温和气单胞菌RC-07-KA株溶血素基因克隆、表达及活性检测

为研究温和气单胞菌(A.sobria)溶血素基因的特性,本研究根据GenBank登录的气单胞菌属溶血素基因序列设计一对引物,经PCR扩增得到大小约1.5kb的A.sobria RC-07-KA株溶血基因片段,将该片段克隆到pGEM-T载体,测序结果显示:溶血素基因片段大小为1 467 bp,编码487个氨基酸残基,遗传进化分析结果表明该基因与气单胞菌属中的A.hydrophila菌株Sb (AY611033)、NLEPA-1607 (AF410466)、AEF (HM853019),A.sobria菌株357 (AY157998)、人源分离株(EF620533)和A.salmonicida菌株17-2 (X65048)的溶血素基因亲缘关系较近,同源性大于95％,而与其他菌株的同源性较低.通过构建溶血素重组表达质粒pET-HIy,诱导表达并通过western blot鉴定表达蛋白,结果显示重组菌能高效表达重组溶血素,而且纯化的重组溶血素具有溶解鲤鱼红细胞的活性.为该菌进一步深入研究奠定了基础.

Cloning and expression of hemolysin gene from Aeromonas sobria and its haemolysis reactivity

To study the characteristics of Aeromonas sobria hemolysin,the hemolysin gene was amplified by PCR with a pair designed according to the sequences of A.sobria strain RC-07-KA available in GenBank.The PCR product was cloned into pGEM-T vector for sequencing and the result showed that the hemolysin gene of A.sobria strain RC-07-KA was 1,467 bp encoding 487 amino acids.The phylogenetic analysis showed that the hemolysin gene of A.sobria strain RC-07-KA shared over 96% identity with A.hydrophila strain Sb(AY611033),NLEP A-1607(AF410466),AEF(HM853019),A.sobria strain 357(AY157998),A.salmonicida strain 17-2(X65048) and a Homo sapiens isolate(EF620533).Moreover,the recombinant hemolysin was expressed based on pET28 vector and identified by western blot with anti-His tag monoclonal antibody.Hemolysis test results demonstrate that the recombinant hemolysin had hemolytic reactivity which was able to lyze the red blood cell of Carp.This formed a basis for further study of A.sobria.