人DC-SIGN转基因细胞的构建及鼠抗人DC-SIGN单克隆抗体的研制

发布时间：2018-03-14 03:16

本文选题：DC-SIGN　切入点：DCs　出处：《苏州大学》2009年硕士论文　论文类型：学位论文

【摘要】： 树突状细胞(dendritic cells,DCs)是免疫系统中最主要的抗原递呈细胞(antigen presenting cells,APCs),它具有强大的抗原处理和递呈能力。由于其分布广泛,可以第一时间发现并处理外来抗原并将其递呈至T细胞,诱导免疫应答或者免疫耐受。DCs功能介导与其表面大量的免疫分子密切相关。 DC-SIGN属于II类C型凝集素,由胞内段、跨膜段和胞外段组成,分子量为44kDa。胞外段只有一个糖基识别序列(carbohydrate recognition domain,CRD),识别一些单糖和寡糖。DC-SIGN主要表达于DCs,是单核细胞来源的DCs(monocyte-derived dendritic cells,Mo-DC)表面重要的标记分子。DCs表面的DC-SIGN与抗原结合后可以介导抗原的内吞,并递呈至T淋巴细胞,诱导免疫应答,而HIV-1的包膜糖蛋白gp120则可以利用DC-SIGN逃避免疫监视,从而有利于HIV-1对T细胞的感染,此外,DC-SIGN还介导DCs的转运并参与调节免疫应答。因此构建DC-SIGN转基因细胞及研制鼠抗人DC-SIGN单克隆抗体不仅在理论研究中具有重要的意义而且在临床应用中具有重要的价值。本论文分为两个部分: 1.人DC-SIGN基因克隆及其转基因细胞的构建从人外周血Mo-DC中抽提总RNA,采用RT-PCR的方法,把总RNA中的mRNA逆转录成cDNA并大量扩增目的基因。将目的基因装入pGEZ-Term载体中并测序,通过脂质体转染技术将测序正确的重组逆转录病毒载体pGEZ-Term/DC-SIGN与两个辅助病毒载体共转染包装细胞293T,用其培养上清感染L929细胞,72h后,用Zeocin筛选并最终获得了稳定表达DC-SIGN分子的L929基因转染细胞。 2.鼠抗人DC-SIGN单克隆抗体的研制及其鉴定以DC-SIGN转基因细胞株L929/DC-SIGN为免疫原,免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术,将免疫后小鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合,经HAT选择培养,以L929/DC-SIGN细胞株作为阳性筛选细胞株,以转pGEZ-Term的L929/mock细胞作为阴性对照细胞株,经免疫荧光标记分析,对抗体分泌阳性孔内细胞的反复筛选并经多次的克隆化培养,最终获得1株持续、稳定分泌鼠抗人DC-SIGN单克隆抗体的杂交瘤细胞株,命名为4G8。杂交瘤细胞株经体外连续传代(40代)培养,液氮冻存半年后复苏,仍生长良好,稳定分泌抗体。采用本室建立的腹水诱生方法生产单克隆抗体,腹水的产量平均为3.5ml/只小鼠。经Protein G亲和层析柱分离纯化抗体,单克隆抗体纯化后蛋白含量在4mg/ml之上,免疫荧光法分析表明,其效价在1:1000以上,抗体蛋白用于间接免疫荧光分析的用量为0.2～2μg/1×106细胞。核型分析结果显示,杂交瘤4G8的染色体数目超过小鼠体细胞数目,表明杂交瘤4G8为融合体。经快速定性试纸条鉴定,单克隆抗体4G8的重链为IgG1,轻链为κ链。Western blot及流式细胞分析均显示,单克隆抗体4G8能与DC-SIGN分子特异性结合。竞争抑制实验结果表明,单克隆抗体4G8与商品化抗体E021819识别不同的抗原表位。流式细胞分析显示,DC-SIGN分子特异性高表达于Mo-DC,并且随着Mo-DC的成熟表达水平有一定降低;外周血来源的单核细胞、B淋巴细胞、T淋巴细胞均不表达DC-SIGN分子;人B淋巴瘤细胞株Daudi及Raji、人T淋巴瘤细胞株Jurkat、人白血病细胞株K562、肺癌细胞株A549及H1299、人单核来源的THP-1、U937等肿瘤细胞株也均不表达DC-SIGN分子。流式细胞技术分析DC-SIGN分子在单核来源的肿瘤细胞株THP-1、U937上的诱导表达情况,结果显示,经PMA和IL-4联合刺激后THP-1细胞株上有DC-SIGN分子的表达,并且在刺激48h后达到了较高水平,刺激72h后表达水平又有所降低;而U937细胞株上始终未检测到DC-SIGN分子的表达。证实了THP-1细胞株可以作为单核细胞向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.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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