CAJ | 학술논문

为获得具有免疫原性的FMDV VP1蛋白,以O型FMDV重组质粒PMD18T-VP1为模板,利用PCR技术扩增得到O型FMDV VP1基因片段,将此基因片段与原核表达载体pET32a连接,构建重组表达载体,命名为pET-VP1,经PCR和测序鉴定后,用IPTG诱导表达,收集诱导的菌液进行SDS-PAGE电泳和Western-Blot分析.结果显示,在分子量约为45 ku处有1条明显的蛋白条带,且能被口蹄疫阳性血清识别,表达产物通过包涵体纯化后用透析法复性;ELISA检测结果显示,所复性的蛋白具有较高的活性.结果表明,FMDV VP1蛋白在大肠杆菌中得到高效表达,为开发诊断制剂和疫苗的研制打下基础.
위획득구유면역원성적FMDV VP1단백,이O형FMDV중조질립PMD18T-VP1위모판,이용PCR기술확증득도O형FMDV VP1기인편단,장차기인편단여원핵표체재체pET32a련접,구건중조표체재체,명명위pET-VP1,경PCR화측서감정후,용IPTG유도표체,수집유도적균액진행SDS-PAGE전영화Western-Blot분석.결과현시,재분자량약위45 ku처유1조명현적단백조대,차능피구제역양성혈청식별,표체산물통과포함체순화후용투석법복성;ELISA검측결과현시,소복성적단백구유교고적활성.결과표명,FMDV VP1단백재대장간균중득도고효표체,위개발진단제제화역묘적연제타하기출.
The objective of this research is to get the FMDV VP1 protein which be of antigenancy activity.The recombinant expression vector pET-VP1 was constructed by cloning VP1 gene of FMDV into the prokaryotic expression plasmid pET32a from PMD18T-VP1.After the recombinant plasmid was verified by PCR and sequenced analysis,the result showed that VP1 gene was successfully cloned into expression plasmid pET32a,The protein corresponding to the VP1 gene was expressed after induction with IPTG.SDS-PAGE and Western-blot with the product of bacterial culture in different time showed that the molecular mass of the protein was approximately 45 ku,which was identified by positive sera of FMDV;The protein was refolded by dialysis method,and was assayed by ELISA,the result showed that it demonstrates high activity.So we can say that VP1 protein was expressed efficiently in E.coli,and the renaturation VP1 protein can be developed as diagnosis antigen or vaccine.