布鲁氏菌S2株菌壳的制备研究

摘要

Lysis gene E and temperature-sensitive regulatory system XpL/pR-cI857 were amplified by PCR from pBV220 :: E and inserted into shuttle plasmid pBBRlMCS-2 to construct the lysis plasmid pBBRlMCS-E. Plasmid pB-BR1MCS-E was transformed into Brucella suis strain S2 and grown at 28℃ until mid log-phase, following by incubation at 42°C to induce the expression of gene E. Then the Brucella ghosts were generated. The lysis rate was 99. 99%. All viable bacteria could be eliminated by freeze-thawing in hypertonic solution. The Brucella ghosts were shown to be intact cells under electron microscope, with contents released to extracellular region. In conclusion, the Brucella ghosts were successfully generated with lysis gene E, which laid foundation for future development of bacterial ghost vaccine against Brucella infection.%目的 利用温控表达调节系统在布鲁氏菌中诱导表达裂解基因E,制备布鲁氏菌S2株菌壳,监测其裂解效率,并观察茵壳形态.方法 从pBV220::E中PCR扩增温控裂解盒和裂解基因E融合片段,克隆至pBBR1 MCS-2载体上,构建重组裂解质粒pBBRl MCS-E,并将其电转化至布鲁氏菌S2株,构建重组菌S2(pBBRl MCS-E).通过28℃至42℃升温诱导培养制备布鲁氏菌菌壳,绘制生长曲线和裂解曲线,计算裂解率,并电镜观察菌壳形态.结果 成功构建了重组裂解质粒pB-BRlMCS-E并制备了布鲁氏菌菌壳.绘制出了布鲁氏菌裂解曲线,布鲁氏菌菌壳的裂解率达99.99％,经高渗溶液冻融后可完全消除其中残存的活菌.电镜观察表明布鲁氏菌菌壳保持有细胞的基本形态,但由于细胞内容物外流而使细胞发生变形和皱缩.结论 本研究成功制备出了布鲁氏菌菌壳,为进一步研究布鲁氏菌菌壳的特性奠定了基础.