HEK293T细胞敲除TPL2基因促进塞内卡病毒复制的研究

HEK293T cell knockout of TPL2 gene promotes replication of Seneca virus

肿瘤进展位点2(TPL2)在不同病毒感染过程中扮演着不同角色。为探究TPL2对塞内卡病毒(SVA)复制的影响,本研究利用CRISPR/Cas9基因编辑技术构建TPL2基因敲除HEK293T细胞系,以PCR测序和Western-blot对TPL2敲除效率进行双重鉴定,同时检测TPL2缺失对细胞形态和细胞增殖速度的影响。最后,用SVA感染细胞,采用IFA、RT-qPCR、Western-blot和TCID50方法检测病毒增殖水平,综合评价TPL2敲除对SVA复制的影响。结果,获得两株敲除TPL2基因的HEK293T细胞。随机选取B1细胞株(HEK293T-TPL2-/-)进行后续功能评价,发现TPL2敲除后细胞的增殖速度与对照HEK293T细胞无显著差异,均为贴壁、上皮样细胞形态。SVA感染后与对照细胞相比,敲除TPL2基因显著增加病毒拷贝数,病毒mRNA的转录,病毒蛋白的翻译以及子代病毒的毒力。综上所述,本研究成功建立TPL2基因敲除HEK293T细胞系并证明TPL2在SVA感染过程中发挥着抗病毒作用,为进一步揭示TPL2相关免疫反应和SVA感染过程的具体作用机制奠定试验基础并提供良好的细胞模型,也为疫苗生产过程中提高病毒产量提供一种可行性策略。

Tumor progression site 2(TPL2)plays a different role in different viral infections.To investigate the effect of TPL2 on the replication of Seneca virus(SVA),TPL2 knockout HEK293 T cell line was constructed by CRISPR/Cas9 gene editing technique this study.TPL2 knockout efficiency was double identified by PCR sequencing and Western-blot,and the effects of TPL2 deletion on cell morphology and cell proliferation rate were detected.Finally,SVA was used to infect the cells,and IFA,RT-q PCR,Western-blot,and TCID50 methods were used to detect the levels of virus appreciation,and the effect of TPL2 knockout on SVA replication was comprehensively evaluated.In result,two HEK293 T cells with the TPL2 gene knocked out were obtained.B1 cell line(HEK293 T-TPL2-/-)was randomly selected for subsequent functional evaluation.The rate of cell proliferation after TPL2 knockout was not significantly different from that of control HEK293 T cell,both were adherent and epithelial-like cell morphology.Compared with control cells after SVA infection,knockout of the TPL2 gene significantly increased virus copy number,viral m RNA transcription,viral protein translation,and virulence of progeny viruses.In summary,this study successfully establish the TPL2 gene knockout HEK293 T cell line and prove that TPL2 plays an antiviral role during SVA infection,which lay the experimental foundation and provide a good cell model for further revealing the specific mechanism of TPL2 related immune response and SVA infection process,and also provide a feasible strategy for increasing the production of virus in vaccine production process.