超滤过结合密度梯度离心纯化鲍曼不动杆菌噬菌体方法的建立  

Purification of Acinetobacter baumannii bacteriophages by ultra filtration combined with density gradient centrifugation

作　　者：史红艳[1] 余静丹[1] 王丹[1] 逯茵茵[1] 孙延波[1] 

机构地区：[1]吉林大学白求恩医学院病原生物学系,吉林长春130021

出　　处：《中国微生态学杂志》2013年第3期350-352,共3页Chinese Journal of Microecology

基　　金：国家自然科学基金委主任基金资助课题(81150037):噬菌体在肠源性脓毒血症动物模型中的作用

摘　　要：目的探讨获得低浓度内毒素和高滴度鲍曼不动杆菌噬菌体的方法,为制备安全的噬菌体生物制剂提供参考。方法用可截留100 kD以上分子量的超滤离心管浓缩噬菌体裂解液并滤出分子量约为10 kD的内毒素,然后用蔗糖密度梯度离心纯化噬菌体浓缩液;分别测定超滤前、超滤后和纯化后的噬菌体滴度,采用鲎试验测定超滤前后内毒素的浓度,通过SDS-PAGE分析超滤前后和纯化后噬菌体蛋白的纯度。结果经超滤离心法噬菌体滴度从3.9×1010PFU/mL提高至1.68×1012PFU/mL,并可去除99.2%的内毒素;超滤过结合密度梯度离心后的SDS-PAGE可清晰呈现7种蛋白,分子量为29～100 kD。结论超滤过结合密度梯度离心是一种简便、快速浓缩和纯化噬菌体的方法,并可有效地去除裂解液中的内毒素。Objective To explore an efficient method for obtaining Acinetobacter baumannii phage preparation with high titer and low concentration of endotoxin. Methods Phage preparation were concentrated and the endotoxin was eliminated by ultra filtration. The condensed phages were purified by sucrose density gradient centrifugation. The phage titers were determined before and after the ultra fil- tration and purification. The limulus test was employed to examine the endotoxin concentration before and after ultra filtration. The protein patterns of both crude and purified phages were analyzed using SDS-PAGE. Results The titer of phage was dramatically increased from 3.9 × 10^10 PFU/mL to 1.68 × 10^12 PFU/mL and 99.2% of the endotoxin was eliminated by ultra filtration; SDS-PAGE revealed a total of 7 protein bands with molecular weight ranging from 29 - 100 kD after ultra filtration combined with density gradient centrifugation. Conclusion The ultra filtration combined with sucrose density gradient centrifugation is an efficient method for both the purification of phages and the elimination of endotoxin.

关 键 词：噬菌体 超滤过 内毒素 

分 类 号：R378[医药卫生—病原生物学]