细胞信号肽酶复合体亚基1与牛病毒性腹泻病毒复制的相关性分析

doi:10.11843/j.issn.0366-6964.2022.06.020

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (6): 1870-1876.doi: 10.11843/j.issn.0366-6964.2022.06.020

细胞信号肽酶复合体亚基1与牛病毒性腹泻病毒复制的相关性分析

史慧君, 董文丽, 郭妍婷, 袁圆圆, 陈俊贞, 杨莉, 冉多良, 付强*

新疆农业大学动物医学学院，乌鲁木齐 830052

收稿日期:2021-08-09 出版日期:2022-06-23 发布日期:2022-06-25
通讯作者: 付强，主要从事草食家畜病原学研究，E-mail：fq198505@gmail.com
作者简介:史慧君(1987-)，女，新疆阜康人，博士，副教授，主要从事病原微生物致病机制研究，E-mail: shihuijunmm@163.com
基金资助:
国家自然科学基金项目(31902271;32160829)；自治区天山创新团队(2020D14005)；自治区百名博士引进计划

Correlation Analysis of Cell Signal Peptidase Complex Subunit 1 and Bovine Viral Diarrhea Virus Replication

SHI Huijun, DONG Wenli, GUO Yanting, YUAN Yuanyuan, CHEN Junzhen, YANG Li, RAN Duoliang, FU Qiang*

College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi 830052, China

Received:2021-08-09 Online:2022-06-23 Published:2022-06-25

摘要/Abstract

摘要： 旨在探明信号肽酶复合体亚基1(signal peptidase complex subunit 1，SPCS1)影响牛病毒性腹泻病毒(bovine viral diarrhea virus，BVDV)复制的作用。使用Benchling和CHOPCHOP等平台设计靶向SPCS1基因的sgRNA，克隆至慢病毒载体lentiCRISPR v2，连同包装质粒pSPAX2和pMD2.G转染至人胚肾细胞HEK-293T中，包装慢病毒并感染MDBK细胞，用嘌呤霉素筛选5代后获得敲低SPCS1蛋白后稳定表达的细胞，用Western blot检测SPCS1蛋白表达的情况；使用荧光定量PCR和免疫荧光分析检测BVDV感染SPCS1蛋白敲低细胞不同时间后5'非翻译区(untranslated region，UTR)mRNA的水平和双链RNA(double-stranded，dsRNA)积累，利用倒置显微镜观察BVDV致细胞病变(cytopathic effect，CPE)情况，根据Reed-Muench方法测定病毒滴度变化情况。结果：Western blot检测SPCS1蛋白表达量明显下降，成功构建SPCS1敲低(knockdown，KD)细胞；BVDV感染SPCS1 KD细胞后与对照Scramble相比，BVDV 5' UTR mRNA水平和dsRNA量均显著性降低(P < 0.01)，CPE现象推迟并减弱，子代病毒滴度显著性下降(P < 0.01)，最高降低95.8%。SPCS1敲低后显著性影响BVDV复制，为BVDV防控新技术的建立提供理论依据。

关键词: 信号肽酶复合体亚基1, 牛病毒性腹泻病毒, 5'非翻译区, 双链RNA, 复制

Abstract: The present study aimed to investigate the effect of signal peptidase complex subunit 1 (SPCS1) on the replication of bovine viral diarrhea virus (BVDV). The sgRNA targeting SPCS1 gene was designed by using bioinformatics platforms such as Benchling and CHOPCHOP, following with cloning into lentivirus vector lentiCRISPR v2, and transfected into human embryonic kidney cell HEK-293T plus packaging plasmids pSPAX2 and pMD2.G. After lentivirus generation, lentivirus was used to infect MDBK cells, and puromycin was used for continuous screening for 5 generations. The knockdown (KD) of SPCS1 protein was determined by Western blot. At different time intervals after BVDV infected SPCS1 gene knockdown cells, the mRNA level of 5' untranslated region (UTR) and the accumulation level of double-stranded RNA (dsRNA) of BVDV were detected by real-time quantitative PCR and immunofluorescence assay. The cytopathic effect (CPE) was observed under a microscope. The progeny virus suspension was collected and its titer was determined by Reed-Muench method. We observed that the expression of SPCS1 protein decreased significantly, and SPCS1 KD cells were successfully constructed. Compared with the scramble cells, after BVDV infected SPCS1 KD cells, BVDV 5'UTR mRNA level and dsRNA accumulation decreased significantly (P < 0.01), CPE phenomenon was significantly delayed and weakened, and the virus titer of progeny virus decreased significantly (P < 0.01), up to 95.8% reduce. Our findings suggest that SPCS1 KD significantly inhibits BVDV replication, which provides an important target for the establishment of new technology for BVDV prevention and control.

Key words: signal peptidase complex subunit 1, bovine viral diarrhea virus, 5' untranslated region, double-stranded RNA, replication

中图分类号:

史慧君, 董文丽, 郭妍婷, 袁圆圆, 陈俊贞, 杨莉, 冉多良, 付强. 细胞信号肽酶复合体亚基1与牛病毒性腹泻病毒复制的相关性分析[J]. 畜牧兽医学报, 2022, 53(6): 1870-1876.

SHI Huijun, DONG Wenli, GUO Yanting, YUAN Yuanyuan, CHEN Junzhen, YANG Li, RAN Duoliang, FU Qiang. Correlation Analysis of Cell Signal Peptidase Complex Subunit 1 and Bovine Viral Diarrhea Virus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1870-1876.

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细胞信号肽酶复合体亚基1与牛病毒性腹泻病毒复制的相关性分析

Correlation Analysis of Cell Signal Peptidase Complex Subunit 1 and Bovine Viral Diarrhea Virus Replication

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