WO2019061197A1 - 一种扩增tcr全长序列的pcr引物及其应用 - Google Patents

一种扩增tcr全长序列的pcr引物及其应用 Download PDF

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WO2019061197A1
WO2019061197A1 PCT/CN2017/104107 CN2017104107W WO2019061197A1 WO 2019061197 A1 WO2019061197 A1 WO 2019061197A1 CN 2017104107 W CN2017104107 W CN 2017104107W WO 2019061197 A1 WO2019061197 A1 WO 2019061197A1
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primer
pcr
tcr
sequence
round
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PCT/CN2017/104107
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French (fr)
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王飞
周清
王磊
吴靓
赵正琦
杨乃波
李贵波
侯勇
李波
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深圳华大生命科学研究院
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Priority to CN201780095498.6A priority Critical patent/CN111164210A/zh
Priority to PCT/CN2017/104107 priority patent/WO2019061197A1/zh
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  • the invention belongs to the field of molecular biology, and relates to a PCR primer for amplifying a full-length TCR sequence and an application thereof, and particularly relates to a PCR primer for amplifying a full-length TCR sequence, a method for amplifying a full-length TCR sequence and an application thereof.
  • T lymphocyte precursor enters the thymus from the bone marrow and undergoes a positive and negative screening process to develop into mature T cells. During this period, T cells acquire a functional T cell receptor (TCR), express surface molecules of mature T cells (CD4/CD8, etc.) and obtain MHC restriction and autotolerance.
  • T cell type associated with the complexity and diversity of the TCR rearrangements in the thymus, TRB (TCR beta) gene via DJ, VDJ, VDJC region rearrangement eventually produce about 108 different gene rearrangement TRB; TRA (TCR ⁇ ) gene via VJ, VJC region rearrangement, eventually produce about 104 different gene rearrangement TRA.
  • TCR diversity is up to 10 18 due to the rearrangement of TRB and TRA, which determines how the human immune system adapts to environmental changes.
  • M ⁇ Ltiplex PCR combined with high-throughput sequencing technology, by designing multiple primers covering all TRA and TRB typing, capturing the intact TRA and TRB isoforms in the population, and reading the TRA of each subtype by high-throughput sequencing.
  • TRB sequence this technique employs the design of multiplex PCR primers in the TCR V region, such as the T cell repertoire primer Vp1-Vp9 covering all functional TRB rearrangement gene V regions, And T-cell repertoire primers Vp1-Vp24 covering all functional TRA rearrangement gene V regions, combined with TCR constant region C region primers, for multiplex PCR amplification, which can generally perform TCR repertoire sequence amplification of population samples, capture diversity
  • the TCR sequence was subsequently analyzed by combining high-throughput sequencing techniques for T cell diversity.
  • M ⁇ Ltiplex PCR has certain amplification preference, and can not accurately obtain TCR ⁇ / ⁇ pairing sequence information.
  • M ⁇ Ltiplex RT-PCR developed at the single cell level can distinguish the single-cell TCR ⁇ / ⁇ pairing sequence to a certain extent. It is one of the few single-cell TCR ⁇ / ⁇ paired amplification technologies, but the experimental system is relatively complicated. Easy to achieve high throughput sequencing;
  • RACE rapid-amplification of cDNA ends
  • designing primers by constant known sequences in TCR sequences, amplifying TCR gene sequences in populations, and reading each subtype by high-throughput sequencing technology TRA, TRB sequences; gene-specific primers (GSP1 and GSP2) designed based on PCR technology based on a known cDNA sequence, designed for 5' and 3' RACE reactions, obtained by extension to both ends to obtain a complete 3' And the 5' end method.
  • the product of the PCR is specific due to the presence of both primers.
  • This technique is applied to T cell TCR amplification.
  • the TRA or TRB rearrangement gene constant region C region primer is generally designed.
  • the 5' end of the TCR gene is amplified by 5' RACE, and the TCR sequence is interpreted by high-throughput sequencing technology. This technique is commonly used for population sample T cell diversity analysis with a high initial sample size and is difficult to capture for low abundance T cell subsets.
  • DNA barcode combined with high-throughput sequencing technology is to culture T cells with distinct subpopulations in 96-well plates, and to amplify TCRs of different T cell subsets.
  • the specific DNA barcode is combined with the amplification product, and the TCR sequence information and the DNA barcode information are simultaneously read, and the source of the hole corresponding to the TCR is determined by the DNA barcode information.
  • the sample processing procedure of this technical solution is complicated, and it is necessary to pre-group T cells, and the sequencing result is not accurate. Simulate alpha/beta pairing information.
  • TraCeR is an open analytical tool (Michael JTStubbington et al. (2016) Nature Methods, DOI: 10.1038/NMETH.3800) for analysis of T cell single-cell full transcriptome sequences, reconstructed by computer technology.
  • the long TCR ⁇ / ⁇ pairing sequence reveals T cell function specificity.
  • Single-cell full transcriptome data can reveal the transcriptional heterogeneity between cells and the evolutionary process between different cells, thus deducing the T cell subset type.
  • This technology combines single-cell transcriptome data to obtain the ⁇ / ⁇ pairing sequence information of recombinant TCR, which has higher advantages and innovations, but the integrity and accuracy of computer-reconstructed TCR sequences still need to be improved.
  • the present invention provides a PCR primer for amplifying a full-length TCR sequence and an application thereof, thereby realizing full-length amplification of a TCR sequence and obtaining pairing information of an ⁇ / ⁇ subunit in a TCR, and solving the problem.
  • Other existing methods have the disadvantages of high initial sample size, incomplete TCR subtype coverage and preferential amplification, incomplete expansion of TCR sequences, and real-life realization of full-length amplification of TCR sequences at the individual T cell level.
  • the invention provides a PCR primer pair that amplifies a full length TCR sequence, comprising an upstream primer and a downstream primer;
  • the upstream primer is a primer designed according to a piece of linker sequence, the linker sequence being introduced to the first The 3' end of the strand cDNA; the downstream primer is a downstream primer designed according to the C region of the TCR.
