H9 N2亚型禽流感病毒血凝素基因的克隆与表达

Cloning and Expression of Hemagglutinin Gene of H9 N2 Subtype Avian influenza virus

根据已知H9N2亚型禽流感病毒血凝素基因序列,设计、合成PCR引物。自H9N2亚型禽流感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聚合酶扩增血凝素基因,采用Invitrogen定向表达系统进行克隆表达,纯化获得N末端携带多聚组氨酸标签的重组血凝素,分子质量约75 ku。采用阳性血清经免疫印迹及ELISA分析重组血凝素的免疫反应性,结果表明,重组血凝素能与H9N2亚型病毒抗血清发生特异性结合,具有良好的免疫反应性。

A pair of clone primers were designed and synthesized based on hemagglutinin （HA） gene se-quences of known H9N2 subtype avian influenza viruses. HA gene were amplified from total RNA, which had been extracted from allantoic fluid of HgN2 subtype virus inoculated embryo, by reverse transcriptase -polymerase chain reaction using high proofreading polyraerase （Pyobest^TMDNA Polymerase）, and expressed using Invitrogen championTM pET directional TOPO expression system. Recombinant HA containing polyhistidine （6xHis） tag in N-terminal was about 75 ku in size, had been obtained and purified. Its immunoreactivity was analyzed by western blot and ELISA using positive serum. The results showed that recombinant HA can bind to antiserum against HgN2 subtype virus with specificity and possesses good immunoreactivity.