ZIKA病毒E蛋白膜外区(E80)的表达、纯化及鉴定

Expression,Purification and Identification of Extracellular Domain of ZIKA Virus(E80)

目的构建ZIKA病毒E蛋白膜外区(E80)的原核表达质粒,对重组蛋白E80进行表达、纯化和鉴定。方法以重组质粒pCMV-S-ZIKA_ME为模板,利用PCR扩增E80基因片段并将其插入原核表达载体pET-28a,构建重组质粒pET-28a-ZIKA-E80,转入大肠埃希菌BL21(DE3)感受态细胞,经IPTG诱导表达,采用Ni+柱亲和层析法纯化蛋白,Western印迹检测蛋白特异性。结果重组质粒pET-28a-ZIKA-E80经双酶切验证构建正确,且测序结果与原序列一致。E80成功的以包涵体的形式表达,大小为45 kD且与预期一致。亲和层析纯化后纯度达到95%以上,能被ZIKA病毒E蛋白抗体特异性识别。结论成功构建了能高效表达E80的基因工程菌,纯化后的目的蛋白纯度高、特异性好,为ZIKA病毒疫苗和E蛋白的结构与功能的研究奠定了基础。

Objective To construct the prokaryotic expression plasmid of the extracellular do-main of E protein of ZIKA virus(E80),and to identify and purify the recombinant protein E-80.Methods The E80 gene fragment was amplified from the recombinant plasmid pCMV-S-ZIKA_ME by PCR and cloned into the prokaryotic vector pET-28a to construct the recombinant plasmid pET-28a-ZIKA-E80,which was transformed into E.coli BL21(DE3)competent cells.IPTG in-duced the protein expression,and the protein was purified by Ni+column affinity chromatography,and the protein specificity was detected by Western blotting.Results The correct construction of re-combinant plasmid pET-28a-ZIKA-E80 was confirmed by double enzyme digestion and the sequen-cing results were consistent with the original sequence.E80 was successfully expressed in the form of inclusion bodies with a size of 45 kD,which was consistent with expectations.After purification by affinity chromatography,the purity is over 95%,and the protein can be specifically recognized by ZIKA virus E protein antibody.Conclusion The prokaryotic recombinant plasmid with high expression level of E80 was successfully constructed.The purified protein has high purity and specificity,which lays a foundation for the study of ZIKA virus vaccine and the structure and function of E protein.