A型禽流感病毒M2e重组T7噬菌体疫苗构建及免疫原性研究  

Construction and Immunogenicity of Recombinant Bacteriophage T7 Vaccine Expressing M2e Peptides of Avian Influenza Virus

作　　者：徐海[1] 王义伟[1] 唐应华[1] 郑其升[1] 侯继波[1] 

机构地区：[1]江苏省农业科学院国家兽用生物制品工程技术研究中心,南京210014

出　　处：《病毒学报》2013年第4期376-381,共6页Chinese Journal of Virology

基　　金：江苏省农业科技自主创新资金项目CX(11)4073

摘　　要：构建展示A型禽流感病毒M2e多肽的重组T7噬菌体,检测其对SPF鸡的免疫保护效果。比对GenBank近期发表的A型禽流感病毒M2e基因序列进行人工合成并重复至两拷贝,将其克隆到T7Select 415-1b噬菌体多克隆位点,构建重组噬菌体T7-M2e。经PCR鉴定并序列测定筛选阳性重组噬菌体,SDS-PAGE和Westernblot检测重组噬菌体表面M2e多肽。重组噬菌体以1×1010pfu/只剂量免疫SPF鸡,免疫后不同时间段采血通过ELSIA检测血清中抗M2e抗体,免疫荧光检测血清抗体与H9亚型禽流感病毒的结合能力,并以200个EID50/只剂量进行攻毒保护效率检测。成功构建重组噬菌体T7-M2e,插入两拷贝M2e基因获得表面展示,并与M2e抗体有免疫反应活性。噬菌体疫苗免疫后均产生抗M2e抗体,其抗血清能跟病毒粒子特异性结合,攻毒保护率达4/5(80%)。获得了展示禽流感病毒M2e多肽的重组噬菌体,噬菌体疫苗免疫鸡产生较高血清抗体并提供攻毒保护,为新型通用禽流感疫苗研制提供新思路。To construct a recombinant T7 phage expressing matrix protein 2 ectodomain （M2e） peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 × 10^10 pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virusinfected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 （80%）. These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.

关 键 词：禽流感病毒 M2e T7噬菌体 噬菌体疫苗 

分 类 号：S852.65[农业科学—基础兽医学]