[Development of a high-efficient assay for amplifying Vbeta-encoding genes of human T-cell receptor].
OBJECTIVE
To develop an effective assay for amplifying human T-cell receptor (TCR) variable region of beta chain (Vbeta)-encoding genes.


METHODS
Based on the property of the 26 subfamilies of human TCR Vbeta-encoding gene sequence, 34 sets of outer and 37 sets of inner sense primers were divided into 8 degenerate primer groups, and a set of outer and inner antisense primers located in conserved region beta chain (Cbeta) was designed for the amplification of the TCR Vbeta-encoding genes. In addition, a sequencing primer and a sense primer for amplifying Cbeta-encoding genes were designed. CD8 T-cell RNA was extracted and subjected to reverse transcription mediated by poly A, followed by a nested polymerase chain reaction (PCR) to amplify the 26 subfamilies of human TCR Vbeta-encoding genes. Jurkat T lymphoma cells were used as control. T-easy vector was employed to detect DNA sequencing of the cloned target genes.


RESULTS
All the subfamilies of the Vbeta-encoding genes were obtained from CD8 T cells of a healthy donor, except the subfamily of Vbeta8 , was obtained from Jurkat cells. The results were confirmed by DNA sequencing of the cloned genes.


CONCLUSIONS
The nested RT-PCR assay can effectively amplify human TCR Vbeta-encoding genes with broad spectrum, which is helpful for the cloning and functional study of the TCR expressed by antigen-specific cytotoxic T lymphocytes.
Ye
Hai-yan
HY
College of Biotechnology, Guilin Medical University, Guilin 541004, China.
Wang
Lin
L
Fan
Zhen-ping
ZP
Zhong
Yan-wei
YW
Su
He-ling
HL
Liu
Yong-ming
YM
Xu
Dong-ping
DP
D000328:Adult; D003001:Cloning, Molecular; D019674:Genes, T-Cell Receptor beta / Q000235:genetics*; D006801:Humans; D008297:Male; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA