纤连蛋白的分段表达及与乙肝表面抗原相互作用研究

Cloning and expression of fragments of fibronectin to screen hepatitis B virus surface antigen-binding domains

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。

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 eDNA sequences of each domain,each eDNA 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 interaetional domains. Results：Each eDNA 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 beparin-binding domain and carboxyl terminus（ c terminus）.