title: [Development of a high-efficient assay for amplifying Vbeta-encoding genes of human T-cell receptor].
abstract: 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.
authors.affiliation: College of Biotechnology, Guilin Medical University, Guilin 541004, China.
authors.forename: Hai-yan
authors.initials: HY
authors.lastname: Ye
authors.forename: Lin
authors.initials: L
authors.lastname: Wang
authors.forename: Zhen-ping
authors.initials: ZP
authors.lastname: Fan
authors.forename: Yan-wei
authors.initials: YW
authors.lastname: Zhong
authors.forename: He-ling
authors.initials: HL
authors.lastname: Su
authors.forename: Yong-ming
authors.initials: YM
authors.lastname: Liu
authors.forename: Dong-ping
authors.initials: DP
authors.lastname: Xu
mesh_terms: 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