应用CRISPR/Cas9技术敲除SERPINF2基因对牛病毒性腹泻病毒复制的影响

Effects of Knockout of Gene SERPINF2 via CRISPR/cas9 on the Replication of Bovine Viral Diarrhea Virus

应用CRISPR/Cas9基因编辑技术敲除MDBK细胞的SERPINF2基因,以探明SERPINF2基因对牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)复制的影响。采用CRISPR/Cas9技术建立SERPINF2敲除细胞;实时荧光定量PCR、免疫荧光染色、Reed-Muench法等检测BVDV感染SERPINF2敲除细胞后病毒RNA水平、dsRNA积累、病毒滴度和CPE等。构建SERPINF2基因敲除的MDBK细胞SERPINF2 KO,测序27株阳性克隆中有19株基因序列发生缺失(-)和插入(+)等移码突变,敲除效率约为70.37%;实时荧光定量PCR检测BVDV TC株感染MDBK细胞后SERPINF2 mRNA转录水平显著升高;与对照组Scramble细胞相比,BVDV TC株感染SERPINF2 KO细胞后病毒RNA水平和dsRNA积累显著性降低,CPE发生延迟,相同时间导致细胞脱落数量显著减少,BVDV滴度显著性降低。应用CRISPR/Cas9基因编辑技术成功敲除MDBK细胞的SERPINF2基因,建立SERPINF2 KO细胞,试验表明敲除SERPINF2基因显著性抑制BVDV复制。

This work is aimed to knock out the gene SERPINF2 in MDBK cells by CRISPR/cas9 gene editing technology,and to explore the effect of gene SERPINF2 on the replication of bovine viral diarrhea virus(BVDV).CRISPR/Cas9 technology was performed to establish SERPINF2-knockout cells.Quantitative real-time PCR(qRT-PCR),immunofluorescence staining and Reed-Muench assay were applied to detect the viral RNA levels,the accumulation of double strand RNA(dsRNA),viral titer,and cytopathic effect(CPE)in the BVDV infected by SERPINF2-knockout cells.The SERPINF2-knockout MDBK cells SERPINF2 KO were successfully constructed.Of the 27 positive clones,19 clones were sequenced with frame-shift mutations,such as deletion(-)and insertion(+).The knockout efficiency was approximately 70.37%.The SERPINF2 mRNA transcription level significantly increased after the BVDV TC strain infected MDBK cells.Compared with the scramble cells in the control group,viral RNA levels and dsRNA accumulation decreased significantly in the SERPINF2 KO cells infected with BVDV TC,CPE delay occurred,and the number of cells shedding and BVDV titer were in significant decrease at the same time interval.The gene SERPINF2 of the MDBK cells is successfully knocked out using CRISPR/Cas9 gene editing technology,and the SERPINF2 KO cells are established.Knockout of the gene SERPINF2 significantly inhibits BVDV replication.