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目的 探索应用双杂交体系统克隆与乙型肝炎病毒 (HBV)PreS1蛋白结合的肝细胞受体的可行性。方法 构建编码HBVPreS1全序列或部分序列与酵母蛋白GAL 4DNA结合区域融合蛋白的酵母表达质粒 ,应用这些质粒转化酵母报道菌株SFY52 6 ,检测 β 半乳糖苷酶活性作为酵母中转录激活的指标。构建编码PreS1全序列与GAL4DNA结合区域融合蛋白的哺乳动物细胞表达质粒 ,与CAT报道质粒共转染肝癌细胞系Huh 7,使用薄层层析法检测细胞提取物的氯霉素乙酰转移酶活性。结果 全序列PreS1蛋白与GAL4DNA结合区域的融合蛋白在酵母细胞中呈现转录激活功能 ,其转录激活序列被定位于PreS1第 2 1～ 47氨基酸之间。全序列PreS1蛋白与GAL 4DNA结合区域的融合蛋白在哺乳动物细胞中未呈现转录激活功能。结论 HBVPreS1蛋白在酵母细胞中的转录激活功能 ,限制了研究人员应用酵母双杂交体系统克隆与PreS1结合的HBV受体。然而 ,PreS1蛋白在哺乳动物细胞未呈现转录激活功能。哺乳动物细胞双杂交体系统可能是克隆与PreS1蛋白作用的肝细胞受体的可行途径
Objective To explore the feasibility of using a two-hybrid system to clone hepatocyte receptors that bind to preS1 protein of Hepatitis B virus (HBV). Methods The yeast expression plasmid encoding HBV PreS1 full or partial sequence and yeast protein GAL 4 DNA binding region fusion protein was constructed and used to transform yeast reporter strain SFY52 6 to detect β-galactosidase activity as an indicator of transcriptional activation in yeast. A mammalian cell expression plasmid encoding the fusion protein of PreS1 complete sequence and GAL4 DNA binding region was constructed and transfected into the hepatocellular carcinoma cell line Huh 7 with CAT reporter plasmids. The chloramphenicol acetyltransferase activity of cell extracts was determined by TLC. Results The fusion protein of full sequence PreS1 and GAL4 DNA binding region showed transcriptional activation in yeast cells. The transcriptional activation sequence was located between amino acids 21 to 47 of PreS1. The fusion protein of the full-sequence PreS1 protein and GAL4 DNA binding region did not exhibit transcriptional activation in mammalian cells. Conclusion The transcriptional activation of HBV PreS1 in yeast cells restricted the researchers from using yeast two-hybrid system to clone HBV receptor preS1. However, PreS1 does not exhibit transcriptional activation in mammalian cells. Mammalian cell two-hybrid system may be a viable pathway for cloning hepatocyte receptors that interact with PreS1 protein