灿烂弧菌粘附相关因子VsA的表达及功能研究

The expression and function of adhesion related factor VsA in Vibrio splendidus

为了探究灿烂弧菌中Zn^(2+)转运系统的细胞周质组分Vs A的表达与功能,本实验采用PCR克隆vsA基因,实时荧光定量测定vsA基因的表达,利用抗体封闭实验研究Vs A蛋白的功能。生物信息学分析表明Vs A具有结合和转运Zn^(2+)的结构基础。实时荧光定量结果表明,vsA的表达受Zn^(2+)调控,且具有浓度依赖性,0.01 mmol/L的Zn^(2+)可使vsA的表达量下调至对照组的0.5倍,而添加等摩尔量的Cu^(2+)、Fe3+、Mg^(2+)和Ca^(2+)对vsA的表达无明显影响。而0.1μmol/L Zn^(2+)则会诱导vsA的表达。此外,在大肠杆菌Escherichia coli BL21 (DE3)中进行了Vs A的重组表达,制备了Vs A的小鼠多克隆抗体。利用抗体封闭Vs A蛋白可导致灿烂弧菌在Zn^(2+)限制条件下的生长受抑制,以及过氧化氢的能力和粘附效率的降低。本研究获得了灿烂弧菌的vsA基因,其表达受Zn^(2+)浓度调控,Vs A蛋白在灿烂弧菌生长、抗氧化以及粘附宿主细胞过程中发挥作用。

To explore the expression and function of periplasmic protein VsA of Zn2+ transport system in Vibrio splendidus,vsA was cloned using PCR, and its expression was characterized using real-time RT-PCR, and the function of VsA wasdetermined using antibody blocking test. Bioinformatics analysis showed that VsA has the structural basis for the combinationand transportation of Zn2+. Real-time reverse transcriptase PCR showed that expression of vsA was regulated by Zn2+level anddepend on the level of Zn2+. Addition of 0.01 mmol/L Zn2+reduced its expression to 0.5-fold compared to the control sample.While the same concentration of Cu2+, Fe3+, Mg2+and Ca2+showed no effect on the expression of vsA. 0.1 μmol/L Zn2+caninduce the expression of vsA. VsA was also recombined and expressed in Escherichia coli BL21(DE3) and the antiserumagainst VsA was obtained using the purified VsA. Using the antibody blocking method may inhibite the growth of V.splendidus under low Zn2+condition, reduce anti-hydrogen peroxide capacity and adhesion of V. splendidus. In all, vsAgene was cloned in V. splendidus, and its expression was regulated by Zn2+. VsA is involved in the growth, antioxidantactivity, and adhesion activity in V. splendidus.