猪hepcidin基因质粒载体的构建及其在毕赤酵母中的表达

为构建猪hepcidin毕赤酵母(Pichia pastoris)分泌型表达载体,实现其真核表达,本文采用RT-PCR方法获得猪肝脏中的铁代谢调节基因hepcidin,通过连接酶使其与真核表达载体pGAPZaA相连构建重组表达质粒pGAPZaA-hepcidin。利用高压电击处理,使质粒载体转入到毕赤酵母中,用含Zeocin抗生素的YPD平板筛选阳性转化子,并做PCR鉴定。结果表明:hepcidin基因准确地插入表达载体pGAPZaA,并已整合到酵母基因组中,并成功构建了酵母重组表达质粒。

Plasmid Vector Construction of hepcidin Gene in Pig and Its Expression in Pichia pastoris

In order to construct Pichia pastoris secretive expression vector of hepcidin and realize its eukaryotic expression,hepcidin,an iron-metabolism regulating gene,was amplified from pig’s liver by RT-PCR,and the recombinant expression plasmid pGAPZaA-hepcidin was constructed by connecting the obtained hepcidin with eukaryotic expression vector pGAPZaA through ligase.This plasmid vector was transformed into Pichia pastoris by high-voltage electroporation,and the target colonies were selected by Zeocin and PCR detection.The results showed that hepcidin gene was correctly inserted into expression vector pGAPZaA and integrated into the genome of this yeast,and the P.pastoris recombinant expression plasmid of hepcidin was successfully constructed.