Neuritin毕赤酵母分泌表达系统的构建

Construction of Pichia Pastoris Secretory Expression System of Human Neuritin

为构建Neuritin毕赤酵母分泌表达系统,进一步得到高纯度的具有天然活性的Neuritin蛋白提供实验基础。采用人工合成去除信号肽的neuritin基因并在N端引入His标签序列,两端引入Eco RⅠ和NotⅠ酶切位点,克隆至p PIC9K分泌型表达载体。重组质粒经PCR与测序鉴定正确后电击转化至毕赤酵母GS115感受态细胞中,经MD平板与G418平板筛选阳性重组子,抽提毕赤酵母基因组PCR鉴定重组质粒的插入。结果显示,p PIC9K-neuritin重组质粒经测序鉴定完全正确,电转后成功插入毕赤酵母基因组中。由此可知,成功构建Neuritin的毕赤酵母分泌表达系统。

Constructed the pichia pastoris secretorial expression system of human Neuritin for further research on acquiring the Neuritin protein with high purity and natural activity.After removing the signal peptide of neuritin gene and inserting His tag in N-terminal as well as Eco R I and NotⅠ restriction sites in both sides,Neuritin c DNA was co-cloned to the shuttle plasmid p PIC9 K.The recombinant plasmid p PIC9k-neuritin was electrically transformed into pichia pastoris GS115 after the identification by PCR and sequencing analysis.Stably-expressed pichia pastoris recombinant was screened by MD and G418 plate,and then the pchia genome was extracted to identify the recombinant plasmid insertion by PCR analysis.Sequencing analysis showed that the recombinant expression vector of p PIC9 K with neuritin c DNA was correctly constructed and successfully transformed into Pichia pastoris GS115 by electroporation.Pichia pastoris secretary system of neuritin was successfully constructed.