人重组白介素3的中试表达纯化及其在造血干细胞体外增殖中的研究

发布时间：2018-06-16 02:03

本文选题：rhIL-3 + 复性　；参考：《北京协和医学院》2012年硕士论文

【摘要】：目的：大规模表达复性并纯化脐带血干细胞(CBSC)曾殖因子人重组白介素3(rhIL-3),制备其单克隆抗体,研究蛋白及其抗体在干细胞增殖中的作用。方法：构建rhIL-3表达菌株,中试规模表达该蛋白并提取其包涵体,透析法复性蛋白,以离子交换柱层析结合分子筛获得电泳纯的rhIL-3。以纯化的蛋白免疫小鼠,制备杂交瘤,ELISA筛选单克隆抗体分泌株,并以western-blot鉴定抗体特异性。在CBSC增殖实验中,rhIL-3与干细胞因子(SCF,200ng/mL)、fms样酪氨酸激酶受体-3配体(FL,200ng/mL)口促血小板生成素(TPO,50ng/mL)共同作用于CBSC,通过培养后细胞总数和CD34+细胞数变化来检测其对干祖细胞增殖的影响,进一步将rhIL-3与其抗体共同孵育后进行共培养实验,检测其拮抗效果。成果：获取了电泳纯rhIL-3蛋白；制备得到鼠抗rhIL-3单克隆抗体IgG1两株和IgG2b一株；摸索到rhIL-3作用于脐血干祖细胞的最佳增殖条件为15-20ng/mL(无TPO)和50ng/mL(：有TPO)；筛选得到了特异性抑带(?)rhIL-3干祖细胞增殖活性的单克隆抗体两株。结论：研究表明表达纯化的rhIL-3具有良好的促脐带血干祖细胞增殖活性。该rhIL-3及其新获取的单克隆抗体对进一步研究脐带血干细胞增殖的实验室和临床研究有着极为重要的意义。
[Abstract]:Aim: to renaturation and purify recombinant human interleukin 3 rhIL 3 (rhIL 3) from cord blood stem cell (CBSCC), and to prepare its monoclonal antibody to study the role of protein and its antibody in stem cell proliferation. Methods: rhIL-3 expression strain was constructed. The protein was expressed in a pilot scale and its inclusion bodies were extracted. The purified rhIL-3 was obtained by ion exchange column chromatography combined with molecular sieve. Immunized mice with purified protein, hybridoma Elisa was prepared to screen monoclonal antibody secreting strain, and western-blot was used to identify the specificity of antibody. In CBSC proliferation test, rhIL-3 and stem cell factor SCF- (200ng / mL) tyrosine kinase receptor-3 ligand FL200ng / mL) acted on CBSCs. The effects of rhIL-3 on the proliferation of stem progenitor cells were determined by the changes of total cell count and CD34 cell number after culture. Furthermore, rhIL-3 was co-cultured with its antibody to detect its antagonistic effect. Results: electrophoretic pure rhIL-3 protein was obtained, mouse anti-rhIL-3 monoclonal antibody IgG1 strain and IgG2b strain were prepared, and the optimal conditions for rhIL-3 proliferation on umbilical cord blood stem progenitor cells were found to be 15-20 ng / mL (without TPOO) and 50ngmLL / L with TPOO. Two monoclonal antibodies to the proliferation activity of rhIL-3 stem progenitor cells were obtained. Conclusion: the expression and purification of rhIL-3 has a good proliferative activity in cord blood stem progenitor cells. The rhIL-3 and its newly obtained monoclonal antibodies are of great significance to further study the proliferation of umbilical cord blood stem cells in laboratory and clinic.
【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】：R329.2

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