表达绿色荧光蛋白重组猪塞内卡病毒的构建及初步应用

doi:10.11843/j.issn.0366-6964.2021.010.029

Abstract

Abstract: Senecavirus A (SVA), a newly emerged causative agent linked to swine idiopathic vesicular disease and epidemic transient neonatal losses, is spreading worldwide and causing economic loss to the swine industry. Since no commercial vaccine or antiviral drug has been licensed, constructing a reporter virus expressing enhanced green fluorescent protein (EGFP) for high-throughput screening antiviral drugs will be undoubtedly conducive to prevent and control of SVA. In this study, the porcine teschovirus-1 2A gene (P2A) was synthesized and cloned into the C-terminal of EGFP gene of pEGFP-C1 to construct pEGFP-P2A vector. Subsequently, the EGFP-P2A gene was inserted into pwtSVA, the DNA-launched infectious clone of wtCH/HeN-2018, at site between 2A and 2B through homologous recombination to create a recombinant plasmid pSVA-EGFP. The pSVA-EGFP was then directly transfected into PK-15 cells and the recombinant virus rCH/HeN-2018-EGFP caused stable cytopathic effect and strong green fluorescence was harvested after twice blind passages. The genetic stability and growth kinetic of rCH/HeN-2018-EGFP was further investigated via RT-PCR and virus infection assays. Results demonstrated that rCH/HeN-2018-EGFP shared similar growth dynamics with that of the parental virus wtCH/HeN-2018 and rCH/HeN-2018. Moreover, no mutation was detected in the inserted EGFP-P2A gene and cells infected by P10 passage of rCH/HeN-2018-EGFP stably expressed green fluorescence. Finally, the rCH/HeN-2018-EGFP was employed to test the antiviral effect of curcumin and baicalin. Data showed that pretreated with both curcumin (20 μmol·L^-1) and baicalin (110 μmol·L^-1) for 2 hours restricted the replication of rCH/HeN-2018-EGFP in PK-15 cells, with green fluorescence intensity lower than that in DMSO control group. Compared to curcumin, however, baicalin exhibited greater anti-SVA effect, which was further confirmed by inhibiting replication of wtCH/HeN-2018 in vitro. Conclusively, the recombinant rCH/HeN-2018-EGFP virus constructed in this study effectively and stably expressed EGFP during infection and provided a robust tool to screen anti-SVA drugs and proteins.

Key words: Senecavirus A, reverse genetics system, reporter virus, green fluorescent protein, antiviral drug screen

CLC Number:

S852.659.6

ZHANG Xiaozhan, YANG Lei, DENG Tongwei, ZHAO Pandeng, PENG Zhifeng, CHEN Lulu, GUO Yiwen, XIA Yanxun, QIAO Hongxing, BIAN Chuanzhou, WANG Zeng. Development and Application of Recombinant Senecavirus A Expressing the Green Fluorescent Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(10): 2978-2985.

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Development and Application of Recombinant Senecavirus A Expressing the Green Fluorescent Protein

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