单核细胞增生李斯特菌sRNA伴侣分子hfq的基因克隆、表达及纯化  

Gene Cloning,Expression and Purification of sRNA Chaperone hfq of Listeria monocytogenes

作　　者：彭叶龙[1] 乔军[1] 孟庆玲[1] 谢堃[1] 陈诚[1] 刘田莉 马玉[1] 才学鹏[2] 陈创夫[1] 

机构地区：[1]石河子大学动物科技学院,动物疾病防控兵团重点实验室,新疆石河子832003 [2]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《华北农学报》2014年第5期80-84,共5页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31360596;30960274);国家国际科技合作专项(2014DFR31310)

摘　　要：为克隆、表达单核细胞增生李斯特菌sRNA分子伴侣蛋白hfq基因,并纯化重组蛋白,通过PCR的方法从单核细胞增生李斯特菌基因组DNA中扩增出hfq基因,将扩增产物克隆于pMD18-T载体中,测序验证后,再将hfq基因亚克隆至表达载体pET-32a(+)中,成功构建pET-32a(+)-hfq原核表达载体。然后,转化至大肠杆菌BL21(DE3)感受态细胞中,通过IPTG进行诱导表达,对表达蛋白进行可溶性分析,并采用Ni-NTA纯化目的蛋白。结果显示:克隆的hfq基因全长234 bp,编码77个氨基酸,通过SDS-PAGE可以检测到27 kDa的蛋白特异性条带;并从上清中纯化得到了Hfq融合蛋白。为进一步研究该蛋白的生物学功能奠定了基础。In order to clone,express the Listeria monocytogenes sRNA chaprone hfq gene and purify the recombinant protein,the hfq gene was amplified by PCR from LM genome,the amplified products was cloned in pMD18-T vector,sequenced and subcloned in the expression vetor pET-32a（＋）. The recombinant pET-32a（＋）-hfq expression vector was generated successfully and then was transformed into E. coli competent cells of BL21（DE3）,induced by IPTG. The solubility of Hfq protein was analyzed and then the recombinant protein was purified by the NiNTA affinity chromatography. The result showed the hfq gene had a length of 234 bp,encoding 77 amino acids. The specific 27 kDa fusion protein was examined by SDS-PAGE. Finally,the fusion protein was obtained successfully from the supernatant. This study laid the foundation for further study the biological function of the Hfq protein.

关 键 词：单核细胞增生李斯特菌 hfq基因 sRNA伴侣分子 克隆 表达 纯化 

分 类 号：S852[农业科学—基础兽医学] Q78[农业科学—兽医学]