Tat融合蛋白表达载体TAT-c-Myc的克隆化及蛋白表达研究  

Construction of Vector TAT-c-Myc and expression of fusion protein

作　　者：王建军[1] 万志红[2] 赵平[1] 靳雪源[1] 谢国明[2] 辛绍杰[2] 

机构地区：[1]解放军第302医院国际肝病诊疗中心,北京100039 [2]解放军第302医院肝衰竭诊疗与研究中心,北京100039

出　　处：《中华临床医师杂志（电子版）》2015年第5期72-75,共4页Chinese Journal of Clinicians(Electronic Edition)

基　　金：军队"十二五"重大课题(BWS11J075)

摘　　要：目的构建重组表达载体TAT-c-Myc,在E.coli BL21中高效表达并纯化融合蛋白。方法经PCR获得编码人c-Myc的全基因序列,含有设计的限制性酶切位点的产物连接到含有TAT转导结构的原核表达载体PET-28b-TAT-V2上,得到重组表达载体TAT-c-Myc,转化大肠杆菌,应用IPTG诱导TAT-c-Myc融合蛋白的表达。表达产物用SDS-PAGE鉴定,亲和层析柱纯化,并应用Western blot检测蛋白的特异性,融合蛋白转导人皮肤成纤维(HSF)细胞的效果应用免疫荧光检测。结果成功构建了TAT-c-Myc融合蛋白的原核表达载体,在诱导下获得了高效表达。Western blot检测提示融合蛋白有良好的特异性。免疫荧光检测提示融合蛋白具有快速转导入HSF细胞内的能力。结论为进一步的通过蛋白转导方式诱导多能干细胞提供了物质基础。Objective To construct recombinant expression vector TAT-c-Myc and express the fusion protein in E. coli BL21.Methods C-Myc fragment were amplified by PCR. Products containing designed enzyme restriction cutting sites connected to prokaryotic expression vector PET-28b-TAT-V2 which containing TAT transduction structures, and get the recombinant expression vector TAT-c-Myc. The recombinant vector was transformed into E. coli BL and induced with IPTG to express fusion protein. SDS-PAGE method was used to analyze the expression fusion protein. The highly expressed product was purified by affinity chromatography. Western blot was used to identify the specificity of the fusion protein. The immunofluorescence was used to detection the efficiency of fusion protein transducted to HSF cells.ResultsThe recombinant expression vector TAT-c-Myc was correctly constructed and the fusion protein TAT-c-Myc was successfully expressed in prokaryotic cells. Western blotshowed the specificity of the fusion protein. The immunofluorescence suggested the fusion protein has the ability to rapidly be transducted into HSF cells.Conclusion This has provides the material basis for the further induced pluripotent stem cells by protein transduction.

关 键 词：原癌基因蛋白质C-MYC 多能干细胞 蛋白转导结构域: 载体构建 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]