嗜水气单胞菌TPS-30株丝氨酸蛋白酶基因与溶血素基因在大肠杆菌中的融合表达

嗜水气单胞菌丝氨酸蛋白酶和溶血素是该菌重要的致病因子与保护性抗原。致病性嗜水气单胞菌TPS-30株为江浙一带鱼类暴发病病原主要血清型O:9的代表株。研究利用PCR方法扩增嗜水气单胞菌TPS-30株的丝氨酸蛋白酶基因(Spe)和溶血素基因(Hly),将基因Spe和Hly通过柔性片段进行融合,并将融合片段插入pET32a的多克隆位点,构建成重组融合表达载体pET32a-Spe-Hly。将重组载体转化大肠杆菌BL21(DE3),经异丙醛-β-D-硫代半乳糖苷(IPTG)诱导表达,获得融合蛋白Spe-Hly。表达产物经SDS-PAGE检测,显示与预期大小约130kD相吻合的融合蛋白带。纯化融合蛋白并对鲫鱼进行免疫攻毒试验。结果表明,丝氨酸蛋白酶基因和溶血素基因融合表达载体构建成功,并成功获得了融合蛋白Spe-Hly,对鲫鱼的免疫保护率达81.4%。这为基因工程亚单位多价疫苗的开发提供基础。

FUSION EXPRESSION OF SPE GENE WITH HLY GENE OF AEROMONAS HYDROPHILA STRAIN TPS-30 IN ESCHERICHIA COLI

Serine protease (Spe) and hemolysin (Hly) are important pathogenic factors and protective antigens of Aero-monas hydrophila. The pathogen A. hydrophila TPS-30 strain was assigned to serogroup O: 9 of the Sakazaki and Shi-mada (National Institutes of Health) scheme, which caused hemorrhagic septicemia in Jiangsu Province. In this study, two pairs of primers were designed and synthesized according to the sequence of gene Spe and lily. The Spe and lily DNA fragments were amplified by PCR Spe from A. hydrophila TPS-30 genomic DNA respectively. The subsequent sequence analysis revealed the Spe gene of TPS-30 has an open reading frame of 1800 bp encoding a 600 amino-acid protein with a molecular weight of 66.6 kD, the Hly gene of TPS-30 has an open reading frame of 1410 bp encoding a 470 amino-acid protein with a molecular weight of 51.7 kD. Gene Spe and Hly were fused and cloned into expression vector pET32a (+) at the site of polyclone resulting to recombinant vector pET32a-Spe-Hly, gene Hly lying in 5' end and gene Spe in 3' end. The expression vector pET32a-Spe-Hly were transformed into Escherichia coli BL21 (DE3). After engineered, E. coil were incubated in LB medium at 37 ℃ for 18h, 1% inoculums volume were transferred into LB me-dium to incubate. When A_(600) reached 0.5, engineered E. coli were induced by the isopropyl-(IPTG) at ultimate concentration 0.2 mmol/L with 4h. Fusion protein (Hly-Spe) was expressed. The molecular weight of the fusion engineered protein was about 130 kD, which was identified by sodium dodecyl sulfate polyaerylamide gel electrophoresis (SDS-PAGE). The results showed that Spe-Hly fusion protein expression vector pET32a-Spe-Hly was constructed successfully and the Spe-Hly fusion protein was highly expressed in recombinant strain BL21(DE3) (pET32a-Spe-Hly). To test the effectiveness of the recombinant Spe-Hly as a vaccine, Carassius auratus gibelio were immunised with the recombinant Spe-Hly and then challenged with different bacterial strains. The immune fish were significantly protected from challenges by the homologous strain (A. hydrophila TPS-30) and one serologically different strain (A. hydrophila BSK-10), giving RPS values of 81.4% and 73.1%, respectively, as compared with the control groups (injected with Montanide ISA763A and PBS). All these findings suggest that this recombinant Spe-Hly has the potential to be developed into an effective vaccine in fish against the infections of A. hydrophila.