of above-mentioned diffuse large B cell lymphoma related antigen specific TCR V α 6 subfamilies, can be used for further detecting the situation of existing TCR V α 6 subfamily T cells in the patient body on the one hand, to estimate its cellular immune function state, be to be applied in the molecular targeted adoptive immunity cell therapy of preparation diffuse large B cell lymphoma research aspect on the other hand.
The former detection institute employing technology is identical with the technology of above-mentioned detection tcr gene, by the analysis of different time points, can understand the situation of the existing TCR V of patient α 6 subfamily T cells, is used to understand patient's immunological status.
The latter mainly modifies the tcr gene of DLBL correlation antigen specific from body or the normal T cell of recessive allele by transgenic technology, these just can provide the t cell responses of the definite tumour specific antigen of antagonism through the cell of modifying, bring into play the cytotoxic activity that corresponding specific recognition is killed and wounded the DLBL cell thereby possessed, can prepare the CTL product of the anti-DLBL cell of specificity.There is relevant animal experiment study to point out this class technology might bring into play specific immunotherapy effect abroad.The application final purpose of tcr gene provided by the present invention is by DLBL patient is carried out specific active immunotherapy, thereby raising curative effect, remove microresidual disease, recurrence improves long-term survival rate after reducing disease treatment, improving patient's life quality etc., is to have very big marketing using value and and bigger economic results in society.
Description of drawings
Fig. 1 is the genescan figure of DLBL correlation antigen specific TCR V α 6 subfamilies.
Wherein, A is TCR V α 6 subfamily mono-clonal figure; B is the two clone figure of TCR V α 6 subfamilies; C is TCR V α 6 subfamily polyclone figure; D is the few clone figure of TCR V α 6 subfamilies.
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) antigen specific T CR analysis of gene sequences
Analyze the length of CDR3 of TCR V α gene profile and base sequence method be to utilize the CDR3 length of 29 subfamilies of RT-PCR-genescan methods analyst TCR V α.At first collect DLBL patient's peripheral blood, the Ficoll top and bottom process is separated peripheral blood mononuclear cell, leaves and takes 1*10
7Individual PBMCs (peripheral blood mononuclear cell) extracts RNA, and RT-PCR synthesizes cDNA.Simultaneously, in the gene pool of NCBI, find out the gene order of TCR α chain, and mark V district and the C district position in genome of each subfamily.CDNA according to 29 subfamily genes of V α designs corresponding 29 V alpha specific upstream primers, and establishes a C α downstream primer in C α district, the V-N-JC district that produces when utilizing 29 V α of round pcr amplification patient subfamily TCR to reset.As shown in Figure 1, the C α district of each subfamily PCR product of fluorescein Fam mark, genescan is analyzed the expression of each subfamily of T cell, according to different positions, the peak of height and form, size, amount and the homogeneity of expression product, thus determine the T cell clone (generally speaking, each V α subfamily of normal transcription comprises 8～10 peaks, i.e. 8～10 different clones' T cell; The appearance of main peak shows the excessive amplification of identical big or small cDNA, unimodally shows that the dna segment size is identical in the product, all has few clone or mono-clonal T cell mass, and the three points out product respectively from polyclone, few clone and monoclonal T cell).Genescan found that patient's V α 6 subfamily T cells are obvious mono-clonal; The PCR product of V α 6 subfamilies is cut glue purification, and gene order and CDR3 district (the V-N-J district) thereof of V α 6 subfamily TCR found in sequential analysis, is and lymphoid tumor related antigen specific bonded position.
The concrete preparation method of the MR gene order of diffuse large B cell lymphoma (DLBL) correlation antigen specific TCR V α 6 subfamilies:
1, separates peripheral blood mononuclear cell
(1) get lymphocyte separation medium (Ficoll, density 1.077) 4mL and add in the centrifuge tube, the anticoagulation cirumferential blood sample suspension after the dilution is tiled on the parting liquid, with horizontal centrifuge, 2000rpm, centrifugal 15 minutes.
(2) draw middle mononuclearcell layer and be transferred in another centrifuge tube, add 5mL 1640 liquid again, piping and druming gently, with 1500rpm, centrifuge washing 10 minutes is abandoned supernatant, adds 1640 liquid to 2mL, count behind the mixing, and the washed twice that uses the same method, by 2 * 10
7It is standby that/mL adjusts cell concn.
2, RNA extracts (TRIzol kit method extraction method)
(1) will add TRIzol reagent-20 ℃ frozen cell and take out, and put on ice and thaw, add the 0.2mL chloroform behind the mixing, abundant mixing, 4 ℃, centrifugal 30 minutes of 14000rpm.
