Antiserum Preparation Of EPEC Adhesin IntiminC300 And Exploration Of Its Neutralization Ability

Objective:Making prepare EPEC adhesion factor IntiminC300 antiserum,observe whether the antiserum could affect the adhesion of EPEC to Hep-2 cells by in vitro cell experiments in order to make sure whether IntiminC300 can be EPEC’s protective antigen,and laying the foundation for the study of EPEC ’s vaccine components.Method:The IntiminC300 expression plasmid pET32a-intiminC300 was identified by digestion and PCR,and the plasmid was transformed into host strain BL21,Constructng IntiminC300 prokaryotic expression system.The expression of His tag-IntiminC300 fusion protein was induced by IPTG and SDS-PAGE was used to analyze its expression.The fusion protein was purified by adopting NI column affinity chromatography and the purified protein was used as an antigen with assist of adjuvant was carried out to Immune the rabbits.After 4 immunizations,the rabbit’s blood samples were collected from its heart and the antiserum titer was measured by convective immunoassay.Meanwhile the antiserum was used as primary antibody and the expression of EPEC adhesin IntiminC300 was detected by Westen Blotting.Having neutralized EPEC with different dilution of antiserum,the adhesion of Hep-2 cells to neutralized bacteria was observed under the microspe.Results:1.The BL21 / pET32a-intiminC300 prokaryotic expression system was successfully constructed,the expression of His-IntiminC300 fusion protein was detected by SDS-PAGE.The result shows that the fusion protein was expressed both in soluble and in vivo,but mainly in inclusion body.2.His tag protein purification kit(inclusion body protein)was purified to this protein.3.Successfully obtained high titer antiserum(convective immunoassay detection titer up to 1:32).4.In vitro neutralization experiments observed that the antiserum could effectively inhibit the adhesion of EPEC to Hep-2 cells within the dilution factor of 16.Conclusion:EPEC adhesion factor intiminC300 not only has good immunogenicity,but also its antibodies can effectively prevent the adhesion of bacteria to epithelial cells,and its antibodies can protect peple from EPEC infection.Thus,we can conclude that the intiminC300 is an important protective antigen of EPEC and it can be used as an ideal candidate antigen of EPEC vaccine.