中国生物工程杂志

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.