  • the antigen binding site of TCR is extremely diverse, and the mechanism for generating such diversity is mainly derived from the V(D)J recombination of the immunoglobulin gene, and the gene locus encoding the immunoglobulin is composed of a plurality of gene fragments, including Variable segment (V), junction segment (J), and various segments (D) that may exist between them, the alpha subunit is recombined by VJ, and the beta subunit is recombined by VDJ, where J is linked to the constant region (C)
  • V Variable segment
  • J junction segment
  • D various segments
  • the alpha subunit is recombined by VJ
  • the beta subunit is recombined by VDJ
  • J is linked to the constant region (C)
  • the TCR has species diversity, its C region sequence homology is better, so the present invention designs the downstream primer according to the C region, and can be designed at any position from the 5' end to the 3' end of the C region.
  • the designed primers follow general primer design principles, such as GC content 40-60%, no secondary hairpin structure, no primer dimer, etc., and primer design should be designed as far as possible without base polymorphism. .
  • the upstream primer is 18-28 nt in length, for example 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt or 28 nt, preferably 23 nt.
  • the linker sequence is introduced by a template method using a linker primer, which is designed according to the linker sequence, wherein the linker primer can be any fixed length sequence, those skilled in the art.
  • the nucleotide sequence of the adaptor primer of the present invention is shown in SEQ ID NO. 1, and the nucleotide sequence shown in SEQ ID NO. 1 is as follows:
  • nucleotide sequence of the upstream primer of the present invention is shown in SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is as follows:
  • the downstream primer is designed according to the 5' end or the 3' end of the C region of the TCR. Since the platform for post-sequencing is mainly the Miseq sequencing or Sanger sequencing platform, the maximum read length can only be used for the Miseq sequencing platform. 600 bp, and the V(D)J sequence is almost as long, so primers are designed at the 5' end of the C region primer to amplify the full length of V(D)J. If it is a Sanger sequencing platform, it can be at the 3' end. Primers were designed so that sequencing can directly yield the full length of V(D)JC.
  • the downstream primer of the present invention is preferably a nested primer, and the downstream primer includes an external primer, an intermediate primer or an internal primer. Any one or a combination of at least two of the primers, the combination may be, for example, a combination of an external primer and an intermediate primer, a combination of an intermediate primer and an internal primer, a combination of an external primer, an intermediate primer, and an internal primer, preferably an external primer. , an intermediate primer or a combination of internal primers.
  • a three-round nested primer is selected, and the inventors have found that not only the sample with a small initial amount in the single cell of the present application, but also the mechanism for the TCR is so complicated, the result after amplification is very accurate.
  • the full length of the TCR is composed of the full length of the TCR ⁇ gene and the full length of the TCR ⁇ gene or the full length of the TCR ⁇ gene and the full length of the TCR ⁇ gene, and 95% of the TCR is composed of two subunits ⁇ and ⁇ , 5% TCR consists of two subunits, gamma and delta.
  • nucleotide sequences of the external primer, the intermediate primer and the internal primer of the downstream primer which amplifies the full length of the TCR ⁇ gene are shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, respectively;
  • the nucleotide sequence of the primer is as follows:
  • External primer (SEQ ID NO. 3): GCAGACAGACTTGTCACTGG;
  • nucleotide sequences of the external primer, the intermediate primer and the internal primer of the downstream primer which amplifies the full length of the TCR ⁇ gene are shown in SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively;
  • the nucleotide sequence of the primer is as follows:
  • External primer (SEQ ID NO. 6): GAGCAGATTAAACCCGGCCACTT;
  • nucleotide sequences of the external primer, the intermediate primer and the internal primer of the downstream primer which amplifies the full length of the TCR ⁇ gene are shown in SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO.
  • the nucleotide sequence of the primer is as follows:
  • External primer (SEQ ID NO. 9): TGGTCGGGGAAGAAGCCTGTG;
  • nucleotide sequences of the external primer, the intermediate primer and the internal primer of the downstream primer which amplifies the full length of the TCR ⁇ gene are shown in SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, respectively;
  • the nucleotide sequence of the primer is as follows:
  • External primer (SEQ ID NO. 12): GCCTCTGGAATCCTTTCTCTTGACC;
  • the external primer SEQ ID NO. 3, the intermediate primer SEQ ID NO. 4, the internal primer SEQ ID NO. 5, and the external primer SEQ ID of the full length of the amplified TCR ⁇ gene are amplified.
  • the downstream primer is designed according to a position near the 5' end of the C region of 20-30 bp; the external primer for amplifying the full length of the TCR ⁇ gene SEQ ID NO. 6, the intermediate primer SEQ ID NO. 7, and the internal primer SEQ ID NO.
  • the sequence is about 800-900 bp, and the downstream primer is designed to be within 100 bp from the 3' end of the C region.
  • the invention also provides a method of amplifying a full length TCR sequence, comprising the steps of:
  • the cell of the step (1) is a single cell derived from peripheral blood mononuclear cells of peripheral blood.
  • the isolated single cells described in the step (1) are conventional techniques in the art, and are not particularly limited herein.
  • the present invention employs a method comprising the following specific steps: peripheral blood mononuclear cells are stained with antibodies, and flow-through The CD8+ T cells were sorted by a cytometer, single cells were picked up by a micromanipulator, added to the lysate, and rapidly centrifuged to ensure that the cells entered the lysate, immediately placed on dry ice, and the samples were stored at -80 ° C before amplification or In liquid nitrogen.
  • the lysate formulation is as follows:
  • the lysed single cells are preferably subjected to the following steps: a PCR tube containing a single cell is placed in a PCR machine, and the hot lid temperature is set to 75 °C. The single cells were incubated at 72 ° C for 3-5 min, immediately after the completion of the lysis, and placed on ice for 1 min; centrifuged at 10,000 rpm for 4 s for 30 s, and immediately transferred to ice.
  • the system for reverse transcription of mRNA described in step (2) comprises a linker primer (TSO).
  • TSO linker primer
  • the linker primer has a final concentration of 0.5-2 ⁇ M, and may be, for example, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.8 ⁇ M. Or 2 ⁇ M, preferably 1 ⁇ M.
  • the reverse transcription system described in step (2) is as follows:
  • the reverse transcription conditions of the mRNA are: 42 ° C for 90 min 1 cycle; 50 ° C for 2 min, 42 ° C for 2 min, 10 cycles; 70 ° C for 15 min 1 cycle; stored at 4 ° C.
