苦瓜MAP30基因的毕赤酵母表达载体构建及重组菌株的筛选

目的:从苦瓜中克隆MAP30全长基因,并将该基因连接至表达载体pPIC9中,建立酵母菌落PCR筛选方法。方法采用改良SDS法从苦瓜表皮中提取基因组DNA,设计特异性的引物,通过PCR技术扩增出全长861bp的MAP30基因。该基因经XhoⅠ和EcoRⅠ双酶切,连接至毕赤酵母表达载体pPIC9中。重组载体转化GS115菌株,运用菌落PCR鉴定重组菌株。结果:基因测序表明,该基因已成功插入酵母表达载体pPIC9α-factor分泌信号下游,同源性分析表明该基因与GeneBank(AF284811)的核苷酸同源性达99.9%,氨基酸同源性达100%。菌落PCR显示外源基因已整合入酵母GS115菌株中。结论:成功地克隆了MAP30全长基因,并构建了含MAP30基因的重组毕赤酵母表达载体,并获得了整合菌株,为下一步研究奠定了基础。

Construction of Recombinant Vector pPIC9-MAP30 and Screening of Recombinant GS115 Strain in Pichia pastoris System

Objective:MAP30 gene cloning by PCR from total DNA of Momordica charantia L, the construction of recombinant vector Ppic9 - MAP30, and establishing the rapid screening method of recombinant GS115 strain.Method :The total genome DNA was extracted by modified SDS method from Momordica charantia L With designed specific primers, MAP30(Momordica anti - HIV protein of 30 Kd) gene was amplified by PCR.The PCR product was digested by Xho Ⅰ and EcoR Ⅰ ,then ligated to Ppic9 vector.Then the Ppic9 - MAP30 vector was transformed into GS115 strain, and the positive elones were screened by colony PCR.Result:Mter DNA sequencing, MAP30 gene was inserted into Ppic9, Nueleotide acid sequence and amino acid sequence showed 99.88％ and 100％ homology to GeneBand (AF284811) respectively.Four positive strains were identified by colony PCR.Condusion: MAP30 gene was suecessfuUy cloned into Ppic9 vector, and target gene was also integrated into chromosome of GS115 strain.