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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。
The HEK-293-HA cell line stably expressing the influenza virus hemagglutinin protein was obtained. HA gene of influenza B virus was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1 (+) to construct pcDNA3.1 (+) - HA recombinant plasmid. The recombinant plasmids pcDNA3.1 (+) - HA and PLV-EF1a-EGFP (2A) Puro were co-transfected into HEK293 cells by liposome-mediated method. After puromycin selection, the recombinant cell line HEK293 -HA, the expression of HA in the cells was detected by flow cytometry (FCM). The resulting recombinant cells were expanded and cultured for another 15 passages. The stability of HA protein expression was detected by indirect immunofluorescence (IFA) and Western blotting (WB). The results showed that the recombinant plasmid pcDNA3.1 (+) - HA was identified by double enzyme digestion and sequencing. Three highly positive cell lines were obtained. The results of Western blot and IFA showed that the HA protein of the recombinant cells was stably expressed after 15 generations of continuous subculture. The HEK-293-HA cells stably expressing the hemagglutinin protein of influenza B virus have been successfully obtained, which can provide a good foundation for the further study of HA protein of the influenza B virus.