人Oct4与细胞穿膜肽融合蛋白的表达

Expression of human Oct4 and cell penetrating peptide fusion protein

目的通过原核表达获得人Oct4(hOct4)与细胞穿膜肽融合蛋白,优化其表达方法并观察其穿膜效果。方法通过基因工程手段构建了pET原核表达载体,利用BL21(DE3)和Rosetta2(DE3)蛋白表达菌表达蛋白。Ni亲和纯化方法纯化蛋白,蛋白质印迹分析检测融合蛋白组成。将融合蛋白用罗丹明染色后加入人正常皮肤成纤维细胞株BJ观察其穿膜进入细胞情况。结果构建了pET21a(+)-hOct4-11R-His和pET21a(+)-EGFP-11R-His表达载体,转入大肠杆菌中后诱导获得了hOct4-11R-His和EGFP-11R-His融合蛋白,经蛋白质印迹分析检测表明融合蛋白正确。经BJ细胞测试,观察到融合蛋白进入细胞中。结论成功获得了hOct4-11R-His和EGFP-11R-His融合蛋白,且融合蛋白能够高效进入BJ细胞并聚集于细胞核周围。

Objective To express the fusion protein of hOct4 and cell penetrating peptides using prokaryotic expression systems,and to optimize its expression methods and observe the membrane penetrating ability of the fusion proteins.Methods The pET-based prokaryotic expression system was constructed by genetic engineering,and the fusion plasmid was transferred into E.coli BL21（DE3） and Rosetta2（DE3）.The protein was purified by Ni affinity chromatography and identified by Western blotting analysis.The penetrating ability of the Rhodamine-labelled fusion protein was investigated using BJ cells.Results We successfully constructed pET21a（＋）-hOct4-11R-His and pET21a（＋）-EGFP-11R-His vectors.Fusion proteins hOct4-11-His and EGFP-11R-His were generated by transfering the plasmids into E.coli.The fusion protein was verified by Western blotting analysis and was detected in BJ cells.Conclusion We have successfully generated EGFP-11R-His and hOct4-11R-His fusion proteins,and the proteins can effectively enter the BJ cells and locate around the nuclei.