人HVEM基因转染细胞株的建立及其单克隆抗体的研制

发布时间：2018-01-09 18:35

本文关键词：人HVEM基因转染细胞株的建立及其单克隆抗体的研制　出处：《苏州大学》2010年硕士论文　论文类型：学位论文

更多相关文章： HVEM 基因转染 单克隆抗体 T细胞

【摘要】： HVEM(Herpesvirus-entry mediator)是至今发现的唯一一个既能与肿瘤坏死因子超家族成员LIGHT结合,同时也与免疫球蛋白家族成员BTLA结合的共刺激分子,被称为是调节机体免疫应答的分子开关。HVEM是单纯疱疹病毒侵入机体的媒介,是肿瘤坏死因子(TNF)受体超家族中的一员。HVEN既是TNF家族成员LIGHT及LTα的受体,又是单纯疱疹病毒gD糖蛋白(HSV1-gD)、LTα3、BTLA和CD160分子的配体。HVEM既能与LIGHT、LTα3结合产生正性刺激信号促进T细胞的活化与增殖,也能通过与BTLA、CD160的结合产生负性信号从而抑制T细胞活化与增殖。因此,HVEM充当了一个能同时介导正性和负性信号的角色。HVEM与TNFR/TNF超家族和CD28/B7超家族成员之间的交叉作用形成的微观网络跨越了共刺激分子家族内部的相互作用,将共刺激信号和共抑制信号协调地联系在了一起,形成了一个对T细胞免疫应答的调控网络。随着对HVEM功能不断地深入研究,HVEM在细胞和体液免疫中参与调节免疫应答的作用越来越重要。将来可能被广泛应用到自身免疫性疾病、移植排斥、心脑血管疾病及恶性肿瘤等多个临床领域。由此可见,分析和揭示其对免疫应答的调控作用有重要的生物学意义。本研究旨在通过克隆人HVEM基因,建立基因转染细胞株作为免疫原免疫小鼠,研制获得鼠抗人HVEM单克隆抗体,并以表达人HVEM分子的基因转染细胞和鼠抗人HVEM单抗为手段,研究揭示人HVEM分子的生物学特性及其对T细胞增殖作用的效应与方式。一、人HVEM基因的克隆、基因转染细胞株的建立及其生物学功能的初步研究【目的】通过克隆人HVEM编码区全长基因,建立能稳定表达人HVEM分子的基因转染细胞株,为研制鼠抗人HVEM单克隆抗体提供免疫原;并初步探讨基因转染细胞在体外对T细胞活化与增殖的影响。【方法】从此前已插入人HVEM编码区全长基因的pMD18-T/HVEM中用PCR法扩增出目的片段,经EcoR I和BamH I双酶切后插入pIRES2-EGFP真核表达载体中,构建成pIRES2-EGFP/HVEM重组载体,脂质体法以重组载体pIRES2-EGFP/HVEM转染鼠L929细胞。经G418长期加压筛选获得基因转染细胞。免疫荧光标记和流式细胞术分析HVEM分子在基因转染的L929细胞膜上的表达;通过MTT法和酶联免疫吸附测定法(ELISA)测定获得的基因转染细胞L929/HVEM体外对T淋巴细胞增殖及细胞因子分泌的影响。【结果】测序结果表明,获得了HVEM编码区全长基因,针对HVEM全长基因构建的重组载体经双酶切后,能够释放出852bp左右的目的基因片段,DNA测序进一步证明插入载体中的基因片段与HVEM序列完全一致;流式细胞术检测基因转染细胞株L929/HVEM细胞膜上能稳定高表达人HVEM分子。L929/HVEM和T细胞体体外细胞共培养试验表明,与未转染HVEM基因的L929/mock细胞相比,L929/HVEM能显著地促进抗人CD3单抗(mAb)刺激的T细胞增殖,以及促进T细胞对IL-2、IFN-γ、IL-10和TNF-α的分泌。【结论】成功建立了可稳定高表达人HVEM基因转染细胞,为研制抗人HVEM隆抗体提供了有效的免疫原。基因转染细胞L929/HVEM在体外能一定程度地促进抗人CD3单抗刺激的T细胞活化与增殖,并影响细胞因子的分泌。二、鼠抗人HVEM单克隆抗体的研制及生物学特性鉴定【目的】研制鼠抗人HVEM单克隆抗体为探讨人HVEM的分子特性及其介导的正性号对人T淋巴细胞促进作用的效应与方式提供必备的物质手段。【方法】以高表达人HVEM基因转染细胞L929/HVEM为免疫原,常规免疫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与小鼠骨髓细胞SP2/0进行细胞融合,并以基因转染细胞L929/HVEM和L929/mock流式筛选分别阳性、阴性杂交瘤克隆,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,获得一株特异分泌鼠抗人HVEM分子单克隆抗体的杂交瘤细胞株;采用细胞核染色体计数、Ig亚型快速定性试纸法、竞争结合抑制以及Western blot等试验,对获得的杂交瘤细胞株及单克隆抗体进行生物学特性的鉴定;用间接免疫荧光法初步分析单抗对不同来源人肿瘤细胞株的识别作用。【结果】成功获得一株稳定特异分泌鼠抗人HVEM单克隆抗体的杂交瘤细胞株,命名为2B11。经体外连续培养半年以上,液氮冻存后复苏,细胞的生长状态良好,分泌抗体的性能稳定。染色体数目分析显示,杂交瘤细胞株的染色体在100条以上。杂交瘤细胞核型分析显示,杂交瘤细胞株的染色体数目在100条以上,超过小鼠B细胞和SP2/0细胞的染色体数,表明为融合体;经过快速定性试纸分析显示,单抗轻链均为κ链,重链为IgG1亚类; Western blot分析结果显示单抗2B11与HVEM-Fc融合蛋白特异性结合,形成阳性条带;采用本室建立的腹水诱生法制备单抗,腹水形成阳性率约为80%以上,腹水的收获量平均为3.5ml/只。经( NH4)2SO4盐析法纯化单克隆抗体,腹水中抗体蛋白的得率为0.5mg/ml。纯化抗体用于间接免疫荧光检测的用量为0.5~1.0μg/1x106细胞。单抗2B11与不同来源的人肿瘤细胞株作用结果表明在测定的肿瘤细胞株中,单抗检测到白血病来源的细胞株THP-1表达人HVEM分子。【结论】成功获得1株稳定分泌特异性鼠抗人HVEM单抗的杂交瘤细胞株,单抗的研制成功也为揭示HVEM的膜表达特性、深入探讨BTLA/LIGHT/HVEM共信号对T细胞的调节作用及其发挥作用的信号机制提供了必备的物质手段。总之,本论文主要开展了如下工作:克隆获得人HVEM编码区正确的全长基因,建立了基因转染细胞株L929/HVEM,初步探讨了L929/HVEM对T细胞增殖与活化的促进作用;以L929/HVEM为免疫原免疫小鼠研制获得一株特异性分泌鼠抗人HVEM单克隆抗体的杂交瘤细胞株2B11;T细胞体外增殖试验表明,单抗2B11对L929/HVEM介导的促T细胞增殖与细胞因子分泌具有一定的阻断作用。鉴于HVEM在免疫排斥和自身免疫性疾病中起着重要的作用,HVEM基因转染细胞株的建立及鼠抗人HVEM单克隆抗体2B11成功研制为以后的研究工作奠定了必备的物质基础。
[Abstract]:HVEM (Herpesvirus-entry mediator) is found so far only one can with the tumor necrosis factor superfamily member LIGHT and costimulatory molecules also combined with members of the immunoglobulin family BTLA, known as.HVEM molecular switch to regulate the immune response of herpes simplex virus invading media, tumor necrosis factor (TNF) receptor superfamily, a member of the.HVEN family members is TNF LIGHT and LT alpha receptor, and herpes simplex virus gD glycoprotein (HSV1-gD), LT alpha 3, BTLA and CD160 molecular ligand.HVEM can LIGHT and LT alpha 3 combine to produce positive signals to promote the activation and proliferation of T the cell, but also through the combination of CD160 and BTLA, negative signals to inhibit T cell activation and proliferation. Therefore, HVEM can act as a mediating positive and negative signaling roles for.HVEM and TNFR/TNF super family