猪传染性胸膜肺炎放线杆菌“菌影”的制备

Preparation of Actinobacillus pleuropneumoniae ghosts

本试验通过PCR扩增噬菌体PhiX174的裂解基因E,将该基因连接到含有λPL/PR-cI857启动阻遏系统的pBV220载体中,从而使裂解基因E和启动阻遏系统λPL/PR-cI857串联成为温度敏感的裂解盒,构建重组质粒pBV-E。再将含有E基因的裂解盒插入到App-E.coli穿梭载体pGZRS-18中,构建胸膜肺炎放线杆菌打孔质粒。采用电击穿孔法将其转入胸膜肺炎放线杆菌中,含有打孔质粒的胸膜肺炎放线杆菌在28℃条件下生长到对数生长期,升温42℃诱导E基因的表达,制备了胸膜肺炎放线杆菌菌影。电镜观察菌影形态完整,内容物全部被释放到胞外。本试验为进一步研究菌影这一新型菌苗及佐剂奠定了基础。

In the present study, lysis gene E from phage Ph/X174 was amplified by PCR and inserted into pBV220 vector downstream of the k PL/PR-cI857 regulatory system to form the temperature-sensitive lysis cassette. Bacteriolysis plasmid pGZRS-E was produced by cloning the lysis cassette into the pGZRS-18 vector and electrotransformed into App serotype 1 （Appl）. The Appl was then grown at 28 ℃ under aerobic conditions until mid log-phase, followed by incubation at 42 ℃ to induce the expression of gene E. The Appl ghosts were shown to be intact cells under transmission electron microscope, with contents released to extracellular region. The potential application of the App I ghosts was discussed.