甲/戊型肝炎病毒重组抗原基因的克隆及表达

Gene cloning and expression of recombinant antigen of hepatitis A and E virus

摘要: 将甲肝病毒vp1编码基因(aa 24~171)和戊型肝炎病毒ORF2基因(aa 431~615)通过一段编码柔性甘氨酸链(Gly4 Ser Gly4)的基因片断连接,获得编码HAV/HEV融合蛋白基因(AEAg342).将该融合基因插入质粒pBV220,构建表达质粒pBV220-AEAg342.将pBV220-AEAg342转化入大肠杆菌BL21中进行表达,表达量约占菌体总量的20%,经离子交换层析纯化,目的蛋白纯度达90%以上.Western blot证实该蛋白能与甲肝及戊肝患者阳性血清发生特异性反映,为进一步开发双价疫苗和联合诊断试剂奠定了一定基础.

Abstract: The HAV vp1(aa 24—171)cDNA and HEV ORF2(aa 431—615)cDNA were linked via a linker sequence encoding glycine hinge(Gly4 Ser Gly4).The obtained cDNA(AEAg342)that encoding a HAV/HEV fusion protein was inserted into vector pBV220 to construct the expression plasmid pBV220-AEAg342.Then the recombinant plasmid was transformed into E.coli BL21 and induced to express the target protein.The expressed product was about 20% in total bacterial proteins.About 90% of the purity of fusion protein was obtained after purifying by DEAE-Sepharose and CM-Sepharose.It indicated that the fusion protein can produce specific reaction with HAV and HEV positive sera by Western blot.The data provided experimental confirmation for development of bivalent vaccine and serologically diagnostic kit of HAV and HEV.