SARS冠状病毒S2蛋白胞外段的原核表达及其细胞膜融合效应研究  

Prokaryotic expression of S2 extracellular domain of SARS coronavirus spike protein and its fwsion with Hela cell membrane

作　　者：刘芸[1] 刘爱华[2] 邓鹏[1] 吴向玲[1] 李涛[1] 刘亚伟[1] 徐佳[1] 姜勇[1] 

机构地区：[1]南方医科大学病理生理学教研室,广东省功能蛋白质组学重点实验室,广东广州510515 [2]南方医科大学南方医院呼吸科,广东广州510515

出　　处：《南方医科大学学报》2009年第3期381-386,共6页Journal of Southern Medical University

基　　金：国家自然科学基金(30572151);广东省自然科学基金(05004730);广州市科技计划(2007J1-C0301;2002Z3-C7081)

摘　　要：目的构建SARS冠状病毒S2蛋白胞外段(S2ED)和增强型绿色荧光蛋白(EGFP)的融合蛋白表达质粒,获得在原核细胞中表达的目的蛋白。方法在生物信息学预测基础上,利用PCR方法扩增S2ED和EGFP的编码序列,插入到质粒pET-14b中构建融合表达载体,在大肠杆菌中诱导表达融合蛋白His-S2ED-EGFP。采用Ni-NTA亲和树脂纯化融合蛋白可溶部分,利用SDS-PAGE和免疫印迹的方法鉴定纯化后的蛋白,通过荧光显微镜考察S2ED与Hela细胞胞膜能否发生融合效应。结果重组质粒pET-14b-S2ED-EGFP的构建正确无误,并能够在BL21(DE3)中高效表达。纯化后的蛋白与Hela细胞孵育后,未观察到蛋白通过膜融合的过程内化进入细胞。结论S2ED的绿色荧光蛋白融合表达载体构建成功,并在原核细胞中获得了部分可溶的表达和有效的纯化。虽然原核表达的重组蛋白未能在Hela细胞上发挥预期的生物学效应,但是该工作将为加深S2蛋白介导的胞膜融合过程的认识以及未来进行以特异性细胞结合肽为基础的定向靶细胞转运系统研究奠定重要的基础。Objective To construct the expression plasmid of S2 extracellular domain （S2ED） of SARS-coronavirus （SARS- Cov） spike protein （S protein） and enhanced green fluorescent protein （EGFP） to obtain the fusion protein expressed in prokaryotie cells. Methods S2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fi＇actions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion Of S2ED with Hela cell membranes was observed with fluorescent microscope. Results The pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 （DE3）. When incubated with Hela cells, the purified protein could not internalize through membrane fusion. Conclusions The expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.

关 键 词：SARS冠状病毒 S2蛋白 胞外段 蛋白表达 细胞膜融合 

分 类 号：R373[医药卫生—病原生物学]