[Preparation of streptavidin-C3d fusion protein and detection of its function in vitro]

Objective To detect the prokaryotic expression of streptavidin-complement 3d (SA-C3d) fusion protein and verify its function in vitro. Methods The C3d DNA was amplified using C3 cDNA as a template, and the C3d fragment was ligated with the vector plasmid pET-24a-6His-SA-IL15 after the digestion with a one-step cloning method to obtain the SA-C3d prokaryotic expression plasmid. The correctly sequenced plasmid was transformed into expression competent Rosetta to induce protein expression. The target protein was obtained by nickel column affinity chromatography and urea dialyzed refolding. The function of SA was demonstrated by anchoring the biotinylated MB49 cell experiment, and the function of C3d was detected by an experiment that promoted the growth of Raji cells. Results The prokaryotic expression vector of SA-C3d was successfully constructed. The purified target protein was obtained by nickel column purification and dialysis refolding. The protein was specifically bound to biotinylated MB49 cells, which promoted the proliferation of Raji cells in a dose-dependent manner, indicating that the protein SA-C3d had a bifunctional activity. Conclusion The successfully prepared SA-C3d fusion protein can be bound to biotinylated MB49 cells in vitro and promote Raji cell proliferation.