长角血蜱卵cDNA表达文库的构建  

Construction of cDNA Expression Library from Eggs of Haemaphysalis longicornis

作　　者：刘光远[1] 田占成[1] 才学鹏[1] 李知新[1] 谢俊仁[1] 王路[1] 张林[1] 龚真莉[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室/甘肃省动物寄生虫病重点实验室,兰州730046

出　　处：《中国农业科学》2008年第7期2204-2208,共5页Scientia Agricultura Sinica

基　　金：国家科技基础条件平台项目(2005DKA21205-3);国家高新技术研究发展计划("863")项目(2006AA10A207);甘肃省自然科学基金 项目(32S041-A25-035)

摘　　要：【目的】以长角血蜱卵为材料构建高质量的长角血蜱卵cDNA表达文库,为进一步筛选克隆目的基因奠定基础。【方法】在无RNA酶污染的环境下从长角血蜱卵中提取RNA,进而纯化mRNA,采取oligo(dT)引物合成双链cDNA,定向克隆到λSCREEN载体。用PhageMaker extract对其进行体外包装以形成完整的噬菌体,转染大肠杆菌ER1647,测定库容量。并用构建的cDNA文库克隆已知的长角血蜱促卵泡激素基因。【结果】所构建长角血蜱卵cDNA表达文库的基础库容量约为1.38×106PFU,重组率为100%,扩增后文库的滴度为2×109PFU·ml-1。获得了目的基因,其序列与GenBank中的长角血蜱促卵泡激素(DQ248886)的核苷酸序列的同源性为97.8%。【结论】成功构建了长角血蜱卵cDNA表达文库。[Objective] cDNA expression library from eggs of Haemaphysalis longicornis was constructed for further screening and cloning of the potential candidate antigenic genes. [Method] Total RNA was isolated from eggs of H, longicornis, mRNA were purified, and cDNAs were synthesized and ligated to λSCREEN vector. The recombinant phage DNA was packaged by using Phagemark packaging extracts and then transfected to E. coli ER1647 to obtain the cDNA expression library. [Result]The size of the primary cDNA library was 1.38 × 10^6 PFU with titer of the amplified cDNA library of 2 × 10^9 PFU·ml^-1. A full length cDNA encoding follistatin-related protein was cloned from the cDNA library by PCR. [ Conclusion] The cDNA expression library from eggs of H, longicornis was successfully constructed.

关 键 词：长角血蜱 卵 CDNA表达文库 构建 

分 类 号：Q78[生物学—分子生物学]