巨噬细胞激活剂Polytuftsin在毕赤酵母中的表达及其活性检测

发布时间：2018-01-03 12:11

本文关键词：巨噬细胞激活剂Polytuftsin在毕赤酵母中的表达及其活性检测　出处：《中国协和医科大学》2009年硕士论文　论文类型：学位论文

更多相关文章： Polytuftsin 毕赤酵母 巨噬细胞

【摘要】： 目的: 构建可持续分泌表达巨噬细胞激活剂Polytuftsin蛋白的重组毕赤酵母菌株,鉴定所获得的Polyttuftsin纯化重组蛋白的生物学活性。方法: 合成串联编码40个Tuftsin单体以及6His纯化标签的载体质粒pPIC9K-6His-Polytuftsin;PEG1000化学法将线性化后的载体质粒转化入毕赤酵母菌GS115中,筛选鉴定获得高拷贝阳性转化子;甲醇培养基诱导其分泌表达Polytuftsin蛋白,亲和纯化蛋白粗品后鉴定最终得到的纯化蛋白:体内、外水平上分别检测纯化后的Polytuftsin重组蛋白的生物学功能。结果: 1)成功构建了可持续分泌表达Polytuftsin蛋白的重组毕赤酵母菌株,并证实经亲和纯化后得到的Polytuftsin重组蛋白是正确的目的产物,分子量约为20Kda; 2)纯化后的Polytuftsin重组蛋白能够提高鼠源巨噬细胞分泌NO、TNF-α因子的能力,较对照组相比,提高比率可达到43%;与此同时,还可提高人源巨噬细胞分泌NO因子能力,较对照组相比,提高比率可达到56%; 3)纯化后的Polytuftsin重组蛋白能够提高小鼠腹腔巨噬细胞的杀瘤能力,杀伤率可达到50%;利用Polytuftsin纯化蛋白刺激后的人源巨噬细胞对人胰腺癌细胞系JF305的杀伤活力最大,可达31%; 4)体内小鼠L1210白血病模型治疗实验结果发现:较对照组相比,给予Polytuftsin重组蛋白后的小鼠出腹水时间延迟,且小鼠生存期延长,平均达到21天。结论: 经毕赤酵母表达系统获得的Polytuftsin重组蛋白,可以有效激活巨噬细胞,是一种新型的巨噬细胞蛋白激活剂。
[Abstract]:Objective: A recombinant Pichia pastoris strain expressing macrophage activator Polytuftsin protein was constructed. The biological activity of recombinant protein purified by Polyttuftsin was identified. Methods: The vector plasmid pPIC9K-6His-Polytuftsin encoding 40 Tuftsin monomers and 6His purified label was synthesized. The linearized vector plasmid was transformed into Pichia pastoris GS115 by PEG1000 chemical method, and high copy positive transformants were obtained by screening and identification. Methanol medium induced the secretion and expression of Polytuftsin protein. The purified protein was identified as the final purified protein: in vivo. The biological function of purified Polytuftsin recombinant protein was detected in vitro. Results: 1) A recombinant Pichia pastoris strain expressing Polytuftsin protein was successfully constructed. The recombinant Polytuftsin protein obtained by affinity purification was proved to be the correct target product with a molecular weight of about 20kda. 2) the purified Polytuftsin recombinant protein could enhance the ability of murine macrophages to secrete NO-TNF- 伪, and the ratio of increase was 43% compared with the control group. At the same time, the ability of human macrophages to secrete no factor was increased, and the ratio of increase reached 56% compared with the control group. 3) the purified Polytuftsin recombinant protein could improve the tumor killing ability of murine peritoneal macrophages, and the killing rate could reach 50%. Human macrophages stimulated by purified Polytuftsin protein had the highest killing activity against human pancreatic cancer cell line JF305, up to 31%. 4) the experimental results of L1210 leukemia model in vivo showed that: compared with the control group, the ascites time of mice treated with Polytuftsin recombinant protein was delayed. The survival time of mice was prolonged, reaching 21 days on average. Conclusion: The recombinant Polytuftsin protein obtained by Pichia pastoris expression system can effectively activate macrophages and is a new type of macrophage protein activator.
【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】：2009
【分类号】：R329

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