(2) draw upper strata liquid to another centrifuge tube, add 0.5mL Virahol and mixing, room temperature leaves standstill after 10 minutes 4 ℃, centrifugal 30 minutes of 14000rpm.
(3) remove supernatant, and (2～8 ℃, 14000rpm), each mixing is after 30 seconds, centrifugal 10 minutes to wash twice with 1mL 75% ethanol (20 ℃).
(4) remove supernatant, centrifugal again 2～3 minutes, remove remaining ethanol, placed on the super clean bench seasoning 1～1.5 hour.Add ultra-clean water (20～50 μ L) dissolving.
(5) purity of test sample and content in ultraviolet spectrophotometer, 0.8% agarose gel electrophoresis detects integrity and the quality of RNA.
(6) RNA preserves: after the RNA sample adds its 0.1 times of volumetrical 3mol/LNaAC (sodium-acetate) and 2 times of volumetrical ethanol, be stored in-80 ℃ standby.
3, cDNA is synthetic
(1) uses synthetic cDNA first chain of six aggressiveness random primers and ThermoScript II test kit.1 μ LRNA add 16.5 μ L in advance liquid mixture prepared comprising 7 μ L H
2O, 2 μ L10 * PCR-Buffer, 2 μ L0.1mmol/L DTT, 5 μ L2.5mmol/L dNTP and 0.5 μ L Rand.Hexmer.
(2) in 73 ℃ after 3 minutes, placed ice 30 seconds.Low-temperature and high-speed placed ice 2～3 minutes in centrifugal 30 seconds.
(3) behind adding 0.6 μ L Rnasin (33U/ μ L) and the 0.5 μ LSuperscript (200U/ μ L), centrifugal 30 seconds of low-temperature and high-speed.
(4) room temperature left standstill 10 minutes; 42 ℃, more than 40 minutes; 83 ℃, 5 minutes ,-20 ℃ of preservations were standby.
4, the synthetic quality examination of cDNA
The cDNA of all samples is detected β through PCR
2M gene (primer sequence sees Table 1) and determine its synthetic quality.Total reaction system 20 μ L, upstream and downstream primer 0.5 μ mol/L wherein, dNTP 0.1mmol/L, Taq polysaccharase 1.0U, MgCL
21.5mmol/L, 10 * PCR damping fluid, 2 μ L, cDNA product 1 μ L and dH
2O is provided with the positive and negative control simultaneously.In German Biometra pcr amplification instrument, carry out the PCR reaction.
Reaction conditions: 94 ℃ 1 minute (3 minutes first), 58 ℃ 1 minute, 72 ℃ 1 minute (last 6 minutes), totally 35 circulations are stored in 4 ℃ at last.Get 8 μ LPCR products and carry out electrophoresis, β with 1.5% (w/v) sepharose
2M positive person can further study, and it is synthetic that negative patient is considered as not having cDNA, and discarding need not.
5,29 subfamilies of pcr amplification TCR V α
With 29 corresponding specificity upstream primers of TCR V α subfamily gene design, and at a C α district design downstream primer (primer sequence sees Table 1).PCR carries out according to a conventional method.Total reaction system 20 μ L wherein contain arbitrary V α primer (one of 24 V β subfamilies) and C α primer 0.5 μ mol/L, and are surplus the same.
Reaction conditions: 94 ℃, 1 minute (3 minutes first), 60 ℃, 1 minute and 72 ℃, 1 minute (last 6 minutes) carry out 40 circulations altogether, and last PCR product is stored in 4 ℃.Get 8 μ L PCR product electrophoretic analysis results.
6, T cell clone analysis
(1) mark PCR product
The reaction system of 10 μ L contains the unlabelled PCR product of 2.5 μ L, 0.15 μ mol/L C α-fam primer, 1.5mmol/L MgCl
2, 0.1mmol/L dNTP, 0.75U Taq polysaccharase, 1 μ L10 * PCR damping fluid and dH
2O.PCR carries out 35 circulations altogether, and each circulation comprises: 94 ℃, 1 minute (3 minutes first), 66 ℃, 1 minute and 72 ℃, 1 minute (last 6 minutes), last PCR product is stored in 4 ℃.
(2) genescan sample preparation
Fluorescein-labeled PCR product (2 μ L) adds in the 10 μ L mixed solutions that high-quality deionized formamide (Hi-DiFormamide) and Genescan-500 LIZ molecular weight standard product prepare in 20: 1 ratios.
