重叠延伸PCR法克隆人脂联素基因及其在毕赤酵母中表达

Cloning of Human Adiponectin Gene by PCR-driven Overlap?Extension and Expression in Pichia pastoris

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DH5a, 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

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 recombinant expression vector pPIC3.5K-ADPN. The recombinant plasmid was transformed into Pichia pastoris GS115 by electroporation, then the recombinant strain was identified by PCR and Southern blotting. After induction by methanol, ADPN was expressed in GS115, then the protein was identified by Western blotting. The results showed that the ADPN was expressed successfully. The optimum conditions of expression were 30°C and 1% methanol inducing 48 h.

Key words:human adiponectin, PCR-driven overlap?extension, P. pastoris, Southern blotting, Western blotting