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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hIgG1Fc基因一起装入真核表达载体pcDNA3中 ,瞬时转染 2 93T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 FccDNA阅读框完整 ,连接部位序列正确 ;ELISA和SDS PAGE证实LIGHT 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
OBJECTIVE: To construct eukaryotic expression plasmid of human LIGHT Fc gene and express the purified human LIGHT Fc fusion protein with high biological activity. Methods: The full length LIGHT gene was amplified by PCR from cDNA library and introduced into cloning vector. After the sequencing was confirmed, continue to amplify the cDNA of the outer membrane by PCR, and introduce the CD137 signal peptide gene by the overlap extension technique (SOE). The SOE product was inserted into the eukaryotic expression vector pcDNA3 together with the hIgG1Fc gene and transiently transfected into 293T cells. The expression of SOE and hIgG1Fc gene was detected by sandwich ELISA. The protein was purified by protein A affinity chromatography, Cells are target cells and tested for their biological activity. Results: The LIGHT and LIGHT FccDNA constructs were constructed and sequenced correctly. The expression and purity of LIGHT Fc fusion protein were confirmed by ELISA and SDS PAGE. MTT demonstrated that human LIGHT Fc protein had growth inhibitory effect on some tumor cell lines. CONCLUSION: The purified hLIGHT Fc fusion protein with anti-tumor activity was obtained. To explore the role of LIGHT in the regulation of immune homeostasis, the kinetics and signal transduction mechanism after binding to the corresponding receptor and the exploration of other biological functions of LIGHT A solid experimental foundation