论文部分内容阅读
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
In order to make rBPIm2 3 recombinant antimicrobial protein secreted expression in the Pichia pastoris expression system, to construct synBPIm60 0 yeast expression vector. The synBPI2 0 0 gene fragment was amplified by using the pUC18 synBPI41 4 plasmid as a template, and the synBPI1 85 bp fragment was digested with XhoI / EcoRI to obtain the BPIm42 0 gene fragment by digesting the PBV2 20 BPIm60 0 with EcoRI / SalI. The gene fragments were ligated and cloned into the pPICZα yeast expression vector. The ligation product was transformed into E. coli TOP 10F ’and positive transformants were selected on low salt LB solid media containing Zeocin. Positive colonies were identified by colony PCR, enzyme digestion analysis and sequencing identification, the results were in line with expectations. Prompt: pPICZα synBPIm60 0 yeast expression vector was successfully constructed