(3) genescan (being undertaken) by operational guidance
1) is ready to instrument and changes water and damping fluid by operational guidance.
2) encapsulating: encapsulating took out POP-4 (Performance OptimizedPolymer-4) glue from 4 ℃ of refrigerators in preceding 2 hours and rose again 1～2 hour, glue is drawn in the storage glue 5mL syringe (the mL number of extraction is about 0.5+0.07x and does the sample number of times), be inverted and discharge bubble in the syringe, load syringe.
3) go up sample: the sample got ready in 94 ℃ of sex change 4 minutes, was placed cooled on ice 2 minutes then immediately.Sample after the sex change adds 96 orifice plates, directly is loaded into 3100 genetic analyzers and carries out electrophoresis.
4) operation: automatic sampler can move on to sample panel the position of 4 samples will analyzing automatically, and the CCD detector is converted to fluorescent signal electrical signal and it is sent to the computer workstation that 3100 data acquisition softwares have been installed in the electrophoresis process.
5) data processing: just be stored in the lane database of computer after data processing is finished and show with the form of electrophoresis signal figure.The X-axis of electrophoresis signal figure is represented the time, and Y-axis is represented relative intensity of fluorescence, several pulsating fluorescein signals of marker DNA that are used for that draw simultaneously among each figure, and a dna segment is represented at each peak among the electrophoresis signal figure.Finishing the data of processing is automatically extracted from database and is analyzed.
6) interpretation of result: open GeneMapper SoftwareTM v3.5, the raw data that is stored in the computer hard disk data storehouse is added analysis software, the length of assay products and fluorescein intensity.
7, nucleotide sequence analysis
(1) the E.Z.N.A. gel reclaims the test kit purified pcr product
1) cuts glue: the well that the PCR product is all added 1～2% sepharose, when electrophoresis when being enough to separate target DNA, downcut the sepharose piece that contains the purpose band with clean scalpel down in ultraviolet lamp, the gel piece of cutting-out places the 1.5mL centrifuge tube.
2) reclaim the purifying of test kit explanation carrying out PCR product by the E.Z.N.A. gel, add 500 μ LBinding Buffer earlier, hatch to gel piece for 55～65 ℃ and dissolve fully; After the gel lysate adds that HiBind DNAspin-column high speed is centrifugal and removes waste liquid, add 300 μ LBinding Buffer more successively, 700 μ LSPW Buffer wash film; High speed centrifugation adds 30 μ L DNA ElutionBuffer (being added on the filter membrane) after drying the filter membrane of spin-column, and room temperature leaves standstill 5min, can get the PCR product of purifying behind the high speed centrifugation 1min.
3) the PCR product of getting behind the 3 μ L purifying carries out 1% agarose gel electrophoresis, observes purification effect; Remaining PCR purified product vacuum lyophilization concentrates about about 20min to 10 μ L.
(2) BigDye mark
With the PCR product behind the BigDye Terminator v3.1 Cycle Sequencing kit mark purifying, reaction system is: PCR product 2.4 μ L behind BigDye Terminator 1 μ L, corresponding unidirectional primer (2 μ mol/L) 1.6 μ L, the purifying, reaction conditions: 96 ℃ of 1min, (96 ℃ of 10sec, 50 ℃ of 5sec, 60 ℃ of 4min) 25 circulations.
(3) order-checking
With order-checking product moisturizing to the 20 μ L of the 5 μ L systems of the good BigDye of mark, change the centrifuge tube of 1.5ml over to, add 2 μ L 3M NaAc (pH 5.2), 2 μ L 125mM EDTA-Na more successively
2, 50 μ L, 95% ethanol, lucifuge is placed 15min under the room temperature of vortex vibration back; The centrifugal 20min of 5415 type high speed tabletop centrifuge 2800g carefully draws supernatant and discards along the offside of centrifugal sediment, adds the freezing ethanol of 250 μ L70%, and the centrifugal 5min of 2800g abandons supernatant; To manage lid and open and place on the hot-plate, 94～95 ℃ of 1min make its drying.Add the high-quality deionized formamide of 10 μ L, blow and beat mixing repeatedly, change in the PCR pipe of 0.2 μ L, 95 ℃ of sex change 4min on the PCR instrument, put rapidly then cool off at least 4min in the ice after, be loaded on the 3100 dna sequence analysis instrument and carry out capillary electrophoresis.
