Xiao Shengxiang, Chu Yonglie, Peng Xuanxian, Wang Cuiling, Xiao Shengbin, Wu Yanhong, Cao Zhenping
Laboratory of Molecular Biology, Institute of Dermatology, The Second Hospital, Xi?an Jiaotong University, Xi'an 710004, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2002 Jun;16(2):154-6.
To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two hybrid system.
Yeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526. Reporter gene product ?galactosidase activity was assayed as a measure of transcription activation in yeast. Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh?7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin?layer chromatography.
The fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain present transcriptional activation function in yeast. The transcription activating sequence is localized to the 21 to 47 amino acids of Pre S1 protein Fusion proteins of full length Pre S 1 and GAL 4 DNA binding domain do not show transcriptional activation function in mammalian cells.
The transcriptional activating sequence of HBVPre S1 protein in yeast overlaps the hepatocyte receptor binding site. The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers?from using yeast two hybrid system to clone HBV receptor interacting with Pre S1 protein. However, the Pre S1 protein does not show transcriptional activation function in mammalian cells. Mammalian two?hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein.
通过双杂交系统探索克隆与乙肝病毒前S1蛋白相互作用的肝细胞受体的可行性。
构建编码乙肝病毒前S1全长或部分片段与酵母蛋白GAL4 DNA结合结构域融合蛋白的酵母表达质粒，并用于转化酵母报告菌株SFY526。测定报告基因产物β-半乳糖苷酶活性，作为酵母中转录激活的指标。构建编码前S1全长与GAL4 DNA结合结构域融合蛋白的哺乳动物表达质粒，并与CAT报告质粒一起共转染肝癌细胞系Huh-7。通过薄层层析法测定细胞提取物中的CAT活性。
前S1全长蛋白与GAL4 DNA结合结构域的融合蛋白在酵母中呈现转录激活功能。转录激活序列定位于前S1蛋白的第21至47个氨基酸。前S1全长与GAL4 DNA结合结构域的融合蛋白在哺乳动物细胞中未显示转录激活功能。
乙肝病毒前S1蛋白在酵母中的转录激活序列与肝细胞受体结合位点重叠。乙肝病毒前S1蛋白在酵母中的转录激活功能可能会阻碍研究人员利用酵母双杂交系统克隆与前S1蛋白相互作用的乙肝病毒受体。然而，前S1蛋白在哺乳动物细胞中未显示转录激活功能。哺乳动物双杂交系统可能是克隆与前S1蛋白相互作用的乙肝病毒肝细胞受体的一种实用方法。
