克氏肺炎杆菌NiFe-氢酶基因的克隆与序列分析

Cloning and Sequence Analysis of the Gene Encoding NiFe-hydrogenase from Klebsiella pneumoniae

摘要:

采用CLUSTAL-W软件对Swiss-Prot蛋白数据库中已报道的NiFe-氢酶大亚基氨基酸序列进行比对分析，找到保守区并根据此设计兼并引物。利用其中一对引物进行PCR得到一条大小约为1000bp的DNA序列，并根据此序列设计引物进行反向PCR得到整个NiFe-氢酶的序列。再利用生物信息学软件对此氢酶的序列进行二、三级结构预测及大小亚基的对接（docking）。结果表明克氏肺炎杆菌的NiFe-氢酶属于一类膜结合放氢酶（Ech 氢酶）。

Abstract:

Degenerate PCR primers were designed by multiple alignment of the protein sequences of known structural genes encoding the catalytic subunits of NiFe-hydrogenases obtained from Swiss-Prot Protein Sequence Database through CLUSTAL-W software and compared for conserved sequence motifs. An amplified PCR product 1 kb in size was obtained from the genomic DNA of Klebsiella pneumoniae using a set of degenerate primers, and then inverse PCR technique was used to obtain the full hydrogenase coding region. A predicted secondary structure and 3D structural model were constructed by homology modeling and docking. On the basis of these results, it was inferred that NiFe-hydrogenase from Klebsiella pneumoniae belongs to the membrane-bound H₂ evolving hydrogenase group (Ech hydrogenase group).