青蒿素与牛病毒性腹泻病毒NS5B蛋白的分子对接预测及其抗病毒作用

【目的】 采用分子对接以及体外试验确定青蒿素对牛病毒性腹泻病毒（Bovine viral diarrhea virus，BVDV）复制的抑制作用，为抗病毒药物和制剂开发提供新思路。【方法】 以BVDV-NS5B蛋白为作用靶点，通过PDB数据库检索蛋白三维晶体结构，并对其结构进行适当修饰处理；检索TCMSP数据库中的青蒿素结构，应用Autodock软件将两者进行分子对接及结合能打分。随后采用CCK-8试剂盒测定青蒿素对MDBK细胞的最大安全浓度；将选定浓度的青蒿素进行梯度稀释，采用先加病毒后加药物、先加药物后加病毒、中药和病毒同时作用的3种不同加药方式进行药物抗病毒试验，确定对病毒的最佳药物抑制浓度、预防浓度和杀灭浓度。应用实时荧光定量PCR法检测3种药物作用方式下BVDV的拷贝数，进一步明确药物对BVDV复制的作用。【结果】 分子对接数据表明青蒿素与BVDV-NS5B存在相互作用，结合自由能为-28.6748 kJ/mol。青蒿素在MDBK细胞上的最佳药物安全浓度为100 μmol/L。3种作用方式下青蒿素浓度为100 μmol/L时均可有效影响BVDV的复制，青蒿素对BVDV的抑制作用最为明显。【结论】 青蒿素可与BVDV-NS5B蛋白靶点互作，并能在MDBK细胞上有效抑制BVDV的复制，本研究为抗BVDV中药筛选奠定了基础。

Molecular Docking Prediction of Artemisinin and NS5B Protein of Bovine Viral Diarrhea Virus and Its Antiviral Effect

【Objective】 The study was aimed to determine the inhibitory effect of artemisinin on Bovine viral diarrhea virus (BVDV) replication using molecular docking as well as in vitro tests, and provide new ideas for antiviral drug and formulation development.【Method】 BVDV-NS5B protein was used as the target, and the three-dimensional crystal structure of BVDV-NS5B protein was retrieved through the PDB database, and the structure was appropriately modified.The structure of artemisinin from TCMSP database was retrieved, and Autodock software was applied to perform molecular docking and score the binding energy.The maximum safe concentration of artemisinin for MDBK cells was subsequently determined using a CCK-8 kit.The selected concentrations of artemisinin were subjected to gradient dilution, and three different ways of spiking drugs, including adding virus before adding drug, adding drug before adding virus, and the simultaneous action of traditional Chinese medicine and virus, were used for drug antiviral test to determine the optimal drug inhibitory concentration, prophylactic concentration and killing concentration against virus.Real-time quantitative PCR was used to measure the copy number of BVDV under the three modes of drug action to further define the effect of drugs on BVDV replication.【Result】 Molecular docking data indicated that artemisinin interacted with BVDV-NS5B with a binding free energy of -28.6748 kJ/mol.The optimal drug safety concentration of artemisinin on MDBK cells was 100 μmol/L.The three modes of action could effectively affect the replication of BVDV, and artemisinin had the most obvious inhibitory effect on BVDV.【Conclusion】 Artemisinin could interact with the NS5B protein target of BVDV, and could effectively inhibit the replication of BVDV in MDBK cells.This study laid a foundation for the screening of anti BVDV traditional Chinese medicine.