Cloning And Expression Of Human TRP - 2 CDNA

Tyrosinase related protein-2 (TRP-2) is a important member of the tyrosinase related protein family. It referred to as DOPAchrome tautomerase and catalyses the conversion of DOPA chrome to 5,6-dihydroxyindole-2-carboxylic acid as part of the pathway of melanin production. It is related to the pathogenesis of vitiligo and there exists the cross-reactivity between TRP-2 and Tyrosinase. It is of great importance to find out the antigenic epitope of TRP-2 as an antigen in pigmental cells, hi order to set up a foundation to investigate the epitope of TRP-2, we studied on the cloning and expression of human TRP -2 code gene.Total RNA was isolated from cultured human pigmental cells and mRNA was reversly transcribed into cDNA. Then PCR was used to amplify the TRP -2 coding region. The PCR product was cloned into pUC19 plasmid and sequenced, then subcloned into vector pGEX-4T-1. The TRP-2 protein was expressed hi E coli of DH5 a as fusion protein with glutathioneS-transferase (GST) induced by IPTG. The fusion protein was purified by glutathione resin column.Experimental result was: l.TRP - 2 coding region was cloned into pUC19 and the sequence was confirmed; 2.Fusion expressing vector of pGEX -4T-1/TRP-2 was successfully constructed; 3.GST/TRP-2 fusion protein was correctly expressed.We successfully cloned the human TRP - 2 gene and constructed GST/TRP-2 fusion expressing vector. GST/TRP-2 fusion protein was correctly expressed in E coli, which is the basis for investigation of TRP-2 protein epitopes and pathogenesis of vitiligo.