寨卡病毒非结构蛋白1的真核表达纯化和免疫反应性鉴定

目的 在HEK293F细胞中表达寨卡病毒(Zika Virus,ZKV)的非结构蛋白1(Nonstructural Protein 1,NS1),并分离纯化得到重组ZKNS1以及验证其免疫反应性。方法 根据GenBank中的ZKNS1序列合成基因,在基因上下游分别引入信号肽和组氨酸标签,通过NotI和XbaI双酶切将基因克隆至pcDNA31(+)载体;将重组载体转染HEK293F细胞,重组蛋白用镍亲和柱层析纯化;免疫印迹法(Western blot)鉴定重组ZKNS1与寨卡病毒阳性人血清的免疫反应性。结果 成功构建了ZKNS1的真核表达载体pcDNA31(+)-ZKNS1;在HEK293F细胞中重组ZKNS1以分泌形式表达于细胞培养上清中,经纯化后获得了纯度较高的分子量约为46kDa的重组蛋白;Western blot结果显示重组ZKNS1与寨卡病毒阳性人血清能发生特异性结合。结论 成功构建ZKNS1的真核表达载体,获得了免疫反应性较高的纯化的重组ZKNS1,为进一步建立ELISA检测方法及制备单克隆抗体奠定了基础。

Eukaryotic expression,purification and immunoreactivity identification of the nonstructural protein 1 of Zika virus

Objective To express and purify the nonstructural protein 1 of Zika virus(ZKNS1)in HEK293F cells,and to verify the immunoreactivity of the recombinant ZKNS1.Methods The gene was synthesized based on the ZKNS1 sequence in GenBank,and a signal peptide and a histidine label were introduced into the upstream and downstream of the gene,respectively.The gene was cloned into the pcDNA3.1(+)vector by double restriction enzyme digestion.The recombinant vector was transfected into HEK293F cells,and the recombinant ZKNS1 was purified by Ni2+ affinity column chromatography.The immunoreactivity of the recombinant ZKNS1 and the Zika virus positive human serum were verified by Western blot.Results The eukaryotic expression vector pcDNA3.1(+)-ZKNS1 was constructed successfully.The recombinant ZKNS1 was expressed in HEK293F cells,secreted into the medium,and purified to obtain a recombinant protein with a molecular weight of approximately 46 kDa.The Western blot result verified the immunoreactivity of the recombinant ZKNS1,which was specific reactivity with the Zika virus positive human serum.Conclusion The eukaryotic expression vector of ZKNS1 is successfully constructed,and the purified recombinant ZKNS1 with high immunoreactivity is obtained,which lays the foundation for establishing the ELISA detection method and preparing monoclonal antibodies.