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目的利用丙型肝炎病毒非结构基因NS3的真核表达载体(pCMV-Tag1-NS3)在HEK293细胞中进行表达。方法通过酶切分析和DNA序列分析鉴定pCMV-Tag1-NS3重组载体构建正确后,通过脂质体介导转染HEK293细胞,用RT-PCR和Western blotting证实丙型肝炎非结构蛋白NS3基因(HCV-NS3)表达。结果 RT-PCR结果显示HCV-NS3基因的mRNA在转染的HKK293细胞中表达,Western blotting结果也显示蛋白质能正确表达。结论成功构建了实现HCV-NS3过表达的稳定HEK293细胞系,为获得HCV非结构蛋白3的重组蛋白以及早期诊断试剂的研制与筛选药物提供了基础。
Objective To express HEK293 cells by using eukaryotic expression vector (pCMV-Tag1-NS3) of non-structural gene of hepatitis C virus NS3. Methods The recombinant plasmid pCMV-Tag1-NS3 was identified by restriction enzyme digestion and DNA sequencing. HEK293 cells were transfected by lipofectamine 2000. The NS3 gene of non-structural hepatitis C (HCV -NS3) expression. Results The results of RT-PCR showed that the mRNA of HCV-NS3 gene was expressed in transfected HKK293 cells. Western blotting also showed that the protein was correctly expressed. Conclusion The stable HEK293 cell line that can overexpress HCV-NS3 was successfully constructed, which provided the foundation for the development of the recombinant protein and the early diagnosis of HCV non-structural protein 3 and the screening of drugs.