淬灭酶AiiO-AIO6酶学性质及对嗜水气单胞菌毒力因子的表达调控

将N-乙酰高丝氨酸内酯酶基因(aiiO-AIO6)插入表达载体pET28a构建了原核表达载体pET28a/ aiiO-AIO6.转化大肠杆菌后筛选具有活性的重组子并对纯化的目的蛋白AiiO-AIO6的酶学性质进行了研究,同时研究了纯化的酶液对嗜水气单胞菌ATCC7966溶血素( Hemolysin,Hem)、细胞肠毒素(Cytotonic enterotoxin,Ast)、胞外蛋白酶(Extracellular protease,Ep)、丝氨酸蛋白酶(Serine protease,Sp)及磷脂酶Pho( Phospholipase Al,Pho)等5个毒力因子表达的影响.结果表明,pET28a/aiiO-AIO6在大肠杆菌中大量表达N-乙酰高丝氨酸内酯酶,纯化后的N-乙酰高丝氨酸内酯酶的最适作用条件为pH8.0,30℃,在pH 6.0 ～11.0的范围内能够稳定的存在,在80℃条件下保温30 min后酶活力能够维持在65％以上；且该酶对多种金属离子、化合物和中性蛋白酶都具有很好的抗性；该酶对多种底物都具有一定的降解作用；以3-oxo-C8-HSL为底物时的km值为0.015 mmol/L；比活力为583.33 U/mg.N-乙酰高丝氨酸内酯酶在培养8h及12 h时对嗜水气单胞菌5个毒力因子的表达量都具有明显的下调趋势(P＜0.05).本实验通过测定AiiO-AIO6的酶学性质及证明AiiO-AIO6可以通过降解嗜水气单胞菌产生的信号分子来抑制其毒力因子的表达,从而为将其应用于水产环境及致病性嗜水气单胞菌的生物防治奠定理论基础.

Characteristics of quenching enzyme AiiO-AIO6 and its effect on Aeromonas hydrophila virulence factors expression

The prokaryotic expression vector pET28a/aiiO-AIO6 was constructed by inserting the aiiO-AIO6 gene into expression vector pET28a and transformed into E.coli.Subsequently,the recombinants with activity of N-acyl homoserine lactonase were investigated.After purification with nickel column,enzymatic characteristic of the target protein was researched.Moreover,the effects of purified enzyme solution on the expression of Hemolysin,Cytotonic enterotoxin,Extracellular protease,Serine protease,Phospholipase A1,ea t...