Tat融合蛋白表达载体TAT-KLF4的克隆化及蛋白表达研究  

Construction of TAT-KLF4 vector and expression of fusion protein

作　　者：王建军[2] 万志红 赵平 靳雪源 谢国明 辛绍杰 

机构地区：[1]解放军医学院,北京100853 [2]解放军第302医院

出　　处：《中华实验和临床病毒学杂志》2015年第2期180-182,共3页Chinese Journal of Experimental and Clinical Virology

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-KLF4,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KLF4的开放读码框基因序列,酶切并连接到原核表达载体PET-28b-TAT-V2上,得到重组表达载体TAT-KLF4,转化大肠埃希菌,IPTG诱导TAT-KLF4融合蛋白的表达.表达产物用SDS-PAGE鉴定,亲和层析柱纯化融合蛋白,并应用Western Blot检测蛋白的特异性,应用免疫荧光检测融合蛋白转导HSF细胞的效果.结果 成功构建了TAT-KLF4融合蛋白的原核表达载体,在诱导下获得了高效表达并纯化了融合蛋白,Western Blot实验提示获得了特异性的融合蛋白.免疫荧光提示融合蛋白可快速转导入HSF细胞内.结论 为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-KLF4 and express the Fusion Protein in E.coli BL21.Methods The open reading frame（ORF） of KLF4 gene was amplified by PCR.The PCR product was digested with restriction enzymes then was inserted into PET-28b-TAT-V2 which contained protein transduct domain of TAT.The recombinant vector was transfected into E.coli BL21 and the expression of the TAT-KLF4 fusion protein was induced with IPTG.The fusion protein was analyzed by using SDS-PAGE and purified by the 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.Results The recombinant expression vector TAT-KLF4 was correctly constructed and fusion Proteion TAT-KLF4 was successfully expressed in prokaryotic cells.Western blotting showed the specificity of the fusion protein.The immunofluorescence prompt fusion protein transduced into HSF cells quickly.Conclusion This study provides the material basis for further induction of the induced pluripotent stem cells by protein transduction.

关 键 词：蛋白质KLF4 干细胞 蛋白质结构四级 遗传载体 PROTEIN KLF4 

分 类 号：R3411[医药卫生—基础医学]