A型口蹄疫病毒结构蛋白VP1的表达·纯化及活性检测

Expression,Purification and Activity Detection of VP1 of A-type FMDV

[目的]表达口蹄疫病毒结构蛋白VP1,并对其进行纯化和相关活性的检测。[方法]以A型口蹄疫病毒结构蛋白VP1重组质粒pGEM-VP1为模板,用特异性表达引物扩增其编码区,经酶切后与pGEX-4T-1、pPROExHTb等原核表达载体相连,转化大肠杆菌BL21(DE3)pLysS表达菌株,经IPTG诱导表达,以实现VP1蛋白的表达,SDS-PAGE鉴定融合蛋白的表达并对2种载体重组菌进行超声裂解,取上清和沉淀电泳,用金属螯合亲合层析法对His-VP1进行纯化。[结果]SDS-PAGE电泳分析显示pPRO-VP1诱导后目的条带为33ku,与预期结果相符;目的蛋白纯化后,在电泳图片上显示较为清晰的单一条带;His-VP1可与A型口蹄疫病毒豚鼠灭活阳性血清发生特异性的反应。[结论]表达及纯化了具有高特异性的A型口蹄疫病毒结构蛋白VP1,为深入研究结构蛋白VP1在口蹄疫病毒致病机制中的作用奠定了基础。

[Objective]The research aimed to induce the expression of FMDV structural protein VP1 in E.coli and purify the protein,then detect the activity.[Method]The fragment coding VP1 was amplified by PCR and doubly digested with BamHⅠand XhoⅠ,then cloned into expression vector pGEX-4T-1 and pPROExHTb respectively to get recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1.The recombinant plasmid pGEX-4T-1-VP1 and pPROExHTb-VP1 was transformed into E.coli BL21（DE3） and induced by IPTG,fusion protein was identified by SDS-PAGE.After fusion protein was purified,it was used for detecting the activity and specificity by ELISA and western blot.[Result]SDS-PAGE demonstrated that the fusion protein GST-VP1 was expressed with the expected molecular weight of His-VP1 of 33 Ku.After purification,a single clear band of GST-VP1 fusion protein appeared in SDS-PAGE gel,the same to His-VP1 fusion protein.His-VP1 fusion protein reacted with inactivated serum against guinea pigs infected with FMDV type A specifically.[Conclusion]His-VP1 fusion protein with high affinity and specificity was prepared successfully,which would lay a foundation for further understanding of the roles of structural protein VP1 in pathogenic mechanism in FMDV infection.