抗醛糖还原酶（AR）单克隆抗体的制备及初步应用

发布时间：2018-07-10 01:53

本文选题：醛糖还原酶 + 醛糖还原酶相似蛋白　；参考：《东南大学》2005年硕士论文

【摘要】： 醛糖还原酶(aldose reductase, AR)属于醛—酮还原酶家族,来自不同哺乳动物的AR有80%以上的氨基酸序列相同,这种序列的保守性提示这种蛋白质可能在细胞中起着重要的生理作用。AR生理功能还不是很清楚,有文献报道AR在糖尿病并发症的发生与发展、动脉粥样硬化过程中起着重要作用,AR及ARL-1与肝癌的发生发展及其抗药性之间也可能存在某种关系。AR及ARL-1与人类原发性肝癌的关系是近年来研究的一个新热点。为了进一步研究AR蛋白的功能,以及探索AR与ARL-1在相关疾病中的作用,本实验制备了抗AR单克隆抗体(monoclonal antibody, mAb),与我室制备的抗醛糖还原酶相似蛋白(aldose reductase-like protein,ARL-1)mAb的特性进行比较,并初步探讨其应用价值。提取正常人胎盘总RNA,经RT-PCR获得AR基因,构建重组质粒pGEX-4T-1(His)6C -AR,转化入E.coli Rosetta诱导表达GST-AR蛋白,用纯化的GST-AR蛋白免疫BALB/c小鼠,采用杂交瘤技术制备mAb。由于AR和ARL-1的氨基酸序列71%同源,本实验用同样方法诱导表达AR与ARL-1氨基酸序列差异较大的一段蛋白GST-dAR(第80～142氨基酸),以筛选特异性强的抗AR mAb。并参考相关软件(Clustalx、Antheprot)分析AR的抗原性,将AR分为四段,采用同样基因重组方法诱导表达AR四段截短蛋白GST-dA1(第1～79氨基酸)、GST-dA2(第80～99氨基酸)、GST-dA3(第111～142氨基酸)、GST-dA4 (第143～316氨基酸),以分析制备的抗AR mAb与AR抗原的结合部位。采用间接ELISA法、Western blot免疫印迹试验对抗AR mAb进行筛选和鉴定。利用抗AR mAb和抗ARL-1 mAb,采用Western blot的方法检测正常人肝脏组织、肝癌及癌旁组织中AR与ARL-1蛋白的表达。结果获得5株稳定分泌抗AR蛋白的mAb的杂交瘤细胞系,分别命名为ARB3、ARE6、AR7B3G4、ARF10、ARH8。经鉴定,该5株抗AR mAb的Ig亚类均为IgG1,轻链属于κ型;均可与胎盘组织中的AR蛋白起反应,而与GST-ARL-1、GST蛋白无交叉反应。用AR全长蛋白和AR五段截短蛋白分别鉴定5株mAb,证实所获得的mAb可以针对AR至少三个不同的抗原表位,即ARB3和ARE6分泌的mAb识别AR第1～79位氨基酸,AR7B3G4分泌的mAb识别AR第111～142位氨基酸,ARF10和ARH8分泌的mAb识别AR第143～316位氨基酸。Western blot结果提示:用AR7B3G4分泌的mAb及抗ARL-1 mAb检测的11例肝癌组织中,45.5%(5/11)存在AR高表达, 54.5%(6/11)存在ARL-1高表达,且ARL-1高表达的肝癌组织AR不表达,AR高表达的肝癌组织ARL-1不表达;在相应癌旁组织中AR和ARL-1不表达或表达较弱(11例);在正常肝组织(3例)中未检测到AR与ARL-1蛋白表达。以上结果说明利用该mAb检测AR蛋白在肝癌患者中存在高表达。本研究表达了AR全长及五段截短蛋白,成功制备了5株特异性抗AR蛋白的mAb,可以分别识别至少三个不同的AR抗原表位。有利于更好地分析AR蛋白的功能;将抗AR mAb与抗ARL-1 mAb的特性进行比较,联合应用抗AR mAb和抗ARL-1 mAb,可能找到一个新的、特异性高的肝癌早期诊断指标,将有助于肝癌的早期诊断,从而提高肝癌的生存率,改善其预后。也为进一步研究AR及ARL-1与其他疾病的关系,以及大规模流行病学调查提供一个有力工具。
[Abstract]:Aldose reductase (aldose reductase, AR) belongs to the aldehyde - ketone reductase family, the AR from different mammals has more than 80% of the same amino acid sequence. The conservativeness of this sequence suggests that the protein may play an important physiological role in the cell,.AR physiological function is not clear, and there is a literature report that AR is in diabetic complications. The occurrence and development of atherosclerosis play an important role in the process of atherosclerosis. The relationship between AR and ARL-1 with the development of liver cancer and its resistance may also have a relationship between.AR and ARL-1 and human primary liver cancer in recent years. In order to further study the function of AR egg white, and explore the related diseases of AR and ARL-1. In this experiment, the anti AR monoclonal antibody (monoclonal antibody, mAb) was prepared and compared with the properties of aldose reductase-like protein, ARL-1 mAb, which was prepared in my room, and its application value was preliminarily discussed.
