轮状病毒外壳蛋白VP4在大肠埃希菌中的表达

Expression of coat protein VP4 of rotavirus SA11 in E.coli

目的克隆表达轮状病毒融合素蛋白VP4。方法以轮状病毒SA11株细胞培养物提取总RNA为模板,经RT-PCR获得VP4全长基因片段cDNA,将其重组于pET-30a(+)表达载体,转化大肠埃希菌BL21(plysS),异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经SDS-PAGE和Western blot鉴定,并用镍柱纯化。结果重组载体所含目的基因片段的序列与文献公布序列相符,IPTG诱导表达的目的蛋白主要以包涵体形式存在;SDS-PAGE检测表达产物与目的蛋白大小一致,为88kD,Western blot检测发现目的蛋白所带组氨酸标签可与标签抗体反应;镍柱纯化获得RV SA11 VP4融合素蛋白,纯度达90%。结论构建了表达载体pET-30a(+)-VP4,经诱导表达、纯化,获得了RV融合素蛋白VP4。

Objective To express VP4 protein of rotavirus in prokaryotic expression system.Methods Total RNA were extracted from MA104 cells infected with rotavirus.Total length cDNA of VP4(2331 bp)was amplified by RT-PCR.After sequencing,it was cloned into expression vector pET-30a(+).VP4 protein was expressed in E.coli BL21(plysS)induced by IPTG and was analyzed by SDS-PAGE and Western blot.VP4 protein was purified by Ni-NTA.Results The whole VP4 gene was successfully cloned and was expressed abundantly mainly in the form of incusion bodies.Molecular weight of expressed products is 88 kD analyzed by SDS-PAGE.By consequence analysis of Western blot,we found VP4 protein with a six-His tag can bind with anti-His antibody.By Ni-NTA purification and dialysis,we acquired VP4 protein with more than 90% purity.Conclusions The rotavirus SA11 VP4 protein was successfully expressed by prokaryotic expression system.