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目的提纯甲型副伤寒沙门菌鞭毛蛋白并鉴定。方法选用甲型副伤寒沙门菌,经自制的液体培养基扩大培养后,酸裂解法初步分离出鞭毛蛋白,用AKTAExplorer蛋白纯化系统除盐,弱阴离子交换层析纯化。纯化的蛋白用SDS-PAGE、Westernblot和磷钨酸负染扫描电子显微镜(scanningelectronmicrocopy,SEM)观察并摄片鉴定。考马斯亮蓝法测定所获得鞭毛蛋白的产量。结果SDS-PAGE提示纯化的鞭毛蛋白为一条相对分子质量(Mr)为52×103蛋白带;免疫印迹试验亦提示条带在Mr52×103处;SEM观察发现该鞭毛蛋白呈丝状;三者均证实该蛋白为同一均质蛋白。蛋白定量测得每克湿重的细菌可提取(4.8±0.5)mg鞭毛蛋白。结论经酸裂解法可获得高产量的鞭毛蛋白,且易于纯化和鉴定。
Objective To purify and identify Salmonella paratyphi A flagellin protein. Methods The Salmonella paratyphi A strain was cultured in homemade liquid medium. The flagellin protein was isolated by acid-cleavage method and purified by AKTAExplorer protein purification system and weak anion exchange chromatography. The purified protein was observed by scanning electron microscopy (SEM) and identified by SDS-PAGE, Western blot and phosphotungstic acid staining. Coomassie brilliant blue method was used to determine the production of flagellin. Results SDS-PAGE indicated that the purified flagellin was a 52 × 103 protein with a molecular weight of 52 × 103. Immunoblotting also indicated that the band was at Mr52 × 103. SEM showed that the flagellin was filamentous. Confirmed that the protein is the same homogeneous protein. Protein quantification Per gram of wet bacteria can extract (4.8 ± 0.5) mg of flagellin. Conclusion The high yield of flagellin was obtained by acid cleavage and was easily purified and identified.