Prokaryotic Expression And Immunological Identification Of F4AC Fimbrial Subunit FAEG Of ETEC

Objective To establish a foundation for preparing the vaccine to prevent ETEC infection of young animals,F4ac fimbrial subunit gene faeG of Enterotoxi- genic Escherichia coli(ETEC) was cloned and expressed.The expressed product was identified for antigenicity and immunogenicity.Methods The faeG gene fragment of ETEC(C83902) genomic DNA was amplified by PCR and inserted into prokaryotic expression plasmid pGEX-6P-1. The constructed recombinant plasmid was defined as pGEX-faeG.The constructed recombinant plasmid pGEX-faeG was transformed into E.coli BL-21 and expressed under induction of IPTG.The expressed product was determined for form and relative molecular mass by SDS-PAGE and identified for antigenicity by Western blot.The mice were immunized with the inclusion-bodies extracted from the sonicated bacterial cells.The IgG and IgA levels were determined in sera,faces and intestines mucus to identify the immunogenicity of the expressed product.The mice were immunized with the recombinant protein FaeG to conduct the animal protection experiment of the immunization. Results The sequence of amplified faeG gene was about 786bp and shared 96% identity with the pulished sequence in GeneBank.Both PCR and restriction enzyme analysis showed that recombinant plasmid pGEX-faeG contained the insertion element which sequences were complete.The expressed product with a relative molecular mass of 53kD existed mostly in the form of inclusion body.Western blot showed specific reaction of the expressed recombinant FaeG protein with the F4ac-positive sera of pigs.The IgG and IgA levels of mice immunized with FaeG were significantly higher than those control mice with PBS and GST.The mice immunized with FaeG were resistant to attack by virulent strain of Escherichia coli C83902 at least about 1MLD.Conclusion The recombinant plasmid pGEX-faeG contained the F4ac fimbrial subunit gene faeG of ETEC was constructed and expressed successfully. The recombinant protein showed good antigenicity and immunogenicity,and might be used for the development of ETEC vaccine to prevent ETEC infection of young animals.