摘要： 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5 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.
关键词:
口蹄疫病毒,
VP1基因,
原核表达,
蛋白复性
Abstract: The objective of this research is to get the FMDV VP1 protein which be of antigenancy activity.The recombinant expression vector pET2VP1 was constructed by cloning VP1 gene of FMDV into the prokaryotic expression plasmid pET32a from PMD18T2VP1.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.SDS2PAGE and Western2blot 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.
Key words:
FMDV,
VP1 gene,
Prokaryotic expression,
Protein renaturation
中图分类号:
杨苏珍, 张改平, 鲍登克, 乔松林, 万博, 樊剑鸣, 职爱民, 李学伍. O型口蹄疫病毒VP1蛋白的原核表达及功能鉴定[J]. 华北农学报, 2009, 24(6): 11-14. doi: 10.7668/hbnxb.2009.06.003.
YANG Su-zhen, ZHANG Gai-ping, BAO Deng-ke, QIAO Song-lin, WAN Bo, FAN Jian-ming, ZHI Ai-min, LI Xue-wu. Prokaryotic Expression and Activity Analysis of VP1 Gene of Foot-and-Mouth Disease Virus Serotype O[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2009, 24(6): 11-14. doi: 10.7668/hbnxb.2009.06.003.