流感病毒神经氨酸酶广谱抑制多肽的筛选研究  

Screening of Peptides as Broad-spectrum Neuraminidase Inhibitors against Influenza Viruses

作　　者：匡治州[1] 郑丽舒[1] 段招军[1] 漆正宇[1] 瞿小旺[1] 刘文培[1] 张万菊[1] 李丹地[1] 高寒春[2] 侯云德[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]北京金迪克生物技术研究所,北京100176

出　　处：《病毒学报》2007年第3期165-171,共7页Chinese Journal of Virology

摘　　要：以纯化的流感病毒颗粒H1N1、H3N2为靶点对噬菌体展示的随机12肽库进行了筛选。经ELISA、神经氨酸酶抑制活性鉴定,获得52个阳性噬菌体克隆。根据测序和氨基酸序列分析,挑选出不同克隆间同源性最高的6条多肽进行化学合成。神经氨酸酶抑制活性实验显示,6条多肽中54-N1和69-N2的抑制活性最强,对N1、N2和乙型流感病毒三种流感病毒神经氨酸酶均有明显的抑制活性,54-N1的IC50值为7.486~14.693μmol/L,69-N2的IC50值为6.605~13.007μmol/L。同时,54-N1和69-N2可抑制流感病毒在MDCK细胞中的生长。病毒空斑抑制实验显示,54-N1和69-N2可明显抑制空斑形成的大小和数目。通过分析,54-N1的IC50值为18.38-31.76μmol/L,69-N2的IC50值为16.56-25.49μmol/L。两条多肽的浓度在高达10mmol/L时仍对细胞无任何毒性。54-N1和69-N2作为新的流感病毒神经氨酸酶抑制剂,与现有NA抑制剂Oseltamivir进行了比较和讨论,为进一步研制局部应用的广谱抗流感病毒的鼻腔喷雾剂奠定了基础。In this study, a random duodecimal peptides phage display library was probed with influenza A viruses H1N1 and H3N2, resulting in the identification of 52 positive phage clones by using ELISA and neuraminidase inhibition assay. Based on analysis of peptide sequences displayed by positive phage clones, we picked up six peptides to synthesize which are conserved among several clones, respectively. Among these peptides, 54-N1 and 69-N2 had strong inhibitory activity against influenza A viral neuraminidase N1 and N2 as well as influenza type B neuraminidase, with ICs0 7. 486-- 14. 693btmol/L for 54-N1 and ICs0 6. 605-13. 007μmol/L for 69-N2. Peptides 54-N1 and 69-N2 inhibited plaque formation of influenza A and B viruses, with IC50 ranging from 18.38 to 31.76btmol/L and 16. 56 to 25.49μmol/L, respectively. No cytotoxicity was observed with 54-N1 and 69-N2 at up to 10mmol/L. Also, 54-N1 and 69-N2 were compared with Oseltamivir by neuraminidase inhibition assay and plaque formation assay. 54-N1 and 69-N2 peptides therefore have potential values in development of nasal spray as new NA inhibitors for broad-spectrum antivirus therapy.

关 键 词：流感病毒 神经氨酸酶抑制剂 噬菌体展示 

分 类 号：R373.1[医药卫生—病原生物学]