大黄鱼源溶藻弧菌的鉴定及其菌蜕制备  

Identification of Vibrio alginolyticus isolated from large yellow croaker(Pseudosciaena crocea) and generation of V. alginolyticus ghosts

作　　者：曹际 马林 张文畅 张舒六 张嘉俊 李槿年[1] 黄安宁[1] 

机构地区：[1]安徽农业大学动物科技学院人兽共患病重点实验室,安徽合肥230036

出　　处：《微生物学通报》2018年第1期129-137,共9页Microbiology China

基　　金：国家自然科学基釐(31672698);安微农业大学研究生创新基釐(2017yis-15);安徽省大学生创新创业训练计划(AH201510364019)~~

摘　　要：【背景】菌蜕是诱导Phi X174噬菌体裂解基因E(Lysis E)在革兰氏阴性菌中表达后所获得无细胞内容物的细菌空壳。菌蜕生物安全性高,能以类似活菌方式诱导机体产生良好的系统和黏膜免疫应答。【目的】对分离自患溃疡病大黄鱼肝脏中的病原菌株16-3进行种属鉴定,利用温控调节表达系统控制Phi X174噬菌体裂解基因E在该菌株中的表达来制备菌蜕,为防控鱼类溶藻弧菌感染提供有效手段。【方法】采用形态特征观察、生理生化特性测定及16S r RNA基因序列分析等方法对菌株16-3进行鉴定;构建温控裂解质粒p BV220-Lysis E,并将其电转至溶藻弧菌菌株16-3,形成重组溶藻弧菌菌株16-3(p BV220-Lysis E);将不同起始浓度的重组溶藻弧菌培养物同时进行42°C升温诱导,比较其溶菌动力曲线和裂解效率的差异;在最佳条件下制备溶藻弧菌菌株16-3菌蜕,电镜观察其形态与结构,采用倾注平板法测定冻干菌蜕中的活菌数。【结果】综合菌株16-3在形态、生理生化及16S r RNA基因系统发育等方面的特性,确定其为溶藻弧菌;构建了温控裂解质粒p BV220-Lysis E和重组溶藻弧菌菌株16-3(p BV220-Lysis E);溶藻弧菌菌株16-3菌蜕制备的最佳条件是选择起始浓度OD600为0.3的菌液进行诱导,诱导3 h后即可收获菌蜕,其裂解效率为96.9%,但经冻干处理后的菌蜕无活菌残留;电镜观察发现菌株16-3菌蜕保持原细胞的基本形态,但细胞表面有明显的溶菌孔道,且由于细胞内容流失而使细胞表面发生皱缩。【结论】制备出溶藻弧菌菌株16-3菌蜕,为其作为疫苗或疫苗递送载体奠定了基础。[Background] Bacterial ghosts（BGs） are empty and intact bacterial envelopes of Gram-negative bacteria that are produced by controlled expression of the phage Phi X174 lysis E gene. BGs have higher biological safety, which can induce good systemic and mucosal immune response in a similar way of live bacteria. [Objective] To identify the pathogenic strain 16-3 isolated from the liver of large yellow croaker（Pseudosciaena crocea） with ulcerative disease and to generate bacterial ghosts from this strain by the temperature-controlled expression of cloned bacteriophage Phi Xl74 lysis gene E. This study will provide an effective means to prevent and control the infection of Vibrio alginolyticus in aquaculture fish species. [Methods] Strain 16-3 was identified by morphological, physiological and biochemical characteristics and 16 S r RNA gene sequence analysis. Temperature-controlled lysis plasmid p BV220-Lysis E was constructed and then electroporated into V. alginolyticus strain 16-3 to form recombinant strain 16-3（p BV220-Lysis E）. Different initial concentration cultures of recombinant strain 16-3（p BV220-Lysis E） were induced at 42 °C to compare the differences of lysis kinetics curves and lysis rate among them. V. alginolyticus strain 16-3 ghosts were generated under the optimum condition, and then their morphology and structure were observed by electronic microscope, and the viable cell counts in the lyophilized ghosts were measured using a pour plate method. [Results] The pathogenic strain 16-3 was identified as V. alginolyticus according to morphological features, physiological and biochemical characteristics an d 16 S r RNA gene phylogenetic analysis. The temperature-controlled lysis plasmid p BV220-Lysis E and recombinant V. alginolyticus strain 16-3（p BV220-Lysis E） were constructed, respectively. The optimum conditions for V. alginolyticus strain 16-3 ghosts generation were as follows： the induction was carried out by selecting the initial concentration of cultures reached an OD600 of

关 键 词：溶藻弧菌 鉴定 温控裂解质粒 菌蜕 

分 类 号：S943[农业科学—水产养殖]