Preparation and immunologenicity study of HpaA-CtxB fusion protein to Helicobacter pylori in mice / 中国免疫学杂志

ABSTRACT

Objective:To explore the immune response in mice fed orally with the conjugated antigen of HpaA-CtxB(HCTB).Methods:A recombinant strain which could express bivalent antigen of HpaA and CtxB subunit was constructed. HpaA and CtxB gene was amplified by PCR. The DNA products of HpaA and CtxB were inserted into a prokaryotic expression vector pQE-30 respectively, and then translated into E.coli strain DH5? to express HCTB fusion protein. Its immunogenicity was analyzed by Western blot. After purification, to fed mice by oral immunization. The change of antigen-specific ASC antibody(IgG and IgA) in the mucosa of the animals were detected by ELISPOT and ELISA assay. Results:HCTB fusion gene was sequenced as 1 161 bp, the fusion protein encoded polypeptides of 387 amino acid residues.The molecular weight was 40 kD analysed by SDS-PAGE. The level of soluble expression product was about 41.67% of total cell protein. After affinity chromatography, the purity of fusion protein was above 92%.Western blot analysis confirmed that fusion protein could be specifically recognized by the serum of anti-HpaA and anti-CT. Antigen-specific ASC and antibody response in animals immunized with HCTB or HpaA was determined by ELISPOT and ELISA. The results showed that the number of sIgA and IgG-ASC increased significantly in PP and gastric mucosa, especially those of the sIgA-ASC by orally immunization with HCTB. The levels of specific antibody were also higher than those of controls. Conclusion:The results indicated that the oral immunization with HCTB induces effective mucosal immune respones and produced higher levels sIgA. The recombinant fusion protein HCTB can be used as an effective oral vaccine for prevention and treatment of infection of Hp.