The primer sequence that table 1 RT-PCR experiment is related
| Primer | Sequence |
| Vα1 Vα2 Vα3 Vα4 Vα5 Vα6 | 5’GGCATTAACGGTTTTGAGGCTGGA3’ 5’CAGTGTTCCAGAGGGAGCCATTGT3’ 5’CCGGGCAGCAGACACTGCTTCTTA3’ 5’TTGGTATCGACAGCTTCACTCCCA3’ 5’CGGCCACCCTGACCTGCAACTATA3’ 5’TCCGCCAACCTTGTCATCTCCGCT3’ |
| Vα7 Vα8 Vα9 Vα10 Vα11 Vα12 Vα13 Vα14 Vα15 Vα16 Vα17 Vα18 Vα19 Vα20 Vα21 Vα22 Vα23 Vα24 Vα25 Vα26 Vα27 Vα28 Vα29 Cα1 Cα-Fam β 2-M5′ β 2-M3′ | 5’GCAACATGCTGGCGGAGCACCCAC3’ 5’CATTCGTTCAAATGTGGGCAAAAG3’ 5’CCAGTACTCCAGACAACGCCTGCA3’ 5’CACTGCGGCCCAGCCTGGTGATAC3’ 5’CGCTGCTCATCCTCCAGGTGCGGG3’ 5’TCGTCGGAACTCTTTTGATGAGCA3’ 5’-TTCATCAAAACCCTTGGGGACAGC-3’ 5’-CCCAGCAGGCAGATGATTCTCGTT-3’ 5’-TTGCAGACACCGAGACTGGGGACT-3’ 5’-TCAACGTTGCTGAAGGGAATCCTC-3’ 5’-TGGGAAAGGCCGTGCATTATTGAT-3’ 5’-CAGCACCAATTTCACCTGCAGCTT-3’ 5’-ACACTGGCTGCAACAGCATCCAGG-3’ 5’-TCCCTGTTTATCCCTGCCGACAGA-3’ 5’-AGCAAAATTCACCATCCCTGAGCG-3’ 5’-CCTGAAAGCCACGAAGGCTGATGA-3’ 5’-TGCCTCGCTGGATAAATCATCAGG-3’ 5’-CTGGATGCAGACACAAAGCAGAGC-3’ 5’-TGGCTACGGTACAAGCCGGACCCT-3 5’-AGCGCAGCCATGCAGGCATGTACC-3’ 5’-AAGCCCGTCTCAGCACCCTCCACA-3’ 5’-TGGTTGTGCACGAGCGAGACACTG-3’ 5’-GAAGGGTGGAGAACAGATGCGTCG-3’ 5’-GTTGCTCCAGGCCGCGGCACTGTT-3’ 5’-Fam-ATACACATCAGAATCCTTACTTTG-3’ 5’-TACACTGAATTCACCCCCAC 5’-CATCCAATCCAAATGCGGCA |
(2) antigen-specific tcr gene transduction technology
1, the structure of recombinant plasmid TCR V α 6 (MR)-pIRES
*
(1) amplification of goal gene
According to relevant TCRV α 6 subfamily gene orders (the comprising complete CDR3 district) design of DLBL primer, have on the upstream primer of TCR V α 6 gene orders in the patient's sample that increases and have EcoR I restriction enzyme site (corresponding) on XhoI restriction enzyme site, the downstream primer with the restriction enzyme site of insertion site A among the carrier for expression of eukaryon pIRES.Press BD Advantage
TM2 PCR test kit specification sheetss carry out the PCR reaction: PCR total reaction system 50 μ L, wherein contain 10 * BD Advantage, 2 PCR Buffer, upstream and downstream primer each 0.5 μ mol/L, dNTP Mix 0.2mmol/L, 50 * BD Advantage, 2 PolymeraseMix, 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.Amplified production is identified with 1% (w/v) agarose gel electrophoresis.
