以迎霜品种茶树基因组为模板,利用高保真聚合酶pfu,通过PCR方法扩增得到茶树PPO基因的编码区序列。通过设计酶切引物将其成功融合至真核表达载体pPICZa中,构建出茶多酚氧化酶的融合蛋白真核表达载体pPICZa-PPO,并将其转化进入巴斯德毕赤酵母（Pichia pastoris）GS115中,成功筛选出多个阳性转化子。对阳性转化子进行甲醇诱导,采用Western-Blotting方法在培养液中成功检测到诱导表达的目的蛋白。酶活检测结果也显示诱导产物具有较高的相对酶活性,能够正确发挥酶蛋白的生物功能。
The polyphenol oxidase (PPO) cds sequence was amplified by PCR from the genome of Yingshuang tea plant with the high fidelity polymerase pfu. The cds sequence was recombinanted into the eukaryotic expression vector pPICZa with the designed primer, and the fusion protein eukaryotic expression vector of tea polyphenol oxidase “pPICZa-PPO” was succeeded to be constructed, and then it was transformed into Pichia pastoris GS115. Therefore, several positive converters were screened. After those positive converters were induced by methanol,the target protein was successfully detected in the culture medium by using Western-Blotting method. The detection result of enzyme activity showed that the induced products had a relatively high enzyme activity and can correctly play their biological activity.
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