pPICZα-synBPI_(m600)酵母表达载体的构建和鉴定

为使rBPIm2 3 重组抗菌蛋白在毕赤酵母表达系统中分泌表达 ,拟构建synBPIm60 0 酵母表达载体。以pUC18 synBPI41 4 质粒为模板扩增synBPI2 0 0 基因片段 ,经XhoI/EcoRI双酶切获得synBPI1 85bp基因片段 ;EcoRI/SalI双酶切PBV2 2 0 BPIm60 0 质粒获得BPIm42 0 基因片段 ;将上述 2个基因片段连接后定向克隆到pPICZα酵母表达载体上。连接产物转化E .coliTOP 10F′ ,在含Zeocin的低盐LB固体培养基上筛选阳性转化子。阳性克隆菌经菌落PCR法鉴定、酶切分析和测序鉴定 ,结果与预期相符。提示 :成功构建了pPICZα synBPIm60 0 酵母表达载体

Construction and Identification of pPICZα-synBPI_(m600) Recombinant Expression Vector

In order to express rBPI m23 recombinant bactericidal protein in Pichia pa storis, synBP I m600 expression vectors were constructed. synBPI 200 bp fragment was amplified by PCR from pUC18-synBPI 414 vector, then the product was diges ted by XhoI and EcoRI to obtain synBPI 185 bp fragment; PBV220-BPI m600 recombinant expressing vector was digested by EcoRI and SalI to obtain BPI m420 bp fragment; the above two fragments were linked and cloned in pPICZα e xpression vector to form intact pPICZα-synBPI m600 recombinant expression vector. After E.coli TOP 10 F' was transformated by pPICZα-synBPI m600 vector, positive cloning strains were selected by low salt LB medium with Zeocin and id entificated by PCR amplication, restriction analysis by XhoI/EcoRI/SalI and DNA sequencing. The results show that intact pPICZα-synBPI m600 recombinant expression vectors are constructed successfully.