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  本文關(guān)鍵詞：人HVEM基因轉(zhuǎn)染細(xì)胞株的建立及其單克隆抗體的研制　出處：《蘇州大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HVEM 基因轉(zhuǎn)染 單克隆抗體 T細(xì)胞

【摘要】： HVEM(Herpesvirus-entry mediator)是至今發(fā)現(xiàn)的唯一一個既能與腫瘤壞死因子超家族成員LIGHT結(jié)合,同時也與免疫球蛋白家族成員BTLA結(jié)合的共刺激分子,被稱為是調(diào)節(jié)機(jī)體免疫應(yīng)答的分子開關(guān)。HVEM是單純皰疹病毒侵入機(jī)體的媒介,是腫瘤壞死因子(TNF)受體超家族中的一員。HVEN既是TNF家族成員LIGHT及LTα的受體,又是單純皰疹病毒gD糖蛋白(HSV1-gD)、LTα3、BTLA和CD160分子的配體。HVEM既能與LIGHT、LTα3結(jié)合產(chǎn)生正性刺激信號促進(jìn)T細(xì)胞的活化與增殖,也能通過與BTLA、CD160的結(jié)合產(chǎn)生負(fù)性信號從而抑制T細(xì)胞活化與增殖。因此,HVEM充當(dāng)了一個能同時介導(dǎo)正性和負(fù)性信號的角色。HVEM與TNFR/TNF超家族和CD28/B7超家族成員之間的交叉作用形成的微觀網(wǎng)絡(luò)跨越了共刺激分子家族內(nèi)部的相互作用,將共刺激信號和共抑制信號協(xié)調(diào)地聯(lián)系在了一起,形成了一個對T細(xì)胞免疫應(yīng)答的調(diào)控網(wǎng)絡(luò)。隨著對HVEM功能不斷地深入研究,HVEM在細(xì)胞和體液免疫中參與調(diào)節(jié)免疫應(yīng)答的作用越來越重要。將來可能被廣泛應(yīng)用到自身免疫性疾病、移植排斥、心腦血管疾病及惡性腫瘤等多個臨床領(lǐng)域。由此可見,分析和揭示其對免疫應(yīng)答的調(diào)控作用有重要的生物學(xué)意義。本研究旨在通過克隆人HVEM基因,建立基因轉(zhuǎn)染細(xì)胞株作為免疫原免疫小鼠,研制獲得鼠抗人HVEM單克隆抗體,并以表達(dá)人HVEM分子的基因轉(zhuǎn)染細(xì)胞和鼠抗人HVEM單抗為手段,研究揭示人HVEM分子的生物學(xué)特性及其對T細(xì)胞增殖作用的效應(yīng)與方式。 一、人HVEM基因的克隆、基因轉(zhuǎn)染細(xì)胞株的建立及其生物學(xué)功能的初步研究 【目的】通過克隆人HVEM編碼區(qū)全長基因,建立能穩(wěn)定表達(dá)人HVEM分子的基因轉(zhuǎn)染細(xì)胞株,為研制鼠抗人HVEM單克隆抗體提供免疫原;并初步探討基因轉(zhuǎn)染細(xì)胞在體外對T細(xì)胞活化與增殖的影響。 【方法】從此前已插入人HVEM編碼區(qū)全長基因的pMD18-T/HVEM中用PCR法擴(kuò)增出目的片段,經(jīng)EcoR I和BamH I雙酶切后插入pIRES2-EGFP真核表達(dá)載體中,構(gòu)建成pIRES2-EGFP/HVEM重組載體,脂質(zhì)體法以重組載體pIRES2-EGFP/HVEM轉(zhuǎn)染鼠L929細(xì)胞。經(jīng)G418長期加壓篩選獲得基因轉(zhuǎn)染細(xì)胞。免疫熒光標(biāo)記和流式細(xì)胞術(shù)分析HVEM分子在基因轉(zhuǎn)染的L929細(xì)胞膜上的表達(dá);通過MTT法和酶聯(lián)免疫吸附測定法(ELISA)測定獲得的基因轉(zhuǎn)染細(xì)胞L929/HVEM體外對T淋巴細(xì)胞增殖及細(xì)胞因子分泌的影響。 【結(jié)果】測序結(jié)果表明,獲得了HVEM編碼區(qū)全長基因,針對HVEM全長基因構(gòu)建的重組載體經(jīng)雙酶切后,能夠釋放出852bp左右的目的基因片段,DNA測序進(jìn)一步證明插入載體中的基因片段與HVEM序列完全一致;流式細(xì)胞術(shù)檢測基因轉(zhuǎn)染細(xì)胞株L929/HVEM細(xì)胞膜上能穩(wěn)定高表達(dá)人HVEM分子。