λpL/pR-cI857温控系统的改造及其对大肠杆菌菌蜕制备的影响

Mutation of λpL/pR-cI857 system for production of bacterial ghost in Escherichia coli

摘要:

菌蜕是革兰氏阴性菌在噬菌体Phix174的溶菌基因E的作用下形成的细菌空壳，通常由λpL/pR-cI857温控系统通过调控E基因的表达制备。通过重叠PCR技术，将λpL/pR-cI857温控系统中λpR启动子的第2个操纵基因 (OR2) 的第9位碱基由T突变为C (T→C)。溶菌试验结果表明，突变后的λpL/pR-cI857系统在37 ℃时仍能稳定抑制基因E的表达，对大肠杆菌的溶菌率为99.9％。通过对λpR启动子的改造，扩大了λpL/pR-cI857温控系统的温度调控范围，为菌蜕疫苗的研制提供了参考。

Abstract:

Bacterial ghost is intact envelope of Gram-negative bacteria, which is produced by the function of the lysis gene E from bacteriophage PhiX174. The expression of the lysis gene E is usually controlled by the thermosensitive λpL/pR-cI857 promoter. In this study, we described a mutation (T→C) at the ninth nucleotide of the OR2 in the λpR promoter of the λpL/pR-cI857 system by overlap PCR. The bacteriolytic assay showed that the mutation in the λpL/pR-cI857 system enhanced the temperature of repressing the expression of gene E up to 37 ℃. The lysis efficiency of altered λpR promoter in Escherichia coli DH5α and avian pathogenic E. coli DE17 was up to 99.9%. The expanded range of temperature will benefit for the production of bacterial ghost.