【摘要】 目的:克隆纤连蛋白(fibronectin)各功能区域基因,在大肠杆菌中表达以筛选与HBsAg相互作用的区域。方法:将纤连蛋白分成7个片段,设计引物,利用RT-PCR从HepG2.2.15中扩增出相应的PCR产物,连接到表达载体pGEX4T-1中,用IPTG诱导目的蛋白在E.coli中表达,通过Western印迹分析对表达产物进行鉴定。融合蛋白纯化后制备蛋白芯片,与HBsAg杂交,筛选纤连蛋白中与HBsAg相互作用的区域。结果:经RT-PCR从HepG2.2.15中扩增到各结构域片段的cDNA,序列分析均正确;SDS-PAGE分析表明,各结构域片段的可溶性表达产物均占细菌可溶性蛋白的10%以上;Western印迹分析提示,诱导表达产物可与GST单抗发生特异性反应。经制备蛋白芯片并杂交,发现片段FN1、FN2信号最强。结论:成功地表达了纤连蛋白各片段,蛋白芯片杂交结果显示纤连蛋白可能是通过Ⅱ型肝素结合区及其羰基端(c端)与HBsAg相互作用。更多还原

【Abstract】 Objective:To clone and express the genes of fragments of fibronectin in E.coli for screening HBsAg-binding domains.Methods:Fibronectin was divided into seven domains,and primers were designed according to cDNA sequences of each domain,each cDNA was amplified by RT-PCR from HepG2.2.15 cells and cloned into expression vector pGEX4T-1.The constructed expression vectors were transformed into E.coli and induced to express the recombinant proteins.The fusion proteins were identified by Western blot using anti-GST monoclonal antibody.After purification,the fusion proteins were immobilized onto the aldehyde-coated glass slides in an array format,which could be used for detection by HBsAg to screen interactional domains.Results:Each cDNA was cloned from HepG2.2.15 cells,sequence analysis revealed identity to the GenBank report.The soluble expression products accounted for above 10% of soluble proteins of the bacteria.Western blot showed that the recombinant proteins can be recognized by GST monoclonal antibody.FN1 and FN2 showed the most intensive signals in protein screening.Conclusion:Fibronectin may interact with HBsAg by its heparin-binding domain and carboxyl terminus(c terminus).更多还原