【摘要】 为研究Ｈｅｙｍａｎｎ肾炎致病原的病理表型和免疫复合物形成机理，应用ＤＮＡ重组技术，将Ｈｅｙｍａｎｎ肾炎（ＨＮ）致病原受体相关蛋白（ＲＡＰ）的ｃＤＮＡ克隆到表达质粒ｐＧＥＸ中，构建重组表达质粒ｐＧＥＸ－Ｒ９３，经限制性内切酶图谱分析，ＤＮＡ测序，鉴定插入子ＲＡＰ－ｃＤＮＡ正确克隆到ｐＧＥＸ表达框架后，转化大肠杆菌ＤＨ５α，经ＩＰＴＧ诱导，高效表达了谷胱甘肽巯基转移酶－受体相关蛋白（ＧＳＴ－ＲＡＰ）融合蛋白，蛋白产量占大肠杆菌总蛋白量的３９．４％。经ＧＳＴ－Ｓｅｐｈｏｓｅ４Ｂ亲和层析得到高度纯化的ＧＳＴ－ＲＡＰ。免疫印迹分析证明，针对ＧＳＴ－ＲＡＰ的抗血清能特异地识别肾皮质天然抗原４４０００α蛋白，免疫组化分析显示ＧＳＴ－ＲＡＰ抗血清特异性识别大鼠肾近曲小管刷状缘抗原。更多还原

【Abstract】 Tostudythepathologicalphenotypeofpathogenandthemechanismoftheimmunocom-plexinHeymannnephritis,weutilisedDNArecombinanttechnique,toconstructplasmidpGEX-R93byinsertingthecDNAencodingofreceptor-associatedproteinofHeymannnephritis(HN)pathogenicepi-topeintothepGEX.ThepGEX-R93codingregionfragmentwasconfirmedbyenzymecutingandse-quencinganalysis.AfterthepGEX-R93wastransfectedintoDH5α,theexpressionofGST-RAPfu-sionproteinwasinducedbyisopropylthio-β-D-galactosidearetheoverproducedproteinconsituted39.4%ofthetotalcelularprotein.WesternblotanalysisshowedthattheantiserumforGST-RAPrecognizedthe44000αproteinbandofnaturalantigeninHN.Inaddition,immunofluorescenceanalysisshowedthattheantiserumalsodistinctivelyrecognizedtheciliatedmembraneantigenoftheproximalconvolutedtubuleoftheratkidney.更多还原