重组多肽酶联免疫吸附法检测抗LKM-1抗体的初步应用

Detection of anti-LKM-1 antibody by recombinant fusion peptide in enzyme-linked immunosorbent assay:a preliminary study

目的:制备人自身抗原细胞色素P4502D6(CYP2D6)257-351位氨基酸片段融合蛋白作为自身抗原,探讨ELISA检测抗LKM-1抗体的敏感性和特异性。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,经GST亲和层析法进行纯化后,免疫印迹法鉴定抗原性,ELISA检测抗LKM-1抗体阳性血清及部分其他结缔组织病患者血清中的抗LKM-1抗体。结果:重组融合蛋白在宿主菌中获得表达,免疫印迹法鉴定表明其能与标准抗LKM-1抗体阳性血清反应,而与正常血清、其他抗血清无反应。在26份抗LKM-1抗体阳性血清中,6份抗HCV抗体阳性血清用重组多肽ELISA检测有5份呈阳性,其余20份血清用重组多肽ELISA检测均呈阳性;20份其他结缔组织病患者血清用重组多肽ELISA检测均为阴性。结论:重组的257-351位氨基酸片段是CYP2D6抗原的主要抗原表位区域,以重组多肽为基质ELISA检测抗LKM-1抗体的敏感性较高,为进一步研究抗体水平与临床病情变化的相关性奠定了基础。

Objective： To detect anfi-LKM-1 antibody with enzyme-linked immunosorbent assay （ELISA） using a recombinant fusion peptide which comprises 257-351 amino acid fragment of CYP2D6 as antigen. Melhods： We obtained CYP2D6 cDNA fragment by means of PCR, using total liver eDNA library as the template. The PCR products were ligated into pEGH expressing vector to construct the recombinant expressing vector with high efficiency in Saceharomyees Cerevisiae Y258. The positive clones were identified by PCR reaction and then induced by galactose. Glutathione-Sepharose 4B was used for purifcafion of recombinant CYP2D6 protein. After affinity purification, the antigenicity was identified with Western blot. Senma samples from 26 patients who were positive for anti-LKM-1 antibedy,20 patients with other connective tissue disorder（CTD） and 30 normal controls were retrospectively tested with ELISA. Results：A fusion peptide was expressed and purified.The antigenicity was confirmed with Western blot using standard of anti-LKM-1 antibody-positive serum. Of the 26 serum samples which are positive for anti-LKM-1 antibody,5 of 6 samples positive for anti-HCV antibody also recognized the recombinant fusion peptide with ELISA,only one serum sample which was showed positive anti-HCV antibody displayed a negative result in ELISA assay.All other 20 patients with positiv anti-LKM-1 antibody were shown positive in ELISA assay using this recombinant peptide. All the serum samples from patients with other CrD were negative in ELISA assay. Conclusion：The recombinant antigen fragment contains major epitope regions in natural CYP2D6 antigen. Detection of anti-LKM-1 antibody with ELISA using the recombinant peptide can improve the sensitivity and has a potential role in determining its clinical association.