Expression, purification of epitope peptide of human melanin-concentrating hormone receptor 1 and its antigenicity detection in vitiligo patients / 中华皮肤科杂志

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.

Construction of eukaryotic expressing vector of human MCHR1 and establishment of stable transfectant HEK293 cell line / 西安交通大学学报(医学版)

Objective To construct eukaryotic expressing vector of human melanin-concentrating hormone receptor1(MCHR1) and transfect HEK293 cells so as to establish stable HEK293 cell line.Methods The full-length MCHR1 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and was inserted into pcDNA3.1(+).After the identification of digestion and sequencing on the recombinant pcDNA3.1(+)/MCHR1,we transfected the recombinant into HEK293 cell by lipofectamineTM2000.After screening culture by G418,stable transfected HEK293 cell line was established,and the expression of MCHR1 was identified by RT-PCR and Western blotting.Results The eukaryotic expression vector pcDNA3.1(+)/MCHR1 was constructed,stable transfected HEK293 cell line was established,and MCHR1 protein was expressed successfully.Conclusion The construction of the eukaryotic expression vector pcDNA3.1(+)/MCHR1 and the establishment of stable transfected HEK293 cell line have provided solid experiment foundation for further studies on the function of MCHR1.