Expression of CTB-p277 Fusion Gene in E. coli and Analysis of ITS Immunogenecity

To enhance the immunogenicity of polypepetide epitopes,a fusion gene CTB-p277,in which p277(from Shock heat protein 60 of human) was fused to the 3' end of CTB gene,was constructed and cloned into pET28a(+) to obtain a prokaryotic expression vector pET28a-CTB-p277.Subsequently the recombinant plasmid pET28a-CTB-p277 was transformed into E.coli strain BL21(DE3).After being induced with 2% lactose for 5h,the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel(12%) electrophoresis(SDS-PAGE),and the results indicated that the recombinant protein CTB-p277 was expressed and accumulated as inclusion bodies.The recombinant CTB-p277 protein accumulated to the level of 40% total bacterial protein.About 99.1% purity of the recombinant protein was obtained following washing,denaturing and purifying via DEAE-cellulose chromatography.After the inclusion bodies were refolded in vitro,the recombinant protein represented assembled pentamers.After mucous immunity for KK-Ay mice with CTB-p277 fusion protein,the results of ELISA analysis demonstrated that the fusion protein could be recognized with the anti-p277 antibody;blood glucose concentration and body mass were decreased correspondingly.In addition,the results of the GM1-ELISA assay showed that the protein could bind to monosialoganglioside specifically.