Recombinant expression of Helicobacter pylori GroEL protein and immunoprotection of the expression product in mice / 中华微生物学和免疫学杂志

ABSTRACT

Objective To determine the sequence diversity in groEL gene of Helicobacter pylori isolates,and the antigenicity,immunoreactivity and immunoprotection of recombinant expressed GroEL (rGroEL) against H.pylori-infection in mice.Methods The sequences of groEL genes from H.pylori isolates were detected by PCR and sequencing.A prokaryotic expression system of H.pylori groEL gene using pET42a and E.coli BL21DE3 was generated.SDS-PAGE and Gel Image Analyzer were used to measure the yield of rGroEL and Ni-NTA affinity chromatography was applied to extract the expressed rGroEL.The antigenicity and immunoreactivity of rGroEL were examined using ELISA and Western blot assay.The membrane location of GroEL was determined by ELISA using whole cells of H.pylorias the coated antigen.The immunoprotection of rGroEL was detected in H.pylori-infected mouse model.Results All the 35 tested H.pylori isolates possess the groEL gene with 99.4％ to 100％ sequence identities.The yield of rGroEL expression occupied 56％ of the total bacterial proteins and the extracted rGroEL showed a single band in gel after SDSPAGE.rGroEL could induce the production of high-titer IgG in rabbits or mice.The lgG against whole cell of H.pylori(Hp-IgG) and rGroEL-IgG could recognize as well as combine with rGroEL protein.GroEL is a superficial protein antigen of H.pylori.The immunization with 50,100 or 200 μg rGroEL prevented 50.0％ (6/12),75.0％ (9/12) or 91.7％ (11/12) mice from infection of H.pylori strain SS1.The immunoprotective rate ( 91.7％ ) due to the immunization of 200 μg rG roEL was significantly higher than that ( 50.0％ ) of 50 μg rGroEL ( P＜0.05 ).Conclusion The GroEL protein is a sequence-conserved,immunogenic and immunoprotective superficial antigen of H.pylori,which can be used as the immunogen candidate for developing genetically engineering vaccines.