弗氏志贺菌2a T32株asd基因缺失突变体的构建

Construction of Δasd mutant of Shigella flexneri 2a strain T32

构建痢疾弗氏志贺菌 2aT32的asd基因全缺失突变体。方法 :采用全菌PCR技术 ,从痢疾弗氏 2aT32株染色体扩增了asd基因及其上下游各约 50 0bp的染色体DNA序列 ,并将其克隆至 pUC18,测定其核苷酸序列 ,在体外用ctxB基因置换了asd基因 ,然后将其克隆至自杀载体pXL2 75。通过细菌交配和体内同源重组 ,用ctxB基因完全取代痢疾弗氏 2aT32株染色体上的asd基因。结果与结论 :构建成asd基因全缺失而稳定表达CtxB的T32突变株FWL0 1,为进一步用载体 /宿主平衡致死系统构建多价菌苗候选株打下了基础。

Objective: To construct Δasd mutant of Shigella flexneri 2a strain T32. Methods: Asd gene and its 3′,5′chromosomal DNA sequences(～500 bp,respectively) were obtained from S.flexneri 2a strain T32 by the whole cell PCR, cloned into pUC18, and sequenced. In vitro, asd gene was replaced by ctxB gene, and then the ctxB with asd′s flanks was inserted into the suicide vector pXL275. By bacterial mating and in vivo homologous recombination,asd gene of S.flexneri 2a strain T32 was completely replaced by ctxB gene. Results and Conclusions: A Δasd mutant of T32, FWL01, was constructed. This mutant could express CtxB stably. This work provides the basis for constructing a multivalent vaccine with the vector/host balanced lethal systems in the future. [