禽多杀性巴氏杆菌C48-1株OmpA基因表达与抗原性鉴定

Prokaryotic expression and antigenicity analysis of the Omp A gene of avian Pasteurella multocida strain C48-1

为了研究禽多杀性巴氏杆菌(Pm)外膜蛋白A(Omp A)的免疫原性,根据C48-1菌株Omp A基因序列设计一对特异性引物,采用PCR方法扩增去信号肽的成熟Omp A基因.将去信号肽的成熟Omp A基因扩增片段双酶切后,克隆到p GEX-6p-1表达载体中,构建重组表达质粒p GEX-Omp A,转化宿主菌BL21并经异丙基硫代半乳糖苷(IPTG)诱导表达.SDS-PAGE的结果显示,融合蛋白GST-Omp A大小约为63 ku,与预期的分子质量相符.免疫印迹分析结果表明,该融合蛋白与鸡抗Pm血清具有明显的免疫反应,说明Omp A具有良好的抗原性,这为禽Pm Omp A在免疫防御研究中的应用奠定了基础.

According to the nucleotide sequencing result of outer membrane protein A（ Omp A） gene of Pasteurella multocida（ Pm）C48-1 strain,a pair of primers were designed. The mature Omp A gene of C48-1 strain was amplified by polymerase chain reaction technique and then cloned into prokaryote expression vector p GEX-6p-1 for sequence analysis. The recombinant plasmid,p GEXOmp A was then transformed into E. coli BL21. After induction with Isopropyl β-D-1-Thiogalactopyranoside（ IPTG）,SDS-PAGE and western blotting results showed that the fusion protein GST-Omp A was about 63 ku and could be detected by chick serum against Pm. So,it was suggested that the Omp A protein was a immunodominance antigen in the process of Pm infection and could be used in the study of immune protection against Pm.