山羊Klf4 基因克隆、原核表达和His-Klf4融合蛋白纯化

生物技术通报 ›› 2013, Vol. 0 ›› Issue (2): 80-85.

山羊Klf4 基因克隆、原核表达和His-Klf4融合蛋白纯化

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

（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）

收稿日期:2012-08-08 修回日期:2013-02-27 出版日期:2013-02-26 发布日期:2013-02-27
作者简介:辛桂瑜，女，硕士，研究方向：动物干细胞与生物技术；E-mail ：xgy_4321@163.com
基金资助:
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）

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

Xin Guiyu¹ Wang Lixia¹ Ye Xinhui¹ Shen Kuikui¹ Fu Xinhui¹ Li Gonghe¹ Zhang Ming^1，2 Lu Shengsheng^1，2 Lu Kehuan^1，2 Zheng Xibang¹

（1. College of Animal Science and Technology，Guangxi University，Nanning 530004 ；2. State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources，Nanning 530004）

Received:2012-08-08 Revised:2013-02-27 Published:2013-02-26 Online:2013-02-27

摘要/Abstract

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。

关键词: Klf4, 分子克隆, 原核表达, 蛋白纯化, 山羊

Abstract: The paper was to clone Klf4 gene of Capra hircus, and construct recombinant plasmid pET30a-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 ：（1）the Klf4 gene was successfully cloned from promodial genital ridges of fetal Capra hircus; the open reading frame（ORF）of Capra hircus Klf4 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 hircus 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-Klf4 fusion protein was purified under denature condition.

Key words: Klf4, Molecular cloning, Prokaryotic expression, Protein purification, Capra hircus

辛桂瑜, 王丽霞, 叶新慧, 申魁魁, 符欣蕙, 李恭贺, 张明, 卢晟盛, 卢克焕, 郑喜邦. 山羊Klf4 基因克隆、原核表达和His-Klf4融合蛋白纯化[J]. 生物技术通报, 2013, 0(2): 80-85.

Xin Guiyu, Wang Lixia, Ye Xinhui, Shen Kuikui, Fu Xinhui, Li Gonghe, Zhang Ming, Lu Shengsheng, Lu Kehuan, Zheng Xibang. Cloning and Prokaryotic Expression of Capra hircus Klf4，and Purification of His-Klf4 Fusion Protein[J]. Biotechnology Bulletin, 2013, 0(2): 80-85.

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