戊型肝炎病毒ORF2重组蛋白的克隆表达及初步应用

Expression and diagnostic utility of recombinant ORF2 protein from hepatitis E virus

目的 研制戊型肝炎病毒重组抗原诊断试剂。方法 用融合蛋白表达载体 pGEX 4T 3 ,表达戊型肝炎病毒第二开放读码框架 (ORF2 )近 3′端 492bp片段。结果 经酶切、聚合酶链反应(PCR)及序列分析鉴定 ,证实重组表达质粒 ( pGEX 4T 3 HEV)构建成功。重组体菌株经异丙基 β D硫代半乳糖苷 (IPTG)诱导表达 ,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳 (SDS PAGE) ,在 46× 10 3处见一蛋白条带。该蛋白经Westernblot分析 ,能与 1∶10 0抗 HEV阳性血清起特异性反应 ;应用于ELISA诊断 ,与Genelabs公司重组抗原抗 HEVELISA试剂盒比较 ,一致率为 96 .9%。结论 该克隆表达产物可用于制备戊型肝炎重组抗原诊断试剂。

Objective To obtain recombinant antigen for developing anti-HEV ELISA kit. Methods The nucleotide of HEV ORF2 extending 492 bp was digested with BamH Ⅰ and EcoR Ⅰ and inser-ted into an expression vector pGEX-4T-3 digested with the same restriction endonucleases. The expression plasmid was named pGEX-4T-3-HEV and transformed into Escherichia coli TG-1. The recombinant strains induced with IPTG to express recombinant protein, which was confirmed by Western blot ana-lysis using serum of patient with hepatitis E. Results The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR), and sequencing, respectively. An about 46×10 3 additional protein band was demonstrated by SDS-PAGE. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera 1∶100 diluted. This protein was used as an immunodiagnostic target in ELISA to identify anti-HEV from 97 sera isolated from the patients with hepatitis E. The result showed that the consistent rate with Genelabs anti-HEV ELISA kit was 96.9%. Conclusions The recombinant protein is a candidate for developing anti-HEV ELISA kit.