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目的应用酵母双杂交技术及免疫共沉淀技术证实HCV非结构蛋白4A(HCV NS4A)与钙离子信号调节亲环素配体(CAML)的相互作用。方法对CAML编码基因进行克隆,并构建其酵母表达载体.在酵母细胞中与HCV NS4A进行回交验证后,构建HCV NS4A及CAML真核表达载体,转染293细胞,行免疫共沉淀实验及Western印迹检测。结果成功克隆CAML基因,并构建CAML的酵母表达载体,在酵母细胞中回交验证HCV NS4A与CAML的相互作用,构建HCV NS4A及CAML的真核细胞表达载体,并利用免疫共沉淀技术验证了两者的相互作用。结论CAML与HCV NS4A的相互作用可能对HCV感染细胞的钙离子浓度、细胞凋亡等生物过程产生影响,从而在HCV感染慢性化过程中发挥作用。
Objective To verify the interaction between HCV NS4A and calcium signaling regulatory cyclophilin ligand (CAML) by yeast two-hybrid technique and co-immunoprecipitation. Methods The CAML gene was cloned and its yeast expression vector was constructed.After the HCV NS4A was verified in yeast cells, the HCV NS4A and CAML eukaryotic expression vector was constructed and transfected into 293 cells. The co-immunoprecipitation assay and Western Blot detection. Results The CAML gene was successfully cloned and the CAML yeast expression vector was constructed. The interaction between HCV NS4A and CAML was confirmed by backcrosses in yeast cells, and the eukaryotic expression vector of HCV NS4A and CAML was constructed. The co-immunoprecipitation Interaction of people. Conclusion The interaction between CAML and HCV NS4A may affect the biological processes such as Ca 2+ concentration and apoptosis of HCV infected cells and play a role in the process of chronic HCV infection.