犊牛口腔黏膜上皮细胞T7噬菌体展示文库的构建

中国畜牧兽医

犊牛口腔黏膜上皮细胞T7噬菌体展示文库的构建

韩岩岩¹，宋德光²

（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）

收稿日期:2013-10-22 出版日期:2014-05-20 发布日期:2014-06-25
作者简介:韩岩岩（1984—），女，黑龙江人，博士，研究方向：分子营养和营养相关疾病。
基金资助:
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）。

Construction of Calves Oral Mucosal Epithelial Cells T7 Phage Display Library

HAN Yan-yan¹，SONG De-guang²

(1.School of Public Health, Zunyi Medical University, Zunyi 563003, China; 2.College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062, China)

Received:2013-10-22 Online:2014-05-20 Published:2014-06-25

摘要/Abstract

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Hind Ⅲ酶消化双链cDNA末端接头，使双链cDNA 2端含有EcoRⅠ和Hind Ⅲ黏性末端。所有处理后的双链cDNA，经Mini Column 纯化，收集400 bp以上的双链cDNA片段，将其连接带有EcoRⅠ和Hind Ⅲ末端的T7 Select 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1.3×10⁷ PFU/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400 bp。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。

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

Abstract: 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, mRNA was separated and purified by mRNA isolation kit and the ds cDNA were synthesized by reverse transcription. ds cDNA ends were ligated EcoR Ⅰ/Hind Ⅲ linkers, then ds cDNA were digested by EcoRⅠ/Hind Ⅲ, so that we got ds cDNA ends containing EcoR Ⅰ/Hind Ⅲ sticky ends. All digested ds cDNA were separated by Mini Column, only ds cDNA 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 library. The results indicated that the titer of un-amplified library was 1.3×10⁷ PFU/mL, and the recombination rate was 95.8%. After amplification, titer of library was 2.6×10¹⁰ PFU/mL. Randomly picked 100 plaques were identified by PCR, 95% of the inserted cDNA 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.

Key words:

calves oral mucosal epithelial cells; T7 phage display library; vesicular stomatitis virus

韩岩岩，宋德光. 犊牛口腔黏膜上皮细胞T7噬菌体展示文库的构建[J]. 中国畜牧兽医.

HAN Yan-yan，SONG De-guang. Construction of Calves Oral Mucosal Epithelial Cells T7 Phage Display Library[J]. .

参考文献

1 刘太峰，孟庆峰，段小波，等.cDNA文库构建方法的研究进展［J］.中国畜牧兽医，2012，39（7）：40～43.
2 闫守庆, 张玉静. 噬菌体表面呈现技术及在疫苗研制中的应用［J］. 黑龙江医学, 2003, 27（2）:110～121.
3 沈倍奋. 分子文库［M］. 北京:科学出版社, 2001.
4 侯云德. 分子病毒学［M］. 北京: 科学出版社, 1990.
5 殷震, 刘景华. 动物病毒学［M］. 第2版. 北京: 科学出版社，1997.
6 魏东芝, 赖敏. 噬菌体抗体库筛选技术［J］. 生命科学, 2000, 12（3）: 134～137.
7 Arap M. Phage display technology—Applications and innovations［J］.Genetics and Molecular Biology, 2005, 28(1): 1～9.
8 Benhar I. Biotechnological applications of phage and cell display［J］.Biotechnology Advances, 2012, 19(1): 1～33.
9 Danner S, Belasco J G. T7 phage display: A novel genetic selection system for cloning RNA-binding proteins from cDNA libraries［J］. Proc Natl Acad USA, 2011， 98(23): 12954～12959.
10 Georgel P, Jiang Z, Kunz S, et al. Vesicular stomatitis virus glycoprotein G activates a specific antiviral Toll-like receptor 4-dependent pathway［J］. Virology, 2007, 362(2): 304～313.
11 Petra W, Lechner M, Merschak P,et al. Molecular cloning of a novel lipocalin-1 interacting human cell membrane receptor using phage display［J］. J Biol Chem, 2001， 276(23): 20206～20212.
12 Rose N F, Roberts A, Buonocore L, et al. Glycoprotein exchange vectors based on VSV allow effective boosting and generation of neutralizing antibodies to a primery isolate of human immunolo deficiency virus［J］. J Virol, 2000, 74(23): 109～113.
13 Santi E, Capone S, Mennuni C,et al.Bacteriophage lamda display of complex cDNA libraries: A new approach to functional genomics［J］. J Mol Biol, 2000， 296: 497～506.