鳗弧菌溶血素基因的克隆及突变株的构建

PCR扩增出鳗弧菌M3溶血素基因的保守区序列，根据保守区序列设计引物，利用反向PCR和巢式PCR扩增出溶血素基因的部分序列，结合构建的鳗弧菌M3的基因组文库，克隆出溶血素基因的全长序列。根据基因推测的氨基酸编码序列与已发表的血清型为A的鳗弧菌PT84057有86％的相似性。用自杀质粒对鳗弧菌M3染色体上的溶血素基因进行了插入突变，将突变株对金鱼进行肌肉注射，结果发现，突变株的毒力没有发生显著的变化，证明所克隆的溶血素基因对鳗弧菌M3的毒力没有直接的贡献。

Cloning and Mutation of Hemolysin Gene from Vibrio anguillarum M3

The conserved region of hemolysin gene from Vibrio anguillarum M3 was amplified by normal PCR. Part of the hemolysin gene was cloned using inverse PCR and nested PCR.A genomic library was made by linked with adaptors after genomic DNA was digested by EcoRV and the whole gene was amplified. The deduced amino acid sequence was 86% identical to that of V. anguillarum PT84057. A chromosomal hemolysin mutant was made via the integration of foreign DNA into the hemolysin gene. The mutated strain was screened by antibiotic and verified by the sequencing of PCR product. The change of virulence was investigated by injecting the mutant and parent strain into fish respectively. No significant change was found, which indicates that hemolysin does not contribute to the virulence of V.anguillarum M3 directly.