假病毒技术在抗H5N1禽流感病毒中药筛选中的应用及评价  

Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5N1 avian influenza virus

作　　者：刘红[1] 张国良[1] 沈丽[2] 曾贞[2] 周保罗[3] 刘成海[2] 聂广[1] 

机构地区：[1]深圳市第三人民医院中西医结合科,广东深圳518020 [2]上海中医药大学曙光医院肝病研究所,上海201203 [3]中国科学院上海生命科学研究院,上海200025

出　　处：《中西医结合学报》2010年第11期1036-1040,共5页Journal of Chinese Integrative Medicine

基　　金：国家自然科学基金资助项目(No.30873264)

摘　　要：目的:建立基于假病毒技术的抗H5N1禽流感病毒中药筛选方法,利用血清药理学的方法系统评价攻下方、解毒方、凉血方和扶正方4类中药复方体外对H5N1禽流感病毒活性的影响及其机制。方法:构建整合H5N1禽流感病毒血凝素(hemagglutinin,HA)蛋白的假病毒,雄性Wistar大鼠25只灌胃不同中药制备复方中药血清。采用犬肾(Madin-Darby canine kidney,MDCK)细胞和人胚肾细胞293T为研究对象,将不同浓度药物血清分别与假病毒或靶细胞体外培养后,观察药物血清对假病毒活性的直接抑制作用,通过流式细胞术检测药物血清对H5N1禽流感病毒HA蛋白表达的影响。结果:HA蛋白特异性中和抗体2B4在50μL(抗体总量500μg)、25μL(抗体总量250μg)及12.5μL(抗体总量125μg)的剂量条件下均可以有效抑制假病毒的感染活性,从而证实利用假病毒技术筛选药物的方法的可行性。与对照血清相比,4种复方中药含药血清体外对假病毒均无明显抑制作用(P>0.05),也不能有效预防假病毒感染(P>0.05),不能抑制H5N1流感病毒HA蛋白的表达。结论:假病毒技术是一种安全、高效、精确的筛选抗H5N1禽流感病毒中药的方法;4类复方中药含药血清并不含有作用于H5N1假病毒HA蛋白及唾液酸α-2,3半乳糖苷受体的有效成分或单体。Objective： To establish a method based on a pseudotyped virus assay for screening drugs for anti-H5N1 avian influenza virus,and then to evaluate anti-virus activity of traditional Chinese herbal compounds with the function of purgation,detoxification,cooling the blood and reinforcing the healthy qi based on seropharmacology.Methods： Pseudotyped viruses expressing H5N1 virus hemagglutinin were constructed.Herb-medicated sera were prepared by feeding Wistar rats traditional Chinese herbal compounds.Then inhibitory effects of different drug-medicated sera against pseudotyped virus were evaluated based on incubating serum with pseudotyped viruses or target cells such as Madin-Darby canine kidney cells and human embryonic kidney cell line 293T cells,and the expression of hemagglutinin（HA） protein was detected by flow cytometry.Results： Pseudotyped virus could be neutralized by specific 2B4 monoclonal antibody at doses of 50 ？？（500 ？？）,25 ？？（250 ？？） and 12.5 ？？（125 ？？）,which showed that it was a feasible assay to screen drugs.No significant difference was observed in inhibition against H5N1 pseudotyped virus between herb-medicated sera and control serum（P0.05）,meanwhile,the herb-medicated sera also could not inhibit HA expression.Conclusion： Pseudotyped virus assay is a safe,effective,and exact method for screening anti-H5N1 avian influenza virus herbs,and herb-medicated sera prepared by administration of herbs with the function of purgation,detoxification,cooling the blood and reinforcing the healthy qi do not contain active ingredient or monomer which can inhibit HA and sialic acid-？？2,3-galatose.

关 键 词：假病毒 H5NI禽流感 中草药 

分 类 号：R285[医药卫生—中药学]