下调bla表达提高pcDNA3·1+在重组菌中的稳定性  

Enhanced Stability of Plasmid pcDNA3.1+ within Salmonella typhimurium by Downregulation of the Ampicillin Resistance Gene Expression

作　　者：张晓明 焦新安 唐丽华 潘志明 黄金林 刘秀梵 

机构地区：[1]扬州大学,扬州225009

出　　处：《微生物学通报》2005年第5期51-55,共5页Microbiology China

基　　金：国家自然科学基金项目(No.30425031;30170700);全国优秀博士学位论文作者专项资金项目(No.FANEDD200358)~~

摘　　要：高拷贝质粒pcDNA3·1+在鼠伤寒沙门氏菌SL7207中不稳定。通过去除pcDNA3·1+中氨苄青霉素抗性基因(bla基因)的启动子序列,仅保留其核糖体结合位点,构建了新质粒pmcDNA3·1+。SL7207(pmcDNA3·1+)在含与不含氨苄青霉素的培养基中均能稳定保留其质粒。SL7207(pmcDNA3·1+)的β-内酰胺酶活力明显低于SL7207(pcDNA3·1+),且接种SL7207(pmcDNA3·1+)的液体培养基中的氨苄青霉素不易被降解。腹腔接种小鼠7d后,脾脏中SL7207(pmcDNA3·1+)的质粒稳定性仍保持在95%以上,而SL7207(pcDNA3·1+)免疫后第3d99%丢失质粒。所以,通过降低bla基因的表达水平,为解决高拷贝质粒在沙门氏菌中的稳定性问题提供了新方法。The high-copy-number plasmid peDNA3.1 ＋ is unstable within Salmonella typhimurium. A novel plasmid pmcDNA3.1 ＋ was constructed by removing the promoter sequence of ampieillin resistance gene （ bla gene） in plasmid pcDNA3.1 ＋. In contrast to peDNA3.1 ＋ , pmcDNA3.1 ＋ was stable within Salmonella typhimurium SL7207 in LB medium with or without ampieillin. Further experiments showed the β-lactamase activity of Salmonella typhimurium SL7207 （ pmcDNA3.1 ＋ ） was apparently lowered than that of Salmonella typhimurium SL7207 （pcDNA3. 1 ＋ ） and the high ampieillin concentration was maintained longer in LB medium culturing Salmonella typhimurium SL7207 （ pmeDNA3. 1 ＋ ） . When mice were administered with Salmonella typhimurium SL7207 （pmcDNA3. 1 ＋ ） intraperitoneally, more than 95% of Salmonella cells separated from the spleen still harbored the plasmid pmcDNA3. 1 ＋ 7 days later; but 99% of Salmonella cells lost the plasmid peDNA3.1 ＋ at day 3 in mice innoeulated with Salmonella typhimurium SL7207 （ peDNA3. 1 ＋ ） . By lowering the expression of bla gene, the rapid decomposition of ampieillin in LB medium was avoided and the metabolic pressure was relieved for the host cells. This method offers a solution for the problem of the instability of high-copy-number plasmid within Salmonella typhimurium.

关 键 词：沙门氏菌 高拷贝质粒 质粒稳定性 Β-内酰胺酶 

分 类 号：S852.4[农业科学—基础兽医学] Q789[农业科学—兽医学]