The total RNA of normal human placenta was extracted and the AR gene was obtained by RT-PCR. The recombinant plasmid pGEX-4T-1 (His) 6C -AR was constructed and transformed into E.coli Rosetta to induce the expression of GST-AR protein. The purified GST-AR protein was used to immunize BALB/c mice. The 71% homology of the amino acid sequence was prepared by hybridoma technique. GST-dAR (eightieth to 142 amino acids) with a large difference in the sequence of RL-1 amino acids was used to screen specific strong anti AR mAb. and to analyze the antigenicity of AR by reference software (Clustalx, Antheprot). AR was divided into four segments, and the same gene recombination method was used to induce the expression of AR four truncated protein GST-dA1 (first to 79 amino acids), GST-dA2 (eightieth to 99). Amino acids), GST-dA3 (111st to 142 amino acids) and GST-dA4 (143rd to 316 amino acids) were used to analyze the binding sites of anti AR mAb and AR antigens. Indirect ELISA, Western blot immunoblotting test was used to screen and identify AR mAb. The expression of AR and ARL-1 protein in liver and adjacent tissues obtained 5 hybridoma cell lines that secreted the mAb of anti AR protein, named ARB3, ARE6, AR7B3G4, ARF10, and ARH8., the 5 AR mAb Ig subclasses were all kappa type. No cross reaction. 5 strains of mAb were identified with AR full length protein and AR five segment truncated protein respectively. It was proved that the obtained mAb could identify at least three different epitopes of AR, that is, ARB3 and ARE6 secreted mAb to identify AR first to 79 amino acids, AR7B3G4 secreted mAb AR 111st to 142 amino acids, and 143rd to 316 secreted. .Western blot results indicated that 45.5% (5/11) had high expression of AR in 11 cases of liver cancer detected by AR7B3G4 secreted mAb and anti ARL-1 mAb, and 54.5% (6/11) had high expression of ARL-1, and ARL-1 high expression of liver cancer tissues was not expressed. The expression of AR and ARL-1 protein was not detected in normal liver tissue (3 cases). The above results showed that the high expression of AR protein in liver cancer patients was detected by using this mAb.
In this study, the total length of AR and five segments of truncated protein were expressed. 5 mAb specific anti AR proteins were successfully prepared, and at least three different AR epitopes could be identified respectively. It was beneficial to better analyze the function of AR protein; compare the anti AR mAb with the properties of the anti ARL-1 mAb, and the combination of anti AR mAb and ARL-1 anti ARL-1, may find a new one. The early diagnosis of high specific liver cancer will help the early diagnosis of liver cancer, improve the survival rate and improve the prognosis of HCC. It also provides a powerful tool for further study of the relationship between AR and ARL-1 and other diseases, as well as a large-scale epidemiological survey.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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