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Prokaryotic Expression of VEGF165 and Preliminary Screening of Anti-VEGF Hybridoma Cell Lines |
BAO Xin-chen1, LI Zi1, GAO Xiang-dong1, LU Xiao-dong2, XU Chen2 |
1. College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China;
2. Beijing Tri-Prime Genetic Engineering Incorporated, Beijing 102600, China |
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Abstract Vascular endothelial growth factor (VEGF) plays a key role in physiological and pathological angiogenesis and is a potent and critical target as blocking tumor growth and metastasis. The process described in this report involves a complex auto-induced expression in Escherichia coli and a downstream purification process consisting of protein refolding and three chromatography steps in order to obtain the functional rhVEGF165. Biological activity of the purified 38kDa homodimer was verified by the induction of the proliferation of human umbilical vein endothelial cells (HUVECs). The EC50 for this effect was 2.4ng/ml. Finally, three Anti-VEGF hybridoma cell lines were obtained after immunization, fusion and preliminary screening.
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Received: 25 April 2011
Published: 25 September 2011
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[1] Folkman J. Tumor angiogenesis: therapeutic implications. New Engl J Med, 1971, 285(21):1182-1186.
[2] Ferrara N. The role of VEGF in the regulation of physiological and pathological angiogenesis. Mechanisms of Angiogenesis, 2005,(94):209-231.
[3] Ferrara N. Vascular endothelial growth factor as a target for anticancer therapy. The oncologist, 2004, 9(Supplement 1):2.
[4] Hsu J Y, Wakelee H A. Monoclonal antibodies targeting vascular endothelial growth factor: current status and future challenges in cancer therapy. Bio Drugs, 2009, 23(5):289-304.
[5] 崔斐, 李军, 罗荣城. 贝伐单抗在肿瘤临床治疗中的研究进展. 河北医学,2008, 11(6):741-746. Cui F, Li J, Luo R C. Hebei Medicine, 2008, 11(6):741-746.
[6] Robinson C J, Stringer S E. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. Journal of cell science, 2001, 114(Pt 5):853.
[7] 杨佩, 王坤正, 时志斌,等. 血管内皮生长因子在原核细胞中的高效表达, 纯化与活性研究. 南方医科大学学报, 2006, 26(9):1263-1268. Yang P, Wang K Z, Shi Z B, et al. Journal of Southern Medical University, 2006, 26(9):1263-1268.
[8] 周红霞, 周梅仙, 吴洁,等. 人源血管内皮生长因子突变体的克隆, 分离纯化及活性研究. 中国药科大学学报, 2006, 37(3):273-276. Zhou H X, Zhou M X, Wu J, et al. Journal of China Pharmaceutical University, 2006, 37(3):273-276.
[9] 徐静, 李树香, 刘立成,等. 血管内皮细胞生长因子的表达及其单克隆抗体的制备. 中国生物制品学杂志, 2008, 21(11):1002-1005. Xu J, Li S X, Liu L C, et al. Chinese Journal of Biologicals, 2008, 21(11):1002-1005.
[10] 盛世乐, 韩源, 黄钢. 人血管内皮生长因子的克隆表达及免疫学检测工作标准品的制备. 放射免疫学杂志, 2004, 17(5):395-398. Sheng S L, Han Y, Huang G. Journal of Radioimmunology, 2004, 17(5):395-398.
[11] Studier F W. Protein production by auto-induction in high-density shaking cultures. Protein expression and purification, 2005, 41(1):207-234.
[12] Claffey K P, Senger D R, Spiegelman B M. Structural requirements for dimerization, glycosylation, secretion, and biological function of VPF/VEGF. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1995, 1246(1):1-9.
[13] Peretz D, Gitay-Goren H, Safran M, et al. Glycosylation of vascular endothelial growth factor is not required for its mitogenic activity. Biochemical and biophysical research communications, 1992, 182(3):1340-1347.
[14] Fischer B, Sumner I, Goodenough P. Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in Escherichia coli as inclusion bodies. Biotechnology and bioengineering, 1993, 41(1):3-13.
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