中文摘要：

目的克隆表达大鼠长链2一羟酸氧化酶。方法以鼠肾eDNA为模板，扩增大鼠长链2-羟酸氧化酶基因，连接到pET-28a（4-）载体上构建重组质粒pET28a—rHa02，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605nm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—rHa02（β1）和pET28a—rHa02（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K分别为（25．1±1．9）和（24.6±1．2）μmol·L-1；对2-羟基异己酸的k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。

英文摘要：

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 eDNA, and was cloned into pET-28a（ ＋ ） vector as pET28a-rHao2 recombinant plas- mid,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 As0s 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-hydroxyisoeaproie 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-hy- droxy acid oxidase in vitro.