Effects of a Sjogren's syndrome-associated mutation of the TAP2 gene on the diversity of HLA-bound antigen peptides

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1) Preparation of the mutated TAP2 gene.The A to G mutation was introduced at the 1729^<th> base of TAP2 gene by the method of Shuldier. A.R.et al (Analyt. Biochem. 194, 9-15, 1991), and the mutated (TAP2^*Bky2) and wild (TAP2^*b) type cDNA were prepared.2) Esablishment of TAP1 and TAP2 genes transfected cell lines.TAP1 and TAP2 genes (TAP2^*B or TAP2^*Bky2) were transfected to TAP genes-deficient cell line, T2, by electroporation method. Two cell lines. T2/Bky2 and T2/B, which were different only at the codon 577, were established.3) Collection of HLA-bound peptides from the established cell linesAntigen peptides bound to HLA-B5 were collected by anti-HLA column using anti-HLA-B5 monoclonal antibody (4D12). After filtration with 5,000 dalton cut-off filter, peptide fraction was collected.4) Analysis of HLA-bound peptidesThe fraction was applied on a reversed-phase HPLC, and some peaks were collected. The repertory and amino acid sequence of HLA-bound peptides are now further analyzed by mass-spectrometry.