大肠杆菌猪水肿毒素B亚单位基因的克隆与鉴定  

Cloning and Identification of the Shiga-like ToxinⅡvB Gene from an Escherichia coli Strain of O_138 Serotype

作　　者：倪振亚[1] 焦新安[1] 高崧[1] 彭大新[1] 张如宽[1] 刘秀梵[1] 

机构地区：[1]扬州大学农业部畜禽传染病学重点实验室,扬州225009

出　　处：《农业生物技术学报》2000年第1期76-78,共3页Journal of Agricultural Biotechnology

基　　金：农业部九五攻关项目;江苏省"青蓝工程基金"资助

摘　　要：根据已知大肠杆菌志贺氏菌样毒素Ⅱ型变异体Ｂ亚单位（ＳＬＴ－ⅡｖＢ）基因序列，设计并合成 ３条２对引物，以大肠杆菌 Ｏ_１３８基因组为模板，通过聚合酶链式反应（ＰＣＲ）分别扩增出 ２０４ ｂｐ的基因序列及包含一段信号肽的 ２６１ ｂｐ基因序列。将这两段 ＤＮＡ分别克隆进表达性载体质粒ＰＹＡ３３３４的ＥｃｏＲⅠ和ＢａｍＨⅠ位点之间，经地高辛标记探针进行Ｓｏｕｔｈｅｒｎ杂交和酶切分析鉴定，并对两条扩增片段进行序列测定。结果表明，扩增产物的大小与设计相符，扩增与克隆的片段为ＳＬＴ－ⅡｖＢ基因的特异性片段。Under the introduction of two pairs of primers (P1. P2) and (P3. P2), a 204 bp DNA fragment and a 261 hp DNA fragment including the signal sequence of shiga-like toxin II variant B gene were amplified from an E. coli strain of O_138 serotype by the polymerase chain reaction. The agarose electrophoresis analysis and Sourthern assay proved the PCR products were the SIT-ⅡvB gene as expected. Then they were inserted into the expression plasmid vector PYA3334 at the EcoRⅠ and BamHⅠ site. The restriction endonuclease analysis and Sourthern assay indicated that the two fragments had been cloned into plasmid PYA3334 successfully. and the sequence showed that the two fragments were the SLT-ⅡvB gene.

关 键 词：大肠杆菌 克隆 序列测定 仔猪 水肿毒素B亚基 

分 类 号：S852.61[农业科学—基础兽医学] S852.44[农业科学—兽医学]