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D.-I. Ham, J.-A. Kim; Uveitogenicity and Antigenicity of Human RPE65 Fusion Proteins in Rats. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2647.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65 is an uveitogenic antigen, expressed in the retinal pigment epithelial cells. Unlike bovine native RPE65, human native RPE65 protein is difficult to prepare in sufficient amount for the experimental uveitis study, and the production of recombinant protein should be considered. This study examined the uveitogenicity and antigenicity of recombinant human RPE65 fusion proteins in rats.
Glutathione S-transferase and polyhistidine were used as tag proteins to make human RPE65 fusion proteins. Proteins were made with the bacterial culture system, and confirmed by immunoblotting with anti-tag and anti-RPE65 antibodies. Lewis rats were immunized with hRPE65 fusion proteins emulsified with complete Freund’s adjuvant, and treated concurrently with pertussis toxin. Immunization with PBS or S-antigen peptide were used for the negative or the positive control groups. Development of inflammatory disease was examined both clinically and histologically. Antibody production and antigen-specific cellular response were evaluated with ELISA and lymphocyte proliferation assay.
GST-hRPE65 induced mild ocular inflammation in only one of 6 rats. Polyhistidine-hRPE65 induced uveitis in all of 15 rats, and 10 micrograms of polyhistidine-hRPE65 was enough to induce uveitis in all of 6 immunized rats. High titers of IgG anti-RPE65 antibodies were detected in the serum of rats immunized with polyhistidine-hRPE65. Lymphoproliferative responses to the polyhistidine-hRPE65 were observed. Rats immunized with PBS showed no disease and no antibody responses, whereas rats immunized with S-antigen peptide showed ocular inflammation.
Polyhistidine-hRPE65 protein is highly uveitogenic and antigenic in Lewis rats. It might be used in the autoimmune uveitis experiment in which the native human RPE65 protein is needed, but cannot be prepared in sufficient amount.
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