迟缓爱德华菌HB01外膜蛋白OmpS_2基因的克隆表达及其免疫原性研究  

Cloning and expression of a outer membrance protein gene(OmpS_2) of Edwardsiella tarda HB01 and its immunogenicity

作　　者：张亚宁[1] 李晓玥[1] 耿晓娜[1] 赵宝华[1] 

机构地区：[1]河北师范大学生命科学学院,河北石家庄050016

出　　处：《细胞与分子免疫学杂志》2011年第10期1075-1078,1082,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：河北省重大科技攻关项目(06780503)

摘　　要：目的:构建重组菌株BL21(DE3)(pET-28a-OmpS2),制备防治鱼类爱德华氏菌病的基因工程疫苗。方法:根据GenBank报道的致病性迟缓爱德华菌外膜蛋白OmpS2基因(AY078509)序列设计引物,通过PCR扩增得到大小为1284bp的迟缓爱德华菌(E.t.)HB01的外膜蛋白基因OmpS2。将该基因定向克隆到原核表达载体pET-28a(+)中,并转化大肠杆菌BL21(DE3),得到重组菌株BL21(DE3)(pET-28a-OmpS2)。经IPTG诱导后,由SDS-PAGE分析可见Mr在47000的特异蛋白条带。结果:Westernblot检测说明表达的外膜蛋白具有较好的反应原性。将重组菌株BL21(DE3)(pET-28a-OmpS2)制成基因工程疫苗,与嗜水气单胞菌(A.h.)溶血素(Hly)基因工程疫苗联合使用分别免疫小鼠与牙鲆(Paralichthys olivaceus)后,获得了较高的保护率,ELISA实验结果表明两种基因工程疫苗均能产生较高抗体水平。结论:本实验制备的基因工程疫苗能有效的预防鱼类爱德华氏菌病,且保护率高,能够起到免疫预防效果。AIM： To construct a recombinant strain BL21（DE3）（pET-28a-OmpS2）, and obtain a genetic engineering vaccine to provide protective immunity against diseases caused by Edwardsiella tarda. METHODS： According to the GenBank sequences （GenBank Accession No. AY078509）, one pair of primers was designed and the outer membrance protein gene （OmpS2） of Edwardsiella tarda HB01 was amplified by PCR. The OmpS2 gene was cloned in pET-28a vector and transformed into Escherichia coli BL21（DE3）. The OmpS2 protein was highly expressed when the recombinant strain BL21 （DE3） （pET-28a-OmpS2） was induced by IPTG. The expressed protein was 47 kD as estimated by 150 g/L SDS-polyacrylamide gel electrophoresis （SDS-PAGE）. RESULTS： The immunogenicity of the expressed OmpS2 protein was confirmed by Western blotting. Mice and Paralichthys olivaceus were immunized with the genetic engineering vaccines of Edwardsiella tarda and Aeromonas hydrophila, showing promise that all these vaccines have a high protective ability. And the protective ability to Edwardsiella tarda and Aeromonas hydrophila in Paralichthys olivaceus respectively reached 70% and 80%. CONCLUSION： The recombinant strain BL21 （DE3）（pET-28a-OmpS2） could be candidate of genetic engineering vaccine to provide protective immunity against diseases caused by Edwardsiella tarda.

关 键 词：牙鲆 腹水病 迟缓爱德华菌 嗜水气单胞菌 免疫原性 

分 类 号：S941[农业科学—水产养殖]