LIGHT基因的克隆及其可溶性表达

Gene cloning, soluble expression and bioactivity analysis of LIGHT

目的 克隆LIGHT基因 ,构建含有人LIGHT基因的表达载体 ,诱导其在大肠杆菌中可溶性表达 ,并对表达的LIGHT蛋白的生物学活性进行检测。方法 从人的外周血单个核细胞中克隆LIGHT全长cDNA及其胞外区片段 ,并将其胞外区片段亚克隆至原核表达载体pET 11a中 ,筛选阳性重组质粒pET LIGHT ,以IPTG诱导其可溶性表达 ,并以SDS PAGE和Westernblot检测进行分析。表达的蛋白初步纯化后 ,进行生物学活性分析。结果 RT PCR扩增出了LIGHT全长 72 3bp的cDNA。SDS PAGE和Westernblot分析证实重组pET LIGHT质粒可表达出相对分子质量 (Mr)为 19× 10 3的蛋白。可溶性LIGHT重组蛋白可共刺激T细胞的增殖及诱导IFN γ的产生。结论 本实验成功地将LIGHT胞外区片段在大肠杆菌中进行表达 ,表达的蛋白具有生物学功能 。

Objective To clone LIGHT gene, construct LIGHT expressing vector, induce its protein soluble expression and analyze its bioactivity. Methods The full length LIGHT cDNA was amplified by RT PCR from peripheral blood mononuclear cells (PBMCs) and the extracellular fragment of LIGHT was subcloned into pET 11a vector to make a recombinant plasmid pET LIGHT. After the plasmid was induced with IPTG, the expressed protein was analyzed and confirmed by SDS PAGE and Western blot, then its bioactivity was identified. Results A 723bp cDNA was amplified by RT PCR, SDS PAGE and Western analysis showed that a protein with molecular weight of 1.9×10 4 was expressed. Further bioactivity assay indicated that the soluble protein costimulated T cell proliferation and induced IFN γ secretion. Conclusion LIGHT cDNA was successfully cloned and expressed. The bioactivity analysis showed that the protein was involved in the costimulation of T cell response. These results set up a basis for further studies of LIGHT. [