裂殖壶菌鲨烯合酶基因的克隆及在大肠杆菌中的表达.PDF

※生物工程 食品科学 2010, Vol. 31, No. 19 263 1,2 3,2 2, 朱路英 ，朱清华 ，张学成 * (1.鲁东大学生命科学学院，山东 烟台 264025 ；2. 中国海洋大学海洋生命学院，山东 青岛 266003 ； 3.德州学院生物系，山东 德州 253023) 为探讨裂殖壶菌烯合酶基因的克隆，以及在大肠杆菌中的表达。采用简并引物与RACE 相结合的方法从 裂殖壶菌(Schizochytrium limacinum) 中克隆出鲨烯合酶基因。分析表明该基因的cDNA 全长包括 1672 个核苷酸，编 码一个含 446 个氨基酸的开放阅读框；在裂殖壶菌中以单拷贝的形式存在。同源分析显示裂殖壶菌鲨烯合酶的氨基 酸序列中含有 5 个保守基序。将裂殖壶菌鲨烯合酶的 cDNA 序列连接到原核表达载体pGEX-4T-3 上构建融合表达载 体，并在大肠杆菌 BL21 中进行IPTG 诱导表达，SDS及 Western blotting 分析结果显示，诱导表达出的融合 蛋白主要以包涵体的形式存在，其相对分子质量与预期相符；且融合蛋白具有一定的免疫原性。 裂殖壶菌；鲨烯合酶；cDNA 克隆；原核表达 Cloning and Prokaryotic Expression of Squalene Synthase Gene from Schizochytrium limacinum ZHU Lu-ying 1,2 ，ZHU Qing-hua3,2 ，ZHANG Xue-cheng2, * (1. School of Life Sciences, Ludong University, Yantai 264025, China；2. College of Marine Life Sciences, Ocean University of China,Qingdao 266003, China ；3. Department of Biology, Dezhou College, Dezhou 253023, China) Abstract：A full length cDNA of squalene synthase (SQS) gene was isolated from Schizochytrium limacinum using homology- based cloning and RACE methods. Molecular biological analysis showed that the full length cDNA was of 1672 bp, with a 1338 bp open reading frame encoding 446 amino acids, and the SQS gene was a single copy gene in Schizochytrium limacinum. Homology analysis showed that Schizochytrium limacinum SQS cDNA had five consensus regions. The SQS cDNA was constructed into the prokaryotic expression vector pGEX-4T-3 and expressed in E.coli BL21. SDSand Western blotting analyses showed that the recombinant protein was mainly in the form of inclusion body, of which molecular weight was as expected, and