编码1,3-丙二醇氧化还原酶基因的克隆和表达

Cloning and Expressing of 1,3-propanediol Oxidoreductase-encoding Gene

采用PCR法克隆了巴氏梭菌 (Clostridiumpasteurianum)CpN 86菌株编码 1 ,3 丙二醇氧化还原酶基因 (dhaT基因 ) ;完成了dhaT基因测序、表达载体构建和在大肠杆菌中表达 ;分离和纯化了dhaT基因表达的重组蛋白。实验结果 :( 1 )PCR法克隆的dhaT基因和肺炎克雷伯氏菌Klebsiellapneumoniae菌株dhaT基因的序列同源性为 82 9% ;( 2 )dhaT基因表达蛋白的酶活为 1 0 8U mg ;( 3)dhaT基因表达的蛋白分子量为 43kD ;( 4 )Westernblot确定了dhaT基因表达的蛋白和CpN 86菌株天然蛋白有相同的抗原反应。

Based on what mentioned above, the gene encoding 1,3-propanediol Oxidoreductase ( dha T) in Clostridium pasteurianum CpN-86 was cloned by using PCR method. The sequence, expressing vector construction and its expression of dha T in E.coli were accomplished respectively. The recombination protein expressed by dha T was also isolated and purified. The experimental results showed that: (1)The homogeneity of dha T cloned by PCR and that in klebsiella pneumoniae strain was 82.9%; (2)The enzymatic activity of the protein expressed by cloned dha T was 108μM/mg; (3)The molecular weight of the protein was 43 kD ; (4)The protein expressed by dha T has the same antigenicity as the natural protein of CpN-86 through Western blotting.