A组轮状病毒疫苗株LLR VP8＊基因的原核表达及纯化

Expression and purification of the VP8 ＊ protein of group A rotavirus vaccine strain LLR

目的 表达A组轮状病毒疫苗株LLR的VP8＊蛋白,为深入研究LLR的功能特性奠定基础.方法 从兰州生物制品所生产的A组轮状病毒疫苗中提取病毒RNA为模板,经过RT-PCR反应获得轮状病毒LLR株VP8＊基因的cDNA片段,将其克隆到pGEX-4T1表达载体中构建重组质粒pGEX-4T1-VP8＊,测序正确后转化大肠埃希菌BL21（DE3）,通过SDS-PAGE电泳、免疫印迹方法检测IPTG诱导表达后的产物,用Glutathione Sepharose 4B纯化目的蛋白.结果 SDS-PAGE电泳可见大小约为52 000的融合蛋白,Western-Blot显示该蛋白能与GST标签抗体结合.结论 成功构建了包含LLR株VP8＊序列的重组表达载体pGEX-4T1-VP8＊,获得了可溶性GST-VP8＊重组蛋白.

Objective To express the VP8 ＊ protein of the rotavirus vaccine strain LLR in the E.coli with the pGEX4T-1 vector,which is important for the further research of the VP8 ＊ protein functions.Methods The LLR VP8 ＊ gene was obtained by virus RNA extraction and RT-PCR.Then it was cloned in the expression vectors pGEX4T-1.The recombinant plasmid pGEX4T-1-VP8 ＊ was transformed to the E.coli BL21.Then the VP8 ＊-GST fusion protein was expressed and purified by the affinity chromatograph.The protein of interest was validated by SDS-PAGE and Western Blot.Results The molecular weight of the VP8 ＊-GST fusion protein was about 52 000 according to the SDS-PAGE.The bands of both 52 000 and 26 000 were shown in the Western Blot with the antibody against GST.Conclusions The LLR VP8 ＊ gene was obtained and cloned to the pGEX4T-1 vector.Moreover,the solvable VP8 ＊-GST fusion protein was successfully expressed and purified.