sflk1-IFN-γ融合蛋白的分离与纯化

发布时间：2018-01-05 08:07

本文关键词：sflk1-IFN-γ融合蛋白的分离与纯化　出处：《浙江大学》2007年硕士论文　论文类型：学位论文

【摘要】： 恶性肿瘤的生长、浸润和转移必须依靠肿瘤新生血管提供足够的营养，因此抑制或破坏肿瘤血管生成已成为近年来肿瘤治疗的新方法。血管内皮生长因子(VEGF)与表达在血管内皮细胞上的相应受体VEGFR2结合所引起的信号转导是血管生成中的限速步骤，在整个血管生成中发挥着关键作用。可溶性血管内皮生长因子受体2(sVEGFR2，在小鼠中被称为sflk1)可竞争性地结合VEGF，从而阻断VEGF与VEGFR2结合所引起的信号转导，抑制肿瘤细胞诱导的血管生成和肿瘤的生长。IFN-γ是细胞免疫应答中非常重要的效应因子，能诱导CTL应答及Th1细胞的分化，并且尚能直接抑制多种肿瘤的生长，同时它自身也有抑制肿瘤血管生成的作用。本所已经构建了pcDNA3.1(+)／sflk1-IFN-γ重组质粒，获得高效稳定表达的CHO细胞，并对其表达产物sflk1-IFN-γ融合蛋白的生物学活性进行了初步研究。目的：为了进一步研究sflk1-IFN-γ融合蛋白的结构、理化性质、生物学活性以及进行动物试验等，本实验对CHO细胞上清液中sflk1-IFN-γ融合蛋白进行分离与纯化，拟获得纯度较高的融合蛋白，并探讨真核细胞表达所蛋白的分离纯化方法。方法：大规模培养高效稳定表达sflk1-IFN-γ融合蛋白的CHO细胞，收集上清，经超滤、盐析或阴离子交换层析(IEX)方法进行初步的浓缩与分离，再经疏水层析(HIC)进行中度纯化及凝胶过滤(GF)精细纯化并采用SDS-PAGE电泳进行蛋白纯度的鉴定。结果：1．利用低血清(5％FCS)培养表达sflk1-IFN-γ融合蛋白的CHO细胞，收集的上清中总蛋白的浓度为1741±40μg／ml，，sflk1的浓度为55.2±6.70ng／ml，纯度为2.72～3.64×10~(-3)％，IFN-γ的浓度为1.4±0.78ng／ml，纯度为0.03～0.14×10~(-3)％； 2．通过盐析可将细胞上清液中的sflk1-IFN-γ融合蛋白中IFN-γ纯度提高13.25倍，回收率30.02％；利用超滤膜Biomax-30浓缩细胞上清液，可将细胞上清液中的sflk1-IFN-γ融合蛋白中IFN-γ纯度提高3.38倍，回收率35.1％；采用阴离子交换层析可将细胞上清液中的sflk1-IFN-γ融合蛋白中IFN-γ纯度提高4.18倍，回收率48％； 3．经疏水层析中度纯化，可将细胞上清液中的sflk1-IFN-γ融合蛋白中sflk1纯度由4.37×10~(-3)％提高到48×10~(-3)％，回收率19.2％； 4．经凝胶过滤精细纯化，可将疏水层析收集液中的sflk1-IFN-γ融合蛋白中sflk1纯度由48×10~(-3)％提高到2.34％以上。结论：蛋白质初步浓缩后，再经疏水层析及凝胶过滤可将CHO细胞所表达的sflk1-IFN-γ融合蛋白的纯度提高500多倍，sflk1的纯度可达2.34％，本论文中采用的方法对于真核体系表达的蛋白有一定的纯化作用。
[Abstract]:The growth, invasion and metastasis of malignant tumors must depend on tumor neovascularization to provide adequate nutrition. Therefore, inhibiting or destroying tumor angiogenesis has become a new method of tumor therapy in recent years. Vascular Endothelial growth Factor (VEGF). Signal transduction induced by binding to the corresponding receptor VEGFR2 expressed on vascular endothelial cells is a rate-limiting step in angiogenesis. Soluble vascular endothelial growth factor receptor 2sVEGFR2, known as sflk1 in mice, can competitively bind to VEGF. Thus blocking the signal transduction caused by the combination of VEGF and VEGFR2, inhibiting angiogenesis induced by tumor cells and tumor growth. IFN- 纬 is a very important effector factor in cellular immune response. It can induce CTL response and the differentiation of Th1 cells, and can directly inhibit the growth of many kinds of tumors. At the same time, it can inhibit tumor angiogenesis. PcDNA3.1 (rsflk1-IFN- 纬) recombinant plasmid has been constructed to obtain high and stable expression of CHO cells. The biological activity of sflk1-IFN- 纬 fusion protein was studied. Objective: to further study the structure, physicochemical properties, biological activity and animal test of sflk1-IFN- 纬 fusion protein. The fusion protein sflk1-IFN- 纬 from supernatant of CHO cells was isolated and purified in this experiment. The fusion protein with high purity was obtained and the method of purification of the protein expressed by eukaryotic cells was discussed. Methods: CHO cells expressing sflk1-IFN- 纬 fusion protein were cultured on a large scale and supernatant was collected and ultrafiltration. The method of salting out or anion exchange chromatography (IEX) was used to concentrate and separate. The protein was purified by hydrophobic chromatography (HIC) and purified by gel filtration. The purity of the protein was identified by SDS-PAGE electrophoresis. Results: 1. CHO cells expressing sflk1-IFN- 纬 fusion protein were cultured with low serum FCS. The concentration of total protein in the supernatant was 1741 卤40 渭 g / ml. The concentration of sflk1 was 55.2 卤6.70 ng / ml, and the purity was 2.72 卤3.64 脳 10 ~ (-1) ng / ml. The concentration of IFN- 纬 was 1.4 卤0.78 ng / ml. The purity is 0.03 ~ 0.14 脳 10 ~ (10) ~ (-3). 2. The purity of sflk1-IFN- 纬 fusion protein in cell supernatant was increased 13.25 times by salting out, and the recovery rate was 30.02%. The purity of sflk1-IFN- 纬 fusion protein in cell supernatant could be increased by 3.38 times by using ultrafiltration membrane Biomax-30 to concentrate cell supernatant. The recovery rate was 35.1a; The purity of sflk1-IFN- 纬 fusion protein in cell supernatant was increased 4.18 times by anion exchange chromatography. 3. Medium purification by hydrophobic chromatography. The sflk1 purity of sflk1-IFN- 纬 fusion protein in cell supernatant was increased from 4.37 脳 10 ~ (-1) ~ (-3) to 48 脳 10 ~ (10) ~ (-3) and the recovery rate was 19.2%. 4. Purified by gel filtration. The purity of sflk1 in the sflk1-IFN- 纬 fusion protein in hydrophobic chromatography was increased from 48 脳 10 ~ (-1) ~ (-3) to more than 2.34%. Conclusion: the purity of sflk1-IFN- 纬 fusion protein expressed in CHO cells could be increased by more than 500 times after the protein was initially concentrated and then purified by hydrophobic chromatography and gel filtration. The purity of sflk1 can reach 2.34. The method used in this paper can purify the protein expressed in eukaryotic system.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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