小鼠B7-H3基因轉染細胞株和單克隆抗體的研制及其對T細胞的共刺激效應
發(fā)布時間：2018-08-22 20:53
【摘要】： T細胞的活化除了需要通過APC遞呈MHC-抗原肽給抗原特異性T細胞提供第一信號外,還需要表達于免疫細胞表面一系列共刺激分子提供第二信號。如果缺少第二信號,將會導致T細胞的無反應性或免疫耐受。共刺激分子的協(xié)同加強或負性調節(jié)在機體免疫應答的整個過程中起著及其重要的調節(jié)作用。 小鼠B7-H3屬B7超家族新成員。迄今B7-H3的生物學特性和功能研究尚待深入并存在爭議。目前的研究認為,B7-H3在介導T細胞免疫應答中存在2種截然不同的作用:協(xié)同刺激或抑制T細胞免疫應答。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)母細胞瘤細胞的表面標記分子具有診斷和干預靶向價值。進而鑒于B7-H3受體尚未得到最終確認,這也是B7-H3研究中存在的最大困難。 本研究旨在通過建立小鼠B7-H3基因轉染細胞株,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的生物學功能。 本論文分為兩個部分: 一、小鼠B7-H3基因轉染細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源的DC中擴增出小鼠B7-H3編碼區(qū)全長基因。經(jīng)雙酶切后插入真核表達載體pIRES2-EGFP中,構建成重組載體pIRES2-EGFP/B7-H3。通過脂質體法將測序正確的重組載體pIRES2-EGFP/B7-H3轉染293和CHO細胞,G418加壓篩選并經(jīng)過數(shù)次亞克隆。流式細胞術分析顯示B7-H3基因同時轉染的293和CHO細胞膜上能穩(wěn)定高表達小鼠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.以轉基因細胞株293/B7-H3為免疫原,免疫SD大鼠,采用B淋巴細胞雜交瘤技術,將免疫大鼠的脾臟細胞與小鼠骨髓瘤細胞SP2/0進行細胞融合,HAT選擇性培養(yǎng)基培養(yǎng)雜交瘤細胞。以CHO/ B7-H3為陽性篩選細胞,CHO/mock細胞作陰性對照,FCM分析篩選陽性克隆,經(jīng)過亞克隆化培養(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及流式細胞分析均顯示,兩株單抗都能與B7-H3分子特異性結合,且兩株單抗識別不同的抗原表位。體外長期培養(yǎng)和液氮凍存后,復蘇的雜交瘤細胞生長狀態(tài)良好,穩(wěn)定分泌抗體。研究發(fā)現(xiàn)小鼠B7-H3在B細胞、單核細胞和DC細胞上組成性表達,在靜息和活化的T細胞、NK細胞上不表達。小鼠B7-H3蛋白表達在膀胱上皮細胞胞漿和膜上,而在其他正常組織幾乎都不表達。由此表明,所研制的單克隆抗體能運用于流式熒光標記,免疫印跡及免疫組化,具有重要的應用價值。 二、小鼠B7-H3-Fc融合蛋白的表達及其生物學活性的研究 通過重疊PCR技術將小鼠B7-H3胞外段基因和人IgG1重鏈Fc恒定區(qū)基因拼接,按照構建B7-H3基因轉染細胞的方法將重組基因轉染CHO細胞,經(jīng)篩選并獲得CHO/mB7-H3-Fc轉染細胞。該轉基因細胞無血清培養(yǎng)后,收集細胞上清、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CCK-8和ELISA方法檢測發(fā)現(xiàn)該融合蛋白對T淋巴細胞的體外增殖和細胞因子IL-2、IFN-γ的分泌具有明顯的促進作用。在T細胞體外增殖實驗中,單抗的加入可部分阻斷T細胞分泌細胞因子。該融合蛋白識別T細胞上的受體,而與構建的TLT2基因轉染細胞株不結合,提示B7-H3的受體可能不是TLT2分子。同時也表明所研制的單抗具有阻斷功能。 綜上所述,本實驗構建了基因轉染細胞株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T細胞體外增殖及分泌IL-2,IFN-γ。T細胞體外增殖試驗顯示,單抗18F9對B7-H3介導的刺激T細胞分泌細胞因子具有阻斷作用。研究也證實TLT2分子不是小鼠B7-H3的受體。B7-H3作為重要的共刺激分子,在調節(jié)T細胞免疫應答中發(fā)揮了正性共刺激作用�？傊�,上述結果為深入研究B7-H3分子在免疫應答和腫瘤免疫中的重要作用奠定物質基礎。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
本文編號：2198273
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392
【參考文獻】
相關期刊論文 前4條
1 ;B7-H3:Another Molecule Marker for Mo-DCs?[J];Cellular & Molecular Immunology;2005年04期
2 ;Human Recombinant B7-H3 Expressed in E.colt Enhances T Lymphocyte Proliferation and IL-10 Secretion in Vitro[J];Acta Biochimica et Biophysica Sinica;2004年06期
3 邱玉華，張學光，謝煒，朱學東;一種顯著提高小鼠生產單抗腹水產量的新方法[J];中國免疫學雜志;1995年06期
4 ;Relationship between co-stimulatory molecule B7-H3 expression and gastric carcinoma histology and prognosis[J];World Journal of Gastroenterology;2006年03期
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