Hwang G Y, Wang J C, Wu C C
Department of Biology, Tunghai University, Taichung, Taiwan.
Virus Res. 1999 Feb;59(2):203-10. doi: 10.1016/s0168-1702(98)00136-1.
Plasmids containing PCR-amplified hepatitis B virus e antigen (HBeAg) genes (HBeAg-MV and HBeAg-SV) were constructed and expressed in E. coli strain DH5alpha. The induced intracellular glutathione S-transferase (GST) fusion proteins of HBeAg-MV and HBeAg-SV were recovered and purified from bacterial lysates by affinity chromatography with glutathione-sepharose beads. The HBeAg-MV protein contained an additional 19 amino acids at its amino terminus. These two proteins were specifically cleaved from GST by the protease factor Xa and recognized by a monoclonal antibody against HBeAg. HBeAg-MV and HBeAg-SV were found to be the two major components of the post-modified HBcAg during viral infection. The antigenic specificities of the fusion and purified HBeAgs (factor Xa-digested) were confirmed by the Abbott HBe enzyme immunoassay (EIA) detection system. Sera from patients with confirmed hepatocellular carcinoma (HCC) specifically reacted only with HBeAg moiety of fusion proteins. HCC sera bound more strongly to the HBeAg-SV protein than to the HBeAg-MV one. This indicates that HBeAg-SV is either more antigenic than -MV or is the major target protein for the elicitation of antibody production after HBV infection. Thus, the two recombinant HBeAgs expressed and obtained in this study are appropriate immunological agents for the diagnostic detection of hepatitis B virus infection in humans.
构建了含有经聚合酶链反应（PCR）扩增的乙型肝炎病毒e抗原（HBeAg）基因的质粒（HBeAg-MV和HBeAg-SV），并在大肠杆菌DH5α菌株中进行表达。从细菌裂解物中回收并通过谷胱甘肽-琼脂糖珠亲和层析纯化诱导产生的细胞内谷胱甘肽S-转移酶（GST）融合蛋白HBeAg-MV和HBeAg-SV。HBeAg-MV蛋白在其氨基末端含有另外19个氨基酸。这两种蛋白被蛋白酶因子Xa从GST上特异性切割下来，并被抗HBeAg单克隆抗体识别。发现HBeAg-MV和HBeAg-SV是病毒感染期间修饰后核心抗原（HBcAg）的两个主要成分。通过雅培HBe酶免疫测定（EIA）检测系统证实了融合和纯化的HBeAg（经因子Xa消化）的抗原特异性。确诊为肝细胞癌（HCC）患者的血清仅与融合蛋白的HBeAg部分发生特异性反应。HCC血清与HBeAg-SV蛋白的结合比与HBeAg-MV蛋白的结合更强。这表明HBeAg-SV要么比-MV更具抗原性，要么是乙肝病毒感染后引发抗体产生的主要靶蛋白。因此，本研究中表达并获得的两种重组HBeAg是用于人类乙型肝炎病毒感染诊断检测的合适免疫试剂。
