摘要
目的利用酵母双杂合体系筛选HBV前S1结合蛋白，进一步探讨前S1蛋白在HBV感染中的作用。方法PCR扩增HBV前S1基因序列，根据酵母双杂合体系构建饵质粒pAS2-1-preS1，并测定序列；饵质粒转化入酵母菌AH109，Western印迹法证实前S1-BD融合蛋白在酵母细胞中的正确表达。pAS2—1-preS1及正常成人肝细胞cDNA文库共转化酵母细胞，通过选择性培养及β-半乳糖苷酶活性测定筛选阳性菌落，分离实验排除假阳性克隆，扩增阳性克隆的目的基因并测定序列，查询其同源序列，行交合实验进一步证实目的蛋白同前S1蛋白在酵母细胞中能否特异性结合。结果成功构建pAS2-1-preS1饵质粒，Western印迹法证实转化饵质粒的酵母细胞能正确表达前S1-BD融合蛋白，pAS2—1-preS1及正常成人肝细胞cDNA文库共转化酵母细胞后，选择性培养出101个菌落，经β-半乳糖苷酶活性测定及分离实验后筛选出1个阳性克隆，其cDNA插入片段与丝裂原活化蛋白激酶-胞外信号调节蛋白激酶（MAPK—ERK）激酶1（MEK1）基因高度同源。交合实验证实MEK1同前S1蛋白在酵母细胞中可特异性结合。结论酵母双杂合体系证实MEK1可能是一个前S1结合蛋白。
Objective To screen and identify the hepatitis B virus （HBV） preS1-interactive proteins from normal human liver cDNA library by yeast two hybrid system 3 and explore the role of preS1 in the pathogenesis of HBV infection. Methods HBV preS1 gene was amplified by polymerase chain reaction（PCR）, HBV preS1 bait plasmid, named pAS2-1-preS1, was constructed by yeast two hybrid system 3 and verified by auto-sequencing assay, pAS2-1-preS1 was transformed into the yeast AH109 and preS1-BD fusion protein expressed in the yeast cells was confirmed by Western blotting method. Yeast cells were cotransformed with PAS2-1-preS1 and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade2 medium. The second screen was performed with β-gal activity detection and false positive clones were eliminated by segregation analysis. Thereafter, true positive clones were amplified, sequenced and analyzed with bioinformatics. Lastly, mating experiment was performed to confirm the binding of putative proteins with preS1 in the yeast cells, Results Bait plasmid pAS2-1-preS1 was successfully constructed and pAS2-1-preS1 correctly expressed preS1-BD fusion protein in the yeast AH109. One hundred and one clones grew in the selective SC/- trp-leu-his-ade2 medium, and only one clone past through β-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with mitogen activated protein kinasesextracellular signal-regulated kinase kinase I （MEK1）. Furthermore, mating experiment identified that the binding of MEK1 with preS1 protein was specific. Conclusion MEK1 is a candidate protein which can interact with preS1 in vivo by yeast two hybrid system.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2007年第1期20-24,共5页
Chinese Journal of Infectious Diseases
基金
福建省自然科学基金资助项目（C0310018）
