Cre-LoxP系统在炭疽芽胞杆菌基因敲除中的应用及eag基因的敲除

将Cre-LoxP系统应用于Bacillus anthracis中并成功敲除eag基因．以B.anthracis基因组为模板扩增得到上下游同源臂，联合两端带有LoxP位点的壮观霉素抗性基因片段构建好同源重组载体，转化B．anthracis AP422，通过一系列筛选得到带有抗性标记的重组菌．然后，通过转入Cre重组酶表达质粒，去除抗性标记，得到eag基因缺失的重组菌，并在DNA水平、RNA水平和蛋白质水平进行了系统的鉴定．最终建立了Cre-LoxP系统在B．anthracis中的应用方法，并成功敲除eag基因.

Application of Cre-LoxP system in the gene knockout of Bacillus anthracis and the knockout of eag gene

Cre-LoxP homologous recombinant system was used to disrupt eag gene in B. anthracis AP422. To construct the recombinant vector, homologous regions and Spc^r cassette with two loxP sites were amplified from the corresponding templates. The produced shuttle vector was then transformed into B. anthracis AP422. Under the pressure of temperature and antibiotic, homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with spectinomycin (Spc) and X-gal. To remove the Spc^r cassette, a plasmid with Cre recombinase was introduced into the recombinant to delete the relevant DNA fragment, resulting in a single loxP site within the targeted genomic segment. The markerless mutant strains were detected by genome PCR, RT-PCR, total proteins SDS-PAGE analysis and Western blot. The results showed that the eag gene was successfully deleted.