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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。
Objective To obtain the expression of direct hemolytic toxin tdh gene of Vibrio parahaemolyticus in Escherichia coli and to identify the biological activity and immunogenicity of the expressed product. Methods The prokaryotic expression system of tdh gene NWⅡtdh / Trc99A / JM109 was constructed and induced with 0.8mmol / L IPTG at 30 ℃. Protein expression was analyzed by SDS-PAGE; hemolysis was tested for hemolysis activity of the expressed protein. The expressed products were injected intraperitoneally into rabbits and the specific antibodies were detected by tube agglutination test. The hemolytic inhibition test was used to examine the inhibitory effect of the antibody on TDH hemolytic activity in vitro. Results After induction of NWⅡ under the above conditions, TDH was soluble and confluent. SDS-PAGE showed that the relative molecular mass of the expressed protein Mr was about 23000. Thin layer scanning showed that the bacteria in the periplasm of the highest amount of protein, accounting for 13.4% of total protein. Hemolysis tests showed that the expressed protein has hemolytic activity. The corresponding antibodies produced by immunized rabbits with the expressed protein have the effect of inhibiting TDH hemolysis activity in vitro. Conclusion The tdh gene was successfully expressed in the prokaryotic expression system Trc99A / JM109. To produce TDH antigens for large-scale genetic engineering, polyclonal and / or monoclonal antibodies against TDH were prepared and the engineered bacterins were constructed to elucidate the pathogenesis of this bacterium Laid the foundation.