重组芋螺毒素GeXIVAWT的表达、纯化和鉴定

中国生物工程杂志

2012, Vol. 32

Issue (09): 34-40

研究报告

重组芋螺毒素GeXIVAWT的表达、纯化和鉴定

高炳淼, 李宝珠, 吴勇, 林波, 朱晓鹏, 长孙东亭, 罗素兰

海南大学热带生物资源教育部重点实验室海口市海洋药物重点实验室海口 570228

Expression, Purification and Identfication of Recombinant Conotoxin GeXIVAWT

GAO Bing-miao, LI Bao-zhu, WU Yong, LIN Bo, ZHU Xiao-peng, ZHANGSUN Dong-ting, LUO Su-lan

Key Lab for Tropical Biological Resources, Ministry of Education; Key Lab for Marine Drugs of Haikou, Haikou 570228, China

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摘要： 芋螺毒素GeXIVAWT是来自食虫将军芋螺(Conus generalis Lonnaeus)毒管中,具有两对二硫键的28肽。通过化学合成这种芋螺毒素产量低、成本高、难以纯化。利用简单快速的重组表达来生产富含二硫键的芋螺毒素有可能成为有效的新途径。通过对芋螺毒素GeXIVAWT的基因进行密码子优化,人工合成引物来构建表达载体pET22b(+)/pelB-GeXIVAWT。融合了信号肽的重组芋螺毒素以包涵体的形式在大肠杆菌中获得了高效表达。利用低浓度尿素洗涤和超滤管进行纯化无组氨酸标签的重组芋螺毒素pelB-GeXIVAWT,再利用稀释复性的方法将无活性的包涵体转变成具有活性的重组芋螺毒素。复性后的重组蛋白采用反相高效液相色谱进一步纯化后进行了质谱鉴定。活性实验表明重组芋螺毒素pelB-GeXIVAWT具有抑制昆虫细胞Sf-9的生长,为研究芋螺毒素作为生物杀虫剂奠定基础。

关键词： 芋螺毒素; 大肠杆菌; 重组表达; 分离纯化; 生物杀虫剂

Abstract: Conotoxin GeXIVAWT is a 28-amino acid peptide containing two pairs of disulfide bond isolated from the venom of worm-hunting Conus generalis Lonnaeus. Usually, the conotoxins produced by means of chemical synthesis was low yield, and it was difficult to be purified. A simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression was presented. The codons of novel conotoxin GeXIVAWT gene were optimized. Two pairs of primers were generated by chemical synthesis for construction of expression vectors. Recombinant expression vector pET22b(+)/pelB-GeXIVAWT fused with pelB signal peptide was successfully expressed in Escherichia coli. Recombinant pelB-GeXIVAWT without His-tag was purified by low concentrations of urea and ultrafiltration tube. Inactive inclusion bodies were transformed into active recombinant conotoxins by using dilution refolding method. It was further purified using HPLC and identified by mass spectrometry. Renaturing in vitro and biological activity experiment showed that the pelB-GeXIVAWT could inhibited the growth of Spodoptera frugiperda 9 (Sf-9) cell, as a foundation for the study of efficient insecticides.

Key words: Conotoxin Escherichia coli Recombinant expression Purification Biological insecticide

收稿日期: 2012-05-05 出版日期: 2012-09-25

ZTFLH:

Q786

基金资助: 国家国际科技合作专项 (2011DFR31210);国家"863"计划(2012AA021706);长江学者和创新团队发展计划(IRT1123);海南省教育厅高校科学研究(Hjkj2010-02)资助项目

通讯作者: 罗素兰 E-mail: luosulan2003@163.com

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引用本文:

高炳淼, 李宝珠, 吴勇, 林波, 朱晓鹏, 长孙东亭, 罗素兰. 重组芋螺毒素GeXIVAWT的表达、纯化和鉴定[J]. 中国生物工程杂志, 2012, 32(09): 34-40.

GAO Bing-miao, LI Bao-zhu, WU Yong, LIN Bo, ZHU Xiao-peng, ZHANGSUN Dong-ting, LUO Su-lan. Expression, Purification and Identfication of Recombinant Conotoxin GeXIVAWT. China Biotechnology, 2012, 32(09): 34-40.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I09/34

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