H7N9亚型禽流感病毒HA基因mRNA的构建

Construction of mRNA containing HA gene of H7N9 avian influenza virus

H7N9亚型禽流感病毒(AIV)持续给家禽及人类健康造成威胁。为构建H7N9亚型AIV(简称H7N9 AIV)mRNA疫苗,本研究以AIV分离株A/chicken/Yunnan/SD024/2021(H7N9)(简称CK/YN/SD024/2021)的HA基因为目的基因、选择来自人β-球蛋白和非洲爪蟾β-球蛋白的不同编码基因序列作为HA基因的非翻译区(UTR),构建了中间转录质粒pGEM-YN和pXT7-YN,以2个质粒为模板通过体外转录制备了mRNA mH7-YN-pGEM和mH7-YN-pXT7;将2种mRNA转染HEK293T细胞,western blot鉴定结果显示2种mRNA转染的细胞均可检测到约为70 ku的HA蛋白条带和50 ku的HA1蛋白条带;通过间接免疫荧光试验(IFA)确定最适转染剂量和HEK293T细胞中HA蛋白的动态表达,结果显示,3μg mRNA转染106个细胞并于转染24 h后荧光信号强度均最强;将2种mRNA转染鸡胚成纤维细胞(CEF),均可通过IFA检测到HA蛋白的表达。以上结果表明,本研究正确构建了2种含H7N9 AIV HA基因的mRNA,且其均能够在HEK293T细胞和CEF中正确表达HA蛋白,3μg mRNA转染HEK293T细胞可获得最佳的蛋白表达水平,转染24 h后HA蛋白的表达水平达到最高。本研究基于UTR序列的差异首次正确构建了两种能在哺乳动物细胞和禽类细胞中高效表达H7N9 AIV HA蛋白的mRNA,为禽流感mRNA疫苗的研发和免疫效力评估奠定了良好的物质和技术基础。

H7N9 avian influenza virus(AIV)poses an insistent threat to poultry production and public health.In order to develop mRNA vaccine against H7N9 AIV,the intermediate transcription plasmids pGEM-YN and pXT7-YN were constructed by using the gene sequences of humanβ-globin and Xenopus laevisβ-globin as UTRs and the HA gene of AIV strain A/chicken/Yunnan/SD024/2021(H7N9)(CK/YN/SD024/2021)as target gene.The mRNA mH7-YN-pGEM and mH7-YN-pXT7 were prepared by in vitro transcription with above two plasmids as template.The two mRNA were transfected into HEK293T cells respectively,about 70ku HA protein and 50ku HA1 protein of CK/YN/SD024/2021 strain could all be detected by the western blot analysis.Then the optimal transfection dose and the dynamic expression of HA protein was detected by indirect immunofluorescence assay(IFA).The results showed that the strongest fluorescence signal intensity was detected when 106 cells were transfected with 3μg mRNA at 24 hours after mRNA transfection.The two mRNA were transfected into chicken embryo fibroblast(CEF)cells respectively,and the HA protein could be detected by IFA in CEF cells after mRNA transfection.Based on above results,mRNA mH7-YN-pGEM and mH7-YN-pXT7 were constructed correctly and the HA protein of H7N9 AIV CK/YN/SD024/2021 strain was successfully expressed in HEK293T cells and CEF cells.The optimal transfection dose was 3μg,and the expression of HA protein reached its peak at 24 hours in HEK293T cells after transfection.In this study,two mRNA were constructed for the first time based on the different UTR sequences and could express HA protein of H7 subtype AIV effectively in both mammalian cells and avian cells.The work laid a good material and technique foundation for the subsequent development and efficacy evaluation of avian influenza mRNA vaccine.