体外对牛冠状病毒复制具有抑制效应藏药的筛选

doi:10.11843/j.issn.0366-6964.2025.02.031

Abstract

Abstract:

This study was aimed at screening Tibetan medicines exerting inhibitory effects on the in vitro replication of Bovine coronavirus (BCoV). To achieve this, a prokaryotic expression vector encoding the BCoV nucleocapsid (N) protein was constructed. Following induced expression, the purified N protein was used to immunize mice and rabbits, resulting in the production of mouse anti-N protein polyclonal antibodies and rabbit anti-N protein antibodies. Western blot and indirect immunofluorescence assay (IFA) techniques were subsequently developed to assess BCoV replication. The water extract of 21 candidate Tibetan medicines was obtained by water decoction method. The maximum safe concentration of water extract on human colorectal adenocarcinoma cells (HCT-8) was determined by CCK-8. HCT-8 cells infected with BCoV were used as the research object, and BCoV infection group, Tibetan medicine water extract treatment group and ribavirin treatment group were set up respectively. After 24 h of BCoV infection, real-time fluorescence quantitative PCR, Western blot and IFA were used to determine the replication of BCoV in cells. The inhibitory effect of Tibetan medicine on virus replication was statistically analyzed, and Tibetan medicine with significant inhibitory effect on BCoV was screened. The polyclonal antibodies of mouse anti-BCoV N protein and rabbit anti-BCoV N protein were prepared respectively, and the Western blot and IFA methods for detecting BCoV were established. Among the 21 Tibetan medicines, 15 had significant inhibitory effects on BCoV replication (P < 0.01). Among them, the inhibitory effects of three Tibetan medicines, Euphorbia altotibetica Paulsen, Terminalia chebula Retz., and Stellera chamaejasme L. on BCoV replication were better than those of ribavirin. In this study, 15 Tibetan medicines with inhibitory effects on BCoV replication in vitro were screened. Among them, three Tibetan medicines, Euphorbia altotibetica Paulsen, Terminalia chebula Retz., and Stellera chamaejasme L. had significant inhibitory effects, which laid a foundation for the development of BCoV prevention and treatment drugs.

Key words: bovine coronavirus, N protein, polyclonal antibody, Tibetan medicine, viral replication

CLC Number:

ZHAO Long, LIN Jingyi, DOU Wei, XU Tingxuan, GU Qingyun, GAO Haihui, LI Shengqing, GUO Kangkang. In vitro Screening of Tibetan Medicine with Inhibitory Effects on Bovine Coronavirus Replication[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(2): 826-838.

Figures/Tables 7

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References 27

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药物名称 Name of medicine	干粉质量/g Weight of dry powder	药物初始浓度/(mg·mL^-1) Initial drug concentration	最大安全浓度/(mg·mL^-1) Maximum safe concentration
独一味	13.22	55.80	3.49
蓝花荆芥	9.32	38.75	4.84
绿萝花	11.35	91.91	11.49
藏木香	14.66	41.12	20.56
盘花垂头菊	17.11	45.96	1.44
诃子	14.90	1.30	0.16
青藏大戟	18.38	3.95	0.12
乌奴龙胆	12.72	223.21	111.61
短穗兔耳草	15.35	1.30	0.33
五脉绿绒蒿	9.88	208.64	6.52
长松萝	17.26	11.88	1.48
余甘子	15.77	4.88	0.31
蒺藜	10.62	71.25	17.81
铁棒锤	18.39	15.63	0.98
螃蟹甲	16.715	7.42	1.86
甘青青兰	13.99	39.06	4.88
川西獐牙菜	13.24	39.06	9.77
甘青乌头	16.71	14.47	3.67
矮紫堇	17.23	82.25	2.57
瑞香狼毒	15.12	56.88	7.17
虎耳草	12.73	18.59	1.16
利巴韦林	0.10	0.38	0.024

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In vitro Screening of Tibetan Medicine with Inhibitory Effects on Bovine Coronavirus Replication

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