补体C3与BPI活性区融合蛋白（CB）的构建与表达

补体C3和杀菌通透性增加蛋白（BPI）对血液中的病原体均有黏附、促吞噬甚至杀灭作用，但两者的作用机制不同，制备两者活性区融合蛋白，可能具有更好的清除血液病原的作用。通过重叠延伸PCR融合人补体C3的补体受体Ⅰ、Ⅲ两个结合区，同时调取了杀菌通透性增加蛋白（BPI）活性区段rBPI，先后将补体C3活性区与BPI蛋白功能区基因克隆入原核表达载体pET28a中，获得融合蛋白（CB）表达载体pET28-CB，在大肠杆菌中进行了高表达产量、可溶性表达等条件的摸索，CB融合蛋白主要以包涵体形式表达，Western印迹证明CB具有C3的抗原活性，将包涵体蛋白变性与复性后，利用Ni2+固相化的螯合Sepharose Fast Flow亲和层析柱进行浓缩和纯化，最后得到了纯度较高的CB原核表达蛋白。CB融合蛋白的构建和高效表达、纯化为下步探讨其在促进血液病原清除上的功能鉴定和应用奠定了基础。

Construction and expression of C3-BPI fusion protein (CB) in E. coli

The third component of human complement (C3) is an important participant in immune surveillance and immune regulation. There are many binding-sites on the surface of human C3 molecule, especially the binding-sites to complement receptor on blood cell, which induce the regulation of complement, phagocytes, and even bacteriolysis of heterogenous pathogens. So we fused the genes of two C3 binding-sites which adhered to complement receptorⅠ (CRⅠ) and complement receptorⅢ (CRⅢ) by overlap extension PCR. Bactericidal-permeability increasing protein (BPI) is a kind of cation proteins separated from heterophil granulocyte which shows strong attachment and disinfection on G- bacteria, even some fungi and protozoon, especially in total blood, plasma. In this research, the active part of BPI, rBPI was obtained by PCR. Then rBPIs was connected to the genes of human complement C3 binding-sites by gene splicing in target to fusion protein of C3-BPI active part, which was named CB. CB fusion protein was supposed to has the function of killing exogenous pathogens and introducing adherence of red blood cell, phagocyte, etc, which would lead to the fast clearance of pathogens in blood. Then pET28-CB, a prokaryote expression vector of CB was constructed and efficiently expressed in Escherichia coli BL21(DE3). The result of western blot showed the target protein had the activity of combination with C3 antibody, Highly concentrated purified protein was obtained after denaturization and renaturation of extracted inclusion protein. The expression and purification of the CB fusion protein was suitable for further analysis of its function and application.