| 马白细胞介素18成熟蛋白(mEIL-18)基因在大肠埃希氏菌中的高效表达与纯化 |
| |
| 引用本文： | 童铁钢,刘光亮,白宇,肖一红,王群,李少英,孟庆文,吴东来.马白细胞介素18成熟蛋白(mEIL-18)基因在大肠埃希氏菌中的高效表达与纯化[J].畜牧兽医学报,2007,38(3):307-312. |
| |
| 作者姓名： | 童铁钢 刘光亮 白宇 肖一红 王群 李少英 孟庆文 吴东来 |
| |
| 作者单位： | 1. 中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,哈尔滨,150001;中国农业科学院研究生院,北京,100081
2. 中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,哈尔滨,150001 |
| |
| 基金项目： | 黑龙江省自然科学基金项目（ZJN-0602-01） |
| |
| 摘 要： | 用RT—PCR从经ConA刺激的马外周血单个核细胞（PBMC）中扩增出马白细胞介素18（Equine interleukin-18，EIL-18）前体蛋白（precursor EIL-18，pEIL-18）基因的cDNA，然后克隆至载体pCR2．1-TOPO中，鉴定并命名为pCR2．1-pEIL-18。自pCR2．1-pEIL-18中扩增马白细胞介素18成熟蛋白（mature EIL-18，mEIL-18）基因，并将其亚克隆至原核表达载体pET-28a（＋）中。将筛选出的阳性克隆进行测序、诱导表达并纯化其表达产物。结果表明，mEIL-18基因全长474bp，含1个开放阅读框，编码157个氨基酸的成熟蛋白；表达产物以可溶性和包涵体两种形式存在，经SDS-PAGE和Western—blot分析，重组蛋白相对分子量约为20ku，且具有免疫生物学活性。mEIL-18基因在大肠杆菌中高效表达并在非变性和变性条件下采用Ni^＋亲和层析纯化方法获得了高纯度的重组mEIL-18，为探究马白细胞介素18的生物学活性及其应用奠定了基础。
|
| 关 键 词： | mEIL-18 克隆 表达 纯化 |
| 文章编号： | 0366-6964（2007）03-0307-06 |
| 修稿时间： | 2006-04-18 |
Expression of Mature Equine Interleukin 18 in Escherichia coli and Its Purification |
| |
| TONG Tie-gang,LIU Guang-liang,BAI Yu,XIAO Yi-hong,WANG Qun,LI Shao-ying,MENG Qing-wen,WU Dong-lai.Expression of Mature Equine Interleukin 18 in Escherichia coli and Its Purification[J].Acta Veterinaria et Zootechnica Sinica,2007,38(3):307-312. |
| |
| Authors: | TONG Tie-gang LIU Guang-liang BAI Yu XIAO Yi-hong WANG Qun LI Shao-ying MENG Qing-wen WU Dong-lai |
| |
| Institution: | 1. National Key Laboratory of Veterinary Biotechnology , Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China ; 2. Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China |
| |
| Abstract: | Using the total RNA extracted from ConA stimulated equine peripheral blood mononuclear cells(PBMC) as template,the cDNA of interleukin l8 was amplified by RT-PCR.The cDNA was subsequently cloned into the vector pCR2.1-TOPO and sequenced.Then the gene encoding mature equine interleukin 18 was amplified from the recombinant plasmid named pCR2.1-pEIL-18 by polymerase chain reaction(PCR) and subcloned into pET-28a( ).The recombinant plasmid pET-mEIL-18 was transformed into E.coli BL21(DE3) after sequence analysis,and a fusion protein was then expressed and purified.Results showed that the fragment of mEIL-18 gene was 474 bp in length,which contained an open reading frame and encoded the protein of 157 aa,and the expressed product existed in soluble portion and inclusion bodies.The SDS-PAGE and Western-blotting analysis indicated that the fusion protein was 20 ku in molecular weight and had immunological activity.The mEIL-18 was expressed successfully in E.coli BL21(DE3),and purified efficiently using Ni column chromatography in non-denatured and denatured conditions,which lays the foundation for investigating the structure and biological effects of EIL18. |
| |
| Keywords: | mEIL-18 cloning expression purification |
| 本文献已被 CNKI 维普 万方数据 等数据库收录！ |
|
