嗜水气单胞菌若干毒力因子同时缺失的突变株的构建及特性分析

Construction and characterization of some virulence determinants-deficient strain in Aeromonas hydrophila

以携带质粒pAM12 0 (Tcr Tn916 )的大肠杆菌CG12 0株为供体菌 ,采用滤膜接合法与受体菌嗜水气单胞菌J_1株 (cfzr)进行接合转移 ,在含Tc和cfz选择平板上进行筛选。共获接合转移菌落 380 0个 ,其接合频率为 3× 10 - 5(按供体细胞计算 )。任取 38个接合子 ,提取基因组DNA ,以嗜水气单胞菌特异性 16SrDNA引物进行PCR扩增 ,所有接合子均阳性。为证明Tn916确实插入基因组 ,以四环素基因 (tet)引物进行PCR扩增 ,结果所有抗性接合子均扩增出一条特异条带。与亲本J_1株相比 ,所有接合子的主要毒力因子如蛋白酶、溶血素、DNA酶和淀粉酶等均不表达 ,对小鼠失去致病力 ,其LD50 大于 10 9CFU。接合子连传 10次后 ,四环素抗性消失 ,但毒力未恢复 ,说明通过转座子Tn916的插入可获得稳定的无毒嗜水气单胞菌突变株。Tn916引起嗜水气单胞菌毒力性状改变的机制有待研究 ,推测可能与该菌染色体上存在Tn916的热点或毒力岛有关。

Cell conjugation was carried out between the donor E. coli CG120 with pAM120 (Tc r/Tn916) and the recipient Aeromonas hydrophila J-1 by filter mating. 3800 positive clones were gained according to the ability of growth on LB medium with 10 g/mL tetracycline (Tc) and 100 g/mL cephazolin (cfz). Conjugation efficiency was 3×10 -5 per donor. When 16S rDNA PCR amplication was performed with special primers, positive results were gained in all the 38 conjugants. To demonstrate the insertion of Tn916 into the genomes of conjugants, tetracycline gene (tet) PCR amplification was performed. A special band could be gained in the conjugants with Tc r. Compared with the parent J-1 strain, some genes of main virulence determinants, such as proteases, hemolysins, DNase and amylases, could not be expressed in the conjugants. The pathogenicity capability of conjugants was greatly decreased and the 50 lethal dose for mice was more than 10 9CFU. By serially passaged for 10 times, the above virulent characters had not been regained despite the disappearance of Tc r. The results showed that by the insertion of Tn916, stable and avirulent A. hydrophila mutants could be gained. The mechanisms, by which Tn916 induced the changes of virulence characters in A. hydrophila, are not definite. It is suspected that there might exist hot spots of Tn916 or pathogenicity island on the chronosome of A. hydrophila.