[Expression and purification of epitope peptide of human tyrosinase and its antigenicity in vitiligo patients].

OBJECTIVE: To express the epitope peptide of human tyrosinase (TYR), and discuss the application of the peptide in detecting autoantibody of the vitiligo patients. METHODS: The epitope areas 240 - 255, 289 - 294, 295 - 300, 435 - 447, and 461 - 479 of human TYR were synthesized and connected to the vector pGEM-T. The target gene was cloned to the prokaryotic expression vector pGEX-4T-2, which was then transferred to Escherichia coli BL21 host cells. Isopropy-beta-D-thiogalactoside (IPTG) was used to induce the protein expression that was examined with SDS-PAGE and Western blotting. Indirect ELISA was conducted to detect the antigenicity of the peptide in 100 blood specimens of active vitiligo patients and 30 healthy controls. RESULTS: The recombinant expression vector was constructed successfully. The SDS-PAGE and Western blotting results showed expression of the recombinant protein in E. coli. The amount of the recombinant protein reached about 70% of the total mass of bacterial protein with PAGE analysis system. With the glutathione S-transferase (GST) purification kit, the purity of recombinant protein reached over 90%. Indirect ELISA showed that reaction with the target protein was negative in all the 30 healthy controls and was positive in 64 of the 100 active vitiligo patients. CONCLUSION: The epitope peptide of human TRY is expressed successfully, and it has antigenicity in the serum of vitiligo patients.