本文選題：GL50　切入點：基因轉染　出處：《蘇州大學》2011年碩士論文

【摘要】：GL50(CD275, inducible costimulator ligand)分子是B7家族的新成員,與其特異性受體ICOS(CD278, inducible costimulator)分子結合所提供的共刺激信號在T細胞的活化、增殖、細胞因子的分泌以及B細胞的增殖、分化、抗體產(chǎn)生等方面發(fā)揮著重要的調(diào)節(jié)作用。GL50信號參與了機體的移植排斥反應、抗腫瘤免疫、抗感染免疫、自身免疫性疾病和過敏反應等眾多免疫過程。轉染人GL50基因細胞及鼠抗人GL50單克隆抗體(monoclonal antibody, mAb)的研制能夠為進一步研究GL50在機體免疫應答中的作用以及與其他共刺激信號通路之間的關系提供有力的工具,從而深入探討機體免疫應答的本質,同時也為臨床相關的疾病診斷、治療和預防提供了新的手段。 第一部分轉染人GL50基因細胞株的建立及其對T細胞體外增殖與活化的促進作用 從人B淋巴瘤細胞株Daudi中抽提總RNA,采用RT-PCR的方法,把總RNA逆轉錄成cDNA并大量擴增目的基因。將目的基因插入pIRES2-EGFP載體中并測序,通過脂質體轉染技術將重組載體pIRES2-EGFP-GL50轉染L929細胞。用G418篩選并通過流式細胞術與RT-PCR的方法反復鑒定,最終獲得了穩(wěn)定高表達GL50分子的L929/GL50基因轉染細胞。功能分析表明L929/GL50基因轉染細胞能夠促進T細胞增殖,并可以顯著地促進T細胞分泌細胞因子IL-10、IL-4和IL-17,而不影響IL-2的分泌。 第二部分功能性鼠抗人GL50單克隆抗體的研制 以L929/GL50為免疫原,免疫Balb/c小鼠,采用經(jīng)典雜交瘤技術,將免疫后鼠的脾臟細胞與小鼠骨髓瘤細胞株SP2/0進行融合并經(jīng)HAT培養(yǎng)基選擇培養(yǎng)。以L929/GL50細胞作為陽性篩選細胞株,經(jīng)流式細胞術分析,對抗體分泌陽性孔內(nèi)細胞反復篩選并經(jīng)多次的克隆化培養(yǎng),最終獲得2株穩(wěn)定、持續(xù)分泌鼠抗人GL50單克隆抗體的雜交瘤細胞株,分別命名為2B4、4D11。這兩株雜交瘤細胞株經(jīng)體外連續(xù)傳代(50代)培養(yǎng),在液氮凍存半年后復蘇,仍然生長狀態(tài)良好,分泌抗體性能穩(wěn)定。 采用本科室建立的腹水誘生的方法制備單克隆抗體,腹水的平均產(chǎn)量為3~4ml/只小鼠。經(jīng)Protein G親和層析柱分離純化,兩株單克隆抗體純化后蛋白濃度在3~4mg/ml之間,間接免疫熒光標記法分析表明,其效價在1:5000以上,用于間接免疫熒光分析時抗體蛋白的用量為0.2～2μg/1×106細胞。單克隆抗體核型分析的結果顯示,兩株雜交瘤2B4與4D11的染色體數(shù)目都超過小鼠B細胞與SP2/0的染色體數(shù)目,約為100條左右,表明這兩株雜交瘤2B4與4D11為融合體。 使用快速定性試紙條鑒定,單克隆抗體2B4與4D11的重鏈分別為IgG1、IgG2a,兩者輕鏈同為κ鏈。流式細胞術,Dot-blot與酶聯(lián)吸附的方法分析表明,單克隆抗體2B4與4D11均能夠與人GL50分子特異性結合�？贵w的抗原表位競爭抑制實驗結果表明,單克隆抗體2B4與4D11識別相同的抗原表位,與商品化抗體MIH12識別的抗原表位不同。 經(jīng)單抗2B4與4D11鑒定表明,人GL50分子高表達于B淋巴瘤細胞系中的Daudi和Raji,卵巢癌細胞株HO8910,單核細胞來源細胞株THP-1,血管內(nèi)皮細胞株ECV等。但是,健康人外周血中的B淋巴細胞上基本無GL50分子的表達。功能分析表明,特異性GL50mAb 2B4和4D11均可阻斷GL50與ICOS結合從而抑制T細胞在體外的增殖。
[Abstract]:GL50 (CD275 inducible, costimulator ligand) is a new member of the B7 family, and its specific receptor ICOS (CD278 inducible costimulator) combined with molecular costimulatory signal in the activation of T cell proliferation, provided, cytokine secretion and B cell proliferation, differentiation, antibody generation plays an important regulatory role of.GL50 signaling in the body of the transplant rejection, anti tumor immunity, infection, autoimmune diseases and allergic reactions and other immune process. GL50 transfected cells and mouse anti human GL50 monoclonal antibody (monoclonal antibody, mAb) for us to further study on the immune response in GL50 the role of other costimulatory signaling pathway provides a powerful tool, so as to explore the nature of the immune response, but also for the clinical diagnosis of diseases related to the treatment. New means of treatment and prevention are provided.
