禽流感病毒分离鉴定及其NA基因克隆分析

Isolation and Identification of Avian Influenza Virus and Cloning and Analysing Its NA Gene

将疑似禽流感病死鸡病料组织研磨液接种SPF鸡胚分离病毒,尿囊分离毒经电镜观察后,应用特异性RT-PCR方法和血凝及血凝抑制试验进行分型检测,同时对核蛋白全长基因进行扩增测序分析。通过血凝及血凝抑制试验初步证实分离的禽流感病毒为H5亚型,然后对分离株HA基因和NA基因进行RT-PCR分析,确定该分离株禽流感病毒为H5N1亚型。通过NA全基因扩增测序、Blast分析显示该分离株NA基因序列与已发表的毒株基因序列(EU429747)同源性为97%,但基因3′端发生明显变异。

To isolate avian influenza virus from suspective dead chicken caused by AIV,and detect its subtype,the SPF chicken embryo was used.After electron microscope observation of the virus from allantoi fluid,HA and HI experiments and RT-PCR were used to detect its subtype.The result showed that the AIV was confirmed as H5 subtype.RT-PCR was used to detect its HA and NA gene,its subtype was defined as H5N1.The analysis of the isolated AIV NA gene showed that this NA gene hold 97% homology with GenBank strain（EU429747）,but had obvious mutation at 3′end.