南京医科大学学报（自然科学版）2011，Vol.31Issue(5)：651-655,5.
人源抗EGFR抗体-鱼精蛋白融合蛋白的制备及活性分析
Preparation and characterization of human anti- EGFR scFv-protamine fusion protein
摘要
Abstract
Objective: To make a fusion protein constructed of human anti-EGFR scFv/truncated protamine(scFv/tP) gene and analyze the binding activity of the over-expresed protein. Methods:Two pairs of oligonucleotide primers were designed and used to amplify the VH and VL nucleotide sequences,then use VH and VL as template to amplify the scFv. Then the protein gene was cloned into expression vector pBAD-A. The synthesized tP coding sequence was cloned into expression vector pBAD-AscFv,and expressed in E. coli TOPIOF'. The fusion protein was detected by SDS-PAGE and Western Blot,then purified by NiNTA chelating agarose. The antigen binding activity was detected by ELISA. And the DNA binding ability was detected by gel shift assay. Results:Restriction digestion and DNA sequencing proved that scFv/tP was correctly cloned into expression vector. SDS-PAGE and Western Blot results showed that scFv/tP fusion protein was successfully expressed in TOPIOF' . ELISA detection confirmed that the specific antigen binding activity of the fusion protein; and gel shift assay results suggested its DNA binding ability. Conclusion:The scFv/tP fusion protein successfully expressed in E. coli and could specially bind with both EGFR antigen and DNA.关键词
EGFR/单链抗体/鱼精蛋白Key words
EGFR/single chainFv/scFv/tP fusion protein分类
生物科学引用本文复制引用
张峰,王小英,唐奇,刘玉,张慧林,苏亦平,冯振卿,朱进..人源抗EGFR抗体-鱼精蛋白融合蛋白的制备及活性分析[J].南京医科大学学报（自然科学版）,2011,31(5):651-655,5.基金项目
江苏省社会发展项目(BS2007019) （BS2007019）
南京市科技发展项目(200901083) （200901083）
