通过产生的单链抗体可变区片段（scFv）抗体偶联溴化氰活化琼脂糖凝胶亲和纯化Raji细胞中表达的肿瘤坏死因子-α
Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose.
作者信息
Abdolalizadeh Jalal, Majidi Zolbanin Jafar, Nouri Mohammad, Baradaran Behzad, Movassaghpour Aliakbar, Farajnia Safar, Omidi Yadollah
机构信息
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran. ; Student' Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
出版信息
Adv Pharm Bull. 2013;3(1):19-23. doi: 10.5681/apb.2013.004. Epub 2013 Feb 7.
PURPOSE
Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins.
METHODS
In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE.
RESULTS
Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%.
CONCLUSION
These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.
目的
重组肿瘤坏死因子-α（TNF-α）已被用作抗肿瘤药物，用于治疗黑色素瘤和肉瘤患者。它通过作用于肿瘤相关血管来靶向肿瘤细胞抗原。亲和层析蛋白纯化已广泛应用于药用级蛋白质的下游加工。
方法
在本研究中，我们检测了我们所制备的抗TNF-α单链抗体片段（scFv）对Raji细胞产生的TNF-α的纯化潜力。用脂多糖（LPS）诱导Raji细胞表达TNF-α。采用蛋白质印迹法和荧光激活细胞分选（FACS）流式细胞术分析来评估TNF-α的表达。从抗体噬菌体展示文库中筛选出的抗TNF-α scFv与溴化氰活化的琼脂糖4B珠偶联，用于亲和纯化表达的TNF-α，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）评估蛋白质的纯度。
结果
蛋白质印迹法和FACS流式细胞术分析表明Raji细胞成功表达了TNF-α。SDS-PAGE分析显示scFv对TNF-α蛋白的纯化效果良好，纯度超过95%。
结论
这些发现不仅证实了所制备的scFv抗体片段的潜力，而且这种高纯度的重组TNF-α蛋白可应用于各种体外和体内应用。
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