抗艰难梭菌毒素纳米抗体在乳酸乳球菌中重组表达及其功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2018-0287

生物技术通报 ›› 2018, Vol. 34 ›› Issue (10): 87-91.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0287

抗艰难梭菌毒素纳米抗体在乳酸乳球菌中重组表达及其功能分析

武国松, 李杉

华南理工大学生物科学与工程学院,广州 510006

收稿日期:2018-03-28 出版日期:2018-10-26 发布日期:2018-11-07
作者简介:武国松,男,硕士,研究方向：生物化学与分子生物学;E-mail：916019624@qq.com
基金资助:
广东省省级科技项目-国际科技合作领域（2015A050502016）

Recombinant Expression and Functional Analysis of Nano-antibody in Lactococcus lactis Against Clostridium difficile Toxin

WU Guo-song, LI Shan

School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006

Received:2018-03-28 Published:2018-10-26 Online:2018-11-07

摘要/Abstract

摘要： 本研究旨在构建表达抗艰难梭菌毒素的纳米抗体重组乳酸乳球菌。所述的抗体为免疫羊驼后从外周血中分离的天然缺失轻链的纳米抗体（分别命名为E3、AA6）。构建重组质粒pNZ8148- E3-AA6,转化乳酸乳球菌NZ9000后诱导表达,SDS-PAGE及Western blot检测到32 kD的目的蛋白。利用镍柱亲和层析法纯化重组蛋白E3-AA6,BCA试剂盒测定其浓度。通过细胞变圆实验以及CCK8实验鉴定其功能,E3-AA6能够抑制艰难梭菌毒素TcdB的毒性。本实验成功构建了重组表达载体pNZ8148- E3-AA6,通过NICE系统实现了二价抗体的表达,为治疗艰难梭菌引发的感染提供了新的治疗途径。

关键词: 乳酸乳球菌, 纳米抗体, 艰难梭菌感染, NICE系统

Abstract: This study is to construct recombinant Lactococcus lactis expressing nanobody against Clostridium difficile toxin. The nano-antibody is a kind of natural antibody（named E3 and AA6 respectively）which is deficient in light chain and isolated from peripheral blood after immunizing alpaca. The recombinant plasmid pNZ8148-E3-AA6 was constructed and transformed into L. lactis NZ9000 to induce expression. About 32 kD target protein was detected by SDS-PAGE and Western blot. The recombinant protein E3-AA6 was purified by nickel-column affinity chromatography and its concentration was measured by BCA protein assay kit. The cell rounding experiment and CCK8 experiment were used to identify its function,E3-AA6 effectively inhibited the C. difficile toxin TcdB. In conclusion,the pNZ8148-E3-AA6 is successfully constructed and bivalent antibodies E3-AA6 is efficiently expressed in the NICE system,this provides a new therapeutic approach for the treatment of infections by C. difficile.

Key words: Lactococcus lactis, nano antibody, Clostridium difficile, NICE expression

武国松, 李杉. 抗艰难梭菌毒素纳米抗体在乳酸乳球菌中重组表达及其功能分析[J]. 生物技术通报, 2018, 34(10): 87-91.

WU Guo-song, LI Shan. Recombinant Expression and Functional Analysis of Nano-antibody in Lactococcus lactis Against Clostridium difficile Toxin[J]. Biotechnology Bulletin, 2018, 34(10): 87-91.

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抗艰难梭菌毒素纳米抗体在乳酸乳球菌中重组表达及其功能分析

Recombinant Expression and Functional Analysis of Nano-antibody in Lactococcus lactis Against Clostridium difficile Toxin

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