【作者】 马达；

【导师】 谢维；

【作者基本信息】 东南大学 ， 遗传学， 2005， 硕士

【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。更多还原

【Abstract】 AR is a member of AKR1B group of aldoketo reductases. AR is thought to be involved in diabetic complications and Atherosclerosis. ARL-1 is a NADPH-dependent enzyme like AR, which can deoxidize compounds of aldehyde and dextrose. Relevant researches suggest that AR and ARL-1 possibly relate to liver cancers. To study the relation between AR, ARL-1 and hepatoma, the monoclonal antibodies(mAb) against aldose reductase(AR)protein exclusive aldose reductase-like protein (ARL-1) were prepared.In this study, RNA was extracted from human placenta. The AR gene was gained by RT-PCR and was inserted into the E.coli expression vector pGEX-4T-1 (His) 6C. Recombinant AR proteins named GST-AR were expressed. Because the amino acid sequence of ARL-1 was 71% homogenous to that of AR, to select the mAb against AR better, the parts of AR (80～142 aa), which are different from ARL-1, were expressed by the same methods. And it was designated as GST-dAR. GST-AR and GST-dAR were purified by affinity chromatograph. Simultaneously, to analyze the binding sites of the mAb against AR and AR protein, according to the analysis in antigenicity of AR by software (Clustalx、Antheprot), 4 parts of AR were expressed, respectively named GST-dA1(1～79 aa)、GST-dA2(80～99 aa)、GST-dA3(111～142 aa)、GST-dA4 (143～316 aa). AR protein (GST-AR) was used to immunize BALB/c mouse. And mAb was prepared by hybridoma technique. The anti-AR mAb were detected and applied by ELISA and Western blot comparing with anti-ARL-1 mAb. Finally, five clones secreting specific anti-AR mAbs were obtained, named ARB3, ARE6, AR7B3G4, ARF10 and ARH8 respectively. They were all of IgG1, type ofκ. Western blot showed that all the five mAbs bound to GST-AR protein and had no cross-reaction to GST-ARL-1 and GST protein. ARB3 and ARE6 recognized GST-dA1. AR7B3G4 recognized GST-dA3. ARF10 and ARH8 recognized GST-dA4. AR protein was expressed in placenta, fetal tissues including kidney, brain, lung and liver. And higher expression of AR protein or ARL-1 protein in the liver cancer than matched normal tissues adjacent to tumors was detected.All these results suggest that the mAbs be highly specific anti-AR mAbs recognized the 1~79 Amino Acid sites, 111~142 Amino Acid sites, 143~316 Amino Acid sites of AR respectively. AR or ARL-1 protein was possibly overexpressed in liver cancer. The anti-AR mAb, with the anti-ARL-1 mAb protein together, may be a useful tool for further studying the function of AR, the relationship between AR, ARL-1 and HCC and for further investigation of epidemiology.更多还原