  • the PCR amplification is nested PCR, and the number of the nested PCR is 2-3 times, preferably 3 times.
  • the nested PCR specifically includes:
  • the third round of nested PCR is carried out by using the upstream primer and the internal primer as primers and the second round of amplification product obtained in the step (2') as a template to obtain a third round of amplification product.
  • the first round of nested PCR conditions described in step (1') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 55 ° C for 15 s, 72 ° C for 2 min, 25 cycles; 72 ° C for 5 min, 1 cycle ; stored at 4 ° C;
  • the second round of nested PCR conditions described in step (2') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 60 ° C for 15 s, 72 ° C for 2 min, 25 cycles; 72 ° C for 5 min, 1 cycle ; stored at 4 ° C;
  • the third round nested PCR conditions described in step (3') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 60 ° C for 15 s, 72 ° C for 2 min, 35 cycles; 72 ° C for 5 min, 1 cycle ; Store at 4 ° C.
  • the sequencing described in step (4) is sanger sequencing and/or Miseq sequencing.
  • the method for amplifying a full length TCR sequence comprises the following steps:
  • the first round nested PCR conditions described in the step (1') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 55 ° C for 15 s, 72 ° C for 2 min, 25 cycles; 72 ° C for 5 min, 1 cycle; °C;
  • the second round nested PCR conditions described in step (2') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 60 ° C for 15 s, 72 ° C for 2 min, 25 cycles; 72 ° C for 5 min, 1 cycle; °C;
  • the third round nested PCR conditions described in the step (3') are: 95 ° C for 3 min, 1 cycle; 98 ° C for 20 s, 60 ° C for 15 s, 72 ° C for 2 min, 35 cycles; 72 ° C for 5 min, 1 cycle; °C;
  • the invention provides a kit for amplifying a full length TCR sequence, the kit comprising a PCR primer pair that amplifies a full length TCR sequence as described in the first aspect.
  • the present invention provides a PCR primer pair for amplifying a full length TCR sequence according to the first aspect, a method for amplifying a full length TCR sequence according to the second aspect, or a kit according to the third aspect Use for construction of the TCR library.
  • the present invention provides a PCR primer pair for amplifying a full length TCR sequence according to the first aspect or a kit according to the third aspect for preparing an immunological diagnosis treatment and/or prognosis monitoring of a disease. the use of.
  • the present invention provides a PCR primer pair for amplifying a full-length TCR sequence and/or a method for amplifying a full-length TCR sequence according to the first aspect, which achieves full-length amplification of a TCR sequence and obtains ⁇ / ⁇ and a TCR. / or pairing information of ⁇ / ⁇ subunits.
  • the present invention has the following beneficial effects:
  • the primers and methods of the present invention are effective for different kinds of TCRs, and it is not necessary to design a specific upstream primer for a single TCR, and the TCR transcript is first made by SMART template conversion technology by using a linker primer in reverse transcription.
  • the 3' end of the strand cDNA was introduced into a linker sequence, and the subsequent PCR was performed with the upstream primer designed according to the linker sequence, and the downstream primer of the conserved region of the TCR C region was used to successfully amplify the full length of the TCR;
  • the present invention adopts three-round nested PCR to amplify the full length of TCR, and is suitable for samples with template as low as 10 pg of total RNA, and can simultaneously acquire the full length sequence of a single cell TCR, thereby obtaining different TCRs.
  • the ⁇ / ⁇ pairing information or the ⁇ / ⁇ pairing information has high sensitivity to TCR amplification of different antigen-stimulated T cells, complete coverage of subtypes and relatively low cost;
  • the method of the present invention is applicable to cell heterogeneity and T cell subtype grouping, exploring a new immunological mechanism, constructing a large-scale full-length TCR immune group library, facilitating disease diagnosis and health management, and developing neo TCR-in combination with tumor new antigen.
  • Figure 1 shows the design principle of PCR primers for amplifying the full length sequence of TCR
  • Example 2 is a diagram showing electrophoresis results in Example 2 of the present invention, wherein the marker is a 100 bp marker, and the bottom is 100 bp, 200 bp, 300 bp, 400 bp, 500 bp (brightest band), 600 bp, 700 bp, 800 bp, 900 bp, 1000bp and 1500bp;
  • Example 3 is a diagram showing electrophoresis results in Example 3 of the present invention, wherein the marker is a 100 bp marker, and the bottom is 100 bp, 200 bp, 300 bp, 400 bp, 500 bp (brightest band), 600 bp, 700 bp, 800 bp, 900 bp, 1000bp and 1500bp.
  • the linker sequence is introduced into the 3' end of the first strand of cDNA by a template method using a linker primer as shown in SEQ ID NO. 1, SEQ ID NO.
  • the nucleotide sequences shown are as follows:
  • the upstream primer is designed according to the adaptor sequence, which is shown in SEQ ID NO. 2, and the nucleotide sequence shown in SEQ ID NO. 2 is as follows:
  • the downstream primer is designed according to the C region of the TCR gene, and the complete sequence of the C region of the T cell ⁇ and ⁇ chain is as follows:
  • the PE300 sequencing strategy requires that the library Insert length be no more than 600bp, and the TCR ⁇ chain 5' UTR sequence +V+D+J region sequence is just within 600bp, so we The downstream primer is placed 20 to 30 bp near the 5' end of the C region as follows:
  • the method for amplifying a full length TCR sequence comprises the following steps:
  • Peripheral blood mononuclear cells were stained with antibodies, and CD8+ T cells were sorted by flow cytometry. Single cells were picked up by micromanipulator, added to the lysate, and rapidly centrifuged to ensure that the cells entered the lysate and immediately placed in dry ice. Above, the sample is stored at -80 ° C or liquid nitrogen before amplification;
  • the first strand cDNA of all mRNAs is synthesized
  • the upstream primer is SEQ ID NO. 1, and the nucleotide sequence shown in SEQ ID NO. 1 is:
  • the downstream primers are as follows:
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • pre-amplification is carried out according to the following conditions:
  • Example 2 Compared with Example 2, except for the nested primers, other conditions and methods are the same as in Example 2, and the nested primers are as follows:
  • the upstream primer is SEQ ID NO. 1, and the nucleotide sequence shown in SEQ ID NO. 1 is:
  • the downstream primers are as follows:
  • the primers and methods of the present invention are effective for different kinds of TCRs, and do not need to design specific upstream primers for a single TCR, and the TCR transcript is first by using SMART template conversion technology in reverse transcription.
