【摘要】： T細(xì)胞的活化除了需要通過(guò)APC遞呈MHC-抗原肽給抗原特異性T細(xì)胞提供第一信號(hào)外,還需要表達(dá)于免疫細(xì)胞表面一系列共刺激分子提供第二信號(hào)。如果缺少第二信號(hào),將會(huì)導(dǎo)致T細(xì)胞的無(wú)反應(yīng)性或免疫耐受。共刺激分子的協(xié)同加強(qiáng)或負(fù)性調(diào)節(jié)在機(jī)體免疫應(yīng)答的整個(gè)過(guò)程中起著及其重要的調(diào)節(jié)作用。 小鼠B7-H3屬B7超家族新成員。迄今B7-H3的生物學(xué)特性和功能研究尚待深入并存在爭(zhēng)議。目前的研究認(rèn)為,B7-H3在介導(dǎo)T細(xì)胞免疫應(yīng)答中存在2種截然不同的作用:協(xié)同刺激或抑制T細(xì)胞免疫應(yīng)答。此外一些研究提出B7-H3分子可以作為腫瘤如肺癌,前列腺癌以及神經(jīng)母細(xì)胞瘤細(xì)胞的表面標(biāo)記分子具有診斷和干預(yù)靶向價(jià)值。進(jìn)而鑒于B7-H3受體尚未得到最終確認(rèn),這也是B7-H3研究中存在的最大困難。 本研究旨在通過(guò)建立小鼠B7-H3基因轉(zhuǎn)染細(xì)胞株,研制特異性單克隆抗體和融合蛋白,從不同角度探討B(tài)7-H3的生物學(xué)功能。 本論文分為兩個(gè)部分: 一、小鼠B7-H3基因轉(zhuǎn)染細(xì)胞株及其單克隆抗體的研制 1.采用RT-PCR的方法從小鼠骨髓來(lái)源的DC中擴(kuò)增出小鼠B7-H3編碼區(qū)全長(zhǎng)基因。經(jīng)雙酶切后插入真核表達(dá)載體pIRES2-EGFP中,構(gòu)建成重組載體pIRES2-EGFP/B7-H3。通過(guò)脂質(zhì)體法將測(cè)序正確的重組載體pIRES2-EGFP/B7-H3轉(zhuǎn)染293和CHO細(xì)胞,G418加壓篩選并經(jīng)過(guò)數(shù)次亞克隆。流式細(xì)胞術(shù)分析顯示B7-H3基因同時(shí)轉(zhuǎn)染的293和CHO細(xì)胞膜上能穩(wěn)定高表達(dá)小鼠B7-H3分子。為研制小鼠B7-H3單克隆抗體提供了有效的免疫原。2.以轉(zhuǎn)基因細(xì)胞株293/B7-H3為免疫原,免疫SD大鼠,采用B淋巴細(xì)胞雜交瘤技術(shù),將免疫大鼠的脾臟細(xì)胞與小鼠骨髓瘤細(xì)胞SP2/0進(jìn)行細(xì)胞融合,HAT選擇性培養(yǎng)基培養(yǎng)雜交瘤細(xì)胞。以CHO/ B7-H3為陽(yáng)性篩選細(xì)胞,CHO/mock細(xì)胞作陰性對(duì)照,FCM分析篩選陽(yáng)性克隆,經(jīng)過(guò)亞克隆化培養(yǎng),最終獲得2株持續(xù)、穩(wěn)定分泌大鼠抗小鼠B7-H3單克隆抗體的雜交瘤細(xì)胞株(18F9和19F6)。經(jīng)熒光微球法鑒定,兩株單抗重鏈均為IgG2b,輕鏈為κ鏈。Western blot及流式細(xì)胞分析均顯示,兩株單抗都能與B7-H3分子特異性結(jié)合,且兩株單抗識(shí)別不同的抗原表位。體外長(zhǎng)期培養(yǎng)和液氮凍存后,復(fù)蘇的雜交瘤細(xì)胞生長(zhǎng)狀態(tài)良好,穩(wěn)定分泌抗體。研究發(fā)現(xiàn)小鼠B7-H3在B細(xì)胞、單核細(xì)胞和DC細(xì)胞上組成性表達(dá),在靜息和活化的T細(xì)胞、NK細(xì)胞上不表達(dá)。小鼠B7-H3蛋白表達(dá)在膀胱上皮細(xì)胞胞漿和膜上,而在其他正常組織幾乎都不表達(dá)。由此表明,所研制的單克隆抗體能運(yùn)用于流式熒光標(biāo)記,免疫印跡及免疫組化,具有重要的應(yīng)用價(jià)值。 二、小鼠B7-H3-Fc融合蛋白的表達(dá)及其生物學(xué)活性的研究 通過(guò)重疊PCR技術(shù)將小鼠B7-H3胞外段基因和人IgG1重鏈Fc恒定區(qū)基因拼接,按照構(gòu)建B7-H3基因轉(zhuǎn)染細(xì)胞的方法將重組基因轉(zhuǎn)染CHO細(xì)胞,經(jīng)篩選并獲得CHO/mB7-H3-Fc轉(zhuǎn)染細(xì)胞。該轉(zhuǎn)基因細(xì)胞無(wú)血清培養(yǎng)后,收集細(xì)胞上清、超濾濃縮后行經(jīng)Protein G柱純化,獲得純品B7-H3-Fc融合蛋白,經(jīng)Western blot鑒定。通過(guò)CCK-8和ELISA方法檢測(cè)發(fā)現(xiàn)該融合蛋白對(duì)T淋巴細(xì)胞的體外增殖和細(xì)胞因子IL-2、IFN-γ的分泌具有明顯的促進(jìn)作用。在T細(xì)胞體外增殖實(shí)驗(yàn)中,單抗的加入可部分阻斷T細(xì)胞分泌細(xì)胞因子。該融合蛋白識(shí)別T細(xì)胞上的受體,而與構(gòu)建的TLT2基因轉(zhuǎn)染細(xì)胞株不結(jié)合,提示B7-H3的受體可能不是TLT2分子。同時(shí)也表明所研制的單抗具有阻斷功能。 綜上所述,本實(shí)驗(yàn)構(gòu)建了基因轉(zhuǎn)染細(xì)胞株293/B7-H3和CHO/B7-H3;研制2株特異性大鼠抗小鼠B7-H3功能性單克隆抗體和mB7-H3-Fc融合蛋白基因。該融合蛋白能夠促進(jìn)T細(xì)胞體外增殖及分泌IL-2,IFN-γ。T細(xì)胞體外增殖試驗(yàn)顯示,單抗18F9對(duì)B7-H3介導(dǎo)的刺激T細(xì)胞分泌細(xì)胞因子具有阻斷作用。研究也證實(shí)TLT2分子不是小鼠B7-H3的受體。B7-H3作為重要的共刺激分子,在調(diào)節(jié)T細(xì)胞免疫應(yīng)答中發(fā)揮了正性共刺激作用�？傊�,上述結(jié)果為深入研究B7-H3分子在免疫應(yīng)答和腫瘤免疫中的重要作用奠定物質(zhì)基礎(chǔ)。
[Abstract]:In addition to providing the first signal to antigen-specific T cells by APC-presenting MHC-antigen peptides, activation of T cells also requires the expression of a series of costimulatory molecules on the surface of immune cells to provide a second signal. It plays an important regulatory role in the whole process of immune response.
