Primer sets, high-throughput sequencing methods and applications for developing rhesus monkey T-cell immune repertoire

A cellular immunity, rhesus monkey technology, applied in the field of molecular biology, can solve problems such as limited use

Inactive Publication Date: 2017-07-25
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although immune repertoire sequencing technology has been widely used in the field of human basic research, the use of this technology in other model organisms is very limited, limited to mice and zebrafish

Method used

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  • Primer sets, high-throughput sequencing methods and applications for developing rhesus monkey T-cell immune repertoire
  • Primer sets, high-throughput sequencing methods and applications for developing rhesus monkey T-cell immune repertoire
  • Primer sets, high-throughput sequencing methods and applications for developing rhesus monkey T-cell immune repertoire

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] The preparation of T lymphocyte receptor (TCR) genomic DNA sample comprises the following steps:

[0074] (1) Collect 10 milliliters (ml) of fresh peripheral blood samples each, and operate according to the instructions of the LymphoPrep kit (Axis-shield, Cat. No. AS1114544UK) to obtain relatively pure PBMCs;

[0075] (2) Use the PureLink Genomic DNA Mini Kit (Life Technology, Cat.No: K1820-00) kit to extract the genomic DNA of the cells obtained in step (1), and use Nanodrop2000 (Thermo) to measure the concentration and purity of the DNA, and then save the genomic DNA .

Embodiment 2

[0077] Using the primer set for developing rhesus monkey T cell immune repertoire to perform multiplex PCR, construct a TCRβ high-throughput sequencing library, and perform high-throughput sequencing, including the following steps:

[0078] (1) Use the genomic DNA obtained in Example 1 as an amplification template, take TCR primers, and then use QIAGEN’s Multiplex PCR kit (article number: 206143) to configure a multiplex PCR system according to the kit instructions, wherein each forward primer, etc. Molar mixing, the total concentration of primers is 10 micromolar, equimolar mixing of each reverse primer, the total concentration of primers is 10 micromolar, the amount of template can be adjusted, 3ug is used in this example, 1ug ​​can also be made, but the diversity will be decline.

[0079] The upstream primers in this system are equimolar mixtures of primers shown in SEQ ID NO:1 to SEQ ID NO:26; the downstream primers are equimolar mixtures of primers shown in SEQ ID NO:27 t...

Embodiment 3

[0126] Methods for correcting multiplex PCR primer amplification errors include:

[0127] One of the purposes of the present invention is to analyze the abundance of TCR sequences. However, during the PCR amplification process, due to the amplification preferences of PCR primers for different gene fragments, the real situation of the abundance of TCR sequences will be affected.

[0128] In order to assess this type of systematic error, we adopted the method in Robins et al, we extracted the genome of 1 rhesus monkey according to the method in Example 1, divided into 6 tubes equally, and 3 tubes adopted 25 cycles of multiplex PCR (refer to implementation Example 2), 3 tubes were used for 30 cycles, and the amplified products of each tube were sequenced by Illumina Miseq. In the 25-cycle experimental group, a total of 10,345 specific TCR CDR3 sequences were found, and 15.3% were also found in the 30-cycle PCR experiment.

[0129] We analyzed the 19658 sequencing results found in ...

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Abstract

The invention provides a primer set, high-throughput sequencing method and application for developing a rhesus monkey T cell immune repertoire. In the present invention, 26 upstream primer sequences are set for the variable region V region (variable) of the rhesus monkey TCR, and 13 downstream primer sequences are set for the joining region (joining) J region of the TCR, and the peripheral region is amplified by multiplex PCR. Up to 10 in blood or other immune tissues 7 A high-throughput sequencing library was prepared and sequenced for TCR sequences of about the order of magnitude, and a comprehensive analysis of the TCR gene immune library of rhesus monkeys was carried out through bioinformatics. The technical scheme provided by the present invention is of great significance to the construction of rhesus monkey analysis models, which is conducive to promoting the application of rhesus monkey models in the study of autoimmune system diseases and infectious diseases, and is also conducive to human beings to have a deeper understanding of T cell-mediated diseases. and broad awareness.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a primer set for developing a rhesus monkey T cell immune library, a high-throughput sequencing method and an application thereof. Background technique [0002] T cell receptor (TCR) plays a very important role in cellular immunity. TCR is a heterodimer molecule. In the circulatory system, more than 95% of TCRs belong to αβ type. In human peripheral blood, there are more than 2×10 7 Specific TCRα and TCRβ pairing. The complexity of the immune repertoire is generated by the three types of gene segments on the chromosome during the maturation process of lymphocytes, including V, D, J gene rearrangement and insertion and deletion at the junction of the three gene segments. This key region, also known as CDR3, is the main antigen recognition site, also known as complementarity determining region 3 (CDR3), which is the most diverse and complex region in the variable regi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 李周芳贺建奎童寅张萌
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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