重组HEV衣壳蛋白截短体的表达及鉴定

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus

戊型肝炎(HE)是由戊型肝炎病毒(HEV)感染引起的肠道病毒性传染病.HEV是一种无囊膜的单股正链RNA病毒,其编码区由3个开放阅读框(ORF)组成,属戊型肝炎病毒科.HEV衣壳蛋白由ORF2编码.本研究根据编码HEV ORF2 aa382～aa674的核苷酸序列克隆了p293基因,并将其克隆入原核表达载体pET28a,利用大肠杆菌BL21(DE3)对HEV衣壳蛋白截短体(p293)进行了表达.SDS-PAGE和Western blot检测结果表明,在优化的表达条件下(1 mmol/L IPTG,250 r/min,37℃,5 h),重组蛋白p293能够在大肠杆菌内有效表达,目的蛋白约占总蛋白的66.15%.TEM检测结果显示,原核表达的p293能够在体外形成约30～40 nm的病毒样颗粒.免疫印迹和免疫荧光检测结果表明,p293与HEV标准阳性血清具有良好的反应原性和反应特异性.实验结果表明,p293可应用于HEV宿主吸附和病毒装配研究,为HEV的预防与诊断研究奠定了基础.

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus（HEV）. HEV is a nonenveloped virus classified in the family of Caliciviridae. The virus appears to be a polyadenylated, positive-stranded RNA virus with three major open reading frames（ORFs）. The capsid protein of HEV is encoded by the open reading frame 2（ORF2）. We attempted to produce a truncated capsid protein, designed p293, in E. coli. The p293 gene encoding amino acids aa382-aa674 of HEV ORF2 was designed based on the full length of HEV ORF2, cloned into the vector pET28a, and expressed in E. coli strain BL21（DE3）. SDS-PAGE and Western blot analysis demonstrated that the recombinant protein p293 could well expressed in E. coli. Under optimized conditions（1 mmol/L IPTG, 250 r/min, 37℃, 5 h）, the yield of soluble p293 was approximately 66.15% of the total protein. It was observed, by transmission electron microscopy, that p293 expressed in E. coli formed 30-40 nm viral-like particles. The experimental results also showed that p293 reacted with HEV positive serum and had a good reactionogenicity. These results showed that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV, and serve as useful antigen for both diagnostic and vaccine purposes.