摘要
获得稳定表达乙型流感病毒血凝素蛋白的HEK-293-HA细胞株。采用RT-PCR方法扩增乙型流感病毒的HA基因,将其克隆至真核表达载体pcDNA3.1(+)上,构建pcDNA3.1(+)-HA重组质粒。将已鉴定正确的pcDNA3.1(+)-HA质粒与辅助质粒PLV-EF1a-EGFP(2A)Puro通过脂质体介导法共转染HEK293细胞,经嘌呤霉素筛选后得到重组细胞株HEK293-HA,通过流式细胞术法(FCM)来检测细胞中HA的表达情况。所得到的重组细胞经扩大培养后再连续培养15代,采用间接免疫荧光法(IFA)和蛋白免疫印迹法(WB)来检测HA蛋白表达的稳定性。结果表明重组质粒pcDNA3.1(+)-HA经双酶切及测序鉴定正确;共获得3株高表达阳性细胞株。细胞扩大培养后连续传代培养15代后进行Western blot和IFA检测结果表明,重组细胞的HA蛋白得到稳定的表达。已成功获得了能稳定表达乙型流感病毒血凝素蛋白的HEK-293-HA细胞,可为乙型流感病毒HA蛋白的进一步研究提供良好的基础。
We wished to develop a stable cell line that could express hemagglutinin(HA)in the influenza B virus(IBV).The HA gene in the full-length IBV was generated by amplification of the reverse transcriptase-polymerase chain reaction from the IBV.The HA gene was ligated with pcDNA3.1(+)to construct the eukaryotic expression plasmid pcDNA3.1(+)-HA,which was co-transfected into HEK293 cells with a PLV-EF1a-EGFP(2A)Puro plasmid by a liposome-mediated method.The recombinant cell line HEK293-HA was obtained after screening with puromycin and expression of HA protein was determined by flow cytometry.Recombinant cells were subcultured for 15 passages and tested for stability of expression of HA protein by immunofluorescence assays and Western blotting.The recombinant plasmid pcDNA3.1(+)-HA was identified correctly by EcoRI and BamHI endonuclease digestion and sequencing analyses.Three positive cell clones with high expression of HA protein were obtained.Stable expression of HA protein was shown by immunofluorescence assays and Western blotting in recombinant cells after culture for 15 passages.A HEK293-HA cell line for stable expression of the HA protein of the IBV was established:this could provide the foundation for further study of the HA protein of the IBV.
出处
《病毒学报》
CAS
CSCD
北大核心
2017年第4期494-500,共7页
Chinese Journal of Virology
基金
广州市科技计划-健康医疗协同创新重大专项(项目号:201508020252),题目:病毒感染重症救治的特异性被动免疫应用研究及腺病毒单克隆抗体药物研发
广州市科技计划-科学研究专项(项目号:201504010032),题目:人腺病毒和流感病毒动物模型的建立及应用研究~~