鸡胚成纤维细胞酵母双杂交cDNA文库的构建及鉴定  

Construction and characterization of the yeast two-hybrid cDNA library of chicken embryo-fibroblasts

作　　者：段志强[1] 王郁杨[1] 朱艳梅[1] 开妍[1] 胡顺林[1] 王晓泉[1] 刘秀梵[1] 

机构地区：[1]扬州大学兽医学院/农业部畜禽传染病学重点开放实验室,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2011年第2期1-5,共5页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金资助项目(30630048);国家科技支撑计划项目(2010BAD04B01);国家蛋鸡产业技术体系专项资金项目(nycytx-41-G07)

摘　　要：选取对新城疫病毒（NDV）易感的鸡胚成纤维细胞（CEF）,Trizol提取细胞总RNA,用SMART技术合成双链cDNA,然后利用同源重组的方法在酵母细胞内构建CEF的cDNA文库,以寻找与NDV基质（M）蛋白相互作用的宿主细胞蛋白,探讨其相互作用对NDV装配和出芽的影响。结果表明：提取的细胞总RNA的D260 nm/D280 nm为1.96,甲醛变性琼脂糖凝胶显示RNA未发生降解,质量良好。获得的文库容量为3×106cfu,插入的双链cDNA片段大小为0.3~3 kb,平均长度为1.3 kb左右,文库重组率为100%。该文库的构建为发现和研究与NDV M蛋白相互作用的宿主细胞蛋白提供有效工具。In order to seek for the host proteins which interact with the Newcastle disease virus（NDV） matrix（M） protein and to study the influence of the interactions on the assembly and budding of NDV,a cDNA library of chicken-embryo-fibroblast-（CEF） was constructed to achieve this aim.The total RNA of CEF was extracted using Trizol method,and double strands cDNA was synthesized by SMART（Switching mechanism at 5′end of RNA transcript） technique.Then homologous recombination mediated approach was used to construct cDNA library of CEF in yeast cells.The results showed that the ratio of D260 nm to D280 nm value of the extracted RNA was 1.96,and the high pure RNA acquired by this method was complete.The titer of cDNA library was 3×106 cfu with the average of inserted fragments about 1.3 kb,and the recombination rate was 100%.These data indicated that the yeast two-hybrid cDNA library of CEF is successfully constructed and may be useful for screening the host proteins interacting with M protein of NDV.

关 键 词：新城疫病毒 鸡胚成纤维细胞 酵母双杂交 CDNA文库 SMART技术 

分 类 号：Q785[生物学—分子生物学]