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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。
OBJECTIVE: To construct a recombinant strain BL21 (DE3) (pET-28a-OmpS2) for preparation of genetically engineered vaccine against Edwardsiella spp. In fish. Methods: According to the sequence of OmpS2 gene (AY078509) of pathogenicity-causing Edwardsiella invader reported by GenBank, the OmpS2 gene of OmpS2 gene was amplified by PCR and amplified to 1284bp in length. The gene was cloned into the prokaryotic expression vector pET-28a (+) and transformed into E. coli BL21 (DE3) to obtain the recombinant strain BL21 (DE3) (pET-28a-OmpS2). After induced by IPTG, SDS-PAGE analysis showed Mr at 47000 specific protein bands. Results: The Western blot showed that the expressed outer membrane protein had good reactivity. The recombinant strain BL21 (DE3) (pET-28a-OmpS2) was made into a genetically engineered vaccine which was used in combination with Hly genetically engineered vaccine to immunize mice and Paralichthys olivaceus ), A higher protection rate was obtained. ELISA results showed that both of the genetically engineered vaccines could produce higher antibody levels. Conclusion: The genetically engineered vaccine prepared in this experiment can effectively prevent Edwardsiella spp. In fish and has a high protection rate and can play an immunoprophylactic effect.