發(fā)布時間：2018-03-14 03:16

  本文選題：DC-SIGN　切入點：DCs　出處：《蘇州大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 樹突狀細(xì)胞(dendritic cells,DCs)是免疫系統(tǒng)中最主要的抗原遞呈細(xì)胞(antigen presenting cells,APCs),它具有強大的抗原處理和遞呈能力。由于其分布廣泛,可以第一時間發(fā)現(xiàn)并處理外來抗原并將其遞呈至T細(xì)胞,誘導(dǎo)免疫應(yīng)答或者免疫耐受。DCs功能介導(dǎo)與其表面大量的免疫分子密切相關(guān)。 DC-SIGN屬于II類C型凝集素,由胞內(nèi)段、跨膜段和胞外段組成,分子量為44kDa。胞外段只有一個糖基識別序列(carbohydrate recognition domain,CRD),識別一些單糖和寡糖。DC-SIGN主要表達(dá)于DCs,是單核細(xì)胞來源的DCs(monocyte-derived dendritic cells,Mo-DC)表面重要的標(biāo)記分子。DCs表面的DC-SIGN與抗原結(jié)合后可以介導(dǎo)抗原的內(nèi)吞,并遞呈至T淋巴細(xì)胞,誘導(dǎo)免疫應(yīng)答,而HIV-1的包膜糖蛋白gp120則可以利用DC-SIGN逃避免疫監(jiān)視,從而有利于HIV-1對T細(xì)胞的感染,此外,DC-SIGN還介導(dǎo)DCs的轉(zhuǎn)運并參與調(diào)節(jié)免疫應(yīng)答。因此構(gòu)建DC-SIGN轉(zhuǎn)基因細(xì)胞及研制鼠抗人DC-SIGN單克隆抗體不僅在理論研究中具有重要的意義而且在臨床應(yīng)用中具有重要的價值。 本論文分為兩個部分: 1.人DC-SIGN基因克隆及其轉(zhuǎn)基因細(xì)胞的構(gòu)建 從人外周血Mo-DC中抽提總RNA,采用RT-PCR的方法,把總RNA中的mRNA逆轉(zhuǎn)錄成cDNA并大量擴增目的基因。將目的基因裝入pGEZ-Term載體中并測序,通過脂質(zhì)體轉(zhuǎn)染技術(shù)將測序正確的重組逆轉(zhuǎn)錄病毒載體pGEZ-Term/DC-SIGN與兩個輔助病毒載體共轉(zhuǎn)染包裝細(xì)胞293T,用其培養(yǎng)上清感染L929細(xì)胞,72h后,用Zeocin篩選并最終獲得了穩(wěn)定表達(dá)DC-SIGN分子的L929基因轉(zhuǎn)染細(xì)胞。 2.鼠抗人DC-SIGN單克隆抗體的研制及其鑒定 以DC-SIGN轉(zhuǎn)基因細(xì)胞株L929/DC-SIGN為免疫原,免疫BALB/c小鼠,采用B淋巴細(xì)胞雜交瘤技術(shù),將免疫后小鼠的脾臟細(xì)胞與小鼠骨髓瘤細(xì)胞SP2/0進行細(xì)胞融合,經(jīng)HAT選擇培養(yǎng),以L929/DC-SIGN細(xì)胞株作為陽性篩選細(xì)胞株,以轉(zhuǎn)pGEZ-Term的L929/mock細(xì)胞作為陰性對照細(xì)胞株,經(jīng)免疫熒光標(biāo)記分析,對抗體分泌陽性孔內(nèi)細(xì)胞的反復(fù)篩選并經(jīng)多次的克隆化培養(yǎng),最終獲得1株持續(xù)、穩(wěn)定分泌鼠抗人DC-SIGN單克隆抗體的雜交瘤細(xì)胞株,命名為4G8。雜交瘤細(xì)胞株經(jīng)體外連續(xù)傳代(40代)培養(yǎng),液氮凍存半年后復(fù)蘇,仍生長良好,穩(wěn)定分泌抗體。 采用本室建立的腹水誘生方法生產(chǎn)單克隆抗體,腹水的產(chǎn)量平均為3.5ml/只小鼠。經(jīng)Protein G親和層析柱分離純化抗體,單克隆抗體純化后蛋白含量在4mg/ml之上,免疫熒光法分析表明,其效價在1:1000以上,抗體蛋白用于間接免疫熒光分析的用量為0.2～2μg/1×106細(xì)胞。核型分析結(jié)果顯示,雜交瘤4G8的染色體數(shù)目超過小鼠體細(xì)胞數(shù)目,表明雜交瘤4G8為融合體。 經(jīng)快速定性試紙條鑒定,單克隆抗體4G8的重鏈為IgG1,輕鏈為κ鏈。Western blot及流式細(xì)胞分析均顯示,單克隆抗體4G8能與DC-SIGN分子特異性結(jié)合。競爭抑制實驗結(jié)果表明,單克隆抗體4G8與商品化抗體E021819識別不同的抗原表位。流式細(xì)胞分析顯示,DC-SIGN分子特異性高表達(dá)于Mo-DC,并且隨著Mo-DC的成熟表達(dá)水平有一定降低;外周血來源的單核細(xì)胞、B淋巴細(xì)胞、T淋巴細(xì)胞均不表達(dá)DC-SIGN分子;人B淋巴瘤細(xì)胞株Daudi及Raji、人T淋巴瘤細(xì)胞株Jurkat、人白血病細(xì)胞株K562、肺癌細(xì)胞株A549及H1299、人單核來源的THP-1、U937等腫瘤細(xì)胞株也均不表達(dá)DC-SIGN分子。 流式細(xì)胞技術(shù)分析DC-SIGN分子在單核來源的腫瘤細(xì)胞株THP-1、U937上的誘導(dǎo)表達(dá)情況,結(jié)果顯示,經(jīng)PMA和IL-4聯(lián)合刺激后THP-1細(xì)胞株上有DC-SIGN分子的表達(dá),并且在刺激48h后達(dá)到了較高水平,刺激72h后表達(dá)水平又有所降低;而U937細(xì)胞株上始終未檢測到DC-SIGN分子的表達(dá)。證實了THP-1細(xì)胞株可以作為單核細(xì)胞向DCs分化的模型。
[Abstract]:Dendritic cells (dendritic cells DCs) is the main immune system of antigen-presenting cells (antigen presenting cells, APCs), it has powerful antigen processing and presenting ability. Because of its wide distribution, can be the first time to discover and deal with foreign antigens and its presentation to T cells, inducing immune responses or the function of.DCs mediated immune tolerance and surface of immune molecules are closely related.
DC-SIGN belongs to the II class of C type lectin by intracellular domain, transmembrane and extracellular segments, the molecular weight of 44kDa. extracellular domain only a carbohydrate recognition sequence (carbohydrate recognition domain, CRD), the identification of some monosaccharides and oligosaccharides.DC-SIGN mainly expressed in DCs, is monocyte derived DCs (monocyte-derived dendritic cells, Mo-DC) with DC-SIGN and.DCs surface marker antigen important after endocytosis mediated antigen presentation to T cells, and induce immune responses, and HIV-1, the envelope glycoprotein of gp120 DC-SIGN can be used to evade immune surveillance, which is conducive to HIV-1 infection of T cells in DC-SIGN also mediated transport of DCs and involved in the regulation of immune response. So the construction of DC-SIGN transgenic cells and preparation of mouse anti human DC-SIGN monoclonal antibody not only in theoretical research but also has important significance in clinical application. There is an important value.
