Immunochemical and biochemical characterization of immunogenic proteins of Giardia lamblia

Abstract

Giardia lamblia is a protozoan parasite that causes one of the most common gastrointestinal diseases worldwide. It is known that to eliminate the parasite from host intestine is necessary the activation of B‐cell and T‐cell dependent mechanisms. However, little is known about which Giardia antigens stimulate effective immune responses. Our research group, using a model of G. lamblia infection in C3H‐heJ mice, previously described a Giardia protein of 70 kDa (5G8 protein) which was strongly recognized by serum from infected mice. This antigen is expressed both intracellular and on the surface of the trophozoite. Mass spectrometry analysis (ESI‐MS/MS) indicated that 5G8 could be the Binding Immunoglobulin Protein (BIP), a heat shock protein of 70 kDa (HSP 70). The main aim of this study is determine whether the Giardia 5G8 antigen is the BIP protein. We synthesize a recombinant BIP protein (BIPr) in E. coli system and it was purified by immobilized metal affinity chromatography (IMAC). To identify BIP protein, we generated polyclonal antibodies by immunizing C3H‐heN mice with BIPr protein. Finally, we analyze the expression profile of both proteins on Giardia trophozoites by immunochemical and flow cytometry assays. The immunochemical assays showed a differential profile of recognition in the protein extracts of two strains of G. lamblia: GS/M83‐H7 and GS/M83‐H7 5G8 (+). The monoclonal antibody 5G8.B5 recognized a 70 kDa band in the protein extract of Giardia GS/M83‐H7 5G8 (+), while BIP recombinant protein was not recognized. On the other hand, anti‐BIP polyclonal antibodies reacted with a 70 kDa protein band of both Giardia strains at a similar level. Additionally, the Flow Cytometry analysis determine that BIP is expressed in the intracellular compartments of Giardia trophozoites, while 5G8 is strongly expressed on the surface of Giardia trophozoites GS/M83‐H7 5G8 (+). Our data indicate that 5G8 and BIP proteins are different molecules of Giardia. Additional studies are required to determine the nature and function of 5G8 protein.