猪源多杀性巴氏杆菌ompH基因的克隆、表达

Cloning and Expression of the ompH Gene of Pasteurella multocida

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1114bp(ORF为960bp),并克隆到载体pMD18T(TVector),测序表明该基因相当保守。用pET28b构建了原核表达载体pET28bompH,转化BL21并诱导表达,SDSPAGE结果显示表达蛋白约为35ku,与报道大小相近。Westernblot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。

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.