调控表达LacI蛋白的猪霍乱沙门氏杆菌C500Δasd株的构建

Construction of a Salmonella choleraesuis C500Δasd strain with regulation of Lac I protein expression

为了构建具有asd基因缺失和LacI蛋白表达调控的猪霍乱沙门氏杆菌C500株,试验以猪霍乱沙门氏杆菌C500株为载体,对其基因组进行改造,使沙门氏杆菌asd基因缺失,随后将araC PBADLacI序列置于其中,构建包含araC PBAD-LacI基因表达框的猪霍乱沙门氏杆菌C500Δasd基因突变株以及包含Ptrc启动子的EGFP表达质粒pYA4514-EGFP,并验证是否有LacI蛋白表达。结果表明:试验成功构建出包含araC PBAD-LacI基因表达框的猪霍乱沙门氏杆菌C500Δasd基因突变株,以及包含Ptrc启动子的EGFP表达质粒pYA4514-EGFP;Western-blot检测验证了突变菌在阿拉伯糖存在的条件下可大量表达LacI蛋白,而当pYA4514-EGFP质粒上的Ptrc启动子受到抑制时,EGFP蛋白不表达;用IPTG诱导后可以消除LacI对Ptrc启动子的抑制作用,EGFP蛋白表达。说明试验成功构建了调控表达LacI蛋白的猪霍乱沙门氏杆菌C500Δasd基因突变株。

In order to construct Salmonella choleraesuis C500 strain with asd gene deletion and LacI protein expression regulation,Salmonella choleraesuis C500 strain was used as a vector,and the Salmonella asd gene was deleted to modify the genome,and then the araC PBAD-LacI sequence was inserted in C500 strain without asd gene.The Salmonella choleraesuis C500Δasd mutant containing the araC PBAD-LacI gene expression frame and the EGFP expression plasmid pYA4514-EGFP containing the Ptrc promoter were constructed,and then verified whether there was LacI protein expression.The results showed that the Salmonella cholerae C500Δasd mutant containing araC PBAD-LacI gene expression frame and the EGFP expression plasmid pYA4514-EGFP containing Ptrc promoter were successfully constructed.Western-blot results confirmed that the mutant bacteria expressed a large amount of LacI protein in the presence of arabinose,the Ptrc promoter on the pYA4514-EGFP plasmid was inhibited,and the EGFP protein was not expressed.After induction with IPTG,the inhibition effect of LacI on the Ptrc promoter can be eliminated,and EGFP expression can be obtained.The results indicated that Salmonella cholerae C500Δasd mutant strain was successfully constructed,which could be used to express exogenous antigens in the future.