人源天然ScFv噬菌体抗体库的构建及鉴定

中国生物工程杂志

2010, Vol. 30

Issue (05): 18-22

研究报告

人源天然ScFv噬菌体抗体库的构建及鉴定

毛晓燕^*,乔玉玲,卢卫嘉,马瑞,赵红

兰州生物制品研究所兰州 730046

Construct and Characterization of Human Naive ScFv Phage Display Library

MAO Xiao-yan,QIAO Yu-ling,LU Wei-jia,MA Rui,ZHAO Hong

Lanzhou Institute of Biological Products, Lanzhou 730046, China

全文: PDF(570 KB) HTML

摘要：

噬菌体抗体库技术是获得治疗性抗体的一条重要途径。以20份健康人外周血为样本，通过提取淋巴细胞、逆转录－PCR（RTPCR）、抗体可变区基因的扩增、重叠PCR获得单链抗体（ScFv）基因，将ScFv克隆入噬粒载体，通过近300次的电转化获得了库容量为1.3×109的全人源天然ScFv噬菌体抗体库。通过随机挑克隆测序和用5种不同抗原筛选对抗体库进行了初步验证。随机测序表明抗体库具有较好的多样性，用5种不同抗原对其进行筛选，均获得了特异性噬菌体抗体的不同富集，表明成功构建了一个多样性良好的人源天然ScFv噬菌体抗体库。

关键词： 噬菌体展示技术; 抗体库; 单链抗体（ScFv）

Abstract:

Phage display antibody library is an important technology for generating therapeutic antibody.It was reported that the construction and characterization of a large human antibody phage display library. Total RNA was extracted from peripheral blood lymphocytes of 20 healthy donors. VH and VL gene were amplified by RT-PCR. The ScFv (single chain variable fragment) gene were assembled by overlap PCR, and cloned into a phagemid vector by electroporation of E.coli TG1. A large naive human ScFv phage antibody library containing 1.3×109 antibody members was constructed by repetitive electroporation about 300 times. The quality of the library was examined by sequence analysis and selection against 5 different protein antigens. Sequence analysis of the randomly picked 40 clones showed this library had good diversity. Panning of the 5 different antigens all resulted in successful isolation of antigen specific ScFv antibodies. The results indicate that a large nave human ScFv phage antibody library with good diversity was constructed successfully.

Key words: phage display antibody library single chain antibodies (ScFv)

收稿日期: 2009-12-08 出版日期: 2010-05-25

通讯作者: 毛晓燕 E-mail: maoxiaoyan2002@163.com

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引用本文:

毛晓燕乔玉玲卢卫嘉马瑞赵红. 人源天然ScFv噬菌体抗体库的构建及鉴定[J]. 中国生物工程杂志, 2010, 30(05): 18-22.

MAO Xiao-Yan, JIAO Yu-Ling, LEI Wei-Jia, MA Rui, DIAO Gong. Construct and Characterization of Human Naive ScFv Phage Display Library. China Biotechnology, 2010, 30(05): 18-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2010/V30/I05/18

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