一株乳源松鼠葡萄球菌ExhC基因的克隆及序列分析

Cloning and Sequence Analysis of ExhC Gene from a Milked Staphylococcus Sciuri

松鼠葡萄球菌(Staphylococcus sciuri,S.sciuri)是引起奶牛乳房炎(Cow Mastitis)的病原菌之一,其主要致病因子是该菌产生的脱皮毒素C(Exfoliative Toxin C,ExhC)。为了深入了解该基因的分子特征,本试验对从乳房炎患牛牛乳中分离的一株松鼠葡萄球菌ExhC基因(Genbank登录号:MT845354)进行了克隆及序列分析。按GenBank收录的ExhC基因设计并合成引物,利用PCR对ExhC基因进行扩增,并对扩增后的ExhC基因进行测序及分析。结果表明,松鼠葡萄球菌ExhC基因的开放阅读框序列长度为837bp,共编码278个氨基酸。通过Blast模块对ExhC基因进行同源性分析,得到5株相似序列,与ExhC基因序列相比同源性均为99.04%。系统进化树图指示该基因与其余五株相似序列属于不同的分支,遗传距离较远。利用TMHMM对松鼠葡萄球菌ExhC基因837bp序列跨膜区进行预测,结果显示ExhC蛋白的第1~277位氨基酸在细胞膜外,5~25位氨基酸所在位置为跨膜区。通过ExhC氨基酸序列分析和ExhC蛋白二级结构、三级结构预测,推断第97位氨基酸插入Asn以及其他位点氨基酸的突变可能会导致松鼠葡萄球菌功能区变化和毒株毒力的相对变化,这将给松鼠葡萄球菌致病机理的研究奠定基础。

Staphylococcus sciuri(S.sciuri)is the pathogen that causes Cow Mastitis in pig and ExhC produced by this bacterium was the main pathogenic factor.In order to further understand the molecular characteristics of this gene,the ExhC gene of S.sciuri isolated from bovine milk with mastitis was cloned and sequenced.Designed and synthesized primers according to ExhC gene included in GenBank,amplified ExhC gene by PCR,and sequenced the amplified ExhC gene.The results showed that:the length of ORF sequence which is from the ExhC gene of S.sciuri is 837bp,encoding 264 amino acids.Blast analyzed the homology of exhc gene,and obtained 5 similar sequences,which showed 98.92%homology with the exoc gene sequence.Phylogenetic tree analysis showed that the genetic distance of this gene was far from the other five strains.TMHMM was used to predict the transmembrane region of 837bp ExhC gene in S.sciuri.The results showed that the amino acid at positions 1~277 of ExhC protein was located outside the cell membrane,and the amino acid at positions 5~25 were transmembrane regions.By ExhC amino acid sequence analysis,ExhC protein secondary structure and tertiary structure prediction,it was inferred that the mutation of amino acid at the insertion site OF Asn and other amino acid sites might lead to functional area changes and relative changes of virulence of S.sciuri,this will lay the foundation for the research on the pathogenic mechanism of S.sciuri.