溶藻弧菌VcrV基因缺失株的构建及生物学特性

Construction of VcrV-deleted mutant of Vibrio alginolyticus and its biological characteristics

摘要:

为研究溶藻弧菌Ⅲ型分泌系统VcrV基因的功能和生物学特性，采用同源重组技术成功构建了溶藻弧菌缺失株ΔVcrV，并用PCR检测其遗传稳定性。结果显示，缺失株遗传稳定；与野生株相比，生长和自凝集能力无显著变化；但生物膜形成能力降低，半致死剂量升高16.5倍；游动性和涌动性极显著升高，细胞粘附能力极显著降低(P<0.01)，对H₂O₂和NaCl的耐受性降低；对头孢呋辛、麦迪霉素、克林霉素抗生素敏感度上升，对丁胺卡那、多粘菌素B抗生素敏感度降低；活性氧含量极显著降低(P<0.01)，脯氨酸、肽聚糖、β-内酰胺酶、过氧化氢酶、超氧化物歧化酶、谷胱甘肽过氧化物酶指标均极显著升高(P<0.01)。缺失株生物学特性表明，VcrV基因参与溶藻弧菌Ⅲ型分泌系统性的致病性和多种生物学功能。

关键词:溶藻弧菌;Ⅲ型分泌系统;VcrV基因;缺失株;生物学特性

Abstract:

A mutant strain ΔVcrV was constructed by using homologous recombination method for investigating the function of the VcrV gene in Vibrio alginolyticus type Ⅲ secretion system. The genetic stability of ΔVcrV was detected by PCR, and the biological characteristics between the mutant and the wild type strains were compared. ΔVcrV muntat had no significant changes in growth rate and autoagglutination compared with the wild type strain, but the ability to form biofilms was reduced, and the LD₅₀ was increased by 16.5 times. The swimming and swarming motility of the mutant strain ΔVcrV were significantly enhanced, while cell adhesion was significantly reduced than the wild strain (P<0.01). The tolerance of ΔVcrV mutant to H₂O₂ and NaCl was decreased. Compared with that of the wild type strain, the sensitivity of ΔVcrV mutant to cefuroxime, medimycin and clindamycin was increased, but to amikacin and polymxin B was decreased. The reactive oxygen species (ROS) content of ΔVcrV mutant was significantly decreased (P<0.01), and the indexes of proline, peptidoglycan, β-lactamase, catalase, superoxide dismutase and glutathione peroxidase of ΔVcrV mutant were significantly increased than that of the wild type strain (P<0.01). The biological characteristics of ΔVcrV mutant indicated that VcrV gene was involved in pathogenicity and various biological functions of V. alginolyticus type Ⅲ secretion system.

Key words:Vibrio alginolyticus;type Ⅲ secretion system;VcrV gene;mutant strain;biological characteristics