金葡菌野生株肠毒素B基因克隆及分子特征  

Cloning and molecular characteristics of enterotoxin B gene from wild staphylococcus aureus strain

作　　者：高世同[1] 刘会娟[2] 张仁利[1] 秦莉[3] 耿艺介[1] 黄达娜[1] 吴少庭[1] 

机构地区：[1]深圳市疾病预防控制中心,518020 [2]桂林医学院 [3]华中科技大学同济医学院

出　　处：《中国公共卫生》2006年第1期72-73,共2页Chinese Journal of Public Health

摘　　要：目的克隆金黄色葡萄球菌野生株S407030肠毒素B(SEB)基因,并分析其分子特征。方法根据Genebank中金黄色葡萄球菌S6株肠毒素B基因设计1对引物,采用PCR法从金黄色葡萄球菌野生株S407030基因组中扩增目的基因,将目的片段与pMD-18T载体连接构建重组子pMD-18T-SEB,并转化E.coli DH5a;阳性克隆以双酶切鉴定后,双脱氧末端终止法作序列测定分析,并以ExPasy Proteoic Tools蛋白分析软件预测其编码SEB蛋白的二级结构。结果从金黄色葡萄球菌基因组DNA中扩增出约705bp的基因片段,所构建的阳性克隆重组子PCR及双酶切鉴定与预期结果一致;测序结果表明,克隆的SEB基因含705个碱基,与S6株序列相比无碱基的插入或缺失,同源性为98.4%;存在11个碱基变异,其中7个为无义突变,4个为有义突变(突变位点位于第364,699,899,988位,编码氨基酸变化分别为:Ser→Ala、Gln→His、Asn→Ser、Met→Leu);所预测的2菌株SEB蛋白的二级结构基本相同。结论本实验成功克隆了金葡菌野生株S407030SEB基因,其在不同菌株间相对保守;由SEB基因点突变引起的个别氨基酸的改变对SEB生物活性与毒力的影响有待进一步研究。Objective To clone the enterotoxin B gene from a wild staphylococcus aureus strain S407030, and to analyze its molecular characteristics. Methods The Staphylococcal enterotoxin B gene was amplified using PCR mehtod, and ligated with the pMD - 18T plasmid to construct the recombinant rpMD-SEB, Which was transformed into E. coli DH5 α. The positive bacteria clones were identified ty PCR and double enzymes diegestion methods. The inserted SEB gene fragment was finally sequenced and its deduced SEB protein secondary structure analyzed. Results 705 bp SEB gene fragement was correctly cloned. Compared with S. aureus strain S6, the overall nucleotides identity was 98.4 %. Among the 11 different nucleotids, 4 nucleotids leading to the encoding amino residues different. However, the heterogeneity of nucleotides had no significant influence on the deduced SEB protein secondary structures between the S. aureus strain S407030 and S6. Conclusion The Staphylococcal Enterotoxin B gene of S. aureus strain S407030 was successfully cloned, and it was relatively conserved among different S. aureus strains. The problem that the heterogeneity of nucleotides of SEB gene lead to the variety of SEB virulence deserved further study.

关 键 词：金黄色葡萄球菌 肠毒素B 克隆 分子特征 

分 类 号：R373[医药卫生—病原生物学]