基于λ Red 重组系统敲除鼠伤寒沙门菌yejE基因  

Deletion of yejE gene of Salmonella tyhimurium LT2 byλRed recombination system

作　　者：李瑞 肖金雨 娄莉萍 唐田 LI Rui;XIAO Jin-yu;LOU Li-ping;TANG Tian(West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu,Sichuan 610041,China)

机构地区：[1]四川大学华西公共卫生学院(华西第四医院),四川成都610041

出　　处：《现代预防医学》2020年第17期3207-3211,共5页Modern Preventive Medicine

基　　金：中国博士后科学基金(2016M602691)。

摘　　要：目的采用改良的λRed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(ΔclpPΔyejE 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(ΔclpPΔyejE::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(ΔclpPΔyejE::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(ΔclpPΔyejE::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(ΔclpPΔyejE TT-1)。结果通过两轮(一次和二次)λRed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λRed同源重组技术完全适用于沙门菌;通过两轮λRed同源重组,可实现对沙门菌特定靶基因的无痕敲除。Objective To construct yejE gene deletion mutation(TT-54,ΔclpPΔyejETT-1)based on the attenuated Salmonella typhimurium(TT-16,ΔclpPTT-1)with improvedλRed homologous recombination technique,and to lay the foundation of the subsequent research onthe function of yejE gene.Methods 1)Recombinant plasmid PKD46 was induced into the 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 was electro-transformed into TT-51 and selected on tetracycline agarplate.The positive recombinants were recombinant recipient bacteria of the first round(TT-52,ΔclpPΔyejE::tetRATT-1).3)Recombinant plasmid PKD46 was imported into TT-52 and 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 into TT-53,and the yejE gene deletion mutation(TT-54)was obtained by the screening of tetracycline-sensitive plate.Results After two rounds ofλRed recombination,yejEgenedeletionmutantwasobtained andit can be identifiedbyPCR and sequencingtechnique.ConclusionλRed recombination system based on model bacteria(Escherichia coli)is also applicable to Salmonella.The complete knockout of a specific target gene of Salmonella can be achieved through two rounds ofλRed recombination system.

关 键 词：沙门菌 同源重组 菌株构建 yejE基因 

分 类 号：R111[医药卫生—公共卫生与预防医学]