人重组LIGHT-Fc分子的真核表达及其抗肿瘤活性研究

目的：构建人LIGHT-Fc基因的真核表达质粒，并表达获得有较高生物学活性的纯化人LIGHT-Fc融合蛋白。方法：从cDNA文库中PCR扩增LIGHT全长编码基因，并导入克隆载体。测序证实后，继续用PCR扩增其膜外区cDNA，用重叠延伸技术(SOE)引入cD137信号肽基因。将SOE产物与hlgG1Fc基因一起装入真核表达载体pcDNA3中，瞬时转染293T细胞，用夹心ELISA检测其表达情况，经蛋白A亲和纯化，用SDS-PAGE检测其纯度与Mr，并以肿瘤细胞为靶细胞，检测其生物学活性。结果：测序证实构建的LIGHT与LIGHT-Fc cDNA阅读框完整，连接部位序列正确；ELISA和SDS-PACE证实LICHT-Fc融合蛋白的表达与纯度；MTT证明人LIGHT-Fc蛋白对一些肿瘤细胞系具有生长抑制作用。结论：获得了有抗肿瘤活性的纯化hLIGHT-Fc融合蛋白，为探讨LIGHT在免疫自稳调节中的地位、结合相应受体后的动力学与信号转导机制，以及探索LIGHT的其他生物学功能奠定了坚实的实验基础。

Eukaryotic expression of human LIGHT-Fc chimeric molecule and its anti-tumor activity

AIM: Construct eukaryotic expression plasmid of hLIGHT-Fc gene and express hLIGHT-Fc fusion protein with high biological activity. METHODS: Utilizing PCR to clone the human LIGHT cDNA from a normal human activated T cells cDNA library phAD. CAD, then using SOE (splicing by overlap extension) technique to fuse CD137 signal peptide gene into LIGHT extramembrane domain encoding sequence, inserting this recombinated LIGHT cDNA with human IgGI Fc cDNA into the eukaryotic expression plasmid pcDNA3, expressed it transiently in 293T cells, and purified by recombinated protein A affinity chromatography column, at last we checked its Mr, purity and anti-tumor activity. RESULTS: LIGHT-Fc gene's ORF is right and coincident with we expected, then its expression was confirmed by ELISA and SDS-PAGE, and we proved that the purified LIGHT-Fc protein could inhibit the growth of some cultured tumor cell lines by MTT. CONCLUSION: we got a purified recombinated protein of LIGHT-Fc with anti-tumor activity and the anticipated Mr, thus lay a foundation for further works on LIGHT such as its role in immune homeostasis,the mechanism of its receptors' signal transduction, et al.