罗非鱼无乳链球菌α蛋白基因的克隆、鉴定及分子特性分析

Cloning,Identificatin and Molecular Characteristics Analysis of α Protein Gene of Streptococcus agalactiae from Tilapia

本研究克隆了罗非鱼源无乳链球菌HN0101分离株的α蛋白基因,通过生物信息学对其序列分析结果显示:α蛋白基因的ORF由1341个碱基组成,编码一个长度为446个氨基酸的多肽,N-端具有一个保守的YSIRK信号肽序列和一个AlphaC_N超家族结构域,C-末端含有1个保守Gram_pos_anchor超家族结构域,而在中间具有2个重复的Rib结构域;比较不同来源分离株的α蛋白结构,发现主要区别在于Rib结构域重复数目的不同;同源性及系统进化树分析,发现本株菌的α蛋白与已报道的人源A909和鱼源GD201008-001α蛋白聚为一簇,同源性达100%;罗非鱼源无乳链球菌α蛋白为亲水性蛋白,存在一个跨膜区,有37个潜在的磷酸化位点和1个潜在的N-糖基化位点;二级结构分析发现存在较多的无规则卷曲,推测有12个氨基酸区域可能是B细胞抗原表位;当选择大肠杆菌为表达宿主时,该重组蛋白的溶解度高达100%,亚细胞定位显示该蛋白位于细胞壁上。

α protein gene of Streptococcus agalactiae that isolated from tilapia was cloned in this study. Based on the prediction of bioinformatics analysis, the ORF of α protein gene was composed of 1341 nucleotides and encoded a polypeptide of 446 amino acids. The structure domain analysis showed a conserved YSIRK signal peptide sequence and an alpha C_N super family structure domain in N-terminal, a conserved Gram_pos_anchor super family structure domain in C-terminal, and two Rib structure domains in the median region. Moreover, the main difference was the repeat number of Rib as deter- mined by comparing the structures of c~ protein of different origins. The homology of a protein was 100% between the strain A909 （this study） and GD201008-001 based on the results of homologous and phylogenetic analysis. Our results also showed that a protein was a hydrophilic protein with trans-membrane region, and had 37 potential phosphorylation sites and one potential N-glycosylation site. Secondary structure analysis showed lots of random coil. It is predicted that the region of 12 amino acids should be B cell epitopes. When Escherichia coli were selected as expressing hosts, the solubility of the recombinant protein could reach 100%. Subcellular localization showed that the protein located in the cell wall.