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Construction and expression of a single-chain HV antibody-protamine fragment fusion protein 1A8 ScFv/Cκ/P
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- Author:
- No author available
- Journal Title:
- JOURNAL OF THE FOURTH MILITARY MEDICAL UNIVERSITY
- Issue:
- 11
- DOI:
- 10.3321/j.issn:1000-2790.2008.11.003
- Key Word:
- 汉坦病毒;单链抗体;鱼精蛋白;融合蛋白;靶向输送
Abstract： 目的:构建汉坦病毒(Hantavirus, HV)单链抗体/鱼精蛋白片段融合蛋白1A8 ScFv/Cκ/P基因的原核表达载体,蛋白经表达后纯化并进行1A8 ScFv/Cκ/P融合蛋白的功能活性鉴定.方法:采用PCR的方法,扩增单链抗体基因1A8 ScFv和1A8 ScFv/Cκ,将鱼精蛋白片段的编码序列设计入扩增引物,获得HV单链抗体/鱼精蛋白片段的融合蛋白基因1A8 ScFv/Cκ/P. 将获得的重组基因克隆入原核表达载体pET32a,并在大肠杆菌BL21中表达. 表达产物经SDS-PAGE和Western Blot鉴定,用Ni-NTA螯合层析介质进行纯化. 采用ELISA方法分析1A8 ScFv/Cκ/P融合蛋白与HV抗原的结合活性,凝胶迁移阻滞实验检测1A8 ScFv/Cκ/P融合蛋白与质粒DNA的结合活性. 结果:成功构建了HV 1A8 scFv/Cκ/P融合蛋白基因的表达载体,在大肠杆菌中实现了可溶性表达. 通过功能活性测定,纯化的1A8 scFv/Cκ/P融合蛋白既保持了与HV抗原的结合能力,同时又具有与核酸的结合活性. 结论:构建和表达的HV 1A8 scFv/Cκ/P融合蛋白具有HV抗原结合活性和DNA结合的活性.
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