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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。
Objective: To construct the eukaryotic expression vector of HCV-C protein and to express and identify it in human hepatocyte HL-7702. Methods: HCVcore gene fragment was amplified from the recombinant plasmid pBRTM / HCV1-3011 containing the full-length gene of hepatitis C virus to construct a recombinant eukaryotic expression vector pcDNA3.1 (-) / core. Then it was transfected into human normal hepatocyte HL-7702 by cationic multimer, and the expression of HCVC protein was detected by immunohistochemical staining (SP). The expression of HCVC protein was identified by Western blot. Results: The size of the cloned HCV-C gene fragment was 573 bp with the correct sequence. The pcDNA3.1 (-) / core recombinant plasmid was successfully constructed. After transfection with HL-7702 cells, the expression of protein C was detected by SP immunohistochemistry. Westernblot showed that its relative molecular mass (Mr) was about 21,000. CONCLUSION: The constructed eukaryotic expression vector pcD-NA3.1 (-) / core can express HCV-C protein efficiently in human hepatocytes, which lays the foundation for the preparation of the corresponding antibody in the future.