摘要
采用ＰＣＲ技术从Ｅ．ｃｏｌｉ基因组片段中克隆出碱性磷酸酯酶（ＰｈｏＡ）的启动子和信号肽序列．在ＰｈｏＡ启动予５＇端设计了ＥｃｏＲⅠ酶切位点，在信号肽编码序列３＇端设计了ＨｉｎｄⅢ酶切位点．将ＰＣＲ产物酶切后ＥｃｏＲⅠ－ＨｉｎｄⅢ片段克隆至ｐＢＲ３２２的ＥｃｏＲⅠ－ＨｉｎｄⅢ位点，组构出含有ＰｈｏＡ启动子和信号肽序列的分泌表达载体ｐＢＭ－Ｐｈｏ－１．之后将人表皮生长因子的成熟肽基因克隆至该载体，使之在Ｅ．ｃｏｌｉ中获得分泌表达，另采用ｐＩＮⅢ载体系统以分泌方式表达了人表皮生长因子。
The E. coli secrection vector, PBMPho-1 ,had been constructed by amplified the E. coli alkaline phosphatase A (PhoA) promoter-signal sequences by PCR and by subcloning the PCR product, which has EcoRⅠcleavage site at its 5'terminus and has Hind Ⅲ cleavage site at its 3'terminus, into PBR322 precutted by EcoRⅠand Hind Ⅲ. Human epidermal growth factor(hEGF) mature peptide coding sequence was then cloned into the secrtion vector and was expressed in E. coli. The hEGF was also expressed in E. coli by using PIN Ⅲ secretion expression system.
基金
国家博士后科学基金
关键词
表皮生长因子
大肠杆菌
分泌表达载体
Human epidermal growth factor
E. coli secretion vector
Gene expression
