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1、双砷染料四半胱氨酸重组噬菌体准确鉴定细菌的存活状态吴丽娜，杨晓婷，周颖星，黄天逊，郑彦，颜晓梅 *厦门大学化学化工学院化学生物学系，福建 厦门，361005*Email: 采用非跨膜型核酸染料（如propidium iodide, PI），流式细胞术可实现细菌活性的快速检测。然而最新的研究发现，非跨膜型染料的跨膜性能与细菌的生长周期有关，因此，核酸染料的方法并不能准确地分辨活菌和死菌 1。噬菌体是细菌的病毒，能在活的具有感受性的菌体内繁殖。我们实验室通过对噬菌体进行基因改造，构建了双砷染料- 四半胱氨酸重组噬菌体体系，成功地应用于细菌的灵敏、特异检测 2。由于噬菌体只能在活菌中繁殖，而且重组四
2、半胱氨酸标签中的半胱氨酸必须处于还原态才能与双砷染料牢固结合，因此可以利用细菌胞浆的还原环境，通过对重组噬菌体四半胱氨酸的检测实现死菌和活菌的区分。噬菌体入侵活的宿主菌并在其体内快速繁殖，噬菌体衣壳蛋白所表达的四半胱氨酸片断与后续加入的跨膜双砷染料结合，发出强烈的荧光，单个活细菌的信号可用流式细胞仪或荧光显微镜灵敏检测。该方法的确立，将为食品安全、细菌致病机理研究等提供重要的检测手段。关键词：双砷染料；细菌；存活状态；噬菌体；流式细胞仪参考文献1 Shi L. ; Gunther S. ; Hubschmann T. ; Wick L.Y. ; Harms H. ; Muller S. Cyt
3、ometry A 2007, 71, 592-598.2 Wu L.; Huang T.; Yang L.; Pan J.; Zhu S.; Yan X. Angew. Chem. Int. Ed. 2011, 50, 5873 - 5877.致谢：国家自然科学基金（20975087，90913015，21027010，21105082） ，高等学校博士学科点专项基金（20090121110009，20090121120008） 。Tetracysteine-Tagged Phages in Conjunction with Biarsenical Dye for Accurate Asses
4、sment of Bacterial Viability Lina Wu, Xiaoting Yang, Yingxing Zhou, Tianxun Huang, Yan Zhen, Xiaomei Yan*Department of Chemical Biology, College of Chemistry and Chemical engineering, Xiamen University, Xiamen Fujian 361005, ChinaThe viability of bacteria is usually determined by flow cytometry with
5、 commercially available kits that rely on the propidium iodide (PI)-based assessment of membrane integrity. However, recent research poses a query regarding the appropriateness of using PI as a universal indicator for the viability of bacteria. Employing bacteriophages as the specific detection prob
6、es, we have developed a novel strategy (TC-phage-FlAsH) for sensitive and selective bacterial detection. Because phages can only propagate in live bacteria, in present study the TC-phage-FlAsH strategy is exploited for the variability differentiation of bacteria. Upon bacterial infection, phages pro
7、pagate in bacteria and the TC-tag is expressed on the surface of every progeny virion. After staining with a membrane-permeant biarsenical dye, such as fluorescein arsenical helix binder (FlAsH), significant fluorescence enhancement is achieved for the labeled bacteria which are readily detectable by flow cytometry and fluorescence microscopy.