This paper is divided into two parts:
Cloning of DC-SIGN gene from 1. people and construction of transgenic cells
From human peripheral blood Mo-DC total RNA was extracted by RT-PCR method, the total RNA of cDNA mRNA by reverse transcription and amplification of target gene. The sequencing of target gene into pGEZ-Term vector and transfected by liposome technology, the recombinant retroviral vector pGEZ-Term/DC-SIGN and two helper virus vectors were transfected into packaging the culture supernatant of 293T cells, L929 cells infected with 72h, screened by Zeocin and obtained a stable expression of DC-SIGN protein in L929 transfected cells.
Development and identification of 2. mouse anti human DC-SIGN monoclonal antibodies
The DC-SIGN transgenic cell line L929/DC-SIGN as immunogen, BALB/c mice were immunized with B lymphocyte hybridoma technique, the immunized mice spleen cells and mouse myeloma cell SP2/0 by cell fusion, HAT culture, L929/DC-SIGN cells were used as positive screening cell line, with pGEZ-Term and L929/mock as negative control cell line through the analysis, immunofluorescence, antibody secretion of repeated screening positive cell and cloning of multiple culture, 1 seedlings were obtained by continuous, stable hybridoma cell lines secreting anti human DC-SIGN monoclonal antibody, named 4G8. hybridoma cell lines in vitro Subculture (40 generation) culture, frozen in liquid nitrogen save recovery after half a year, still good growth, stable secretion of antibodies.
The relationship of ascites induced production of monoclonal antibody, ascites production averaged 3.5ml/ mice. After Protein G affinity purified antibody chromatography, purified monoclonal antibody protein content on 4mg/ml, immunofluorescence analysis showed that, the antibody titer was more than 1:1000, used for indirect immunofluorescence analysis was 0.2 ~ 2 g/1 * 106 cells. Karyotype analysis showed that chromosome number of hybridoma 4G8 exceeds the number of mouse somatic cells, showed that the hybridoma 4G8 fusion.
The rapid qualitative test strip identification, heavy chain monoclonal antibody 4G8 IgG1 light chain kappa chain.Western blot and flow cytometry analysis showed that monoclonal antibody 4G8 can bind with DC-SIGN specifically. The competitive inhibition experiment results show that the epitope of monoclonal antibody 4G8 with commercial antibody E021819 recognizing different flow. Cytometry showed high specificity, DC-SIGN molecules expressed in Mo-DC, and with the maturity of Mo-DC expression level decrease; mononuclear cells from peripheral blood B lymphocytes, T lymphocytes were not DC-SIGN expression; human B lymphoma cell lines Daudi and Raji, T lymphoma cell line Jurkat, human leukemia cells strain K562, A549 and H1299 in lung cancer cell lines, human monocyte derived THP-1, U937 tumor cell lines also expressed DC-SIGN molecules.
Analysis of DC-SIGN molecules on the monocyte derived tumor cell line THP-1 by flow cytometry, the expression of U937, induced by PMA and the results show that IL-4 combined with THP-1 cells after stimulation with DC-SIGN expression, and reached a high level after 48h stimulation, stimulation of the 72h expression level and decreased; and U937 cell line has not detected the expression of DC-SIGN was confirmed. THP-1 cell line can be used as monocytes to differentiate into DCs model.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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相關(guān)期刊論文 前2條

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2 ;Expression of dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin on dendritic cells generated from human peripheral blood monocytes[J];World Journal of Gastroenterology;2006年03期

本文編號：1609359