伴放线放线杆菌磷酸胆碱的电泳分析

目的 比较3个磷酸胆碱阳性的伴放线放线杆菌菌株在蛋白酶K作用后电泳结果的变化,分析磷酸胆碱抗原在细菌中的附着结构.方法 将培养收集的伴放线放线杆菌破碎处理后,加入蛋白酶K水解,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE),考玛斯亮蓝染色显示蛋白条带的分布情况,免疫印迹法检测磷酸胆碱的分布情况.结果 伴放线放线杆菌的3个菌株SA716、SA1398、SA2791通过SDS-PAGE电泳,可见未经蛋白酶K处理的细菌悬液显示连续分布的蛋白条带,而蛋白酶K处理后,只显示1条蛋白条带,该条带在只有等量蛋白酶K的对照组也出现,而在未经蛋白酶K处理的细菌悬液中无该条带,可以确定这一条带是蛋白酶K,细菌蛋白都已经被分解.未经蛋白酶K处理的菌株通过SDS-PAGE电泳和磷酸胆碱免疫印迹检测,磷酸胆碱显示阳性结果,磷酸胆碱附着结构分子量大小约为9 kDa,而蛋白酶K处理后,显示阴性结果,磷酸胆碱信号消失.结论 伴放线放线杆菌磷酸胆碱信号在蛋白酶K处理后消失,提示该抗原的附着结构为蛋白成分.

Electrophoresis of phosphorylcholine epitope in Aggregatibacter actinomycetemcomitans

Objective To find out the chemical composition of the antigen reacting with TEPC-15 in Aggregatibacter actinomycetemcomitans（Aa）,and the electrophoresis changes of the proteins and anti-TEPC-15 reactivity with and without proteinase K treatment were compared.Methods Bacteria cells were broken by Branson Sonifier,treated with proteinase K and separated on SDS-PAGE.The gels were first analyzed by Coomassie blue staining.Then the electrophoresis results were transferred onto membrane and immunoblot were done by using mouse anti-phosphorylcholine monoclonal antibody TEPC-15 as the primary antiserum and goat anti-mouse IgA as the secondary antiserum.Results After Coomassie blue staining,the sample without proteinase K treatment showed a variety of bands distributed from low to high molecular weight area.The samples treated with proteinase K revealed a single clear band of 29 kDa,which was not detected in the samples without treatment.This 29-kDa band could also be detected in a control sample with the same amount of proteinase K in PBS but no bacteria.Since the molecular size of proteinase K（Sigma） is 28.93 kDa,the result suggests that proteinase K was the source of the 29-kDa band.Immunoblot analysis showed a single band of 9 kDa with anti-TEPC-15 reactivity without proteinase K treatment and the proteinase K treatment eliminated the TEPC-15 reactivity with A.actinomycetemcomitans lysates.Conclusion The Aa antigen for TEPC-15 is a protein,since proteinase K treatment of the cell lysate abolished the reactivity.