CN112143777B

CN112143777B - Primer group for constructing CDR3 region high-throughput sequencing library of human TCR beta and application thereof

Info

Publication number: CN112143777B
Application number: CN202010829545.9A
Authority: CN
Inventors: 宋瑾; 郭静雅; 任树成; 张恒辉; 沈宁; 孔雅娴
Original assignee: Beijing Zhenzhi Medical Technology Co ltd
Current assignee: Beijing Zhenzhi Medical Technology Co ltd
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-07-01
Anticipated expiration: 2040-08-18
Also published as: CN112143777A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a primer group for constructing a CDR3 region high-throughput sequencing library of human TCR beta and application thereof, in particular to a primer group required for constructing a CDR3 region high-throughput sequencing library of human TCR beta and a library preparation method. The primer group provided by the invention can be used in a comprehensive, efficient, convenient and high-sensitivity TCR detection and analysis method, and has the following characteristics: high compatibility to various samples, high standardization and automation of experimental operation process, and simple and rapid analysis process.

Description

Primer group for constructing CDR3 region high-throughput sequencing library of human TCR beta and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a primer set for constructing a CDR3 region high-throughput sequencing library of human TCR beta and application thereof, in particular to a primer set required for constructing a CDR3 region high-throughput sequencing library of human TCR beta and a preparation method of the library.

Background

Lymphocytes display immune function by recognizing specific antigens through their surface antibodies. The specificity of the antigen recognition is embodied in clone level, namely, lymphocytes of the same clone can recognize the same antigen receptor and the same antigen epitope. The T cell antigen receptor (TCR) is a structure that T cells specifically recognize and bind to antigen peptide-MHC molecules, and is a heterodimer, consisting of two distinct subunits. The receptors of 95% of T cells are composed of an alpha subunit and a beta subunit, and the other 5% of receptors are composed of a gamma subunit and a delta subunit, the proportions of which may vary due to ontogeny or disease.

TRB is a gene locus for coding TCR beta, which comprises 4 gene segments of a variable segment (V), a variable segment (D), a connecting segment (J) and a constant region (C), V, D, J, C is divided into a plurality of alleles, gene recombination occurs in the TRB gene locus in the T cell development process, molecular basis is provided for the diversity of the TCR beta, in addition, by the mechanisms of random insertion and deletion of bases near the gene recombination sites and the like, the diversified TCR is finally generated, and the requirement of an organism for identifying various antigens is met.

The CDR3 region of TCR beta is composed of the nucleotide sequence inserted between the tail end of V gene segment, D gene segment, the front end of J gene segment and V, D, J. The rearrangement of the TCR beta chain in preference to the TCR alpha chain in an allelic exclusion manner can better reflect the TCR characteristics of T cells. In addition, the variable region of each subunit of the TCR comprises three highly variable Complementarity Determining Regions (CDRs), the most important CDR3 being responsible for direct binding to polypeptides presented by the MHC, and the sequence being highly variable, so the diversity of the TCR is determined primarily by CDR 3. By sequencing the CDR3 region of TCR beta, the parameters such as diversity of TCR beta in an immune repertoire can be evaluated, and the response generation mechanism and process of an immune system can be further analyzed. Thus, the identification of antigen-specific T cell receptors has also often focused on the detection of the β chain CDR3 coding sequence.

T cell-mediated cellular immunity is one of the adaptive immunizations and is an important pathway for the immune system to recognize and eliminate pathogens and tumor cells. Adoptive T cell Therapy (ACT) that restores the immune surveillance, defense and regulation functions of the body by enhancing T cell mediated adaptive immune responses overcomes the limitations of traditional therapeutic techniques. ACT therapies include Tumor Infiltrating T cell (TIL) therapy, CAR-T cell therapy, and TCR-T (T cell receptor-gene engineered T cells) therapy. The TCR-T therapy is the latest technology in the field of ACT at present, is a means of genetic engineering to directly modify the T cell to recognize the surface receptor TCR of the tumor antigen, thereby strengthening the capability of the T cell to recognize and kill the tumor cell, is widely concerned and becomes a research hotspot, is proved to have good curative effect in the carried clinical test, and the research result is disputed and reported by top-level journals such as Science, Nature and the like. The therapeutic potential of this therapy for infectious diseases and specific viruses has also been explored. Such as HIV, HBV and COVID-19, can be target points of TCR-T, and can be used for treating and controlling infectious diseases as an innovative therapy. In the case of HBV infection, current antiviral therapy merely inhibits viral replication and cures less than 5% of patients. Treatment of these patients with an anti-viral drug in combination with CAR/TCR-T cells may be a viable option. The approach of gene engineering CAR/TCR-T cells by Tan and its research team using mRNA electroporation limits their functional activity to a short time, thus providing enhanced safety profiles suitable for use in patients with chronic viral diseases (gastroenterology, 2019,156(6): 1862-1876). The identification of the antigen-specific TCR is the most key step of TCR-T therapy and drug development, and the method for identifying the high-throughput antigen-specific TCR can greatly shorten the open cycle of the TCR-T therapy and reduce the cost.