(2) construction of recombinant plasmid
A, E.Z.N.A. gel reclaim PCR product (same first part, the step 7) of test kit purifying goal gene TCR V α 6;
B, the TCR V α 6 PCR products behind carrier for expression of eukaryon pIRES and the purifying are cut with the XhoI enzyme respectively, system is: 1. TCR V α 6 PCR products 28 μ L, Tango
TMBuffer 8 μ L, XhoI restriction endonuclease 1 μ L, ultrapure water 3 μ L; 2. pIRES carrier 4 μ L, Tango
TMBuffer 8 μ L, XhoI restriction endonuclease 1 μ L, ultrapure water 27 μ L; Mixing is placed on 37 ℃ of water-bath 3h;
PIRES carrier after C, purifying Xho I enzyme are cut and TCR V α 6 PCR products;
D, will be XhoI enzyme that purifying is good pIRES carrier and the TCR V α 6 PCR products after cutting cut with the EcoRI enzyme more respectively, system is: 1. TCR V α 6 PCR products (Xho I) 28 μ L, Tango
TMBuffer8 μ L, EcoRI restriction endonuclease 1 μ L, ultrapure water 3 μ L; 2. pIRES carrier (Xho I) 28 μ L, Tango
TMBuffer 8 μ L, EcoRI restriction endonuclease 1 μ L, ultrapure water 3 μ L; Mixing is placed on 37 ℃ of water-bath 3h;
PIRES carrier behind E, the double digestion and TCR V α 6 PCR products carry out ligation: system is: the pIRES carrier 2 μ L behind 10 * T4 buffer1 μ L, T4 DNA enzyme 1 μ L, the double digestion, the TCR V α 6 PCR products 6 μ L behind the double digestion, and mixing is placed on 16 ℃ and spends the night;
F, conversion: 1. will connect product (10 μ L) and add in the competence bacillus coli DH 5 alpha (1.5mL centrifuge tube, 100 μ L bacterium liquid ,-70 ℃ of preservations, Beijing Tiangen company), mixing is put 30min in the ice gently; 2. 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;
G, 10 bacterium colonies of random choose are inoculated in 5mL respectively and contain in the LB nutrient solution of penbritin, 37 ℃ of 180rpm jolting overnight incubation, PureLink
TMUltrapure plasmid extraction kit extracting plasmid (5415 type table model high speed centrifuge): 1. centrifugal bacterium liquid 15min under the 3000rpm room temperature, remove supernatant; 2. add 250 μ L and contain the Resuspension Buffer of RNase A, resuspended bacterium; 3. add 250 μ L Lysis Buffer, mixing is placed 5min under the room temperature gently; 4. add 350 μ LPrecipitation Buffer, mixing immediately vibrates; 5. with behind the centrifugal 10min of 11400rpm, supernatant liquor is transferred among the Mini column, the centrifugal 1min of 11400rpm removes waste liquid; 6. add 700 μ L Wash Buffer, the centrifugal 1min of 11400rpm removes waste liquid; 7. again with behind the centrifugal 1min of 11400rpm, Mini column is put into a new 1.5mL centrifuge tube, add the TE Buffer of 65 ℃ of 75 μ L preheatings, place 3min under the room temperature, the centrifugal 2min of 11400rpm, collected solution are and are rich in plasmid solution;
Identify with XhoI and EcoRI double digestion after H, the extraction plasmid DNA, 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, the PCR reaction volume is 25 μ L, 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; Enzyme cuts product and pcr amplification product is identified with 1% agarose gel electrophoresis.Filter out positive colony, again it is carried out two-way order-checking and identify, turn out to be behind the special gene sequence of the present invention the amplification positive colony and extract plasmid, thereby obtain the relevant TCR V α 6-pIRES plasmid of specific CTL.Measure the concentration of recombinant plasmid with ultraviolet spectrophotometer.
2, liposome-mediated recombinant plasmid TCR V α 6-pIRES transfection
(1) liposome Lipofectamine
TM2000 transfection A549 cell strains, Molt4 cell strain transfection A549 cell strain: the A549 cell strain that 1. will be in logarithmic phase is with 4 * 10
5The density of individual cells/well, the IMDM substratum of adding 2mL serum-free antibiotic-free is inoculated 6 well culture plates; 2. get TCR V α 6-pIRES recombinant plasmid (4 μ g) and place the 1.5mL centrifuge tube, add IMDM substratum to the 250 μ L of serum-free antibiotic-free, gently mixing; 3. get 15 μ L liposome Lipofectamine
TM2000 place the 1.5mL centrifuge tube, add IMDM substratum to the 250 μ L of serum-free antibiotic-free, hatch 5min under the room temperature behind the mixing gently; 4. will be 2. and 3. behind the mixing, hatch 20min under the room temperature; 5. will 4. add in (parallel 2 holes of transfection) in the A549 cell culture medium 1., other establishes 1 hole transfection pIRES empty carrier as negative control, and concrete operation method is with the transfection recombinant plasmid; Place 37 ℃, 5%CO
2Cultivating behind the 24h full dose in the incubator, to change liquid be perfect medium, add G418 behind the cultivation 72h and screen (final concentration 600 μ g/mL), changed a subculture in per 4 days, and added the G418 of same concentrations simultaneously, under these screening conditions, cultivate 20d and can obtain stable expression cell line.
