人的核糖体蛋白S3a基因克隆、表达、纯化与亚细胞定位
Cloning,expression,purification and subcellular localization of human ribosomal protein S3a
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摘要: 目的 克隆人核糖体蛋白S3a (Ribosomal protein S3a, RPS3a)基因,并表达、纯化其蛋白,分析RPS3a蛋白的细胞定位,为其功能研究奠定基础。 方法 应用RT-PCR从人胚肾HEK293细胞的总RNA中反转录并扩增出RPS3a基因,克隆到原核表达载体PET-21a中,转化大肠杆菌(Escherichia coli)BL21(DE3)进行蛋白表达。在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下,PET21a-RPS3a融合表达载体在大肠杆菌菌株BL21(DE3)中以可溶性蛋白的形式高效表达RPS3a蛋白,超声破碎细胞,通过Ni2+-NTA亲和层析柱初步纯化,再应用50 kD超滤膜进一步纯化。用Western Blot印迹实验检测纯化后的蛋白。构建绿色荧光蛋白(Enhanced Green Fluorescent Protein,EGFP )的pEGFP-N1-RPS3a表达载体,转染HEK293细胞,通过荧光显微镜观察RPS3a重组荧光蛋白在细胞内的分布。 结果 获得了较高纯度的RPS3a蛋白,亚细胞定位分析表明RPS3a蛋白主要分布于细胞质。 结论 成功克隆了RPS3a基因并表达纯化了其蛋白,确定其亚细胞分布,为进一步研究RPS3a蛋白的性质和功能奠定了基础。Abstract: Objective To express and purify the human ribosomal protein S3a and study the subcellular localization of human ribosomal protein S3a. Methods The RPS3a gene was obtained by RT-PCR, total RNA extracted from human HEK293 cell as the template,and cloned into the prokaryotic expressing vector PET-21a to form PET21a-RPS3a, then transferred into (Escherichia coli)BL21(DE3). PET21a-RPS3a was highly expressed in (Escherichia coli)BL21(DE3) in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG) and most products existed in a soluble form. After ultrasonication, recombinant fusion proteins were purified by Ni2+-NTA affinity chromatography and 50 kD ultrafiltration membrane, the purity of RPS3a was further confirmed by Western Blot analysis. The full sequence of RPS3a gene was recombined in the downstream of the green fluorescent protein gene in the EGFP-N1 vector.The recombined pEGFP-N1-RPS3a vector was transfected into human kidney epithelial cells (293 cells), through the subcellular localization, which was observed RPS3a recombinant fluorescent protein in the cell distribution. Results The higher purity of recombinant fusion protein was obtained. Subcellular localization analysis confirmed that RPS3a recombinant fluorescent protein distributed mainly in the cytoplasm. Conclusion The recombinant fusion protein PET21a-RPS3a was successfully cloned, expressed, purified. Application of subcellular localization raises the foundation to the study on nature and function of human RPS3a protein.
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