耐热碱性磷酸酯酶基因的亚克隆及其在大肠杆菌中的表达

Subcloning and Expression of Thermostable Alkaline Phosphatase Gene in Escherichia Coli

根据耐热碱性磷酸酯酶（ＦＤ－ＴＡＰ）基因的酶切图谱和ＤＮＡ序列，分别在基因的起始密码子和终止密码子处设计了２个突变引物．引物的５’端分别带有Ｎｄｅ Ｉ和ＢａｍＨＩ酶切位点．通过ＰＣＲ从质粒ｐＴＡＰ１１８Ｂ中扩增获得了ＦＤ－ＴＡＰ基因的全序列．经过酶切将基因亚克隆于高表达载体ｐＪＬＡ５０３．重组质粒ｐＴＡＰ５０３Ｆ的ＦＤ－ＴＡＰ在大肠杆菌Ｍｐｈ４４中的表达量比ｐＴＡＰ１１８Ｂ提高了约１０倍．重组酶和原始菌株所产的天然酶耐热性无明显差异．

The coding sequence of the gene for recombinant thermostable alkaline phosphatase (FD-TAP) was amplified from the pTAP118B by polymerase chain reaction (PCR) using two mutagenic primers incorporating Nde I and Burn H I sites at the 5' termini,respectively. By Nde I and BamH I cheavage,the PCR product was then subcloned in-frame into high expression vector pJLA 503. The expression of the recombinant FD-TAP of pTAP503F in E. coli Mph44 was approximately ten-fold of that of pTAP118B. The thermostability of the recombinant FD-TAP was analogous to that of Native FD-TAP produced by the original strain.