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目的通过分子克隆技术,构建人细小病毒B19非结构基因ns1和7.5kugene的原核表达载体,并诱导重组NS1和7.5ku融合蛋白的表达,为蛋白的纯化奠定基础。方法以含有B19病毒全基因组感染性克隆为模板,用PCR法扩增目的基因ns1和7.5kugene,以pET-28a为表达载体,构建重组表达质粒pET28a-ns1和pET28a-7.5ku,转化E.coliBL21(DE3),获得重组工程菌株。经IPTG诱导培养6h后,通过SDS-PAGE、Western blot鉴定表达产物。结果成功构建了pET28a-ns1和pET28a-7.5ku,IPTG诱导能获得较高的目的蛋白。结论人细小病毒B19的非结构基因能在大肠埃希菌中获得成功的表达,为蛋白的纯化和抗体制备奠定基础。
Objective To construct the prokaryotic expression vector of human parvovirus B19 ns1 and 7.5kugene by molecular cloning and to induce the expression of recombinant NS1 and 7.5ku fusion protein and lay the foundation for the purification of the protein. Methods The full-length infectious clone of B19 virus was used as a template to amplify the target genes ns1 and 7.5kugene by PCR. The recombinant plasmid pET28a-ns1 and pET28a-7.5ku were constructed by using pET-28a as the expression vector and transformed into E.coli BL21 (DE3) to obtain a recombinant engineering strain. After induced by IPTG for 6h, the expressed products were identified by SDS-PAGE and Western blot. Results pET28a-ns1 and pET28a-7.5ku were successfully constructed, and induced by IPTG could obtain higher target protein. Conclusion The non-structural gene of human parvovirus B19 can be successfully expressed in Escherichia coli, which will lay the foundation for protein purification and antibody preparation.