The important basis for their role, whether as therapeutic and prophylactic vaccines specifically targeting tumor or pathogen antigens, antigen-specific TCR-T or other specific immune cell therapies targeting specific antigens, Including Checkpoint Inhibitors (ICIs) targeting immune checkpoints, is to mobilize T cells to recognize tumor or pathogen antigens, eliciting tumor-specific killing responses. In the course of immunotherapy, the expansion, persistence and decline of tumor antigen-specific T cell clones can intuitively reflect the effect of immunotherapy. Therefore, the identification method of the tumor antigen specific TCR can not only test whether the immunotherapy effectively stimulates the tumor antigen specific T cell response, but also be used for monitoring the change of the specific T cell in the immunotherapy process, and has the potential value of the concomitant diagnosis of the immunotherapy.

In the prior art, a 5' RACE and a nested PCR method are used for amplifying, building a library, sequencing and analyzing and comparing sequencing results of CDR3 regions of TCR beta. The method of 5' RACE and nested PCR is characterized in that RNA is used as a starting material for immobilization, the sample type is single, the operation requirement is relatively high compared with other technologies, the whole process is complicated, and the repeatability can be influenced. Furthermore, because of the high degree of similarity between TCR sequences, particularly between sequences of V or J genes of the same subtype, detection of TCR sequences for detecting T cell clones of low frequency in a sample has been a great challenge.

Disclosure of Invention

In order to overcome the defects of a TCR detection and identification method for low-frequency T cell clone in the prior art, the invention provides a primer group for constructing a CDR3 region high-throughput sequencing library of human TCR beta and a comprehensive, high-efficiency, convenient and high-sensitivity TCR detection and analysis method, provides an application for identifying tumor antigen specific TCR based on the TCR detection and analysis method, and can provide technical support for development of tumor immunotherapy therapy and accompanying diagnosis research of tumor immunotherapy.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention constructs a primer group of a CDR3 region high-throughput sequencing library of human TCR beta, wherein the target genes and the primer sequences of the primer group are shown in Table 1; wherein, the upstream primer of the primer group comprises TRBV10-1-F, TRBV10-2/3-F, TRBV11-1/2/3-F, TRBV12-3.1/4/5.1-F, TRBV12-3.2/5.2-F, TRBV13-F, TRBV14-F, TRBV15-F, TRBV16-F, TRBV18-F, TRBV19-F, TRBV20-F, TRBV24-F, TRBV25-F, TRBV27-F, TRBV28-F, TRBV29-F, TRBV2-F, TRBV3-F, TRBV30-F, TRBV4-1-F, TRBV4-2/3-F, TRBV5-1-F, TRBV5-4/5/6/8-F, TRBV6-1-F, TRBV6-2/3-F, TRBV6-4.1-F, TRBV6-4.2-F, TRBV6-1.2/5/8-F, TRBV6-1.1/6/7/9-F, TRBV7-1-F, TRBV7-2/4/6/7/8-F, TRBV7-3-F, TRBV7-9-F and TRBV 9-F;

wherein, the nucleotide sequence of TRBV10-1-F is shown in SEQ ID NO. 1; the nucleotide sequence of TRBV10-2/3-F is shown in SEQ ID NO. 2; the nucleotide sequence of TRBV11-1/2/3-F is shown in SEQ ID NO. 3; the nucleotide sequence of TRBV12-3.1/4/5.1-F is shown in SEQ ID NO. 4; the nucleotide sequence of TRBV12-3.2/5.2-F is shown in SEQ ID NO. 5; the nucleotide sequence of TRBV13-F is shown in SEQ ID NO. 6; the nucleotide sequence of TRBV14-F is shown in SEQ ID NO. 7; the nucleotide sequence of TRBV15-F is shown in SEQ ID NO. 8; the nucleotide sequence of TRBV16-F is shown in SEQ ID NO. 9; the nucleotide sequence of TRBV18-F is shown in SEQ ID NO. 10; the nucleotide sequence of TRBV19-F is shown in SEQ ID NO. 11; the nucleotide sequence of TRBV20-F is shown in SEQ ID NO. 12; the nucleotide sequence of TRBV24-F is shown in SEQ ID NO. 13; the nucleotide sequence of TRBV25-F is shown in SEQ ID NO. 14; the nucleotide sequence of TRBV27-F is shown in SEQ ID NO. 15; the nucleotide sequence of TRBV28-F is shown in SEQ ID NO. 16; the nucleotide sequence of TRBV29-F is shown in SEQ ID NO. 17; the nucleotide sequence of TRBV2-F is shown in SEQ ID NO. 18; the nucleotide sequence of TRBV3-F is shown in SEQ ID NO. 19; the nucleotide sequence of TRBV30-F is shown in SEQ ID NO. 20; the nucleotide sequence of TRBV4-1-F is shown in SEQ ID NO. 21; the nucleotide sequence of TRBV4-2/3-F is shown in SEQ ID NO. 22; the nucleotide sequence of TRBV5-1-F is shown in SEQ ID NO. 23; the nucleotide sequence of TRBV5-4/5/6/8-F is shown as SEQ ID NO. 24; the nucleotide sequence of TRBV6-1-F is shown in SEQ ID NO. 25; the nucleotide sequence of TRBV6-2/3-F is shown as SEQ ID NO. 26; the nucleotide sequence of TRBV6-4.1-F is shown in SEQ ID NO. 27; the nucleotide sequence of TRBV6-4.2-F is shown in SEQ ID NO. 28; the nucleotide sequence of TRBV6-1.2/5/8-F is shown in SEQ ID NO. 29; the nucleotide sequence of TRBV6-1.1/6/7/9-F is shown in SEQ ID NO. 30; the nucleotide sequence of TRBV7-1-F is shown in SEQ ID NO. 31; the nucleotide sequence of TRBV7-2/4/6/7/8-F is shown in SEQ ID NO. 32; the nucleotide sequence of TRBV7-3-F is shown in SEQ ID NO. 33; the nucleotide sequence of TRBV7-9-F is shown in SEQ ID NO.34 and the nucleotide sequence of TRBV9-F is shown in SEQ ID NO. 35;

