无乳链球菌LIP蛋白的截短表达及免疫保护性研究

为评价罗非鱼源无乳链球菌(Streptococcus agalactiae)BX2012株脂蛋白(lipoprotein)的免疫原性及其对奥利亚罗非鱼(Oreochromis aureus)和大菱鲆(Scophthalmus maximus)的保护效果,以无乳链球菌脂蛋白基因序列(GenBank序列号:CP000114.1,SAK_0321)的B细胞线性抗原表位区设计特异性引物进行扩增,构建重组表达载体,将截短表达的脂蛋白制备成亚单位疫苗,同时制备灭活疫苗进行免疫比对。结果显示:重组表达载体p ET-32a-LIP342在BL21(DE3)中获得了良好的可溶性表达,分子质量约为30 kDa,纯化使LIP342纯度由41.75%提至87.23%;Western blotting分析显示,LIP342可被兔抗无乳链球菌高免血清特异性识别;LIP342在目前已知不同种属来源或不同血清型的无乳链球菌分离株中同源性为90.35%~100%,在罗非鱼源分离株中同源性为100%;无乳链球菌LIP342蛋白和灭活疫苗均可显著提升奥利亚罗非鱼和大菱鲆血清抗体水平,而LIP342诱导的血清抗体水平均显著高于灭活疫苗;经0.1 mL 1×10~9cfu/mL的无乳链球菌攻毒后,LIP342蛋白和灭活疫苗对供试鱼的累积存活率均显著高于PBS对照组。结果表明,高度保守的LIP342具有较好的免疫原性,可作为无乳链球菌亚单位疫苗候选因子。

Truncated expression of one lipoprotein of Streptococcus agalactiae and its immunoprotection

The antigenicity and immunoprotection to Oreochromis aureus and Scophthalmus maximus of lipoprotein in Streptococcus agalactiae BX2012 strain isolated from tilapia were analyzed.Specific primers were designed according to B cell linear epitopes of lipoprotein gene sequence (GenBank ID: CP000114.1,SAK_0321) predicted by bioinformatics software.The recombinant expression plasmid was structured.Subunit vaccine of lipoprotein after truncated expression and purification was made and researched compared with inactivated vaccine.The results showed that pET-32a-LIP342 prokaryotic expression plasmid was constructed successfully, and soluble recombinant protein with molecular mass of 30 kDa was expressed efficiently in BL21 (DE3).The purity of LIP342 after purification was increased from 41.75% to 87.23%.Homology of LIP342 from all known species or different serotypes S.agalactiae isolates was 90.35%～100%, and 100% among tilapia isolates.LIP342 was distinguished specifically by high immune serum of rabbit anti-S.agalactiae in Western blotting test.Serum antibody levels and cumulative survival rates after challenged of tilapia and turbot immunized by LIP342 and inactivated vaccine were significantly higher than that of PBS control group.What's more, serum antibody levels of tilapia and turbot induced by LIP342 was significantly higher than that of inactivated vaccine.So, LIP342 was proved as a conserved antigen with good immunogenicity and a subunit vaccine candidate to fish against S.agalactiae.