人自身抗原CYP2D6 285 bp基因片段克隆、真核表达及免疫性鉴定

目的:真核表达人自身抗原细胞色素P450 2D6(CYP2D6)显性表位285 bp基因片段,获得具免疫学活性的纯化重组蛋白,为自身免疫性肝炎抗体的检测提供特异性抗原．方法:以肝脏的cDNA混合文库为模板作 PCR,将PCR产物与真核表达载体pEGH共同转化酿酒酵母Y258,碱裂解法进行质粒制备, PCR扩增鉴定．表达载体构建成功后,在半乳糖的诱导下表达产生重组融合蛋白,利用 GST亲和层析法进行纯化．然后对其产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS- PAGE)和蛋白质印迹法(WB)及蛋白质质谱学 (MALDI-TOF)检测．结果:PCR产物约290 bp,与预期285 bp接近． pEGH-CYP2D6重组阳性克隆PCR鉴定与预期大小接近,SDS-PAGE和WB结果显示,融合蛋白Mr37 000,具有天然人自身抗原CYP2D6 的免疫原性．质谱蛋白肽指纹数据通过Mascot 在人类蛋白质数据库中分析比对,与CYP2D6 蛋白同源性最高．结论:成功克隆表达人自身抗原CYP2D6的显性表位,为建立新的自身免疫性肝炎抗体检测方法奠定了基础．

Gene cloning and fusion expression of immunodominant epitope of human autoantigen cytochrome P450 2D6 in Saccharomyces cerevisiae

AIM: To clone and express immunodominant epitope of human autoantigen cytochrome P450 2D6 (CYP2D6) in Saccharomyces Cerevisiae, and establish a new assay for detecting autoantibody LKM-1. METHODS: We obtained CYP2D6 DNA fragment by polymerase chain reaction (PCR), using total liver cDNA library as the template. The PCR products were recombined into pEGH expression vector to construct the high efficiency recombinant expression vector in Saccharomyces Cerevisiae Y258. The positive clones were identified by PCR and induced by galactose. Glutathione-Sepharose 4B was used for purification of recombinant CYP2D6 protein. The expression products were analyzed by SDS-PAGE and Western blot as well as by matrix-assisted laser desorption inoization-time of flight mass spectrometry (MALDI-TOF-MS). RESULTS: The PCR product was about 290 bp in size, which was in accordance with the predicted 285 bp. The pEGH-CYP2D6 positive clone produced a Mr37 000 fusion protein, which was confirmed to have natural immunogenicity of human autoantigen CYP2D6 by SDS-PAGE and Western blot, and MALDI-TOF-MS showed that it also had high similarity with CYP2D6 protein. CONCLUSION: The immunodominant epitope of human autoantigen CYP2D6 is successfully cloned and expressed in Saccharomyces Cerevisiae, which lays a foundation for a new method of autoantibody detection in autoimmune hepatitis.