【作者】 徐耀瑜；

【导师】 邱玉华；

【作者基本信息】 苏州大学 ， 免疫学， 2008， 硕士

【摘要】 本研究在自行成功研制的鼠抗人CD80阻断型单克隆抗体(4E5)的基础上,采用嵌合抗体构建方法,在真核细胞CHO中实现稳定表达,并对嵌合抗体生物学功能进行初步研究。第一部分:CD80鼠-人嵌合抗体的构建及表达从4E5杂交瘤细胞中抽取总RNA,常规逆转录,应用简并引物进行PCR扩增。根据重、轻链基因序列设计引物,应用SMART-PCR方法扩增含信号肽序列的V_H和V_L基因。同时用特异性引物从pIRES/hu5C11质粒中扩增人IgG1γ链的Fc、CH1及κ链的Cκ基因。再利用TP-PCR方法分别将Fc、CH1和含信号肽序列的V_H序列进行拼接得到嵌合重链,Cκ与含信号肽序列的V_L序列进行拼接获得嵌合轻链。构建共表达嵌合重、轻链的重组质粒pIRES/ch4E5。pIRES/ch4E5重组质粒用脂质体法转染293T细胞,经FCM检测培养上清中嵌合抗体的瞬时表达后,再转染CHO细胞,经G418加压筛选,获取持续稳定分泌嵌合抗体的CHO-ch4E5细胞。研究结果表明:成功构建了含嵌合重、轻链基因的真核表达载体pIRES/ch4E5,并分别在293T及CHO细胞中得到瞬时表达及稳定表达。第二部分:CD80鼠-人嵌合抗体生物学功能的初步研究大量收集CHO-ch4E5细胞无血清培养上清,经protein G亲和层析法纯化及Lowry法定量,SDS-PAGE鉴定嵌合抗体的纯度及分子量,FCM分析嵌合抗体对多种肿瘤细胞Daudi、SH2-1及U937等细胞膜型CD80分子的识别。选择天然高表达CD80分子的人B淋巴瘤细胞株Daudi(阳性表达率＞90%)与ch4E5(终浓度为10μg/ml)共培养,MTT和FCM分析ch4E5对Daudi细胞的生长与存活的影响。采用竞争抑制法分析ch4E5与母本抗体的竞争抑制作用及MTT对MLR的影响作用。ELISA检测ch4E5与PBLs共培养后IL-2、INF-γ及IL-10的水平。结果表明:CHO-ch4E5细胞培养上清中嵌合抗体的得率为4～5.8 mg/L。ch4E5能够与4E5相互竞争抑制抗原抗体结合,并有效识别Daudi细胞膜型CD80分子(结合率为95.5%)。ch4E5能有效抑制Daudi细胞体外增殖(P=0.000067＜0.05),并诱导其凋亡。ch4E5抑制PBLs体外增殖(P=0.000012＜0.05),下调分泌IL-2(P=0.000156＜0.05)及INF-γ(P=0.000001＜0.05),上调分泌IL-10(P=0.000035＜0.05)。提示CD80嵌合抗体具有良好的生物学活性。本研究获得的成果:1、成功构建了抗人CD80鼠-人嵌合抗体(ch4E5)真核表达质粒。2、获得了稳定分泌嵌合抗体的细胞株CHO-ch4E5及相应的纯品抗体。3、CD80嵌合抗体可有效抑制天然高表达CD80分子的人B淋巴瘤细胞株Daudi的体外增殖;抑制混合淋巴细胞反应。该抗体在某些肿瘤的免疫治疗及移植抗排异中具有潜在的应用价值。更多还原

【Abstract】 This research was based on the human CD80 antagonist mouse monoclonal antibody (4E5)established by us.We constructed and expressed one mouse-human chimeric antibody against CD80 in CHO cells,and characterized its primary biological function.PartⅠ:construction and expression of mouse-human chimeric antibody against CD80After V_H and V_L gene were amplified by RT-PCR using degenerate primer from 4E5 murine hybridoma cell line,signal peptide sequences of V_H and V_L gene were amplified by SMART-PCR using special primer.Fc,CH1 and Cκgene of human IgG1 were also amplified from pIRES/hu5C11 plasmid.Variable genes containing signal peptide sequences and constant regions of human IgG1 were respectively fused to construct chimeric heavy and light chain by TP-PCR.Chimeric genes were inserted into pIRES to construct co-expressing recombinant plasmid.Recombinant plasmid was firstly transfected into 293T cells using LipofectAMIN kit.Once FCM had detected transient expression of chimeric antibody,recombinant plasmid was transfected into CHO cells with same method,and cells was pressurized by G418 to obtain CHO cell line constantly expressing ch4E5 antibody.Restriction endonuclease digestion and PCR showed that recombinant genes have been cloned into vector pIRES.Chimeric antibody could transiently express in 293T cells.RT-PCR and FCM pointed that chimeric antibody against CD80 specially is stably effectively expressed in CHO cell line successfully. PartⅡ:primary biological function of mouse-human chimeric antibody against CD80CHO-ch4E5 cells supernatant without FCS was purified by protein G affinity chromatography,quantity of purified antibody was detected using Lowry assay,and chimeric antibody was analyzed by SDS-PAGE and FCM.Competition inhibition between ch4E5 and 4E5 was detected by FCM.mCD80 on cancer cells(Daudi,SH2-1 and U937, et al)were identified by ch4E5 antibody,ch4E5 antibody(10μg/ml)how to act on proliferation and apoptosis of Daudi cells(conjunction ratio＞90%)was detected by MTT and FCM.When PBLs were cultured with ch4E5 antibody,change of proliferation and cytokine concentration was detected by MTT and ELISA.Results showed that concentration of chimeric antibody in cells supernatant is 4～5.8mg/L.ch4E5 and 4E5 could compete to bind CD80 antigen with each other,ch4E5 antibody identifies mCD80 on Daudi cells(conjunction ratio 95.5%).When 10μg/ml,ch4E5 effectively inhibits growth(P=0.000067＜0.05)and induces apoptosis of Daudi cells.Proliferation of PBLs in MLR is depressed(P=0.000012＜0.05).Concentration of IL-2(P=0.000156＜0.05)and INF-γ(P=0.000001＜0.05)secreted from PBLs are decreased,and that of IL-10 (P=0.000035＜0.05)is augmented.In conclusion,our study has constructed co-expressing recombinant plasmid pIERS/ch4E5,expressed mouse-human chimeric antibody against human CD80 in CHO cells successfully and set up artwork of cultivation and purification,ch4E5 antibody could depress proliferation of Daudi cells and inhibit mixed lymphocyte reaction(MLR).ch4E5 antibody has potency on tumor immunotherapy and graft rejection.更多还原