Construction and expression of prokaryotic vector encoding extracellular region of murine LIGHT

Objective To construct a prokaryotic expression vector for the extracellular domain of LIGHT (LIGHTECD) and express it in E.coli. Methods Total RNA was extracted from murine immature bone marrow-derived DCs and the cDNA of extracellular domain of LIGHT was amplified by RT-PCR. The recombinant plasmid pET24-LIGHTECD was constructed by cloning the extracellular domain of LIGHT cDNA into the prokaryotic expression vector pET-24a(+). After the recombinant plasmid was identified by restriction endonuclease digestion analysis and DNA sequencing, the pET24-LIGHTECD was transformed into E.coli BL21 and induced with IPTG. The expressed protein was analyzed by SDS-PAGE and Western blotting. Results A 525 bp of LIGHT extracellular domain cDNA was amplified by RT-PCR and the sequence was confirmed by DNA sequencing. SDS-PAGE and Western blot analysis showed that a protein with molecular weight of 20 000 was expressed in E. coli BL21. Conclusion The extracellular domain of LIGHT is cloned and expressed in E.coli successfully, which lays a basis for the further functional research of LIGHT.