摘要
根据GenBank中登录的副猪嗜血杆菌外膜蛋白P5(outer membrane protein P5,OMP5)基因序列设计1对特异性引物,以江西分离株NC0807基因组DNA为模板,扩增出OMP5基因。将其克隆到pET-28a(+)中,构建重组表达质粒pET-28a-OMP5,质粒转化大肠杆菌BL21(DE3),通过SDS-PAGE和Western blotting分析重组蛋白的表达情况和反应原性。重组蛋白经镍柱亲和层析纯化后免疫豚鼠,测定其免疫原性和保护效率。结果表明,重组蛋白在大肠杆菌中获得了高效表达。表达的蛋白分子质量约为43 ku,能被副猪嗜血杆菌阳性血清识别。动物试验结果表明,重组蛋白免疫后能诱导产生高水平的OMP5特异性抗体,并可显著保护豚鼠抵抗副猪嗜血杆菌强毒菌株的攻击,提示OMP5是副猪嗜血杆菌的保护性抗原。
A pair of primers was designed according to Haemophilus parasuis outer membrane protein P5 （OMPS） gene sequences published in the GenBank, and the gene of OMP5 was amplified by PCR from the genomic DNA of H. parasuis strain NC0807. The recombinant plasmid pET-28a-OMP5 was constructed by inserting this fragment into plasmid pET-28a （＋）. After identification, the recombinant plasmid was transformed into E. coli BI221 （DE3）. SDS-PAGE and western blotting analyses revealed that the transformed BL21 （ DE3 ） bacteria could express OMP5 with molecular weight of 43 ku after induced by IPTG. Sera were examined for the OMP5 specific antibody titers and all animals were challenged with lethal doses of the highly virulent H. parasuis strain NC0807 after the guinea pigs were immunized with the puri- fied recombinant protein. The results demonstrated that the recombinant protein could induce the production of a high level of OMP5 specific antibody and provided the guinea pigs with significant protection from H. parasuis infection. It suggested that OMP5 is a protective antigen of H. parasuis.
出处
《畜牧与兽医》
北大核心
2012年第4期26-30,共5页
Animal Husbandry & Veterinary Medicine
基金
江西省科技支撑计划项目(2009BNB05703)
关键词
副猪嗜血杆菌
外膜蛋白P5
原核表达
豚鼠
亚单位疫苗
Haemophilus parasuis
outer membrane protein PS
prokaryotic expression
guinea pig
subunit vaccine
