重叠延伸PCR法克隆人脂联素基因及其在毕赤酵母中表达.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2008, August 25; 24(8): 1480-1484 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究简报 重叠延伸PCR 法克隆人脂联素基因及其在毕赤酵母中 表达 1 2 1 1 1 1 1 张变玲 , 张儒 , 姬婧媛 , 薛柯 , 荆二勇 , 张展鹏 , 尉亚辉 1 西北大学生命科学学院 西部资源生物与现代生物技术省部共建教育部重点实验室, 西安 710069 2 湖南工程学院化学化工系, 湘潭 411104 摘 要: 利用重叠延伸PCR 法克隆出人脂联素基因, 连接至克隆载体pGEM-T 中, 转化大肠杆菌DH5α, 通过测序对其 进行序列分析后, 进一步构建毕赤酵母表达载体 pPIC3.5K-ADPN, 通过电击法转化毕赤酵母 GS115, 转化的重组酵母 菌用甲醇诱导外源基因表达。经PCR 、Southern blotting 鉴定, 获得了重组毕赤酵母菌株; 经甲醇诱导人脂联素基因表 达, Western blotting 杂交鉴定证明人脂联素基因已经在毕赤酵母中成功表达。结果表明重叠延伸PCR 法准确方便克隆出 人脂联素基因, 在30oC, 1% 甲醇诱导48 h, 人脂联素基因在巴斯德毕赤酵母中的表达最佳。 关键词: 人脂联素, 重叠延伸 PCR, 巴斯德毕赤酵母, Southern blotting, Western blotting Cloning of Human Adiponectin Gene by PCR-driven Overlap Extension and Expression in Pichia pastoris 1 2 1 1 1 1 Bianling Zhang , Ru Zhang , Jingyuan Ji , Ke Xue , Eryong Jing , Zhanpeng Zhang , and Yahui Wei 1 1 Key Laboratory of Resource Biology and Biotechnology in Western China, School of Life Science, Northwest University, Xi ’an 710069, China; 2 Department of Chemistry and Chemical Engineering, Hunan Institute of Engineering , Xiangtan 411104,China Abstract: Gene of human adiponectin (ADPN) was cloned by PCR-driven overlap extension. The ADPN gene was linked into pGEM-T vector. After the sequence was determined, the ADPN gene was subcloned into expression vector pPIC3.5K to yield the rec