禽致病性大肠杆菌(APEC)菌蜕的制备研究

Generation of avian pathogenic E.coli ghosts

菌蜕是通过PhiX174噬菌体溶菌基因E的可调控表达而形成的、缺少细胞浆和核酸且无繁殖能力的革兰氏阴性细菌菌体。这种非变性的灭活方式使菌蜕完好保留了细菌的各种抗原成分和免疫黏附分子,具有优良的免疫原性和固有的免疫佐剂性质以及免疫系统靶向性质。菌蜕的这些生物学特点使其在作为新型非变性灭活菌苗、作为递呈异源抗原的重组疫苗以及作为DNA疫苗甚至药物的载体方面都显现出巨大的研究开发前景。本研究成功构建了热敏诱导、pR、pL串联双启动子表达E基因的、具有不同抗性标记和复制起点的系列溶菌载体,比国外用pR或pL单启动子的溶茵载体在K-12株大肠杆菌(如XL1-blue)的溶茵效率高一个指数级。本研究还成功制备出禽致病性大肠杆菌(Avian pathogenic E.coli,APEC)野毒株的菌蜕,为进一步进行菌蜕疫苗的开发研究奠定了技术基础。

Bacterial ghosts are non-living Gram-negative bacterial cell envelopes devoid of cytoplasmic contents, produced by controlled artificial expression of bacteriophage PhiX174 gene E and subsequent lysis of bacterial cells. This non-denaturing inactivation method keeps antigenic determinants and adhesive molecules of bacteria intact, and as a result, bacterial ghosts are excellent candidates of recombinant vaccines presenting heterologous antigens and delivering vehicles of DNA vaccine or drugs. In this study, we constructed a series of lysis vectors harboring different antibiotic markers and replication origins to express lysis gene E under the control of tandem pR and pL double promoters. The lysis and inactivation efficiency of the lysis vectors in E.coli K-12 strain was up to 1 order of magnitude higher than that of previously reported vectors in which only single promoter ofpR or pL was used. Ghosts of a wild strain of avian pathogenic E.coli （APEC） were successfully generated with one of the lysis vectors, which laid foundation for future development of bacterial ghost vaccines.