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

目的:构建人抗EGFR单链抗体(scFv)/鱼精蛋白截短体(truncated protamine,tP)融合蛋白基因,在大肠杆菌中表达并纯化,分析该融合蛋白的生物学活性。方法:设计引物扩增人抗EGFR单链抗体编码序列,将其克隆于原核表达载体pBAD-A;人工合成tP序列,将其克隆于原核表达载体pBAD-AscFv上,构建成scFv/tP融合基因,在大肠杆菌TOP10F′中诱导表达,表达产物经SDS-PAGE和Western blot鉴定后,用His-trap镍亲和层析柱纯化。ELISA分析scFv/tP融合蛋白抗原亲合活性,凝胶迁移阻滞实验检测scFv/tP融合蛋白与DNA的结合活性。结果:成功构建了人源抗EGFR抗体/tP融合基因,经L-Ara诱导后在大肠杆菌中实现了可溶性表达。表达的scFv/tP融合蛋白保持了与抗原的结合活性,同时具有结合DNA的能力。结论:scFv与tP融合后,同时具有与抗原和DNA结合的活性,该scfv/tP融合蛋白为EGFR表达阳性肿瘤的靶向基因治疗奠定了基础。

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-expressed 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 di- gestion 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.