哈维弧菌rbsB基因的克隆与表达

摘要

Vibrio harveyi is one of the most important mariculture pathogens.We amplified the whole rbsB gene(GenBank ID MF797015)sequence of V.harveyi strain 354 which was predicted to encode 292 amino acid with molecular weight of 30.7 kD,iso-electric point of 5.05 and hydrophilic coefficient of 0.043. Thus, the Vh-RbsB may be a hydrophobic protein. Phylogenetic tree con-structed based on RbsB amino acid sequence indicates that Vh-RbsB has the closest relationship with V.owensii CAIM 1854.A re-combinant plasmid pGEX-4t-1-rbsB was constructed and transformed into Escherichia coli BL21(DE3).The relative molecular weight of the recombinant protein was about 59 kD. The highest expression was observed at 37 ℃, 8 h, concentration of IPTG of 0.6 mmol·L–1.%哈维弧菌(Vibrio harveyi)是海水鱼养殖中一种重要的病原菌.该研究根据已知rbsB (核糖体结合蛋白)基因序列设计引物,扩增得到哈维弧菌354(V.harveyi 354)rbsB基因的全序列(GenBank序列号MF797015),预测该序列可编码292个氨基酸,分子量为30.7 kD,理论等电点为5.05,亲水性系数为0.043,为疏水性蛋白.根据RbsB氨基酸构建的系统进化树可以发现哈维弧菌RbsB蛋白和欧文弧菌(V.owensii CAIM 1854)的关系最近.构建了pGEX-4t-1-rbsB重组质粒并转化至大肠埃希菌BL21(DE3)中,得到的重组蛋白的相对分子量约59 kD,在37℃、IPTG浓度为0.6 mmol·L–1诱导8 h时表达量最高.