摘要
在构建鳗鲡病原性嗜水气单胞菌与爱德华氏菌二联外膜蛋白重组表达载体的基础上,采用不同的诱导剂(IPTG)浓度、诱导温度、诱导时间和诱导时菌液浓度对重组表达载体的表达条件进行探讨。结果表明,不同诱导剂(IPTG)浓度、诱导温度、诱导时间和诱导时菌液浓度对该载体的表达无明显影响。在菌液浓度D600nm=0.8时,采用0.25mmol/L的IPTG经16℃诱导培养过夜后表达。表达产物采用KTApurifier-100蛋白质纯化仪,结合His标签柱亲和层析后获得分子质量87.1ku的高纯度蛋白。蛋白经透析复性后免疫鳗鲡以初步研究其免疫原性。结果表明,免疫后第28天,重组蛋白不同剂量注射组的鳗鲡血清中抗体效价均显著高于PBS注射对照组,初步表明该重组表达蛋白具有良好的免疫原性。
On the basis of the construction of an expressed vector which expresses a bivalent out membrane protein（OMP）of Aeromonas hydrophila and Edwardsiella tarda from diseased eels,optimum expression conditions,including different IPTG concentration,temperature,time and bacteria concentration of the recombinant expression vector were studied.The results indicated that different IPTG concentration,temperature,time and bacteria concentration had no significant influence on the expression of the recombinant protein.A bivalent OMP was expressed by using 0.25mmol/L IPTG to induce the tansformant（E.coli BL21）overnight at 16℃in a suitable bacteria concentration（D600nm=0.8）.The bivalent OMP with a molecular weight of 87.1ku was then purified and obtained by Nickel Chelating Affinity Chromatography withKTApurifier-100 protein purification system.After renaturated by dialysis,the purified protein was injected to American eels（Anguilla rostrata）to study its immunogenicity.The results indicated that the titers of serum antibody in eels at 28 dafter immunization,was significantly（P〈0.05）enhanced in groups injected with different doses of the recombinant protein compare with the PBS injection group,indicating good immunogenicity of the recombinant protein.
出处
《华中农业大学学报》
CAS
CSCD
北大核心
2015年第1期96-102,共7页
Journal of Huazhong Agricultural University
基金
国家自然科学基金项目(31001136)
福建省海洋与渔业厅项目(201212140006)
