鳗弧菌膜绑定溶胞壁质转糖酶MltD的表达纯化及生物信息学分析

Expression,Purification and Bioinformatic Analysis of Membrane-Bound Lytic Murein Transglycosylase D(MltD) Protein in Vibrio anguillarum

本研究将新发现的鳗弧菌毒力相关基因mltD(膜绑定溶胞壁质转糖酶基因)克隆于表达载体pET-32a(+),在大肠杆菌BL21(DE3)中诱导表达,并以镍琼脂糖亲和层析柱纯化表达的融合蛋白;经SDS-PAGE分析,纯化的融合蛋白为单一条带,分子量约为70.2 kDa,与理论预测值相符。生物信息学分析表明,鳗弧菌MltD由523个氨基酸组成,与其他弧菌的MltD氨基酸序列有较高的相似性;二级结构以α螺旋和无规则卷曲为主;功能区包括N端的信号肽区域、1个糖基转移酶结构域和C端的3个溶解酶结构域;蛋白的N端具有一段脂蛋白信号肽,信号肽外的其他部分为非细胞质蛋白。鳗弧菌MltD蛋白为亲水性;不稳定系数为27.40,属于稳定性蛋白;整个蛋白分子共有24个可能的抗原决定簇,具有较强的抗原性。推断MltD蛋白为1种外周膜脂蛋白,在弧菌中相对保守,抗原性强,可应用于抗弧菌病疫苗的开发研制。

In this study,membrane-bound lytic murein transglycosylase D（mltD） from Vibrio anguillarum was chosen as the research object.MltD gene was subcloned into an expression plasmid pET-32a（＋）,and overexpressed in Escherichia coli strain BL21（DE3）.The expressed MltD protein was purified by Ni2＋-affinity chromatography.The molecular weight of recombinant protein was assessed to be about 70.2 kD by SDS-PAGE analysis,which was consistent with the therotical value.Bioinformatic analysis showed that the MltD consisted of 523 amino acids,and the amino acid sequence was highly consistent with the MltD proteins in other Vibrio sp..The secondary structure of MltD in Vibrio anguillarum was predominated by α helix and randon coil.The MltD protein comprised a signal peptide,a transglycosylase domain and three LysM domains at C-terminal.Moreover,a lipoprotein signal peptide was at the N-terminal,and the rest was non-cytoplasmic except the signal peptide.The whole protein molecule was stable in physical properties,hydrophilic,and showed solid antigenicity.It was speculated that the MltD protein in Vibrio anguillarum was a peripheral membrane lipoprotein,and relatively conservative in Vibrio sp.,which had strong antigen activity,and could be used in developing of vaccine against vibriosis.