数字农科院2.0

稳定表达TMPRSS2蛋白Vero-E6细胞系的建立及其对新型冠状病毒增殖影响的研究

文献类型： 中文期刊

关键词： 严重急性呼吸综合征冠状病毒2;;跨膜丝氨酸蛋白酶2;;Vero-E6细胞

期刊名称：中国预防兽医学报

ISSN： 1008-0589

年卷期： 2025 年

页码：

收录情况： 北大核心（2023版） ; ; CSCD（2025-2026年度） ; ; 科技核心（2024版） ; ; 农林核心（2024版）

摘要： 为构建稳定表达跨膜丝氨酸蛋白酶2（TMPRSS2）的Vero-E6细胞系，并分析其对新型冠状病毒增殖的影响，本研究采用同源重组的方法，将经PCR扩增获得的TMPRSS2和IRES-mCherry基因克隆至真核表达质粒pCAGGSpuro中，构建重组质粒pCAGGS-TMPRSS2-IRES-mCherry，经酶切和测序鉴定正确。将1μg/mL～10μg/mL嘌呤霉素加入Vero-E6细胞中加压培养14 d，每天观察细胞死亡情况。以细胞100%死亡的最低浓度为嘌呤霉素的最适浓度。结果显示，5μg/mL～10μg/mL嘌呤霉素培养的Vero-E6细胞全部死亡，其余浓度嘌呤霉素处理后仍有细胞存活。因此，确定其最适浓度为5μg/mL。将pCAGGS-TMPRSS2-IRES-mCherry转染Vero-E6细胞，并以5μg/mL嘌呤霉素筛选培养72 h，采用有限稀释法将单克隆细胞传代，7 d后通过荧光显微镜观察，选择红色荧光较强且仅有1个细胞形成的集落传代，将获得的细胞命名为Vero-E6-TMPRSS2。采用RT-PCR和western blot分别检测该细胞及其不同代次细胞（第5、10、15和20代）中TMPRSS2基因的转录和蛋白的表达水平。将嵌合病毒VSVΔG-SBA.1.1-EGFP分别感染VeroE6-TMPRSS2细胞和对照Vero-E6细胞，培养至12 h和24 h时观察荧光，并在感染后不同时间取上清液，采用ReedMuench法测定各时间点病毒的TCID50，绘制病毒的生长曲线。RT-PCR结果显示，Vero-E6-TMPRSS2细胞及其不同代次均扩增出1 596 bp的目的基因片段；Western blot结果显示，该细胞及不同代次的该细胞均在57.7 ku处出现特异性条带，而对照Vero-E6细胞则无任何基因片段和蛋白条带。荧光观察结果显示，VSVΔG-SBA.1.1-EGFP接种后不同时间出现绿色荧光的Vero-E6-TMPRSS2细胞数量均明显多于该病毒接种的Vero-E6细胞；生长曲线结果显示，感染Vero-E6细胞后72 h VSVΔG-SBA.1.1-EGFP的病毒滴度最高达106.5TCID50/mL；而在感染Vero-E6-TMPRSS2细胞后36 h VSVΔGSBA.1.1-EGFP的病毒滴度最高达108.6 TCID50/mL。上述结果表明，本研究首次建立了稳定表达TMPRSS2 基因的Vero-E6-TMPRSS2细胞系，且Vero-E6-TMPRSS2细胞通过表达的TMPRSS2显著促进VSVΔG-SBA.1.1-EGFP的快速增殖与复制，该结果为后续新冠病毒的基础研究及其疫苗的生产奠定了基础并提供了有力的工具。

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