【作者】 李建民；

【导师】 王海涛； 童贻刚；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2004， 博士

【摘要】 本研究选择在中国仓鼠卵巢(chinese hamster ovary，CHO)细胞中表达具有中和活性的抗基孔肯亚(chikungunya，Chik)病毒的嵌合抗体，以图为Chik病的治疗提供被动免疫制剂。为表达嵌合抗体，首先克隆具有中和活性的抗Chik病毒的鼠源单克隆抗体(monoclonal antibody，McAb)VL和VH基因及人工gG1亚类的CH和CK基因，通过融合PCR构建了抗Chik病毒的完整人-鼠嵌合抗体基因。将人-鼠嵌合抗体基因克隆到表达载体的多克隆位点(mutiple cloning site，MCS)，构建了恒定区(constant region，C区)为cDNA序列的表达质粒。将构建的嵌合抗体表达质粒转染CHO／dhfr-细胞，经氨甲喋呤(methotrexate，MTX)加压筛选，获得最高表达量为2μg／ml的细胞株。对此细胞株扩大培养并纯化抗Chik病毒的嵌合抗体。纯化的嵌合抗体在非还原SDS-PAGE中表现为一条约150kD的带，在还原SDS-PAGE中表现为一条约50kD和一条约25kD的带；Western印迹结果显示，全分子蛋白和重链(heavy chain，H链)分子均可与羊抗人IgG Fc发生特异性免疫反应；全分子蛋白和轻链(light chain，L链)分子均可与羊抗人Kappa发生特异性免疫反应。用间接免疫荧光实验(indirect immunofloresence assay，IFA)检测显示，纯化的嵌合抗体与Chik病毒抗原发生强反应，说明表达的嵌合抗体保留了亲本鼠源McAb的抗原结合活性。用不同浓度的纯化嵌合抗体和鼠源McAb进行微量细胞中和实验，结果25μg／ml的嵌合抗体和15μg／ml的鼠源McAb可以保护细胞不出现病变，说明嵌合抗体具有与鼠源McAb相同的生物学活性。为了提高嵌合抗体的表达量，我们尝试用Flp-InTM系统表达嵌合抗体，获得表达量为1μg／ml的稳定表达细胞株。遗憾的是此系统不能进行基因扩增，基于此，我们构建了在染色体的转录活性位点整合了FRT序列的CHO／dhfr-细胞株，命名为CHO／dhfr-FRT+。在此细胞株中，表达了抗chik病毒的嵌合抗体，通过MTX加压筛选，获得表达量为5μg／ml的稳定表达细胞株，是Flp-InTM表达系统的5倍，是CHO／dhfr-表达系统的2． 5倍。有报道称，Ck基因组序列能改善抗体在CHO细胞中的表达。但未见CH基因组序列是否较cDNA序列表达量高的报道。因此克隆人抗体分子IgG1亚类CH区基因组序列并构建了C区为基因组序列的双基因表达质粒，用F1p-InTM系统研究了C区为cDNA序列和基因组序列对抗体表达量的差异，结果显示CH区使用cDNA序列是基因组序列抗体表达量的3倍。更多还原

【Abstract】 The aim of the study is to express human-mouse chimeric antibody against chikungunya(Chik) virus used for a passive drug for the Chik disease.Both Vl and Vh genes were cloned by RT-PCR. Full length H and L chain genes of chimeric antibody were constructed by fusing Vl and Vh with Cr1 and Ck, respectively. The chimeric antibody expression plasmids were constructed by inserting full length H and L genes into the MCSs of the expression vectors.The plasmids constructed were transfected into the CHO/dhfr’ cells, chimeric antibody against Chik virus were delected positive in the supernatant of all the transfected cell cultures. A few high yielding cell clones were obtained by enhancing stepwisely MTX concentration. A stable and high productive cell line with yield of 2mgl-1 was obtained at MTX concentration of 2x10-7 mol/L. The chimeric antibody was purified by affinity chromatography and analyzed by non-reduced and reduced SDS-PAGE respectively. The molecular weights of the bands were consistant with the expected that is 150kD on the non-reduced gel and 50kD and 25kD on the reduced gel. Western blot showed that both the intact antibody molecular and H chain can specifically react with anti-human IgG Fc. Indirect immuno fluorescence assay (IFA) showed that the chimeric antibody has similar capacity of binding Chik virus antigen as mouse McAb. Cell culture neutralizing test (CCNT) with the purified chimeric antibody at six different concentrations from 6.25 to 100 mgl-1 was also undertaken to test whether the antibody is biologically active. The parent mAb was used as a positive control at 4 different concentrations from 7.5 to 60mgl-1. The result showed that the EC50(5O% effect concentration) of purified chimeric antibody was 25mgl-1 while the EC50 of the parent mouse antibody was 15mgl-1.To improve antibody expression in CHO cells, we attempt to express chimeric antibody with Flp-InTM system. A cell line with productivity of 1 ug/ml was attained. But the regret is that the system can ’t be amplified. Based on the above, a CHO/dhfr- cell clone with FRT sequence intergrating on the transcription-active site was constructed. A cell clone with productivity of 5mgl-1 was obtained copared to the Flp-In?system with 1mgl-1 and CHO/dhfr- system with 2mgl-1.Flp-InTM system was also used to clarify whether heavy chain constant region genomic gene can improve the expression of chimeric antibody or not. The result shows that the antibody expression from the plasmid carrying cDNA sequence is higher than that from Cr1 genome containing plasmid.更多还原