山羊Klf4基因克隆、原核表达和His-Klf4融合蛋白纯化  

Cloning and Prokaryotic Expression of Capra hircus Klf4 , and Purification of His-Klf4 Fusion Protein

作　　者：辛桂瑜[1] 王丽霞[1] 叶新慧[1] 申魁魁[1] 符欣蕙[1] 李恭贺[1] 张明[1,2] 卢晟盛[1,2] 卢克焕[1,2] 郑喜邦[1] 

机构地区：[1]广西大学动物科技学院,南宁530004 [2]亚热带农业生物资源保护与利用国家重点实验室,南宁530004

出　　处：《生物技术通报》2013年第2期80-85,共6页Biotechnology Bulletin

基　　金：广西自然科学基金项目(2011GXNSFD018018);亚热带农业生物资源保护与利用国家重点实验室开放课题(SB0907)

摘　　要：旨在克隆山羊Klf4基因,构建重组质粒pET30a-Klf4,通过原核表达技术获得纯化的His-Klf4融合蛋白。从山羊的生殖脊中提取总RNA,用RT-PCR的方法扩增Klf4基因编码区序列,通过TA克隆构建pMD18-T-Klf4重组质粒。酶切鉴定与测序分析后将Klf4的cDNA亚克隆至pET-30a载体,构建重组质粒pET30a-Klf4。酶切鉴定后将其导入大肠杆菌BL21中,用IPTG诱导表达,SDS-PAGE和Western blotting检测确认,镍离子金属螯合亲和层析法纯化His-Klf4融合蛋白。结果表明:(1)从山羊生殖脊中克隆了Klf4基因,其开放阅读框由1 434个核苷酸组成,编码478个氨基酸,而且该序列与绵羊的同源性最高,达到98.5%;(2)经过SignalP 4.0在线分析,Klf4的编码蛋白不存在信号肽;(3)经SDS-PAGE和Western blotting分析表明,重组质粒pET30a-Klf4在大肠杆菌中得以表达;在变性条件下纯化,获得了His-Klf4融合蛋白。The paper was to clone Klf4 gene of Capra hircus, and construct recombinant plasmid pET3Oa-klf4, and finally to prepare the purified His-klf4 fusion protein by means of prokaryotic expression techniques. Total RNA extracted from the genital ridge of fetal Capra hircus, Klf4 cDNA was amplified by RT-PCR and the plasmid pMD18-T-Klf4 was constructed by TA cloning. After restriction endonuclease digestion and sequencing analysis, Klf4 cDNA was subcloned to pET-30a vector to obtain a recombinant plasmid pET30a-Klf4. Transformed into E. coli BL21, the recombinant plasmid pET30a-Klf4 was induced to express by IPTG, which was verified by SDS-PAGE and Western blotting analysis. Finally, the His-Klf4 fusion protein was purified via metal Nickel-chelating affinity chromatography. The results showed that ： （ I ） the Klf4 gene was successfully cloned from promodial genital ridges of fetal Capra hircus ; the open reading frame （ ORF ） of Capra hircus Kff4 is composed of 1434 nucleutide acids, coding 478 amino acids ; comparison of sequence similarity of Capra hircus Klf4 homologue with other vertebrate Klf4 homologues indicated that Capra hireus shared the highest homology （ 98.5% ） with Ovis aries. （ 2 ） Signal peptide prediction by SignalP 4.0 online showed that there was no signal peptide in the coding proteins of Capra hircus Klf4. （ 3 ） SDS-PAGE and Western blotting assay showed that the recombinant plasmid was expressed in E.coli BL21, and His-Kff4 fusion protein was purified under denature condition.

关 键 词：KLF4 分子克隆 原核表达 蛋白纯化 山羊 

分 类 号：S827[农业科学—畜牧学] Q78[农业科学—畜牧兽医]