NP9蛋白的原核表达及纯化

目的构建表达人NP9融合蛋白的重组体并进行融合蛋白的原核表达及NP9蛋白的分离和纯化。方法通过聚合酶链反应（PCR）得到NP9基因的编码区序列，克隆到原核表达载体pGEX-6P-3上构建重组NP9原核表达质粒。转化大肠杆菌后通过SDS-PAGE电泳和Western blotting鉴定融合蛋白表达。使用GSTFF层析柱分离纯化NP9蛋白。结果得到序列正确的NP9基因及重组原核表达质粒pGEX-6P-3-Np9，SDS-PAGE电泳和Western blotting证实重组质粒能够在大肠杆菌内受IPTG诱导表达，层析分离得到分子量为大约14kDa单一的NP9蛋白。结论成功构建了表达NP9融合蛋白的原核表达载体，该栽体能在细菌内表达出GST—NP9融合蛋白．有效分离得到NP9蛋白．

Prokaryotic expression and purification of NP9 protein

[Objective] To construct recombinant plasmid expressing NP9 fusion protein and to isolate the NP9 protein. [Method] The CDS of NP9 gene was obtained by PCR from the recombinant pRC/CMV2-NP9 plasmid, and the CDS was cloned into expression vector pGEX-6p-3. Recombinant expression plasmid was transformed into BL21 to express GST-NP9 fusion protein. The fusion protein was identified through SDS-PAGE electrophoresis and western blotting, then NP9 protein was isolated by GSTrap FF column. [Results] The recombinant E.Coli expression plasmid pGEX-6p-3-NP9 was constructed, SDS-PAGE and western blotting analysis confirmed that E.Coli BI21 transformed with recombinant NP9 plasmid could express GST-NP9 fusion protein and NP9 protein could be successfully isolated from the GST-NP9 fusion protein. [Conclusion] NP9 recombinant expression vector in E.Coli is constructed and can express NP9 fusion protein. NP9 protein is successfully isolated.