一种高效、稳定的可溶性原核表达载体的构建及应用

以O型口蹄疫病毒结构蛋白VP1基因重组克隆载体pGEM-OVP1为模板,设计表达引物,经PCR扩增获得上游带有PelB信号肽编码序列的VP1 C端编码区,将其克隆至该表达载体pET-30a(+)中,构建了可溶性原核表达载体pET-30a-PelB-VP1C.将VP1全长编码区替换VP1C,获得重组原核表达载体pET-30a-PelB-VP1.将两种重组质粒分别转化大肠杆菌工程菌株BL21-(DE3)-plysS,经IPTG诱导表达,实现了VP1全长及其C端的可溶性表达,以金属离子螯合层析法分别对表达的VP1及其C端融合蛋白进行纯化,SDS-PAGE显示纯化的目的蛋白分别在32 ku和20 ku处有单一目的条带,具有较高的纯度.Western blot 分析,VP1蛋白及其C端均可与牛O型口蹄疫病毒阳性血清反应.对重组菌株的连续传代实验证实了该可溶性表达载体的遗传稳定性,显示了该可溶性原核表达载体在蛋白可溶性表达中的应用价值.

Construction and Application of an Efficient,Stable Soluble Prokaryotic Expression Vector

In this study,the recombinant cloning vector pGEM-OVP1 was used as template for PCR to get the coding region of VP1 C terminus,and the coding region of PelB signal peptide coding region was added to the upstream of VP1C,then the fragment was cloned to prokaryotic expression vector to get recombinant expression plasmid pET-30a-PelB-VP1C.The VP1C fragment was replaced by the VP1 coding fragment to get pET-30a-PelB-VP1 expression plasmid.The E.Coli BL21-(DE3)-plysS was transformed respectively and solublely expressed fused VP1 and VP1 C terminus.Purified VP1 and VP1 C terminus were obtained by Ni-NTA His Bind Resin affinity chromatography and a single clear band of 32 ku and 20 ku appeared respectively in the SDS-PAGE gel.Western blot analysis showed that purified VP1 and VP1 C terminus could react with bovine antiserum against FMDV of serotype O.The consecutive culture of recombinant strains showed pET-30a-PelB-VP1C and pET-30a-PelB-VP1 has excellent stability,which had practical value in soluble protein expression.