小鼠抗人CD52单克隆抗体HI186的生物学功能研究及其单链抗体的构建

发布时间：2019-01-05 11:10

【摘要】： 实验目的：初步研究小鼠源性抗CD52单克隆抗体HI186的生物学功能。利用现有小鼠杂交瘤制备HI186单链抗体(ScFv)，结合其生物学功能的研究，为日后应用于临床治疗打下基础。实验方法：1．应用胎盼蓝染色计数活细胞、MTT法检测活细胞和Annexin V染色特异性检测凋亡细胞等方法研究不同克隆株的抗CD52单克隆抗体对部分白血病细胞系的作用，比较其生物学功能和作用的异同。2．RT-PCR的方法克隆小鼠杂交瘤HI186的抗体基因轻、重链可变区基因，，利用overlap-PCR方法构建HI186-ScFv融合基因，定向克隆入原核表达载体pET22b，构建重组质粒pET22b／HI186-ScFv。将重组质粒转染入BL2 1感受态菌株。在IPTG存在条件下，诱导表达HI186单链抗体。所得融合蛋白经复性、纯化后，通过流式细胞术检测其与天然细胞膜CD52蛋白的结合活性和特异性；Western blot印证其与变性线形蛋白结合活性。实验结果： 1．HI186单克隆抗体对于CEM、LCL-H、HPB-ALL、REH等CD52阳性白血病细胞系在体外具有一定的生长抑制和促凋亡作用。 2．扩增HI186重链可变区基因342bp，m186轻链可变区基因339bp。 3．overlap-PCR构建包含linker的HI186ScFv单链抗体融合基因，全长为759bp，经酶切转入pET22b质粒，构建pET22b／HI186-ScFv，在IPTG存在下可诱导高表达HI186-ScFv单链抗体融合蛋白。 4．抗体免疫竞争实验表明HI186-ScFv单链抗体与原鼠源性HI186单克隆抗体具有相同的结合表位，可以抑制HI186单克隆抗体与细胞膜表面CD52天然抗原的结合，同时也可以结合变性的具有良好的结合活性和特异性。实验结论：HI186单克隆抗体生物学实验表明对相应的白血病细胞系具有抑制生长作用，并且具有诱导凋亡作用。在成功构建了HI186-ScFv单链抗体的基础上，应进一步研究其应用的可能性，并据此对其进行继续改造。
[Abstract]:Objective: to study the biological function of murine anti-CD52 monoclonal antibody HI186. The preparation of HI186 single chain antibody (ScFv),) from mouse hybridoma combined with its biological function will lay a foundation for clinical treatment in the future. Methods: 1. To study the effect of monoclonal antibody against CD52 on some leukemia cell lines by using the method of fetal trypan blue staining to count living cells, MTT assay to detect living cells and Annexin V staining to detect specific apoptotic cells, etc. 2.RT-PCR method was used to clone mouse hybridoma HI186 antibody gene with light and heavy chain variable region gene. HI186-ScFv fusion gene was constructed by overlap-PCR method and cloned into prokaryotic expression vector pET22b,. Construction of Recombinant plasmid pET22b/HI186-ScFv. The recombinant plasmid was transfected into BL2 1 competent strain. In the presence of IPTG, the expression of HI186 scFv was induced. The fusion protein was renatured and purified. The binding activity of the fusion protein to the CD52 protein of natural cell membrane and the specific; Western blot activity were determined by flow cytometry to confirm the binding activity of the fusion protein to denatured linear protein. The results showed that 1.HI186 monoclonal antibody could inhibit the growth and promote apoptosis of CD52 positive leukemia cell lines such as CEM,LCL-H,HPB-ALL,REH in vitro. 2.Amplification of HI186 heavy chain variable region gene 342bpnm186 light chain variable region gene 339bp. HI186ScFv single chain antibody fusion gene containing linker was constructed by 3.overlap-PCR. The full length of the fusion gene was 759 BP. The fusion gene was digested into pET22b plasmid. PET22b/HI186-ScFv, could induce high expression of HI186-ScFv single chain antibody fusion protein in the presence of IPTG. 4. The immuno-competitive assay showed that HI186-ScFv single-chain antibody had the same binding epitope with proto-murine HI186 monoclonal antibody, and could inhibit the binding of HI186 monoclonal antibody to the natural CD52 antigen on the surface of cell membrane. At the same time, it can also bind to denaturation with good binding activity and specificity. Conclusion: HI186 monoclonal antibody can inhibit the growth and induce apoptosis of leukemia cell line. Based on the successful construction of HI186-ScFv scFv, the possibility of its application should be further studied and modified accordingly.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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