丙肝病毒非结构蛋白基因3的克隆及其体外诱导表达

Cloning of the non-structural gene 3 of hepatitis C virus and itsinducible expression in cultured cells

目的：研究丙肝病毒非结构蛋白基因３（ｎｓ３基因）在体外真核细胞中的诱导表达。方法：以ＰＣＲ方法从含ＨＣＶｎｓ３基因的重组载体ｐＢｎｓ３中扩增出ｎｓ３基因，并将其插入到克隆载体ｐＧＥＭ－Ｔ中，再与表达载体ｐＭＳＧ重组，以得到重组表达载体ｐＭＳＧ－ｎｓ３。然后采用磷酸钙沉淀法将其转染哺乳动物ＣＨＯ细胞，经地塞米松（ＤＭ）诱导，采用ＥＬＩＳＡ及Ｗｅｓｔｅｒｎ－ｂｌｏｔ技术检测ＮＳ３蛋白的表达水平。结果：用所构建的诱导型表达载体ｐＭＳＧ－ｎｓ３转染ＣＨＯ细胞，经ＤＭ（３×１０－８ｍｏｌ／Ｌ）诱导后可表达ＮＳ３蛋白，其浓度与时间呈正相关。结论：所得到的表达载体在培养细胞中能够有效地表达。

Objective: To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells. Methods: The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEMT. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)inducible expression plasmid pMSGns3. CHO cells were transfected by pMSGns3 by using calcium phosphate precipitated method and cultivated for 12 to 24 h.The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by using ELISA and Westernblot methods. Results: After treatment with 3×10^(-8) mol/L DM,the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown with the delay time of DM treatment. Conclusion: The inducible expressing vector pMSGns3 might be helpful for further studies of the characterizations of the ns3 gene in vivo.