大黄鱼源溶藻弧菌的鉴定及其菌蜕制备

【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。

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

Background] Bacterial ghosts (BGs) are empty and intact bacterial envelopes of Gram-negative bacteria that are produced by controlled expression of the phage PhiX174 lysisE 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 PhiXl74 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 16S rRNA gene sequence analysis. Temperature-controlled lysis plasmid pBV220-LysisE was constructed and then electroporated into V. alginolyticus strain 16-3 to form recombinant strain 16-3(pBV220-LysisE). Different initial concentration cultures of recombinant strain 16-3(pBV220-LysisE) 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 and 16S rRNA gene phylogenetic analysis. The temperature-controlled lysis plasmid pBV220-LysisE and recombinant V. alginolyticus strain 16-3(pBV220-LysisE) 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 0.3. The bacterial ghosts could be harvested after induced for 3 h and the lysis rate of strain 16-3 ghosts was 96.9%. However, there was no residual bacteria found on plates with lyophilized 16-3 ghosts inoculation. Electron micrographs clearly showed that no gross alterations in cellular morphology compared to unlysed cells except for the obvious lysis pore on the cell surface and cell shrinkage due to the loss of cytoplasmic materials. Conclusion] V. alginolyticus strain 16-3 ghosts were generated in this study, which provided a basis for the further use as a vaccine or vaccine delivery vector.