摘要
根据已知H9N2亚型禽流感病毒神经氨酸酶(NA)基因序列设计,合成克隆引物。自H9N2亚型病毒感染的鸡胚尿囊液中提取总RNA,反转录后采用高可信度DNA聚合酶(PyobestTMDNAPolymerase)扩增NA基因,采用Invitrogen定向表达系统(ChampionTMpETdirectionalTOPOexpressionsystem)进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组NA,分子量约54·7ku。经免疫印迹及ELISA分析重组NA的免疫反应性和免疫动物分析其免疫原性,结果表明:重组NA能与H9N2亚型病毒抗血清发生特异性结合,且其免疫动物后能诱导机体产生特异性抗体,具有良好的抗原性。
A pair of clone primers were designed and synthesized based on neuraminidase （NA） gene sequences of known H9N2 subtype avian influenza viruses. NA gene were amplified from total RNA, which had been extracted from allantoie fluid of H9N2 subtype virus inoculated embryo, by reverse transeriptase-polymerase chain reaction using high proofreading polymerase （Pyobest^TM DNA Polymerase）, and the NA protein was expressed using Invitrogen champion^TM pET directional TOPO expression system. Recombinant NA containing polyhistidine （6xHis） tag in N-terminal was about 54.7 ku in size, had been obtained and purified. Its immunoreactivity had been analyzed by western blot and ELISA and its immunogenicity had been tested by immunizing animal. The recombinant NA could not only bind to antiserum against H9N2 subtype virus with specificity but also elicit specific antibody in immunizing animal, and possessed good antigenicity.
出处
《畜牧与兽医》
北大核心
2006年第9期4-7,共4页
Animal Husbandry & Veterinary Medicine
基金
云南省科技攻关项目(2004NG-01)
农业部948项目(2004-Z41)。
