LIGHT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：2018-05-26 21:21

本文选题：LIGHT + HVEM　；参考：《苏州大学》2010年硕士论文

【摘要】： 正性与负性共信号分子共同参与,实现了对T细胞有效和适度免疫应答的精确调控作用。共信号分子根据结构主要分为免疫球蛋白(CD28/ B7)超家族、肿瘤坏死因子(TNF/TNFR)超家族以及细胞因子超家族。人LIGHT(TNFSF14)属于TNF家族成员,其与TNFR超家族受体分子HVEM作用可介导促进T细胞活化与增殖的正性共刺激信号。近期研究表明,HVEM也可作为配体与免疫球蛋白超家族成员BTLA或CD160作用介导对T细胞的抑制作用,并且相关研究揭示了HVEM/LIGHT和BTLA(CD160)/HVEM间也存在信号的双向性。因此,LIGHT/HVEM/BTLA(CD160)构成了共信号分子中一个微观的免疫调节网络,探讨和分析LIGHT/HVEM信号的特性及其对T细胞的共刺激作用与机制具有重要的理论意义。本研究旨在建立L929/LIGHT基因转染细胞株,以此为手段探讨人LIGHT分子体外对T细胞的共刺激效应,通过制备鼠抗人LIGHT单克隆抗体和人HVEMIg融合蛋白,分析其对LIGHT/HVEM效应的阻断,从而为阐明LIGHT/HVEM信号对T细胞的协同刺激作用与机制,进一步深入探讨LIGHT/HVEM/BTLA(CD160)对T细胞复杂的调控机理提供有价值的实验数据。一.人LIGHT基因转染细胞株的构建及其对T细胞的共刺激作用 RT-PCR从经PHA活化的T细胞中克隆人LIGHT编码区全长基因,双酶切后插入真核表达载体pIRES2-EGFP构建重组子pIRES2-EGFP-LIGHT,测序正确。脂质体法以重组表达载体转染小鼠L929细胞。G418加压筛选并经过数次亚克隆,建立了L929/LIGHT基因转染细胞株,流式细胞术检测结果显示,L929/LIGHT细胞膜上能稳定高表达人LIGHT分子;体外细胞共培养试验表明,与未转染LIGHT基因的L929/mock细胞相比,L929/LIGHT可显著地促进抗人CD3单抗(mAb)刺激的T细胞增殖;同时L929/LIGHT亦能明显促进T细胞对IL-2、IFN-γ和IL-10的分泌。二.鼠抗人LIGHT单抗和人HVEMIg融合蛋白的研制及其生物学特性 1.以L929/LIGHT细胞为免疫原免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术,将免疫小鼠的脾脏细胞和小鼠骨髓瘤细胞SP2/0进行融合,HAT选择性培养基培养杂交瘤细胞。以L929/LIGHT为阳性筛选细胞,L929/mock作阴性对照,FACS分析筛选阳性克隆,经过4次亚克隆化培养,最终得到一株持续分泌抗人LIGHT单克隆抗体的杂交瘤细胞株(7A8),经快速定性试纸条分析法结果显示,单抗7A8重链属于IgG2b,轻链为κ。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;2.将人IgG(Fc)基因编码区基因与人HVEM胞外段基因融合,按照构建L929/LIGHT基因转染细胞的技术和方法将HVEMIg融合基因导入真核细胞,筛选并亚克隆获得CHO/HVEMIg转染细胞。利用流式细胞术、SDS-PAGE和Western-blot进行鉴定,结果显示,HVEMIg融合蛋白分泌表达于CHO/HVEMIg基因转染细胞的培养上清中。大规模细胞培养后再浓缩上清,通过protein G亲和层析技术获得纯品HVEMIg融合蛋白,流式检测结果,纯化的蛋白与其配体LIGHT具有良好的结合能力。T细胞体外增殖实验表明,抗LIGHT单抗和HVEMIg融合蛋白的加入可部分阻断由基因转染细胞协同CD3mAb对T细胞增殖的促进作用,并下调细胞因子的分泌。由此提示LIGHT/HVEM通过介导正性共刺激作用参与活化T细胞的调节。综上所述,本实验成功构建了L929/LIGHT基因转染细胞株,该转染细胞表达的膜型人LIGHT对T细胞体外增殖和细胞因子分泌具有显著的促进作用;成功地研制一株鼠抗人LIGHT单抗7A8和HVEMIg融合蛋白。T细胞体外增殖试验显示,单抗7A8和HVEMIg对L929/LIGHT介导的刺激T细胞增殖和细胞因子分泌分别具有部分阻断作用,单抗7A8为鼠抗人LIGHT的阻断型抗体。上述结果为深入探讨LIGHT分子的共刺激作用及LIGHT/HVEM/BTLA(CD160)调节网络在T细胞免疫应答中的调控作用及干预策略提供了有价值的物质基础和实验参数。
[Abstract]:Both positive and negative co signalling molecules participate in the precise regulation of the effective and moderate immune responses of T cells. Common signal molecules are divided into immunoglobulin (CD28/ B7) superfamily, tumor necrosis factor (TNF/TNFR) superfamily and cytokine superfamily. Human LIGHT (TNFSF14) belongs to the TNF family members, and TNFR (TNFR) is a member of the TNF family. The role of the superfamily receptor molecule HVEM can mediate the positive co stimulation signal that promotes the activation and proliferation of T cells. Recent studies have shown that HVEM can also be used as a ligand to inhibit the inhibition of T cells by the role of BTLA or CD160 in a member of the immunoglobulin superfamily, and the related studies have revealed the presence of signals between HVEM/ LIGHT and BTLA (CD160) /HVEM. Therefore, LIGHT/HVEM/BTLA (CD160) constitutes a microscopic immunomodulatory network in common signal molecules. It is of great theoretical significance to explore and analyze the characteristics of LIGHT/HVEM signals and their co stimulation and mechanism for T cells.
The purpose of this study was to establish a L929/LIGHT gene transfected cell line to explore the co stimulation effect of human LIGHT molecules on T cells in vitro. By preparing mouse anti human LIGHT monoclonal antibody and human HVEMIg fusion protein, the blocking of LIGHT/HVEM effect was analyzed, thus the synergistic stimulation and mechanism of LIGHT /HVEM signal to T cells were elucidated. Further study of LIGHT/HVEM/BTLA (CD160) provides valuable experimental data for the complex regulation mechanism of T cells.
Construction of human LIGHT gene transfected cell line and its costimulatory effect on T cells
