鳗源嗜水气单胞菌L316主要外膜蛋白基因克隆和序列分析

The cloning and sequence anlysis of major outer membrane protein gene of Aeromonas hydrophila

根据已发表嗜水气单胞菌(AH)的外膜蛋白基因(Omp)的核苷酸序列设计1对特异性引物,应用PCR 技术,扩增AHL316的主要外膜蛋白基因(Momp),经T/A克隆,插入到pGEM—T系列载体上进行序列测定,结果表明Momp基因最长的开放阅读框(ORF)为1 035 nt,编码由分子量为36 kD的主要外膜蛋白(MOMP),其与国外AH分离株AF146597的Omp基因的核苷酸的同源性达96%,氨基酸的同源性达98%。应用分子生物学软件分析表明此主要外膜蛋白前24个氨基酸是较强的疏水性区域,可形成信号肽,有跨膜区,经与其他外膜蛋白系统发育进化分析表明,该蛋白属于ompA蛋白家族。

A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene （omp） of Aeromonas hydrophila. With the specific primers, a target fragment about 1. lkb was amplified from Aeromonas hydrophila L316 via PCR. The target fragment was inserted into the linearized pGEM -T easy vector. After enzyme restriction and sequencing analysis, the nucleotide data had been further analyzed by DNAman and ClutalW software. The analysis results showed that the cloned DNA fragment had the longest open reading frame （ORF） of 1035nt, it predicted to be encoded a 344 -AA protein with the molecular weight of 36kD. Hydrophobicity analysis suggested that the protein was highly hydrophilic, especialy at the first 24 amino-acid, this region could be function as signal peptide. The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene, and the alignment of the amino acid sequence was 98%. Campared with other amio acid sequences of outer membrane proteins, major outer menbrane protein of Aeromonas hydrophila L316 belongs to the ompA family.