the downstream primers of the primer group comprise TRBJ1-1-R, TRBJ1-2-R, TRBJ1-3-R, TRBJ1-4-R, TRBJ1-5-R, TRBJ1-6-R, TRBJ2-4-R, TRBJ2-1-R, TRBJ2-2-R, TRBJ2-3-R, TRBJ2-5-R, TRBJ2-6-R and TRBJ 2-7-R;

wherein, the TRBJ1-1-R has a nucleotide sequence shown as SEQ ID NO. 36; the TRBJ1-2-R has a nucleotide sequence shown as SEQ ID NO. 37; the TRBJ1-3-R has a nucleotide sequence shown as SEQ ID NO. 38; the nucleotide sequence of TRBJ1-4-R is shown in SEQ ID NO. 39; the TRBJ1-5-R has a nucleotide sequence shown as SEQ ID NO. 40; the nucleotide sequence of TRBJ1-6-R is shown in SEQ ID NO. 41; the nucleotide sequence of TRBJ2-4-R is shown in SEQ ID NO. 42; the nucleotide sequence of TRBJ2-1-R is shown in SEQ ID NO. 43; the TRBJ2-2-R has a nucleotide sequence shown as SEQ ID NO. 44; the nucleotide sequence of TRBJ2-3-R is shown in SEQ ID NO. 45; the nucleotide sequence of TRBJ2-5-R is shown in SEQ ID NO. 46; the nucleotide sequence of TRBJ2-6-R is shown in SEQ ID NO. 47; the nucleotide sequence of TRBJ2-7-R is shown in SEQ ID NO. 48.

TABLE 1 primer set target genes and primer sequences

The invention also provides application of the primer group, in particular application in preparing a product for constructing a CDR3 region high-throughput sequencing library of human TCR beta or a product for identifying tumor or pathogen antigen specific TCR.

Preferably, the above applications involve various methods, but not limited to the following methods.

The invention provides a method for constructing a CDR3 region high-throughput sequencing library of human TCR beta, which comprises the following steps:

1) obtaining DNA and/or RNA of a sample, and reversely transcribing the RNA into cDNA;

2) preparing a gene library:

2-1) amplification of TCR β fragments: performing PCR amplification on the DNA and/or cDNA obtained in the step 1) by using a multiplex PCR method to obtain a CDR3 beta multiplex amplicon product, wherein the multiplex PCR amplification primer is the primer set of claim 1;

2-2) digestion part primer sequence: digesting the CDR3 beta multiple amplicon product obtained in the step 2-1) to obtain a digested target product;

2-3) linker and tag: connecting joints and labels on the digested target product;

2-4) fragment purification: purifying the product obtained in the step 2-3);

2-5) library quantification: carrying out QPCR (quantitative polymerase chain reaction) amplification on the purified DNA connected with the joint and the label to obtain a CDR3 beta amplicon library, and analyzing the library concentration;

3) high-throughput sequencing to identify libraries: identifying and analyzing the amplicon sequence by high throughput sequencing;

4) sequencing data analysis: sequencing, downloading data, and performing quality control, comparison and assembly on the data.

4. The method of claim 3, wherein the step 4) sequencing data analysis comprises the steps of:

a) quality control: filtering the low-quality sequences and the false positive sequences;

b) comparing the filtered sequencing readings with V, D, J of the TCR and a C gene reference genome file to determine the position of each reading on the genome; wherein different reads having the same nucleotide at the same position are considered to be part of the same clonotype; the reads that qualify for read quality and contain the sequence of the CDR3 region are retained.

c) And for the sequences successfully aligned, continuing to assemble, and collating the alignment assembly results with qualified depth to further obtain the number of sequencing strips corresponding to each CDR3 beta sequence, so as to obtain the proportion of the T cell clone corresponding to each CDR3 beta in the detection sample.

The method according to the invention, wherein the low quality sequence of step a) comprises a sequence with low quality reads greater than 50%, a sequence with linker sequences greater than 30%, off-target sequences, sequences with uncorrectable reads due to PCR errors; the false positive sequence refers to a sequence that repeats in multiple different samples.

