丙型肝炎病毒C区截短型基因原核表达载体的构建及在大肠杆菌中的表达

Cloning of the truncated gene of hepatitis C virus core and expression in E.coli

目的建立稳定表达丙型肝炎病毒(HCV)核心蛋白的原核表达系统,获得高产量的纯化核心蛋白。方法应用多聚酶链反应(PCR),以HCV-H株全长cDNA序列为模板,扩增获得C区羧基端部分缺失的基因片段,克隆入原核表达载体pBVIL1,构建原核表达载体pBVIL1-Ct,转化HB101宿主菌,通过温度诱导表达截短型C蛋白。结果扩增得到目的基因长度为462bp,构建pBVIL1-Ct表达载体,在HB101宿主菌中通过温度诱导获得稳定表达,表达蛋白占菌体总蛋白含量的24%。Western-Blot及ELISA检测证实表达产物可与HCV患者阳性血清发生特异性结合反应。结论羧基端部分缺失的HCV C区基因片段可在大肠杆菌中稳定表达并具有良好的反应原性。

Objective To construct a steady expression system of HCV truncated Core gene in E. coll. Methods The cDNA sequence encoding truncated HCV Core gene which lacked parts of the carboxyl-terminal were amplified by PCR and inserted into plasmid pBVIL1; the resulting expression vector was transformed into HB101 E. coli and the protein was expressed by temperature induction. Results The length of amplified truncated Core gene was 462 bp; the yield of truncated Core protein produced in pBVIL1 was 24% of the total proteins. The protein produced was detected by SDS-PAGE, western blot and ELISA. The results showed specific immunoreactivity with serum from patients with hepatitis C in western blot and ELISA assay. Conclusion Truncated HCV Core gene which lacked parts of the carboxyl-terminal was expressed in E. coli and exhibited immunogenicity.