| 单增李斯特氏菌溶血素基因的克隆及原核表达 |
| |
| 引用本文： | 吴晓薇,徐成刚,马保华,江经纬,叶贺佳,区燕宜,徐小芹,廖明.单增李斯特氏菌溶血素基因的克隆及原核表达[J].中国动物检疫,2011,28(8):46-50. |
| |
| 作者姓名： | 吴晓薇 徐成刚 马保华 江经纬 叶贺佳 区燕宜 徐小芹 廖明 |
| |
| 作者单位： | 1. 华南农业大学兽医学院,广东广州,510642
2. 南海出入境检验检疫局,广东佛山,528200 |
| |
| 基金项目： | 广东出入境检验检验局科技项目 |
| |
| 摘 要： | 参考GenBank收录的单增李斯特菌Hly基因序列,设计1对引物,采用PCR技术扩增出单增李斯特氏菌的溶血素基因Hly(不含有信号肽部分),得到一条1590bp的条带。将其连入pMD18-T载体,经酶切、PCR鉴定和序列测定法进行鉴定。测序正确后,将该基因插入到pET-28a中构建原核表达载体pET-28a-sHly,将重组质粒转化到大肠杆菌BL21(DE3),经IPTG诱导,将诱导产物用SDS-PAGE和Western-blot鉴定。结果显示,Hly基因可以在大肠杆菌中获得表达,表达产物分子质量约为65kU,与预期蛋白质分子质量大小一致。经Western-blotting鉴定可知,诱导表达产物以可溶形式存在,可被兔抗LM阳性血清特异识别,具有较好的抗原活性,为进一步研制基于溶解素蛋白的诊断抗原和特异性单克隆抗体,开展LM的致病与免疫机理研究奠定基础。
|
| 关 键 词： | 单核细胞增生性李斯特氏菌 溶血素基因 克隆 原核表达 |
Cloning and Prokaryotic Expression of Hemolysin Gene of Listeria monocytogenes |
| |
| Wu Xiaowei,Xu Chenggang,Ma Baohu,Jiang Jingwei,Ye Heji,Qu Yanyi,Xu Xiaoqin,Liao Ming.Cloning and Prokaryotic Expression of Hemolysin Gene of Listeria monocytogenes[J].China Journal Of Animal Quarantine,2011,28(8):46-50. |
| |
| Authors: | Wu Xiaowei Xu Chenggang Ma Baohu Jiang Jingwei Ye Heji Qu Yanyi Xu Xiaoqin Liao Ming |
| |
| Institution: | Wu Xiaowei1,2,Xu Chenggang1,3,Ma Baohua4,Jiang Jingwei1,Ye Hejia1,Ou yanyi1,Xu Xiaoqin1,Liao Ming1,3 (1.College of veterinary medcine,South China Agricultural University,Guangzhou 510642,China,2.Guangdong Entry-Exit Inspection and Quarantine Bureau,Guangzhou 510623,3. Guangdong Key lab of prevention and control of zoonosis,4Nanhai Entry-Exit Inspection and Quarantine Bureau,Foshan,Guangdong 528200,China) |
| |
| Abstract: | In this study, the Hly gene ofListeria monocytogenes train C53005 was amplified by PCR using designed primers. Then, the gene was cloned into pMD18-T vector, and indentified by digestion with restriction endonuclease, PCR and sequencing. After the sequences were confirmed correct, a prokaryotic expression vector was constructed by inserting the gene into pET-28a vector. The recombinant plasmid pET-28a- silly was transformed into E.coli BL21 （DE3） competent cells, and was induced with 1PTG. The expressed product was analysed by SDS-PAGE and Western-blot. In result, the Hly gene was highly expressed in E.coli and the expressed protein was 65 Ku in size as expected. Western-blot test demonstrated that the expressed protein could specifically react with rabbit anti-LM serum. The results showed the expressed protein was in soluble form with satisfactory antigenicity. |
| |
| Keywords: | Listeria monocytogenes Hemolysin Gene cloning Prokaryotic expression |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
| 点击此处可从《中国动物检疫》浏览原始摘要信息 |
| 点击此处可从《中国动物检疫》下载免费的PDF全文 |
|
