本文選題：AOPP + 單克隆抗體　； 參考：《南方醫(yī)科大學》2011年碩士論文

【摘要】：晚期氧化蛋白產(chǎn)物(Advanced oxidation protein products, AOPP)是體內氧化應激過程中生成的一類含雙酪氨酸的蛋白質交聯(lián)物,血漿AOPP以次氯酸(HOCl)氧化修飾的白蛋白為主。循環(huán)AOPP水平增高最先在慢性腎功能衰竭、腹膜透析的患者中被發(fā)現(xiàn),隨后在糖尿病、肥胖或代謝綜合征、動脈粥樣硬化等多種常見疾病中被證實。AOPP增高還見于類風濕性關節(jié)炎、潰瘍性結腸炎等免疫炎癥性疾病和惡性腫瘤(結腸、直腸癌、乳腺癌等),因此AOPP被認為是體內氧化應激的敏感生物學標志。 近年不斷增加的研究證據(jù)表明,血漿AOPP水平增高促使該分子在腎臟和血管等組織中沉積并與慢性腎臟病(chronic kidney disease, CKD)及動脈粥樣硬化病變的發(fā)生發(fā)展密切相關。慢性AOPP在體內堆積促進糖尿病動物模型的腎臟的炎癥反應和腎組織損傷,促使CKD動物模型的腎臟炎癥和纖維化,并促使高脂血癥動物發(fā)生動脈粥樣硬化性病變。這些研究結果均表明,AOPP潴留不僅是反映體內氧化應激的指標,其本身可能是一類促進組織炎癥和纖維化的生物致病分子。深入探討AOPP在組織中的分布及其與病變的關系對于闡明AOPP致病的分子基礎至關重要,并有可能為上述疾病的發(fā)生發(fā)展提供新的生物學標志或干預靶標。 然而,目前檢測組織中AOPP水平尚缺乏理想方法。檢查外周血AOPP則通常采用比色分析法以340nm處吸光度值以反映標本中AOPP水平,這是基于AOPP結構含雙酪氨酸,在酸性條件下于340nm處有一特異吸收峰。此法雖然簡便,但易受血脂、纖維蛋白的干擾而影響其準確性；且不能用于對病變組織或細胞中的AOPP定位。 我們擬建立免疫學檢查方法克服上述缺點。采用體外次氯酸修飾的白蛋白作為抗原,成功制備了特異性識別AOPP的單克隆抗體,通過Western Blot、免疫組織化學染色首次證實單抗識別血清和沉積于腎臟中的AOPP,初步建立雙單抗、單多抗及多單抗夾心ELISA法鑒定人工制備的AOPP,為深入研究AOPP這種內源性致病分子提供了必要工具。此外,我們利用噬菌體肽庫篩選技術對AOPP模擬表位進行了初步探討。第一章特異性識別天然AOPP單克隆抗體制備和鑒定 以次氯酸(HOCl)氧化不同種屬血白蛋白(包括人血清白蛋白HSA、小鼠血清白蛋白MSA、牛血清白蛋白BSA、兔血清白蛋白RSA)和人纖維蛋白原、人IgG、人低密度脂蛋白(LDL),按蛋白：HOCl摩爾比為1：140等體積混合,室溫放置30分鐘。制備的AOPP在PBS中透析24小時,除去游離的HOCl。AOPP含量通過測定酸性條件下340nm的吸光度,以氯胺T為標準測得。用AOPP-MSA為免疫原加佐劑免疫BALB/c小鼠,取小鼠脾細胞與骨髓瘤細胞(NS-1)融合,按常規(guī)方法制備雜交瘤。以AOPP-HSA為抗原包被酶標板,并以未氧化正常HAS做平行篩選以排除與正常蛋白的交叉反應。間接ELISA鑒定篩選特異性識別AOPP-HSA的雜交瘤克隆,所獲兩株命名為3F2和4C5；擴大培養(yǎng)后制備腹水,用辛酸硫酸銨沉淀法、HItrap Protein G親和層析柱從腹水中純化抗體�？贵w類別鑒定顯示3F2為IgG1,4C5為IgG2a。 間接ELISA結果顯示2株單抗均能與不同種屬來源的氧化型白蛋白(AOPP-HSA、AOPP-BSA、AOPP-MSA、AOPP-RSA)、HOCl-人纖維蛋白原、HOCl-人IgG、HOCl-LDL特異性結合,而與未氧化蛋白及Cu2+氧化的LDL、糖基化蛋白均無交叉反應。競爭ELISA鑒定表明這兩株單抗分別識別AOPP不同位點。Western Blot結果顯示,在變性與非變性條件下,3F2可特異性結合人工制備的AOPP-HSA、HOCl-IgG、HOCl-人纖維蛋白原。 單抗3F2亦可識別天然血漿與組織中的AOPP。能在一定程度上區(qū)分血液透析患者和正常人血漿中AOPP含量的差別以及慢性腎衰患者透析前后血漿AOPP含量的不同。免疫組織化學法檢查顯示3F2可特異性結合沉積于大鼠模型及各種慢性腎臟病患者腎組織中的AOPP,這種結合可被外源AOPP阻斷。此外,3F2可阻斷AOPP誘導小鼠單核細胞株RAW264.7細胞胞內氧化反應簇(ROS)生成。 第二章建立雙抗體夾心ELISA檢測血漿AOPP的嘗試 以HItrap Protein G柱純化抗體3F2和4C5,純度達95%以上并按照操作流程標記生物素。分別以雙單抗、多單抗夾心、單多抗夾心ELISA方法檢測人工制備的AOPP并進行了體系的優(yōu)化。在抗體包被濃度為1μg/ml,封閉液為0.25%酪蛋白,洗液與抗體稀釋液均為0.5%PBST,檢測抗體濃度為0.5μg/ml時即可良好檢測標準品。