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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。
Myc-R9-EGFP fusion protein was constructed, expressed, purified and identified to verify its transduction activity in cultured fibroblasts. Total RNA was extracted from the primordial genital ridge of fetal pigs and the first strand of the cDNA was reverse transcribed. The specific primers of 9-arginine (R9) were designed and the c-Myc gene was amplified from cDNA and cloned into pET-28a-EGFP vector. After digestion and sequencing, the recombinant plasmid was transformed into E. coli BL21 . After induced by IPTG and purified by Ni 2+ affinity chromatography, the protein expression products were detected by SDS-PAGE and Western blot. Myc-R9-EGFP protein was used to observe the transduction activity of porcine fetal fibroblasts in vitro . The results showed that the prokaryotic expression vector of Myc-R9-EGFP fusion protein was constructed correctly, and the target protein was highly expressed after induction. The size of the fusion protein fragment was confirmed by SDS-PAGE and Western blot, and in vitro fibroblasts Confirmed its high transduction activity. This study laid the foundation for the further study of the biological characteristics of Myc protein and the establishment of induced pluripotent stem cells (iPS cells) using recombinant proteins.