基于Red同源重组和高效自杀性载体系统构建肠炎沙门氏菌突变株方法的比较  

Comparison of two methods for constructing Salmonella enteritidis mutants:Red recombination system and suicide vector system

作　　者：朱春红[1] 孙晓庆[1] 何素芬[1] 朱国强[1] 

机构地区：[1]扬州大学兽医学院,江苏扬州225009

出　　处：《中国预防兽医学报》2009年第2期81-84,98,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30571374;30771603)

摘　　要：Red同源重组系统和高效自杀性载体系统都能对革兰氏阴性菌染色体直接进行修饰,本试验利用上述2种系统敲除不同来源肠炎沙门氏菌菌株SEF14菌毛操纵子亚单位sefD或sefA基因,并对构建肠炎沙门氏菌突变株方法进行比较。本研究成功构建了国际、国内标准株,鸡源和人源国内分离株sefD和sefA缺失株各4株。Red同源重组系统中能将PCR产物一步法直接转化肠炎沙门氏菌,通过同源重组序列发生同源重组交换,但对PCR产物和感受态细胞的质量要求很高,一次重组效率很低,而二次重组过程则相对简单、高效。自杀性载体系统若将构建好的含同源DNA片段的重组质粒转化宿主菌,其一次重组效率高,二次重组也只需蔗糖筛选传代丢失抗性质粒。Red同源重组系统将在染色体中残留FRT位点;而自杀性载体系统在染色体中可不留任何痕迹。所以2个系统都能对不同源肠炎沙门氏菌染色体进行直接修饰,而且易删除抗性选择标志,与传统的高效自杀性载体系统比较,Red同源重组系统更方便快捷,省时省力。Both the Red recombination and suicide vector systems can be use to modify gram-negative bacteria chromosome. In this study Salmonella enteritidis （X） mutants for knocking out the sefA or sefD gene from sef/4 gene cluster were obtained by means of the two systems. Four mutants for X AsefD and X AsefA were successfully generated. Both of the two systems can be used to delete the target genes in Salmonella entertidis chromosome, without any resistant genes remained. Compared with the sui- cide vector system, the Red recombination system need high quality PCR product and high quality competent cells to generate the mutants, and the mutant rate obtaind by this system was very lower than that of other system during the first recombination reaction. Whereas, for the suicide vector systems recombination plasmid had to be constructed and the FRT sites would be remained in the mutant chromosome.

关 键 词：肠炎沙门氏菌 同源重组 SEF14菌毛 sefA sefD 

分 类 号：R516.4[医药卫生—内科学]