and CD28/B7 family The formation of micro network interactions between family members across the costimulatory molecule interaction within the family, the costimulatory signals and signaling coordination together, forming a on the T cell immune response regulatory network. With the function of HVEM constantly in-depth study, participate in the regulation of HVEM and immune response is more important in cellular and humoral immunity. It may be widely applied to autoimmune diseases, future graft rejection, cardiovascular and cerebrovascular diseases and malignant tumors and other clinical areas. Therefore, analyzing and revealing the significance of the regulation of the immune response. This study aims to clone human HVEM the establishment of gene transfected cells as immunogen were prepared from mouse anti human HVEM monoclonal antibody, and the expression of gene transfected cells and mouse anti human HVEM molecule HVEM As a means of monoclonal antibody, the study reveals the biological characteristics of human HVEM and its effect and mode on the proliferation of T cells.
First, cloning of human HVEM gene, establishment of gene transfected cell line and preliminary study on its biological function
[Objective] by cloning the full-length gene of human HVEM coding region, we established a gene transfected cell line that stably expressed human HVEM molecule, and provided immunogen for developing mouse anti human HVEM monoclonal antibody, and preliminarily discussed the effect of gene transfected cells on T cell activation and proliferation in vitro.
[method] from the previous PCR method has been inserted into the full-length human HVEM gene encoding the pMD18-T/HVEM amplified fragment by EcoR I, and BamH I digestion and then inserted into the eukaryotic expression vector pIRES2-EGFP, construct pIRES2-EGFP/HVEM recombinant vector by liposome with the recombinant vector pIRES2-EGFP/HVEM was transfected into mouse L929 cells. After G418 long-term pressure to obtain gene transfected cells. Screened by immunofluorescence and flow cytometry analysis of the expression of HVEM molecule in the cell membrane of L929 gene transfection on; determination by MTT assay and enzyme-linked immunosorbent assay (ELISA) to obtain the gene effects of transfected L929/HVEM cells in vitro on T cells proliferation and cytokine secretion.
[results] the sequencing result showed that the full-length HVEM gene encoding region, for the construction of recombinant vector of HVEM gene by restriction enzyme digestion, can release a gene fragment of about 852bp, DNA sequencing further proved that the inserted gene fragment completely consistent with HVEM sequence in the carrier; flow cytometry in transfected cells L929/HVEM cells could stably express Master HVEM molecules.L929/HVEM and T cells in vitro cell co culture experiments show that, compared with non transfected HVEM L929/mock cells, L929/HVEM can significantly promote the anti human CD3 monoclonal antibody (mAb) stimulated T cell proliferation, and promote IFN- gamma T cells to IL-2, secretion of IL-10 and TNF- alpha.
[Conclusion] successfully established a stable high surface Master HVEM transfected cells, for the preparation of anti human HVEM monoclonal antibody provides an effective immunogen. Gene transfection of L929/HVEM cells to some extent promote stimulation of anti human CD3 monoclonal antibody and T cell activation and proliferation in vitro, and the effect of cytokine secretion.
Two, the development of mouse anti human HVEM monoclonal antibody and identification of its biological characteristics
[Objective] to prepare mouse anti human HVEM monoclonal antibody, and provide necessary material means for exploring the molecular characteristics of human HVEM and the positive effect of positive T on the promotion of human lymphocytes.
