CAJ | 학술논문

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.
목적 구건중조표체재체TAT-KLF4,재E.coli BL21중고효표체병순화융합단백.방법 경PCR획득편마인KLF4적개방독마광기인서렬,매절병련접도원핵표체재체PET-28b-TAT-V2상,득도중조표체재체TAT-KLF4,전화대장애희균,IPTG유도TAT-KLF4융합단백적표체.표체산물용SDS-PAGE감정,친화층석주순화융합단백,병응용Western Blot검측단백적특이성,응용면역형광검측융합단백전도HSF세포적효과.결과 성공구건료TAT-KLF4융합단백적원핵표체재체,재유도하획득료고효표체병순화료융합단백,Western Blot실험제시획득료특이성적융합단백.면역형광제시융합단백가쾌속전도입HSF세포내.결론 위통과단백전도방식유도다능간세포제공료물질기출.
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.