囊泡运输蛋白SEC22B敲低对牛病毒性腹泻病毒复制的影响研究

doi:10.3969/j.issn.1674-7968.2023.04.011

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摘要牛病毒性腹泻病的感染情况日趋严重,给养殖业造成了严重的损失,目前我国缺乏有效的治疗手段,导致感染模式复杂化,其致病机制和病毒-宿主之间相互作用研究也尚不清楚。为了探究囊泡运输蛋白 (vesicle transport protein homolog, SEC22B)敲低对牛病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV)复制的影响,根据 GenBank 数据库中 SEC22B 基因序列,使用 Benchling 平台设计向导 RNA (small guide RNA, sgRNA),克隆至 LentiCRISPR v2 载体后进行慢病毒包装,使用 CRISPR/Cas9 基因编辑技术敲低胎牛肾细胞(madin-darby bovine kidney cells, MDBK)中的 SEC22B 基因表达,使用 Western blot 鉴定 SEC22B 基因敲低(knockdown, KD)情况;BVDV 感染 SEC22B KD 细胞不同时间后,分别使用 qPCR 和免疫荧光染色检测 BVDV 5' UTR mRNA 水平变化和双链 RNA (double-stranded RNA, dsRNA)量,使用 Reed- Muench 法测定 BVDV 感染不同时间后子代病毒滴度变化。Western blot 检测发现,与对照 Scramble 细胞相比,SEC22B KD 细胞中 SEC22B 蛋白表达量显著降低 ;与 BVDV 感染 Scramble 细胞相比,BVDV 感染 SEC22B KD 细胞后 5' UTR mRNA 水平和 dsRNA 量均极显著降低(P<0.01),同时,子代病毒滴度极显著下降(P<0.01)。结果表明,敲低 SEC22B 基因后显著抑制 BVDV 复制,本研究为 BVDV 防控新方法的建立提供理论依据。

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	张成远
	袁圆圆
	郭妍婷
	杨莉
	冉多良
	付强
	史慧君

关键词 ：囊泡运输蛋白(SEC22B), 牛病毒性腹泻病毒(BVDV), CRISPR/Cas9基因编辑技术, 基因敲低, 复制

Abstract：The infection of bovine viral diarrhea is becoming more and more serious, which has caused serious losses to the breeding industry. At present, the lack of effective treatment leads to the complicated infection pattern in China, and the research on the pathogenesis and virus-host interaction is still unclear. To investigate the effect of vesicle transport protein (SEC22B) deletion on Bovine viral diarrhea virus (BVDV) replication, according to the SEC22B gene sequence in GenBank database, small guide RNA (sgRNA) was designed using Benchling platform and cloned into LentiCRISPR V2 vector for lentivirus packaging. The SEC22B gene in madin-darby bovine kidney cells (MDBK) was knocked down (KD) by CRISPR/Cas9 gene editing technique, and identified by Western blot. After BVDV infection with SEC22B KD cells at different times, the changes of 5' UTR mRNA level and accumulation of double-stranded RNA (dsRNA) were detected by qPCR and immunofluorescence staining, respectively. Reed-Muench method was used to measure the titer change of progeny virus after BVDV infection at different time. The results showed that compared with scramble cells, the protein expression level of SEC22B in SEC22B KD cells was significantly decreased by Western blot. Compared with scramble cells infected with BVDV, the level of 5' UTR mRNA and dsRNA of SEC22B KD cells were extremely significantly decreased (P<0.01) after BVDV infection, and the titer of progeny virus was extremely significantly decreased (P<0.01). The results showed that knockdown SEC22B gene significantly inhibited the replication of BVDV. This study provides a target for the establishment of new methods for the prevention and control of BVDV.

Key words： Vesicle transport protein (SEC22B) Bovine viral diarrhea virus (BVDV) CRISPR/Cas9 gene editing technology Gene knockdown Replication

收稿日期: 2022-04-14

ZTFLH:

S852.65

通讯作者: * shihuijunmm@163.com

引用本文:

张成远, 袁圆圆, 郭妍婷, 杨莉, 冉多良, 付强, 史慧君. 囊泡运输蛋白SEC22B敲低对牛病毒性腹泻病毒复制的影响研究[J]. 农业生物技术学报, 2023, 31(4): 784-790.
ZHANG Cheng-Yuan, YUAN Yuan-Yuan, GUO Yan-Ting, YANG Li, RAN Duo-Liang, FU Qiang, SHI Hui-Jun. Effect of Vesicle Transport Protein SEC22B Knockdown on the Replication of Bovine viral diarrhea virus. 农业生物技术学报, 2023, 31(4): 784-790.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.04.011 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I4/784

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