溶藻弧菌Ⅲ型分泌系统注射装置蛋白VscO的原核表达及免疫原性Prokaryotic Expression and Immunogenicity of the Type Ⅲ Secretion System Injectisome Protein VscO from Vibrio alginolyticus
庞欢瑛,周泽军,丁燏,黄郁葱,吴灶和,简纪常
摘要(Abstract)：
为探究溶藻弧菌(Vibrio alginolyticus)ZJ03株Ⅲ型分泌系统(Type III secretion system,T3SS)注射装置蛋白VscO作为疫苗候选抗原的可能性,根据GeneBank上登陆的溶藻弧菌VscO序列(NO.KJ179947),设计1对带酶切位点的特异性引物,PCR扩增vscO基因,序列分析结果显示,该基因全长462 bp,理论分子质量为18.430ku。将vscO基因定向插入原核表达载体pET-28a(+),构建重组表达质粒pET-vscO。用异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,可在大肠杆菌(Escherichia coli)BL21(DE3)中表达分子质量约为22 ku的VscO融合蛋白,且该蛋白主要以包涵体形式存在。VscO蛋白表达和纯化的最优条件为:0.1 mmol/L IPTG、37℃条件下诱导4 h,咪唑洗脱浓度为400 mmol/L。用纯化后的融合蛋白免疫SPF级小鼠,获得高效多克隆抗体。Western-blotting结果表明,鼠抗VscO血清既能与重组VscO蛋白发生反应,也能与分离自溶藻弧菌约22 ku的天然蛋白发生反应,提示T3SS注射装置蛋白VscO可能是溶藻弧菌的重要保护性抗原之一。
关键词(KeyWords)： 溶藻弧菌;vscO基因;Ⅲ型分泌系统;原核表达;免疫原性
基金项目(Foundation): 国家科技支撑计划(2012BAD17B02、2012 BAD17B03);; 广东省自然科学基金(S2013040014562)
作者(Author): 庞欢瑛,周泽军,丁燏,黄郁葱,吴灶和,简纪常
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