鸡胚成纤维细胞cDNA T7噬菌体表达文库的构建和鉴定.pdfVIP

2008年第 7期 鸡胚成纤维细胞 cDNA T7噬茵体 表达文库的构建和鉴定 赵绪永 。，张改平 ，朱礼倩 ，李清州 (1．河南农业大学，河南 郑州 450002；2．河南省农业科学院，河南 郑州 450002； 3．郑州牧业工程高等专科学校，河南 郑州 450011) 摘要：构建鸡胚成纤维细胞(CEF)T7噬茵体表达型cDNA文库，为筛选和研究 CEF上病毒受体 分子奠定基础。提取纯化 CEF的mRNA，反转录合成双链 cDNA，补平末端，连接 EcoR I／Hind Ⅲ接头，双酶切，凝胶过滤层析除去小于 400 bp的cDNA片段，合成双链 cDNA定向与T7噬茵 体左右臂连接，体外包装后建成 cDNA表达文库。测定文库大小、重组率，并以PCR鉴定 cDNA 插入片段的大小。结果表明，构建的cDNA文库滴度为2．2×10 pfu／mL、扩增后滴度为3．5×10 pfu／mL，重组效率达 98 、插入片段多在0．4～3 kb之间，平均插入片段长约 1．3 kb。表明所建文 库具有很高的质量，从而为筛选目的 cDNA克隆奠定了基础。 关键词：鸡胚成纤维细胞(CEF)；T7噬茵体；cDNA表达文库 中图分类号：$831 文献标识码：A 文章编号：1004—3268(2008)07—0100—04 Construction and Characterization of A T7 Phage Display cDNA Library from CEF ZHAO Xu—yong ，ZHANG Gai—ping ，ZHU Li-qian ，LI Qing-zhou 01．Henan Agricultural University，Zhengzhou 450002，China；2．Henan Academy of Agricultural Sciences，Zheng- zhou 450002，China；3．Zhengzhou College of Animal Husbandary Engineering，Zhengzhou 450011，China) Abstract：For further screening of the cellular receptor of IBDV，a cDNA library was constructed in this paper．Purified mRNAs were extracted from CEF cells by using a single roUnd of chroma— tography on oligo(dT)cellulose．First and second strand of the cDNA were synthesized by reverse transcription．After blunting the cDNA terraini with T4 DNA polymerase．the cDNA fragments were ligated with directional EcoR I／HindⅢ Linkers，and the cDNA was subsequently digested with HindⅢ and EcoR I．Then，the small cDNA products( 400 bp)were removed by gel fil～ tration and the remaining products were ligated with the arms of T7 vector．After ligation and packaging in T7 packaging extracts in vitro，the phages were plated to determine the library titer． The pha