驼源天然单域重链抗体库的构建与鉴定

中国生物工程杂志

2011, Vol. 31

Issue (04): 31-36

研究报告

驼源天然单域重链抗体库的构建与鉴定

涂追^1,2, 许杨^1,2, 刘夏^1,2, 何庆华^1,2, 陶勇²

1. 南昌大学食品科学与技术国家重点实验室南昌 330047;
2. 南昌大学中德联合研究院南昌 330047

Construction and Biopanning of Camelid Naïve Single-domain Antibody Phage Display Library

TU Zhui^1,2, XU Yang^1,2, LIU Xia^1,2, HE Qing-hua^1,2, TAO Yong²

1. State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China;
2. Jiangxi-OAI Joint Research Institute, Nanchang University, Nanchang 330047, China

全文: PDF(1180 KB) HTML

摘要：

从未经主动免疫的健康羊驼(Lama pacos)外周血淋巴细胞中提取总RNA,反转录后作为第一轮PCR的模板。根据重链抗体保守区域设计引物,经巢式PCR法扩增获得了全套重链抗体可变区基因,将其克隆至噬菌粒pHEN1,电转化大肠杆菌TG1得到初级抗体库NAL,含有2×10⁷个独立克隆,菌落PCR和Hinf I酶切分析结果显示,克隆效率大于97%,文库的多样性良好。辅助噬菌体救援后,得到噬菌体展示文库命名为NA-PDL,滴度达10¹³CFU/ml。以真菌毒素人工抗原DON-MBSA为目标抗原,对NA-PDL进行了淘选,第二轮洗脱物中,阳性克隆率达36.4%,提示针对目标抗原的噬菌体颗粒得到了有效富集,文库NA-PDL多样性较好,为后续淘选针对特定抗原的单域重链抗体奠定了基础。

关键词： 羊驼; 纳米抗体; 单域重链抗体; 脱氧雪腐镰刀菌烯醇; 噬菌体文库

Abstract:

The objective is to construct a camelid nave single-domain heavy chain antibody phage display library. Total RNA was purified from 30ml blood of two healthy non-immune alpacas (Lama pacos) and directly used for complementary DNA (cDNA) synthesis. Three sets of primers were designed based on the conserved region of heavy-chain antibody. The repertoire of VHH coding sequence was amplified by nested PCR, and the PCR products were cloned into a phagemid vector pHEN1. By electroporation of E.coli TG1, the primary library (designate NAL) was obtained containing more than 10⁷ independence clones. After helper phage rescue, the phage display library (designate SNA-PDL) was generated with a titre up to 10¹³ CFU/ml. The library exhibited high diversity as judged by the Hinf I restriction pattern. Solid phage biopanning against artificial antigen DON-MBSA showed significant enrichment of binding phage particles. The positive rate of panning round two was 36.4%. The data indicated that a nave single-domain antibody phage display library was constructed, which has good diversity and would be useful for generating VHHs with specific binding affinity.

Key words: Alpaca(Lama pacos) Nanobodies VHH Deoxynivalenol Phage display library

收稿日期: 2010-11-22 出版日期: 2011-04-26

ZTFLH:

Q789

基金资助:

食品科学与技术国家重点实验室目标导向资助项目(SKLF-MB-201002)、国家中小企业创新基金(09C26213604449)、江西省教育厅青年科学基金(GJJ09442)资助项目

通讯作者: 许杨 E-mail: xuyang1951@yahoo.com.cn

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引用本文:

涂追, 许杨, 刘夏, 何庆华, 陶勇. 驼源天然单域重链抗体库的构建与鉴定[J]. 中国生物工程杂志, 2011, 31(04): 31-36.

TU Zhui, XU Yang, LIU Xia, HE Qing-hua, TAO Yong. Construction and Biopanning of Camelid Naïve Single-domain Antibody Phage Display Library. China Biotechnology, 2011, 31(04): 31-36.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I04/31

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