致病性哈维氏弧菌溶血素基因克隆及其检测

从山东沿海的发病鲈鱼（Lateolabrax japonicus）分离到1株致病性哈维氏弧菌（Vibrio harveyi），从该菌的染色体DNA扩增出一条长约1．4kb的特异性片段。DNA序列分析表明，该克隆片段含有完整的1254bp溶血素基因，该溶血素基因与哈维氏弧菌VIB645的溶血素基因VhhA和VhhB的相似性分别为99．0％和98．5％；与副溶血弧菌（V．parahaemolyticus）热稳定性溶血素基因（TDH）的相似性为74．5％；与拟态弧菌（V．mimicus）、创伤弧菌（V．vulnificus）、霍乱弧菌（V．chderae）磷脂酶基因的序列相似性分别为57．4％、59．2％、53．0％；与最小弧菌、创伤弧菌、霍乱弧菌、霍氏弧菌（V．hollisae）、河流弧菌（V．tguvialis）、鳗弧菌（V．anguillarum）的溶血素基因的相似性仅为19．9％～24．8％。根据溶血素基因的保守区段，设计了1对特异引物。分析表明，该引物能特异检测哈维氏弧菌。其可检测的DNA最小量为0．001ng。用该方法对55株不同来源的患病鱼分离的疑似病原菌进行检测，结果检出8株哈维氏弧菌，检出率为14．55％，从健康动物中检出数占所检弧菌数的6．25％，海洋环境为10．53％，海水养殖环境为26．53％，表明哈维氏弧菌在不同的海水环境及健康海洋动物中普遍存在，在养殖水体中的数量高于其他海水环境。

Cloning of hemolysin gene from a pathogenic Vibrio harveyi SF1 and its detection in diseased marine animals and marine environments

A pathogenic Vibrio harveyi strain SF1 was originally isolated from diseased seabass(Lateolabrax japonicus) in China.The hemolysin gene was cloned by PCR amplification from chromosomal DNA of Vibrio harveyi SF1.The nucleotide sequence was determined.The ORF of the gene consisted of 1?254?bp,which encoded a putative polypeptide of 418 amino acids.The nucleotide sequence was highly identical to those of Vibrio harveyi 645,and the similarities were 99.0% and 98.5%.The similarities with thermolabile hemolysin genes of Vibrio parahaemolyticus was 74.5%.The similarities with lecithinase genes of V.mimicus,V.vulnificus and V. cholerae were 61.3%,62.4% and 58.3% respectively,but the nucleotide sequence was very different from the hemolysin genes of V.mimicus,V.hollisae,V.anguillarum,V.vulnificus and V.cholerae(with similarity of 19.9%-24.8%).A pair of specific primers were designed to detect hemolysin gene of Vibrio harveyi.The target gene was detected from 13 Vibrio harveyi strains but not from 21 other bacteria strains.The detection limit of the PCR assay was 0.001?ng of DNA.Eight isolates of Vibrio harveyi were detected from 55 strains isolated from diseased marine animals,with a positive rate of 14.55%.The distribution of the bacteria in different environment was investigated.The positive rates of 10.53% and 26.53% were obtained from marine environment and aquaculture marine environment respectively.The results show that Vibrio harveyi exists widely in marine environment and health marine animals,with a high rate in aquaculture environment,which may compose of potential pathogens in marine animals.