维氏气单胞菌JLL-1株外膜蛋白的原核表达及其免疫原性检测  

Prokaryotic expression and immunogenicity detection of outer membrane protein of Aeromonas veronii JLL-1 isolated from common carp

作　　者：张冬星[1] 康元环[1] 陈亨利[1] 张海月[1] 田佳鑫[1] 单晓枫[1] 钱爱东[1] 

机构地区：[1]吉林农业大学动物科学技术学院,吉林长春130118

出　　处：《中国兽医学报》2017年第11期2131-2136,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31201927);吉林省重点科技攻关项目(20150204065NY)

摘　　要：为研究维氏气单胞菌(Aeromonas veronii)外膜蛋白(outer membrane protein,OMP)的免疫原性,本试验对A.veronii的OmpAⅠ基因进行克隆,构建了原核表达质粒pET-30a(+)-OmpAⅠ,并转入大肠杆菌BL21(DE3),将其表达后纯化重组蛋白,利用SDS-PAGE和Western blot分析鉴定。将灭活苗、弗氏佐剂(OmpAⅠ)、纯化蛋白及PBS缓冲液分别免疫鲤鱼后,检测每周各试验组鱼体血清非特异性免疫指标(酸性磷酸酶、碱性磷酸酶、超氧化物歧化酶活力)及特异性IgM抗体水平变化情况,并于加强免疫2周后对鲤鱼进行攻毒保护性试验。结果显示:重组蛋白免疫鲤鱼后能产生明显的免疫反应,保护率在77%以上;受免鲤鱼血清中IgM抗体水平、酸性磷酸酶、碱性磷酸酶及超氧化物歧化酶活力活力较对照组有显著提高。结果表明,重组蛋白OmpAⅠ具有较好的免疫原性和保护作用,为进一步研究外膜蛋白基因工程疫苗的开发奠定基础。To investigate the immunogenicity of Aeromonas veronii outer membrane protein in this study. The OrnpA I gene was amplified from the genomic DNA of A. veronii. Prokaryotic expression vector pET-30a （＋）was used to construct a recombinant expression vector pET-g0a （＋）- OmpA I ,which was transformed into Escherichia coli BL21（DE3）. The expressed recombinant protein was purified and identified by Western blot. Using the inactivated bacteria,Freund＇s adju- vant, purified protein and sterile PBS immunized carp, respectively. The acid phosphatase,alkaline phosphatase,superoxide dismutase activities and IgM antibody levels were tested weekly. Two weeks after the booster immunization, carps were challenged by artificial infection. The results showed that significant immunity were induced by the recombinant protein, and the recorded relative percent survivals （RPS）of the recombinant protein group was 77%. There were significantly higher IgM antibody levels, acid phosphatase, alkaline phosphatase and superoxide dismutase activities in the immunized fish compared with the control group. The results of this study showed that recombinant protein OmpA I has a strong immunogenicity and protective effect,laying the foundation for further study on outer membrane protein genetic engineering vaccine.

关 键 词：雏氏气单胞菌 外膜蛋白AI 原核表达 免疫原性 

分 类 号：S852.61[农业科学—基础兽医学]