Expression, purification of epitope peptide of human melanin-concentrating hormone receptor 1 and its antigenicity detection in vitiligo patients
- VernacularTitle:人黑素浓集素受体1抗原表位肽的表达、纯化及其在白癜风患者中的检测
- Author:
Cuiping GUAN
;
Miaoni ZHOU
;
Fuquan LIN
;
Wen XU
;
Weisong HONG
;
Lifang FU
;
Aie XU
- Publication Type:Journal Article
- Keywords:
Vitiligo;
Melanin-concentrating hormone receptor 1;
Cloning,molecular
- From:
Chinese Journal of Dermatology
2010;43(12):851-854
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express and purify the epitope peptide of human melanin-concentrating hormone receptor 1, and to evaluate its performance in the detection of autoantibodies in vitiligo patients. Methods The target gene encoding the epitope peptide of human melanin-concentrating hormone receptor 1 was synthesized, cloned to prokaryotic expression vector pGEX-4T-2 which was then transferred to E. coli BL21. The protein expression was induced by isopropy-β-D-thiogalactoside (IPTG) and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Blocking ELISA was carried out with membrane proteins extracted from melanocytes as the blocking antigen. The antigenicity of the peptide was detected in sera from 100 patients with progressive vitiligo and 30 healthy human controls. Results The recombinant expression vector was successfully constructed, and the target protein was successfully expressed in E.coli, which was evidenced by SDS-PAGE and Western blot. With the glutathione S-transferase (GST) purification kit, the purity of the recombinant protein reached 100% when the sampling weight was less than 0.625 μg.The binding of the target protein with serum IgG antibodies from vitiligo patients could be blocked by natural membrane antigen of melanocytes. Of the 100 sera from patients with progressive vitiligo, 36 were reactive with the target protein. Conclusions The epitope peptide of human melanin-concentrating hormone receptor 1 has been successfully expressed and purified. The purified protein can bind with serum IgG antibodies from vitiligo patients, and may be applied to the detection of autoantibodies against human melanin-concentrating hormone receptor 1.
