耐热碱性磷酸酯酶的基因克隆及在大肠杆菌中的表达  

Thermostable Alkaline Phosphatase from Thermus sp. FD3041: Cloning of the Gene and Expression in Escherichia coli

作　　者：袁有忠[1] 盛小禹[1] 吕鸿雁 佟石[1] 毛裕民[1] 

机构地区：[1]复旦大学遗传学研究所遗传工程国家重点实验室,上海200433

出　　处：《Acta Genetica Sinica》1998年第4期375-380,共6页

基　　金：国家自然科学基金(39380012)~~

摘　　要：以pUC118质粒为载体,以E.coil TGl为受体,构建了产耐热碱性磷酸酯酶(FD-TAP)的菌株Thermus sp.FD3041的染色体基因文库。在包含1.2万个转化子的文库中,90％的转化子插入有3～10kb的外源DNA片段。用菌落原位碱性磷酸酯酶显色法从文库中筛选到5个阳性克隆。对其中的1个克隆(pTAP362)所产的TAP进行了研究,发现克隆的TAP与出发菌株所产的TAP的热稳定性、最适反应温度和最适pH等性质都相同。通过做物理图谱和测定部分缺失的质粒的TAP活性,将TAP基因定位于2kb的BamHⅠ-HindⅢ DNA片段上。模拟PCR实验表明该酶可用于PCR扩增产物的检出。A genomic library of Thermus sp. FD3041 which produces thermostable alkaline phosphatase (FD-TAP) was constructed with the vector pUC118 and the host E. coli TG1. 3～10kb inserted fragments of foreign DNA were identified in 90 percent of the 12 000 clones thus obtained. Five positive clones were detected after screening the plated library by the method of colony coloration for TAP in situ. Preliminary analysis of the enzyme expressed from one recombinant plasmid pTAP362 showed that the properties of the recombinant enzyme, such as the thermal stability and optimal temperature of reaction, were identical to those of the native enzyme. The gene of FD-TAP was located on the 2.0kb BamHI-HindⅢ fragment of the pTAP362, determined by its physical map and the change of enzyme activity in different partially deleted plasmids. Results of thermostability experiment in PCR thermal cycle showed that the FD-TAP would be suitable for labelling of primers and detection of PCR amplified products.

关 键 词：基因克隆 表达 大肠杆菌 FD-TAP 

分 类 号：Q78[生物学—分子生物学]