之后,我們又試用了競爭ELISA的方法,以人工制備的AOPP-HSA 1.25μg/ml包被酶標板,倍比稀釋AOPP-HSA (0-4μg/ml)、慢性腎衰患者、健康成人血清與0.5μg/ml Bio-3F2等體積混勻后加入板孔中,最后加入HRP-親和素。標準曲線的擬合度良好,而且各種抗體組合的雙夾心ELISA均可特應性地檢出人工體外制備的AOPP,且敏感度可達ng/ml水平但是腎衰患者與正常人的測定值之間沒有統(tǒng)計學意義。因此又嘗試用敏感性更強的時間分辨熒光檢測法檢測患者血清AOPP,按照普通夾心ELISA的條件摸索,即以4C51μg/ml包被酶標板,酪蛋白封閉后,加入不同濃度的標準品AOPP-HSA,反應30min后加入Bio-3F2 0.5μg/ml,30min后加入銪標記的親和素,15min后加入增強液,37℃反應5min后多功能酶標儀測值。所建立的標準曲線已經(jīng)符合臨床檢測的需要,但是在血清樣本的檢測上面,還是存在一定的問題,最佳的包被與檢測條件還要改變。 第三章AOPP表位模擬序列的篩選 鑒于已確定的自然和人工制備的AOPP均為在體內外次氯酸氧化的白蛋白、脂蛋白或其他蛋白,即已形成非天然抗原表位。本章工作的目的旨在探索體內是否有自然存在的類似于AOPP表位的序列,如果存在,將會對機體產(chǎn)生何種影響。 我們以親和層析純化的抗AOPP多克隆抗體為靶標,對噬菌體隨機十二肽庫進行了三輪篩選后,鑒定出18個陽性克隆,均可與多抗產(chǎn)生較強的結合,且不為吸板克隆。挑選6個陽性克隆經(jīng)DNA測序得到三條序列：GWLEENLFDHTR、SSALNDMLRDQR、DSLQDMLADEWQ。上述序列中具有近似的LXDMLXD (X為任意氨基酸)核心序列,提示這一核心序列有可能是模擬AOPP共同表位的優(yōu)勢序列。其次又以抗AOPP單克隆抗體3F2為靶標,篩選噬菌體十二肽庫,經(jīng)三輪篩選、ELISA鑒定獲得18個與3F2特異結合的噬菌體克隆,但這些陽性克隆與抗AOPP多抗和4C5單抗無交叉反應。DNA測序得到SSYSQDAATLRL、LNTPYRNQLAYP、LPYFDSYDSALP AHPMPMTQLFTS四條序列中未發(fā)現(xiàn)保守序列。最后,在以單抗4C5為靶分子篩選噬菌體十二肽庫,經(jīng)ELISA鑒定得到的12個克隆與4C5特異結合,其中一個克隆(No.2)與多抗及單抗3F2較強結合。DNA測序得到的三種序列,LSPLPHPLTRTV、QNLPEWLALHLD、RASPDLLEHQLM,但未發(fā)現(xiàn)保守序列。采用NCBI數(shù)據(jù)庫(http://www.ncbi.nlm.nih.gov/blast)的Blastp程序在蛋白質水平上對篩選的所有序列分別進行同源性搜索,其結果顯示這些序列存在于某些真菌屬、藻類的蛋白分子上,而并不存在于正常的人類與哺乳動物蛋白分子中。即模擬AOPP抗原表位的序列并不存在于已知的正常人與哺乳動物蛋白分子中,其意義在于避免了與AOPP表位相似而導致的后續(xù)事件,維持了機體內環(huán)境的穩(wěn)定；也由此支持AOPP并非人體與動物的正常結構。
[Abstract]:The advanced oxidation protein product (Advanced oxidation protein products, AOPP) is a class of protein crosslinked with dityrosine produced in the process of oxidative stress in the body, and the plasma AOPP is dominated by hypochloric acid (HOCl) oxidized albumin in plasma. The increased level of circulating AOPP is the first in patients with chronic renal failure and peritoneal dialysis, which is found in patients with peritoneal dialysis. In many common diseases, such as diabetes, obesity or metabolic syndrome, atherosclerosis, and other common diseases, the increase of.AOPP is also found in rheumatoid arthritis, ulcerative colitis and other immune inflammatory diseases and malignant tumors (colon, rectal cancer, breast cancer, etc.), so AOPP is recognized as a sensitive biological marker of oxidative stress in the body.
