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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 ,这为进一步的LIGHT基因的功能研究打下了基础
Objective To clone the LIGHT gene and construct an expression vector containing human LIGHT gene. The LIGHT gene was induced to express soluble in Escherichia coli and the biological activity of LIGHT protein was detected. Methods The full-length LIGHT cDNA and its extracellular region were cloned from human peripheral blood mononuclear cells. The extracellular region of the LIGHT cDNA was subcloned into the prokaryotic expression vector pET 11a. The positive recombinant plasmid pET LIGHT was selected and induced by IPTG Soluble expression, and SDS PAGE and Western blot detection analysis. The expressed protein was initially purified and analyzed for biological activity. Results The cDNA with LIGHT full length of 72 3bp was amplified by RT PCR. SDS PAGE and Western blot analysis confirmed that the recombinant pET LIGHT plasmid expressed a protein with a relative molecular mass of 19 × 10 3. Soluble LIGHT recombinant protein can co-stimulate the proliferation of T cells and induce IFNγ production. Conclusion The LIGHT extracellular region was successfully expressed in E. coli and the expressed protein had biological functions, which laid the foundation for further study on the function of LIGHT gene