  • the 3' end of the strand cDNA introduces a linker sequence, and the subsequent PCR uses the primer designed according to the linker sequence as the upstream primer, and the downstream primer of the conserved region of the TCR C region is used to successfully amplify the full length of the TCR.
  • the present invention adopts Three-round nested PCR, which can amplify the full length of TCR and is suitable for samples with template as low as 10pg total RNA.

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Abstract

本发明公开了一种扩增TCR全长序列的PCR引物对及其制备方法和应用，包括上游引物和下游引物：所述上游引物根据一段接头序列设计，所述接头序列通过接头引物TSO引入到第一链cDNA的3'端；所述下游引物根据TCR的C区设计。

Description

一种扩增TCR全长序列的PCR引物及其应用 技术领域
本发明属于分子生物学领域，涉及一种扩增TCR全长序列的PCR引物及其应用，具体涉及一种扩增TCR全长序列的PCR引物，扩增TCR全长序列的方法及其应用。
背景技术
T淋巴细胞前体从骨髓进入胸腺，经历阳性和阴性筛选过程，发育为成熟的T细胞。期间，T细胞获得功能性T细胞受体(T cell receptor，TCR)，表达成熟T细胞的表面分子(CD4/CD8等)且获得MHC限制性和自身耐受性。T细胞种类复杂多样性与TCR重排相关，在胸腺中，TRB(TCRβ)基因经过D-J，V-D-J，V-D-J-C区域重排，最终产生约108不同TRB重排基因；TRA(TCRα)基因经过V-J，V-J-C区域重排，最终产生约104不同TRA重排基因。在人体外周血中，TCR多样性由于TRB、TRA的重排多达1018，决定着人的免疫系统如何适应环境的变化。
目前，对TCR的主要鉴别方法之一是对α和β链进行测序。传统免疫学研究中，基于群体细胞测序获得的数据很难实现对细胞异质性和不同细胞亚群的特性分析。现有技术中目前采用的具体实验技术如：
(1)MμLtiplex PCR联合高通量测序技术，通过设计覆盖所有TRA、TRB分型的多重引物，在群体中捕获尽量完整的TRA、TRB亚型，高通量测序读取每一个亚型的TRA、TRB序列；该技术采用在TCR V区设计多重PCR引物，如针对覆盖所有功能TRB重排基因V区的T细胞repertoire引物Vp1-Vp9，以 及覆盖所有功能TRA重排基因V区的T细胞repertoire引物Vp1-Vp24，结合TCR恒定区C区引物，进行多重PCR扩增，该技术一般可以进行群体样本的TCR repertoire序列扩增，捕获多样性的TCR序列，后续通过联合高通量测序技术对T细胞多样性进行分析。但在不同T细胞亚型间，MμLtiplex PCR有一定的扩增偏好性，不能准确的得到TCRα/β配对序列信息。进一步在单细胞层面开发的MμLtiplex RT-PCR，可以一定程度的区分单细胞TCRα/β配对序列，是目前为数不多的一种单细胞TCRα/β配对扩增技术，但实验体系相对复杂，不易于实现高通量测序；
(2)RACE(rapid-amplification of cDNA ends)联合高通量测序技术，通过TCR序列中恒定已知序列设计引物，在群体中扩增TCR基因序列，高通量测序技术读取每个亚型的TRA、TRB序列；基于PCR技术基础上由已知一段cDNA序列，设计5’和3’RACE反应的基因特异性引物(GSP1和GSP2)，通过往两端延伸扩增而获得完整的3’和5’端的方法。由于两个引物的存在，PCR的产物是特异性的。该技术应用于T细胞TCR扩增，一般设计TRA或TRB重排基因恒定区C区引物，通过5’RACE扩增TCR基因5’端的序列，通过高通量测序技术，从而解读TCR序列。该技术通常用于群体样本T细胞多样性分析，起始样本量较高，对于低丰度的T细胞亚群较难捕获。
(3)群体高通量测序：DNA条形码结合高通量测序技术是在96孔板中培养具有明显亚群的T细胞，针对不同T细胞亚群的TCR进行扩增。在高通量测序过程中，特异性的DNA条形码与扩增产物结合，同时读取TCR序列信息和DNA条形码信息，通过DNA条形码信息确定TCR对应的孔来源。然而，该技术方案样本处理流程复杂，需要预先对T细胞进行分群，且测序结果不能准确 模拟α/β配对信息。
(4)TraCeR是一种开放型分析工具(Michael J.T.Stubbington等，(2016)Nature Methods，DOI：10.1038/NMETH.3800)，用于分析T细胞单细胞全转录组序列，通过计算机技术重构全长TCR α/β配对序列，揭示T细胞功能特异性。单细胞全转录组数据可以揭示细胞之间的转录异质性和不同细胞之间的演化进程，从而推算出T细胞亚群类型。该技术结合单细胞转录组数据，获得重组的TCR的α/β配对序列信息，具有较高的优势和创新，但是计算机重构的TCR序列的完整性和准确性仍有待于提高。
这些技术无法准确区分T细胞TCR亚群及TCRα/β配对关系，而要进一步理解T细胞反应机理，需要对不同T细胞亚群内部和相互间的表型差异进行更精确的识别。因此，在单细胞水平进行免疫细胞分析越来越广泛被应用于免疫学研究中。然而，目前没有十分有效的技术可以在单细胞层面实现TCRα/β配对测序，且覆盖已知的所有TRA、TRB亚型。
