[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.


CONCLUSIONS
The epitope peptide of human TRY is expressed successfully, and it has antigenicity in the serum of vitiligo patients.
Department of Dermatology, 3rd Hospital of Hangzhou, Hangzhou 310009, China.
Cui-ping
CP
Guan
Miao-ni
MN
Zhou
Yong-wei
YW
Li
Xiao-dong
XD
Wei
Ai-E
AE
Xu
D000293:Adolescent; D000328:Adult; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000276:immunology*; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014820:Vitiligo / Q000235:genetics / Q000276:immunology*
D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase