丙型肝炎病毒核心蛋白基因重组质粒的构建及其在真核细胞中的表达

Construction of the hepatitis C virus core gene recombinant plasmid and its expression in hepatoblastoma 7721 cells

目的：构建包含丙型肝炎病毒（ＨＣＶ）核心蛋白（Ｃ）基因片段的重组真核表达载体，并在肝细胞癌细胞株７７２１细胞中表达。方法：将从ｐＢＲＴＭＨＣＶ１－３０１１质粒切下的ＨＣＶＣ基因片段插入ｐｃＤＮＡ３质粒的ＣＭＶ启动子下游，构建真核表达质粒ｐｃＤＮＡＨＣＶ－Ｃ，然后，采用脂质体转染技术，转染７７２１细胞进行瞬时表达，转染细胞裂解煮沸后，通过ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎｂｌｏｔ检测表达的核心抗原。结果：用限制性内切酶酶切后，片段大小与计算值相符。Ｗｅｓｔｅｒｎｂｌｏｔ证实，表达抗原的Ｍｒ约为２２０００。结论：ＨＣＶＣ基因能够插入ｐｃＤＮＡ３真核表达载体，并使其在真核细胞中表达，为进一步ＨＣＶ基因疫苗的研制和探讨抗ＨＣＶ感染打下了基础。

Aim: To construct the recombinant plasmid containing hepatitis C virus core gene and express it in hepatoblastoma 7721 cells. Methods: The HCV C gene coding region cutted from the pBRTMHCV1 3011 plasmid was inserted into the eukaryotic expression plasmid pcDNA3 to construct the recombinant plasmid pcDNAHCV C which was then expressed transiently using lipofectamine technique under the control of CMV promoter of pcDNA3 in the 7721 cells. Results: The enzyme cutting identification showed that HCV C gene fragment was cloned into pcDNA3 eukaryote vectors. The expressed product with a relative molecular mass ( M r) about 22 000 was detected by SDS PAGE and Western blot. Conclusion: HCV C gene fragment may be inserted into eukaryote expression vector pcDNA3 and expressed in hepatoblastoma 7721 cells. It provided us a possible method for the further study of gene immunization in developing HCV genetic vaccine and treating HCV infection.