【摘要】 目的克隆表达大鼠长链2-羟酸氧化酶。方法以鼠肾cDNA为模板,扩增大鼠长链2-羟酸氧化酶基因,连接到pET-28a(+)载体上构建重组质粒pET28a-rHao2,转化表达宿主Rosetta(DE3)。利用IPTG诱导重组蛋白的表达,并通过Ni柱纯化。通过监测DCIP在605 nm处吸光度值的变化来测定其对经典底物的酶动力学参数,验证活性。结果成功构建了表达质粒pET28a-rHao2(β1)和pET28a-rHao2(β2),并诱导表达了大鼠长链2-羟酸氧化酶两种同工酶β1和β2,其对2-羟基辛酸的Km分别为(25.1±1.9)和(24.6±1.2)μmol.L-1;对2-羟基异己酸的Km分别为(24.6±2.3)和(22.4±1.6)μmol.L-1。结论成功克隆表达了大鼠长链2-羟酸氧化酶两种同工酶β1和β2,获得了纯度高,活性好的重组酶,可为其体外底物及抑制剂的筛选提供模型。更多还原

【Abstract】 OBJECTIVE To clone and express rat long-chain 2-hydroxy acid oxidase.METHODS Rat long-chain 2-hydroxy acid oxidase gene was amplified from rat kidney cDNA,and was cloned into pET-28a(+) vector as pET28a-rHao2 recombinant plasmid,which was then transformed into Excherichia coil strain Rosetta(DE3).The expression of recombinant protein was induced by IPTG,and was purified by Ni-NTA purification system.The kinetic parameters of classic substrates were determined by measuring the change of DCIP in A605 to identify the enzyme activities.RESULTS The pET28a-rHao2(β1) and pET28a-rHao2(β2) recombinant plasmids were successfully constructed,and the rat long-chain 2-hydroxy acid oxidase isozymes β1 and β2 were successfully induced and expressed.The Km values of 2-hydroxyoctanoic acid were(25.1±1.9) and(24.6±1.2) μmol·L-1 for the rat long-chain 2-hydroxy acid oxidase isozymes β1 and β2 respectively.The Km values of(S)-(-)-2-hydroxyisocaproic acid were(24.6±2.3) and(22.4±1.6) μmol·L-1 respectively.CONCLUSION Rat long-chain 2-hydroxy acid oxidase isozymes β1 and β2 were successfully cloned and expressed with high purity and good enzyme activities.It can be used as a model to screen the substrates and inhibitors of 2-hydroxy acid oxidase in vitro.更多还原