| 羊种布鲁氏菌LpxB基因的克隆、原核表达及其蛋白的生物信息学分析 |
| |
| 引用本文： | 聂鑫,赵天靖,曹瑞勇,彭冬梅,李国华,李亚颖,徐开莲,朱华培,庞峰,王凤阳,杜丽.羊种布鲁氏菌LpxB基因的克隆、原核表达及其蛋白的生物信息学分析[J].中国畜牧兽医,2016,43(7):1681-1687. |
| |
| 作者姓名： | 聂鑫 赵天靖 曹瑞勇 彭冬梅 李国华 李亚颖 徐开莲 朱华培 庞峰 王凤阳 杜丽 |
| |
| 作者单位： | 海南大学农学院, 海南省热带动物繁育与疫病研究重点实验室, 海口市动物基因工程重点实验室, 海口 570228 |
| |
| 基金项目： | 国家自然科学基金（31460670） |
| |
| 摘 要： | 试验旨在克隆羊种布鲁氏菌LpxB基因并进行原核表达和蛋白的生物信息学分析。以布鲁氏菌M5-90株基因组为模板，参照GenBank中M5-90株基因组DNA序列，用DNAMAN软件设计1对引物，通过聚合酶链式反应（PCR）扩增得到大小为1 188 bp的LpxB基因，将其连接入pMD20-T载体上，构建pMD20-T-LpxB重组质粒，将其转化到E.coli DH5α感受态细胞中，经BamH Ⅰ和 Xho Ⅰ双酶切鉴定正确后扩大培养。将BamH Ⅰ和 Xho Ⅰ双酶切获得的LpxB片段连接入pET-28a，构建重组质粒pET-28a-LpxB，转化到E.coli BL21（DE3）中，双酶切鉴定正确后扩大培养。经IPTG诱导其表达，用SDS-PAGE和Western blotting对蛋白进行鉴定。运用DNAMAN、BioEdit等软件对LpxB基因编码的氨基酸序列进行分析。结果表明，本研究成功克隆了LpxB基因并进行了蛋白表达，在LpxB蛋白二级结构中，α-螺旋、伸展链、β-折叠和无规卷曲分别占52.41%、14.94%、8.10%和24.55%。
|
| 关 键 词： | 羊种布鲁氏菌 LpxB基因 克隆 原核表达 生物信息学分析 |
| 收稿时间： | 2015-11-16 |
Cloning,Prokaryotic Expression and Bioinformatics Analysis of LpxB Gene of Brucella melitensis |
| |
| NIE Xin,ZHAO Tian-jing,CAO Rui-yong,PENG Dong-mei,LI Guo-hua,LI Ya-ying,XU Kai-lian,ZHU Hua-pei,PANG Feng,WANG Feng-yang,DU Li.Cloning,Prokaryotic Expression and Bioinformatics Analysis of LpxB Gene of Brucella melitensis[J].China Animal Husbandry & Veterinary Medicine,2016,43(7):1681-1687. |
| |
| Authors: | NIE Xin ZHAO Tian-jing CAO Rui-yong PENG Dong-mei LI Guo-hua LI Ya-ying XU Kai-lian ZHU Hua-pei PANG Feng WANG Feng-yang DU Li |
| |
| Institution: | Key Laboratory of Animal Genetic Engineering of Haikou City, Key Laboratory of Tropical Animal Reproduction & Breeding and Epidemic Disease Research of Hainan Province, College of Agriculture, Hainan University, Haikou 570228, China |
| |
| Abstract: | The study was aimed to clone and express LpxB gene,and perform the bioinformatics analysis of protein.The genomic DNA of Brucella melitensis M5-90 was used as template.According to the genome sequence of M5-90 on GenBank,a pair of primers was designed.LpxB gene,which was 1 188 bp,was amplified by PCR,and was ligated into pMD20-T vector.The constructed recombinant plasmid pMD20-T-LpxB was transformed into E.coli DH5α.The recombinant plasmid was confirmed by endonuclease digestion and sequencing.The coding region of LpxB from pMD20-T was digested by BamHⅠ and XhoⅠ.Then,the fragment was inserted into prokaryotic expression vector pET-28a,and the positive plasmid was named pET-28a-LpxB.The pET-28a-LpxB was transformed into E.coli BL21 (DE3).The expressed protein was identified by SDS-PAGE and Western blotting.DNAMAN and BioEdit softwares were used to analyze the sequence of amino acids encoded LpxB gene.The results showed that the CDS of LpxB was successfully cloned and expressed.The secondary structure of LpxB protein consisted structure α -helix,extended strand,β-turn and random coil which accounted for 52.41％,14.94％,8.10％ and 24.55％,respectively. |
| |
| Keywords: | Brucella melitensis LpxB gene cloning prokaryotic expression bioinformatics analysis |
| 本文献已被 CNKI 等数据库收录！ |
| 点击此处可从《中国畜牧兽医》浏览原始摘要信息 |
| 点击此处可从《中国畜牧兽医》下载免费的PDF全文 |
|
