| Author |
Hu, M. D. Yang, Y. He, B. F. Yao, W. Wang, Q. Xu, J. C. Wang, G. S. Mao, M. Xu, J. |
| Description |
Country affiliation: China Author Affiliation: Hu MD ( Department of Respiratory Medicine, Respiratory Research Institute, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.); Wang GS ( Department of Respiratory Medicine, Respiratory Research Institute, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.); Xu J ( Department of Respiratory Medicine, Respiratory Research Institute, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.); Yao W ( Department of Respiratory Medicine, Respiratory Research Institute, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.); He BF ( Department of Respiratory Medicine, Respiratory Research Institute, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.); Yang Y ( Department of Respiratory Medicine, Respiratory Research Institute, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China.); Mao M ( Department of Respiratory Medicine, The 324th PLA Hospital, Chongqing, China.); Wang Q ( Department of Cardiology, The 59th PLA Hospital, Kaiyuan, Yunnan, China.); Xu JC ( Department of Respiratory Medicine, Respiratory Research Institute, The Second Affiliated Hospital, Third Military Medical University, Chongqing, China jianchengxucn@yeah.net.) |
| Abstract |
The aim of this study was to separate, purify, and identify Salmonella paratyphi A flagellin, and to prepare its antisera. Primary flagellin was isolated from S. paratyphi A using the acid lysis method. The flagellin was purified with weak anion exchange chromatography and the protein was identified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and negative staining with phosphotungstic acid with scanning electron microscopy (SEM). The production of the obtained flagellin was then quantified. New Zealand white rabbits were then immunized with the isolated flagellin, the presence of serum anti-flagellin antibodies was assessed with the immunoblot test, and its potency was determined with the double immunodiffusion test. The results of SDS-PAGE showed that the molecular weight (m.w.) of the purified flagellin was 52 x 10(3). The immunoblot test also showed a band at 52 x 10(3) m.w. The SEM results showed that the flagellin was filamentous. These three results showed that the protein was homogeneous. The protein quantification analysis found that 4.8 ± 0.5 mg flagellin could be extracted per 1 g wet weight bacteria. The titer of the anti-flagellin antiserum was 1:64. Through this method, we obtained high productions of flagellin, which could be easily purified, identified, and prepared into high titer antiserum. |
| Subject Keyword |
Flagellin Immunology Isolation & Purification Immune Sera Salmonella Paratyphi A Metabolism Animals Blotting, Western Chromatography, Ion Exchange Complex Mixtures Electrophoresis, Polyacrylamide Gel Ultrastructure Microscopy, Electron, Scanning Rabbits Research Support, Non-u.s. Gov't Discipline Genetics Discipline Molecular Biology Discipline Bioinformatics |
