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目的克隆与丙型肝炎病毒(HCV)核心蛋白结合的肝细胞中的未知蛋白基因。方法应用酵母双杂交系统3.构建HCV核心蛋白诱饵质粒,转化酵母AH109后与含文库质粒的酵母Y187进行配合,在营养缺陷培养基上进行双杂交筛选。筛选出30个阳性克隆,对既能在4缺营养缺陷培养基上生长(SD/-Trp-Leu-Ade-His)又能在涂有X-α-半乳糖(X-α-gal)的四缺培养平皿上长出蓝色菌落,提取此酵母克隆的质粒转化大肠杆菌后进行测序,进行生物信息学分析.将HCV核心蛋白基因分为大、中、小三段,与所克隆的未知功能基因回交,以证实其相互作用的部位。结果分析的结果显示,其中1号克隆的测序结果与GenBank中的1个未知功能序列有99％的同源性,初步证明了此基因与HCV核心蛋白有结合活性(GenBank号:AY032593).HCV核心蛋白的全长、2/3,1/3三段,回交显示三段均能在酵母中与此未知基因具有结合作用。结论 HCV核心蛋白结合蛋白肝细胞基因克隆成功,核心蛋白与此未知基因的作用部位应是氨基端.此项研究结果,为以后研究此基因在HCV致病机制以及在肝细胞中的生理功能提供了线索。
Objective To clone an unknown protein gene in hepatocytes that binds to the hepatitis C virus (HCV) core protein. Methods Yeast two-hybrid system was used to construct bait plasmids for HCV core protein. After transformed into yeast AH109, yeast bait plasmid Y177 was combined with yeast strain Y187 to screen for two-hybrid screening on auxotrophic medium. Thirty positive clones were screened for their ability to grow on both auxotrophic medium (SD / -Trp-Leu-Ade-His) and X-α-galactose Four lacking culture plates grew blue colonies, the plasmid was extracted and transformed into Escherichia coli for bioinformatics analysis.The HCV core genes were divided into large, medium and small segments, and the cloned unknown function Gene backcross to confirm the site of their interaction. The results of the analysis showed that the sequencing result of clone No. 1 showed 99% homology with an unknown functional sequence in GenBank, and it was preliminarily proved that the gene has the binding activity with HCV core protein (GenBank No. AY032593) .HCV The full length of the core protein, two thirds and one third of the three segments, backcrossing showed that all three segments could bind to this unknown gene in yeast. Conclusion The cloning of hepatocyte gene of HCV core protein-binding protein was successful and the site of interaction between the core protein and this unknown gene should be the amino-terminal region. The results of this study will provide theoretical basis for further study on the pathogenesis of HCV and the physiological function in hepatocytes Clues.