犊牛口腔黏膜上皮细胞T7噬菌体展示文库的构建  

Construction of Calves Oral Mucosal Epithelial Cells T7 Phage Display Library

作　　者：韩岩岩[1] 宋德光[2] 

机构地区：[1]遵义医学院公共卫生学院,贵州遵义563003 [2]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国畜牧兽医》2014年第5期49-52,共4页China Animal Husbandry & Veterinary Medicine

基　　金：贵州省科学技术基金(黔科合J字[2013]2313号);2013年遵义医学院博士启动基金(F-584)

摘　　要：试验旨在筛选水泡性口炎病毒的受体,构建犊牛口腔黏膜上皮细胞T7噬菌体展示文库。通过用Trizol试剂提取犊牛口腔黏膜上皮细胞总RNA,然后分离和纯化mRNA,经反转录合成双链cDNA。在双链cDNA末端加上定向EcoRⅠ/HindⅢ接头,然后用EcoRⅠ和HindⅢ酶消化双链cDNA末端接头,使双链cDNA 2端含有EcoRⅠ和HindⅢ黏性末端。所有处理后的双链cDNA,经Mini Column纯化,收集400bp以上的双链cDNA片段,将其连接带有EcoRⅠ和HindⅢ末端的T7Select 10-3b载体,经体外包装后,以BLT5403为受体菌构建T7噬菌体展示文库。经测定未扩增文库的滴度为1.3×107PFU/mL,重组率为95.8%,扩增后文库滴度为2.6×1010 PFU/mL。随机挑取100个噬菌斑进行PCR鉴定,95%的插入片段大于400bp。结果表明成功构建了犊牛口腔黏膜上皮细胞T7噬菌体展示文库。To screen receptor of vesicular stomatitis virus （VSV）, the calves oral mucosal epithelial cells T7 phage display library was constructed. The total RNA of calves oral mucosal epithelial cells was extracted using Trizol reagent. Then, mR- NA was separated and purified by mRNA isolation kit and the ds cDNA were synthesized by reverse transcription, ds eDNA ends were ligated EcoR Ⅰ/Hind Ⅲ linkers, then ds cDNA were digested by EcoR Ⅰ/Hind Ⅲ, so that we got ds eDNA ends containing EcoR Ⅰ/Hind Ⅲ sticky ends. All digested ds cDNA were separated by Mini Column, only ds eDNA fragments more than 400 bp were collected. Then, the collected ds cDNA were ligated into the T7 Select 10-3b vector. After packaging in vitro, the recombinant T7 Select 10-3b vectors were transformed into BLT5403, so we could construct the T7 phage display li- brary. The results indicated that the titer of un-amplified library was 1.3 ×10^ PFU/mL, and the recombination rate was 95.8%. After amplification, titer of library was 2.6 ×10^10 PFU/mL. Randomly picked 100 plaques were identified by PCR, 95 % of the inserted eDNA fragments were longer than 400 bp in length. These results indicated that the calves oral mucosal epithelial cells T7 phage display library was successfully constructed.

关 键 词：口腔黏膜上皮细胞 T7噬菌体展示文库 水泡性口炎病毒 

分 类 号：Q78[生物学—分子生物学]