副猪嗜血杆菌菌影的制备  

Preparation of Haemophilus parasuis ghosts

作　　者：胡明明[1] 常月红[1,2] 张艳禾[1] 司薇[1] 谢芳[1] 于申业[1] 刘慧芳[1] 刘思国[1] 沈楠[1] 王春来[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物细菌病研究室,黑龙江哈尔滨150001 [2]东北农业大学,黑龙江哈尔滨150030

出　　处：《中国预防兽医学报》2011年第1期11-14,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点实验室基本科研业务费(NKLVBP200822)

摘　　要：为研制副猪嗜血杆菌(H.parasuis)菌影疫苗,本实验通过PCR扩增噬菌体PhiX174的裂解基因E,将其连接到含有λPL/PR-cI857启动阻遏系统的pBV220载体中,使裂解基因E与λPL/PR-cI857串联成为温度敏感的裂解盒,构建重组质粒pBV-E。并将含有E基因的裂解盒插入pMD18-T载体中,构建H.parasuis打孔质粒(pMD-E)。采用电击穿孔法将其转入E.coliβ2155中,利用接合的方式将打孔质粒转入H.parasuis 5型菌株中。将含有打孔质粒的H.parasuis在28℃培养至对数生长期,升温到42℃诱导E基因表达,制备H.parasuis菌影。电镜观察菌影形态完整,内容物全部被释放到胞外。本实验为进一步研究菌影疫苗奠定了基础。To develop a Haemophilus parasuis ghost vaccine, in the present study, lysis gene E from phage PhiX174 was amplified by PCR and inserted into pBV220 vector under the control of the h PL/PR-cI857 temperature-sensitive promoter element to eonstructe recombinant plasmid pBV-E. The E gene lysis cassette was subcloned into the pMD18-T vector to generate a bacteriolysis plasmid pMD-E, which was electrotransformed into E. coli β 2155. Then the pMD-E was transformed into serotype 5 H. parasuis by conjugation experiment with the E. coil β 2155. H. parasuis 5 containing the pMD-E at mid log-phase incubated under 28℃ was shifted to 42 ℃ to induce the expression of E gene to pruduce the H.parasuis 5 ghosts, which were proved by observation under transmission electron microscope and live bacteria counting. This experiment laid a foundation on the study of bacterial ghost as a novel bacteria vaccine.

关 键 词：副猪嗜血杆菌 菌影 裂解基因 

分 类 号：S852.61[农业科学—基础兽医学]