细粒棘球蚴全长cDNA文库的构建及初步鉴定

CONSTRUCTION AND PRIMARY IDIFICATION OF FULL-LENGTH cDNA LIBRARY OF ECHINOCOCCUS GRANULOSUS

目的 分离目的基因构建cDNA文库的经典方法繁琐且不易得到全长cDNA(3’和 5’端 ,尤其是基因的 5’非翻译区 )。本研究通过构建细粒棘球蚴全长cDNA文库 ,筛选全长目的基因 ,研究其结构和功能。方法 采用TRIZOL试剂提取E .granulosus总RNA ,用ClonTech公司的SMARTTMcDNA文库构建试剂盒构建E .granulosus原头节全长cDNA文库 ,文库的包装使用EICENTERTECHNOLOGIES公司提供的包装蛋白。结果与结论 成功构建细粒棘球蚴全长cDNA文库 ,文库滴度为 1.4× 10 7pfu/ml。文库的重组效率达到 99%以上 ,插入片段长度约为 1.1kb。

Aim The classical methods of constructing cDNA library for isolating and cloning genes, were complicating, hard work, and difficult to obtain full-length genes which contain 3' and 5'-untranslated regions, especially for those 5'-untranslated regions.To construct a full-length cDNA library of Echinococcus granulosus for screening the genes and studying the structure and function of genes.Method The total RNA was isolated by using the TRI zol reagent (GibcoBRL).Using “SMART” method for constructing a full-length cDNA libraries from Echinococcus granulosus.Result and Conclusion The full-length cDNA librarie of Echinococcus granulosus have been constructed.The packaging efficiency and the recombinant rate were about 1.4×10 7pfu/ml and 99% clones.The average length of the cDNA inserts was about 1.1kb.