哈维氏弧菌PspF基因的克隆及原核表达分析Cloning and Prokaryotic Expression of PspF Gene from Vibrio harveyi
马少鸿,黄郁葱,简纪常,蔡双虎
摘要(Abstract)：
【目的】对哈维氏弧菌(Vibrio harveyi)ZJ0603的PspF基因进行克隆与原核表达分析,优化表达条件。【方法】克隆哈维氏弧菌菌株ZJ0603的FspF基因,对其编码蛋白进行理化性质、信号肽、亚细胞定位、二级结构及三级结构分析,构建表达载体pET28a-PspF,经BamHI和XhoI双酶切和测序鉴定正确后转入表达菌株大肠杆菌BL21,对表达重组菌株进行表达条件优化及Western Blot鉴定。【结果与结论】PspF基因的开放阅读框(ORF)全长为1 179 bp,编码336个氨基酸,分子质量为38.04 ku,理论等电点5.27,不稳定系数41.97,总平均亲水性-0.382,PspF蛋白整体表现为亲水性蛋白。成功构建含pET28a-PspF的表达菌株,其最佳诱导条件为37℃下异丙基-β-D-硫代半乳糖苷(IPTG)0.2 mmol/L诱导6 h,其表达蛋白为38 ku。Western Blot结果表明PspF重组蛋白成功获得,蛋白同源性高,具有作为抗弧菌病疫苗的潜力。
关键词(KeyWords)： 哈维氏弧菌;PspF基因;原核表达;表达优化
基金项目(Foundation): 广东省科技计划(2016A020209010);; 广东省自然科学基金(2017A0303030975)
作者(Author): 马少鸿,黄郁葱,简纪常,蔡双虎
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