丙型肝炎病毒核心蛋白在QSG7701细胞中的表达和鉴定

Expression and identification of the HCV core protein in the human hepatocyte QSG7701

目的:进行丙型病毒肝炎核心蛋白真核表达载体的构建,并在人源肝细胞QSG7701中进行表达与鉴定。方法:从含有丙肝病毒全长基因的重组质粒pBRTM/HCV1-3011质粒中扩增出HCV核心(core)基因片段,构建pcDNA3.1(-)/core重组真核表达质粒。然后采用阳离子多聚体将其转染人肝细胞QSG7701,用免疫组织化学SP法检测丙型肝炎病毒核心蛋白的表达,再通过Westernblot印迹法进行鉴定。结果:所克隆的core片段大小正确,序列正确;成功的构建了pcDNA3.1(-)/core重组表达质粒,瞬时转染QSG7701细胞,用SP免疫组化检测到了核心蛋白的表达,Westernblot印迹法显示其分子量约为21000。结论:丙型病毒肝炎核心蛋白的真核表达载体pcDNA3.1(-)/core在人肝细胞中能有效表达HCV核心蛋白,从而为进一步研究和解析核心蛋白在肝细胞癌变机制中的所起的作用提供了良好的核心蛋白表达系统,同时也为开发丙型肝炎DNA疫苗提供了前期条件。

Objective：To construct a recombinant plasmid enconding HCV core gene and induce the expression of core protein in the human hepatecyte and identify the core protein. Methods： HCV core gene was clone by using PCR from plasmid pBRTM/HCV1-3011 which concludes the full length of HCV gene. We reeombined the core segment with expression plasmid pcDNA3.1 （-） to construct eukaryotic plasmid pcDNA3.1（-）/core.Then the plasmid pcDNA3.1（-）/core was transfected into human hepatocytes by using poly-eation. The expression of core protein was detected by immunochemical staining method and Western blot.Results：The clone core segment had correct length and sequence the QSG7701 cells were successfully transfected and expressed core protein. Conclusion： We have successfully constructed eukaryotic expression plasmid pcDNA3.1（-）/core which included the HCV core gene.And the vector successfully expressed core protein in human hapatocytes these result provide a good core protein expression system for the study of the hepatoeareinogesis of the core protein and lay foundation for developing the HCV DNA vaccine.