[method] with high surface Master HVEM transfected cells L929/HVEM as immunogen, BALB/c mice were immunized with B, lymphocyte hybridoma technique, the immunized mouse spleen cells and bone marrow cells of mice SP2/0 cell fusion, and gene transfection of L929/HVEM and L929/ mock flow cytometry screening were positive and negative clones, analysis by indirect immunofluorescence and flow cytometry, repeated screening and multiple cloning culture, obtained a strain specific secretion of mouse anti human HVEM monoclonal antibody hybridoma cell lines; the nucleus chromosome count, Ig subtype rapid qualitative method, Western and blot competitive binding inhibition test, identification of biological characteristics of hybridoma cell lines and monoclonal antibody obtained; preliminary analysis to identify the role of different sources of monoclonal antibody on human tumor cell lines by indirect immunofluorescence.
[results] successfully obtained a stable secretion of specific mouse anti human HVEM monoclonal antibody hybridoma cell lines, named 2B11. after culture in vitro for more than half a year, frozen in liquid nitrogen after recovery, growth is good, stable secretion of antibodies. Chromosome analysis showed that the hybridoma chromosome in 100 above. Hybridoma cell karyotype analysis showed that chromosome number of hybridoma cell lines in more than 100, more than the chromosome number of mouse B cells and SP2/0 cells, showed that fusion; after a rapid qualitative analysis showed that the monoclonal antibody light chain were kappa chain, heavy chain IgG1 subtype; Western blot analysis showed that mAb 2B11 and HVEM-Fc fusion protein specific binding, the formation of positive bands; the relationship of ascites induced preparation of monoclonal antibodies, the positive rate of ascites formation is about 80% more than the average amount of ascites harvest was 3.5ml / (NH4). After purification of monoclonal antibody 2SO4 salting out method, antibody in ascites was purified 0.5mg/ml. antibody for indirect immunofluorescence detection of the amount of 0.5~1.0 g/1x106 cells. Monoclonal antibody 2B11 and different sources of human tumor cell lines in effect results showed that tumor cell lines were detected, monoclonal antibody to leukemia the source of THP-1 cells expressing human HVEM.
[Conclusion] successfully obtained 1 strains stably secreting specific mouse anti human HVEM monoclonal antibody hybridoma cell strain, monoclonal antibody developed also to reveal the HVEM membrane expression characteristics, in-depth study of BTLA/LIGHT/HVEM signal regulating effect on T cells and its signal mechanism provides the necessary material means.
In conclusion, this paper carried out the following work: get the correct clone gene HVEM encoding region, established the gene transfected cell line L929/HVEM, discussed the L929/HVEM on T cell proliferation and activation effect; with L929/HVEM for the development of immunization of mice and obtained a strain specific secretion of mouse anti human HVEM monoclonal antibody the hybridoma cell strain 2B11; cell proliferation test showed that T, mAb 2B11 can block certain effect on promoting T cell proliferation and cytokine mediated L929/HVEM secretion. In view of the fact that HVEM plays an important role in immune rejection and autoimmune diseases, and establishment of rat HVEM gene transfected cell line anti human HVEM the monoclonal antibody 2B11 was successfully developed for future research work has laid the foundation for.

【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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