新基因TMBP-1的全长cDNA克隆

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

目的 :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C 91,应用RACE成功克隆新基因TMBP - 1cDNA全长序列。它拥有一个 969bp的开放读码框架 ,编码 32 3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 5kb ;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。

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.