CAJ | 학술논문

　　目的构建重组表达载体TAT-OCT4，在E.coli BL21中高效表达并纯化融合蛋白。为进一步通过蛋白转导方式诱导多能干细胞提供物质基础。方法经RT-PCR获得编码人OCT4的全基因序列，连接到原核表达载体TAT-V2上，得到重组表达载体TAT-OCT4，转化大肠埃希菌，异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-thiogalactopyranoside，IPTG)诱导TAT-OCT4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人皮肤成纤维(human skin fibroblasts，HSF)细胞的效果。结果成功构建了TAT-OCT4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鉴定正确。免疫荧光提示融合蛋白可快速转导入HSF细胞内。结论 TAT-OCT4融合蛋白可以安全，高效地转导入HSF细胞中。
　　목적구건중조표체재체TAT-OCT4，재E.coli BL21중고효표체병순화융합단백。위진일보통과단백전도방식유도다능간세포제공물질기출。방법경RT-PCR획득편마인OCT4적전기인서렬，련접도원핵표체재체TAT-V2상，득도중조표체재체TAT-OCT4，전화대장애희균，이병기-β-D-류대필남반유당감(isopropylβ-D-thiogalactopyranoside，IPTG)유도TAT-OCT4융합단백적표체。표체산물용SDS-PAGE감정，친화층석주순화융합단백，병응용Western Blot검측단백적특이성，응용면역형광검측융합단백전도인피부성섬유(human skin fibroblasts，HSF)세포적효과。결과성공구건료TAT-OCT4융합단백적원핵표체재체，재유도하획득료고효표체병순화료융합단백，Western Blot감정정학。면역형광제시융합단백가쾌속전도입HSF세포내。결론 TAT-OCT4융합단백가이안전，고효지전도입HSF세포중。
Objective To provide the material foundation for inducing pluripotent stem cells through protein transduction by constructing the recombinant expression vector TAT-OCT4 which can highly express and purify fusion protein in E.coli BL21. Methods The coding human OCT4 gene sequence was obtained by RT-PCR amplification and linked to the prokaryotic expression vector TAT-V2 for the construction of recombinant vector TAT-OCT4. E.coli BL21 was transformed into TAT-OCT4. Expression of TAT-OCT4 was induced with IPTG and identified by SDS-PAGE. The fusion protein was purified by affinity chromatography, its specificity was assayed by Western blot, and its effect on transduction of HSF cells was detected by immunofluorescence. Results The prokaryotic expression vector of TAT-OCT4 fusion protein was successfully constructed, which could highly express and purify the fusion protein as shown by Western blot. Immunofluorescence indicated that the fusion protein could be rapidly transduced into HSF cells. Conclusion TAT-OCT4 fusion protein can be safely and effectively transduced into HSF cells.