Increasing research evidence in recent years suggests that the increase in plasma AOPP levels causes the molecules to be deposited in the kidneys and blood vessels and is closely related to the development of chronic kidney disease (chronic kidney disease, CKD) and atherosclerotic lesions. Chronic AOPP is accumulated in the kidney of the diabetic animal model by accumulation of chronic AOPP in the body. Renal tissue injury leads to renal inflammation and fibrosis in the CKD animal model and causes atherosclerotic lesions in hyperlipidemia animals. These results show that AOPP retention is not only an indicator of oxidative stress in the body, but also may be a kind of biological pathogeny to promote tissue inflammation and fibrosis. The deep study of AOPP The distribution in the tissue and its relationship with the lesions are essential to elucidate the molecular basis of AOPP's pathogenicity, and may provide new biological markers or intervention targets for the development of the above diseases.
However, there is no ideal method for detecting the level of AOPP in the tissue at present. The detection of AOPP in peripheral blood is usually used to reflect the AOPP level at 340nm by colorimetric analysis. This is based on the AOPP structure containing double tyrosine and a specific absorption peak at 340nm under the acidic condition. This method is simple but easy to be affected by blood lipids and fibrin Interference can affect its accuracy, and can not be used to locate AOPP in pathological tissues or cells.
We intend to establish an immunological method to overcome these shortcomings. The monoclonal antibodies specific to AOPP are successfully prepared by using the albumin modified with hypochloric acid as antigen. Through the Western Blot, the immuno histochemical staining for the first time for the identification of the serum and the AOPP in the kidney of the kidney is first confirmed by immunohistochemical staining. The identification of artificial AOPP by the sandwich ELISA method of monoclonal antibody has provided a necessary tool for the in-depth study of AOPP endogenous pathogenic molecules. In addition, we used phage peptide library screening technique to investigate the AOPP mimic epitopes. Chapter 1 specifically identified the preparation and identification of the natural AOPP monoclonal antibody.
Hypochloric acid (HOCl) was used to oxidize different species of blood albumin (including human serum albumin HSA, mouse serum albumin MSA, bovine serum albumin BSA, rabbit serum albumin RSA) and Human Fibrinogen, human IgG, human low density lipoprotein (LDL), mixed in volume of protein: HOCl mole ratio of 1:140, at room temperature for 30 minutes. The prepared AOPP was permeable in PBS. After 24 hours, the free HOCl.AOPP content was removed by the determination of the absorbance of 340nm under acid condition. The standard of chloramine T was measured with the standard of chloramine T. AOPP-MSA was used as immunogen to immunization BALB/c mice, and the spleen cells of mice were fused with myeloma cells (NS-1), and the hybridoma was prepared by the conventional method. HAS was used in parallel screening to eliminate cross reaction with normal proteins. Indirect ELISA identification and screening of hybridoma clones specifically identified AOPP-HSA, two named 3F2 and 4C5; expanded culture to prepare ascites, ammonium sulfate precipitation, HItrap Protein G affinity chromatography column to purify antibodies from ascites. Antibody category identification showed 3F2 IgG1,4C5 is IgG2a.
The results of indirect ELISA showed that 2 mAbs could be combined with AOPP-HSA, AOPP-BSA, AOPP-MSA, AOPP-RSA, HOCl- Human Fibrinogen, HOCl- human IgG, HOCl-LDL specificity, but no cross reaction with the oxidized protein and Cu2+ oxidation LDL, and glycosylated proteins. Competitive ELISA identification showed that these two monoclonal antibodies were respectively The identification of.Western Blot at different sites of AOPP showed that under denaturing and non denaturing conditions, 3F2 could be specifically combined with artificial AOPP-HSA, HOCl-IgG, and HOCl- Human Fibrinogen.