The method according to the present invention, wherein preferably, the sample of step 1) is whole blood or FFPE; wherein, when the sample is whole blood, PBMC cells in the whole blood sample are separated, and DNA is extracted from the PBMC cells.

In addition, according to the method of the invention, the detection sensitivity can reach 10^-5The frequency, i.e., only one in a ten-thousandth of the CDR3 β sequence in the sample can be stably and accurately detected.

Alternatively, the use of the above-described product for the preparation of a product for identifying a TCR specific for a tumour or pathogen antigen may comprise the steps of:

1) labelling of tumor or pathogen antigen specific T cells: there are two procedures, but not limited to, one is a T cell sample activated by tumor or pathogen antigen impact, and antigen-specific T cells can be distinguished by labeling functional factors (e.g., IFN γ, etc.) activated by immune in T cells; the other is an artificially synthesized antigen epitope-MHC-dye complex multimer, which specifically binds to a TCR capable of recognizing the antigen epitope, to thereby label antigen-specific T cells.

2) Isolation and purification of tumor or pathogen antigen specific T cells: flow cytometry or magnetic beads can be used to separate and enrich the successfully labeled T cells.

3) The method for constructing the CDR3 region high-throughput sequencing library of the human TCR beta provided by the invention is adopted to carry out nucleic acid extraction, library construction and sequencing on a T cell sample which is separated and purified and has specificity to a tumor or pathogen antigen.

4) Tumor or pathogen antigen specific T cell clone identification: based on the previous sequencing data, tumor or pathogen antigen specific T cell clones were further identified. The specific T cell clone, which may be multiple or single, is filtered by the frequency and ranking of T cell clones in the sample to ensure that the true clones of specific T cells are maximally retained.

The primer group provided by the invention can be used in a comprehensive, efficient, convenient and high-sensitivity TCR detection and analysis method, and has the following characteristics: high compatibility to various samples, high standardization and automation of experimental operation process, and simple and rapid analysis process. The method for constructing the library has the advantages of low initial library construction amount, high sensitivity and low requirement on samples; can be compatible with the biological information analysis requirement of the tumor or pathogen antigen specific TCR.

Drawings

FIG. 1 shows the high throughput sequencing of the CDR3 region of the source TCR β of the present invention;

FIG. 2A is a library of 50ng initial amounts of DNA from peripheral blood samples according to example 2 of the present invention;

FIG. 2B shows the library profile of 250ng initial amount of DNA in a peripheral blood sample according to example 2 of the present invention;

FIG. 2C shows the library profile of an initial amount of 500ng DNA in a peripheral blood sample according to example 2 of the present invention;

FIG. 3 shows the results of Pearson correlation analysis of the initial amount of RNA in the cell line samples of example 2 of the present invention, wherein p.ltoreq.0.01;

FIG. 4 is a graph showing the batch-to-batch reproducibility of cell line samples according to example 3 of the present invention, wherein p.ltoreq.0.01;

FIG. 5 shows the cloning frequency of Jurkat cell RNA at different dilution ratios in example 4 of the present invention;

FIG. 6 shows the AFP and MAGEA1 tumor-specific CD8+ MHC multimer + T cell flow assay of hepatoma patients according to example 5 of the present invention;

FIG. 7 is a flow assay of CMV-positive donor CMV-specific CD8+ MHC multimer + T cells of example 6 of the present invention.

Detailed Description

The experimental method, the detection method and the preparation method disclosed by the invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture and recombinant DNA technology in the technical field and the conventional technology in the related field. The method can be specifically carried out according to a specific method listed in a molecular cloning experimental manual (fourth edition, J. SammBruk et al), or according to a kit and a product instruction; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The specific technical scheme of the invention is as follows:

the method for constructing the CDR3 region high-throughput sequencing library of the human TCR beta specifically comprises the following steps if shown in FIG. 1:

1) the related sample is whole blood and/or FFPE, if the sample is whole blood, PBMC cells in the whole blood sample are separated firstly;

2) obtaining sample nucleic acid, wherein the nucleic acid is DNA and/or RNA, and if the nucleic acid is RNA, the nucleic acid is firstly subjected to reverse transcription to be cDNA;

3) preparing a gene library:

3-1) amplification of TCR β fragments: performing PCR amplification on the DNA and/or cDNA obtained in the step 2) by adopting a PCR reaction system comprising a PCR primer mixed solution to obtain a CDR3 beta amplicon product, wherein the PCR amplification primer is the primer shown in the table 1;

3-2) digestion part primer sequence: digesting the multiple amplicon product obtained in the step 3-1) by using a FuPa reagent to obtain a digested target product;

3-3) linker and tag: then, connecting a linker and a label to the target product digested by the FuPa reagent;

3-4) fragment purification: purifying the product obtained in the step 2-3) by using magnetic beads and 70% ethanol;

3-5) library quantification: then, carrying out QPCR amplification on the DNA connected with the joint and the label to obtain a CDR3 beta amplicon library, analyzing the library concentration, and if the library concentration is not less than 0.5 ng/mu L, continuing the next step;

