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

Abstract

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

CLC Number:

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

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