The monoclonal antibody 3F2 can also identify the difference in the content of AOPP in the plasma and the normal human plasma in a certain degree between the natural plasma and the tissue AOPP., and the difference in the plasma AOPP content before and after the dialysis in the patients with chronic renal failure. The immuno histochemical examination shows that 3F2 can be specifically deposited in the rat model and various chronic renal diseases. AOPP in the renal tissue of the patient can be blocked by exogenous AOPP. In addition, 3F2 can block the formation of the intracellular oxidation reaction cluster (ROS) in the RAW264.7 cells of the mononuclear cell line of mice induced by AOPP.
The second chapter is to establish a double antibody sandwich ELISA for the detection of plasma AOPP.
The purified antibody 3F2 and 4C5 were purified with HItrap Protein G column. The purity of the antibody was over 95% and the biotin was marked in accordance with the operation process. The artificial AOPP was detected by double monoclonal antibody, multi monoclonal antibody sandwich and single multi antibody sandwich ELISA. The antibody package was concentrated in 1 u g/ml, the sealing solution was 0.25% casein, and the lotion and antibody diluent were all For 0.5%PBST, the standard samples were detected when the antibody concentration was 0.5 u g/ml. After that, we tried to try the competitive ELISA method. The artificial AOPP-HSA 1.25 mu g/ml package was mixed with the enzyme standard plate, the double dilution AOPP-HSA (0-4 u g/ml), the chronic renal failure patients, the healthy adult blood clear and 0.5 u g/ml Bio-3F2. After adding HRP- avidin, the standard curve is well fitted, and the double sandwich ELISA of various antibody combinations can detect the AOPP prepared in vitro, and the sensitivity can reach ng/ml level, but there is no statistical significance between the renal failure patients and the normal people. Test the patient serum AOPP, in accordance with the condition of ordinary sandwich ELISA, that is to pack the enzyme label with 4C51 mu g/ml, after the casein is closed, adding different concentration of standard AOPP-HSA, 30min after 30min adding Bio-3F2 0.5 u g/ml, 30min after adding europium avidin, 15min after adding the enhancement solution, and the reaction 5min after 37 degrees. The standard curve set up is in line with the needs of clinical testing, but there are still some problems in the detection of serum samples, and the best package is changed with the testing conditions.
Third chapter selection of AOPP epitope simulation sequence
Given that both naturally and artificially prepared AOPP are albumin, lipoprotein, or other proteins that are oxidized by hypochloric acid in vivo and in vivo, that is, the epitopes of non natural antigens have been formed. The purpose of this chapter is to explore whether there is a natural existence of a sequence similar to the AOPP epitope in the body, and what effect it will have on the body if it exists.
The anti AOPP polyclonal antibody purified by affinity chromatography was used as a target. After three rounds of screening of the phage random twelve peptide library, 18 positive clones were identified, which could produce a strong combination with polyclonal and not to be cloned. 6 positive clones were selected and three sequences were sequenced by DNA sequencing: GWLEENLFDHTR, SSALNDMLRDQR, DSLQDMLADEWQ. The above sequence has an approximate core sequence of LXDMLXD (X as any amino acid), suggesting that this core sequence may be the dominant sequence to simulate the common epitopes of AOPP. Secondly, the phage twelve peptide library is screened with anti AOPP monoclonal antibody 3F2 as the target, and 18 specific phage clones which are specifically combined with 3F2 have been obtained by three rounds of screening and ELISA identification. These positive clones were sequenced with anti AOPP polyclonal and 4C5 McAb free reaction.DNA sequencing, and no conservative sequences were found in the four sequences of SSYSQDAATLRL, LNTPYRNQLAYP, LPYFDSYDSALP AHPMPMTQLFTS. Finally, the phage twelve peptide library was screened with mAb 4C5 as the target molecule, and 12 clones identified by ELISA were specifically combined with 4C5, one of which was cloned. No.2) three sequences, LSPLPHPLTRTV, QNLPEWLALHLD, RASPDLLEHQLM, were obtained with strong combination of mAb and MAb 3F2, but no conservative sequence was found. The Blastp program using the NCBI database (http://www.ncbi.nlm.nih.gov/blast) was homologous to the selected sequences at the protein level. The results showed that these sequences were homologous to the selected sequences at the protein level. The sequence exists on some fungi, the protein molecules of algae, and not in normal human and mammalian protein molecules. That is, the sequence of analog AOPP epitopes does not exist in known normal and mammalian protein molecules, the significance of which is to avoid subsequent events similar to the AOPP epitopes and maintain the machine. The stability of the body environment also supports that AOPP is not the normal structure of human beings and animals.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392.1

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本文編號：1911406