摘要
目的探讨Heymann肾炎（HN）受体相关蛋白（RAP）羧基端抗原决定簇在HN发病机制中的作用。方法通过PCR及分子克隆技术，构建RAP全长和羧基端多肽的原核融合表达载体，表达融合蛋白，制备针对RAP全长和羧基端融合蛋白的抗血清，免疫荧光法检测其在肾组织中的定位。利用该抗血清制备两种HN模型，测定24h尿蛋白定量，免疫荧光法检测肾组织中nephrin表达和分布，并比较组间差异。结果成功构建了重组原核表达载体pGEX-4T-1-RAP1083/324，表达了RAP全长（相对分子质量，70×10^3）和羧基端（40×10^3）的融合蛋白，并制备了相应的抗血清，免疫荧光显示制备的两种抗血清均对肾小管上皮细胞有高亲和性。利用两种抗血清成功制备了HN模型，模型组大鼠24h尿蛋白定量分别达（21．31±4．15）mg和（19．054±3．72）mg，与正常对照组相比，差异有统计学意义（P〈0．01）。nephrin在RAP324诱发的大鼠HN肾组织中表达较RAP1083嘲中减弱明显。结论Heymann肾炎RAP羧基端存在病理性抗原决定簇，能够诱发大鼠HN模型。nephrin在两种HN中表达减少，推测RAP作用机制与减少肾小球内nephrin的表达有关。
Objective To explore the role of C-terminal antigenic determinant of Heymann nephritis receptor associated protein（RAP） in Heymann nephritis pathogenesis. Methods The full-length RAP and C-terminal polypeptide karyogamy expression vector was constructed by PCR and molecular cloning to express fusion protein. Antiserum to the full-length RAP and C-terminal fusion protein was prepared and orientated in kidney tissue by immunofluoreseenee. Two kinds of Heymann nephritis animal models were made with these antisera. We measured the 24 h urine protein quantitation, the expression and distribution of nephrin in kidney tissue and compared the data between the two groups. Results Recombinant protokaryon expression vector pGEX-4T-1-RAP1083/324 was successfully constructed and expressed the full-length RAP（70 × 10^3） and C-terminal fusion protein （40 ×10^3 ）. We found that two kinds of prepared relevant antisera strongly expressed in the renal tubular epithelial cell by immunofluorescenee. Heymann nephritis animal models were also sueeessfully made. The 24 h urine protein quantitation in two model rats groups were （21.31 ± 4.15 ） mg and （19.05 ± 3.72） mg respeetively. The 24 h urine protein quantitation in model groups were higher than that in the normal group （P 〈 0.01 ）. The expression of nephrin in RAP1083 induced model rats＇ glomerulus was lower than the RAP324 induced group. Conclusion Pathological antigenic determinant exists in the Heymann nephritis RAP C-terminal and can induee rat Heymann nephritis models. Nephrin expression reduced in two kinds of Heymann nephritis. It indicated that the mechanism of RAP might be related to the low expression of nephrin in glomerulus.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2009年第10期914-918,共5页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金（30670986）
江苏省自然科学基金（BK2006059）
