诺瓦克病毒广州株N蛋白基因的克隆、表达及抗原性鉴定

Cloning, expression and immunocharacterization of the capsid protein of human Norwalk virus Guangzhou strain NVgz01

目的克隆、表达和鉴定诺瓦克病毒广州株NVgz01全长N蛋白。方法在成功构建诺瓦克病毒广州株全基因组克隆并测序的基础上,将全长N蛋白基因克隆到表达载体pET28a(+)上,经IPTG诱导表达后,利用Ni2+亲和层析柱对重组N蛋白进行纯化,并用Western blotting和ELISA方法检测其抗原性。结果重组诺瓦克病毒N蛋白基因在大肠杆菌中可以高效表达,其相对分子质量为62×103,可在Ni2+亲和层析柱上高度纯化;经抗原检测试剂盒检测和免疫学方法分析,均表明重组N蛋白具有良好的抗原性。结论本研究克隆和表达了诺瓦克病毒广州株的全长N蛋白,为诺瓦克病毒诊断试剂和疫苗的开发等进一步的研究奠定了基础。

Objective To clone, express and characterize the capsid protein of human Norwalk virus Guangzhou strain- NVgz01. Methods On the basis of successful construction of full-genome clones and sequence analysis of human norovirus Guangzhou strain NVgz01, the full capsid gene was ligated into pET28a （＋） for expression. After IPTG induction, the recombinant protein was purified through metal （Ni^2＋） chelating affinity chromatography. Western blotting and enzyme-linked immunosorbent assay （ELISA） were used to determine the antigenicity of the recombinant protein. Results The recombinant capsid gene was overexpressed in E.coli, yielding the recombinant protein with relative molecular mass of 62 ×10^3 that was highly purified through metal （Ni^2＋） chelating affinity chromatography. IDEIA Norovirus Kit and immunoassay showed that the recombinant protein had good antigenicity. Conclusion The capsid gene of norovirus Guangzhou strain has been cloned and expressed, which can be useful for developing diagnostic reagents or vaccine of norovirus.