摘要
目的将戊型肝炎病毒ORF3基因转染HeLa细胞，建立能稳定表达ORF3蛋白（pORF3）的细胞株。方法将ORF3基因片段克隆至真核表达质粒pcDNA3，构建重组质粒pcDNA3-ORF3。电穿孔法将其导入HeLa细胞中，G418筛选得到稳定抗性的细胞株，并用RT-PCR和Western印迹法分别从mRNA水平和蛋白水平检测ORF3的表达。结果成功构建了重组表达质粒pcDNA3-ORF3，并筛选获得能稳定表达pORF3蛋白的HeLa细胞株。结论pORF3蛋白能够在HeLa细胞中稳定表达，为进一步研究其生物学功能奠定了实验基础。
Objective To transfer the gene of hepatitis E vires ORF3 gene into HeLa cell line and establish a stable expression system. Method The recombinant plasmid pcDNA3-ORF3 was constructed by insertion of ORF3 gene fragment into eukaryotic expression vector PeDNA3. pcDNA3-ORF3 was transferred into HeLa cell line by electrotransfer, and the positive cell clones were selected with G418. The stable expression of pORF3 was determined by RT-PCR and western blot respectively on its mRNA and protein levels. Results PeDNA3-ORF3 was successfully constructed with DNA recombination technique. After C,418 selection, the HeLa cell lines transferring pcDNA3-ORF3 were obtained. Abundant pORF3 stable expression in HeLa cell line was confirmed by RT-PCR and western blot. Conclusions pORF3 gene can be transferred and stably expressed in HeLa cell line. The pORF3 stable expression system establishes the experiment foundation for further research on its biological function.
出处
《国际流行病学传染病学杂志》
CAS
2008年第6期372-374,共3页
International Journal of Epidemiology and Infectious Disease
基金
浙江省医药卫生青年人才专项基金（2006QN003）,浙江省自然科学基金（Y206384）