发明内容
针对现有技术中的缺陷，本发明提供了一种扩增TCR全长序列的PCR引物及其应用，以实现TCR序列的全长扩增并获得TCR中α/β亚基的配对信息，解决了现有其他方法起始样本量高、TCR亚型覆盖不完整和偏好性扩增、TCR序列扩增不完整等缺点，真正意义上的实现人单个T细胞层面的TCR序列全长扩增。
第一方面，本发明提供了一种扩增TCR全长序列的PCR引物对，包括上游引物和下游引物；
所述上游引物为根据一段接头序列设计的引物，所述接头序列引入到第一 链cDNA的3’端；所述下游引物为根据TCR的C区设计的下游引物。
本发明中，TCR的抗原结合位极为多样化，产生这种多样性的机理主要源于免疫球蛋白基因的V(D)J重组，编码免疫球蛋白的基因位点由许多基因片段构成，包括可变段(V)、连接段(J)以及之间可能存在的多样段(D)，α亚基由VJ重组产生，β亚基则由VDJ重组产生，其中J与恒定区(C)相连，尽管TCR具有种类多样性，但是其C区序列同源性较好，所以本发明将下游引物根据C区进行设计，可以在C区的5’端到3’端中的任意位置进行设计，再通过在TCR的3’端加入一段接头序列，同时根据其设计上游引物，通过这样的上游和下游引物就可以获得完整的V(D)J序列，从而得到TCR全长。
本发明中，所述设计引物遵循一般引物设计原则，例如GC含量40-60％、无二级发夹结构、无引物二聚体等，引物设计应该尽量设计在没有碱基多态性的位置。
根据本发明，所述上游引物的长度为18-28nt，例如可以是18nt、19nt、20nt、21nt、22nt、23nt、24nt、25nt、26nt、27nt或28nt，优选为23nt。
根据本发明，所述接头序列是利用接头引物通过转换模板法引入的，所述上游引物是根据所述接头序列设计的，其中所述接头引物可以是任意一段固定长度的序列，本领域技术人员可以根据需要进行设计，本发明的接头引物的核苷酸序列如SEQ ID NO.1所示，所述SEQ ID NO.1所示的核苷酸序列如下：
Figure PCTCN2017104107-appb-000001
本发明的上游引物的核苷酸序列如SEQ ID NO.2所示，所述SEQ ID NO.2所示的核苷酸序列如下：
Figure PCTCN2017104107-appb-000002
根据本发明，所述下游引物根据TCR的C区的5’端或3’端进行设计，由于后期测序的平台主要是Miseq测序平或Sanger测序平台，针对Miseq测序平台，因为最大读长只能600bp，而V(D)J序列差不多已经有这么长，所以在C区引物的5′端设计引物，扩增得到V(D)J全长；如果是Sanger测序平台，则可以在3′端设计引物，这样测序可以直接得到V(D)JC全长。
根据本发明，由于本发明的TCR检测为单细胞裂解后进行检测，起始样本浓度比较低，本发明所述下游引物优选为巢式引物，所述下游引物为包括外部引物、中间引物或内部引物中的任意一种或至少两种的组合，所述组合例如可以是外部引物和中间引物的组合，中间引物和内部引物的组合，外部引物、中间引物和内部引物的组合，优选为外部引物、中间引物或内部引物的组合。
本发明中，选用三轮巢式引物，发明人发现不仅能够针对本申请单细胞中的起始量小的样本，且其针对TCR如此复杂的机构，扩增后的结果非常准确。
根据本发明，所述TCR全长由TCRα基因全长和TCRβ基因全长组成或由TCRγ基因全长和TCRδ基因全长组成，95％的TCR由α和β两个亚基组成，5％的TCR由γ和δ两个亚基组成。
根据本发明，扩增TCRα基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5所示；
所述引物的核苷酸序列如下：
外部引物(SEQ ID NO.3)：GCAGACAGACTTGTCACTGG；
中间引物(SEQ ID NO.4)：TGGATTTAGAGTCTCTCAGCTGGTACACG；
内部引物(SEQ ID NO.5)：GGTACACGGCAGGGTCAGGGTTC；
根据本发明，扩增TCRα基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8所示；
所述引物的核苷酸序列如下：
外部引物(SEQ ID NO.6)：GAGCAGATTAAACCCGGCCACTT；
中间引物(SEQ ID NO.7)：GGATTCGGAACCCAATCAC；
内部引物(SEQ ID NO.8)：TCGACCAGCTTGACATCACAGGAACT；
根据本发明，扩增TCRβ基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.9、SEQ ID NO.10和SEQ ID NO.11所示；
所述引物的核苷酸序列如下：
外部引物(SEQ ID NO.9)：TGGTCGGGGAAGAAGCCTGTG；
中间引物(SEQ ID NO.10)：TCTGCTTCTGATGGCTCAAACACAGC；
内部引物(SEQ ID NO.11)：TTCTGATGGCTCAAACACAGCGA；
根据本发明，扩增TCRβ基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.12、SEQ ID NO.13和SEQ ID NO.14所示；
所述引物的核苷酸序列如下：
外部引物(SEQ ID NO.12)：GCCTCTGGAATCCTTTCTCTTGACC；
中间引物(SEQ ID NO.13)：ACCAGCACRGCATACADGG；
内部引物(SEQ ID NO.14)：TCTCATAGAGGATGGTGGCAGACAGG.
