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  本文關(guān)鍵詞： 重組人干細胞因子 原核表達 單克隆抗體 臍帶血體外擴增　出處：《北京協(xié)和醫(yī)學院》2012年碩士論文　論文類型：學位論文

【摘要】：目的：通過大腸桿菌原核表達系統(tǒng),由包涵體經(jīng)純化復(fù)性后獲取重組人干細胞因子(recombinant human stem cell factor, rhSCF)蛋白,同時制備相應(yīng)的單克隆抗體,以新鮮臍血中分離的單核細胞(mononuclear cell, MNC)對SCF及其單抗的活性進行檢測。 方法：通過大腸桿菌表達系統(tǒng),經(jīng)IPTG誘導表達獲取SCF包涵體,經(jīng)透析復(fù)性,CM-Sepharose純化后獲得蛋白。用Ficoll密度梯度離心法從臍帶血中提取MNC,利用免疫磁珠分選法(magnetic cell sorting, MACS)分選其中具有CD34+抗原表位的細胞,根據(jù)之前實驗結(jié)果,使用一定濃度的血小板生成素(thrombopoietin、FLT-3配體(FLT-3ligand, FL)及實驗獲取的重組人干細胞因子(rhSCF)組合對臍帶血進行七天體外擴增以檢測rhSCF生物活性。制備篩選得到高滴度單克隆細胞株,使用該單克隆抗體通過臍帶血體外擴增觀察該單抗對rhSCF生物活性的抑制作用。培養(yǎng)七天后通過流式細胞術(shù)檢測CD34+陽性細胞比例。使用肽圖分析對rhSCF進行結(jié)構(gòu)和功能研究。 結(jié)果：成功使用IPTG在大腸桿菌BL21誘導表達rhSCF蛋白,包涵體經(jīng)復(fù)性及CM-Sepharose純化獲得高純度的rhSCF蛋白。篩選獲得高滴度的rhSCF單克隆抗體細胞株23C8。干細胞體外擴增實驗表明rhSCF蛋白FL及TPO聯(lián)用,具有協(xié)同刺激CD34+細胞體外擴增的作用；單克隆抗體23C8可以特異性的抑制rhSCF促進干細胞體外擴增的生物活性。肽圖分析結(jié)果表明,rhSCF的活性位點位于第93-165位氨基酸序列中。 結(jié)論：成功建立rhSCF原核表達、復(fù)性和純化體系。rhSCF蛋白具有良好生物學活性,與同類標準品相比無明顯差異；獲得可分泌高滴度單克隆抗體的23C8單克隆細胞株；實驗表明單克隆抗體23C8的結(jié)構(gòu)域可結(jié)合rhSCF的生物活性位點,能特異性抑制rhSCF的活性,阻止CD34+細胞體外擴增。rhSCF同MAb結(jié)合的活性位點位于第93-165位氨基酸序列中。該課題為造血干／祖細胞體外擴增體系的優(yōu)化研究奠定了基礎(chǔ)。同時為該細胞因子的臨床應(yīng)用提供了有價值的參考。
[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.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392.12

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本文編號：1506697