畜牧兽医学报 ›› 2005, Vol. 36 ›› Issue (7): 701-704.doi:

• 兽医 • 上一篇    下一篇

猪源多杀性巴氏杆菌ompH基因的克隆、表达

吴 斌;唐先春;陈焕春;王大鹏   

  1. 华中农业大学动物病原微生物实验室, 武汉 430070
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2005-07-25 发布日期:2005-07-25

Cloning and Expression of the ompH Gene of Pasteurella multocida

WU Bin;TANG Xian-chun;CHEN Huan-chun;WANG Da-peng
  

  1. Animal Pathogenic Microorganism Lab, Huazhong Agricultural University, Wuhan 430070, China
  • Received:1900-01-01 Revised:1900-01-01 Online:2005-07-25 Published:2005-07-25

摘要: 利用已分离的菌株030224HB,根据NCBI上的序列(U52208)设计了一对引物,用PCR方法扩增了猪源多杀性巴氏杆菌的外膜蛋白基因(ompH),扩增的片段大小为1 114 bp(ORF为960 bp),并克隆到载体pMD18-T(T-Vector),测序表明该基因相当保守。用pET-28b构建了原核表达载体pET28b-ompH,转化BL21并诱导表达,SDS-PAGE结果显示表达蛋白约为35 ku,与报道大小相近。Western-blot结果表明表达的蛋白质具有生物学活性,然后用所表达的蛋白做了ELISA检测方法的初步探讨。

Abstract: Using the isolated strain of 030224HB and a set of specific primers, the ompH gene of P. multocida was amplified by PCR. The primers were designed based on the published sequences of the ompH gene. The 1 114 bp amplified DNA fragment was cloned into pMD18-T. The sequencing result indicated that this gene was quite conservative. The ompH gene in pMD18-T was ligated into pET28b to get the expressing vector of pET28b-ompH which was then transformed into E.coli BL21. The result of SDS-PAGE shows the expressed protein is about 35 ku, resembled to the published researches. Western-blot results indicates that this protein possesses biological activity. Using the expressed protein, ELISA to detect pasteurellosis of pigs is expected to develop.