稳定表达乙型流感病毒血凝素蛋白细胞的构建

发布时间：2018-03-29 02:17

本文选题：乙型流感病毒　切入点：血凝素(HA)蛋白　出处：《病毒学报》2017年04期

【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。
[Abstract]:A stable HEK-293-HA cell line expressing hemagglutinin protein of influenza B virus was obtained. The HA gene of influenza B virus was amplified by RT-PCR and cloned into eukaryotic expression vector pcDNA3.1 (). The recombinant plasmid pcDNA3.1 was constructed. The identified pcDNA3.1 was co-transfected into HEK293 cells by liposome mediated method. The recombinant cell line HEK293-HAwas screened by purine mycin, and the expression of HA was detected by flow cytometry (FCM). Indirect immunofluorescence assay (IFA) and Western blot (Western blot) were used to detect the stability of HA protein expression. The results showed that the recombinant plasmid pcDNA3.1was correctly identified by double enzyme digestion and sequencing. Three high expression positive cell lines were obtained. The results of Western blot and IFA analysis showed that the cells were cultured for 15 consecutive generations. HEK-293-HA cells expressing hemagglutinin protein of influenza B virus have been successfully obtained, which can provide a good basis for the further study of HA protein of influenza B virus.
【作者单位】：广州医科大学生命科学学院医学遗传学与细胞生物学教研室;广州呼吸疾病研究所呼吸疾病国家重点实验室广州医科大学;广东和信健康科技有限公司;
【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~~
【分类号】：R373.13

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