葡萄糖氧化酶基因片段的克隆与序列分析

Cloning of Glucose Oxidase Gene Fragment & Its Sequence Analysis

根据Gen Bank发布的葡萄糖氧化酶基因序列设计PCR扩增引物,从筛选的1株可以产生葡萄糖氧化酶的菌株中克隆得到葡萄糖氧化酶基因片段,将该片段与p MD20-T载体连接后转化至大肠埃希菌DH5α中,测序并进行序列比对分析。结果表明,该克隆片段属于氧化还原酶超家族,且与同属氧化还原酶超家族中的Aspergillus niger strain BT18葡萄糖氧化酶相似度达到92%。从蛋白质预测的三级结构可以看出有FAD和NAG结合位点,说明该GOD片段理论上可以表达出GOD的主要功能。可以推断该克隆的基因片段为葡萄糖氧化酶基因片段。

According to sequences of glucose oxidase（ GOD） gene released by Gen Bank,a pair of PCR proliferation primers was designed and cloned into a GOD gene fragment from a GOD producing Aspergillus sp. strain x-1. The fragment was transferred into E. coli DH 5α competent cells after linked with p MD20-T vector,sequenced and carried out sequence comparison and analysis. The results showed that the cloned fragment belonged to superfamily of oxidoreductase. And having as high as 92% similarity with GOD from the same oxido-ruductase superfamily of Asperigillus niger strain BT18. It can be seen there were FAD-binding sites and NAG-binding sites in its protein pretested tertiary structure,indicated that the GOD fragment theoretcally could express the main function of GOD. Therefore,it can be inferred that the cloned gene fragment to be the fragment of GOD gene.