嗜水气单胞菌aer毒素基因的克隆及核苷酸序列分析

Cloning and sequence analysis of an aer toxin gene of Aeromonas hydrophila

以自行分离的嗜水单胞菌分离株AHCS02为研究对象,分析其产生的aer毒素基因结构。根据G enB ank中aer毒素的基因序列(M 16495),设计扩增编码aer毒素成熟蛋白基因的引物,以AHCS02的基因组DNA为模板,PCR扩增出长度为1 332 bp的核苷酸片段,进行pM D 18-T载体克隆。酶切鉴定后进行序列测定和分析,结果表明扩增的片段与M 16495的同源性达92%,将测得的序列登录G enB ank数据库,所得的序列编号为AY 136943。分析推导其核苷酸编码的氨基酸序列显示:其编码443个氨基酸,为aer毒素成熟蛋白的基因,与M 16495相比,氨基酸差异主要集中在422氨基酸残基至436氨基酸残基的位置上,在这个位置上共有8个氨基酸残基发生变化,其中在435和437氨基酸残基之间有一个氨基酸密码子丢失,氨基酸的同源性达95.5%。证明aer毒素基因的5′端保守,3′端变异较大。

A pair of primers was designed according the aer gene sequences in Genbank （accession No. M16495）. With the specific primers, one target fragment about 1 332 bp was amplified from Aerrnonas hydrophila strain AHCS02 genomic DNA via PCR, which was isolated in Sijiazi reservoir, Jilin, China. The PCR products were cloned into pMD18-T vector, the amplified aer gene was sequenced and analyzed. The sequence was sent to the Genbank （accession No. AY136943） and compared with the corresponding regions of the M16495 by DNA tools software. The nucleotide sequence showed 92% homology to the sequence of M16495. The nucleotide sequence was predicted to encode a 443-aa （amino acid） protein with the molecular weight of 53.9 ku and encode the mature aer toxin protein.