Transfection Molt4 cell strain: the Molt4 cell strain that will be in logarithmic phase is with 2 * 10
6The density of individual cells/well is inoculated 6 orifice plates, gets 4 μ gTCR V α 6-pIRES recombinant plasmid and liposome Lipofectamine
TM2000 mix back transfection Molt4 cell strain, and method is with transfection A549 cell strain.
3, the evaluation of TCR V α 6 genetic expressions after the recombinant plasmid transfection
(1) RT-PCR and real-time quantitative PCR detect the expression of TCR V α 6 mRNA
After the transfection 7 days, each hole collects 3 * 10
6Cell (the A549 cell strain needs trysinization) extracts total RNA, synthetic cDNA respectively with TRIZol test kit and SurperScriptII reverse transcription test kit, through pcr amplification house-keeping gene β
2M detects synthetic quality (same first part, the step 7) of cDNA; Carry out pcr amplification with corresponding TCRV α 6 primers and corresponding C α primer, transfection empty carrier group is as negative control, and pcr amplification system and amplification condition are ditto described.
(2) indirect immunofluorescence and flow cytometer detect the expression of recombinant plasmid
After the transfection 10 days, collect 1 * 10
6Cell (A549 cell strain need trysinization), behind twice of the PBS washed cell of 0.1mol/L, mark rat anti people TCR V α monoclonal antibody (is anti-, dilution in 1: 100) is hatched 1h for 37 ℃; Behind the PBS washed cell three times, the mouse IgG of flag F ITC-rabbit Chinese People's Anti-Japanese Military and Political College antibody (two is anti-, dilution in 1: 50) is hatched 1h for 37 ℃; Behind the PBS washed cell three times, under fluorescent microscope, observe recombinant plasmid expression in the cell strain after transfection; Or directly use FITC-rat anti people TCR V α 6 monoclonal antibody marks, and hatch 30min for 37 ℃, PBS washed cell once back carries out the expression that streaming detects recombinant plasmid with flow cytometer.
(3) hybridization of the some marking and Western Blot detect TCR V α 6 proteic expression
After the transfection 20 days, collect 2 * 10
6The cell of stably express recombinant plasmid extracts total protein with RIPA protein extraction test kit, and vacuum lyophilization concentrates back Coomassie brilliant blue standard measure; The same albumen that extracts cord blood cells (TCR V α 6 subfamilies are expressed all positive) is as positive control, the negative contrast of the albumen of the cell of transfection empty carrier.
1) some marking hybridization
A, get NC film 2cm * 4cm, divide little lattice, put film respectively with the micro sample-adding rifle, the protein sample of recombinant plasmid group, positive controls, negative control group is put respectively 3 times, each 1 μ L; Carry out 2 parallel reactors;
B, with behind the blocking Reagent envelope film 4h with wash buffer washing 2 times, each 5min;
C, 1: 500 rat anti people TCR V α 6 monoclonal antibodies of adding (with blocking Reagent dilution) wash 2 times with wash buffer after 4 ℃ of overnight incubation, at every turn 5min;
D, 1: 500 the mouse IgG of HRP-rabbit Chinese People's Anti-Japanese Military and Political College antibody of adding (with blocking Reagent dilution) incubated at room 2h are with wash buffer washing 3 times, 5min at every turn;
E, the NC film is immersed DAB substrate solution (lucifuge) colour developing.
