Construction And Preliminary Application Of The Expression Of Two Different Outer Membrane Proteins Of Avian E. Coli Fusion Strains

Abstract: Using supersonic lysis and Sarkosyl extract outer membrane protein(Omp) of major serotypes Avian pathogenic Escherichia coli isolated from dead chicken and embryo originated from feeding region of Shaanxi province. The Omp patterns were analyzed by SDS-PAGE. Three different Omp patterns were identified with these 18 strains, of which, four O1,four O2,four O78 and three O89 strains, all appear 2 Omp patterns(Omp-1,2 pattern),three O75 strains appear 3 Omp patterns(Omp-1,2,3pattern).The results indicate that different serotype strains have the same Omp patterns, and the same serotype strains may be have different Omp patterns.Protoplasts were prepared with lysozyme and EDTA, and regenerated in the hypertonic nutrition agar(HNA),the optimal concentraction and time of enzyme were 6mg/mL and 25 min respectively; Combine 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 was genetic stable.Vaccine of Aluminum Hydroxide-Adjuvanted was prepared with fusion strains. Compared with monovalence vaccine, antibody titer and regression are not different greatly after vaccinaction. However, challenge test of mixed serotypes domenstrated the protection rate of fusion vaccine increase 13%~33% than monovelent vaccine, 60%~66% than controls, which suggest that fusion strain vaccine has better immunogenicity and protection.