CN102212888A - High throughput sequencing-based method for constructing immune group library - Google Patents
High throughput sequencing-based method for constructing immune group library Download PDFInfo
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- CN102212888A CN102212888A CN2011100651973A CN201110065197A CN102212888A CN 102212888 A CN102212888 A CN 102212888A CN 2011100651973 A CN2011100651973 A CN 2011100651973A CN 201110065197 A CN201110065197 A CN 201110065197A CN 102212888 A CN102212888 A CN 102212888A
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Abstract
The invention relates to a high throughput sequencing-based method for constructing an immune group library. Multiple primers are designed according to the law of the rearrangement of B chain genes of a T cell antigen receptor to perform amplification on a nucleic acid sequence, and the sequencing is performed by combining the high throughput sequencing technology and utilizing a sequencing primer to find a feasible method for measuring the immune group library. The method comprises the following steps of: 1, separating a T cell sub-group from peripheral blood; 2, extracting messenger ribonucleic acid; 3, performing the amplification on a target fragment by utilizing multiple primers; 4, performing high throughput sequencing by utilizing the sequencing primer; 5, performing variable-diversity-joining (VDJ) fragments comparison on sequencing data; and 6, analyzing a complementarity determining region 3 (CDR 3) sequence to obtain the complete immune group library. In the method, the immune group library of the specific T cell sub-group in the peripheral blood is constructed by utilizing multiple primers and combining the high throughput sequencing technology. The high throughput sequencing-based method for constructing the immune group library is suitable for biomedical research or basic biological research of immunohistochemistry.
Description
Technical field
The invention belongs to biological technical field, relate to immunology and high-flux sequence aspect.
Background technology
(T cell antigen receptor TCR) is body immune system specific recognition and the key molecule of catching the specific antigenic foreign matter to TXi Baoshouti.The T cell antigen receptor is made up of alpha and two polypeptide chains of beta, every polypeptide chain is also as the segmental polypeptide chain of IgF ab, be folded into two functional zone-variable regions and constant region, be embedded in the T cytolemma, the T cell discerns and catches various antigenic substances by it.
According to the antigen receptor that detects 95% mature T cells surface and the structure similar of light chain immunoglobulin and heavy chain, form heterodimer by two peptide chains of alpha, beta, V district (variable region) and C district (constant region) are all arranged on every peptide chain.
The germline gene of TXi Baoshouti is made up of the gene fragment of a plurality of separations, and their random groups in the gene rearrangement process are combined into multifarious generation the genetics basis is provided.Gene rearrangement is a stochastic process, and different T cell clones produces special genes through gene rearrangement, the segmental connection of generation different genes.During V district gene rearrangement, DJ, VDJ or VJ connection when taking place in specific V, D or J fragment (joining region), because the maneuvering ability or the inaccuracy of reading frame, certain variation (skew) can take place in the VDJ junction cause nucleotide deletion, cause the change of nucleotide sequence, codon, the variation that causes V district amino acid to be formed at last, thus the specificity of change TXi Baoshouti has increased its diversity.
The present invention utilizes multi-primers and in conjunction with high throughput sequencing technologies, makes up the immune group storehouse of specific T cells subgroup in the peripheral blood.It is a kind of immune group base construction method based on high-flux sequence.Be applicable to the biomedical research or the fundamental biological knowledge research of immune group.
Summary of the invention
The rule that the present invention resets according to TXi Baoshouti beta chain gene, the design multi-primers increases to nucleotide sequence, then in conjunction with high throughput sequencing technologies, utilizes sequencing primer to check order, thereby has found a kind of method of feasible mensuration immune group storehouse.Implementation step is as follows, sees Fig. 1:
Step 1: from peripheral blood, separate the T cell subsets.
Step 2: extract messenger RNA(mRNA).
Step 3: utilize multi-primers amplification purpose segment.
Step 4: utilize sequencing primer to carry out high-flux sequence.
Step 5: sequencing data carries out the VDJ comparison.
Step 6: analyze CDR3 (being the junction of VDJ) sequence, obtain a complete immune group storehouse.
The feature of present method: the rule that the present invention resets according to TXi Baoshouti beta chain gene, the design multi-primers increases to nucleotide sequence, in conjunction with high throughput sequencing technologies, utilize sequencing primer to check order then, thereby found a kind of method of feasible mensuration immune group storehouse.
Aspect novelty, it is characterized in that: our method has been introduced the multi-primers method of design, and the high-flux sequence method, and the result is more comprehensive, helps setting up a complete reliable immune group storehouse.
Embodiment
CD4, CD8 positive t lymphocytes with the people is example below, is described further
Step 1: the T cell subsets of from human peripheral, using paramagnetic particle method separation of C D4, CD8.
Step 2: extract messenger RNA(mRNA).
Step 3: utilize multi-primers amplification purpose segment.All primer sequences are seen Fig. 2.
Step 4: utilize sequencing primer to carry out high-flux sequence.
Step 5: utilize blast comparison instrument (http://www.ncbi.nlm.nih.gov/blast/) to V, the comparison of D fragment sequence.
Step 6: according to the result of comparison, reconstruct CDR3 sequence from sequencing data.Finally, obtain a complete immune group storehouse.
More than be the description of this invention and non-limiting, based on other embodiment of inventive concept, all among protection scope of the present invention.
