抗人CD44单克隆抗体HI313生物学功能研究及其单链抗体的构建

发布时间：2018-01-02 12:37

本文关键词：抗人CD44单克隆抗体HI313生物学功能研究及其单链抗体的构建　出处：《中国协和医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 实验目的:探讨小鼠抗人CD44单克隆抗体(HI313)对髓系白血病细胞增殖、分化的影响。构建HI313单链抗体(ScFv),为进一步治疗性抗体开发打下基础。实验方法:通过流式细胞仪检测,比较小鼠抗人CD44单克隆抗体HI313与HI44a的亲合力及竞争抑制作用;以胎盘兰计数和MTS法检测HI313对白血病细胞系增殖抑制的影响;Annexin V/PI和The Cycle TESE~(TM) PLUSDNA Reagent Kit检测HI313对NB4细胞凋亡及细胞周期影响;以及HI313对AML病人血细胞诱导分化作用;RT-PCR的方法克隆小鼠杂交瘤HI313的抗体轻、重链可变区基因,利用overlap-PCR方法构建HI313-ScFv融合基因,定向克隆入原核表达载体pET22b,构建重组质粒pET22b/HI313-ScFv。将重组质粒转染入BL21感受态菌株,在IPTG存在条件下,诱导表达HI313单链抗体。所得融合蛋白经复性、镍柱纯化后,通过流式细胞术检测其与天然细胞膜CD44蛋白的结合活性和特异性。实验结果:通过THP-1和KG1a细胞的检测,HI313克隆的相对亲合力分别为1.2nM和1.34nM;HI44a克隆的相对亲和力分别为3.5nM和5.89nM;免疫竞争实验表明HI313与HI44a识别不同抗原表位;HI313单克降抗体对NB4白血病细胞系具有强烈的抑制增殖作用,并使NB4细胞周期停留在G0/G1期;对AML病人血细胞诱导分化作用结果显示HI313可使髓系表面的成熟标志表达量增加。扩增HI1313重链可变区基因357bp,HI313轻链可变区基因339bp;成功构建HI313-ScFv单链抗体,能与KG1a细胞表面抗原结合。实验结论:HI313单克隆抗体具有高亲和力;且与HI44a克隆结合的不是一个抗原表位;HI313对NB4细胞具有增殖抑制作用,作用机制可能与阻滞细胞周期于G0/G1期相关;HI313能有效诱导AML病人血细胞的分化,并对AML病人的各亚型有一定的促分化作用,提示此抗体具有针对AML进行靶向治疗的潜力,为HI313人抗体的进一步基因工程改造及临床应用奠定了基础。
[Abstract]:Objective: To explore the effect of murine anti human CD44 monoclonal antibody (HI313) on proliferation and differentiation of myeloid leukemia cells, and construct HI313 single chain antibody (ScFv), so as to lay a foundation for further development of therapeutic antibodies.
Methods: by flow cytometry, affinity and competition comparison of mouse monoclonal antibody HI313 against human CD44 and HI44a inhibition; HI313 detected by trypan blue counting and MTS method in inhibiting proliferation of leukemia cell lines; Annexin V/PI and The Cycle TESE~ PLUSDNA Reagent Kit (TM) effect on the cell cycle and the detection of HI313 the apoptosis of NB4 cells; and HI313 of AML patients blood cell differentiation induced by RT-PCR antibody; cloned mouse hybridoma HI313 light and heavy chain variable gene, construct HI313-ScFv fusion gene by overlap-PCR method and cloned into the prokaryotic expression vector pET22b to construct recombinant plasmid pET22b/HI313-ScFv. plasmid was transfected into BL21 competent strains and in the condition of IPTG, induced expression of HI313 scFv. The fusion protein after refolding, nickel column purification, by detecting the natural fine flow cytometry The binding activity and specificity of CD44 protein in the cell membrane.
Results: the detection of THP-1 and KG1a cells, the relative affinity of HI313 clones were 1.2nM and 1.34nM; the relative affinity of HI44a clones were 3.5nM and 5.89nM; immune competition experiments show that HI313 and HI44a recognize different antigen epitope; monoclonal antibody HI313 to inhibit the proliferation of NB4 strong leukemia cell line NB4, and the cell cycle arrest in the G0/G1 phase of AML patients; blood cell differentiation results show that HI313 can make the mature myeloid surface marker expression increased. Amplification of HI1313 gene of heavy chain variable region of 357bp gene, 339bp HI313 light chain variable region; the successful construction of HI313-ScFv single chain antibody binds to KG1a cell surface antigen.
Conclusion: HI313 monoclonal antibody with high affinity; and combined with HI44a cloning is not an epitope; HI313 can inhibit the proliferation of NB4 cells. The mechanism may be related to cell cycle arrest in G0/G1 phase; HI313 can effectively induce the differentiation of blood cells in AML patients, and AML patients have different subtypes differentiation of the antibody against AML with the targeted therapeutic potential, laid the foundation for further improvement of genetic engineering HI313 antibody and its clinical application.

【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】：2008
【分类号】：R392

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