6株水产动物源气单胞菌安徽分离株的毒力基因的克隆与序列分析

Cloning and sequence analysis of virulence genes of six Aeromonad strains isolated from aquatic animals in Anhui

根据气单胞菌主要黏附素基因（ahal）、溶血素基因（hly）和细胞兴奋性肠毒素基因（alt）完整开放阅读框（0RF）设计3对特异性引物，对6株气单胞菌安徽分离株进行ahal、hly和alt基因的PCR扩增、克隆和测序。测序结果显示，安徽分离株aim、hly和alt基因的ORF大小分别为1056--1068bp、1482bp和1104N1107bp，各编码351-355、493和367-368个氨基酸。序列分析显示：（1）属于alt^＋aim^＋hly^＋毒力基因型的3个安徽分离株间aha1核苷酸序列和推测的氨基酸序列同源性均很高，分别为98.8％-99．3％和97％-97．6％，且与国内参考株mh／Trionyx sinensis／Guangzhou（Guangdong）／AF276639的同源性高达98．5％-99％和96.8％-97．6％。而alt^＋ahal^＋hly^-HA6安徽分离株与国外参考株Ah／Fish／Barcelona（Spain）／AF183931间的核苷酸序列和氨基酸序列同源性较高，分别为95.8％和97．2％。（2）不同表型种气单胞菌安徽分离株之间及其与国内外其他分离株之间的hly核苷酸序列和推测的氨基酸序列同源性均较高，分别介于88．4％-100％之间和90.8％-98．7％之间。（3）5个安徽分离株（RA16、CA1、HA6、HA7和GA1）之间及其与惟一参考株之间的alt核苷酸序列和推测的氨基酸序列同源性分别高达93．5％-99．9％和80．2％-98．3％，但与BA17分离株间的核苷酸序列同源性（81．6％-81．7％）和推测的氨基酸序列同源性（77．5％-84．9％）均较低。这些结果表明，气单胞菌ahal和alt基因在不同毒力基因型间存在一定差异性，hly基因在不同表型种间存在较高保守性，该基因可作为研制气单胞菌基因工程亚单位疫苗的候选成分。

Three different sets of specific oligonucleotide primers were designed according to the ORF sequences of aha1, hly and alt genes of Aeromonads. Three virulence genes of six strains of Aeromonads isolat- ed from aquatic animals in Anhui were amplified by PCR technique respectively. The PCR products were cloned into pMD-18T vectors and then sequenced. The results show that ahal gene of strains HA7, BA17, GA1 and HA6 contain an ORF of 1 056 - 1 068 bp, encoding 351 - 355 amino acids. Hly gene ORF of strains HA7, BA17 and GA13 are all 1 482 bp in length, encoding 493 amino acids. Air gene ORF of six isolates are 1 104 - 1 107 bp in length, encoding 367 - 368 amino acids. Sequence homology comparison shows that the homology at the nucleotide levels for the ahal gene are 98.8 % - 99.3 % and that of the deduced amino acid sequences are 97% - 97.6% among three srtains belonging to alt^＋ ahal^＋ hly^＋ virulence genotypes. The nucleotide and deduced amino acid sequences homology of the ahal gene are 98.5 % - 99 % and 96.8 % - 97.6 % between three strains belonging to alt^ ＋ ahal ^＋ hly^ ＋ virulence genotypes and reference strain Ah/Trionyx sinensis/Guangzhou（Guangdong）/AYE76639, but only 80.9 % - 81.5 % and 72.9 % - 73.6 % between the three virulence strains and strain HA6 （alt^＋ ahal^＋ hly^-）. Ahal gene of strain HA6 shares 95.8% nucleotide identity and 97.2% deduced amino acid identity with reference strain Ah/Fish/ Barcelona（Spain）/AF183931. The nucleotide homology of hly gene among different phenospecies is 88.4% - 100 % and deduced amino acid homology is 90.8 % - 98.7 %. The nucleotide and deduced amino acid sequence homology of alt gene among strains HA6, HA7, BA16, CA1 and GA1, and the only reference strain A/Diarrheal disease/Texas（America）/L77573 are 93.5 % - 99.9 % and 80.2 % - 98.3 %, but the sequence homology is only 81.6% -81.7% and 77.5% -84.9% between the fine strains and strain BA17 strain BA17 at the nucleotide and amino acid levels respectively. All of these results reveal that there are some vari- ation in ahal and alt gene among different virulence genotypes. Hly gene is widely distributed in different phenospecies and shares quite high similarity in nucleotide and deduced amino acid sequences, so hly can be used as a candidate for gene engineered sudunit vaccine.