新基因TMBP-1的全长cDNA克隆

Molecular cloning of a novel mouse full- length eDNA TMBP- 1

关键词:  抑制性差减杂交  RACE  cDNA克隆  新基因

Key words:  SSH  RACE  cDNA cloning  novel gene

摘要:

克隆胸腺细胞发育相关新基因。方法：本研究建立了抑制性差减杂交技术(SSH)，构建了胸腺基质细胞系cDNA差减杂交文库。应用cDNA末端快速扩增方法(RACE)进一步克隆新基因的cDNA全长序列。结果：筛选两个不同胸腺基质细胞系的差异表达基因，获得了新基因cDNA片段C91，应用RACE成功克隆新基因TMBP-1 cDNA全长序列。它拥有一个969 bp的开放读码框架，编码323个氨基酸。同源序列比较发现，它编码一个未知功能的膜结合蛋白。Northem杂交分析显示，mRNA转录本长度为1.5 kb；在两种胸腺基质细胞系中均有表达。新基因的cDNA全长序列，已被GenBank所接受。结论：抑制性差减杂交技术是克隆新基因片的有效方法；成功克隆新基因TMBP-1的cDNA全长序列

Abstract:

Objective:To clone and characterize novel genes which might be involvde in T cell development. Methods:A subtracted cDNA library of mouse thymic stromal cell lines was constructed using suppression subtractive hybridization with modification.RACE was used to clone the full-length cDNA of novel gene.Results: A novel gene fragment C91 was obtained and the full-length cDNAs of C91 cloned using RACE.Sequence of the novel gene revealed that it has an open reading frame of 969bp encoding a 323 aa protein of unknown functions. Northern blot analysis of C91 showed that the mRNA transcript was 15 kb and expressed in the two thymic stromal cell lines investigated.The full-length cDNA C91 has been accepted by GenBank. Conclusion: suppression subtractive hybridization is an effective method to clone novel genes; a novel gene full-length cDNA TMBP-1 was cloned.