The full length gene of human LIGHT coding region was cloned from the T cells activated by PHA, and the recombinant plasmid was inserted into the eukaryotic expression vector pIRES2-EGFP after double enzyme digestion to construct the recombinant pIRES2-EGFP-LIGHT, and the sequencing was correct. The liposome method was transfected with the recombinant expression vector to transfect the mouse L929 cell.G418 by compression screening and after several subclones, the L929/LIGHT gene transfected cells were established. The results of flow cytometry showed that the L929/LIGHT cell membrane could stabilize the high apparent LIGHT molecule on the membrane, and in vitro cell co culture test showed that L929/LIGHT could significantly promote the proliferation of T cells stimulated by the human CD3 monoclonal antibody (mAb) compared with the L929/mock cells without LIGHT gene, and L929/LIGHT also significantly promoted T cells to IL-2, IFN-. The secretion of gamma and IL-10.
Two. Preparation and biological characteristics of mouse anti human LIGHT monoclonal antibody and human HVEMIg fusion protein
1. BALB/c mice were immunized with L929/LIGHT cells. The B lymphocyte hybridoma technique was used to fuse the spleen cells of the mice and the murine myeloma cells SP2/0. The hybridoma cells were cultured on the HAT selective medium. The positive cells were screened with L929/LIGHT as positive, L929/mock was negative control, FACS analysis screened positive clones and passed 4. The hybridoma cell line (7A8) sustained secretory anti human LIGHT monoclonal antibody was obtained. The rapid qualitative test paper analysis showed that the heavy chain of McAb 7A8 was IgG2b and the light chain was kappa. After long-term culture and liquid nitrogen cryopreservation, the resuscitation clutter cells grew well, stable secreted antibodies; 2. the human Ig was Ig. G (Fc) gene coding region gene and human HVEM extracellular gene fusion gene, according to the construction of L929/LIGHT gene transfection cells technology and methods to transfect HVEMIg fusion gene into eukaryotic cells, screening and subcloning to obtain CHO/HVEMIg transfected cells. Using flow cytometry, SDS-PAGE and Western-blot to identify, the results show that HVEMIg fusion protein secretion. It was expressed in the culture supernatant of CHO/HVEMIg gene transfected cells. After large-scale cell culture, the purified supernatant was re concentrated and the pure HVEMIg fusion protein was obtained by protein G affinity chromatography. The flow test results, the purified protein had a good binding ability with the ligand LIGHT, and the.T cell proliferation experiment showed that the anti LIGHT monoantibody and HVEMIg were melted. The addition of synprotein can partially block the synergistic effect of gene transfected cells on the proliferation of T cells with CD3mAb and down regulate the secretion of cytokines, thus suggesting that LIGHT/HVEM participates in the regulation of activated T cells by mediating positive co stimulation.
To sum up, the L929/LIGHT gene transfected cell line was successfully constructed in this experiment. The membrane type human LIGHT expressed by the transfected cell has a significant promotion effect on the proliferation and cytokine secretion of T cells in vitro. A mouse anti human LIGHT monoclonal antibody 7A8 and HVEMIg fusion protein.T in vitro proliferation test was successfully developed, and the monoclonal antibody 7A8 and HVEMIg pairs L9 were shown. 29/LIGHT mediated stimulation of T cell proliferation and cytokine secretion have partial blocking effect respectively. The monoclonal antibody 7A8 is a blocking antibody against human LIGHT. The above results are valuable for exploring the co stimulation of LIGHT molecules and the regulatory role of LIGHT/HVEM/BTLA (CD160) Regulation Network in T cell immune response and intervention strategies. The material basis and experimental parameters.
【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

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