Sox2-R_9-EGFP融合蛋白的表达及穿膜能力鉴定

Expression of Sox2-R_9-EGFP Fusion Protein and Validation of its Transduction Activity

构建、表达、纯化、鉴定Sox2-R9-EGFP融合蛋白,验证其在体外培养的成纤维细胞中的转导活性。以胎猪原始生殖嵴为材料提取总RNA,反转录成cDNA第一链,设计带9聚精氨酸(R9)的特定引物,从cDNA中扩增出猪Sox2基因,克隆到pET-28a-EGFP载体中,经酶切和测序鉴定后,重组质粒转化大肠杆菌BL21,经IPTG诱导表达,Ni2+柱亲和层析纯化和SDS-PAGE检测表达产物后,将Sox2-R9-EGFP蛋白作用于体外培养的猪胎儿成纤维细胞中以观察其转导活性。成功构建Sox2-R9-EGFP融合蛋白原核表达载体,纯化后蛋白经SDS-PAGE检测证实融合蛋白片段大小正确,加到培养的猪胎儿成纤维细胞中可观察到Sox2-R9-EGFP融合蛋白能进入细胞,且部分可定位于细胞核。获得了Sox2-R9-EGFP原核蛋白表达载体,R9能介导Sox2蛋白进入细胞,为进一步利用重组蛋白建立诱导性多潜能干细胞(Induced pluripotent stem cells,iPS细胞)的研究奠定了基础。

To construct,express,purify,identify the Sox2-R9-EGFP fusion protein and validate its transduction activity in the cultured porcine embryo fibroblasts（PEFs）.cDNA of pig Sox2 gene was amplified by RT-PCR with specific primers of 9 arginines（R9） from the primordial genital ridges and inserted into vector pET-28a-EGFP.After restriction endonuclease digestion and DNA sequencing confirmation,the recombinant plasmid was then transformed into BL21（Escherichia coli） strain.After IPTG induction,the expressed Sox2-R9-EGFP was purified by Novagen His-Bind kit and confirmed by SDS-PAGE.Then fusion proteins were added to cultured porcine embryo fibroblasts to validate its transduction activity.The prokaryotic expression vector for the Sox2-R9-EGFP fusion protein was successfully constructed,and the target fusion proteins were efficiently induced to express by IPTG.The purified Sox2-R9-EGFP was validated by SDS-PAGE.And purified fusion proteins could enter cultured PEFs efficiently,and parts of them were located in cell nuclear.The prokaryotic expression vector for the Sox2-R9-EGFP is obtained.The transduction activity of Sox2-R9-EGFP fusion protein to cells is proved in this experiment and it greatly facilitates the further study of establishing induced pluripotent stem cells by recombinant protein.