2)Western Blot
A, SDS-PAGE electrophoresis: protein concentrate sample, positive control and the negative control of transfection recombinant plasmid group are distinguished application of sample in 15%SDS-PAGE glue (the about 100 μ g of sample on every hole), for dying maker in advance, carry out 3 parallel reactors with PageRulerPrestained Protein Ladder; Behind constant voltage 60V electrophoresis 30min, change constant voltage 100V 1h;
B, separate the wherein SDS-PAGE glue of 1 parallel reactor, after Coomassie brilliant blue dyeing, use glacial acetic acid: meoh eluate repeatedly rinsing until there being clear band to show;
C, commentaries on classics film: pvdf membrane soaks 1min with methyl alcohol in advance, and being dipped into changes standby in the film liquid again; Filter paper that cuts and sponge also are dipped into changes 10min in the film liquid; The SDS-PAGE glue of 2 parallel reactors in addition of downcutting and corresponding pvdf membrane put into to be changeed film liquid and soaks a moment; Sponge-filter paper-pvdf membrane-SDS-PAGE glue-filter paper-sponge is put on the positive plate successively glue size=film＞filter paper; With the commentaries on classics film liquid of 4 ℃ of precoolings, constant voltage 40V, 90min (the wet commentaries on classics);
D, envelope film: with washing 2 times each 5min with wash buffer behind the blocking Reagent envelope film 4h;
The expression of E, detection people β-actin albumen (internal reference): wherein the pvdf membrane of 1 parallel reactor adds mouse anti human β-actin monoclonal antibody (with blocking Reagent dilution) of 1: 800, wash 2 times each 5min after 4 ℃ of overnight incubation with wash buffer; HRP-goat anti-mouse igg antibody (with blocking Reagent dilution) the incubated at room 2h that adds 1: 1000, with wash buffer washing 3 times, each 5min;
The expression of F, testing goal albumen TCR V α 6: 1: 800 rat anti people TCR V α 6 monoclonal antibodies of pvdf membrane adding (with blocking Reagent dilution) with other 1 parallel reactor, wash 2 times each 5min with washbuffer after 4 ℃ of overnight incubation; The mouse IgG of HRP-rabbit Chinese People's Anti-Japanese Military and Political College antibody (diluting) the incubated at room 2h that adds 1: 1000 with blockingReagent, with wash buffer washing 3 times, each 5min;
G, chemoluminescence method develop: pvdf membrane is changed among the LumiGLO Working Reagent over to incubated at room 3min in the darkroom; With tweezers pvdf membrane is taken out, drop removes unnecessary reagent; Pvdf membrane is put into plastics film, and guarantee does not have bubble between pvdf membrane and the plastics film; The plastics film that will contain pvdf membrane is put into X line exposing magazine, presses X line film (exposure 30s), the X line film that obtains developing after conventional development, the photographic fixing.
4, the normal human T-cell of recombinant plasmid TCR V α 6-pIRES transfection makes up specific CTL
(1) the normal human T-cell of recombinant plasmid TCRV α 6-pIRES 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 4 μ gTCR V α 6-pIRES recombinant plasmid and liposome Lipofectamine
TM2000 mix the normal human T-cell of back transfection, and working method is ditto described, cultivate the T cell that obtains relevant TCR V α 6 genes of antigen expressed specific CTL after increasing, and the expression of detection validation recombinant plasmid (method is the same).
(2) cytotoxic activity experiment
(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.Being the effector cell with the normal T cell of transfection recombinant plasmid, the normal T cell of transfection empty carrier respectively, is target cell with DLBL cell strain, Raji cell, Molt4 cell (negative control), and 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 2500rpm 30s 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 light close (opticaldensity, OD) value, mensuration wavelength 490nm, reference wavelength 670nm.
CTL cytotoxic activity calculation formula:
CTL 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)
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
The Second Affiliated Hospital of Guangzhou Medical School
＜120〉the MR gene order and the application of diffuse large B cell lymphoma related antigen specific TCR V α 6 subfamilies
<130>42
<160>2
<170>PatentIn version 3.2
<210>1
<211>333
<212>DNA
<213>Artificial sequence
<220>
＜223〉fill the air the MR gene order of large B cell lymphoma correlation antigen specific TCR V α 6 subfamilies
<400>1
tccgccaacc ttgtcatctc cgcttcacaa ctgggggact cagcaatgta tttctgtgca 60
atgagggatt actctggggc tgggagttac caactcactt tcgggaaggg gaccaaactc 120
tcggtcatac caaatatcca gaaccctgac cctgccgtgt accagctgag agactctaaa 180
tccagtgaca agtctgtctg cctattcacc gattttgatt ctcaaacaaa tgtgtcacaa 240
agtaaggatt ctgatgtgta tatcacagac aaaactgtgc tagacatgag gtctatggac 300
ttcaagagca acagtgccgc ggcctggagc aac 333
<210>2
<211>111
<212>PRT
<213>Artificial sequence
<220>
＜223〉fill the air large B cell lymphoma correlation antigen specific TCR
The protein of the MR gene order translation of V α 6 subfamilies
<400>2
Ser Ala Asn Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met
1 5 10 15
Tyr Phe Cys Ala Met Arg Asp Tyr Ser Gly Ala Gly Ser Tyr Gln Leu
20 25 30
Thr Phe Gly Lys Gly Thr Lys Leu Ser ValIle Pro Asn Ile Gln Asn
35 40 45
Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys
50 55 60
Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln
65 70 75 80
Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met
85 90 95
Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn
100 105 110
Claims (3)
1. the MR gene order of Diffuse Large B-Cell Lymphoma correlation antigen specific TCR V α 6 subfamilies, it is characterized in that: described MR gene order is as follows: TCCGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATGTATTTCTG TGCAATGAGGGATTACTCTGGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGA CCAAACTCTCGGTCATACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTG AGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCA AACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGC TAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCCGCGGCCTGGAGCAAC.
