大肠杆菌O157:H7的sRNA伴侣蛋白Hfq的基因克隆、表达及纯化  

Cloning,Expression and Purification of sRNA Chaperone Hfq of EHEC O157:H7

作　　者：刘晓霞[1,2] 马延[2] 魏波[2,3] 周围[2] 廖翔[2] 高原[2] 刘虎岐[1] 岳俊杰[2] 梁龙[2] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]军事医学科学院生物工程研究所,北京100071 [3]内蒙古农业大学动物科学与医学学院,内蒙古呼和浩特010018

出　　处：《生物技术通讯》2011年第2期178-181,共4页Letters in Biotechnology

摘　　要：目的:克隆、表达并纯化肠出血性大肠杆菌(EHEC)O157:H7的sRNA伴侣蛋白Hfq。方法:利用PCR方法从EHEC O157:H7基因组中扩增出基因hfq,并插入含6×His标签序列的原核表达载体pET28a(+)的多克隆位点中,构建重组表达质粒pET28a(+)-hfq,以重组质粒转化大肠杆菌BL21(DE3),诱导目的基因表达,采用Western印迹法进行鉴定,通过Ni2+螯合柱纯化获得目的蛋白。结果:质粒酶切鉴定结果表明pET28a(+)-hfq重组质粒构建成功;对目的蛋白进行了融合表达,SDS-PAGE显示相对分子质量约17×103的蛋白特异表达条带;对表达产物进行亲和纯化,从上清中获得了Hfq的融合蛋白。结论:得到了sRNA伴侣蛋白Hfq,为进一步筛选与该蛋白有相互作用的sRNA,研究sRNA的功能奠定了基础。Objective：To express and purify small RNA（sRNA） chaperone Hfq of EHEC O157：H7 in Escherichia coli.Methods：The hfq gene coding Hfq protein was amplified by PCR from EHEC O157：H7 genome and inserted into plasmid pET28a（＋） containing 6×His Tag sequence to construct a recombinant vector pET28a（＋）-hfq.The recombinant vector was transformed into host strain E.coli BL21（DE3）.The fusion Hfq was expressed by IPTG induction and detected by SDS-PAGE and Western blot.The fusion protein was purified by Ni2＋-chelating chromatography.Results：The expression vector was digested by restriction enzyme,and the consequent electrophoresis showed the correct length DNA fragment,therefore,the plasmid pET28a（＋）-hfq was correctly constructed.The 17 kD fusion protein expressed in E.coli BL21（DE3） was examined by SDS-PAGE.Finally,the purified fusion protein was successfully obtained from supernatant.Conclusion：The sRNA chaperone Hfq of EHEC O157：H7 was obtained,which will lay the foundation for screening the interacting sRNA and further research on sRNA＇s function.

关 键 词：大肠杆菌O157∶H7 SRNA Hfq蛋白 基因克隆 表达 

分 类 号：Q78[生物学—分子生物学]