单增李斯特菌p60蛋白的表达、纯化以及免疫原性鉴定

Objective：p60 is Listeria monocytogenes-specific protein on the cell surface.To provide specific immunogen for monoclonal antibody against protein p60, we clone and prokaryotic express protein p60 from Listeria monocytogenes (LM) ,and identify the immunogenicity of p60.Method: Designing the primers of iap gene by biological software; the iap gene was amplified by PCR,and sucloned into expression vector PET28a（+）to obtain pET-28a-iap expression plasmid;then transformed into E.coliBL21 and protein p60 was expressed through ITPG induction.The p60 protein was purified by Ni2+ -chelating affinity column chromatography;identifing the immunogenicity by Western blotting and ELISA with antiserum against LM. The purified protein was used as antigen for immunization of Balb/c;testing the titre of the antiserum and the cross reactivity by ELISA.Result: It was demonstrated by agarose gel electrophoresis that the cloned iap gene was 1443 bp in length and the protein sequence got about 99% homology with EGD, the original strains of LM,which published in GenBank. SDS-PAGE indicated that the molecular weight of recombinant p60 protein was about 60 kD.Western blotting and ELISA showed that the recombinant protein p60 was able to bind specificly with antiserum against LM. The p60 antiserum were obtained with a titer as high as 1∶8,000 after the third immunization of mice. The antibodies had low cross reaction with L.welshimeri and L. innocua.Conclusion:The immunogenity of p60 was identified by analysis with LM antiserum,and the antiserum against p60 was identified preliminarily;these will provide specific immunogen for monoclonal antibody against protein p60.