本课题组早期研究结果表明，炭疽芽胞杆菌BA2380蛋白可能与炭疽芽胞杆菌毒力有关，因而有必要对其功能进行深入研究。选取炭疽芽胞杆菌A16D2株为出发菌株，以其BA2380基因为目的缺失基因，参照A16D2株基因组序列及质粒pSET4s序列，利用软件设计上下游同源臂及抗性基因引物，用本实验室改造的“Golden Gate”克隆方法将3个片段同时连入温敏型穿梭载体pKMBK中(本实验室构建的受体质粒)，从而构建基因打靶质粒。将该基因打靶质粒导入炭疽芽胞杆菌A16D2感受态细胞中，利用同源重组原理，筛选获得炭疽芽胞杆菌A16D2 BA2380基因缺失突变株，并对其进行验证。结果验证了本课题组构建的“Golden Gate”克隆体系进行多片段克隆的高效性，也为后续探索其基因功能奠定了基础。

The early results of our laboratory showed that BA2380 protein may be related to the virulence of Bacillus anthracis (B. anthracis). The goal of this study is to knock out the BA2380 gene in B．anthracis A16D2 strain. The downstream and upstream sequences of BA2380 and spectinomycin resistance gene (spc) were obtained with polymerase chain reaction (PCR) respectively and used in the construction of plasmid pK2380usd for gene replacement. pK2380usd was introduced into B. anthracis A16D2 competent cells, and the BA2380 gene deletion was obtained by antibiotic screening and PCR identification. The results laid a foundation for the subsequent exploration of gene function.