鸭多杀性巴氏杆菌外膜蛋白H基因的表达及抗原性鉴定

Expression and antigenicity analyasis of the Omp H Gene of Pasteurella multocida

为了对多杀性巴氏杆菌(Pm)外膜蛋白基因H(OmpH)的免疫学活性进行鉴定,本研究根据已发表的Pm70株序列(AE004439)设计了两对引物,用PCR方法扩增了鸭多杀性巴氏杆菌C48-102株的Omp H,扩增片段为1180bp(ORF为1056bp)。Omp H与GenBank中登录的C44-1、P-1059、P52、X-73、PM-70、serogroup D的序列比对结果表明:Omp H在核苷酸水平上同源性为82.8%～99.2%;在氨基酸水平上同源性为82.1%～99.1%。扩增去信号肽的Omp H基因,构建了原核重组表达质粒pHT-Omp H,转化BL21并诱导表达,SDS-PAGE结果表明:表达蛋白约为41ku,与预期的分子量大小相符;western blot结果表明具有良好的免疫学活性,这为以重组OmpH蛋白建立针对鸭Pm的血清学检测方法奠定了基础。

To evaluate the immunologic competence of the recombinant Omp H protein of Pasteurella multocida（Pm） expressed in E.coli,the Omp H gene of C48-102 strain was amplified by PCR using a set of primers based on the sequences of Pm70 in GenBank.The 1180 bp PCR fragment contained an open reading fragment of 1056 bp,which shared sequence identities of 82.6 %-99.2% at nucleotide level and 82.1 %-99.1 % at amino acid level with C48-102,C44-1,P-1059,P52,X-73,PM-70 and serogroup D,respectively.The Omp H fragment with deleted signal peptide was cloned into the expression vector of pHT-Omp H and expressed in E.coli BL21.SDS-PAGE showed that the expressed protein was about 41 ku and possessed biological activity of Omp H gene by western blot detection.