采用同源重组技术构建金黄色葡萄球菌sae基因缺失突变株

Construction of a Sae-Deleted Mutant of Staphylococcus aureus by Homologous Recombination

针对金黄色葡萄球菌sae基因前后两段序列设计两对引物,PCR扩增出sae基因上下游同源臂序列,克隆到穿梭载体pBT2中;两段序列之间用来自质粒p646的Em抗性基因片段连接,作为筛选标记,从而构建同源重组穿梭质粒pBT2Δsae;将pBT2Δsae电转化到金黄色葡萄球菌菌株RN4220中,40℃经过七轮培养,进行抗性培养基筛选和PCR验证,以及RT-PCR观察基因表达水平。获得一株金黄色葡萄球菌sae基因缺失突变株RNΔsae,为进一步研究sae基因的调控机制等提供有用的实验材料。

In this study,two pairs of primers were designed according to the upstream and downstream sequence of sae gene and used to amplify the homologous arms by PCR.Then the upstream and downstream homologous arms were cloned into the shuttle vector pBT2 with the Em resistance gene fragment from the plasmid p646 inserted as a selection marker between the two sequences.The plasmid pBT2Δsae was constructed.The homologous recombination vector was subsequently transformed into S.aureus RN4220 by electroporation,S.aureus RNΔsae deletion mutant was successfully selected by homologous recombination at 40 ℃ and confirmed by PCR.Subsequently,the sae gene expression level was also valuated by RT-PCR.This study provided the useful tool for further exploring the regulation mechanism of sae gene in S.aureus.