小鼠B7-H3基因转染细胞株和单克隆抗体的研制及其对T细胞的共刺激效应
发布时间：2018-08-22 20:53
【摘要】： T细胞的活化除了需要通过APC递呈MHC-抗原肽给抗原特异性T细胞提供第一信号外,还需要表达于免疫细胞表面一系列共刺激分子提供第二信号。如果缺少第二信号,将会导致T细胞的无反应性或免疫耐受。共刺激分子的协同加强或负性调节在机体免疫应答的整个过程中起着及其重要的调节作用。 小鼠B7-H3属B7超家族新成员。迄今B7-H3的生物学特性和功能研究尚待深入并存在争议。目前的研究认为,B7-H3在介导T细胞免疫应答中存在2种截然不同的作用:协同刺激或抑制T细胞免疫应答。此外一些研究提出B7-H3分子可以作为肿瘤如肺癌,前列腺癌以及神经母细胞瘤细胞的表面标记分子具有诊断和干预靶向价值。进而鉴于B7-H3受体尚未得到最终确认,这也是B7-H3研究中存在的最大困难。 本研究旨在通过建立小鼠B7-H3基因转染细胞株,研制特异性单克隆抗体和融合蛋白,从不同角度探讨B7-H3的生物学功能。 本论文分为两个部分: 一、小鼠B7-H3基因转染细胞株及其单克隆抗体的研制 1.采用RT-PCR的方法从小鼠骨髓来源的DC中扩增出小鼠B7-H3编码区全长基因。经双酶切后插入真核表达载体pIRES2-EGFP中,构建成重组载体pIRES2-EGFP/B7-H3。通过脂质体法将测序正确的重组载体pIRES2-EGFP/B7-H3转染293和CHO细胞,G418加压筛选并经过数次亚克隆。流式细胞术分析显示B7-H3基因同时转染的293和CHO细胞膜上能稳定高表达小鼠B7-H3分子。为研制小鼠B7-H3单克隆抗体提供了有效的免疫原。2.以转基因细胞株293/B7-H3为免疫原,免疫SD大鼠,采用B淋巴细胞杂交瘤技术,将免疫大鼠的脾脏细胞与小鼠骨髓瘤细胞SP2/0进行细胞融合,HAT选择性培养基培养杂交瘤细胞。以CHO/ B7-H3为阳性筛选细胞,CHO/mock细胞作阴性对照,FCM分析筛选阳性克隆,经过亚克隆化培养,最终获得2株持续、稳定分泌大鼠抗小鼠B7-H3单克隆抗体的杂交瘤细胞株(18F9和19F6)。经荧光微球法鉴定,两株单抗重链均为IgG2b,轻链为κ链。Western blot及流式细胞分析均显示,两株单抗都能与B7-H3分子特异性结合,且两株单抗识别不同的抗原表位。体外长期培养和液氮冻存后,复苏的杂交瘤细胞生长状态良好,稳定分泌抗体。研究发现小鼠B7-H3在B细胞、单核细胞和DC细胞上组成性表达,在静息和活化的T细胞、NK细胞上不表达。小鼠B7-H3蛋白表达在膀胱上皮细胞胞浆和膜上,而在其他正常组织几乎都不表达。由此表明,所研制的单克隆抗体能运用于流式荧光标记,免疫印迹及免疫组化,具有重要的应用价值。 二、小鼠B7-H3-Fc融合蛋白的表达及其生物学活性的研究 通过重叠PCR技术将小鼠B7-H3胞外段基因和人IgG1重链Fc恒定区基因拼接,按照构建B7-H3基因转染细胞的方法将重组基因转染CHO细胞,经筛选并获得CHO/mB7-H3-Fc转染细胞。该转基因细胞无血清培养后,收集细胞上清、超滤浓缩后行经Protein G柱纯化,获得纯品B7-H3-Fc融合蛋白,经Western blot鉴定。通过CCK-8和ELISA方法检测发现该融合蛋白对T淋巴细胞的体外增殖和细胞因子IL-2、IFN-γ的分泌具有明显的促进作用。在T细胞体外增殖实验中,单抗的加入可部分阻断T细胞分泌细胞因子。该融合蛋白识别T细胞上的受体,而与构建的TLT2基因转染细胞株不结合,提示B7-H3的受体可能不是TLT2分子。同时也表明所研制的单抗具有阻断功能。 综上所述,本实验构建了基因转染细胞株293/B7-H3和CHO/B7-H3;研制2株特异性大鼠抗小鼠B7-H3功能性单克隆抗体和mB7-H3-Fc融合蛋白基因。该融合蛋白能够促进T细胞体外增殖及分泌IL-2,IFN-γ。T细胞体外增殖试验显示,单抗18F9对B7-H3介导的刺激T细胞分泌细胞因子具有阻断作用。研究也证实TLT2分子不是小鼠B7-H3的受体。B7-H3作为重要的共刺激分子,在调节T细胞免疫应答中发挥了正性共刺激作用。总之,上述结果为深入研究B7-H3分子在免疫应答和肿瘤免疫中的重要作用奠定物质基础。
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前4条
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 邱玉华，张学光，谢炜，朱学东;一种显著提高小鼠生产单抗腹水产量的新方法[J];中国免疫学杂志;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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