乙型肝炎病毒全S蛋白与纤维蛋白原α链的相互作用  

Interaction between fibrinogen alpha chain and the whole S protein of hepatitis B virus

作　　者：周飞[1,2,3] 任建林[1,2,3] 卢雅丕[1,2,3] 陈美娅 许鸿志[1,2,3] 潘金水 蔡稼燕[1,2,3] 董菁 

机构地区：[1]厦门大学附属中山医院消化内科 [2]厦门大学消化疾病研究所 [3]厦门市消化疾病中心,福建省厦门市361004

出　　处：《世界华人消化杂志》2008年第23期2581-2586,共6页World Chinese Journal of Digestology

基　　金：厦门市首批重大疾病科研攻关项目;No.WKZ0501;厦门市卫生局医学科研立项项目;No.WSK0506;厦门大学引进人才科研启动基金;No.Z03109;福建省青年科技人才创新项目;福建省高校新世纪人才创新资助项目~~

摘　　要：目的:筛选人肝细胞cDNA文库中与乙型肝炎病毒(HBV)全S蛋白相互作用蛋白的基因,并反向验证HBV全S蛋白候选结合蛋白之间相互作用.方法:将全S基因定向克隆到酵母表达载体pDEST32,构建正向筛选的诱饵质粒并Westernblot法验证其在酵母中的表达.将诱饵质粒与人肝细胞cDNA文库质粒共同转化MaV203酵母细胞,在人肝细胞cDNA文库筛选候选结合蛋白,提取阳性菌落质粒测序,并分析其生物学性质.将筛选出的纤维蛋白原α链中下游序列及不同全S变异株基因,分别定向克隆到pDEST32及pDEST22载体中,利用Westernblot法验证表达.将诱饵质粒与猎物质粒共同转化MaV203酵母细胞,以反向酵母双杂交方法验证初筛结果的可靠性及正确性.结果:正向的酵母双杂交实验,经初筛和再转染实验纤维蛋白原α链可与HBV全S蛋白发生相互作用.再以纤维蛋白原α链为靶基因设计诱饵质粒,以四种变异的HBV全S蛋白为靶基因设计猎物质粒,反向酵母双杂交法证实维蛋白原α链中下游可与不同全S变异体(总差异率2%)发生相互作用,纤维蛋白原α链与全S蛋白的结合域可能为病毒蛋白的前268aa.结论:纤维蛋白原α链中下游可与HBV全S蛋白产生特异性结合,其结合域可能与病毒蛋白的前268aa产生相互作用.AIM： To screen the proteins interacting with the whole S protein of hepatitis B virus （HBV） from hepatocyte cDNA library by yeast two-hybrid system, and to validate interacting behavior between fibrinogen alpha chain and the whole S protein by reverse yeast two-hybrid.METHODS： The whole S gene of HBV was cloned into yeast expression vector pDEST32 to construct a bait plasmid, which was verified by Western blot. The bait plasmid and prey plasmids inserted liver cDNA fragments were cotransformed into yeast cells MaV203 by Liacmediated transformation. Diploid yeasts were plated on synthetic dropout nutrient medium to screen positive colonies. After extracting and sequencing of prey plasmids from positive colonies, the inserted sequences were bioinformatically analyzed. For reverse yeast two-hybrid, the bait plasmid expressing partial fibrinogen alpha chain and four prey plasmids expressing the whole S protein mutants were re-combined. The reconstituted bait plasmid was cotransformed into yeast cells MaV2O3 with the four prey plas- mids, respectively. Diploid yeasts were plated on synthetic dropout nutrient medium and X-gal assay was performed to validate the interacting behavior. RESULTS： By yeast two-hybrid technique, prey plasmids that were inserted partial gene coding 266-644 amino acid of fibrinogen alpha chain had a positive reaction with bait plasmid coding the whole S protein of HBV. By reverse yeast twoybrid, fibrinogen alpha chain protein in- teracted with the four whole S protein mutants. The protein binding domain of the whole S protein might be the leading 268 amino acids. CONCLUSION： Fibrinogen alpha chain protein may bind the whole S protein of HBV. The interacting domain is in the 266-644 amino acids of fibrinogen alpha chain and the frontal 268 amino acids of the whole S protein, respectively.

关 键 词：乙型肝炎病毒 全S基因 纤维蛋白原α链 酵母细胞双杂交技术 

分 类 号：R373.21[医药卫生—病原生物学]