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目的构建炭疽芽孢杆菌(Bacillus anthracis)A16R株lysA基因缺失突变株,为后续的定量蛋白质组学研究奠定基础。方法以炭疽杆菌活疫苗A16R株lysA基因为目的缺失基因,利用软件设计上下游同源臂以及抗性基因的引物,用同源重组酶将3个片段连入质粒中,构建重组质粒,并将重组质粒导入炭疽杆菌A16R感受态细胞中,筛选炭疽杆菌A16R株lysA基因缺失突变株,对其进行验证。最后绘制缺失突变株和野生株生长曲线并进行生理生化分析。结果成功构建了重组质粒,经同源重组后获得lysA基因缺失突变株。鉴定表明目的基因已经丢失。结论成功获得炭疽杆菌A16R株lysA基因缺失突变株,为定量蛋白质组学研究奠定了基础,也为炭疽杆菌重要基因功能的研究建立了良好的技术平台。
Objective To construct the deletion mutant of lysA gene of Bacillus anthracis A16R strain and lay the foundation for the subsequent quantitative proteomics research. Methods The deletion gene of lysA gene of A16R strain of Bacillus anthracis live vaccine was designed. The primers of upstream and downstream homology arms and resistance genes were designed by software. Three fragments were inserted into plasmid by homologous recombinase to construct recombinant plasmids. The recombinant plasmids were introduced into the B. anthracis A16R competent cells, and the deletion mutant of the lysA gene of Bacillus anthracis A16R strain was screened. Finally, the growth curves of the mutant strains and the wild strains were plotted and analyzed for physiological and biochemical analysis. Results The recombinant plasmids were successfully constructed and the lysA gene deletion mutant was obtained after homologous recombination. Identification shows that the target gene has been lost. Conclusion The deletion of lysA gene of Bacillus anthracis A16R strain was successfully obtained, which laid the foundation for the quantitative proteomics research and established a good technical platform for the study of the important gene function of B. anthracis.