罗非鱼源无乳链球菌cpsE基因的克隆和分子特性分析

Cloning and molecular characterization of cpsE gene of Streptococcus agalactiae isolated from tilapia

利用特异性引物,采用PCR扩增出分离自患病罗非鱼无乳链球菌强毒株的cpsE基因,将其克隆到pMD19-T载体上,通过菌落PCR鉴定和利用限制性内切酶BamHⅠ和HindⅢ对重组质粒进行双酶切鉴定之后送测序公司测序,并利用生物信息学软件C lustal X 2.0、MEGA4.1、B ioed it 7.0、TMHMM、NetPhos 2.0、NetNG lyc 1.0、SignalP 3.0 Server、PSIpred、SAM_T08以及CUSP等分析cpsE基因的分子特性。结果显示,罗非鱼源无乳链球菌cpsE编码氨基酸序列具有高度保守性,与人源、动物源无乳链球菌亲缘性达100%,具有1个参与催化糖基元转运的G lycosyltransferases超级家族结构域,具有与蛋白翻译后修饰功能相关的磷酸化位点3个,编码多肽链中亲水区大于疏水区,且存在跨膜区。密码子偏爱性分析表明,罗非鱼源无乳链球菌cpsE基因密码子使用频率差异较大,密码子偏爱性更接近真核生物。

The cpsE gene of Streptococcus agalactiae virulent strain isolated from tilapia was amplified by PCR with specific primers and cloned into pMD19-T vector.The recombinant plasmid was strongly confirmed by PCR combined with restriction enzyme digestion（BamHⅠ and HindⅢ）.Molecular characterization analysis of cpsE gene was performed by bioinformatics tools like Clustal X 2.0,MEGA 4.1,Bioedit 7.0,TMHMM,NetPhos 2.0,NetNGlyc 1.0,SignalP 3.0 Server,PSIpred,SAM_T08 and CUSP.Results indicated that amino acid sequence deduced by cpsE of tilapia S.agalactiae was highly conserved and has revealed a surprising degree（100%）of homology among strains isolated from human and other mammals.Polypeptide analyzed in this study contained a Glycosyltransferases superfamily conserved domain that functioned as enzyme that catalyzed the transfer of sugar moieties from activated donor molecules to specific acceptor molecules.Moreover,the polypeptide possessed 3 phosphorylation sites which related to post-translational modification.The hydrophilic regions were larger than hydrophobic regions and transmembrane domain was predicted to exist in polypeptide chain.The analysis of codon bias demonstrated the codon usage frequency of cpsE of tilapia S.agalactiae was distinctly different and it preferred to perform in eucaryote.