CAJ | 학술논문

[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33 Ku,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.
[목적]표체구제역병독결구단백VP1,병대기진행순화화상관활성적검측.[방법]이A형구제역병독결구단백VP1중조질립pGEM-VP1위모판,용특이성표체인물확증기편마구,경매절후여pGEX-4T-1、pPROExHTb등원핵표체재체상련,전화대장간균BL21(DE3)pLysS표체균주,경IPTG유도표체,이실현VP1단백적표체,SDS-PAGE감정융합단백적표체병대2충재체중조균진행초성렬해,취상청화침정전영,용금속오합친합층석법대His-VP1진행순화.[결과]SDS-PAGE전영분석현시pPRO-VP1유도후목적조대위33 Ku,여예기결과상부;목적단백순화후,재전영도편상현시교위청석적단일조대;His-VP1가여A형구제역병독돈서멸활양성혈청발생특이성적반응.[결론]표체급순화료구유고특이성적A형구제역병독결구단백VP1,위심입연구결구단백VP1재구제역병독치병궤제중적작용전정료기출.
[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 BamH Ⅰ and 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.