青海血蜱cDNA表达文库的构建  

Construction of cDNA Expression Library of Haemaphysalis qinghaiensis

作　　者：高金亮[1] 关贵全[1] 马米玲[1] 刘志杰[1] 党志胜[1] 刘爱红[1] 李文卉[1] 任巧云[1] 罗建勋[1] 殷宏[1] 

机构地区：[1]中国农业科学院兰州兽医研究所,兰州730046

出　　处：《畜牧兽医学报》2005年第11期1187-1191,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30270992);国家高技术研究发展计划(863计划)(2001AA249081)

摘　　要：在无RNA酶污染的环境下摘取半饱血青海血蜱唾液腺、马氏管和卵巢等器官,从中提取RNA,进而纯化mRNA,采用oligo(dT)引物合成双链cDNA,并在其两端加EcoRⅠ/HindⅢ定向接头。将所产生的cDNA分子定向克隆到具有EcoRⅠ/HindⅢ黏性末端的λSCREEN载体的两臂之间。用PhageMaker extract对以上连接产物进行体外包装以形成完整的噬菌体,并用之转染大肠杆菌ER1647,从而构建成青海血蜱的cDNA表达文库。经测定,所构建青海血蜱cDNA文库的库容量约为2×106PFU/mL,重组率为100%,扩增后文库的滴度为8×109PFU/mL。通过对该文库的免疫学筛选,获得一个编码青海血蜱肌球蛋白碱性轻链的全长cDNA序列。所有结果显示,青海血蜱cDNA表达文库已成功构建。Total RNA were isolated from organs such as salivary glands, Malpighian tubules, ovaries dissected from partially engorged Haemaphysalis qinghaiensis. Subsequently mRNA were purified. A library of oligo（dT）-primed cDNA with added directional EcoR Ⅰ/Hind Ⅲ linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoRⅠ/HindⅢ arms of the λSCREEN vector. The recombinant phage DNA was packaged by using PhageMaker packaging extracts, resulting in a primary cDNA library with a size of 2.0×10^6 PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 8×10^9PFU. A full-length cDNA encoding myosin light chain alkali of Haemaphysalis qinghaiensis was screened from the expression library using rabbit anti-Haemaphysalis qinghaiensis differential proteins serum. The results suggested that the cDNA expression library was constructed successfully.

关 键 词：青海血蜱 CDNA表达文库 构建 

分 类 号：S852.746[农业科学—基础兽医学] Q785[农业科学—兽医学]