耐热DNA聚合酶基因的克隆及在大肠杆菌中的表达

Cloning and Expression of Thermostable DNA Polymerase Gene in Escherichia coli

用ＰＣＲ法从水生栖热菌菌株ＹＴ－１中扩增耐热ＤＮＡ聚合酶基因，得到２．５ｋｂ的ＤＮＡ片段，扩增片段重组到ｐＵＣ１８中测序证实为Ｔａｑ ＤＮＡ聚合酶基因，将该片段重组到ｐＢＶ２２１温控表达质粒中，在大肠杆菌中表达出９４ｋＤａ的重组蛋白，１００ｍｌ培养物的细胞产酶为１．５×１０＆５ｕ，表达的蛋白能催化ＰＣＲ反应的进行。

Thermostable DNA polymerase gene had been amplified from Thermus aquaticus YT-1 using PCR technique. The amplified 2. 5kb DNA fragement was inserted into pUCl8 and confirmed to be thermostable DNA polymerase gene by restriction mapping and DNA sequencing. The insert fragement was then combined into an expression vector, pBV221. This recombinant plasmid overexpressed a 94kDa of recombinant protein in E. colt. 100ml of E. colt culture could yield 1. 5X 105 units of Taq DNA polymerase, which could be applied in the PCR.