CD80鼠—人嵌合抗体的构建、表达及生物学功能的初步研究

发布时间：2018-04-29 16:03

本文选题：CD80 + 嵌合抗体　；参考：《苏州大学》2008年硕士论文

【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。
[Abstract]:On the basis of the mouse anti-human CD80 blocking monoclonal antibody 4E5), the stable expression of chimeric antibody in eukaryotic CHO was achieved by using chimeric antibody construction method, and the biological function of chimeric antibody was preliminarily studied. Part one: construction and expression of murine-human chimeric antibody against CD80 Total RNAs were extracted from 4E5 hybridoma cells and were amplified by PCR using degenerate primers. According to the sequence of heavy and light chain genes, primers were designed and amplified by SMART-PCR method. At the same time, specific primers were used to amplify human IgG1 纬 chain Fctch1 and 魏 chain C 魏 genes from pIRES/hu5C11 plasmids. Using TP-PCR method, the chimeric heavy chain C 魏 and the signal peptide sequence VL sequence were spliced to obtain the chimeric light chain. The recombinant plasmid pIRES/ch4E5.pIRES/ch4E5 was constructed and transfected into 293T cells by liposome method. The transient expression of chimeric antibody in the supernatant was detected by FCM, and then transfected into CHO cells. CHO-ch4E5 cells secreting chimeric antibodies were obtained. The results showed that the eukaryotic expression vector pIRESr / ch4E5 containing chimeric weight and light chain gene was successfully constructed, and transient expression and stable expression were obtained in 293T and CHO cells, respectively. Part two: a preliminary study on the biological function of mouse human chimeric antibody against CD80 A large number of supernatants of serum-free culture of CHO-ch4E5 cells were collected and purified by protein G affinity chromatography. The purity of chimeric antibodies and the recognition of cell membrane CD80 molecules such as Daudio SH2-1 and U937 were identified by Lowry quantitative SDS-PAGE. The natural human B-lymphoma cell lines with high expression of CD80 (positive rate > 90) and ch4E5 (final concentration 10 渭 g / ml) were cocultured. The effects of ch4E5 on the growth and survival of Daudi cells were analyzed. The competitive inhibition of ch4E5 and maternal antibody and the effect of MTT on MLR were analyzed by competitive inhibition method. Elisa was used to detect the levels of IL-2INF- 纬 and IL-10 after co-culture of ch4E5 and PBLs. The results showed that the yield of chimeric antibody in the supernatant of Cho ch4E5 cell culture was 45.8 mg/L.ch4E5, which could bind to 4E5. The Daudi cell membrane type CD80 molecule (the binding rate was 95.5%).ch4E5) could effectively inhibit the proliferation of Daudi cells in vitro and induce its apoptosis. Ch4E5 could inhibit the proliferation of PBLs in vitro and down-regulate the secretion of IL-2(P=0.000156 < 0.05), and INF- 纬 -P0. 0001 < 0. 05, and up-regulate the secretion of IL-10(P=0.000035 < 0. 05. The results suggest that CD80 chimeric antibody has good biological activity. The result of this study was: 1, successfully constructed the eukaryotic expression plasmid of anti-human CD80 murine-human chimeric antibody ch4E5). The cell line CHO-ch4E5 and the corresponding purified antibody, .3mCD80 chimeric antibody, which secreted the chimeric antibody stably, could effectively inhibit the natural high surface. Proliferation of human B lymphoma cell line Daudi with CD80 molecule in vitro; Inhibition of mixed lymphocyte reaction. This antibody has potential application value in immunotherapy and transplantation of some tumors.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【参考文献】