The first part of the transfected human GL50 gene cell line and its promoting effect on the proliferation and activation of T cells in vitro
From human B lymphoma cell line Daudi total RNA was extracted by RT-PCR method, the total RNA reverse transcription into cDNA and amplification of the target gene. The target gene was inserted into pIRES2-EGFP vector and sequenced, the recombinant vector pIRES2-EGFP-GL50 was transfected into L929 cells by liposome transfection technique. Screened by G418 and by FCM and RT-PCR. The repeated identification, finally obtained stable high expression of GL50 L929/GL50 gene transfected cells. Functional analysis showed that L929/GL50 gene transfection can promote the proliferation of T cells and T cells can significantly promote the secretion of cytokines IL-10, IL-4 and IL-17, and does not affect the secretion of IL-2.
Development of the second part of functional mouse anti human GL50 monoclonal antibody
Using L929/GL50 as immunogen to immunize Balb/c mice, using classical hybridoma technique, the immune rats spleen cells and mouse myeloma cell line SP2/0 were fused and the HAT selective culture medium. L929/GL50 cells were used as positive screening cell lines by flow cytometry analysis of antibody secreting positive cell repeatedly screening and cloning by repeated training, finally obtained 2 strains of hybridoma cell lines stably, continuous secretion of mouse anti human GL50 monoclonal antibody, named 2B4,4D11. two hybridoma cell lines were passaged (50 generation) cultured, cryopreserved in liquid nitrogen after half a year, still good growth state, secretion antibody stability.
Methods by using an established ascites induced by monoclonal antibody, the average yield of ascites were 3~4ml/ mice. By Protein G affinity chromatography column separation and purification, two strains of monoclonal antibody purified protein concentration between 3~4mg/ml, indirect immunofluorescence analysis showed that, the titer was more than 1:5000, used for indirect immunofluorescence analysis when the antibody concentration is 0.2 ~ 2 g/1 * 106 cells. The results showed that the karyotype analysis of monoclonal antibodies, chromosome number and chromosome number of two hybridoma cell lines 2B4 and 4D11 are more than B cells in mice with SP2/0, about 100 or so, that these two strains of hybridoma 2B4 and 4D11 fusion.
The use of rapid qualitative test strip identification, heavy chain of monoclonal antibody 2B4 and 4D11 were IgG1 and IgG2a, both with light chain kappa chain. Flow cytometry, Dot-blot method and enzyme-linked immunosorbent analysis showed that monoclonal antibodies 2B4 and 4D11 were capable of binding with specific GL50 molecular epitopes. Competitive inhibition experiment showed that 2B4 and 4D11 identify the same monoclonal antibody epitopes, and commercialization of MIH12 antibodies recognize different epitopes.
The monoclonal antibody 2B4 and 4D11 identification showed that GL50 molecule is highly expressed in B lymphoma cell lines Daudi and Raji ovarian cancer cell lines HO8910, monocyte derived cell lines THP-1 and ECV in vascular endothelial cells. However, healthy human peripheral blood B lymphocytes in the fundamental expression of GL50 molecules function analysis showed that the specific GL50mAb 2B4 and 4D11 inhibited GL50 binding with ICOS to inhibit the proliferation of T cells in vitro.

【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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