利用SMART技术构建中国人肝组织cDNA酵母双杂交文库

目的: 针对人类肝脏蛋白质组计划中大规模基因克隆及构建肝脏蛋白相互作用图谱的研究需要，改造酵母双杂交载体pPC86和pDBleu，并构建中国人肝组织cDNA文库，为肝脏蛋白质组计划的研究奠定基础。 方法: 设计并合成特定寡核苷酸序列，经过处理形成具有黏性末端的双链DNA片段，插入到pPC86、pDBLeu载体相应的酶切位点处，从而获得改造的新载体。利用改造后的载体、中国人正常肝组织mRNA和Clontech公司SMART文库构建试剂盒构建NpDBLeu-cDNA文库。 结果: 酶切鉴定和DNA测序证实改造后载体插入位点和序列正确，成功获得新载体NpPC86和NpDBLeu。滴度测试表明，所构建NpDBLeu肝组织cDNA文库容量为1.93×10⁶ cfu，转化子重组效率为95.2%，扩增文库达到109cfu·mL^-1。 结论: 利用SMART技术成功构建了中国人肝组织cDNA酵母双杂交文库。

Construction of cDNA library of Chinese liver tissues using SMART technique

Objective According to the need of large-scale gene cloning and the construction of protein interaction map in human liver proteome project(HLPP),to reconstruct the yeast two-hybrid plasmids pPC86and pDBLeu, and then set up the Chinese liver cDNA library,and lay the foundation for further study of HLPP.Methods The specific oligonucleotides were designed and synthesized.They were treated to form double-strand DNA fragments with specifc sticky end,and inserted into pPC86or pDBLeu vector at the appropriate res...