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    大牡蛎弧菌S2胶原酶基因的克隆、表达及其对菲律宾蛤仔基因表达的调控

    Cloning and expression of the collagenase gene from Vibrio crassostreae S2 and its regulation on gene expression in Manila clam Ruditapes philippinarum

      摘要
    • 摘要:
      目的 菲律宾蛤仔（Ruditapes philippinarum）是中国重要的水产养殖贝类，然而细菌性疾病频发严重制约其育苗产业发展。大牡蛎弧菌（Vibrio crassostreae）S2是菲律宾蛤仔育苗过程中重要的病原菌，本研究旨在鉴定大牡蛎弧菌S2的致病因子，并筛选可抑制致病因子的物质。
      方法 设计引物进行聚合酶链式反应（PCR）克隆基因，并将基因重组至表达载体pET28-a(+)中，转入表达菌株大肠杆菌（Escherichia coli）BL21(DE3)，经表达、复性与纯化获得重组胶原酶（rColVc）。通过注射实验分析rColVc致病性，利用转录组分析胶原酶对菲律宾蛤仔基因表达的调控作用。最后以胶原酶活性为指标，筛选对胶原酶具有抑制活性的小分子药物。
      结果 大牡蛎弧菌S2胶原酶基因colVc编码含有277个氨基酸的蛋白质。表达菌株BL21/pET28colVc可大量表达胶原酶，纯化了重组的蛋白酶，将rColVc注射至菲律宾蛤仔中可导致20%的菲律宾蛤仔死亡。胶原酶注射菲律宾蛤仔后，导致132个基因差异表达，下调的差异表达基因显著富集于DNA重组、DNA整合、DNA代谢等生物学过程，以及肌动蛋白结合、细胞骨架蛋白结合等分子结合功能。KEGG富集分析表明，胶原酶组菲律宾蛤仔中下调的差异表达基因主要富集于肌动蛋白细胞骨架调控通路，提示胶原酶在弧菌致病中起重要作用。核黄素、姜黄素、原花青素、盐酸巴马汀、2’-羟基查尔酮、氯己定和苯扎氯铵均对rColVc产生抑制作用。
      意义 本研究首次揭示了大牡蛎弧菌S2胶原酶对菲律宾蛤仔的致病性及基因表达的调控作用，并为其育苗过程中弧菌病的防控提供了潜在靶位点。

       

      Abstract:
      Objective Manila clam (Ruditapes philippinarum) is a key aquaculture shellfish species in China, however, frequent bacterial diseases severely constrain its hatchery production. Vibrio crassostreae S2 is an important pathogen during clam larval rearing. The purpose of this study is to identify the pathogenic factor of V. crassostreae and screen for chemicals that can inhibit the pathogenic factor.
      Methods The collagenase gene (colVc) was cloned using PCR with designed primers, ligated into the expression vector pET-28a(+), and expressed in Escherichia coli BL21(DE3). Pathogenicity was assessed through injection assay, and transcriptomic data was used to reveal the regulation of collagenase on gene expression in clams. Small molecule inhibitors were screened, based on the ability to inhibit the enzymatic activity of collagenase.
      Results The colVc gene encoded a protein containing 277 amino acids, without the classic domain. The expression strain BL21/pET28colVc produced recombinant collagenase rColVc, which induced more than 20% mortality in clams upon injection. Transcriptomic data identified 132 differentially expressed genes (DEGs). The downregulated DEGs were enriched in BPs of DNA recombination, DNA integration, and DNA metabolic processes; MFs of actin binding and cytoskeletal protein binding. KEGG enrichment analysis showed that the downregulated DEGs in the collagenase-treated group of clam were primarily enriched in the actin cytoskeleton regulation pathway, implicating the critical role of collagenase in virulence. Finally, using the enzymatic activity of the collagenase as the target, the compounds, riboflavin, curcumin, proanthocyanidins, palmatine hydrochloride, 2’-hydroxychalcone, chlorhexidine, and benzalkonium chloride were tested to exhibit inhibitory effects on enzymatic activity of collagenase.
      Significance The study is the first time to elucidate the pathogenicity of collagenase in V. crassostreae S2 and explore its regulation on gene expression of Manila clam, providing the potential target for controlling vibriosis in clam hatcheries.

       

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