可溶型小鼠细胞因子LIGHT/TNFSF14的原核表达、纯化及活性验证  

Prokaryotic Expression,Purification and Activity Verification of Soluble Murine Cytokine LIGHT/TNFSF14

作　　者：颜晓庆 刘飞 刘庆亚 寇艳波 YAN Xiaoqing;LIU Fei;LIU Qingya(Xuzhou Medical University,Xuzhou 221004,China)

机构地区：[1]徐州医科大学,江苏徐州221004

出　　处：《中国医学创新》2018年第32期27-31,共5页Medical Innovation of China

基　　金：国家自然科学基金青年基金(81700774);徐州医科大学优秀人才科研启动基金(53591518)

摘　　要：目的:前期研究发现小鼠细胞因子LIGHT(TNFSF14)能够抑制脂肪前体细胞分化,表达纯化LIGHT并验证其活性,将为进一步明确其调控脂肪前体细胞分化的机制提供坚实的基础。方法:以小鼠脾脏总cDNA为模板,通过PCR扩增得到LIGHT胞外段编码序列,并将其克隆至pGEX4T-1,经菌液PCR、限制性内切酶酶切及测序验证后获得重组质粒pGEX4T-1-LIGHTc。重组质粒转化至大肠杆菌BL21(DE3),经IPTG诱导蛋白表达后SDS-PAGE检测菌体破碎液上清中可溶型LIGHTc的存在。含有重组蛋白的上清经亲和纯化后获得GST-LIGHTc重组蛋白,重组蛋白经凝血酶消化后得到游离型LIGHTc单体,最后通过结肠癌细胞生长抑制实验验证LIGHTc单体的活性。结果:通过大肠杆菌异源表达和亲和纯化获得了GST-LIGHTc重组蛋白,经凝血酶处理后得到有活性的游离型小鼠LIGHT蛋白。结论:通过大肠杆菌异源表达及亲和纯化获得有活性的小鼠LIGHTc重组蛋白,为进一步研究LIGHT影响脂肪前体细胞分化的分子机制奠定了重要的材料基础。ObjectiveIn our previous research,we find the murine LIGHT（TNFSF14） can inhibit preadipocytes differentiation to mature adipocytes.To futher illuminate the mechanism how LIGHT inhibits preadipocytes differentiation,the extracellular section coding sequence of LIGHT was cloned and heterologously expressed in E.coli.Method：The LIGHT coding sequence was amplified from spleen total cDNA by PCR and then cloned into prokaryotic expression vector pGEX4T-1.After verified with restriction enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21.The transformed BL21 was treated with IPTG to induce the expression of recombinant protein GST-LIGHTc.Before activity detection,the GST-LIGHTc protein was digested by thrombin to aquire dissociated LIGHTc.Result：Gst-lightc recombinant protein was obtained by heteroexpression and affinity purification of escherichia coli,and the active free mouse LIGHT protein was obtained by thrombin treatment.Conclusion： The soluble recombinant murine LIGHTc with biological activity is obtained with E.coli heterologous expression and affinity purification.This will lay the foundation for illuminating the mechanism of LIGHT regulating preadipocytes differentiation.

关 键 词：LIGHT 肿瘤坏死因子 异源表达纯化 亲和纯化 

分 类 号：R589.2[医药卫生—内分泌]