Prokaryotic expression of the extracellular portion of mouse FcγRIIb and the functions of the expressed proteins in a mouse model of SLE

Objective To clone and express the extracellular portion of mouse Fcγ receptor Ⅱ-b (FcγRⅡb) and to analyze the functions of the expressed proteins in a mouse model of systemic lupus erythematosus (SLE). Methods The gene fragment encoding FcγRⅡb was amplified by PCR, and then inserted into the prokaryotic expression vector pET-32a(+ ) to construct the recombinant expression plasmid pET-FcγRⅡb. The expression plasmid was identified with restriction enzymes and then sent to the Shanghai Bio-Engineering Co. LTD for further sequencing analysis. The transformed Escherichia coli (E.coli) BL21 (DE3) strains carrying the recombinant expression plasmid pET-FcγRⅡb were induced by isopropyl β-D-1-thiogalactoside (IPTG). The expressed fusion proteins were analyzed by Western blot assay and purified with purification kits. The immune complex (IC)-binding ability of FcγRⅡb was measured by ELISA. MRL/lpr mice with SLE in both the prevention (12 weeks old, n=40) and the treatment groups (19 weeks old, n=40) were randomly divided into four groups including 60 μl (4.8 μg) treatment group, 120 μl (9.6 μg) treatment group, 180 μl (14.4 μg) treatment group and PBS treatment group with 10 mouse in each group. The MRL/lpr mice with SLE were injected with the fusion protein through tail vein once a week for four consecutive weeks. Serum samples were collected from each mouse after one week of observation. The levels of FcγRⅡb in soluble form in mice form both the prevention and treatment groups as well as the levels of anti-double stranded DNA antibodies were detected by ELISA. Results The gene encoding FcγRⅡb was amplified and the recombinant expression plasmid pET-FcγRⅡb was successfully constructed. The recombinant proteins were expressed in the prokaryotic expression system, and then successfully purified. The recombinant proteins could bind to IC. Compared with the corresponding PBS control group, the levels of FcγRⅡb in soluble form were increased in mice from both prevention and treatment groups after treating with various concentrations of the recombinant protein (P<0.05). Significant differences in the levels of FcγRⅡb were found among mice of the same age after treating with different concentrations of the recombinant protein (P<0.05). Compared with the corresponding PBS control group, the levels of anti-double stranded DNA antibodies were decreased in mice from both prevention and treatment groups after treating with various concentrations of the recombinant protein (P<0.05). The levels of anti-double stranded DNA antibodies were gradually decreased along with the increasing dosage of protein (P<0.05). Conclusion The extracellular portion of murine FcγRⅡb in soluble form was successfully expressed. The recombinant proteins played a certain role in the prevention and treatment of SLE in a mouse model. Key words: Mouse FcγRⅡb; Soluble; Treatment and prevention; Systemic lupus erythematosus