| 重组多肽酶联免疫吸附法检测抗LKM-1抗体的初步应用 |
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| 引用本文： | 王文凯,李永哲,段秀杰.重组多肽酶联免疫吸附法检测抗LKM-1抗体的初步应用[J].中国免疫学杂志,2008,24(11). |
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| 作者姓名： | 王文凯 李永哲 段秀杰 |
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| 作者单位： | 1. 江苏大学附属人民医院检验科,镇江,212002
2. 中国医学科学院中国协和医科大学北京协和医院检验科,北京,100730 |
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| 摘 要： | 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。
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| 关 键 词： | 重组 表位 抗LKM-1抗体 酶联免疫吸附测定 |
Detection of anti-LKM-1 antibody by recombinant fusion peptide in enzyme-linked immunosorbent assay:a preliminary study |
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| WANG Wen-Kai,LI Yong-Zhe,DUAN Xiu-Jie.Detection of anti-LKM-1 antibody by recombinant fusion peptide in enzyme-linked immunosorbent assay:a preliminary study[J].Chinese Journal of Immunology,2008,24(11). |
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| Authors: | WANG Wen-Kai LI Yong-Zhe DUAN Xiu-Jie |
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| Institution: | WANG Wen-Kai,LI Yong-Zhe,DUAN Xiu-Jie.Affiliated People\'s Hospital,University of Jiangsu,Zhenjiang 212002,China |
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| Abstract: | Objective:To detect anti-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.Methods:We obtained CYP2D6 cDNA fragment by means of PCR,using total liver cDNA library as the template.The PCR products were ligated into pEGH expressing vector to construct the recombinant expressing vector with high efficiency in Saccharomyces Cerevisiae Y258.The positive clones were identified by PCR reaction and then i... |
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| Keywords: | Recombinant Epitopes anti-LKM-1 antibody ELISA |
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