辛德毕斯病毒E2包膜蛋白可溶性表达条件的优化

中国生物工程杂志

2011, Vol. 31

Issue (02): 62-68

技术与方法

辛德毕斯病毒E2包膜蛋白可溶性表达条件的优化

李江姣, 朱武洋, 何英, 梁国栋

中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室北京 100052

Optimizing the Soluble Expression Conditions of Sindbis Virus E2 Envelope Protein

LI Jiang-jiao, ZHU Wu-yang, HE Ying, LIANG Guo-dong

State Key Laboratory for Infectious Disease Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China

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摘要：

目的:通过优化表达条件,提高辛德毕斯病毒E2包膜蛋白胞外区的可溶性表达量。方法:构建含E2基因胞外区的重组表达质粒pGEX-6p-1-E2,筛选合适宿主菌和诱导温度,并构建5种分子伴侣共表达系统(即pG-KJE8、 pGro7、pKJE7、 pG-Tf2和pTf16 5种分子伴侣质粒分别与重组表达质粒pGEX-6p-1-E2共表达),筛选最适分子伴侣质粒。结果:(1)E2蛋白的表达量在E.coli BL21、BL21 (DE3) pLysS、Rosetta (DE3)及Origami B (DE3) 4种表达菌中没有明显差别;(2)16℃诱导时E2蛋白在上清中可溶性表达量最高;(3)分子伴侣质粒pG-Tf2使目的蛋白的可溶性表达量提高了15.7%,作用最为显著。结论:通过优化表达条件及使用分子伴侣共表达系统提高了E2蛋白的可溶性表达,为进一步E2蛋白的相关研究奠定了基础。

关键词： 辛德毕斯病毒; E2包膜蛋白; 分子伴侣; 可溶性表达

Abstract:

To improve the soluble expression level of extracellular domain of E2 envelope protein of Sindbis Virus (SINV) in prokaryotic expression system, the expression conditions were optimized. The extracellular domain of E2 gene was cloned from cDNA of SINV and sub cloned into expression vector pGEX-6P-1. The recombinant plasmid pGEX-6p-1-E2 was constructed and transformed into four host bacteria respectively. The optimum host bacteria and induction temperature were selected by SDS-PAGE assay. The molecular chaperone co-expression system was constructed and the optimum molecular chaperone vector was screened. The results were described as follows: the E2 gene was amplified and cloned into vector pGEX-6p-1 successfully; the quantity of E2 expression was similar in four host bacteria; the soluble expression level of E2 was highest when induced at 16℃; in the molecular chaperone co-expression system, the five vectors containing different molecular chaperones (pG-KJE8、 pGro7、pKJE7、 pG-Tf2 and pTf16) improved the soluble expression level of E2 in varying degrees when co-expression with recombinant plasmid pGEX-6p-1-E2, and pG-Tf2 was confirmed to contain the optimum molecular chaperones combination and made the soluble expression level of E2 improved 15.7%. These results can provide a potentia1 value for further structural and functional studies of E2.

Key words: Sindbis virus E2 envelope protein Molecular chaperone Soluble expression

收稿日期: 2010-08-31 出版日期: 2011-02-18

ZTFLH:

Q786

基金资助:

国家自然科学基金(30170046;30970160)、"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(2008ZX10004-001;2009ZX10004-705)资助项目

通讯作者: 梁国栋 E-mail: gdliang@hotmail.com

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引用本文:

李江姣, 朱武洋, 何英, 梁国栋. 辛德毕斯病毒E2包膜蛋白可溶性表达条件的优化[J]. 中国生物工程杂志, 2011, 31(02): 62-68.

LI Jiang-jiao, ZHU Wu-yang, HE Ying, LIANG Guo-dong. Optimizing the Soluble Expression Conditions of Sindbis Virus E2 Envelope Protein. China Biotechnology, 2011, 31(02): 62-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I02/62

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