文档介绍：HBV preS1基因酵母表达载体的构建及表达
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作者:张曦,蔺淑梅,吴列秀,陈天艳,叶峰,赵英仁,张树林
【摘要】目的构建HBV preS1基因的酵母表达载体,探讨HBV preS1蛋白的功能。方法以HBV ayw亚型全长质粒PCP10为模板,聚合酶链反应(polymerase chain reaction, PCR)扩增HBV preS1基因,克隆到pGEMT载体中命名为TpreS1,以NcoⅠ和MluⅠ双酶切TpreS1后回收与酵母表达载体pSos连接,对重组质粒进行序列测定后命名为pSospreS1,经醋酸锂法将其转化酵母菌cdc25(a),提取酵母蛋白质进行Western免疫印迹分析。结果成功构建了HBV preS1基因的酵母表达载体,Western免疫印迹分析显示HBV preS1基因在酵母细胞中正确表达。结论 pSospreS1的构建为通过SOS招募系统(sosrecruitment system, SRS)筛选与HBV preS1蛋白相互作用的蛋白和进一步探讨HBV preS1蛋白在HBV致病中的作用奠定了基础。
【关键词】乙肝病毒 preS1蛋白酵母表达载体
ABSTRACT: Objective To construct the yeast expression vector of HBV preS1 gene for investigating the potential role of preS1 protein in mediating the attachment of HBV particles to human hepa
tocytes. Methods PCR was performed to amplify HBV preS1 gene from the plasmid PCP10/HBV ayw subtype containing the whole fragment of HBV, and the PCR product was cloned into pGEMT vector and then named TpreS1. HBV preS1 gene was cut from TpreS1 by NcoⅠ and MluⅠ, and cloned into yeast expression plasmid pSos; the reconstructed plasmid was tested by autosequencing assay and named pSospreS1. pSospreS1 was transformed into yeast cell cdc25(a) by LiAc mediated transformation, and the yeast protein was isolated and analyzed by Western blot. Results The yeast expression vector of HBV preS1 gene was constructed essfully, and the presence of HBV preSl protein in yeast cells was confirmed by Western blot analysis. Conclusion Reconstruction of pSospreS1 has laid a foundation for better understanding of the mechanism of HBV preS1 protein in viral endocytosis and is helpful in seeking the preS1 related protein that is supposed to interact with preS1 protein in vitro by sosrecruitment system.
KEY WORDS: hepa
titis B virus; preS1 protein; yeast expression vector
乙型肝炎病毒(hepatitis B virus, HBV)是一种严重危害人类健康的重要病原体,其感染人体后可引起急、慢性乙型肝炎,并与肝硬化、肝癌密切相关。和其他病毒一样,HBV也是通过与靶细胞表面的相应受体结合而进入细胞,从而实现对机体的感染。preS1区是HBV与肝细胞膜的直接结合位点。目前认为preS1蛋白与HBV的急性感染[1]、介导入胞