Prokaryotic Expression and Activity Identification of Rat sFcγRIIb Gene

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.022

Abstract

Abstract: FcγRIIb is an important inhibitory receptor with immune negative regulation function. The purpose of this study is to clone the rat FcγRIIb gene,construct the prokaryotic expression system of sFcγRIIb,and prepare the recombinant rat sFcγRIIb protein. The extracellular domain gene of FcγRIIb was cloned from RBL-2H3 cells using RT-PCR and the recombinant expression plasmid containing gene sFcγRIIb was constructed and transferred to Escherichia coli. The recombinant protein was purified by nickel column affinity chromatography and identified by Western blotting and ELISA after refolding. As results,the gene sFcγRIIb was successfully cloned,and the prokaryotic expression vector sFcγRIIb-pET17b was constructed and transferred into E. coli BL21（DE3）. By optimizing the expression system,the protein expression efficiency was the highest when the IPTG concentration was 1.0 mmol/L,and the induction time was 4 h. The recombinant proteins were mainly the inclusion bodies and dissolved by 8 mol/L urea. Then the purified recombinant protein was obtained by Ni-NTA column affinity chromatography. The results of Western blotting,ELISA and competitive ELISA showed that the recombinant protein sFcγRIIb after refolding was recognized by specific antibodies and combined with IgG. Conclusively,the prokaryotic expression system of gene sFcγRIIb was successfully established,and the recombinant protein with biological function was prepared.

Key words: inhibitory receptor, gene FcγRIIb, prokaryotic expression, recombinant protein

ZHANG Yan-fen¹, LIU Li-peng², CHEN Yao², CUI Zhe², BI Ke-wei², WANG Nan-nan², LIU Zhong-cheng². Prokaryotic Expression and Activity Identification of Rat sFcγRIIb Gene[J]. Biotechnology Bulletin, 2016, 32(9): 165-171.

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