PDT/GR-ΔLBD融合蛋白的表达、纯化及穿膜特性的鉴定  

Expression,purification and cell penetrativity of fusion protein PDT/GR-ΔLBD

作　　者：张方[1] 施毅[1] 李子玲[1] 胡波[2] 宋勇[1] 

机构地区：[1]南京军区南京总医院呼吸内科,南京210002 [2]南京军区联勤部卫生部,南京210002

出　　处：《解放军医学杂志》2011年第1期27-30,共4页Medical Journal of Chinese People's Liberation Army

基　　金：中国博士后科学基金(20070411050);江苏省博士后科研资助计划(0701023B)

摘　　要：目的构建穿膜肽(PDT)和缺失结合配体结构域的小鼠糖皮质激素受体(GR-ΔLBD)融合基因表达载体,经原核表达、纯化后,进行穿膜特性的鉴定。方法质粒pEGFP-GR-ΔLBD经双酶切,获得目的基因片段GR-ΔLBD,将该基因片段亚克隆入原核表达载体pGEX-PDT,获得pGEX-PDT/GR-ΔLBD融合基因表达载体。将表达载体转化大肠杆菌,经IPTG诱导表达、谷胱甘肽-琼脂糖树脂处理、谷胱甘肽洗脱液洗脱后,获得PDT/GR-ΔLBD融合蛋白,SDS-PAGE分析融合蛋白;采用荧光素FITC体外标记PDT/GR-ΔLBD融合蛋白(终浓度分别为0、500、1000nmol/L),与小鼠肺上皮细胞TC-1共培养2h,在荧光显微镜下观察其穿膜特性。结果成功构建了融合基因原核表达载体pPDT/GR-ΔLBD,并能表达蛋白产物,大小约78.6kD,与融合蛋白PDT/GR-ΔLBD的理论计算值(80kD)相符。随着PDT/GR-ΔLBD浓度的增加,进入TC-1上皮细胞的PDT/GR-ΔLBD明显增多,并可在胞核中浓聚。结论 PDT/GR-ΔLBD融合蛋白具有良好的可溶性和穿膜特性,为进一步研究PDT/GR-ΔLBD融合蛋白在抗炎过程中的作用奠定了基础。Objective To construct the fusion gene expression vector of penetrating peptide（PDT） and the glucocorticoid receptor lack of ligand binding domain（GR-ΔLBD）,and evaluate the prokaryotic expression,purification and cell penetrativity of fusion protein PDT/GR-ΔLBD.Methods The target gene fragment GR-ΔLBD was obtained from plasmid pEGFP-GR-ΔLBD by double digestion,and sub-cloned into the prokaryotic expression vector pGEX-PDT to construct the fusion gene expression vector pGEX-PDT/GR-ΔLBD.PDT/GR-ΔLBD fusion protein was obtained after the expression vector was transformed into E.coli,followed by sequential induction with IPTG,treatment with glutathione-agarose resin and elution with glutathione.SDS-PAGE was performed to determine the expression of PDT/GR-ΔLBD fusion protein,and it which was diluted into a final concentration of 0,500 and 1000nmol/L,labeled with fluorescein FITC and co-cultivated with TC-1 cells for 2 hours,and the penetrativity was observed by fluorescence microscopy.Results The successfully constructed prokaryotic expression vector pPDT/GR-ΔLBD had the capacity of expressing protein,and it was 78.6kD in molecular weight,which was consistent with the theoretical value（80kD） of the fusion protein PDT/GR-ΔLBD.PDT-GR-ΔLBD,penetrating the nuclear membrane in a concentration-dependent manner,was concentrated within nuclei.Conclusion PDT/GR-ΔLBD fusion protein,with good solubility and cell penetrativity,paves the way for further research on its anti-inflammatory effects.

关 键 词：受体 糖皮质激素 配体结合结构域 胞间信号肽类和蛋白质类 人工基因融合 

分 类 号：R394.26[医药卫生—医学遗传学]