[Expression and purification ficolin 3 and its role for RAW264.7 macrophage activation in vitro]

To establish a prokaryotic expression plasmid of ficolin-3, express and purify the fusion protein His-ficolin 3 in E.coli, and explore the role of ficolin-3 in the activation of RAW264.7 macrophages.

Using the cDNA from human peripheral blood mononuclear cells as template, human ficolin-3 cDNA was amplified by PCR and inserted into vector pET22b. The recombinant plasmid pET22b-ficolin 3 was identified by digestion of XhoI and SalI and confirmed by DNA sequencing. Thereafter, the protein His-ficolin 3 was induced and purified in E.coli BL21. His-fincolin 3 at different concentrations was added into RAW264.7 cells. With the protein His as control, the effects of His-fincolin 3 on the phagocytosis of RAW264.7 cells to chicken red blood cells (CRBCs) and on the production of IL-6, IL-12 and TNF-α were tested by flow cytometry and ELISA, respectively.

Enzyme digestion and sequencing confirmed that recombinant plasmid pET22b-ficolin 3 was established as expected. SDS-PAGE showed the expression of soluble His-ficolin 3 of Mr 33 000. The purity of the products was over 90%. Western blotting further identified His-ficolin 3. Compared with His protein, His-ficolin 3 improved the phagocytosis of RAW264.7 cells in a dose-dependent manner, and it raised the secretion of IL-6, IL-12 and TNF-α in RAW264.7 cells.

The plasmid pETb-ficolin 3 was cloned successfully and the purity of the protein His-ficolin 3 was over 90%. His-ficolin 3 could induce the activation of RAW264.7 cells obviously.