The construction, expression and activity examinations of 6B11ScFv/murine GM-CSF fusion protein

Objective To construct expression vector which expresses fusion protein of antiidiotypic single chain antibody 6B11ScFv and murine GM CSF(6B11mGM) for observing its possible immune reactions in vivo. Methods Using DNA recombinant techniques, the murine GM CSF cDNA gene was recombined to 6B11ScFv and they were cloned into expression vector pET 30a(+) to produce insoluble protein. Inclusion bodies collected after the breakage of bacteria through sonication were subjected to repeatedly washing. Inclusion bodies solubilized in the resence of 8 mol·L -1 urea were diluted with renaturation solutions so that folding process could be initiated. The purity was examined by SDS PAGE, ELISA and cell proliferation assay were used to determine the activities of fusion protein. Results 6B11mGM fusion proteins were obtained with a purity of over 90%. The optimum conditions were 1mmol·L -1 and 5 mmol·L -1 for the concentrations of GSSG and GSH, respectively, with 48 hours at 10℃ as renatured time. Fusion protein could specifically react with the primary anti ovarian carcinoma monoclonal antibody(COC166 9) and rat anti mouse GM CSF monoclonal antibody, respectively, and fusion proteins could also stimulate murine GM CSF dependent cell line NFS 60 cells to proliferate. Conclusion Fusion proteins 6B11mGM expressed as inclusion bodies can keep the activity of both the proteins and provid foundation to research the immunoreactions in vivo.