重组人干细胞因子的表达、复性、纯化及其对脐带血体外扩增作用研究

发布时间：2018-02-12 22:23

本文关键词： 重组人干细胞因子原核表达单克隆抗体脐带血体外扩增　出处：《北京协和医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的：通过大肠杆菌原核表达系统,由包涵体经纯化复性后获取重组人干细胞因子(recombinant human stem cell factor, rhSCF)蛋白,同时制备相应的单克隆抗体,以新鲜脐血中分离的单核细胞(mononuclear cell, MNC)对SCF及其单抗的活性进行检测。方法：通过大肠杆菌表达系统,经IPTG诱导表达获取SCF包涵体,经透析复性,CM-Sepharose纯化后获得蛋白。用Ficoll密度梯度离心法从脐带血中提取MNC,利用免疫磁珠分选法(magnetic cell sorting, MACS)分选其中具有CD34+抗原表位的细胞,根据之前实验结果,使用一定浓度的血小板生成素(thrombopoietin、FLT-3配体(FLT-3ligand, FL)及实验获取的重组人干细胞因子(rhSCF)组合对脐带血进行七天体外扩增以检测rhSCF生物活性。制备筛选得到高滴度单克隆细胞株,使用该单克隆抗体通过脐带血体外扩增观察该单抗对rhSCF生物活性的抑制作用。培养七天后通过流式细胞术检测CD34+阳性细胞比例。使用肽图分析对rhSCF进行结构和功能研究。结果：成功使用IPTG在大肠杆菌BL21诱导表达rhSCF蛋白,包涵体经复性及CM-Sepharose纯化获得高纯度的rhSCF蛋白。筛选获得高滴度的rhSCF单克隆抗体细胞株23C8。干细胞体外扩增实验表明rhSCF蛋白FL及TPO联用,具有协同刺激CD34+细胞体外扩增的作用；单克隆抗体23C8可以特异性的抑制rhSCF促进干细胞体外扩增的生物活性。肽图分析结果表明,rhSCF的活性位点位于第93-165位氨基酸序列中。结论：成功建立rhSCF原核表达、复性和纯化体系。rhSCF蛋白具有良好生物学活性,与同类标准品相比无明显差异；获得可分泌高滴度单克隆抗体的23C8单克隆细胞株；实验表明单克隆抗体23C8的结构域可结合rhSCF的生物活性位点,能特异性抑制rhSCF的活性,阻止CD34+细胞体外扩增。rhSCF同MAb结合的活性位点位于第93-165位氨基酸序列中。该课题为造血干／祖细胞体外扩增体系的优化研究奠定了基础。同时为该细胞因子的临床应用提供了有价值的参考。
[Abstract]:Objective: to obtain recombinant human stem cell factor human stem cell factor (rhSCF) protein from E. coli prokaryotic expression system, and to prepare corresponding monoclonal antibody. The activity of SCF and its monoclonal antibodies was detected by mononuclear cells (MNCCs) isolated from fresh umbilical cord blood. Methods: the inclusion bodies of SCF were obtained by the expression system of Escherichia coli and induced by IPTG. Ficoll density gradient centrifugation was used to extract MNCs from cord blood. The cells with CD34 antigen epitopes were sorted by immunomagnetic bead sorting. Umbilical cord blood was amplified in vitro with a certain concentration of thrombopoietinnin-FLT-3 ligand (FLT-3) and recombinant human stem cell factor (rhSCF) for seven days to detect the bioactivity of rhSCF. High titer monoclonal cell lines were obtained. The inhibitory effect of the monoclonal antibody on the biological activity of rhSCF was observed by using umbilical cord blood amplification in vitro. After 7 days of culture, the proportion of CD34 positive cells was detected by flow cytometry. The structure and function of rhSCF were studied by peptide analysis. Results: IPTG was successfully used to induce the expression of rhSCF protein in Escherichia coli BL21. High purity rhSCF protein was obtained by renaturation and CM-Sepharose purification, and high titer rhSCF monoclonal antibody cell line 23C8. the results of stem cell amplification in vitro showed that the combination of rhSCF protein FL and TPO could stimulate CD34 cells to expand in vitro. Monoclonal antibody 23C8 could specifically inhibit the biological activity of rhSCF to promote the expansion of stem cells in vitro. The results of peptide map analysis showed that the active site of rhSCF was located in the amino acid sequence at the 93-165 position. Conclusion: the prokaryotic expression, renaturation and purification system of rhSCF. RhSCF protein has good biological activity, and there is no significant difference compared with the same standard, the 23C8 monoclonal cell line which can secrete high titer monoclonal antibody is obtained. The results showed that the domain of monoclonal antibody 23C8 could bind to the bioactive site of rhSCF and specifically inhibit the activity of rhSCF. The active site of inhibiting the expansion of CD34 cells with MAb in vitro is located in the amino acid sequence at the 93-165 position. This subject lays a foundation for the optimization of the in vitro amplification system of hematopoietic stem / progenitor cells, and also provides a basis for the clinical application of the cytokine. The application provides a valuable reference.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.12

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