斑点叉尾鮰源海豚链球菌DGX07株simA基因克隆、鉴定及分子特性分析

以Primer p14(5’-GATCAAGTCC-3’)为引物对分离自患病斑点叉尾海豚链球菌强毒株DGX07进行RAPD分析。同时参照GenBank中海豚链球菌simA基因序列设计特异性引物,以DGX07基因组DNA为模板,扩增出约1 500 bp的simA基因,并将其克隆到pMD19-T载体上,之后对重组质粒进行PCR和双酶切(BamHⅠ+XhoⅠ)鉴定,鉴定正确后送测序公司测序。RAPD分析结果显示,DGX07和标准菌株均能扩增出750 bp大小的条带。通过生物信息学软件对测序结果分析显示,simA基因全长1 566 bp,由521个氨基酸组成,与海豚链球菌simA和simB亲缘性达100%,存在1个由41个氨基酸组成的信号肽,具有Bap31和Gram_pos_anchor两个超家族的保守结构域;具有与蛋白翻译后修饰功能相关的磷酸化位点22个和N-糖基化位点2个,编码多肽链中亲水区大于疏水区,是一种膜外蛋白,并具有多个抗原优势位点区域。密码子偏爱性分析表明,斑点叉尾源海豚链球菌simA基因密码子使用频率差异较大,密码子偏爱性与酵母较为接近。获得GenBank登录号为JF330100。

Cloning,identification and molecular characteristics analysis of simA gene of a Streptococcus iniae strain DGX07 from channel catfish

The RAPD analysis of Streptococcus iniae virulent strain DGX07 isolated from channel catfish(Ictalurus punctatus)was conducted by PCR with Primer p14(5′-GATCAAGTCC-3′).The simA gene of DGX07 was amplified by PCR with specific primers and cloned into pMD19-T vector.The positive plasmid was selected and identified by PCR and digestion of double restriction enzyme(BamHⅠ+XhoⅠ).Molecular characterization analysis of simA gene was performed by bioinformatics tools.The results showed that S.iniae simA amino acid sequence was highly conservative and with 100% homology to the simA and simB of S.iniae isolated.The polypeptide contained a signal peptide comprising 41 amino acids and Bap31 and Gram_pos_anchor functional domains.The polypeptide had some important sites related to post-translational modification,including 22 phosphorylation sites and 2 N-glycosylation sites.The hydrophilic regions were larger than hydrophobic regions in polypeptide chain.The S.iniae simA was extramembranous protein with multiple antigen advantaged regions.The analysis of codon bias indicated the codon usage frequency of S.iniae simA different,and the codon bias of S.iniae simA preferred the yeasts.Gene accession number:JF330100.