筛选用转座子Tn916诱变的具有免疫原性的嗜水气单胞菌蛋白酶缺失株  

Screening the reduced virulent protease-deficient mutant of Aeromonas hydrophila by Tn 916 transposon insertions

作　　者：储卫华[1] 陆承平[1] 

机构地区：[1]南京农业大学农业部动物疫病诊断与免疫重点实验室,江苏南京210095

出　　处：《水产学报》2001年第3期244-248,共5页Journal of Fisheries of China

基　　金：国家九五攻关子课题(96-005-03-01)

摘　　要：以携带质粒pAM120(Apr、Tcr/Tn916)的大肠杆菌CG120株为供体菌，与受体菌嗜水气单胞菌J-1株，采用滤膜接合法进行接合转移，在选择平板(含Tc、cfz和脱脂奶)上进行筛选，筛选出6株蛋白酶缺失株(ECPase-)，ECPase-菌株其生长速度，对正常鲫血清的抗性降低，用脱脂奶平板法及底物法检测，蛋白酶产量明显降低。与亲本J-1株相比，突变株致病力降低，对鲫的半数致死量大于108(J-1株对鲫的半数致死量为5×103)。用ECPase-突变株MJ-1株的18h培养物(5×106CFU)接种健康鲫，不产生亲本J-1株引起的病变及死亡。用MJ-I活菌免疫鲫，在第5周产生较高的凝集抗体，且对强毒株J-1的攻击有保护作用。表明蛋白酶是嗜水气单胞菌的一个重要的毒力因子，ECPase-突变株仍具有免疫原性。Cell conjugation was carried out between the donor E. coli CG1 20( pAM 120: Apr, Tcr/ Tn916) and the recipient Aeromonas hydrophila J- 1 by filter mating. 6 protease- deficient mutant strains were screened by inoculation on TSA with 1 .5% skim milk. MJ-1 ,one of the six mutants was further characterized by its growth rate, serum resistance and degree of virulence (LD50). Unlike the parent strain, the growth rate and serum resistance of MI-i were decreased significantly, and there was no lesions in Carrassius auratus ibebio caused by parent strain J- I after intramuscular injection with 5 × 1 0~6 CFU germs of MJ- I. The 50% lethal dose of MJ-1 for the fish was more than 10~8CFU. MJ-1 was tested in fish vaccination experiment, and its antibody evoked highest degree after 5 weeks intraperitoneal injection. After being challenged with the parent strain J-1, the survival abilities of vaccinated fish were higher than that of the unvaccinated control group. It shows that the protease is a virulent factor of Aeromoncts hydrophila and the immunogenicity of ECPase - mutant MJ-1 remains.

关 键 词：嗜水气单胞菌 胞外蛋白酶 Tn916诱变 免疫原性 鱼类 毒力 

分 类 号：S941.42[农业科学—水产养殖]