H5N1亚型禽流感病毒神经氨酸酶基因的克隆与表达

Cloning and Expression of Neuraminidase Gene of H5N1 Subtype Avian Influenza Virus

根据已知H5N1亚型禽流感病毒神经氨酸酶基因(na)序列设计、合成克隆引物。自H5N1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神经氨酸酶,分子量约53.8kDa。分析重组NA的免疫反应性和免疫原性,结果表明:重组NA能与H5N1亚型病毒抗血清发生特异性结合,且其免异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 Avian influenza virus H5N1 subtypes. NA gene were amplified from total RNA, which had been extracted from allantoic fluid of H5N1 subtype virus inoculated embryo, by reverse transcriptase-polymerase chain reaction using high proofreading polymerase （PyobestTM DNA Polymerase）, and expressed using Invitrogen championTM pET directional TOPO expression system. 53.8kDa recombinant NA fusion with polyhistidine （6xHis） tag in N-terminal was expressed and purified. Its immunoreactivity and immunogenicity had been analyzed. The results showed the recombinant NA could not only bind to antiserum against H5N1 subtype virus with specificity but also elicit specific antibody in immunizing animal, and possessed good antigenicity.