| Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. Recently, its incidence rate gradually increased. Although the exact pathogenesis is not known, a lot of investigations were inclined to the autoimmune hypothesis. Recent research showed that the pigmented lesions present destruction of melanocytes and a cellar infiltrate. The evidences are that autoantibodies against melanocyte cell surface antigens present in the circulation of most patient with vitiligo, the level of vitiligo antibodies correlates with the extent of depigmentation and activity of the disease. Although the pathogensis of autoimmne disease can't be interpreted presently, on the basis of immunology, an important link is the lost of immune tolerance to some certain autoantigens. "Toboo strains" in lymphocytes lose tabu and the B/T lymphocytes directed to specific autoantigens appear accordingly. Removing these pathogenic lymphocytes is an exploration of treatment in the autoimmune disease.We have succeeded in identifing the antibody of IgG and IgM by immunochemical analysis and demonstrating its functional capacity of killingpigment cells in vitro by a mechanism of complement-dependent cytotoxicty. We have screened vitiligo IgG serum at high liter using live cell ELISA. From western blot, the melanocytes'antigens conjugating vitiligo autoantibodies were detected. There approximate MWs were 150, 90, 75, 50 and 40-45 KD. The antigens of 150, 90 and 75KD have been purefied primarily by immunoprecipitation. Presently, our investigative group is cloning cDNA of the antigens related to antibodies in patients with vitiligo. If we identify the epitopes, we can make chromatography with the cloned epitope peptide to remove the correlated autoantibodies and inject epitope peptide linked with toxic molecules to the body of vitiligo to clear the pathogenic lymphocyte strains. These may be successful methods in treating vitiligo.Tyrosinase related protein 1 (TRP-1) is a kind of glucoprotein which locates in melanocytes abundantly. As a member of the tyrosinase related protein family, it acts as a key enzyme in the constructive metabolism of melanin. Lots of reaserch work have demonstrated that TRP-1 which acts as a autoantigen is related to the autoimmune cause of vitiligo and plays an important role in the antineoplastic reaction of malignant melanoma(MM).A rat B cell epitope of TRP-1 has been found,but we know none of its human B cell epitope till now.Human B cell epitope of TRP-1 is of importance not only to learn deeply the pathogenesis of vitiligo but also to instruct the immune therapy of vitiligo,melanoma.Based on the rat B cell epitope of TRP-1,we have cloned a sequence encoding TRP-1 from human melanoctes'cDNA and expressed it efficiently by coupling GST in Kcoli JM109. Compared with the rat's,the sequence had the antoploidy of 90% and included the rat's. When we detected the vitiligo serum with TRP-1 coupling GST, the positive rate werelow.The reason maybe that the TRP-1 expressed in JM109 has the first grade structure only and lake the necessary interspace structure which is important in maintenance the antigenicity. For this reason, we want to expressed TRP-1 in pastoris.For the TRP-1 expressed in pastoris smoothly,the antibody to TRP-1 was conducted firstly. The molecular weight of TRP-1 is 14KD and the GST is 26KD which is bigger than TRP-1. To avoid the influence of GST, we subcloned the TRP-1 to the vector pRSETA owning 6His.The molecular weight of 6His was only 6KD, it could reduced the passive influence on TRP-1 in maximal limitation.After confirmed by SDS-PAGE,the fusion protein pRSEA/TRP-1 was cut,frozen and crushed.The gel injected the rabbit and the antiserum could help the indentification of the 6His-TRPl expressed in Pichia pastoris.Utilizing 6His-TRPl from Pichia,we detected the titer of antiserum by ELISA,the maximum was l:32000.At the same time,the polyclonal antibody against pRSETA/TRP-1 showed high specificity to 6His...
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