Study On OMP Of Predominant Types Of Pathogenic Escherichia Coli From Chongqing Piglet

Pathogenic Escherichia coli(E.coli)is the most popular pathogeny bacteria in clinic with serious harm to the development of husbandry.The problem of bacterial drug resistance becomes increasingly outstanding,because of the extensive use of antibacterials In order to effectively prevent this disease,various kinds of gene engineering bacterial vaccine and multivalence inactivated vaccine have been developed at home and abroad.The poor immue effect has caused great difficulties for the effective control of Colibacillosis because of the multiplicity of serotype and diversity of epidemic strains.It is reported that the OMP with favourable immunogenicity could speed up macropHage absorbing antigen,activate proliferative response of lymphocyte,stimulate humoral immunity and cell immunity and counteract the aggression of homologous and allogenetic bacterium.91 in 173 pathogenic E.coli strains isolated in our lab were identified serotype.The predominant types are O₁₀₁,O₈,O₂₀,O₆₄,O₄₅and O₁₄₉.In this research,we choose the OMP of predominant serotypes of(O₈,O₂₀and O₁₀₁)strains from Chongqing Piglet to study for the development of subunit vaccine ofE.coli OMP in future.1 Analysis of OMP by SDS-PAGEOMP was extracted by dealing with N-sacrosyl and ultracentrifugation from predominant serotypes of isolated strains including five O₈ strains,five O₂₀strains and six O₁₀₁strains.The variability of OMP of each strain was analyzed by SDS-PAGE.The result showed that sixteen E.coli strains emerged five OMP types:the O₈,O₂₀and O₁₀₁strains emerged two,three and two OMP types,respectively.2 Preparation antiserum of whole cell protein and OMP of predominant serotypes of E.coli strainsHealthy rabbits were immuned by whole cell protein and OMP of the separation E.coli strains. The antibody was detected by agar diffusion test after seven or ten days after immuned three times. Antibody titer was determined by indirect ELISA after separated antiserum when the serum titer arrived 1:16.The result of the study showed whole cell protein antiserum titer of(O₈,O₂₀, O₁₀₁)strains were 1:2500,1:3000 and 1:3000,respectively.The OMP antiserum titer of(O₈,O₂₀. O₁₀₁)strains of E.coli are 1:1500,1:2000 and 1:2500,respectively.3.Analysis of OMP by Western-blottingOMP was analyzed by Western-blotting using whole cell protein pooled serum.The result of the study showed that all the OMP had immunogenicity.In all sixteen strains,seven strains(one O₈ strain,two O₂₀strains,four O₁₀₁strains)emerged one immune strap while nine strains(four O₈ strain, three O₂₀strains,two O₁₀₁strains)emerged two or three immune strap.The OMP of eleven strains(including three O₈ strain,three O₂₀strains and five O₁₀₁strains)had the common immune straps in the position of 34kD.Meanwhile,two O₈ strains and two O₂₀strains had the common straps in the position of 36kD.The anti-OMP positive serum of(O₈,O₂₀,O₁₀₁strains)were used for western blotting analysis on E.coli OMP.The result showed that different OMP appeared a same band around 34kDa when they reacted with anti-OMP positive serum within their serotype,but little bands appeared with anti-OMP positive serum if out of their serotype.4.Analysis of OMP of O₂₀strain by Two-dimensional electropHoresis and Western-blottingOMP of O₂₀strain was analyzed by two-dimensional electrophoresis.Self-made anti-OMP positive serum of O₂₀strain and single factor serum of O₂₀strain were used for western blotting.The result showed that OMP of O₂₀strain appeared lots of immune reaction points when it reacted with self-made anti-OMP positive serum,but only appeared four immune reaction points when it reacted with single factor serum of O₂₀strain.