Claims (1)
1. the present invention's rule of resetting according to TXi Baoshouti B chain gene, the design multi-primers increases to nucleotide sequence, in conjunction with high throughput sequencing technologies, utilizes sequencing primer to check order, thereby has found a kind of method of feasible mensuration immune group storehouse.The present invention mainly comprises following flow process:
Step 1: from peripheral blood, separate the T cell subsets;
Step 2: extract messenger RNA(mRNA);
Step 3: utilize multi-primers amplification purpose segment;
Step 4: utilize sequencing primer to carry out high-flux sequence;
Step 5: sequencing data carries out the VDJ comparison;
Step 6: analyze the CDR3 sequence.Obtain a complete immune group storehouse.
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105095687A (en) * | 2015-06-26 | 2015-11-25 | 南方科技大学 | Method and terminal for analyzing immune repertoire |
| CN106156539A (en) * | 2015-03-27 | 2016-11-23 | 深圳华大基因科技有限公司 | The method and apparatus analyzing the immunity difference of individual two class states |
| CN106156536A (en) * | 2015-04-15 | 2016-11-23 | 深圳华大基因科技有限公司 | The method and system that sample immune group storehouse sequencing data is processed |
| CN106755410A (en) * | 2016-12-23 | 2017-05-31 | 孙涛 | A kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence |
| CN108004304A (en) * | 2017-12-20 | 2018-05-08 | 北京旌准医疗科技有限公司 | A kind of Clonal method for detecting lymphocyte related genes and resetting |
| CN108804874A (en) * | 2018-06-15 | 2018-11-13 | 广州华银医学检验中心有限公司 | Immune group library analysis of biological information flow based on molecular labeling |
| CN109979528A (en) * | 2019-03-28 | 2019-07-05 | 广州基迪奥生物科技有限公司 | A kind of analysis method of unicellular immune group library sequencing data |
| CN111518196A (en) * | 2020-05-23 | 2020-08-11 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
| 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 |
| CN113150113A (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 |
| CN113214383A (en) * | 2020-05-23 | 2021-08-06 | 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 |
| WO2021237932A1 (en) * | 2020-05-23 | 2021-12-02 | 湖南源品细胞生物科技有限公司 | Tcr enriched clonotypes and acquisition method and application therefor |
| CN114107458A (en) * | 2013-03-15 | 2022-03-01 | 血统生物科学公司 | Method for sequencing an immune repertoire |
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Cited By (22)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107458A (en) * | 2013-03-15 | 2022-03-01 | 血统生物科学公司 | Method for sequencing an immune repertoire |
| CN106156539A (en) * | 2015-03-27 | 2016-11-23 | 深圳华大基因科技有限公司 | The method and apparatus analyzing the immunity difference of individual two class states |
| CN106156539B (en) * | 2015-03-27 | 2018-09-14 | 深圳华大基因科技有限公司 | The method and apparatus of the immunity difference of the individual two class states of analysis |
| CN106156536B (en) * | 2015-04-15 | 2018-11-13 | 深圳华大基因科技有限公司 | The method and system that sample immune group library sequencing data is handled |
| CN106156536A (en) * | 2015-04-15 | 2016-11-23 | 深圳华大基因科技有限公司 | The method and system that sample immune group storehouse sequencing data is processed |
| CN105095687A (en) * | 2015-06-26 | 2015-11-25 | 南方科技大学 | Method and terminal for analyzing immune repertoire |
| CN105095687B (en) * | 2015-06-26 | 2018-10-26 | 南方科技大学 | A kind of immune group library data analysing method and terminal |
| CN106755410A (en) * | 2016-12-23 | 2017-05-31 | 孙涛 | A kind of method for detecting T cell and B cell immune group storehouse simultaneously based on high-flux sequence |
| CN106755410B (en) * | 2016-12-23 | 2019-12-06 | 孙涛 | Method for simultaneously detecting T cell and B cell immune repertoire based on high-throughput sequencing |
| CN108004304A (en) * | 2017-12-20 | 2018-05-08 | 北京旌准医疗科技有限公司 | A kind of Clonal method for detecting lymphocyte related genes and resetting |
| CN108004304B (en) * | 2017-12-20 | 2021-04-23 | 北京旌准医疗科技有限公司 | Method for detecting clonality of lymphocyte related gene rearrangement |
| CN108804874A (en) * | 2018-06-15 | 2018-11-13 | 广州华银医学检验中心有限公司 | Immune group library analysis of biological information flow based on molecular labeling |
| CN108804874B (en) * | 2018-06-15 | 2019-04-23 | 广州华银医学检验中心有限公司 | Immune group library analysis of biological information method based on molecular labeling |
| CN109979528A (en) * | 2019-03-28 | 2019-07-05 | 广州基迪奥生物科技有限公司 | A kind of analysis method of unicellular immune group library sequencing data |
| CN109979528B (en) * | 2019-03-28 | 2021-06-25 | 广州基迪奥生物科技有限公司 | Analysis method of single cell immune repertoire sequencing data |
| 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 |
| CN113150113A (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 |
| CN113214383A (en) * | 2020-05-23 | 2021-08-06 | 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 |
| CN113248592A (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 |
| WO2021237932A1 (en) * | 2020-05-23 | 2021-12-02 | 湖南源品细胞生物科技有限公司 | Tcr enriched clonotypes and acquisition method and application therefor |
| CN111518196A (en) * | 2020-05-23 | 2020-08-11 | 湖南源品细胞生物科技有限公司 | TCR enrichment clone type and acquisition method and application thereof |
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Application publication date: 20111012 |