Cloning of extracellular domain fragment of human LIGHT gene and expression of fusion protein

Objective: To clone human LIGHT extracellular domain ( hsLIGHT) gene, construct its recombinant expression plasmid and induce the expression of fusion protein using a prokaryotic expression system. Methods:The hsLIGHT gene fragment was amplified by RT-PCR from HL60 cells and subcloned into the prokaryotic expression plasmid pGEX-4T-2 to form pGEX-4T-2/hs-LIGHT. After recombinant plasmid was induced by different concentrations of IPTG,the expressed proteins were analyzed by SDS-PAGE and confirmed by Western blot. Results:The sequence of hsLIGHT gene amplified by RT-PCR was the same as the sequence of LIGHT extracelluar domain in gene map of Genbank;SDS-PAGE and Western blot analysis showed that a protein was expressed,the molecular weight of this protein was 47 000,which was the same as the fusion protein GST-hsLIGHT. Conclusion;The hsLIGHT was cloned and its recombinant expression plasmid was constructed successfully. The fusion protein GST-hsLIGHT was successfully expressed in the prokaryotic expression system E. coli BL21 induced by IPTG. This research laid a foundation for further studying on its antitumor effects and exploring new ways of immunotherapy of tumor.