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| 产志贺毒素大肠杆菌毒素基因的敲除 | |
| 引用本文： | 潘虹,朱善元,成大荣,王晨娟,左伟勇,洪伟鸣,贾宁.产志贺毒素大肠杆菌毒素基因的敲除[J].扬州大学学报(农业与生命科学版),2012,33(1):1-5. |
| 作者姓名： | 潘虹 朱善元 成大荣 王晨娟 左伟勇 洪伟鸣 贾宁 |
| 作者单位： | 1. 甘肃农业大学动物医学院,甘肃兰州,730070 2. 江苏畜牧兽医职业技术学院,江苏泰州,225300 3. 扬州大学兽医学院,江苏扬州,225009 |
| 基金项目： | 江苏省农业科技支撑计划项目 |
| 摘 要： | 根据GenBank上致仔猪水肿病大肠杆菌志贺毒素(Stx2e)A单位基因序列(M21534)以及Red重组系统试剂盒提供的FRT-PGK-gb2-neo-FRT序列设计1对引物;引物的5′端与Stx2e基因同源,3′端与FRT-PGK-gb2-neo-FRT序列同源。使用合成的引物利用PCR扩增FRT-PGK-gb2-neo-FRT序列片段。将扩增的PCR片段转化带有pRedET重组质粒的产志贺毒素大肠杆菌(STEC)S451521菌株(F18+)的感受态细胞中,通过同源重组技术替换Stx2eA单位基因的805～1 152bp区域。通过卡那霉素抗性以及PCR检测获得同源重组的STEC后,将708-FLPe质粒进一步转化重组菌,利用Flp重组酶去除了重组菌的卡那霉素抗性。另外,研究还发现Stx2e基因在STEC基因组中并非单拷贝。 |
| 关 键 词： | 大肠杆菌 仔猪水肿病志贺毒素 基因敲除 |
Knockout of the gene coding for Stx2e of Shiga toxin-producing Escherichia coli |
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| PAN Hong , ZHU Shan-yuan , CHENG Da-rong , WANG Chen-juan , ZUO Wei-yong , HONG Wei-ming , JIA Ning.Knockout of the gene coding for Stx2e of Shiga toxin-producing Escherichia coli[J].Journal of Yangzhou University:Agricultural and Life Science Edition,2012,33(1):1-5. | |
| Authors: | PAN Hong ZHU Shan-yuan CHENG Da-rong WANG Chen-juan ZUO Wei-yong HONG Wei-ming JIA Ning |
| Institution: | 1(1.Coll of Vet Med,Gansu Agric Univ,Lanzhou 730070,China; 2.Jiangsu Anim Husb and Vet Coll,Taizhou 225300,China; 3.Coll of Vet Med,Yangzhou Univ,Yangzhou 225009,China) |
| Abstract: | A set of primers homologous to shigatoxin 2e(Stx2e) gene and FRT-PGK-gb2-neo-FRT gene,were designed and synthetized according to the published sequences of stx2e A(accession No.M21534) and FRT-PGK-gb2-neo-FRT gene.The FRT-PGK-gb2-neo-FRT gene was then amplified by PCR with the primers.The PCR products were introduced into Shiga toxin-producing Escherichia coli(STEC) S451521 which has plasmid pRedET by electroporation.With the help of Red recombinant system,the 805—1 152 bp region of Stx2e A genes were replaced.Following the recombinations of STEC selected by kanamycin-resistance and identification of PCR,the plasmid 708-FLPe was introduced into the recombinant to eliminate the kanamycin gene by Flp recombinase.In addition,the data also revealed that the Stx2e genes were not single copy in STEC genome. |
| Keywords: | Escherichia coli Shiga toxin2e knockout of gene |
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