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H. Alizadeh, H. Li, S. Neelam, J. Y. Niederkorn; Cloning and Characterization of the Acanthamoeba Mannose-Induced Protein. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5534.
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We have identified the mannose-induced protein (MIP-133) from Acanthamoeba castellanii trophozoites. MIP-133 plays an important role in the pathogenesis of Acanthamoeba infections by facilitating invasion and inducing apoptosis of corneal epithelial cells. The aim of this study is to describe the isolation and molecular cloning of a MIP-133 from A. castellanii.
The protein was verified and isolated by 2D gel electrophoresis and six peptides were obtained and the amino acid sequences were characterized by mass spectrometry. Similarity search of the deduced amino acid sequences to published sequences were performed using Acanthamoeba genomic database Cross combination of deduced primers with Acanthamoeba genomic library resulted in several genes that had homology with 631 residue sequences of Acanthamoeba genes. This gene sequence did not match with any known genes. Deduced amino acid sequence from the protein sequence resulted in five peptide sequences that were indicative of a high-scoring match with these deduced proteins. After TA cloning and gene sequencing, the gene fragments were confirmed. Six clones were confirmed with in-frame sequences and were ligated into the pQE-16 vector for protein expression using TAGZyme systems MIP-133 with a 6-histidine tag added to the C terminus was expressed in E. coliM15 (pREP4) cells. Cell cultured supernatants were chromatographed on a nickel-affinity column (1-ml Ni-NTA resin, Qiagen). Supernatants were eluted with elution buffer. Recombinant MIP-133 protein (rMIP-133) was tested by ELISA using chicken anti-Acanthamoeba MIP-133 .MIP-133 protein isolated by FPLC was used as a positive control.
ELISA analysis indicated that anti-MIP antibody specifically bound to the rMIP-133 protein eluted from cell culture supernatants. By contrast, pre-immune serum did not bind to rMIP-133 protein. Cells transfected with the empty vector alone or pQE-16 did not react with the anti-MIP-133 antibody. Acanthamoeba MIP-133 protein isolated by FPLC specifically reacted with anti-MIP-133 antibody (positive control). Hybridomas were produced as previously described. Three cell lines, each originating from different primary wells, produced antibody against rMIP-133 protein.
These results suggest that the anti-MIP-133 antibody specifically recognized rMIP-133 expressed in E. coli. Experiments are in progress to select the clones by limiting dilution to produce a monoclonal anybody against rMIP-133 protein and to characterize the antibody by western blot and by its capacity to block MIP-133 activity in vitro and in vivo.
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