副溶血性弧菌直接溶血毒素基因的原核表达及产物的性质

Expression of thermostable direct hemolysin gene of Vibrio parahaemolyticus in E.coli and activity of ex-pressed product

目的实现副溶血性弧菌直接溶血毒素tdh基因在大肠杆菌中的表达,鉴定表达产物的生物学活性和免疫原性。方法构建tdh基因的原核表达系统NWⅡtdh/Trc99A/JM109,并在30℃条件下,用0.8mmol/LIPTG诱导表达。用SDS-PAGE分析蛋白表达;溶血试验检测表达蛋白的溶血活性。将表达产物腹腔注射免疫兔,用试管凝集反应试验检测特异性抗体。用溶血抑制试验检测该抗体体外对TDH溶血活性的抑制作用。结果NWⅡ在上述条件下诱导后,TDH呈可溶性、融合性表达。SDS-PAGE表明,表达蛋白的相对分子质量Mr约23000。薄层扫描显示,细菌的周质腔中的蛋白量最高,占总蛋白的13.4%。溶血试验表明,表达的蛋白具有溶血活性。用表达蛋白免疫兔产生的相应抗体,在体外具有抑制TDH溶血活性的作用。结论tdh基因在原核表达系统Trc99A/JM109中获得成功表达,为基因工程大规模制备TDH抗原,制备抗TDH的多克隆和/或单克隆抗体,构建基因工程菌苗,阐明该菌的致病机制打下了基础。

Aim To realize the expression of tdh gene in E.coli and identify the bioactivity of the expr essed product.Methods Recombinant bacterial NWⅡ(tdh /Trc99A /JM109)was induced by 0.8mmol /L IPTG at the temperature of 30℃.Hemol ytic test was done in vitro.The rabbits were immunized with NWⅡ.Agglutination test was used to dete ct the polyclonal antibodies.The hemo lytic suppression test was used to detect suppressive effect of the ant ibodies on hemolytic activity of TDH.Results SDS -PAGE analysis of bacterial crude extracts showed that the relative molecular mass(M r )of the expressed protein in soluble a nd fused form was 23000.The amount of exogenous protein in periplasmic space was 13.4%of the total bacterial prot ein.The result of hemolytic testshowed that the protein had hemolyti c activity.The serum of immunized rabbits could suppress the hemolyti c activity of TDH.Conclusion The above findings suggested that tdh ge ne had been successfully expressed i n Trc99A /JM109and paid the preliminary foundation for preparing anti -TDH poly -and /or monoclonal antibod ies used to the diagnosis,con-structing the vaccine candidate and elucidating pathogenesity of Vibrio parahaemolyticus.