本发明中，所述扩增TCRα基因全长的外部引物SEQ ID NO.3，中间引物SEQ ID NO.4，内部引物SEQ ID NO.5和扩增TCRβ基因全长的外部引物SEQ ID  NO.9，中间引物SEQ ID NO.10，内部引物SEQ ID NO.11针对Miseq测序平台，由于最大读长只能600bp，TCR 5’端UTR序列+V+D+J区序列正好在600bp以内，所述下游引物为根据靠近C区的5’端20-30bp的位置进行设计的；所述扩增TCRα基因全长的外部引物SEQ ID NO.6，中间引物SEQ ID NO.7，内部引物SEQ ID NO.8和扩增TCRβ基因全长的外部引物SEQ ID NO.12，中间引物SEQ ID NO.13，内部引物SEQ ID NO.14针对Sanger测序平台，由于最大读长800bp，TCR全长序列约800-900bp，所述下游引物为根据靠近C区的3’端100bp以内进行设计的。
第二方面，本发明还提供了一种扩增TCR全长序列的方法，包括如下步骤：
(1)裂解细胞；
(2)将得到的mRNA进行反转录，得到第一链cDNA；
(3)以步骤(2)得到的第一链cDNA为模板，以第一方面所述的上游引物和下游引物进行PCR扩增；
(4)测序验证，得到所述TCR的全长序列。
根据本发明，步骤(1)所述的细胞为单细胞，所述单细胞来源于外周血的外周血单个核细胞。
优选地，步骤(1)所述的分离单细胞为本领域的常规技术，在此不做特殊限定，本发明采用方法，包括如下具体步骤：外周血单个核细胞经抗体染色后，利用流式细胞仪分选出CD8+T细胞，利用显微操作仪挑取单细胞，加入裂解液中，快速离心保证细胞进入裂解液，立即放入干冰上，扩增之前将样品保存于-80℃或者液氮中。
优选地，所述裂解液配方如下表所示：
Figure PCTCN2017104107-appb-000003
为了保证mRNAs都从单细胞中释放，所述裂解单细胞优选为如下步骤：将装有单细胞的PCR管置于PCR仪内，热盖温度设为75℃。将单细胞72℃孵育3-5min，裂解完成后立即置于冰上1min；10000rpm 4℃离心30s，后立即转至冰上。
根据本发明，步骤(2)所述的mRNA反转录的体系包括接头引物(TSO)。
根据本发明，所述接头引物的终浓度为0.5-2μM，例如可以是0.5μM、0.6μM、0.7μM、0.8μM、0.9μM、1μM、1.2μM、1.3μM、1.5μM、1.6μM、1.8μM或2μM，优选为1μM。
优选地，步骤(2)所述的反转录体系如下表所示：
Figure PCTCN2017104107-appb-000004
Figure PCTCN2017104107-appb-000005
根据本发明，所述的mRNA反转录的条件为：42℃ 90min 1循环；50℃ 2min，42℃ 2min，10循环；70℃ 15min 1循环；保存在4℃。
根据本发明，步骤(3)所述的下游引物为巢式引物时，所述PCR扩增为巢式PCR，所述巢式PCR的次数为2-3次，优选为3次。
根据本发明，所述巢式PCR具体包括：
(1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
(2’)以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
(3’)以所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物。
优选地，步骤(1’)所述的第一轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，55℃ 15s，，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
优选地，步骤(2’)所述的第二轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
优选地，步骤(3’)所述的第三轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，，72℃ 2min，35个循环；72℃ 5min，1循环；保存在4℃。
根据本发明，步骤(4)所述的测序为sanger测序和/或Miseq测序。
作为优选技术方案，所述扩增TCR全长序列的方法包括如下步骤：
(1)裂解细胞；
(2)将得到的mRNA进行反转录，得到第一链cDNA；
(3)以步骤(2)得到的第一链cDNA为模板，以权利要求1所述的上游引物和所述的巢式引物为下游引物进行PCR扩增，具体包括：
(1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
步骤(1’)所述的第一轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，55℃ 15s，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
(2’)以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
步骤(2’)所述的第二轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
(3’)以所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物；
步骤(3’)所述的第三轮巢式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，72℃ 2min，35个循环；72℃ 5min，1循环；保存在4℃；
(4)sanger和/或Miseq测序验证，得到所述TCR的全长序列。
第三方面，本发明一种扩增TCR全长序列的试剂盒，所述试剂盒包含如第一方面所述的扩增TCR全长序列的PCR引物对。
第四方面，本发明提供如第一方面所述的扩增TCR全长序列的PCR引物对、第二方面所述的扩增TCR全长序列的方法或如第三方面所述的试剂盒用于TCR文库构建的用途。
第五方面，本发明提供如第一方面所述的扩增TCR全长序列的PCR引物对或如第三方面所述的试剂盒用于制备疾病的免疫学诊断治疗和/或预后监控的药物的用途。
本发明提供如第一方面所述的扩增TCR全长序列的PCR引物对和/或扩增TCR全长序列的方法，实现了TCR序列的全长扩增并获得了TCR中α/β和/或γ/δ亚基的配对信息。
与现有技术相比，本发明具有如下有益效果：
(1)本发明引物和方法对不同种类的TCR均有效，不需要针对单一TCR设计特异性的上游引物，通过在反转录过程中，采用接头引物通过SMART模板转换技术使得TCR转录本第一链cDNA的3’端引入一段接头序列，后续PCR均以根据接头序列设计的上游引物，同时配合TCR C区保守区的下游引物，成功扩增出TCR全长；
(2)本发明采用三轮巢式PCR，对TCR全长进行扩增，适用于模板低至10pg总RNA的样品，能同时获取某一个单细胞的TCR的全长序列，从而可以获得不同TCR的α/β配对信息或γ/δ配对信息，对不同抗原刺激的T细胞TCR扩增灵敏度高、亚型覆盖完整且成本相对较低；
(3)本发明方法适用于细胞异质性和T细胞亚型分群，探索免疫学新机制，构建大规模全长TCR免疫组库，方便疾病诊断及健康管理，结合肿瘤新抗原开发neo TCR-T肿瘤免疫细胞疗法，进行肿瘤免疫治疗，癌症或自体免疫疾病预后监测，有助于指导医生用药和科学研究等。
附图说明
图1为扩增TCR全长序列的PCR引物设计原理；
图2是本发明实施例2中的电泳结果图，其中，marker为100bp marker，从下到上为100bp、200bp、300bp、400bp、500bp(最亮条带)、600bp、700bp、800bp、900bp、1000bp和1500bp；
图3是本发明实施例3中的电泳结果图，其中，marker为100bp marker，从下到上为100bp、200bp、300bp、400bp、500bp(最亮条带)、600bp、700bp、800bp、900bp、1000bp和1500bp。
