RESUMO
Objective To construct a recombinant expressing plasmid of the hy1 gene of Enterococcus faecium and to express the recombinant Hy1 protein in E.coil.To explore the immune response in mice fed orally with Hyl protein.Methods hy1 gene was amplified by polymerase chain reaction(PCR)and inserted into a prokaryotic expression vector pQE-30.The recomhinant plasmids were transfected into DH5_?to express Hy1 fusion proteins,which were purified by Ni~--column. Western blot was employed to confirm the immunogenicity of the purified protein.Mice were immu- nized by feeding with the fusion protein.The concentrations of antigen-specific antibody in the serum, mucosal fluid and faces were detected by enzyme-linked immunosorbent assay(ELISA).The role of these antibodies in the anti-infection response was evaluated after the mice were challenged with TX0016.Results hy1 gene was sequenced as 1662 bp,the fusion protein encoding polypeptides of 553 amino acid residues.The relative molecular weight was 60 000 when it was determined by sodium dodecylsulfatepo-lyacry-lamide gel electropboresis(SDS-PAGE).The dissolvable expression protein accounted for 38% of total cell protein.After processed by affinity chromatography,the purity of fusion protein was above 92%.Western blot analysis confirmed that fusion protein could be specifically recognized by the anti-TX0016 serum.The concentrations of serum IgA,serum IgG,faeces sIgA and intestmucosal fluid sIgA was 0.365?0.048,0.431?0.064,0.743?0.056 and 1.112?0.113 respectively in hy1 groups and 0.051?0.013,0.098?0.019,0.102?0.032 and 0.187?0.051 respectively in control group.The differences were statistically significant.The mice survival rate after TX0016 challenge was 70% in hyl group and 50% in control group.There was significant difference between these two groups.Conclusion The results indicate that oral immunization with hyl can induce effective mueosal immune response and produce high level sIgA.