哈维氏弧菌胞外蛋白酶基因的克隆表达及免疫原性

CLONING,EXPRESSION,IMMUNOGENICITY OF EXTRACELLULAR PROTEINASE FROM VIBRIO HARVEYI

根据实验室分离的大黄鱼弧菌病主要病原菌哈维氏弧菌(Vib rio harveyi)GYC1108-1株的胞外蛋白酶基因序列,设计胞外蛋白酶基因(ΔProA)特异性引物,扩增获得1552bp的ΔProA基因,克隆于pUC57-T载体;将ΔProA基因亚克隆到原核表达载体pET-28a进行融合表达,SDS-PAGE电泳检测发现,ΔProA融合蛋白经IPTG诱导后在大肠杆菌中以包涵体形式表达,分子量大小约55kD,诱导5h的表达蛋白产量约占细菌总蛋白的21%。Western blot分析,表达的55kD蛋白具有较高免疫原性。用纯化的ΔProA融合蛋白对大黄鱼进行免疫试验,结果免疫保护率达到75%。

Vibrio harveyi is a causative agent of vibriosis that infects large yellow croaker （Pseudosciaena crocea） as well as some other marine fish and brings severe loss to their culture. The extracellular proteases of V. harveyi have a role in the virulence of the organism and are potential candidates for vaccine development. In this study, the specific primers of extracellular protease （△ProA） gene excluding the region coding for signal peptide were designed according to the sequence of V. harveyi strain GYC1108-1 △ProA gene （GeneBank No.： EF126129）, and a piece of DNA sequence about 1552 bp was amplified by PCR from genomic DNA of GYC1108-1 strain. The △ProA gene was cloned into pUC57-T easy vector and sequenced. Then the △ProA gene fragment were subcloned into pET-28a（＋） to construct expression plasmid pET28a-△ProA. It expressed a 55 kD fusion protein HIS-△ProA in E. coli TG1 when induced by isopropyl thiogalactoside （IPTG）. SDS-PAGE analysis showed that the recombinant protein was expressed in the form of inclusion body and accumulated up to 21% of the total bacteria protein. It revealed that, by western blot analysis, the fusion proteins △ProA had immunoreactivity. The purified fusion protein △ProA used as antigen to immunize large yellow croaker （Pseudosciaena crocea） and mice, the relative percentage survival （RPS） were 75% and 91.6% respectively. This result indicates that the △ProA may be one of the important protective antigens of V. harveyi, and may play a role in protecting fish from infection of V. harveyi.