金黄色葡萄球菌SEB基因的克隆及其系统发生分析

目的克隆和测定金黄色葡萄球菌深圳分离株S4070肠毒素B（SEB）基因序列,并作系统发生分析。方法采用PCR法从金黄色葡萄球菌S4070基因组中扩增出SEB基因片段,纯化后与pMD-18T载体连接构建重组子pMD-18T-SEB,并转化E.coli DH5α;阳性克隆质粒经双酶切法鉴定后,采用双脱氧末端终止法作序列测定,并以BLAST和MEGA4软件分析其分子特征。结果从金黄色葡萄球菌基因组DNA中扩增出约704bp的基因片段,阳性克隆质粒鉴定结果与预期一致;序列测定结果显示,所克隆的SEB基因片段含704个碱基,与GenBank中17株金黄色葡萄球菌SEB基因序列比对,无碱基的插入或缺失,同源性均为98%以上;基于SEB基因序列,采用邻位连接法（Neigh bor-joining）构建系统发生树,深圳S4070株与日本分离株NN35、NN37、NN41-F1D、NN41-F1F和NN41-F1G以及印度分离株DQ535901、DQ535902的遗传距离小,同处在一个分支群上。结论实验成功克隆了金葡菌深圳株S4070 SEB基因,不同分离株间SEB基因序列相对保守,本分离株与日本和印度分离株遗传关系较近。

Cloning of the enterotoxin B gene and its phylogenetic analysis of Staphylococcus aureus strains

Objective To clone and analyze the enterotoxin B gene sequence from a wild Staphylococcus aureus strain S4070. Methods The staphylococcal enterotoxin B（SEB） gene was amplified by PCR method, and ligated with the pMD-18T plasmid to construct the recombinant rPMD-SEB, which was transformed into E.coil DH5α. The positive bacteria clones was identified by double enzymes digestion method. The inserted SEB gene fragment was finally sequenced and analyzed with BLAST and MAGA4 software. Results The amplified SEB DNA sequence was 704bp in length. Aligning it with the SEB sequences of 17 S.aureus strains deposited in GenBank, the overall nucleotides identity was above 98%. Based on the SEB DNA sequence, phylogenetic analysis was conducted by Neighbor-joining（NJ） method, and the result indicated that S.aureus strain S4070 has close association with Japanese isolates （NN35, NN37, NN41-F1D, NN41-F1F, NN41-F1G） and Indian isolates （DQ535901, DQ535902）. Conclusion Staphylococcal enterotoxin B gene of S.aureus strain S4070 was successfully cloned, and it was relatively conserved among different S.aureus strains.