B7-H3 is a new member of the B7 superfamily in mice. Up to now, the biological characteristics and functions of B7-H3 have not been studied thoroughly and controversial. Current studies suggest that B7-H3 has two distinct roles in mediating T-cell immune responses: co-stimulating or inhibiting T-cell immune responses. Surface markers of cancer, prostate cancer, and neuroblastoma cells have diagnostic and interventional targeting values. And since B7-H3 receptors have not yet been identified, this is the biggest difficulty in B7-H3 research.
The aim of this study was to establish a mouse B7-H3 gene transfected cell line and develop specific monoclonal antibodies and fusion proteins to explore the biological functions of B7-H3 from different perspectives.
This thesis is divided into two parts.
First, the mouse B7-H3 gene transfected cell line and the preparation of its monoclonal antibody.
1. The full-length gene of mouse B7-H3 coding region was amplified from mouse bone marrow-derived DC by RT-PCR. The recombinant vector pIRES2-EGFP/B7-H3 was constructed by inserting into eukaryotic expression vector pIRES2-EGFP after double enzyme digestion. The recombinant vector pIRES2-EGFP/B7-H3 with correct sequence was transfected into 293 and CHO cells by liposome method. The recombinant vector pIRES2-EGFP/B7-H3 was screened by G418 and pressurized. Flow cytometry analysis showed that B7-H3 gene transfected 293 and CHO cell membrane could stably and highly express mouse B7-H3 molecule. It provided an effective immunogen for the preparation of mouse B7-H3 monoclonal antibody. 2. SD rats were immunized with transgenic cell line 293/B7-H3 as immunogen, and the rats were immunized with B lymphocyte hybridoma technique. The spleen cells were fused with mouse myeloma cells SP2/0 and hybridoma cells were cultured in HAT selective medium.
B7-H3 was positive screening cells, CHO/mock cells were negative control, FCM analysis was used to screen positive clones. After subcloning culture, two hybridoma cell lines (18F9 and 19F6) secreting rat anti-mouse B7-H3 monoclonal antibodies were obtained. The two monoclonal antibodies were identified by fluorescent microspheres as IgG2b and light chain as kappa chain. Flow cytometry analysis showed that both McAbs could specifically bind to B7-H3 molecules and recognized different epitopes. After long-term culture in vitro and cryopreservation in liquid nitrogen, the resuscitated hybridoma cells grew well and secreted antibodies steadily. The expression of B7-H3 protein in the cytoplasm and membrane of bladder epithelial cells was almost not expressed in other normal tissues. The results showed that the monoclonal antibody could be used in flow cytometry, immunoblotting and immunohistochemistry.
Two, the expression and biological activity of mouse B7-H3-Fc fusion protein.
The murine B7-H3 extracellular segment gene and human IgG1 heavy chain Fc constant region gene were spliced by overlapping PCR. The recombinant gene was transfected into CHO cells according to the method of constructing B7-H3 gene transfected cells. The CHO/mB7-H3-Fc transfected cells were screened and obtained. After serum-free culture, the supernatant of the transfected cells was collected and concentrated by ultrafiltration and then transfected by Protein G column. The fusion protein B7-H3-Fc was purified and identified by Western blot. CCK-8 and ELISA assays showed that the fusion protein could promote the proliferation of T lymphocytes and the secretion of cytokines IL-2 and IFN-gamma in vitro. The fusion protein recognizes receptors on T cells and does not bind to the constructed TLT2 gene transfected cell lines, suggesting that the receptor of B7-H3 may not be a TLT2 molecule.
To sum up, two transgenic cell lines 293/B7-H3 and CHO/B7-H3 were constructed, and two specific rat anti-mouse B7-H3 functional monoclonal antibodies and mB7-H3-Fc fusion protein genes were developed. It has been proved that TLT2 is not the receptor of B7-H3 in mice. B7-H3, as an important costimulatory molecule, plays a positive costimulatory role in regulating T cell immune response. In conclusion, these results provide a basis for further study of the important role of B7-H3 in immune response and tumor immunity. A qualitative basis.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

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本文編號(hào)：2198273