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

doi:10.13560/j.cnki.biotech.bull.1985.2020-0886

摘要/Abstract

摘要：

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。

关键词: α2抗纤溶酶, 牛病毒性腹泻病毒, CRISPR/Cas9

Abstract:

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.

Key words: SERPINF2, bovine viral diarrhea virus, CRISPR/Cas9

付强, 陈俊贞, 郭妍婷, 姚刚, 冉多良, 史慧君. 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[J]. 生物技术通报, 2021, 37(5): 267-272.

FU Qiang, CHEN Jun-zhen, GUO Yan-ting, YAO Gang, RAN Duo-liang, SHI Hui-jun. Effects of Knockout of Gene SERPINF2 via CRISPR/cas9 on the Replication of Bovine Viral Diarrhea Virus[J]. Biotechnology Bulletin, 2021, 37(5): 267-272.

图/表 8

表1 sgRNA序列

Table 1 SgRNA sequences

向导RNA sgRNA	序列 Sequence （5'→3'）
SERPINF2	CACCGCATCCTGTCACCTCTGAGTG
SERPINF2	AAACCACTCAGAGGTGACAGGATGC
Scramble	CACCGCACTACCAGAGCTAACTCA
Scramble	aaacTGAGTTAGCTCTGGTAGTGC

图1 lentiCRISPR v2质粒酶切鉴定结果 M：D2000 DNA Marker;1：酶切lentiCRISPR v2载体

Fig.1 Identification of lentiCRISPR V2 plasmid by enzyme digestion M: D2000 DNA marker, 1: enzyme cutting lentiCRISPR v2 vector

图2 MDBK细胞SERPINF2基因敲除效率检测结果 WT：野生型;-：碱基缺失;+：碱基插入;数字：碱基个数;（数字）：缺失或插入等突变的克隆数

Fig.2 Efficiency Detection of SERPINF2 gene knockout in the MDBK cells WT：wild type, -：base deletion, +：base insertion, Number：base number, (Number)：Clone number of mutation such as deletion or insertion

图3 qRT-PCR检测BVDV感染MDBK细胞后SERPINF2 mRNA转录水平

Fig.3 Transcription levels of SERPINF2 mRNA in the MDBK cells infected by BVDV via qRT-PCR

图4 qRT-PCR检测BVDV感染SERPINF2 KO细胞后BVDV RNA水平

Fig.4 qRT-PCR detection of BVDV RNA level in the SERPINF2 KO cells infected with BVDV

图5 免疫荧光染色检测BVDV感染SERPINF2 KO细胞后BVDV双链RNA含量结果

Fig.5 Detection of BVDV dsRNA accumulation in the SERPINF2 KO cells infected with BVDV by immunofluorescence staining

图6 BVDV感染SERPINF2 KO细胞后病毒滴度检测结果

Fig.6 Detection results of virus titer in the SERPINF2 KO cells infected with BVDV

图7 BVDV感染SERPINF2 KO细胞后致细胞病变效应检测结果

Fig.7 Detection results of cytopathic effect in the SERPINF2 KO cells infected with BVDV

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