具体实施方式
下面结合附图和实施例对本发明作进一步的详细说明。可以理解的是，此处所描述的具体实施例仅仅用于解释本发明，而非对本发明的限定。另外还需要说明的是，为了便于描述，附图中仅示出了与本发明相关的部分而非全部结构。
实施例1 TCR引物设计
引物设计的原理如图1所示，利用接头引物通过转换模板法在cDNA第一链的3’端引入接头序列，所述接头引物如SEQ ID NO.1所示，所述SEQ ID NO.1所示的核苷酸序列如下：
Figure PCTCN2017104107-appb-000006
根据接头序列设计上游引物，所述上游引物如SEQ ID NO.2所示，所述SEQ ID NO.2所示的核苷酸序列如下：
Figure PCTCN2017104107-appb-000007
所述下游引物根据TCR基因C区进行设计，所述T细胞α和β链的C区完整序列如下：
TCRA C区：
Figure PCTCN2017104107-appb-000008
TCRB C区：
Figure PCTCN2017104107-appb-000009
由于Miseq测序平台和Sanger测序平台的不同，所述Miseq测序平台和 Sanger测序平台的引物分别设计。
(1)Miseq测序
因Miseq目前最长的读长为300bp/reads，采用PE300的测序策略，要求文库Insert长度不超过600bp，TCR αβ链5’端UTR序列+V+D+J区序列正好在600bp以内，因此我们将下游引物设置在靠近C区的5’端20～30bp的位置，如下所示：
表1 人T细胞C区半长引物序列信息
Figure PCTCN2017104107-appb-000010
(2)Sanger测序
表2 人T细胞C区半长引物序列信息
Figure PCTCN2017104107-appb-000011
Figure PCTCN2017104107-appb-000012
实施例2 TCR在Miseq测序平台测序
所述扩增TCR全长序列的方法，包括如下步骤：
(1)第一链cDNA合成：
a)配制裂解液，所述裂解液配方如下表所示：
Figure PCTCN2017104107-appb-000013
配制时，按样品数110％配制(如有10个细胞样品，则配制11管的量)。配制好的裂解液吹打混匀后分装到洁净0.2ml离心管中，14000rpm，4℃离心离心30s(将液滴离心到管底并去除气泡)；冰盒放置，待后续加入细胞；
b)单细胞分离
外周血单个核细胞经抗体染色后，利用流式细胞仪分选出CD8+T细胞，利用显微操作仪挑取单细胞，加入裂解液中，快速离心保证细胞进入裂解液，立即放入干冰上，扩增之前将样品保存于-80℃或者液氮中；
c)细胞裂解
将0.2ml PCR管置于PCR仪内，72℃，3min孵育(细胞mix样本增至5min)，热盖温度为75℃，裂解完成后立即置于冰上1min；10000rpm 4℃离心30s，后立即转至冰上；此步后，所有mRNAs都从单细胞中释放，并且Oligo-dT引物也已与mRNAs结合；
d)mRNA反转录
配制反转录体系如下：
Figure PCTCN2017104107-appb-000014
配制时，按样品数+0.5个配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次加入到上步离心管中；
吹打混匀、瞬时离心后，按如下条件进行反转录反应(75℃热盖)：
Figure PCTCN2017104107-appb-000015
Figure PCTCN2017104107-appb-000016
所有mRNAs的第一链cDNA合成完毕；
(2)巢式PCR
采用实施例1中的引物，
上游引物为SEQ ID NO.1，SEQ ID NO.1所示的核苷酸序列为：
下游引物如下：
Figure PCTCN2017104107-appb-000017
a)第一轮PCR
以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巢式PCR，得到第一轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104107-appb-000018
Figure PCTCN2017104107-appb-000019
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取15μL加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104107-appb-000020
b)第二轮PCR
以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巢式PCR，得到第二轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104107-appb-000021
Figure PCTCN2017104107-appb-000022
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24μL加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104107-appb-000023
c)第三轮PCR
以所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巢式PCR，得到第三轮扩增产物；
按下表配制PCR体系：
Figure PCTCN2017104107-appb-000024
配制时，按样品数+0.5配制(如有9个细胞样品，则配制9.5管的量)。配制好的Mix充分混匀后，依次取24μL加入到上步离心管中；
吹打混匀、瞬时离心后，按下述条件预扩增：
Figure PCTCN2017104107-appb-000025
(3)电泳检测
巢式PCR完成后电泳检测，采用2％琼脂糖凝胶，取15μL产物，加3μL Loading buffer混匀，130V电泳45min。目的条带切胶回收，结果如图2所示，得到了完整的TCR的αβ链。
(4)将加收产物进行TA克隆和测序，测序结果如下：
α链序列
α1：V12+J21
Figure PCTCN2017104107-appb-000026
Figure PCTCN2017104107-appb-000027
α2：V5+J36
Figure PCTCN2017104107-appb-000028
β链序列(V27+J2)
Figure PCTCN2017104107-appb-000029
将上述结果整理如下表3所示：
表3
Figure PCTCN2017104107-appb-000030
Figure PCTCN2017104107-appb-000031
由表3可知，样品A的α、β型别分别是：
α：V12J21和V5J36
β：V27J2。
可见，采用Miseq测序平台，测序可以直接得到V(D)J全长。
实施例3 TCR在Sanger测序平台测序
相比于实施例2，除了巢式引物，其他条件与方法与实施例2相同，所述巢式引物如下：
采用实施例1中的引物，
上游引物为SEQ ID NO.1，SEQ ID NO.1所示的核苷酸序列为：
下游引物如下：
Figure PCTCN2017104107-appb-000032
电泳检测
巢式PCR完成后电泳检测，采用2％琼脂糖凝胶，取15μL产物，加3μL Loading buffer混匀，130V电泳45min。目的条带切胶回收，结果如图3所示，得到了完整的TCR的αβ链。
送到公司去完成TA克隆和测序，测序结果如下：
α链序列
α1：V12-2+J21
Figure PCTCN2017104107-appb-000033
β：V27+J2-7
