Heymann肾炎致病原受体相关蛋白羧基端病理性抗原决定簇的定位

IDENTIFICATION OF A PATHOGENIC EPITOPE ON COOH TERMINAL OF THE 44 kd RECEPTOR ASSOCIATED PROTEIN (RAP) OF HEYMANN NEPHRITIS ANTIGENIC COMPLEX

目的：探讨Ｈｅｙｍａｎｎ肾炎（ＨＮ）致病原受体相关蛋白（ＲＡＰ）羧基端１０８个氨基酸多肽链上是否存在致肾炎的抗原决定簇。方法：应用ＮｅｓｔｅｄＰＣＲ技术，扩增了编码ＲＡＰ羧基端１０８个氨基酸多肽链的ｃＤＮＡ。应用基因重组及亚克隆技术，高效表达了特异性的ＲＡＰ融合蛋白（ＲＡＰ－ｆｐ），并制备了针对该ＲＡＰ－ｆｐ的多抗血清。结果：该多抗血清体外免疫荧光定位于肾小管刷状缘。体内结合试验的直接免疫荧光和免疫电镜表明，在肾小球足突细胞和肾小球基膜之间，形成了典型的被动型Ｈｅｙｍａｎｎ肾炎（ＰＨＮ）免疫复合物。结论：本实验说明天然羧基端ＲＡＰ在体内能被循环抗体结合，在原位形成免疫复合物。首次证明了在ＲＡＰ羧基端１０８个氨基酸多肽链上至少存在一个病理性抗原决定簇。

OBJECTIVE Heymann nephritis is an animal model for the study of human membranous glomerulonephritis.Recent data showed that the disease is initiated by the binding of circulating antibodies to membrane protein,the Heymann nephritis antigen complex (HNAC), which is a heterodimer composed of a large subunit designated as gp330 and a small subunit designated as receptor associated protein (RAP). We perfoumed the present study to investigate if there is a pathogenic determinant on the COOH teminal 108 amino acids polypeptide of RAP. METHODOLOGY Gene reconstruction and nested PCR techniques were used on expression of COOH teminal 108 aminoacids RAP fp. Immunoflurence and immunoelectronic microscopy techniques were used on kidney from rat models indrced by administration of anti RAP fp serum. RESULTS Indirect immunoflurence and immunoelectronic microscopy showed that when rabbit anti RAP fusion protein antibody was injected intravenously into normal rat,typical deposits of PHN were found at 5 days after injection. CONCLUSIONS We conclude that at least one nephrogenic epitope is presented within the COOH teminal 108 amino acids polypeptide of RAP molecules.