【作者】 陈昌友；

【导师】 邱玉华；

【作者基本信息】 苏州大学 ， 免疫学， 2011， 硕士

【摘要】 CD80(也称B7-1)是B7家族重要的共刺激分子。人CD80分子编码基因定位于人类染色体3q13.3-q21区内,含有1491bp。CD80是分子量约为44-54kD的Ⅰ型跨膜糖蛋白,主要表达在专职性抗原提呈细胞(antigen-presenting cell,APC)树突状细胞、单核-巨噬细胞及B淋巴细胞。CD80分子的配体为表达在T淋巴细胞表面的CD28和CTLA-4。CD80与CD28结合为T细胞的活化提供第二信号,促进T细胞的增殖与分化;而与CTLA-4结合介导抑制信号,从而下调或终止T细胞效应。若缺乏此共刺激信号,抗原特异性的第一信号非但不能有效激活特异性T细胞,反而导致T细胞无能,或者耐受。研究表明,CD80介导的信号通路不仅能调节T细胞活化过程,而且还参与许多疾病的发生发展及预后状况。单链抗体(single-chain variable fragment,ScFv)是一种小分子的抗体,含有完整的抗原结合位点,由重链可变区(the variable heavy chain,VH)和轻链可变区(the variable light chain,VL)通过一段连接肽连接而成,大小为26-28kDa。VH和VL之间的连接肽长度为10-25个氨基酸,最为普遍的为(Gly4Ser)3十五肽。Diabody是单链抗体的二聚体形式,是通过将单链抗体VH和VL之间的连接肽缩短至3-10个氨基酸时,可促进分子间VH和VL的配对而形成。第一部分抗人CD80单链抗体(ScFv)的研制及生物学功能初步研究1.目的:构建及表达抗人CD80单链抗体(ScFv),鉴定其抗原抗体结合活性,并初步研究其生物学功能。2.方法:采用RT-PCR法从分泌鼠抗人CD80 McAb的杂交瘤细胞株(4E5)中克隆VH和VL基因。用重叠延伸拼接PCR方法构建具有前导肽的L-VH-Linker-VL单链抗体基因,并克隆至pIRES2-EGFP表达载体,脂质体法转染中华仓鼠卵巢细胞(CHO),G418加压筛选。纯化抗人CD80-ScFv,分析其效价,并鉴定其对膜型CD80的识别。竞争抑制实验分析抗人CD80-ScFv与相应抗原的结合能力。MTT法体外分析抗人CD80-ScFv对Raji细胞体外增殖的抑制作用和对CD80介导的共刺激信号的阻断作用。3.结果:构建的抗人CD80-ScFv基因全长为828bp,经测序含有信号肽和连接肽编码基因。实验获得稳定分泌细胞株(命名为SA-Ⅱ)。上清中抗体得率为15.12mg/L,相对效价为2.0μg/5×105个细胞。该抗体与天然表达CD80分子的Raji和Daudi细胞阳性结合率分别为96.6%和95.0%。抗人CD80-ScFv对鼠源亲本抗体4E5与抗原结合的竞争抑制率达98.67%,并能抑制Raji细胞的体外增殖,而且能阻断CD80介导的共刺激信号转导,抑制PBMC的增殖,增殖率下降43.48%。4.结论:成功构建的表达抗人CD80-ScFv的细胞株,其分泌的抗体具有良好的生物学活性。该抗体的研制在CD80及相关分子的基础或/及临床研究中具有重要的应用价值。第二部分抗人CD80双价抗体(diabody)的研制及生物学功能初步研究1.目的:构建及表达抗人CD80双价抗体(diabody),并初步研究其生物学功能。2.方法:PCR法获得轻重链可变区基因,构建表达载体pIRES2- EGFP/diabody。将pIRES2-EGFP/diabody转染中华仓鼠卵巢细胞(CHO),G418加压,筛选稳定高表达细胞株,扩大培养并收集上清,经镍柱纯化,并经变性及非变性SDS-PAGE电泳对抗体分子的双体性进行鉴定。以鼠源性亲本抗体4E5为对照,测定抗人CD80-diabody的效价。采用免疫荧光及流式细胞术(FCM)分析抗体与细胞膜型CD80分子的结合活性及与鼠源性亲本抗体4E5的竞争抑制效应;MTT法分析该抗体对天然高表达CD80的Raji细胞体外增殖的影响,同时分析其对CD80介导的共刺激信号阻断作用的影响。3.结果:成功构建抗人CD80-diabody表达载体,基因全长为798bp,含有信号肽和Gly4Ser连接肽编码基因。获得了稳定分泌双价抗体的细胞株(命名为SA-Ⅲ),培养上清中纯化抗体的得率为15mg/L,PAGE结果显示其为一个二聚体,Mr约为53kD(a抗人CD80-ScFv Mr约为27kDa)。抗人CD80-diabody的效价为1.5μg/5×105个细胞,与L929-CD80、Raji及Daudi的阳性结合率分别为96.5%、98.1%及93.0%;该抗体对鼠源亲本抗体4E5与抗原结合的抑制率为97.11%,对Raji细胞体外增殖的抑制率达31.27%。同时也能阻断CD80介导的共刺激信号。与单链抗体相比,亲和活性明显提高。4.结论:成功获得了分泌抗人CD80-diabody的CHO细胞株,该抗体具有良好的生物学活性,亲和活性较单链抗体也明显提高,为进一步对CD80分子的研究奠定了基础。更多还原

