玉米赤霉烯酮降解酶多拷貝表达载体构建及其毕赤酵母高效表达.docVIP

多拷贝玉米赤霉烯酮降解酶在毕赤酵母的分泌表达 王义春 江均平* （农业部农产品加工综合性重点实验室，中国农业科学院农产品加工研究所，北京 100193） 摘要：本研究通过多拷贝克隆技术实现玉米赤霉烯酮（Zearalenone, ZEN）降解酶基因（zlhy-6）在巴斯德毕赤酵母中的高效分泌表达。基因序列根据酵母密码子偏爱性优化后与α因子信号肽编码序列一起合成（即α-zlhy-6），插入到 pA0815 中，并构建 pA0815-（α-zlhy-6）4 多拷贝表达载体，转化毕赤酵母 GS115 后进行诱导表达，并对表达产物进行鉴定。结果表明，酶切证实成功构建了α-zlhy-6多拷贝毕赤酵母表达载体 pAO815-（α-zlhy-6）4，利用 SDS法检测到表达产物在 30 kDa 附近出现条带，与预期值相符。活性测定表明重组酶具有较高降解 ZEN 的能力。重组酶对水溶液中 ZEN 的降解率可达到 97.5% 以上，对玉米碴中 ZEN 的降解率达到 75.51% 左右。 关键词：玉米赤霉烯酮；降解；基因表达；毕赤酵母 Construction of a Multi-Copy Secretory Expression Vector and Expression of ZEN Degradation Enzyme in Pichia pastoris Wang Yichun, Jiang Junping* (Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Institute of Agro-Products Processing Science Technology, Chinese Academy of Agricultural Sciences, Beijing, 100193) Abstract: In this work, a multi-copy method for secreted expression of Zearalenone degradation enzyme encoded by zlhy-6 was developed. Zlhy-6 optimized according to codon preference of Pichia pastoris was syntheticed along with alpha (α) signal peptide gene and cloned into pAO815. The multi-copy expression vector pAO815-(α-zlhy-6)4 was constructed. The recombinant was transformed into P. pastoris strain GS115 for induction expression and then the activity of secreted products was identified. According to the results, a new multi-copy vector pAO815-(α-zlhy-6)4 was successfully constructed and was capable of secreting recombinant enzyme efficiently, which was confirmed by enzyme digestion and SDSrespectively. The recombinant enzyme possessed high activity of ZEN-degradation. The degradation rate of 97.5% and 75.51% were achieved in the aqueous solution and in the corn, respctively. Key words: Zearalenone; Degradation; Gene expression; Pichia pastoris; 玉米赤霉烯酮(Zearalenone，ZEN)，又称F-2毒素，2,4-二羟基苯甲酸内酯类化合物，主要由禾谷镰刀菌产生的一种霉菌毒素。ZEN具有雌激素活性，对生殖发育系统有毒害作用。摄入ZEN超标的母猪会出现外阴和乳腺肿大甚至阴道及直肠脱垂的症状[1]。ZEN有致癌性，能导致DNA收敛、染色体断裂[2]，还可能通过雌激素受体通路和抑制凋亡促进人神经母细胞瘤 SK-N-SH 细胞的体外增殖[3]。Jian Ying等[4]研究发现ZEN能导致老鼠变态数精子增加、