鸭源大肠杆菌ZYD_2与多杀性巴氏杆菌YB_2原生质体融合菌株的鉴定分析

以新霉素标记的ZYD2(NEOs)和YB2(NEOr)作为亲本菌株,培育具有双亲本菌株免疫原性的融合菌株。在溶菌酶作用下,制备两亲本菌株原生质体,聚乙二醇4000诱导原生质体发生融合,利用NEO标记及亲本菌株培养条件不同筛选阳性融合子。ZYD2和YB2可分别得到95.32%和95.25%的原生质体制备率以及32.19%和33.52%的再生率。在含新霉素的麦康凯平板上传代培养获得5株稳定遗传的融合菌株。5株融合菌株可同时凝集双亲本菌株的鸭抗阳性血清。染色镜检观察融合菌株为革兰氏阴性中等大小杆菌。利用双重PCR检测,2株检测到双亲本菌株部分外膜蛋白A(OmpA)基因和部分外膜蛋白H(OmpH)基因,2株只检测到部分OmpA基因,另1株检测不到任何条带。测序结果表明检测到的部分基因片段与亲本菌株的相关基因片段同源性为100%。微量凝集反应显示,外周血可以检测到双亲本菌株抗体,抗体效价在二次免疫后可以维持较稳定水平。结果表明获得5株具有双亲本菌株免疫原性且可稳定遗传的融合菌株。

Generation and identification of the protoplast fusion strains of duck Escherichia coli ZYD2 and Pasteurella multocida YB2

The study was aimed at constructing the fusion strain with desired properties of two parental strains Escherichia coli ZYD2(NEOs) and Pasteurella multocidaYB2(NEOr).The parental strains ZYD2 and YB2 were treated with lysozyme and EDTA to remove the cell wall.The protoplasts were induced for fusing by polyethylene glycol 4000(PEG4000).The rate of protoplast formation of the two parental strains were 95.32% and 95.25% and the rate of regeneration were 32.19% and 33.52%,respectively.Five potential fusants had stable inheritance such as biochemical activities,morphology and serological characteristic,et al.Duplex PCR method was used to detect the part of the OmpA gene and OmpH gene,with 2 of them observed in the two of the strains,while with only the OmpA gene or none obtained in others.Sequence alignment showed that the identity of the target genes of fusions were all 100% compared to the parental strains.The antibodies of the fusions could be tested in the peripheral blood of ducks immunized with inactivated fusion strain vaccine by the microagglutination test.Five protoplast fusions of E.coli and P.multocida were achieved.