H5N1型禽流感病毒广谱中和单克隆抗体的筛选及其中和机制初步研究  

Broad-spectrum Neutralizing Monoclonal Antibodies Against H5N1 Avian Influenza A Viruses and Primary Research on The Mechanism

作　　者：张晓[1] 曾晓燕[2] 刘哲[1] 金秋[1] 徐言[1] 冯振卿[1] 焦永军[2] 

机构地区：[1]南京医科大学卫生部抗体技术重点实验室,南京210029 [2]江苏省疾病预防控制中心卫生部肠道病原微生物重点实验室,南京210009

出　　处：《生物化学与生物物理进展》2011年第6期519-527,共9页Progress In Biochemistry and Biophysics

基　　金：江苏省自然科学基金(BK2008446;BK2009431)资助项目;江苏省卫生厅重点科研项目(H200857)~~

摘　　要：利用杆状病毒-昆虫细胞表达H5N1型禽流感病毒的血凝素蛋白(HA),纯化后的重组蛋白HA免疫小鼠并制备杂交瘤单克隆抗体,用H5N1型禽流感病毒的全病毒进行筛选,成功地获得了抗H5N1型禽流感病毒血凝素蛋白HA的单抗.MDCK细胞微量中和试验表明,单抗8G10D7可对clade2和clade9的H5N1型禽流感病毒起中和作用.Western-blot及血凝抑制实验进一步证明了该单抗的结合位点位于HA蛋白的HA1亚基上.鸡胚感染病毒预防试验结果表明,8G10D7对禽来源的和人来源的H5N1型禽流感病毒均可达到100%的保护率;在治疗试验组中,8G10D7对禽来源的病毒感染具有较高的保护率,可达100%,对人来源的H5N1型禽流感病毒最高也可达87.5%的保护率.该抗体的获得不仅为H5N1型高致病性禽流感病毒的预防和治疗带来了希望,同时其中和位点的发现也为以后亚单位疫苗的研制提供新的思路.Avian influenza is a highly contagious disease of birds caused by A influenza viruses. The circulation in humans by the highly pathogenic H5N1 avian flu in the past few years have caused most pandemics and have heightened fear that the next influenza pandemic is due. Antibodies could be used as an efficient anti-virus agent in clinical therapy. The full-length HA of the A/Jiangsu/1/2007（H5N1） about 1.7 kb was amplified, subcloned to the pFastBac vector and recombinant bacmid DNA was selected. The recombinant HA was expressed and purified HA about 70 ku was used as the antigen to immunize Balb/c mice. The whole H5N1 virus was used to select 5 mono-antibodies （mAbs）, and all of them were tested using microneutralization assays. 8G10D7, one of the antibodies, had broad neutralizing effect against clade 2 and clade 9 H5N1 avian influenza A viruses, and the IC50 was from 1 ： 256 to 1 ： 64. When detected with 8G10D7, all 4 viruses showed 70 ku and 43 ku protein band, which confirms that the binding site of the scFv antibodies were located at the HA1 domain. The nucleuses of MDCK cells infected by 4 viruses were colored purple, and red around the nucleus. 8G10D7 showed HI activity to the 4 viruses, the HI has a positive correlation with neutralization concentration IC50, which also further confirms that the binding site of the scFv antibodies were located at the HA1 domain. When the mAb 8G10D7 was used for the study of prophylaxis and therapeutic effect on influenza A viruses infection in an embryonated chicken eggs model. It had a complete 100% protection effect on the H5N1 viruses in avian host in the prophylactic and therapeutic groups. The 100% preventive protection effect could be reached when challenged with H5N1 avian influenza A viruse in human host in the prophylactic groups, and there is also a 87.5% protection effect with H5N1 viruses in human host in the therapeutic groups. Thereby, the study suggests that the mAb 8G10D7 could be used in therapies to counter the H5N1 influenza A virus, and the ep

关 键 词：H5N1型禽流感病毒 杆状病毒.昆虫细胞表达系统 单克隆抗体 微量中和 半数细胞培养物感染量 血凝抑制 

分 类 号：R392.11[医药卫生—免疫学]