HCV-C蛋白抗原在人肝细胞株中的表达和鉴定

目的:构建HCV-C蛋白基因的真核表达载体,并在正常人肝细胞HL-7702中表达与鉴定。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HCVcore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正常肝细胞HL-7702,用免疫组化染色(SP)法检测HCVC蛋白的表达,并通过Westernblot进行鉴定。结果:所克隆的HCV-C基因片段的大小为573bp,序列正确。成功地构建了pcDNA3.1(-)/core重组表达质粒。以其转染HL-7702细胞后,用SP免疫组化染色法检测到了C蛋白的表达。Westernblot显示,其相对分子质量(Mr)约为21000。结论:构建的真核表达载体pcD-NA3.1(-)/core在人肝细胞中能有效表达HCV-C蛋白,为以后相应抗体的制备打下了基础。

Expression and identification of HCV core protein in human hepatocytes

AIM: To construct the recombinant plasmid of HCV core protein, and to express and identify it in normal human hepatocyte HL-7702. METHODS: HCV core gene was cloned by using PCR from plasmid pBRTM/HCV1-3011 which included the full length of HCV gene. The core segment with expression plasmid pcDNA3.1(-) was recombined to construct eukaryotic expression plasmid pcDNA3.1(-)/core, which was then transfected into human hepatocytes by using poly-cation. The expression of core protein was detected by immunochemical staining and Western blot. RESULTS: The length and sequence of the cloned core segment were correct. The transfected HL-7702 cells expressed the core protein. CONCLUSION: The eukaryotic expression plasmid pcDNA3.1(-)/core including HCV core gene is successfully constructed. The effective expression of HCV core protein in human hepatocytes is useful for further development of HCV core antigen.