论文部分内容阅读
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 a method for obtaining low concentration of endotoxin and high titer Acinetobacter baumannii phage to provide a reference for the preparation of safe phage biological preparation. Methods The phage lysate was concentrated in an ultrafiltration centrifuge tube with a molecular weight of more than 100 kD and the endotoxin with a molecular weight of about 10 kD was filtered off. The phage concentrate was then purified by sucrose density gradient centrifugation. The purified phage titer was determined by 鲎 test before and after ultrafiltration endotoxin concentration by SDS-PAGE analysis before and after ultrafiltration and purified phage protein purity. Results The titer of phage was increased from 3.9 × 1010 PFU / mL to 1.68 × 1012 PFU / mL by ultrafiltration centrifugation and 99.2% of the endotoxin could be removed. Seven proteins were clearly expressed by SDS-PAGE after ultrafiltration and density gradient centrifugation , The molecular weight of 29 ~ 100 kD. Conclusion Ultrafiltration combined with density gradient centrifugation is a simple, rapid method for the concentration and purification of phage, and can effectively remove endotoxin in lysates.