免疫球蛋白A在人肾小球系膜细胞中的合成与分泌

发布时间：2018-02-10 21:36

本文关键词： 人肾小球系膜细胞免疫球蛋白A 蛋白质谱 DNA测序　出处：《北京大学学报(医学版)》2017年06期 　论文类型：期刊论文

【摘要】：目的:探讨人肾小球系膜细胞(human mesangial cells,HMCs)中是否表达免疫球蛋白A(immunoglobulin A,Ig A)。方法:培养人HMCs细胞系,以免疫荧光染色检测Igα、Igκ、Igλ在系膜细胞中的表达;以反转录聚合酶链式反应(reverse transcription polymerase chain reaction,RT-PCR)和DNA测序检测HMCs中Igα、Igκ、Igλ恒定区转录本表达;以Western blot方法检测细胞裂解液及培养上清液中Igα、Igκ、Igλ蛋白水平表达;以jacalin为配体的亲和层析纯化上清液蛋白质,以蛋白质谱检测纯化的蛋白氨基酸序列。结果:免疫荧光染色检测到HMCs细胞浆Igα、Igκ、Igλ的阳性表达;RT-PCR检测到细胞内Igα1、Igα2重链、Igκ轻链、Igλ轻链恒定区转录本表达,且其核苷酸序列与美国国立生物技术信息中心(National Center of Biotechnology Information,NCBI)数据库相应的mRNA序列比对,同源性分别达到99%、97%、98%和97%;Western blot检测到细胞内Igα1、Igα2和Igλ蛋白水平的表达,Igκ目前检测为阴性;细胞上清液中可见完整Ig A分子表达;以jacallin为配体亲和层析纯化的培养上清液中的Ig A,蛋白质谱测序所得的氨基酸片段序列与NCBI数据库比对,显示HMCs分泌的蛋白质与B细胞表达的Igα1恒定区52~104之间53个氨基酸、154~221之间68个氨基酸、以及276~327之间52个氨基酸的序列完全相同,与Igα2重链恒定区52~113之间62个氨基酸、151~204之间54个氨基酸、以及251~314之间64个氨基酸的序列完全相同,提示系膜细胞可以将合成的Ig A分泌到胞外。结论:HMCs可能合成并分泌Ig A。
[Abstract]:Objective: to investigate the expression of immunoglobulin A immunoglobulin A (Ig A) in human Mesangial cells (mesangial cells). Methods: human HMCs cell line was cultured and the expression of Ig 伪 Ig 魏 Ig 位 in Mesangial cells was detected by immunofluorescence staining. Reverse transcription polymerase chain reactionation (RT-PCR) and DNA sequencing were used to detect the expression of Ig 伪 transcription 魏 Ig 位 constant region in HMCs, and the expression of Ig 伪 Ig 魏 Ig 位 protein in cell lysate and culture supernatant was detected by Western blot method. Supernatant proteins were purified by affinity chromatography with jacalin as ligand. Results: the positive expression of Ig 伪 + Ig 魏 Ig 位 in the cytoplasm of HMCs cells was detected by immunofluorescence staining. RT-PCR was used to detect the expression of Ig 伪 1, Ig 伪 2 heavy chain and Ig 魏 light chain constant region transcripts of Ig 位 in HMCs cells. The nucleotide sequence was aligned with the corresponding mRNA sequence in the National Center of Biotechnology Information (NCBI) database of the National Biotechnology Information Center, and the homology reached 990.97% and 97% respectively. The expression of Ig 伪 2 and Ig 位 protein in the cells was negative at present. The complete expression of IgA was found in the supernatant, and the sequence of the amino acid fragment was compared with that of the NCBI database, which was purified by affinity chromatography with jacallin as the ligand, and the sequence of the amino acid fragment of the supernatant was compared with that of the NCBI database. The results showed that the protein secreted by HMCs was identical to that of Ig 伪 1 expressed in B cells. The sequence of 53 amino acids between 53 amino acids and 158 amino acids in the constant region of Ig 伪 1, and between 2766 and 327 amino acids, was the same as that in B cells. The sequence of 64 amino acids and 64 amino acids between 62amino acid and 151C204 in the constant region of heavy chain of Ig 伪 2 is identical to that in the region of Ig 伪 2, suggesting that the synthesized IgA can be secreted into extracellular by Mesangial cells. Conclusion\\\;
【作者单位】：北京大学第三医院肾内科;北京大学基础医学院免疫学系;
【分类号】：R446.6

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