L929/HVEM和T細(xì)胞體體外細(xì)胞共培養(yǎng)試驗(yàn)表明,與未轉(zhuǎn)染HVEM基因的L929/mock細(xì)胞相比,L929/HVEM能顯著地促進(jìn)抗人CD3單抗(mAb)刺激的T細(xì)胞增殖,以及促進(jìn)T細(xì)胞對IL-2、IFN-γ、IL-10和TNF-α的分泌。 【結(jié)論】成功建立了可穩(wěn)定高表達(dá)人HVEM基因轉(zhuǎn)染細(xì)胞,為研制抗人HVEM隆抗體提供了有效的免疫原�；蜣D(zhuǎn)染細(xì)胞L929/HVEM在體外能一定程度地促進(jìn)抗人CD3單抗刺激的T細(xì)胞活化與增殖,并影響細(xì)胞因子的分泌。 二、鼠抗人HVEM單克隆抗體的研制及生物學(xué)特性鑒定 【目的】研制鼠抗人HVEM單克隆抗體為探討人HVEM的分子特性及其介導(dǎo)的正性號對人T淋巴細(xì)胞促進(jìn)作用的效應(yīng)與方式提供必備的物質(zhì)手段。 【方法】以高表達(dá)人HVEM基因轉(zhuǎn)染細(xì)胞L929/HVEM為免疫原,常規(guī)免疫BALB/c小鼠,采用B淋巴細(xì)胞雜交瘤技術(shù),將免疫小鼠的脾臟細(xì)胞與小鼠骨髓細(xì)胞SP2/0進(jìn)行細(xì)胞融合,并以基因轉(zhuǎn)染細(xì)胞L929/HVEM和L929/mock流式篩選分別陽性、陰性雜交瘤克隆,經(jīng)間接免疫熒光標(biāo)記和流式細(xì)胞術(shù)分析、反復(fù)篩選和多次克隆化培養(yǎng),獲得一株特異分泌鼠抗人HVEM分子單克隆抗體的雜交瘤細(xì)胞株;采用細(xì)胞核染色體計數(shù)、Ig亞型快速定性試紙法、競爭結(jié)合抑制以及Western blot等試驗(yàn),對獲得的雜交瘤細(xì)胞株及單克隆抗體進(jìn)行生物學(xué)特性的鑒定;用間接免疫熒光法初步分析單抗對不同來源人腫瘤細(xì)胞株的識別作用。 【結(jié)果】成功獲得一株穩(wěn)定特異分泌鼠抗人HVEM單克隆抗體的雜交瘤細(xì)胞株,命名為2B11。經(jīng)體外連續(xù)培養(yǎng)半年以上,液氮凍存后復(fù)蘇,細(xì)胞的生長狀態(tài)良好,分泌抗體的性能穩(wěn)定。染色體數(shù)目分析顯示,雜交瘤細(xì)胞株的染色體在100條以上。雜交瘤細(xì)胞核型分析顯示,雜交瘤細(xì)胞株的染色體數(shù)目在100條以上,超過小鼠B細(xì)胞和SP2/0細(xì)胞的染色體數(shù),表明為融合體;經(jīng)過快速定性試紙分析顯示,單抗輕鏈均為κ鏈,重鏈為IgG1亞類; Western blot分析結(jié)果顯示單抗2B11與HVEM-Fc融合蛋白特異性結(jié)合,形成陽性條帶;采用本室建立的腹水誘生法制備單抗,腹水形成陽性率約為80%以上,腹水的收獲量平均為3.5ml/只。經(jīng)( NH4)2SO4鹽析法純化單克隆抗體,腹水中抗體蛋白的得率為0.5mg/ml。純化抗體用于間接免疫熒光檢測的用量為0.5~1.0μg/1x106細(xì)胞。單抗2B11與不同來源的人腫瘤細(xì)胞株作用結(jié)果表明在測定的腫瘤細(xì)胞株中,單抗檢測到白血病來源的細(xì)胞株THP-1表達(dá)人HVEM分子。 【結(jié)論】成功獲得1株穩(wěn)定分泌特異性鼠抗人HVEM單抗的雜交瘤細(xì)胞株,單抗的研制成功也為揭示HVEM的膜表達(dá)特性、深入探討B(tài)TLA/LIGHT/HVEM共信號對T細(xì)胞的調(diào)節(jié)作用及其發(fā)揮作用的信號機(jī)制提供了必備的物質(zhì)手段。 總之,本論文主要開展了如下工作:克隆獲得人HVEM編碼區(qū)正確的全長基因,建立了基因轉(zhuǎn)染細(xì)胞株L929/HVEM,初步探討了L929/HVEM對T細(xì)胞增殖與活化的促進(jìn)作用;以L929/HVEM為免疫原免疫小鼠研制獲得一株特異性分泌鼠抗人HVEM單克隆抗體的雜交瘤細(xì)胞株2B11;T細(xì)胞體外增殖試驗(yàn)表明,單抗2B11對L929/HVEM介導(dǎo)的促T細(xì)胞增殖與細(xì)胞因子分泌具有一定的阻斷作用。鑒于HVEM在免疫排斥和自身免疫性疾病中起著重要的作用,HVEM基因轉(zhuǎn)染細(xì)胞株的建立及鼠抗人HVEM單克隆抗體2B11成功研制為以后的研究工作奠定了必備的物質(zhì)基礎(chǔ)。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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