本文選題：醛糖還原酶 + 醛糖還原酶相似蛋白��； 參考：《東南大學(xué)》2005年碩士論文

【摘要】： 醛糖還原酶(aldose reductase, AR)屬于醛—酮還原酶家族,來自不同哺乳動(dòng)物的AR有80%以上的氨基酸序列相同,這種序列的保守性提示這種蛋白質(zhì)可能在細(xì)胞中起著重要的生理作用。AR生理功能還不是很清楚,有文獻(xiàn)報(bào)道AR在糖尿病并發(fā)癥的發(fā)生與發(fā)展、動(dòng)脈粥樣硬化過程中起著重要作用,AR及ARL-1與肝癌的發(fā)生發(fā)展及其抗藥性之間也可能存在某種關(guān)系。AR及ARL-1與人類原發(fā)性肝癌的關(guān)系是近年來研究的一個(gè)新熱點(diǎn)。為了進(jìn)一步研究AR蛋白的功能,以及探索AR與ARL-1在相關(guān)疾病中的作用,本實(shí)驗(yàn)制備了抗AR單克隆抗體(monoclonal antibody, mAb),與我室制備的抗醛糖還原酶相似蛋白(aldose reductase-like protein,ARL-1)mAb的特性進(jìn)行比較,并初步探討其應(yīng)用價(jià)值。 提取正常人胎盤總RNA,經(jīng)RT-PCR獲得AR基因,構(gòu)建重組質(zhì)粒pGEX-4T-1(His)6C -AR,轉(zhuǎn)化入E.coli Rosetta誘導(dǎo)表達(dá)GST-AR蛋白,用純化的GST-AR蛋白免疫BALB/c小鼠,采用雜交瘤技術(shù)制備mAb。由于AR和ARL-1的氨基酸序列71%同源,本實(shí)驗(yàn)用同樣方法誘導(dǎo)表達(dá)AR與ARL-1氨基酸序列差異較大的一段蛋白GST-dAR(第80～142氨基酸),以篩選特異性強(qiáng)的抗AR mAb。并參考相關(guān)軟件(Clustalx、Antheprot)分析AR的抗原性,將AR分為四段,采用同樣基因重組方法誘導(dǎo)表達(dá)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),以分析制備的抗AR mAb與AR抗原的結(jié)合部位。采用間接ELISA法、Western blot免疫印跡試驗(yàn)對(duì)抗AR mAb進(jìn)行篩選和鑒定。利用抗AR mAb和抗ARL-1 mAb,采用Western blot的方法檢測(cè)正常人肝臟組織、肝癌及癌旁組織中AR與ARL-1蛋白的表達(dá)。結(jié)果獲得5株穩(wěn)定分泌抗AR蛋白的mAb的雜交瘤細(xì)胞系,分別命名為ARB3、ARE6、AR7B3G4、ARF10、ARH8。經(jīng)鑒定,該5株抗AR mAb的Ig亞類均為IgG1,輕鏈屬于κ型;均可與胎盤組織中的AR蛋白起反應(yīng),而與GST-ARL-1、GST蛋白無交叉反應(yīng)。用AR全長蛋白和AR五段截短蛋白分別鑒定5株mAb,證實(shí)所獲得的mAb可以針對(duì)AR至少三個(gè)不同的抗原表位,即ARB3和ARE6分泌的mAb識(shí)別AR第1～79位氨基酸,AR7B3G4分泌的mAb識(shí)別AR第111～142位氨基酸,ARF10和ARH8分泌的mAb識(shí)別AR第143～316位氨基酸。Western blot結(jié)果提示:用AR7B3G4分泌的mAb及抗ARL-1 mAb檢測(cè)的11例肝癌組織中,45.5%(5/11)存在AR高表達(dá), 54.5%(6/11)存在ARL-1高表達(dá),且ARL-1高表達(dá)的肝癌組織AR不表達(dá),AR高表達(dá)的肝癌組織ARL-1不表達(dá);在相應(yīng)癌旁組織中AR和ARL-1不表達(dá)或表達(dá)較弱(11例);在正常肝組織(3例)中未檢測(cè)到AR與ARL-1蛋白表達(dá)。以上結(jié)果說明利用該mAb檢測(cè)AR蛋白在肝癌患者中存在高表達(dá)。 本研究表達(dá)了AR全長及五段截短蛋白,成功制備了5株特異性抗AR蛋白的mAb,可以分別識(shí)別至少三個(gè)不同的AR抗原表位。有利于更好地分析AR蛋白的功能;將抗AR mAb與抗ARL-1 mAb的特性進(jìn)行比較,聯(lián)合應(yīng)用抗AR mAb和抗ARL-1 mAb,可能找到一個(gè)新的、特異性高的肝癌早期診斷指標(biāo),將有助于肝癌的早期診斷,從而提高肝癌的生存率,改善其預(yù)后。也為進(jìn)一步研究AR及ARL-1與其他疾病的關(guān)系,以及大規(guī)模流行病學(xué)調(diào)查提供一個(gè)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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R392

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本文編號(hào)：2111564