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目的构建带有MERS-Co V N(MERS冠状病毒核衣壳蛋白)基因片段的p ET-22b+原核表达载体,并表达和纯化核衣壳蛋白(NP)。方法通过PCR技术扩增NP蛋白基因片段,插入到原核表达载体p ET-22b+上。使用核酸电泳、测序以及小量诱导表达确认所构建克隆的正确性,然后将重组质粒转化进大肠杆菌BL21a(DE3)中,并在大肠杆菌BL21a(DE3)中大规模诱导表达NP蛋白,使用AKTA纯化系统经强阳离子交换层析纯化出NP蛋白。结果测序和小量诱导结果表明,扩增的NP蛋白片段序列正确,所构建的p ET-22b+-NP克隆可在大肠杆菌BL21a(DE3)的包涵体和细胞质中表达;通过阳离子交换层析和浓缩后,蛋白的纯度和浓度都得到明显提高。结论纯化出高纯度、高浓度的NP蛋白,为NP蛋白功能性研究提供重要基础。
Objective To construct p ET-22b + prokaryotic expression vector with MERS-Co V N (MERS coronavirus nucleocapsid protein) gene fragment and express and purify nucleocapsid protein (NP). Methods The NP gene fragment was amplified by PCR and inserted into prokaryotic expression vector p ET-22b +. The correctness of the constructed clones was confirmed by nucleic acid electrophoresis, sequencing and small amount of induced expression. The recombinant plasmids were then transformed into E. coli BL21a (DE3) and expressed in large scale in E. coli BL21a (DE3), using AKTA The purification system was purified by strong cation exchange chromatography NP protein. Results Sequencing and small amount of induction showed that the sequence of amplified NP protein fragment was correct. The constructed p ET-22b + -NP clone was expressed in inclusion bodies and cytoplasm of E. coli BL21a (DE3) After concentration, protein purity and concentration have been significantly improved. Conclusion Purification of high purity and high concentration of NP protein provides an important foundation for the functional study of NP protein.