乙型肝炎病毒preS1基因真核表达载体的构建及表达

Construction of Hepatitis B Virus PreSl Gene Expression Vector and Its Expression in Eukaryotic Cells

目的:构建含preSl基因真核表达质粒,探讨乙型肝炎病毒preSl基因在HBV入胞机制中的作用。方法:PCR法扩增含EcoR I与Pst I酶切点的preSl基因序列,PAS2-1载体及preSl基因PCR产物双酶切,经T_4DNA连接酶将两者连接并转化到大肠杆菌JM105,对重组质粒经序列测定,命名为PAS 2-1-preSl。经乙酸锂转化法将重组质粒转化入酵母菌AH109,Western Blot法证实重组质粒在酵母细胞中的表达。结果:已构建的质粒PAS2-1-preSl经序列测定会有完整的preSl基因片段,转入酵母后经Western Blot证实酵母细胞正确表达preSl-BD融合蛋白。结论:PAS2-1-preS1的构建为通过酵母双杂交体系筛选体内与preSl蛋白相互作用的preSl相关蛋白,为进一步深入探讨preSl在HBV致病机制中的作用奠定了基础。

Obiective: The study was designed to construct the eukaryotic expression vector of hepatitis B virus preS1 gene for investigating the role of preSl gene in the infection of HBV. Methods: PreSl gene including EcoR I and Pst I endoen-zyme sites were obtained by PCR. Vector PAS2-1 and PCR product of preSl gene were connected by T4DNA ligase and transferred to Eco-JM105 after double enzyme digestion. Reconstitute plasmid sequence was tested by auto-sequencing assay and named PAS2-1-preS1. Reconstituted plasmid was transformated into the yeast cell AH109 by Liac-mediated transtormation and the preS1-BD fusion protein expressed in the yeast cell was confirmed by Western blot. Results: The reconstituted plasmid PAS2-1-preS1 including the anticipated fragment of preS1 gene was proved by auto-sequencing assay. Western blot showed that reconstitute plasmid PAS2-1-preS1 can correctly express the preSl-BD fusion protein in the yeast cell. Conclusion: Reconstitu-tion of PAS2-1-preS1 vector laid a foundation for better understanding of the mechanism of HBV preSl protein in viral endocy-tosis and was helpful for seek the preS1-related protein that was supposed to react with preSl protein in vivo by yeast two-hybrid system.