表达绿色荧光蛋白重组猪塞内卡病毒的构建及初步应用

塞内卡病毒A（SVA）是新近发现的一种能够引起猪特发性水疱病及新生仔猪死亡为主要临床特征的病原微生物。该病呈世界性分布，给多个国家的养猪业造成了一定的经济损失。鉴于目前国内外尚无可用SVA商品化疫苗和抗病毒药物上市，本研究拟在实验室前期对SVA研究的基础上建立稳定表达绿色荧光蛋白报告基因的重组病毒，为体外高通量筛选具有抗SVA活性药物提供一种简单快速有效的工具。本研究在pEGFP-C1载体EGFP基因末端引入猪捷申病毒1型的2A基因，构建EGFP-P2A融合基因过度质粒，随后分别设计带有同源臂的克隆引物，通过同源重组技术将EGFP-P2A基因插入SVA毒株CH/HeN-2018基因组2A和2B基因间，并经测序鉴定，成功构建重组载体pSVA-EGFP。将pSVA-EGFP转染PK-15细胞，盲传至P2代，获得致使感染细胞病变明显、病变时间稳定的重组SVA荧光毒rCH/HeN-2018-EGFP。通过绿色荧光观察、RT-PCR和生长曲线测定，评估了重组毒株rCH/HeN-2018-EGFP和亲本毒株生长特性。并进一步通过药物细胞毒性试验和病毒感染试验对rCH/HeN-2018-EGFP作为潜在抗SVA药物筛选工具进行评价。结果表明，rCH/HeN-2018-EGFP与亲本毒株wtCH/HeN-2018和rCH/HeN-2018在PK-15细胞中具有相似的生长特性，且遗传稳定，在P10代能够稳定表达绿色荧光，插入的EGFP-P2A基因没有出现突变现象。抗SVA病毒感染试验结果表明，用终浓度20 μmol·L^-1的姜黄素和110 μmol·L^-1黄芩苷预处理PK-15细胞2 h，通过观察荧光细胞的数量直观判断两种药物均能够抑制rCH/HeN-2018-EGFP在细胞中的复制，其中黄芩苷抑制效果极显著。随后在亲本毒株wtCH/HeN-2018感染试验进一步证实了黄芩苷对SVA病毒复制抑制的效果。本研究所构建的携带EGFP基因的重组SVA毒株能够高效稳定地表达绿色荧光蛋白，可以作为一种工具报告病毒应用于抗SVA药物和蛋白的筛选过程。

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

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.