Myc-R9-EGFP融合蛋白的表达及穿膜能力鉴定

Expression of Myc-R_9-EGFP Fusion Protein and Validation of Its Transduction Activity

构建、表达、纯化和鉴定Myc-R9-EGFP融合蛋白,验证其在体外培养成纤维细胞中的转导活性。从胎猪原始生殖嵴中提取总RNA,反转录cDNA第一链。设计带九聚精氨酸(R9)的特定引物,从cDNA中扩增出猪c-Myc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,Ni 2+柱亲和层析纯化、SDS-PAGE和Western blot检测蛋白表达产物后,将Myc-R9-EGFP蛋白作用于体外培养的猪胎儿成纤维细胞以观察其转导活性。结果显示:Myc-R9-EGFP融合蛋白原核表达载体构建正确,目的蛋白在诱导后获得了高效表达,经SDS-PAGE和Western blot检测证实融合蛋白片段大小正确,并在体外培养的成纤维细胞中证实了其高转导活性。本实验为进一步研究Myc蛋白的生物学特性和利用重组蛋白建立诱导性多潜能干细胞(iPS细胞)的研究奠定了基础。

To construct, express, purify and identify the Myc-Rg-EGFP fusion protein and vaIidate its transduction activity in the cultured porcine embryo fibroblasts, cDNA of pig c-Myc gene was amplified by RT-PCR with specific primers of 9 arginine （R9） from the primordial genital ridges and inserted into prokaryotic expression vector pET- 28a-EGFP. After DNA sequencing confirmation, the recombinant plasmid was then transformed into BL21 （Esche- richia cold strain. After IPTG induction, the target fusion protein was efficiently induced to express, successfully purified by Novagen His-Bind kit, identified by SDS-PAGE and Western blotting. Finally, its high transduction ac- tivity in the porcine embryo fibroblasts was validated. The purified Myc-Rg-EGFP fusion protein and the validation of its transduction activity in fibroblasts have provided an experimental foundation for further studies on the biological characterization of Myc protein, and soundly facilitated the further study of establishing pig induced pluripotent stem cells by recombinant protein.