Tat融合蛋白表达载体TAT-OCT4的克隆化及蛋白表达研究

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。

Cloning of TAT fusion protein expression vector TAT OCT4 and its protein expression

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.