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目的:证实所分离的拟态弧菌为TCP+株,并建立TCP菌毛的提纯方法,为系统地测定其生物学特性,并继而全面揭示拟态弧菌致病机理和研制TCP菌苗奠定理论基础。方法:采用PCR技术、电镜技术和细菌学方法对该拟态弧菌进行毒素共调菌毛(TCP)检测及分离纯化。结果:10 g湿菌体依次经过热洗脱法分离菌毛、30%饱和硫酸铵盐析沉淀蛋白和聚丙烯酰胺凝胶回收纯化蛋白,获得1 mg纯化TCP蛋白,该蛋白的相对分子量为20.14 KD。结论:该菌株为TCP+株;建立了聚丙烯酰胺凝胶回收技术提纯菌毛,取得了良好效果。
OBJECTIVE: To confirm that the isolated Vibrio mimicus is a TCP + strain and to establish a purification method for TCP fimbriae, in order to systematically determine its biological characteristics, and then reveal the pathogenesis of V. mimicus and lay the theoretical foundation for the development of TCP vaccine. Methods: PCR-based, electron microscopy and bacteriological methods were used to detect toxin-co-immunoprecipitated pilus (TCP) and isolated and purified. Results: 10 g wet bacterial cells were isolated by hot elution followed by centrifugation and 30% saturated ammonium sulfate salting-out of precipitated protein and polyacrylamide gel to recover the purified protein to obtain 1 mg of purified TCP protein. The relative molecular weight of this protein was 20.14 KD. Conclusion: The strain is TCP + strain. Polyacrylamide gel recovery technology was used to purify the pili and achieved good results.