摘要
为构建鼠伤寒沙门氏菌Χ4550FlhD基因缺失所致的鞭毛缺失株,采用PCR技术从减毒鼠伤寒沙门氏菌Χ4550基因组DNA中扩增出了鞭毛控制操纵子基因(FlhD)两侧翼基因片段,作为FlhD基因敲除所需的上臂和下臂的同源序列;以pKD4质粒为模板,扩增了卡那霉素(Km)抗性基因,并分别克隆入pMD18-T载体中,获得了重组质粒pMD-FlhD-U、pMD-FlhD-D和pMD-Km。将获得的上臂和下臂基因以及卡那霉素抗性基因通过拼接,构建重组质粒pMD-ΔFlhD/Km。将重组片段ΔFlhD/Km亚克隆入pG-MB151自杀质粒,并转化大肠杆菌χ7213,获得重组菌χ7213(pGMB151-ΔFlhD/Km)。将此细菌作为供体菌,与受体菌鼠伤寒沙门氏菌Χ4550进行固相杂交,经同源重组和抗生素筛选,获得鼠伤寒沙门氏菌Χ4550(ΔFlhD/Km)鞭毛缺失株。通过PCR扩增、动力学鉴定及电镜观察,显示鼠伤寒沙门氏菌Χ4550的FlhD基因已被成功敲除。证实,运用同源重组的方法可成功地敲除鼠伤寒沙门氏菌Χ4550的FlhD基因,并导致其鞭毛缺失,为鼠伤寒沙门氏菌鞭毛的功能研究奠定基础。
To construct flagella mutant by knocking out FlhD gene of Salmonella typhimurium （S. ty-phimurium） X4550,two flank sequences of FlhD gene were amplified by PCR from the genomic DNA of S. typhimurium X4550,which were necessary for suicide plasmid for knocked out FlhD gene as the up and down arm sequences,and a kanamycin-resistant（Km^R） gene was amplified from plasmid pKD4. These am-plified genes were subcloned into pMD18-T vector respectively,and then pMD-FlhD-U,pMD-FlhD-D and pMD-Km plasmids were constructed. The up and down arm gene fragments and the Km^R gene were con-nected through restriction enzyme sites to construct recombinant plasmid pMD-△FlhD/Km. The suicide plasmid pGMB151-△FlhD/Km targeting FlhD gene was developed following △FlhD/Km was subcloned in-to the suicide plasmid pGMB151, then transformed into Escherichia coli γ7213, and the recombinant was designated as γ7213 （pGMB151-△FlhD/Km）. The recombinant γ7213 （pGMB151-AFlhD/Km） as donor bacteria was conjugated with receptor bacteria S. typhimurium X4550. The flagella-deficient mutants X4550（△FlhD/Km） of S. typhimurium were obtained after two homologous recombinations and screening by antibiotics. In result, PCR identification, mobility identification and electron microscope observation indi-cated that the FlhD gene controlling the growth of flagella was successfully knocked out, and flagella-deficient mutant was successfully constructed. It was concluded that the FlhD gene of S. typhimurium X4550 could be knocked out by homologous recombination, which resulted in the deletant of flagella, and these results provided essential base for further study the function of flagella of S. typhimurium.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2010年第1期1-6,共6页
Chinese Veterinary Science
基金
国家自然科学基金项目(30871860)
国家科技支撑计划项目(2007BAD40B01)
江苏省自然科学基金项目(BK2007511,BK2008011)
