制备禽致病性大肠杆菌菌蜕的三种方法比较  

Comparison of three methods for preparation of bacterial ghosts from avian pathogenic Escherichia coli

作　　者：胡剑刚 董洪亮[2] 付立霞[3] 左佳坤 吴小卡 米荣升[2] 黄燕[2] 陆珂[2] 陈兆国[2] 韩先干[2] 胡世君[1] Jian＇gang Hu;Hongliang Dong;Lixia Fu;Jiakun Zuo;Xiaoka Wu;Rongsheng Mi;Yan Huang;Ke Lu;Zhaoguo Chen;Xian＇gan Han;Shijun Hu(College of Animal Science, Southwest University, Chongqing 402460, China;Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences Shanghai 200241 China;Jiangsu Key Laboratoo, of Zoonosis, College of Animal Science and Technology, Yangzhou Univetwity, Yangzhou 225009, Jiangsu, China)

机构地区：[1]西南大学动物科学学院,重庆402460 [2]中国农业科学院上海兽医研究所,上海200241 [3]扬州大学动物科学与技术学院江苏省人畜共患病学重点实验室,江苏扬州225009

出　　处：《生物工程学报》2017年第12期2009-2016,共8页Chinese Journal of Biotechnology

基　　金：上海市科技兴农重点攻关项目(No.2015HNG1-9);国家自然科学基金(No.31370045);江苏省人兽共患病学重点实验室项目(No.R1510)资助~~

摘　　要：菌蜕(Bacterial ghosts)是一种只含有细菌内、外膜结构的细菌空壳,可作为新型疫苗和传递载体。本研究通过3种方式制备禽致病性大肠杆菌(Avian pathogenicity Escherichia coli,APEC)分离株DE17的菌蜕,评价不同的菌蜕制备方法。结果表明,利用噬菌体Phi X174的裂解基因E构建的溶菌质粒pBV220-E制备DE17菌蜕,对DE17菌株裂解率可达99.9%,扫描电镜观测结果表明,在DE17两端或中部形成可见的跨膜孔道,呈现典型的菌蜕结构。利用合成的细胞穿透肽MAP(KLALKLALKALKAALKLA)作用于DE17制备菌蜕,结果表明,10μmol/L的MAP可实现对OD_(600)=0.1的DE17完全灭活,扫描电镜虽未看到明显的跨膜孔道,但细菌的膜结构呈现沟壑状,而构建的可表达MAP的溶菌质粒pBV220-MAP并不能实现对DE17的裂解作用。本研究通过比较分析不同APEC菌蜕的制备方式,为进一步研究菌蜕疫苗和提高菌蜕疫苗的生物安全性提供参考。Bacterial ghosts are bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology, which can be used as a new vaccine and delivery vector. In this study, a clinical isolate of avian pathogenic Escherichia coli（APEC） strain DE17 was used to prepare bacterial ghost through three different ways. The results showed that the cleavage efficiency of DE17 bacterial ghost was 99.9% with the lysis plasmid containing the Phi X174 lysis gene E. Scanning electron microscopy showed that transmembrane tunnels were formed in the middle or both ends of the cell envelope of DE17. Furthermore, the DE17 bacterial ghost was prepared with one of cell penetrating peptides（CPPs） named MAP（KLALKLALKALKAALKLA）, which will completely inactivate DE17（OD600=0.1） by 10 μmol/L MAP. The cell envelope showed a gully-like structure and obvious transmembrane tunnels were not found through the SEM. However, the DE17 could not be lysed by importing the lysis plasmid（p BV220-MAP）, which was used to express MAP. The present study will benefit for research on bacterial ghost preparation methods and provide a reference for biosafety of bacterial ghost vaccines.

关 键 词：裂解基因E 菌蜕 细胞穿透肽 禽致病性大肠杆菌 

分 类 号：S859.797[农业科学—临床兽医学]