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目的 纯化副溶血性弧菌直接溶血相关毒素 (TRH)蛋白 ,鉴定表达产物的生物学活性和免疫原性 ,为构建副溶血性弧菌快速检测方法奠定物质基础。方法 构建trh基因的原核融合表达系统PRⅡ (trh/pGEX 3X/DE3) ,在 31℃条件下 0 6mmol/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。将表达产物皮下注射免疫兔 ,溶血抑制实验检测该抗体体外对TRH溶血活性的抑制作用。结果 PRⅡ在上述条件下诱导后 ,TRH呈可溶性、融合性表达。溶血试验表明 ,表达的蛋白具有溶血活性。用表达的蛋白免疫兔产生的相应抗体 ,在体外具有抑制TRH溶血活性的作用。结论 trh基因在原核融合表达系统pGEX 3X/DE3中获得成功表达 ,并得到纯度较高的TRH蛋白 ,为基因工程大规模制备高纯度TRH抗原、制备抗TRH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奠定了基础
Objective To purify the direct hemolytic related toxin (TRH) protein of Vibrio parahaemolyticus, and to identify the biological activity and immunogenicity of the expressed product, so as to lay a material foundation for the rapid detection of Vibrio parahaemolyticus. Methods The prokaryotic expression system PR Ⅱ (trh / pGEX 3X / DE3) of trh gene was constructed and induced by 0 6mmol / L IPTG at 31 ℃. Protein expression was analyzed by SDS PAGE and hemolysis was tested for hemolysis activity of the expressed protein. The expression product was injected subcutaneously into rabbits, and the hemolytic inhibition test was used to test the inhibitory effect of the antibody on TRH hemolytic activity. Results After induced by PRⅡ under the above conditions, TRH was soluble and confluent. Hemolysis tests showed that the expressed protein has hemolytic activity. The corresponding antibodies produced by immunization of rabbits with the expressed protein have the effect of inhibiting TRH hemolytic activity in vitro. Conclusion The trh gene was successfully expressed in the prokaryotic expression system pGEX 3X / DE3, and the highly purified TRH protein was obtained. For the purpose of genetic engineering, high purity TRH antigen was prepared in large scale and polyclonal and / or monoclonal antibody against TRH , Construction of genetically engineered vaccine, clarify the pathogenic mechanism of the bacteria laid the foundation