双峰驼源天然噬菌体纳米抗体展示库的构建及抗GDH纳米抗体筛选 <sup>*</sup>

doi:10.13523/j.cb.20181207

中国生物工程杂志

2018, Vol. 38

Issue (12): 49-56 DOI: 10.13523/j.cb.20181207

技术与方法

双峰驼源天然噬菌体纳米抗体展示库的构建及抗GDH纳米抗体筛选 ^*

方媛¹,徐广贤^1,^2,^**(

),王羡¹,王红霞¹,潘俊斐¹

1 宁夏医科大学临床医学院银川 750004
2 宁夏医科大学总医院银川 750004

Construction of Camelid Natural Nanobody Phage Display Library and Screening for Anti-GDH Nanobody

FANG Yuan¹,XU Guang-xian^1,^2,^**(

),WANG Xian¹,WANG Hong-xia¹,PAN Jun-fei¹

1 Clinical Medical Colleges,Ningxia Medical University,Yinchuan 750004,China
2 General Hospital of Ningxia Medical University,Yinchuan 750004,China

全文: PDF(1253 KB) HTML

摘要：

目的构建噬菌体天然纳米抗体展示库,以期用于筛选不同抗原分子的纳米抗体筛选平台,并用艰难梭菌谷氨酸脱氢酶(GDH)抗原筛选靶向GDH的纳米抗体,对所构建的噬菌体天然纳米抗体展示库进行验证。方法采用Oligo DT提取双峰骆驼脾脏总RNA进行反转录,通过巢氏PCR获取全套重链可变区基因,将其构建到噬菌粒pCANTAB5E载体,经多次电转化至E.coil TG1构建初级噬菌体抗体库,经辅助噬菌体拯救后构成噬菌体展示库,并对噬菌体展示库的库容及多样性进行分析和鉴定。同时以GDH为靶向抗原对文库进行淘筛,计算淘筛回收率,并对第三轮淘筛后平板的单克隆进行ELISA鉴定。结果构建的天然噬菌体纳米抗体库的插入率为95%左右,随机挑取的9个克隆氨基酸同源性为66.17%,经MEGA分析后具有较好的多样性,同时经辅助噬菌体拯救后,得到的噬菌体展示库滴度为4×10 ¹²CFU/ml。在三轮淘筛过程中,回收率逐步升高,噬菌体得到了有效的富集,同时对阳性克隆进行测序及分析,最终得到2条抗GDH纳米抗体序列。结论成功构建了双峰驼源天然噬菌体纳米抗体展示文库且多样性良好,为后续筛选其他的靶向抗原奠定了基础,同时筛选获得两条抗GDH纳米抗体序列,为制备艰难梭菌谷氨酸脱氢酶诊断抗体提供技术支撑。

关键词： 纳米抗体; 噬菌体展示技术; 艰难梭菌谷氨酸脱氢酶

Abstract:

Objective To construct a natural nanobody phage display library as a nanobody platform for screening different antigens.Using GDH antigen to screen and obtain single domain antibody variable region gene (VHH) of the camel targeting GDH.Methods: Isolate the total RNA from the camel by using Oligo DT and synthesize the cDNA.The genes of variable domain of heavy chain(VHH) were amplified by nested PCR,and then were ligated with the vector pCANTAB5E.Next the recombinant vector cloned into TG1 to construct the phage display library ,and make the analysis and identification for the library.Using GDH to screening anti-GDH nanobodies,and identified the monoclonal by phage-ELISA.Result: The positive rate of the phage display library is 95%,the amino acid homology of nine randomly colonies is 66.17%,and it has a good variety after MEGA analyzing.After helper phage rescue,the titer of the phage display library is 4×10 ¹²CFU/ml.And after three times round of screening,it showed significant enrichment of binding phage.In the end,two nanobodies sequence were obtained by sequencing positive clonoies. Conclusion: Successfully constructed a natural camelid nanobody phage display library,and the good diversity laid a foundation for selecting other nanobodies.Two GDH specific sequences derived from camel are obtained through phage display library,which can be used to prepare the diagnostic antibodies.

Key words: Nanobodies Phage display technology GDH

收稿日期: 2018-07-30 出版日期: 2019-01-10

ZTFLH:

Q511

基金资助: * 宁夏回族自治区重点研发计划重大(重点)项目(2018BEG02002);* 宁夏科技创新领军人才培养项目(KJT2015020)

通讯作者: 徐广贤 E-mail: 599040064@qq.com

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引用本文:

方媛,徐广贤,王羡,王红霞,潘俊斐. 双峰驼源天然噬菌体纳米抗体展示库的构建及抗GDH纳米抗体筛选 ^*[J]. 中国生物工程杂志, 2018, 38(12): 49-56.

FANG Yuan,XU Guang-xian,WANG Xian,WANG Hong-xia,PAN Jun-fei. Construction of Camelid Natural Nanobody Phage Display Library and Screening for Anti-GDH Nanobody. China Biotechnology, 2018, 38(12): 49-56.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20181207 或 https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I12/49

表1 巢氏PCR反应引物设计

图1 第一轮PCR琼脂糖凝胶电泳图

图2 第二轮PCR琼脂糖凝胶电泳图

图3 倍比稀释法测定救援后的噬菌体展示文库滴度

图4 菌落PCR鉴定噬菌体抗体展示库插入率

图5 MEGA系统进化树分析

图6 VHH抗体库中9个随机克隆氨基酸多序列分析

表2 三轮筛选后噬菌体的富集度变化

图7 Phage-ELISA 检测噬菌体多克隆上清

图8 噬菌体单克隆ELISA鉴定及A450较高样品孔

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