4) identifying the library by high-throughput sequencing; the amplicon sequence information contained in the CDR3 β amplicon library and the relative proportion of each amplicon, i.e., the proportion of different T cell clones in the sample, were identified and analyzed by high throughput sequencing; the high-throughput sequencing in the step 4) can use a high-throughput sequencing method which is conventional in the field, and can also use the primer group for high-throughput sequencing;

5) sequencing data analysis: the sequencing off-line data needs quality control, comparison and assembly:

a) quality control mainly comprises filtration of low-quality and false positive sequences. The low quality sequence comprises a sequence with a quality value of less than Q30 and more than 50% of bases (i.e. bases with a misidentification probability of more than 0.1%), a sequence with a proportion of linker sequences of more than 30%, off-target sequences, uncorrectable reads due to PCR errors; false positive sequences are sequences that occur repeatedly in a number of different samples, are blacklisted and filtered out with consideration as background noise.

b) The filtered sequencing reads were aligned to V, D, J for TCR and the C gene reference genome file to determine where each read should be on the genome. Different reads are considered to be part of the same clonotype if they have the same nucleotide at the same position. Only reads that are of acceptable quality and contain important information (e.g., CDR3 region sequences, information on the V, D, J genotype, etc.) will be retained.

c) For successfully aligned sequences, assembly is continued. The alignment assembly results with qualified depth (reading at least more than or equal to 5) are collated, and the number of sequencing strips corresponding to each CDR3 beta sequence is further obtained, so that the proportion of T cell clones corresponding to each CDR3 beta in the detection sample is obtained.

Example 1 establishment of TCR sequencing library

The procedure for building a TCR sequencing library using whole blood samples or FFPE sections is as follows:

step 1 separation of PBMC cells in Whole blood

PBMC cells in whole blood were isolated using Ficoll separation.

Step 2, extracting total RNA or DNA in a sample

The extraction method for extracting the total RNA in the PBMC by using the Trizol method comprises the steps of extracting the total RNA of an FFPE section by using a kit, measuring the concentration of the extracted total RNA by using a nanodrop one ultramicro ultraviolet spectrophotometer, and displaying the OD260/280 to be between 1.8 and 2.0 by using a detection result. The integrity of the RNA was determined using Agilent 2100.

The DNA of PBMC and FFPE slices is extracted by using the kit, the concentration of the extracted RNA is determined by using nanodrop one, and the detection result shows that OD260/280 is between 1.8 and 2.0.

Step 3 reverse transcription of total RNA:

if total RNA is extracted from FFPE sample, it is not heat treated before reverse transcription, heated at 80 deg.C for 10min, and cooled to room temperature. If a visible pellet appears after thawing in the 5 × reverse transcription buffer, the resuspended pellet is vortexed or pipetted at pre-incubation with aspiration. Preparing a reaction system: 2. mu.L of 5 XTRT buffer, 1. mu.L of 10 XTRT, total RNA (2ug), and pure nuclease-free water to 10. mu.L. Fully and evenly mixing by vortex, centrifugally collecting liquid to the bottom of the tube or evenly mixing by sucking and beating. The reaction tube was placed on a PCR instrument and the reaction program was 25 ℃ for 10min, 25 ℃ for 60min, 85 ℃ for 5min, 4 ℃ Hold. The sample can be kept in the PCR apparatus at 4 ℃ for 16 h. If the experiment is not continued, the cDNA sample is stored in a refrigerator at 4 ℃. After the reaction, the reaction mixture was centrifuged thoroughly, and the liquid was collected at the bottom of the tube and the next step was continued.

Step 4 amplification reaction of CDR3 beta target fragment of TCR: the amplification primers are shown in Table 1.

SR-RNA amplification reaction: the PCR tubes were pre-cooled on ice. The 5 x HiFi Mix was placed on ice, mixed by gentle vortexing, and then centrifuged briefly to collect the solution. A PCR system was prepared as follows: cDNA 10. mu.L, 5 XHiFi Mix 4. mu.L, Primer Mix 4. mu.L, nucleic-Free Water 2. mu.L. And (4) buckling a tube cover, and fully whirling and centrifuging to mix the solution uniformly. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and circulating for 25 times; extension at 72 ℃ for 10min, and holding at 4 ℃. The FFPE samples were cycled 28 times.

SR-DNA amplification reaction: the PCR tubes were pre-cooled on ice. dNTP Mix was diluted to 7.5mM at a concentration of 25 mM. The 5 x HiFi Mix was placed on ice, mixed by gentle vortexing, and then centrifuged briefly to collect the solution. A PCR system was prepared as follows: DNA 100. mu.g, 5 XHiFi Mix 4. mu.L, Primer Mix 4. mu.L, dNTP Mix 2. mu.L, Nuclean-Free Water to 20. mu.L. And (4) buckling a tube cover, and fully whirling and centrifuging to mix the solution uniformly. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and circulating for 25 times; extension at 72 ℃ for 10min, and holding at 4 ℃. The FFPE samples were cycled 28 times.

Step 5, digestion amplification product reaction:

the FuPa reagent was placed on ice, mixed gently with vortexing, and the liquid was collected instantaneously. Carefully open the PCR tube and add 2. mu.L of FuPa reagent. The total reaction volume was about 22. mu.L. The PCR reaction conditions are as follows: 10min at 50 ℃; keeping at 55 deg.C for 10min, 60 deg.C for 20min, and 4 deg.C.

