【作者】 孙艳；

【导师】 曾毅； 李泽琳；

【作者基本信息】 北京工业大学 ， 生物学， 2017， 硕士

【摘要】 H1N1属于正粘病毒科（Orthomyxoviridae）,甲型流感病毒属（Influenza AVirus）,其抗原极易发生变异,人类通常无法获得持久的免疫力,一旦爆发,将会对人类健康和社会经济造成巨大的损失。离子通道阻断剂和神经氨酸酶抑制剂是目前上市的主要抗流感病毒药物,但是近年来不断有耐药毒株的报道。流感病毒的核蛋白（Nucleoprotein,NP）在病毒复制周期的各个环节都起着重要作用,并且序列保守,抗原结构十分稳定,是一个非常理想的药物作用靶点。另外,天然中药在我国有着悠久的历史文化,其来源广、价格低、毒副作用小、不易产生耐药性,在抗流感病毒药物研发中具有广阔的发展前景。本研究从细胞和分子水平研究天然植物SY提取和分离产物抗H1N1甲型流感病毒的作用及其初步的作用机理,并分离鉴定出以NP蛋白为靶点的有效活性成分。天然植物SY分别用30%乙醇、60%乙醇和90%乙醇三种不同溶剂渗漉提取,共得到三种粗提物。H1N1流感病毒感染MDCK细胞,加入三种粗提物,ELISA方法测定粗提物对流感病毒的抑制作用,发现SY粗提物均对H1N1的增殖有一定抑制作用,其中60%的乙醇提取物抑制效果最明显,对H1N1病毒的半数抑制浓度(IC₅₀)为1.394±0.11 mg/mL,同时测定了SY-60粗提物对细胞的毒性作用,其细胞的半数毒性浓度(TC₅₀)为41.3±3.7 mg/mL。随后运用表面等离子共振技术（SPR）对SY粗提物进行作用靶点的研究,初步探讨其作用机制。由于NP蛋白在病毒基因组复制、转录和翻译等过程中起着重要的作用,序列保守,抗原结构十分稳定,是一个理想的药物作用靶点。将NP蛋白与血凝素HA蛋白固定到CM5芯片上,牛血清白蛋白为空白对照,用BIAcore 3000 SPR仪器检测提取物与靶蛋白的结合情况。结果显示60%的乙醇提取物与NP蛋白响应值最高,与细胞水平抗病毒作用结果一致,提示天然植物SY的抗病毒作用机制与NP蛋白有关,选择NP作为最终的靶点蛋白。接下来,利用蛋白氨基偶联柱对60%乙醇提取物进行针对NP靶点活性成分的分离富集。将1-2 mg的NP蛋白偶联至蛋白偶联柱后,加入60%的醇提物孵育,与NP结合的成分被吸附柱吸附富集,然后洗脱液洗脱结合产物,分段收集洗脱成分,每段收集约1 mL,得到10管富集洗脱产物,按先后顺序编为G1到G10。各段收集得到的富集产物再次用SPR技术检测其与NP蛋白的亲和性,其中G3和G4管与NP的结合值最高。ELISA细胞药效学实验结果表明G3、G4两管富集产物均具有较好的抗病毒效果。将富集了活性成分的G3、G4管物质,进行高效液相色谱和质谱分析,计算出它们的准分子量,在化合物数据库中进行数据比对,推测出5种可能的化合物。最后利用DS分子对接软件将这5种化合物与NP蛋白对接,结果显示化合物1、3、5能与NP对接,化合物5对接评分最高。提示这3种化合物对H1N1流感病毒有着潜在的抑制效果,其中化合物5的抑制效果最好。最后,对化合物单体5进行细胞药效学的检测,结果表明其具有一定的抗病毒作用,IC₅₀为190.9±11.05μg/mL。更多还原

【Abstract】 H1N1 belongs to Orthomyxoviridae,Influenza A Virus,and its antigens are susceptible to mutations.Human beings are usually unable to obtain lasting immunity.Once it outbreaks,it will cause huge human health and social economy loss.Ion channel blockers and neuraminidase inhibitors are currently the main anti-influenza virus drugs,however,some virus has shown drug resistance already.Influenza virus nucleoprotein（NP）plays an important role in all aspects of the viral replication cycle,and the sequence is conserved and the antigen structure is very stable,it is a very ideal drug target.In addition,natural Chinese medicine has a long history and culture in China,its wide source,low price,little side-effect,not easy to produce resistance,has broad prospects for development in the anti-influenza virus drug research and development.In this study,the effects of natural herb SY extractions and separations anti-H1N1influenza virus and its preliminary mechanism were studied at the leval of cell and molecular,and the active ingredients targeting NP protein were isolated and identified.The natural herb SY was extracted with 30%ethanol,60%ethanol and 90%ethanol.Three kinds of crude extracts were obtained by penetration method with three different solvents.The H1N1 influenza virus infected MDCK cells in the presence of the three crude extracts.The results showed that all the extracts had inhibitory effect on the virus,60%of the ethanol extract showed the best inhibiton on the H1N1 virus with,the half inhibitory concentration(IC₅₀)was 1.394±0.11 mg/m L.It’s half toxicity concentration(TC₅₀)of cells was 41.3±3.7 mg/mL.Then,the affinity of the extracts with NP protein was determined by the surface plasmon resonance（SPR）instrument BIA3000.The protein was immobilized on the CM5 chip with bovine serum albumin as the blank control.The results showed that 60%ethanol extract and NP protein had the highest response value,which was consistent with the anti-virus effect at the cell level.The results implied that the antiviral mechanism of 60%ethanol extract was related to NP protein.The active components of 60%ethanol extract on the NP protein were separated and enriched using the protein amino-coupled column.The 1-2 mg of NP protein was incubated with the protein coupling column,then 60%of the alcohol extract was added.The NP-bound components were adsorbed by the column.The elution buffer eluted the column and the combination ingredients were collected in to the EP tube with 1 mL each seperately.The binding affinity of the collection with the NP protein was tested by SPR technique,in which the binding value of G3 and G4 demonstrated significantly increased.G3 and G4 also showed inhibition on influenza virus at the cell level.The ingredients of G3 and G4 were detected by HPLC-MS.The data were alligned with the Chinese Academy of Sciences chemical database and the laboratory of Chinese medicine composition database,and five possible compounds were deduced.Finally,the five compounds were docked with NP protein by DS molecular docking software.The results suggested that these three compounds have a potential inhibitory effect on H1N1 influenza virus,and compound 5 might have the best inhibitory effect with the highest score.The test of compound 5 on H1N1 virus showed a good antiviral activity with IC₅₀ of 190.9±11.05μg/m L.更多还原