摘要
目的:克隆泛素—核糖体蛋白S27a基因并在大肠杆菌细胞内高效表达,并纯化该蛋白。方法:从HL-60细胞中提取总RNA,通过RT-PCR扩增获得泛素/核糖体蛋白S27a的编码区cDNA,将HindⅢ/NdeⅠ双酶切的PCR产物连接到同样双酶切的GST融合表达载体pGEXGST构建为融合表达质粒pGEXGST-S27a,转化感受态的大肠杆菌JM109实现质粒的扩增与纯化,经DNA测序确证后再转化感受态表达菌BL21,表达并纯化出融合蛋白。结果:(1)PCR扩增获得长约560bp的泛素—核糖体蛋白S27a基因片段。(2)SDS-PAGE证明,泛素—核糖体蛋白S27a-GST融合蛋白的分子量为43kDa。(3)通过过柱法纯化出泛素—核糖体蛋白S27a-GST融合蛋白。结论:成功克隆出泛素-核糖体蛋白S27a的基因,并在表达菌BL21中可见GST融合表达,并纯化出该融合蛋白,为进一步研究其功能获得了候选蛋白分子。
Objective： To clone and express the ubiquitin - ribosomal protein S27a protein from HL- 60 cells in E. coli., purification the fusing protein in vitro. Methods： Extract total RNA from HE- 60 cells. The target ubiquitinribosomal protein S27a＇s cDNA was obtained by RT- PCR amplifying the total RNA. Hind Ⅲ/Nde Ⅰ- digested PCR products were directionally cloned into fusing vector pGEXGST which had been digested by Hind Ⅲ/Nde Ⅰ. The recombinant plasmid was named as pGEXGST - S27a plasmid which followed by Hind Ⅲ/Nde Ⅰ - digested determined and DNA sequenced. The pGEXGST - S27a plasmids were transformed into the competent B1221 and the fusing proteins were obtained and purified. Resuh： The ubiquitin- ribosomal protein S27a＇ s cDNA fragment （about 560 bp） was obtained by PCR and the GST fusion protein expressed in the transformants was approximate 43kDa as determined in SDS - PAGE analysis. The GST fusing protein was purificated by GST eolunms. Conclusion： The ubiquitin - ribosomal protein 827a was highly expressed in E. coll. and the GST fusing protein was abtained and purified.
出处
《赣南医学院学报》
2005年第5期589-592,共4页
JOURNAL OF GANNAN MEDICAL UNIVERSITY
