核糖体展示口蹄疫单链抗体库的构建

中国生物工程杂志

2011, Vol. 31

Issue (7): 98-103

技术与方法

核糖体展示口蹄疫单链抗体库的构建

杨双, 马军武, 周广青, 林密, 冯霞, 刘涛, 代鹏

中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部畜禽病毒学重点开放实验室, 农业部兽医公共卫生重点开放实验室兰州 730046

Construction of Ribosome Display Single Chain Antibody Library of Foot and Mouth Disease

YANG Shuang, MA Jun-wu, ZHOU Guang-qing, LIN Mi, FENG Xia, LIU Tao, DAI Peng

Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Science, State Key Laboratory of Veterinary Etiological Biology, National Foot-and-Mouth Disease Reference Laboratory, Lanzhou 730046, China

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摘要：

目的:构建库容量大、多样性好的核糖体展示口蹄疫单链抗体(scFv)库。方法: 分离口蹄疫病毒免疫的兔脾细胞,提取总RNA,用RT-PCR扩增兔抗体的重链可变区(VH)基因和轻链可变区(VL)基因,同时扩增作为间隔区的兔抗体Ck基因;采用重叠延伸PCR (简称SOE-PCR)技术连接VH-VL基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接pMD18-T载体转化E.coli DH5α大肠杆菌,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达8.21×10¹³的兔源口蹄疫核糖体展示scFv库。结论: 构建的大容量兔源性口蹄疫核糖体展示抗体库可以成为进一步筛选特异性口蹄疫单链抗体的实验平台,为开发诊断性口蹄疫单链抗体奠定了很好的实验基础。

关键词： 核糖体展示技术; 重叠延伸PCR; 单链抗体库; 口蹄疫病毒

Abstract:

Objective: To construct a rabbit source high-capacity and good-diversity ribosome display single chain antibody (scFv) library of FMDV. Methods: The isolated spleen cells from rabbit immuned by the FMDV Virus,and extracted RNA for amplifying rabbit VH gene, VL gene and Ck gene as the spacer by RT-PCR,and then connect VH-VL gene by overlapping extension PCR (referred to as SOE-PCR). After that,the elements for ribosome display such as T7 promoter and ribosome binding site were introduced after SOE products were amplified. Finally, the ribosome display scFv library template was constructed in vitro. Moreover, the ribosome display templates were connected to pMD18-T vector into E.coli DH5α, and the positive clones were picked sequencing to identify the ScFv assembly. Results: We successfully constructed a rabbit source ribosome display scFv library whose capacity is 8.21 × 10¹³ of FMDV. Conclusion: The rabbit-derived high-capacity ribosome display antibody library can be a good platform for screening specific foot and mouth disease, and lay a good experimental basis for the measuring test of FMDV.

Key words: Ribosome display technology Overlapping extension PCR scFv library FMDV

收稿日期: 2011-01-26 出版日期: 2011-07-25

ZTFLH:

Q78

基金资助:

质检公益性行业科研专项(2007GYJ024,2007GYJ023)、国家质检总局科技计划项目(2010IK211)资助项目

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引用本文:

杨双, 马军武, 周广青, 林密, 冯霞, 刘涛, 代鹏. 核糖体展示口蹄疫单链抗体库的构建[J]. 中国生物工程杂志, 2011, 31(7): 98-103.

YANG Shuang, MA Jun-wu, ZHOU Guang-qing, LIN Mi, FENG Xia, LIU Tao, DAI Peng. Construction of Ribosome Display Single Chain Antibody Library of Foot and Mouth Disease. China Biotechnology, 2011, 31(7): 98-103.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I7/98

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