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目的探讨乙型肝炎病毒(HBV)PreS1蛋白与人类初期多肽相关复合体α亚单位(nascent-polypeptide-asso-ciated complex alpha polypeptide,NACA)之间的结合作用。方法采用交合实验验证PreS1蛋白和NACA在酵母细胞内的结合作用,利用离体结合实验证实二者在体外的结合作用,免疫共沉淀实验证实二者在哺乳动物细胞中的特异性结合。结果携带NACA基因的酵母同携带preS1基因的酵母交合后发生反应,LacZ活性检测菌落呈现蓝色,离体结合实验Western印迹中出现特异性结合活性反应条带,提示PreS1蛋白和NACA在体外存在直接相互作用,免疫共沉淀实验在13×103处出现蛋白条带,提示在哺乳动物细胞水平仍能检测到PreS1蛋白和NACA的特异结合。结论NACA与PreS1蛋白在酵母细胞和哺乳动物细胞中均存在特异性结合,推测NACA可能通过与PreS1蛋白的结合影响病毒黏附及反式激活等过程。
Objective To investigate the binding effect of preS1 protein of hepatitis B virus (HBV) with the nascent polypeptide-associated complex alpha polypeptide (NACA). Methods The interaction between PreS1 protein and NACA in yeast cells was verified by cross-fertilization experiments. The in vitro binding of both PreS1 protein and NACA was verified by in vitro binding experiments. The co-immunoprecipitation assay confirmed the specific binding between PreS1 and NACA in mammalian cells. Results The yeast carrying NACA gene reacted with the yeast carrying preS1 gene, and the LacZ activity-detecting colonies presented a blue color. The specific binding activity bands appeared in the Western blot of the binding experiments in vitro, suggesting that PreS1 protein and NACA existed directly in vitro Interaction and co-immunoprecipitation showed protein bands at 13 × 103, suggesting that specific binding of PreS1 protein to NACA could still be detected at mammalian cells. Conclusion NACA and PreS1 protein both have specific binding in yeast cells and mammalian cells. It is speculated that NACA may affect virus adhesion and transactivation by binding to PreS1 protein.