表达两种不同外膜蛋白的禽大肠杆菌融合菌株的构建

对从陕西省主要养鸡地区病死鸡及鸡胚中分离的致病性禽源大肠杆菌,用SDS-PAGE进行Omp分型。采用不同浓度溶菌酶作用不同时间裂解细胞壁,制备原生质体,在高渗平板上再生培养;在药敏试验的基础上,结合Omp型分析,选择具有交叉耐药且Omp型不同的菌株,利用PEG诱导进行原生质体融合,在含双抗的高渗平板上筛选融合株,对不同代次的融合株分别进行革兰氏染色镜检、生化鉴定、血清型鉴定和Omp型分析。结果表明,所构建的融合菌株染色镜检和生化特性均符合大肠杆菌特征,75%融合子可凝集2亲本的O抗原,25%凝集其中1个亲本的O抗原;外膜蛋白型表现为1个或2个亲本的带型,但都有不同程度的增强。经稳定性检验证明其为遗传学上稳定的融合子。

Establishment of avian Escherichia coli fusion strain expressing two different Omp

The outer membrane protein (Omp) patterns were analyzed by SDS-PAGE of major serotypes avian pathogenic Escherichia coli isolated from dead chicken and embryo originated from feeding region of Shaanxi province.Protoplasts were prepared with lysozyme and EDTA,and regenerated in the hypertonic nutrition agar (HNA);Combining antibiotic sensetivity with Omp patterns,we choose two strains that have cross-drug resistance and different Omp patterns as parental strains,protoplast fusion was induced by PEG,and screen fusion strains in HNA containing two antibiotic.Fusion strains were tested through Gram stain,biochemical character,serotype identification and Omp patterns.The results show that:Gram stain and biochemical character consistent with that of Escherichia coli;75% fusion strain agglutinate two parental O antigen;Omp patterns appear as one or two parental patterns,but all have been amplified.Fusion strain was genetically stable.