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Construction and expression of human anti-HER2 ScFv/Ck/tP fusion protein in E.coli and identification of its activity
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 7
- DOI:
- 10.3321/j.issn:1000-2790.2007.07.002
- Key Word:
- HER2;单链抗体;鱼精蛋白
Abstract： 目的:构建人抗HER2单链抗体(ScFv)/人轻链恒定区(Ck)/鱼精蛋白截短体(tP)融合蛋白基因,在大肠杆菌中表达、纯化并分析该融合蛋白的活性.方法:设计引物扩增人抗HER2单链抗体e23sFv基因和轻链恒定区编码序列(Ck),连接成ScFv/Ck片段,人工合成tP序列,连接于ScFv/Ck末端,构建成ScFv/Ck/tP融合基因,将其克隆入原核表达载体pET32a后,在大肠杆菌BL21(DE3)LysS中诱导表达,表达产物经SDS-PAGE和Western Blot鉴定后,用Ni-NTA螯合层析介质纯化.细胞ELISA分析ScFv/Ck/tP融合蛋白抗原亲合活性,凝胶迁移实验检测ScFv/Ck/tP融合蛋白与DNA的结合活性.结果:成功构建了人抗HER2 ScFv/Ck/tP融合基因,经IPTG诱导后在大肠杆菌中实现了可溶性表达.表达的ScFv/Ck/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Ck,tP融合后,同时具有抗原和DNA结合活性,为该ScFv用于HER2阳性肿瘤的靶向基因治疗奠定了基础.
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