本文關鍵詞：sflk1-IFN-γ融合蛋白的分離與純化　出處：《浙江大學》2007年碩士論文　論文類型：學位論文

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【摘要】： 惡性腫瘤的生長、浸潤和轉移必須依靠腫瘤新生血管提供足夠的營養(yǎng)，因此抑制或破壞腫瘤血管生成已成為近年來腫瘤治療的新方法。血管內皮生長因子(VEGF)與表達在血管內皮細胞上的相應受體VEGFR2結合所引起的信號轉導是血管生成中的限速步驟，在整個血管生成中發(fā)揮著關鍵作用�？扇苄匝軆绕どL因子受體2(sVEGFR2，在小鼠中被稱為sflk1)可競爭性地結合VEGF，從而阻斷VEGF與VEGFR2結合所引起的信號轉導，抑制腫瘤細胞誘導的血管生成和腫瘤的生長。IFN-γ是細胞免疫應答中非常重要的效應因子，能誘導CTL應答及Th1細胞的分化，并且尚能直接抑制多種腫瘤的生長，同時它自身也有抑制腫瘤血管生成的作用。本所已經(jīng)構建了pcDNA3.1(+)／sflk1-IFN-γ重組質粒，獲得高效穩(wěn)定表達的CHO細胞，并對其表達產物sflk1-IFN-γ融合蛋白的生物學活性進行了初步研究。 目的：為了進一步研究sflk1-IFN-γ融合蛋白的結構、理化性質、生物學活性以及進行動物試驗等，本實驗對CHO細胞上清液中sflk1-IFN-γ融合蛋白進行分離與純化，擬獲得純度較高的融合蛋白，并探討真核細胞表達所蛋白的分離純化方法。 方法：大規(guī)模培養(yǎng)高效穩(wěn)定表達sflk1-IFN-γ融合蛋白的CHO細胞，收集上清，經(jīng)超濾、鹽析或陰離子交換層析(IEX)方法進行初步的濃縮與分離，再經(jīng)疏水層析(HIC)進行中度純化及凝膠過濾(GF)精細純化并采用SDS-PAGE電泳進行蛋白純度的鑒定。 結果：1．利用低血清(5％FCS)培養(yǎng)表達sflk1-IFN-γ融合蛋白的CHO細胞，收集的上清中總蛋白的濃度為1741±40μg／ml，，sflk1的濃度為55.2±6.70ng／ml，純度為2.72～3.64×10~(-3)％，IFN-γ的濃度為1.4±0.78ng／ml，純度為0.03～0.14×10~(-3)％； 2．通過鹽析可將細胞上清液中的sflk1-IFN-γ融合蛋白中IFN-γ純度提高13.25倍，回收率30.02％；利用超濾膜Biomax-30濃縮細胞上清液，可將細胞上清液中的sflk1-IFN-γ融合蛋白中IFN-γ純度提高3.38倍，回收率35.1％；采用陰離子交換層析可將細胞上清液中的sflk1-IFN-γ融合蛋白中IFN-γ純度提高4.18倍，回收率48％； 3．經(jīng)疏水層析中度純化，可將細胞上清液中的sflk1-IFN-γ融合蛋白中sflk1純度由4.37×10~(-3)％提高到48×10~(-3)％，回收率19.2％； 4．經(jīng)凝膠過濾精細純化，可將疏水層析收集液中的sflk1-IFN-γ融合蛋白中sflk1純度由48×10~(-3)％提高到2.34％以上。 結論：蛋白質初步濃縮后，再經(jīng)疏水層析及凝膠過濾可將CHO細胞所表達的sflk1-IFN-γ融合蛋白的純度提高500多倍，sflk1的純度可達2.34％，本論文中采用的方法對于真核體系表達的蛋白有一定的純化作用。
[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- 緯 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- 緯) recombinant plasmid has been constructed to obtain high and stable expression of CHO cells. The biological activity of sflk1-IFN- 緯 fusion protein was studied. Objective: to further study the structure, physicochemical properties, biological activity and animal test of sflk1-IFN- 緯 fusion protein. The fusion protein sflk1-IFN- 緯 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- 緯 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- 緯 fusion protein were cultured with low serum FCS. The concentration of total protein in the supernatant was 1741 鹵40 渭 g / ml. The concentration of sflk1 was 55.2 鹵6.70 ng / ml, and the purity was 2.72 鹵3.64 脳 10 ~ (-1) ng / ml. The concentration of IFN- 緯 was 1.4 鹵0.78 ng / ml. The purity is 0.03 ~ 0.14 脳 10 ~ (10) ~ (-3). 2. The purity of sflk1-IFN- 緯 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- 緯 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- 緯 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- 緯 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- 緯 fusion protein in hydrophobic chromatography was increased from 48 脳 10 ~ (-1) ~ (-3) to more than 2.34%. Conclusion: the purity of sflk1-IFN- 緯 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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

相關期刊論文 前1條

1 陳浩,陳于紅,朱德煦,劉建寧;重組蛋白質純化技術[J];中國生物工程雜志;2002年05期

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