【Abstract】 CD80,also called B7-1,is one important costimulatory molecule of B7 family.Human CD80 molecule,mainly expressed in APC such as dendritic cells, mononuclear phagocytes and B cells,is a 44-54kDaⅠtype transmember protein, whose encoding genes are directed at human chromosome 3q13.3-q21, containing 1491bp. CD80 ligand is CD28 and CTLA-4.While combination with CD28 can support a second set of signals for T cell activation and promote the proliferation and differiation of T cells,bind with CTLA-4 strongly diminishs T cells response but maintains T cell homeostasis.If T cells activation lacks the second signals,there is a serious problem that antigen peptide-MHC complexes can not activate specific T cells effectively,but conversely make T cells anergy or tolerance.Now it was demonstrated that CD80 is not merely the critically regulatory molecule in the course of T cell activiation,but involved in many diseases.Single-chain variable fragment(ScFv) is a small recombinant antibody fragment, containing the complete antigen binding site, which includes the variable heavy (VH) and variable light (VL) domains of an antibody. The VH domain is linked to a VL domain by an introduced ?exible polypeptide linker,whose length is commonly 10-25 amino acids.The common peptide linker is (Gly4Ser)3.Diabody,a dimer of ScFv,is formed by shorting the polypeptide linker between VH and VL to 3-10 amino acids,which can promote pair between intermolecules. Objective: To construct and express the single chain variable fragment (ScFv) of monoclonal antibody against human CD80 and study its biological functions.Methods: The VH and VL genes were cloned by RT-PCR from a murine hybridoma cell line 4E5,which produces monoclonal antibody (McAb) aginst human CD80 antign.Splicing by overlapping extension PCR (SOE-PCR) was used to splice the VH and VL genes to construct L-VH-Linker-VL CD80-ScFv genes, which were cloned into the eukaryotic expressing vector pIRES2-EGFP. CHO cells were transfected by pIRES2-EGFP/ScFv plasmids through the liposome-mediated method and selected by G418.Anti-human CD80-ScFv was purified from culture supernatant.Then the experiments evaluated the potency and analyzed the identification of ScFv to membrane CD80;competitive inhibition experiment was applied to analyze the binding ability of anti-human CD80-ScFv with the corresponding antigen. The inhibitory effect of anti-human CD80-ScFv on in vitro proliferation of Raji cells and the effect of ScFv on costimulatory signals mediated by CD80 were detected by MTT methods.Results: The ScFv genes consist of 828bp and include genes encoding the signal peptide and linker. The highly secreting and stable cells were harvested. About 15.12 milligram ScFv antibodies, whose relative potency is 2.0μg/5×105cells,were purified from one liter culture supernatantat,and its positive binding rate with Raji and Daudi is 96.6% and 95.0% respectively and cannot bind with Jurkat cells.The competitive inhibition rate of anti-human CD80-ScFv against murine parent antibody 4E5 is 98.67%,and anti-human CD80-ScFv could inhibit growth of Raji cells in vitro.In addition to them ,it can also make the proliferation rate of PBMC decline by 43.48% via blocking costimulatory signals mediated by CD80.Conclusion: Anti-human CD80-ScFv has been successfully expressed in CHO cells(named SA-Ⅱ) and could identify membrane CD80 molecule in the cell surface and mediate the related biological functions. Its preparation possess an important value on fundamental and clinical research of CD80 and related molecules.To construct and express anti-human CD80 bivalent dimer(diabody) and study its biological functions.Methods: VH and VL genes were cloned by PCR,and then cloned into the eukaryotic expressing vector pIRES2-EGFP to construct recombinant pIRES2- EGFP/ diabody. CHO cells was transfected by above plasmids through the liposome-mediated methods and selected by G418 to harvest stably highly secreting cell lines.Anti-human CD80-diabody was purified from culture supernatant by Ni catridges.Reducing and non-reducing SDS-PAGE was used to analyze its bivalent.Next the potency of diabody was detected by FCM.Then the experiments analyzed the identification of diabody to membrane CD80 and competitive inhibition against parent antibody 4E5 via immunofluorescence and FCM ; The effect of anti-human CD80-diabody on proliferation of Raji cells which express CD80 higtly and naturely,was determined by MTT methods and we also researched blockade of diabody on CD80-mediated costimulatory signal simultaneously.Results: It was demonstrated that pIRES2-EGFP/diabody was constructed successfully,concluding 798bp.One cell lines named SA-Ⅲ,which can secret the diabody stably were generated.About 15 milligram diabody were purified from one liter culture supernatantat.Mr of the dimer was about 53kDa(anti-human CD80-ScFv Mr is about 27kDa).And the potency of anti-human CD80-diabody is 1.5μg/5×105 cells in comparation with murine parent antibody 4E5,and its positive binding rate with L929-CD80,Raji and Daudi cells is 96.5%,98.1% and 93% respectively,but diabody don’t bind with Jurkat cells.The diadoby not only can inhibit combination of 4E5 with CD80 antigen and its inhibition rate is 97.11%,but also inhibit in vitro proliferation of Raji cells and corresponding rate is 31.27%.Finally the antibody can also block costimulatory signal.Compared with ScFv,the affinity of diabody has improved obviously.Conclusion: Anti-human CD80-diabody secreting cell SA-Ⅲwas generated and the diabody have good biological function. The affinity of diabody improved clearly.All those supported a base for future study of CD80.更多还原