一种核糖体展示单链抗体文库的构建与鉴定

中国生物工程杂志

2009, Vol. 29

Issue (04): 51-55

研究报告

一种核糖体展示单链抗体文库的构建与鉴定

年四季²,袁青³,殷幼平^1,王中康¹

1. 重庆大学生物工程学院基因工程研究中心2. 四川省泸州医学院病原生物教研室3. 四川省泸州医学院免疫学教研室

Construction and identification of single chain Fv fragment library used for ribosome display

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摘要： 用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠，提取小鼠脾细胞mRNA，RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL），采用linker (Gly4Ser)3连接VH和VL，构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109，随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示，用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右，扩增的VH大小为350bp左右，VL的大小为650bp左右，linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示，单链抗体文库多样性好。以Xac为靶，筛选到了抗Xac的特异性抗体，说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。

关键词： 单链抗体;抗体文库;柑桔溃疡病菌;核糖体展示

Abstract: The mice were immunized with Xanthomonas axonopodis pv. citri (Xac) and the mRNA of spleen cells from immunized mice were extracted. The variable fragment of heavy chain (VH) and light chain (VL) were amplified by RT-PCR and the linker (Gly4Ser)3 were used to ligate VH and VL. Then the single chain Fv fragment (scFv) library was constructed which would be used to screen positive scFvs by ribosome display. To analyze the diversity of the developed scFv library, the DNA of library was translated to Escherichia coli JM109, and the positive clones were selected by random and then sent to sequence. The results showed that the serum titer of immunized mice was about 2500×. The size of amplified VH were 350 bp and VL was 650 bp, and that of constructed scFv library DNA was 1200 bp. The scFv library were diversity and specific scFvs against Xac were selected from the constructed scFv library. It showed that the scFv library could be used to select positive scFvs against Xac by ribosome displaly.

收稿日期: 2008-11-11 出版日期: 2009-04-27

基金资助:

省部基金

通讯作者: 王中康

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引用本文:

年四季,袁青,殷幼平,王中康. 一种核糖体展示单链抗体文库的构建与鉴定[J]. 中国生物工程杂志, 2009, 29(04): 51-55.

NIAN Si-Ji- Yuan-Jing- Yan-Yao-Beng- Wang-Zhong-Kang. Construction and identification of single chain Fv fragment library used for ribosome display. China Biotechnology, 2009, 29(04): 51-55.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I04/51

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