Figure PCTCN2017104107-appb-000034
将上述结果整理如下表4所示：
表4
TCR样品 V J αβ链
B V12-2 J21 α
  V27 J2-7 β
由表4可知，样品B的α、β型别分别是：
α：V12-2J21，β：V27J2-7。
可见，采用Sanger测序平台，测序可以直接得到V(D)JC全长。
综上所述，本发明引物和方法对不同种类的TCR均有效，不需要针对单一TCR设计特异性的上游引物，通过在反转录过程中，采用SMART模板转换技术使得TCR的转录本第一链cDNA的3’端引入一段接头序列，后续PCR均以根据接头序列设计的引物为上游引物，同时配合TCR C区保守区的下游引物，成功扩增出TCR全长，不仅如此，本发明采用三轮巢式PCR，对TCR全长进行扩增，适用于模板低至10pg总RNA的样品，能同时获取某一个单细胞的TCR的全长序列，从而可以获得不同TCR的α/β配对信息或γ/δ配对信息，对不同抗原刺激的T细胞TCR扩增灵敏度高、亚型覆盖完整且成本相对较低。
上述仅为本发明的较佳实施例及所运用技术原理。本领域技术人员会理解，本发明不限于这里所述的特定实施例，对本领域技术人员来说能够进行各种明显的变化、重新调整和替代而不会脱离本发明的保护范围。因此，虽然通过以上实施例对本发明进行了较为详细的说明，但是本发明不仅仅限于以上实施例，在不脱离本发明构思的情况下，还可以包括更多其他等效实施例，而本发明的范围由所附的权利要求范围决定。

Claims (15)

  1. 一种扩增TCR全长序列的PCR引物对，其包括上游引物和下游引物；
    所述上游引物为根据一段接头序列设计的引物，所述接头序列引入到第一链cDNA的3’端；
    所述下游引物为根据TCR的C区设计的下游引物。
  2. 根据权利要求1所述的PCR引物对，其特征在于，所述接头序列是利用接头引物通过转换模板法引入。
  3. 根据权利要求1或2所述的PCR引物对，其特征在于，所述接头序列的长度为18-35nt。
  4. 根据权利要求3所述的PCR引物对，其特征在于，所述接头序列的长度为28nt。
  5. 根据权利要求1-4任一项所述的PCR引物对，其特征在于，所述接头序列的核苷酸序列如SEQ ID NO.1所示；
    优选地，所述上游引物的核苷酸序列如SEQ ID NO.2所示；
    优选地，所述下游引物根据TCR的C区的5’端或3’端进行设计；
    优选地，所述下游引物为巢式引物；
    优选地，所述下游引物包括外部引物、中间引物或内部引物中的任意一种或至少两种的组合，优选为外部引物、中间引物或内部引物的组合。
  6. 根据权利要求1-5任一项所述的PCR引物对，其特征在于，所述TCR全长由TCRα基因全长和TCRβ基因全长组成或由TCRγ基因全长和TCRδ基因全长组成；
    优选地，扩增TCRα基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.3、SEQ ID NO.4和SEQ ID NO.5所示；
    优选地，扩增TCRα基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8所示；
    优选地，扩增TCRβ基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.9、SEQ ID NO.10和SEQ ID NO.11所示；
    优选地，扩增TCRβ基因全长的下游引物的外部引物、中间引物和内部引物的核苷酸序列分别如SEQ ID NO.12、SEQ ID NO.13和SEQ ID NO.14所示。
  7. 一种扩增TCR全长序列的方法，其使用如权利要求1所述的PCR引物对，其特征在于，包括如下步骤：
    (1)裂解细胞；
    (2)将得到的mRNA进行反转录，得到第一链cDNA；
    (3)以步骤(2)得到的第一链cDNA为模板，以权利要求1所述PCR引物对中的上游引物和下游引物进行PCR扩增；
    (4)测序验证，得到所述TCR的全长序列。
  8. 根据权利要求7所述的方法，其特征在于，步骤(2)所述的mRNA反转录的体系包括接头序列。
  9. 根据权利要求8所述的方法，其特征在于，所述接头序列的终浓度为0.5-2μM，优选为1μM；
    优选地，步骤(2)所述的mRNA反转录的条件为：42℃ 90min 1循环；50℃ 2min，42℃ 2min，10循环；70℃ 15min 1循环；保存在4℃。
  10. 根据权利要求7-9任一项所述的方法，其特征在于，步骤(3)所述的下游引物为巢式引物，所述PCR扩增为巢式PCR；
    优选地，所述巢式PCR的次数为2-3次，优选为3次；
    优选地，所述巢式PCR具体包括：
    (1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巣式PCR，得到第一轮扩增产物；
    (2’)以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮扩增产物为模板，进行第二轮巣式PCR，得到第二轮扩增产物；
    (3’)以所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巣式PCR，得到第三轮扩增产物；
    步骤(1’)所述的第一轮巣式PCR条件为：95℃ 3min，1循环；98℃ 20s，55℃ 15s，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
    优选地，步骤(2’)所述的第二轮巣式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，72℃ 2min，25个循环；72℃ 5min，1循环；保存在4℃；
    优选地，步骤(3’)所述的第三轮巣式PCR条件为：95℃ 3min，1循环；98℃ 20s，60℃ 15s，，72℃ 2min，35个循环；72℃ 5min，1循环；保存在4℃；
    优选地，步骤(4)所述的测序为sanger测序和/或Miseq测序。
  11. 根据权利要求8或9所述的构建TCR文库的方法，其包括如下步骤：
    (1)裂解细胞；
    (2)将得到的mRNA进行反转录，得到第一链cDNA；
    (3)以步骤(2)得到的第一链cDNA为模板，以权利要求1所述的上游引物和所述的巢式引物为下游引物进行PCR扩增，具体包括：
    (1’)以所述上游引物和所述外部引物为引物，以第一链cDNA为模板，进行第一轮巣式PCR，得到第一轮扩增产物；
    (2’)以所述上游引物和所述中间引物为引物，以步骤(1’)得到的第一轮 扩增产物为模板，进行第二轮巣式PCR，得到第二轮扩增产物；
    (3’)以所述上游引物和所述内部引物为引物，以步骤(2’)得到的第二轮扩增产物为模板，进行第三轮巣式PCR，得到第三轮扩增产物；
    (4)sanger和/或Miseq测序验证，得到所述TCR的全长序列。
  12. 根据权利要求7-11任一项所述的方法，其特征在于，步骤(1)所述的细胞为单细胞；
    优选地，步骤(1)所述单细胞来源于外周血的外周血单个核细胞。
  13. 一种扩增TCR全长序列的试剂盒，其特征在于，所述试剂盒包含如权利要求1-6中任一项所述的扩增TCR全长序列的PCR引物对。
  14. 一种如权利要求1-6中任一项所述的扩增TCR全长序列的PCR引物对、如权利要求7-12中任一项所述的扩增TCR全长序列的方法或如权利要求13所述的试剂盒用于TCR的建库的用途。
  15. 一种如权利要求1-6中任一项所述的扩增TCR全长序列的PCR引物对或如权利要求13所述的试剂盒用于制备疾病的免疫学诊断治疗和/或预后监控的药物的用途。
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