Method for detecting genetic constitution of clone-specific T lymphocyte TCR BV CDR3

Abstract

The invention relates to a genetic constitution detecting method for a cloned specific T lymphocyte TCR BV CDR3 gene, which comprises the following steps: a single nucleus cell of peripheral blood or a general RNA in tissue sample is extracted and reverse-transcribed to cDNA. The upper reach primer of the TCR BV twenty four families and a common lower reach primer BC are designed according to TCRBV gene; the cDNA is expanded using real-time fluorescent quantification PCR with dye method. The PCR product is analyzed with the melting curve; the negative primary derivative (-dF/dT) of fluorescence intensity change of the PCR product is graphed vs. the temperature (Tm); the peak graph of BV each family is obtained; the BV family PCR product showing single peak is purified and processed with serial analysis; the sequencing results are compared with the TCR Beta chain standard gene sequence. The distribution of the cloned specific T lymphocyte in tissue or peripheral blood is judged according to the appearing situation of single peak in BV each family peak graph; the genetic constitution of the cloned specific TCR BV CDR3 is determined combining the sequencing results. The genetic constitution detecting method for the cloned specific T lymphocyte TCR BV CDR3 gene has important significance in evaluating diseases related to specific cloned T cell.

Description

A method for detecting TCRBVCDR3 gene composition of clone-specific T lymphocytes technical field The invention belongs to the technical field of biomedicine, relates to molecular immunology and molecular biology, in particular to a detection method, in particular to a method for detection of peripheral blood clone-specific T lymphocytes. Background technique T cell receptor (TCR) is an important molecule on the surface of T lymphocytes to recognize and bind antigens and participate in specific cellular immunity. The TCR of 95% T lymphocytes in normal human peripheral blood is a heterodimer composed of two polypeptide chains α and β through disulfide bonds. The α chain is formed by the rearrangement of AV, AJ, and AC in the germline gene. The nucleotide sequence inserted at the end of AV, the front end of AJ and the middle constitutes the complementarity determining region 3 (CDR3) of the variable region of the TCR α chain. The β chain is formed by the germline gene. The ...

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However, these methods cannot provide information on the TCR molecular signa

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