2. a kind of MR gene order of filling the air large B cell lymphoma correlation antigen specific TCR V α 6 subfamilies according to claim 1 is characterized in that: the described protein of stating the MR gene order translation of filling the air large B cell lymphoma correlation antigen specific TCR V α 6 subfamilies is as follows: SANLVISASQLGDSAMYFCAMRDYSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQL RDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN.
3. the application of the MR gene order of the described diffuse large B cell lymphoma related antigen specific TCR of claim 1 V α 6 subfamilies in preparation diffuse large B cell lymphoma specific molecular target adoptive immunity medicine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100259947A CN101225388A (en) | 2008-01-24 | 2008-01-24 | MR gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 6 subfamily and uses thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008100259947A CN101225388A (en) | 2008-01-24 | 2008-01-24 | MR gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 6 subfamily and uses thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101225388A true CN101225388A (en) | 2008-07-23 |
Family
ID=39857593
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008100259947A Pending CN101225388A (en) | 2008-01-24 | 2008-01-24 | MR gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 6 subfamily and uses thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101225388A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150114A (en) * | 2020-05-23 | 2021-07-23 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
| CN113248593A (en) * | 2020-05-23 | 2021-08-13 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
| WO2022194033A1 (en) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | Peripheral blood tcr marker for diffuse large b-cell lymphoma, and detection kit and use therefor |
-
2008
- 2008-01-24 CN CNA2008100259947A patent/CN101225388A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150114A (en) * | 2020-05-23 | 2021-07-23 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
| CN113248593A (en) * | 2020-05-23 | 2021-08-13 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
| WO2022194033A1 (en) * | 2021-03-15 | 2022-09-22 | 成都益安博生物技术有限公司 | Peripheral blood tcr marker for diffuse large b-cell lymphoma, and detection kit and use therefor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mathewson et al. | Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis | |
| Sutton et al. | Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans | |
| Nelson | Immunobiology of the Macrophage | |
| Antonsson et al. | The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses | |
| Qu et al. | Gamma-ray resistance of regulatory CD4+ CD25+ Foxp3+ T cells in mice | |
| Nairn et al. | Immunological reactivity in patients with carcinoma of colon | |
| CN102203290A (en) | Molecular marker for cancer stem cell | |
| CN1444043A (en) | Individuation specific immunocytofunction determination method | |
| Proctor et al. | Targeting replication stress using CHK1 inhibitor promotes innate and NKT cell immune responses and tumour regression | |
| CN101225388A (en) | MR gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 6 subfamily and uses thereof | |
| CN102161982B (en) | Monoclonal antibodies (McAb) against human CXCR3 molecules and application thereof | |
| CN102183655A (en) | New molecular marker CUEDC2 (CUE Domain Containing 2) protein for prognosis judgement in endocrine therapy of breast cancer | |
| CN103499685A (en) | Immunofluorescence diagnostic reagent and preparation thereof and application for clinical diagnosis of human urinary tract pathogen infection | |
| CN101182519A (en) | P gene order of diffused large B cell lymphoid tumor related antigen specific TCR V Alpha 23 subfamily | |
| CN101225389A (en) | VR gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 23 subfamily and uses thereof | |
| CN101235373A (en) | RG gene sequence for diffusivity large B cell lymphoma correlation antigen specific TCR Vbeta13 subfamily and application thereof | |
| CN105233280A (en) | DC-based glioma holoantigen vaccine and preparation method thereof | |
| CN106199005B (en) | The active method of the anti-colorectal cancer of lymphocyte is detected using CD137 expression quantity | |
| CN102768283A (en) | Kit for detecting or supplementarily detecting medullary leukemia cell differentiation stages | |
| CN101182518A (en) | Y gene order of diffused large B cell lymphoid tumor related antigen specific TCR V Beta 13 subfamily | |
| CN101225390A (en) | MS gene order of diffuse large B cell lymphoma related antigen specific TCR V alpha 6 subfamily and uses thereof | |
| Nossal | The case history of Mr TI Terminal patient or still curable? | |
| CN105062975A (en) | Imatinib drug-resistant gastrointestinal stromal tumor cell line and nude mice transplantation tumor model construction method | |
| CN109061191A (en) | Application of the S100P albumen as marker in diagnostic activities tuberculosis | |
| CN108424933A (en) | A kind of primary multiple myeloma mouse model construction method and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Open date: 20080723 |