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目的:构建人源性抗HER2单链抗体(scFv)/精氨酸九聚体(9R)融合蛋白基因,在大肠杆菌里表达纯化并检测该融合蛋白的活性。方法:采用PCR的方法,扩增融合基因scFv-9R,将获得的基因克隆入原核表达载体pQE30,在大肠杆菌M15中表达,表达产物经SDS-PAGE和Western blot鉴定,通过Ni-NTA螯合层析纯化,透析复性,超滤浓缩。ELISA分析scFv-9R融合蛋白抗原亲和活性,凝胶迁移实验检测scFv-9R融合蛋白与siRNA结合活性。结果:成功构建了人源性抗HER2 scFv-9R融合基因,经IPTG诱导后在M15中以包涵体形式表达。表达的目的蛋白scFv-9R能与HER2抗原结合,同时具有siRNA结合能力。结论:scFv-9R具有结合抗原与siRNA双重活性,为靶向递送siRNA的研究奠定了基础。
Objective: To construct a human anti-HER2 scFv / arginine 9mer (9R) fusion protein gene, express and purify the fusion protein in Escherichia coli and test its activity. Methods: The fusion gene scFv-9R was amplified by PCR. The obtained gene was cloned into the prokaryotic expression vector pQE30 and expressed in E. coli M15. The expressed product was identified by SDS-PAGE and Western blot, and was purified by Ni-NTA chelation Chromatography purification, dialysis refolding, ultrafiltration concentration. ELISA analysis scFv-9R fusion protein antigen affinity activity, gel migration assay scFv-9R fusion protein and siRNA binding activity. Results: The human anti-HER2 scFv-9R fusion gene was successfully constructed and expressed as inclusion bodies in M15 after induced by IPTG. The expressed protein scFv-9R can bind to HER2 antigen and bind to siRNA. Conclusion: scFv-9R has dual activity of binding antigen and siRNA, which lays the foundation for the research of targeted delivery of siRNA.