维氏气单胞菌TH0426株外膜蛋白lamB基因的原核表达及其免疫原性分析  

Prokaryotic expression and immunogenicity detection of outer membrane protein lamBgene of Aeromonas veronii TH0426

作　　者：康元环[1] 陈亨利[1] 张冬星[1] 张海月[1] 安鼎杰 田佳鑫[1] 贾俊鹏 孙武文 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12期2333-2338,2343,共7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资助项目(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lamB基因进行了克隆,构建了原核表达质粒pET-30a(+)-lamB,并转化大肠杆菌BL21(DE3),将其表达后纯化重组蛋白Maltoporin,利用SDS-PAGE和Western blot进行分析鉴定。将灭活菌体+弗氏佐剂、纯化的Maltoporin蛋白、Maltoporin蛋白+弗氏佐剂及PBS分别免疫鲤鱼后,检测每周各试验组鱼体血清的免疫指标:酸性磷酸酶、碱性磷酸酶、超氧化物歧化酶活力以及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Maltoporin免疫鲤鱼后能产生明显的免疫反应,保护率在84.2%以上;免疫后鲤鱼血清中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较对照组有显著提高。结果表明,重组蛋白Maltoporin具有较好的免疫原性和保护作用,为进一步研究A.veronii外膜蛋白基因工程疫苗奠定一定的基础。To investigate the immunogenicity of Aeromonas veronii outer membrane protein, the lamb gene was amplified from the genomic DNA of A. veronii TH0426 in this study. Prokaryotic expression vector pET-30a（＋） was used to construct a recombinant expression vector pET-30a （ ＋ ）-lam B, which was transformed into Escherichia coli BL21 （DE3）. The expressed recombinant protein was purified and identified by Western blot. Total four groups fish were immunized with the inactivated bacteria, purified protein Maltoporin, Freund＇s adjuvant and Maltoporin, and sterile PBS,respectively. The activities of acid phosphatase, alkaline phosphatase, superoxide dismutase and IgM were tested weekly. Two weeks after the booster immunization, the carps were challenged by artificial infection. The results showed that significant immunity were induced by the recombi- nant protein,and the recorded relative percent survivals（RPS） of the recombinant protein grou：, was 84.2 %. There were significantly higher levels of IgM, acid phosphatase, alkaline phosphatase and superoxide dismutase in the immunized fish compared with the control group. The results showed that recombinant protein Maltoporin has a strong immunogenicity and protective effect, laying the foundation for further study of outer membrane protein genetic engineering vaccine.

关 键 词：鲤鱼 维氏气单胞菌 外膜蛋白Maltoporin 原核表达 免疫原性 

分 类 号：S852.61[农业科学—基础兽医学] R535[农业科学—兽医学]