Step 6, connecting the amplified product with a joint label:

and (3) connecting the product obtained in the step (5) with a joint label. Where [ ACGTCCTG ] and [ TCGCCTTA ] are tag sequences, including but not limited to sequencing tag sequences of illumia, [ ] the sequences can be any 8bp short sequence that is validated to distinguish between different samples. The following solutions were added sequentially to the PCR tube: digest amplification 22. mu.L, Ligase Buffer 4. mu.L, DNA Ligase 2. mu.L, Barcode adapter mix 2. mu.L, and finally DNA Ligase was added. The PCR tube was placed in a PCR instrument and the following reaction program was run: 30min at 22 ℃; keeping at 68 deg.C for 5min, 72 deg.C for 5min, and 4 deg.C.

Step 7 library purification

The specific operation steps of the purification are as follows: fresh 70% ethanol was prepared before each experiment. The library in the PCR tube was centrifuged and collected at the bottom of the tube and the product was transferred to a new EP tube. mu.L (1.5 sample volume) of library purified magnetic beads was added, thoroughly pipetted and mixed, and incubated at room temperature for 5 min. The EP tube was placed in a magnetic stand and allowed to stand at room temperature for 2min, and the supernatant was aspirated away to avoid touching the magnetic beads. Add 150. mu.L of 70% ethanol, shake the PCR tube gently to wash the beads, place the EP tube in a magnetic stand, stand at room temperature for 2min, aspirate the supernatant, and repeat this step 1 time. The EP tube was kept in a magnetic stand and the beads were air dried at room temperature for 2-5 min.

Step 8 library quantification:

the EP tube with the purified library was removed from the magnetic rack, 50. mu.L of Low TE was added, the EP tube was placed in the magnetic rack, allowed to stand at room temperature for 2min, and the supernatant was pipetted into a new EP tube. Use of

qPCR quantification kit determined the concentration of each library. Taking 2 μ L of the supernatant obtained in step 6, thenThen combined with 198. mu.L of water, i.e., diluted 100-fold. Negative control NTC and standard E.coli DH10B (standard concentrations of 6.8pM, 0.68pM and 0.068pM) were set. The following reaction system was prepared in a PCR tube:

Master Mix 10μL，20×Ion

assay 1. mu.L, sample 9. mu.L. The QPCR instrument setting conditions are as follows: ROX was chosen as a passive reference dye, a reaction volume of 20 μ Ι _, FAM dye/MGB as TaqMan probe reporter/quencher, and standard cycling conditions were chosen. QPCR reaction conditions were: heating the cover at 105 ℃; at 50 ℃ for 2 min; at 95 ℃ for 2 min; 95 deg.C, 15s, 60 deg.C, 1min, 40 cycles, 4 deg.C, and holding. After qPCR was performed, the average concentration of the undiluted library was calculated by multiplying the determined concentration x 100.

The library was quality checked using Agilent 2100.

Example 2 different initial amounts of RNA and cell line sample batch replicates for FFPE samples

Selecting 1 sample of whole blood with better DNA extraction quality, performing PCR amplification on target sequences with DNA initial amounts of 50ng, 250ng and 500ng, constructing library and sequencing, and obtaining library with better quality as low as 50ng under the condition of better DNA quality (FIGS. 2A-C)

1 cell line sample is selected, the library is repeatedly established for 2 times in the same batch, and 2 libraries are obtained in total to complete sequencing. After obtaining the expression level (nrpm) of the target sequence in each library, Pearson correlation analysis was performed on 2 libraries, the correlation coefficient R was above 0.9, and the consistency was very high (see fig. 3 for details).

Example 3 batch-to-batch reproducibility of cell samples

Selecting 1 cell line sample, building a library under the same experimental condition and the same initial library building amount (10ng), obtaining 2 libraries in different batches, and obtaining 2 sequencing data by 2 times of computer sequencing. The expression level (nrpm) of the target sequence in each library was obtained, and Pearson correlation analysis was performed on 2 libraries, with a correlation coefficient R of 0.9 or more and extremely high consistency (see FIG. 4).

Example 4 high sensitivity and stability of TCR assay

Selecting a Jurkat cell line specifically expressing a certain TCR receptor subtype and a normal human fresh peripheral blood sample, extracting RNA, diluting the Jurkat cell line in a gradient way, mixing the RNA with the normal human fresh peripheral blood sample, establishing a library by adopting the method of example 1 for sequencing, wherein the specific dilution ratio and the cloning sequence are shown in the following table 2, A is the normal human peripheral blood sample, B is the Jurkat cell line sample, and obtaining the RNA expression level (support reading) of a target sequence. The experimental result shows that the Jurkat cell line sample can still detect the existence of the subtype when being diluted to one hundred thousand content, which indicates that the detection has enough sensitivity. And the batch-to-batch repeatability of the same sample is high, which indicates that the technology has higher stability and repeatability. See figure 5 for details.

