基于λ Red重组系统敲除鼠伤寒沙门菌yejE基因

目的 采用改良的λ Red同源重组技术，以减毒沙门菌TT-16（ΔclpP TT-1）为前体菌，构建敲除yejE基因的减毒沙门菌TT-54（ΔclpP ΔyejE TT-1），为后续研究yejE基因的功能奠定基础。方法 （1）将重组质粒PKD46导入减毒沙门菌TT-16（ΔclpP TT-1），获得一次重组受体菌TT-51（ΔclpP PKD46 TT-1）；（2）通过PCR扩增获得yejE基因的一次同源重组打靶片段，将其导入TT-51（ΔclpP PKD46 TT-1），利用四环素平板筛选，获得重组菌TT-52（ΔclpP ΔyejE::tetRA TT-1）；（3）将重组质粒PKD46导入重组菌TT-52，获得同源重组受体菌TT-53（ΔclpP ΔyejE::tetRA PKD46 TT-1）；（4）将PCR扩增获得的yejE基因二次同源重组打靶片段导入TT-53（ΔclpP ΔyejE::tetRA PKD46 TT-1），通过四环素敏感平板筛选，获得敲除了yejE基因的减毒沙门菌TT-54（ΔclpP ΔyejE TT-1）。结果 通过两轮（一次和二次）λ Red同源重组，减毒沙门菌TT-16（ΔclpP TT-1）的yejE基因被成功敲除，PCR及测序鉴定结果符合预期。结论 基于模式菌（大肠杆菌）所构建的λ Red同源重组技术完全适用于沙门菌；通过两轮λ Red同源重组，可实现对沙门菌特定靶基因的无痕敲除。

Deletion of yejE gene of Salmonella tyhimurium LT2 by λRed recombination system

To construct yejE gene deletion mutation ( TT － 54，ΔclpPΔyejETT － 1 ) based on the attenuatedSalmonella typhimurium ( TT － 16，ΔclpPTT － 1) with improved λＲed homologous recombination technique，and to lay thefoundation of the subsequent research onthe function of yejE gene. Methods 1) Ｒecombinant plasmid PKD46 was induced intothe attenuated Salmonella typhimurium( TT － 16，ΔclpPTT － 1) to obtain the recombinant recipient bacteria for the first round( TT － 51，ΔclpP PKD46 TT － 1) . 2) The homologous fragment of yejE gene constructed by PCＲ amplification technique waselectro － transformed into TT － 51 and selected on tetracycline agarplate. The positive recombinants were recombinant recipientbacteria of the first round ( TT － 52，ΔclpPΔyejE: : tetＲATT － 1) . 3) Ｒecombinant plasmid PKD46 was imported into TT － 52and secondary recombinant recepient bacteria for the second round of recombination ( TT － 53，ΔclpPΔyejE: : tetＲAPKD46 TT－ 1) was obtained. 4) The secondary linear target fragment of the yejE gene obtained by PCＲ amplification was imported intoTT － 53，and the yejE gene deletion mutation ( TT － 54) was obtained by the screening of tetracycline － sensitive plate. ＲesultsAfter two rounds of λＲed recombination， yejEgenedeletionmutantwasobtained andit can be identifiedbyPCＲ andsequencingtechnique. Conclusion λＲed recombination system based on model bacteria ( Escherichia coli) is also applicableto Salmonella. The complete knockout of a specific target gene of Salmonella can be achieved through two rounds of λＲedrecombination system