戊型肝炎病毒结构基因cDNA的克隆与表达

Cloning and expression of cDNAs from hepatitis E virus structural gene.

作者信息

Ruan Bing, Zumbika Edward, Wang Shu-Ying, Chen Yong, Ma Yi-Lin, Chen Ya-Gang, Liu Ke-Zhou

机构信息

First Affiliated Hospital, Medical College of Zhejiang University, Hangzhou 310003, China.

出版信息

Hepatobiliary Pancreat Dis Int. 2003 Aug;2(3):387-90.

PMID:14599945

Abstract

OBJECTIVE

To obtain recombinant antigen for development of anti-HEV ELISA kit and vaccine against hepatitis E virus infection.

METHODS

A 492 base cDNA was collected from 5-terminus of open reading frame 2 (ORF2) among epidemic hepatitis E virus (HEV) isolated from Xinjiang, Western China. The fragment was digested with BamH I and EcoR I, and inserted into vector pGEX-4T-3 which was also digested by the same enzyme. The recombinant plasmid was transformed into E.coli TG-1 and the fusion protein expressed was confirmed by Western blot analysis using serum of a patient with hepatitis E.

RESULTS

The recombinant plasmid was identified and confirmed with enzyme digestion, polymerase chain reaction (PCR) and sequencing, respectively. A protein band of about 46 kDa was demonstrated by SDS-PAGE and designated GST-pORF2. The result of Western blot analysis suggested that the fusion protein reacted with anti-HEV positive sera at a dilution of 1:100.

CONCLUSION

The recombinant protein GST-pORF2 may be useful in developing anti-HEV ELISA kit and vaccine against hepatitis E virus infection.

摘要

目的

获取重组抗原，用于开发抗戊型肝炎病毒（HEV）ELISA试剂盒及戊型肝炎病毒感染疫苗。

方法

从中国西部新疆分离的戊型肝炎流行病毒（HEV）开放阅读框2（ORF2）的5'端收集一段492个碱基的cDNA。该片段用BamH I和EcoR I进行酶切，并插入同样经这两种酶酶切的载体pGEX-4T-3中。将重组质粒转化到大肠杆菌TG-1中，并用戊型肝炎患者血清通过蛋白质免疫印迹分析来确认表达的融合蛋白。