人LIGHT胞外区基因的克隆及融合蛋白的表达

目的：克隆人LIGHT胞外区基因（hsLIGHT），构建其原核表达质粒，并诱导其在大肠杆菌中表达融合蛋白。方法：采用RT-PCR方法从HL60细胞中扩增hsLIGHT，将其克隆人原核表达质粒pGEX-4T-2，构建其重组表达质粒pGEX-4T-2／hsLIGHT，以不同浓度IPTG诱导表达，于不同时间经SDS-PAGE和Western blot分析、鉴定。结果：经RT-PCR扩增获得的hsLIGHT序列与Genebank报道的LIGHT基因胞外区序列完全一致；SDS-PAGE和Westernblot分析证实重组质粒可表达出相对分子量为47000的蛋白，与GST-hsLIGHT融合蛋白分子量一致。结论：成功完成了人LIGHT胞外区基因的克隆及其原核表达质粒的构建，在大肠杆菌E-coli BL21中经IPTG诱导表达了融合蛋白GST-hsLIGHT，为进一步探讨LIGHT的抗肿瘤生物学活性、探索肿瘤免疫治疗新方法奠定了基础。

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.