TABLE 2 dilution ratio and cloning sequences

Sample	Variable	CDR3 AA	Supporting readings
				jurkat(B)	TRBV12-3	ASSFSTCSANYGYT	2776095
1A:1B	TRBV12-3	ASSFSTCSANYGYT	1772486
				9A:1B	TRBV12-3	ASSFSTCSANYGYT	520948
99A:1B	TRBV12-3	ASSFSTCSANYGYT	55650
				999A:1B	TRBV12-3	ASSFSTCSANYGYT	4164
9999A:1B	TRBV12-3	ASSFSTCSANYGYT	447
				99999A:1B	TRBV12-3	ASSFSTCSANYGYT	59

Example 5 identification of tumor patient antigen-specific TCRs

Epitope peptides of AFP and MAGEA1 were designed and synthesized, and then prepared into MHC multimers, respectively. CD8+ T cells were sorted in PBMCs of patients treated with reinfused CTLs using the CD8+ T cell isolation kit. The CD8+ T cells of the patient are respectively stained by using the synthesized MHC multimer, and the stained CD8+ T cells are sorted by a flow cytometer to obtain CD8+ MHC multimer + T cells. The CDR3 beta sequence of the TCR of the double positive T cells obtained was detected by the method of the present invention, i.e., the identification of TCR specific for both AFP and MAGEA1 tumor antigens was accomplished (FIG. 6).

The CDR β amino acid sequence of the identified TCR is shown below:

AFP and MAGEA1 specific CDR beta

Antigens	Epitope peptide sequences	HLA type	CDR3 beta sequence	Frequency of
					AFP	GLSPNLNRFL	HLA-A*02	ASSLLLQETQY	0.4843
AFP	FMNKFIYEI	HLA-A*02	ASSLELSETQY	0.2427
					MAGEA1	KVLEYVIKV	HLA-A*02	ASSLYQETQY	0.153

Example 6 identification of Cytomegalovirus (CMV) specific TCR

Epitope peptides of CMV were designed and synthesized, and then prepared as MHC multimers. CD8+ T cells in PBMCs of CMV positive donors were isolated using CD8+ T cell isolation kit. The CD8+ T cells of the patient were stained with synthetic MHC multimers, and stained CD8+ T cells were sorted by flow cytometry to give CD8+ MHC multimer + T cells. The identification of CMV antigen-specific TCRs was accomplished by detecting the CDR3 β sequence of the TCR of the resulting double positive T cells using the method of the invention (fig. 7), and the CDR β amino acid sequence of the identified TCRs is shown below:

TABLE 4 CMV-specific CDR beta

Antigens	Epitope peptide sequences	HLA type	CDR3 beta sequence	Frequency of
					CMV	NLVPMVATV	HLA-A*02	CASRGQGFSYEQYF	0.5214
CMV	NLVPMVATV	HLA-A*02	CASSFLGLNEQFF	0.3332

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited. Although the present invention has been described in detail with reference to the embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Beijing Zhenzhi medical science and technology Limited liability company

<120> primer group for constructing CDR3 region high-throughput sequencing library of human TCR beta and application thereof

<160> 48

<170> SIPOSequenceListing 1.0

<210> 1

<211> 22

<212> DNA

<213> Artificial

<400> 1

ccctcactct ggagtctgct gc 22

<210> 2

<211> 22

<212> DNA

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<400> 2

ccctcactct ggagtcmgct ac 22

<210> 3

<211> 25

<212> DNA

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<400> 3

ctccactctc aagatccagc ctgca 25

<210> 4

<211> 21

<212> DNA

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<400> 4

gatccagccc tcagaaccca g 21

<210> 5

<211> 21

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<400> 5

ggtgcagcct gcagaaccca g 21

<210> 6

<211> 27

<212> DNA

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<400> 6

cattctgaac tgaacatgag ctccttg 27

<210> 7

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<400> 7

ggacgtattc tactctgaag gtgcag 26

<210> 8

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gataacttcc aatccaggag gccgaac 27

<210> 9

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ctgtagcctt gagatccagg ctacg 25

<210> 10

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gcatcctgag gatccagcag gta 23

<210> 11

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cctttcctct cactgtgaca tcgg 24

<210> 12

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cttgtccact ctgacagtga ccagt 25

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ctccctgtcc ctagagtctg cca 23

<210> 14

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gccctcacat acctctcagt acc 23

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gatcctggag tcgcccagc 19

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attctggagt ccgccagc 18

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aactctgact gtgagcaaca tga 23

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<211> 27

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atttcactct gaagatccgg tccacaa 27

<210> 19

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gctcacttaa atcttcacat caattccctg 30

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cggcagttca tcctgagttc taagaa 26

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cttaaacctt cacctacacg ccctg 25

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cttattcctt cacctacaca ccctg 25

<210> 23

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gccagttctc taactctcgc tc 22

<210> 24

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tcaggtcgcc agttccctaa yta 23

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tcgctcaggc tggagtcggc tg 22

<210> 26

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gctggggttg gagtcggctg ctc 23

<210> 27

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cacgttggcg tctgctgtac cct 23

<210> 28

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agtcgcttgc tgtaccctct cag 23

