蜂毒素与基因变构IL-2嵌合蛋白的纯化工艺探索和生物学活性研究

发布时间：2018-06-24 10:11

  本文选题：蜂毒素与基因变构IL-2嵌合蛋白 + 表达　； 参考：《青岛大学》2007年硕士论文

【摘要】： ①目的：将含重组质粒pGEX-4T-2／melittin-~(88)ArgIL-2的大肠杆菌进行表达发酵条件的优化，得到可溶性表达的重组蛋白；分离纯化该重组蛋白，得到高纯度的、足量的、具有生物学活性的蜂毒素与基因变构IL-2fmelittin-~(88)ArgIL-2)嵌合蛋白，探索建立该蛋白的纯化工艺；研究此嵌合蛋白在体外增强免疫功能和抗肿瘤的生物学活性。②方法：将含重组质粒的大肠杆菌DH5a，经IPTG诱导进行高效表达，经反复实验摸索表达出可溶性蛋白；经过亲和层析、凝血酶Thrombin酶切和离子交换层析进行纯化，纯化的重组melittin-~(88)ArgIL-2嵌合蛋白，经SDS-PAGE电泳鉴定蛋白纯度并进行定量；用液相测定法研究嵌合蛋白的抑菌活性，用MTT法研究嵌合蛋白对外周血单个核细胞PBMC增殖、NK细胞杀伤活性、人卵巢癌细胞SKOV_3和Hela细胞生长抑制的作用。③结果：在25℃和IPTG0.8mmol／L条件下，表达出了可溶性蛋白；经过一系列的分离纯化步骤，纯化后的重组melittin-~(88)ArgIL-2嵌合蛋白纯度达95％，蛋白浓度大约200mg／L，建立了该嵌合蛋白的纯化工艺；纯化后的嵌合蛋白在体外能抑制金黄色葡萄球菌和大肠杆菌的生长，能促进PBMC细胞增殖，能增强NK细胞的杀伤活性，能抑制人卵巢癌细胞SKOV3和Hela细胞的生长增殖。④结论：建立了melittin-~(88)ArgIL-2嵌合蛋白的纯化工艺，，该嵌合蛋白在体外具有一定的抑菌作用，具有一定的增强免疫功能和抗肿瘤活性。嵌合蛋白具有能协同基因变构IL-2与蜂毒素增强细胞免疫的作用，并且其抗肿瘤活性得到激活并显著增强。为其在真核细胞系统的表达、纯化、体内的生物学活性研究以及大规模制备的中试放大研究和临床前期研究奠定了基础。
[Abstract]:Objective: to optimize the fermentation conditions of Escherichia coli containing recombinant plasmid pGEX-4T-2 / melittin-88 ArgIL-2, to obtain soluble recombinant protein, to isolate and purify the recombinant protein, and to obtain high purity and sufficient amount of recombinant protein. The bioactive chimeric protein of melittin-88 (IL-2fmelittin-88) chimeric protein with biological activity was studied and the purification process of the protein was explored. To study the enhancement of immune function and biological activity of the chimeric protein in vitro. Methods: the recombinant plasmid containing E. coli DH 5a was highly expressed by IPTG, and the soluble protein was expressed by repeated experiments. The purified recombinant melittin-88 ArgIL-2 chimeric protein was purified by Thrombin digestion and ion exchange chromatography. The protein purity was identified by SDS-PAGE electrophoresis, and the bacteriostatic activity of the chimeric protein was studied by liquid chromatography. MTT assay was used to study the cytotoxicity of chimeric protein on PBMC proliferation and NK cell proliferation of peripheral blood mononuclear cells. The results showed that soluble protein was expressed at 25 鈩

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