Exendin-4基因的克隆及其在毕赤酵母中的表达

摘要

According to the announced sequence of Exendin-4 encoded AAB22006 in the GenBank and the code bias of yeast,the gene of Exendin-4 was modified.The synthesized Exendin-4 gene was cloned into the vector pPIC9K with an a-factor to construct recombinant secreting expressing plasmid of pPIC9K-E4.The pPIC9K-E4 was linearized by Sac I and then transformed into Pichia pastoris GS115 via electroporation.The positive transformants screened on MD plate were transplated to G418 plate with increasing gradient to get the multicopy recombinant.Muts clones screened on MM and MD plates were induced by methanol expressing for 6 days.Samples were got every 24h.Tricine-SDS-PAGE indicated that the highest level of expression appeared after 6 days＇ induction.%根据GenBank中已公布的编号为AAB2200的Exendin-4氨基酸片断,及酵母密码子偏好性,设计了Exendin-4在酵母中的表达序列。利用基因工程技术,将编码Exendin-4的蛋白基因亚克隆到含有分泌信号肽序列的毕赤酵母表达载体pPIC9K中,构建成分泌型重组表达栽体pPIC9K-E4。用电转化法将线性化的pPIC9K-E4转化入毕赤酵母菌株GSll5,转化子经MD平板筛选,得到的阳性菌株再用浓度梯度递增的G418筛选多拷贝重组子。将MM和MD平板筛选得到Muts菌株用甲醇诱导表达6d,每隔24h取样。Tricine-SDS-PAGE检测结果显示,诱导后第6天表达量达到最高。