实验研究
Fuyangbo
2015, 39(12): 0-0.
[摘要] 目的 构建带有MERS-CoV N（MERS冠状病毒核衣壳蛋白）基因片段的pET-22b+原核表达载体，表达和纯化出MERS-CoV N蛋白。方法 通过PCR技术扩增出MERS-CoV N基因片段，插入到原核表达载体pET-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MERS-CoV N蛋白，使用AKTA纯化系统经强阳离子交换层析纯化出MERS-CoV N蛋白。结果 测序和小量诱导结果表明，扩增的MERS-CoV N片段序列正确、构建的克隆能够在大肠杆菌BL21a（DE3）中可溶性和包涵体表达；通过阳离子交换层析和浓缩之后，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MERS-CoV N蛋白，为MERS-CoV N蛋白功能性研究提供重要基础。
[关键词] MERS-CoV N；原核表达纯化；阳离子交换层析
[Abstract] Objective To construct a prokaryotic expression vector pET-22b+ with MERS coronavirus nuclocapsid protein gene and to express and purify MERS-CoV N protein. Methods MERS-CoV N gene amplified by PCR technology was inserted into the prokaryotic expression vector pET-22b+. Recombinant plasmid was confirmed using DNA electrophoresis, sequencing, and small amounts of induced expression and transformed into E.coli BL21(DE3).MERS-CoV N protein was induced expressed in E.coli BL21(DE3) in large scale and purified using the AKTA purification system by cation exchange chromatography. Result The MERS-CoV N gene sequence was proved to be correct by sequencing and the protein expressed in soluble and insoluble form in E.coli BL21(DE3) after IPTG induction. The purity and concentration of recombinant protein had been improved obviously by cation exchange chromatography and enrichment. Conclusion Recombiant MERS-CoV N protein of high purity and concentration was purified and will lay a foundation of MERS-CoV N protein functional research.
[Key words] MERS-CoV N; Prokaryotic expression and purification; Cation exchange chromatography
