重组GST-HBx蛋白在表达过程中断裂状况的研究  

Stabilities and Broken Sites of Truncated GST-HBx Proteins During Prokaryotic Expression

作　　者：蔡雪飞[1] 龚旭阳[1] 杜茜[1] 胡接力[1] 张文露[1] 胡源[1] 黄爱龙[1] 汤华[1] 

机构地区：[1]重庆医科大学感染性疾病分子生物学教育部重点实验室,重庆市400016

出　　处：《医学分子生物学杂志》2011年第5期377-381,共5页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30771924）

摘　　要：目的 在对乙型肝炎病毒非结构蛋白HBx研究的过程中,发现体外表达HBx蛋白产物的稳定性存在差异.为了了解其中的原因,拟通过原核表达方式进行分析.方法 以包含全长HBV DNA的质粒pEco63为模板,通过PCR反应,扩增HBV X基因全长序列（1～154氨基酸）和截短序列（48～104氨基酸,48～146氨基酸,98～146氨基酸）,将这几种不同长度和位置的HBX DNA序列克隆到pGEX4T-2原核表达载体中,并通过下游引物设计中引入6×组氨酸标签序列.结果 最终成功构建N端融合GST蛋白标签序列和C端融合6×组氨酸标签序列的4种HBx重组表达载体.将4种包含不同长度HBx的重组质粒转化大肠埃希菌Rosetta,培养至菌液浓度A值大约0.6左右,加入终浓度0.1 mmol/L的IPTG进行诱导表达.提取全菌蛋白通过Western印迹对HBx各重组蛋白产物的N端和C端进行检测.结论 在分段表达的HBx重组蛋白中发生了多处局部的断裂,而断裂部位主要集中于GST蛋白上.而造成这一结果的原因可能与HBx蛋白功能区域暴露后与GST相互作用有关.Objective HBx is only non-structure protein of hepatitis B virus. This study investigated the mechanism of different stabilities of complete GST-HBx and truncated GST-HBx during prokaryotie expression which was previously reported. Methods To understand the reason, special primers were designed for amplification of HBV X complete and truncated DNA sequence fragments with pEco63 plasmid as template by PCR. 6 His tags included in antisense primer sequence were fused to C-terminal of GST-HBx recombinant plasmids and these fusion proteins were induced to express by IPTG in Rosetta E. coli strain. Results The results of Western blot using GST and 6 His tag monoclonal antibodies indicated that GST region broken contributed to instabilities of truncated GST-HBx proteins. Conclusion Truncated HBx protein C-terminal region interacting with GST specific region would lead to GST protein alternating global form itself, and result in its some internal protease sites exposed. This might cause GST protein being cleaved by endogenic proteases of bacteria.

关 键 词：HBX蛋白 原核表达 GST蛋白 

分 类 号：R373.2[医药卫生—病原生物学]