白癜风患者血清前黑素小体蛋白17自身抗体的检测

Detection of serum autoantibodies against premelanosome protein 17 increases in the vitiligo patients

目的获取重组前黑素小体蛋白17(Pmel 17)并用于检测白癜风患者血清中Pmel 17自身抗体的分布。方法通过RT-PCR从原代黑素细胞中获得人的Pmel 17基因,先后插入克隆载体pMD19-T和表达载体pGEX-4T-1,以构建重组质粒。IPTG诱导Pmel 17蛋白表达后以亲和层析法进行纯化。间接ELISA检测白癜风患者血清中抗Pmel 17自身抗体的阳性率。结果重组质粒经测序,与预期设计完全一致,同时Pmel 17蛋白成功表达和纯化。ELISA检测进展期白癜风患者血清中抗Pmel 17自身抗体阳性率为22%,稳定期白癜风患者阳性率仅为2%,健康对照组未检测到抗Pmel 17自身抗体,进展期和稳定期阳性率差异具有统计学意义。结论成功表达了Pmel 17重组蛋白,证明Pmel 17自身抗体与白癜风的疾病严重程度有一定关系。

Objective To detect the distribution of autoantibodies against premelanosome protein 17 （Pmel 17） in the sera of vitiligo patients with the recombinant Pmel 17 protein. Methods The cDNA encoding human Pmel 17 was amplified by RT-PCR from the primary melanocytes and was cloned into vector pMD19-T and subsequently the expression vector pGEX- 4T-1. After being induced by IPTG, the recombinant Pmel 17 protein was purified by high-performance affinity chromatography. The presence of autoantibodies against Pmel 17 in the sera of vitiligo patients was determined by indirect ELISA coated with recombinant Pmel 17 protein. Results The recombinant Pmel 17 plasmids were right as we expected by DNA sequencing. The recombinant Pmel 17 protein was successfully expressed and purified. The indirect ELISA revealed that, as compared with healthy control group （0/100, 0% ）, positive rate of the serum autoantibodies in the progress vitiligo patients was 22% and positive rate of stable vitiligo patients was only 2% （2/100）. The difference of positive rate between patients with progress and stable vitiligo was statistically significant. Conclusion The recombinant Pmel 17 protein was successfully expressed. The autoantibodies against Pmel 17 is closely related to the severity of vitiligo.