表达Omp1和Omp2的猪大肠埃希菌融合菌株R2的构建

Establishment of Fusion Strain R2 of Swine Escherichia coli Expressing Omp 1 and Omp 2

为了构建猪大肠埃希菌融合菌株,在药敏试验和MIC试验的基础上,对猪大肠埃希菌HY2株(O101,Omp1)和GXA1株(O107,Omp2)进行交叉耐药培育。以HY2(O101,Omp1,Ami r,Czl s)和GXA1(O107,Omp2,Czl r,Ami s)为亲本菌株,采用溶菌酶-EDTA法制备两亲本菌株原生质体,通过聚乙二醇(PEG)助融,进行原生质体融合,得到3株双耐药融合菌株。结果表明,猪大肠埃希菌原生质体制备和再生的最适条件是:溶菌酶浓度为200U/mL,作用时间为25min。筛选得到3株融合菌株R1、R2、R3,均可凝集两亲本的O抗原,其中,R2凝集性更好更稳定。进一步结合SDS-PAGE分析,显示融合菌株R2能较好的表达两亲本菌株形状,经多次传代仍能够稳定遗传。

Combining drug sensitivity test with MIC for development of swine Escherichia coli fusion strains,the cross-drug resistance of swine Escherichia coli HY2 strain （O101, Ompl） and GXA1 strain （O107, Omp2） were developed. With swine HY2 （O101, Ompl, Ami^r, CzP^s ） and GXA1 （ O107, Omp2, Czlr, Amis） as the parental strains, their protoplasts were prepared with lysozyme and EDTA, and the protoplasts fusion was induced with PEG. Three fusion strains were obtained in the experiment. The results showed that the optimal concentration and time of lysozyme were 200 U/mL and 25 min respectively. All the three fusion strains R1,R2 and R3 could agglutinate two parental O antigens,among which R2 strain was more efficient. Combining Omp patterns by SDS-PAGE, fusion strain R2 was genetic stable.