<210> 29

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caggctggtg tcggctgctc c 21

<210> 30

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caggctggag tcagctgctc c 21

<210> 31

<211> 24

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<400> 31

ccactctgaa gttccagcgc acac 24

<210> 32

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gggatccgtc tccactctga mga 23

<210> 33

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gggatccgtc tctactctga aga 23

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caccttggag atccagcgca cag 23

<210> 35

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cttgcactct gaactaaacc tgagctc 27

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caccagactc acagttgtag gta 23

<210> 37

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gaccaggtta accgttgtag gta 23

<210> 38

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<400> 38

aagttggctc actgttgtag gta 23

<210> 39

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agtggaaccc agctctctgt ct 22

<210> 40

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gactcgactc tccatcctag gta 23

<210> 41

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caggctcact gtgacaggta t 21

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gacccggctc tcagtgct 18

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caccgtgcta ggtaagaag 19

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ggctgaccgt actgggtaa 19

<210> 45

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tgacagtgct cggtaagcg 19

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acgcggctcc tggtgctcg 19

<210> 47

<211> 19

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aggctgaccg tgctgggcg 19

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aggctcacgg tcacaggtaa 20

Claims

1. The application of a primer group in preparing a product for constructing a CDR3 region high-throughput sequencing library of human TCR beta or preparing a product for identifying antigen-specific TCR of tumor or pathogen is characterized in that an upstream primer of the primer group comprises TRBV10-1-F, TRBV10-2/3-F, TRBV11-1/2/3-F, TRBV12-3.1/4/5.1-F, TRBV12-3.2/5.2-F, TRBV13-F, TRBV14-F, TRBV15-F, TRBV16-F, TRBV18-F, TRBV19-F, TRBV20-F, TRBV24-F, TRBV25-F, TRBV27-F, TRBV28-F, TRBV29-F, TRBV2-F, TRBV3-F, TRBV30-F, TRBV4-1-F, TRBV4-2/3-F, TRBV5-1-F, TRBV5-4/5/6/8-F, TRBV6-1-F, TRBV6-2/3-F, TRBV6-4.1-F, TRBV6-4.2-F, TRBV6-1.2/5/8-F, TRBV6-1.1/6/7/9-F, TRBV7-1-F, TRBV7-2/4/6/7/8-F, TRBV7-3-F, TRBV7-9-F and TRBV 9-F;

wherein, the TRBJ1-1-R has a nucleotide sequence shown as SEQ ID NO. 36; the TRBJ1-2-R has a nucleotide sequence shown as SEQ ID NO. 37; the nucleotide sequence of TRBJ1-3-R is shown in SEQ ID NO. 38; the nucleotide sequence of TRBJ1-4-R is shown in SEQ ID NO. 39; the TRBJ1-5-R has a nucleotide sequence shown as SEQ ID NO. 40; the nucleotide sequence of TRBJ1-6-R is shown in SEQ ID NO. 41; the nucleotide sequence of TRBJ2-4-R is shown in SEQ ID NO. 42; the nucleotide sequence of TRBJ2-1-R is shown in SEQ ID NO. 43; the nucleotide sequence of TRBJ2-2-R is shown in SEQ ID NO. 44; the nucleotide sequence of TRBJ2-3-R is shown in SEQ ID NO. 45; the nucleotide sequence of TRBJ2-5-R is shown in SEQ ID NO. 46; the nucleotide sequence of TRBJ2-6-R is shown in SEQ ID NO. 47; the nucleotide sequence of TRBJ2-7-R is shown in SEQ ID NO. 48.

2. The use according to claim 1, wherein the method for the preparation of a product for the construction of a high throughput sequencing library of the CDR3 region of human TCR β or a product for the identification of tumor or pathogen antigen-specific TCRs comprises the steps of:

2) preparing a gene library:

2-1) amplification of TCR β fragments: performing PCR amplification on the DNA and/or cDNA obtained in step 1) by using a multiplex PCR method to obtain a CDR3 beta multiplex amplicon product, wherein the multiplex PCR amplification primer is the primer set of claim 1;

2-4) fragment purification: purifying the product obtained in the step 2-3);

3) high-throughput sequencing to identify libraries: identification and analysis of CDR3 β amplicon sequences by high throughput sequencing;

3. The use according to claim 2, wherein the sequencing data analysis of step 4) comprises the steps of:

b) comparing the filtered sequencing readings with V, D, J of TCR and C gene reference genome file to determine the corresponding position of each reading on the genome; wherein different reads having the same nucleotide at the same position are considered to be part of the same clonotype; preserving reads that qualify for read quality and comprise the sequence of the CDR3 region;

4. The use of claim 3, wherein the low quality sequences of step a) comprise sequences with greater than 50% low quality reads, greater than 30% linker sequences, off-target sequences, uncorrectable reads due to PCR errors; the false positive sequence refers to a sequence that repeats in multiple different samples.

5. The use of any one of claims 2-4, wherein the sample of step 1) is whole blood or FFPE; wherein, when the sample is whole blood, PBMC cells in the whole blood sample are separated, and DNA is extracted from the PBMC cells.