摘要
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,并观察菌壳形态。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pBBR1MCS-2载体上,构建重组裂解质粒pBBR1MCS-E,并将其电转化至布鲁氏菌S2株,构建重组菌S2(pBBR1MCS-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-BR1MCS-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。
Lysis gene E and temperature-sensitive regulatory system kpL/pR-cI857 were amplified by PCR from pBV220 ： ： E and inserted into shuttle plasmicl pBBR1MCS-2 to construct the lysis plasmid pBBR1MCS-E. Plasmid pB- BR1MCS-E was transformed into Brucella .sui,s strain $2 and grown at 28℃ until mid log-phase, following by incubation at 42℃ to induce the expression of gene E. Then the Brucella ghosts were generated. The lysis rate was 99. 990/00. All viable bac- teria 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.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2012年第7期679-682,共4页
Chinese Journal of Zoonoses
基金
吉林省科技发展计划资助项目(20100231)~~
关键词
布鲁氏菌
菌壳
裂解质粒
Brucella
bacterial ghosts
lysis plasmid
