H9N2亚型禽流感病毒反向遗传操作平台的建立

ESTABLISHMENT OF REVERSE GENETICS SYSTEM FOR H9N2 SUBTYPE AVAIN INFLUENZA VIRUS

H9N2亚型禽流感病毒(AIV)在自然界中广泛存在和传播,给养禽业造成了巨大损失。为了进一步揭示该病毒的致病机制,本研究采用RT-PCR技术扩增禽流感病毒A/Chicken/Shanghai/1/2006(简称SH1)的PB2、PB1、PA、HA、NP、NA、M、NS 8个基因片段,并分别克隆至PLLB双向表达载体上。采用8质粒系统共转染293T细胞,转染48 h后加入TPCK胰酶作用2 h,将上清液和细胞一同接种9~11日龄SPF鸡胚,并检测其血凝效价。经序列比对,拯救获得的病毒rSH1的8个基因片段序列均与亲本病毒SH1的序列相同。实验结果表明,本研究成功建立了H9N2亚型禽流感病毒反向遗传操作系统,为该病毒的致病机理和传播机制研究等奠定了技术平台。

H9N2 subtype avian influenza virus(AIV)is widespread among chicken populations and causes a large economic loss in the poultry industry.In order to investigate the pathogenesis of H9N2 AIV,here we used the RT-PCR technique to amplify the 8 genes including PB2,PB1,PA,HA,NP,NA,M and NS from A/Chicken/Shanghai/1/2006(H9N2)(SH1),which were cloned into the two-way expression vector PLLB.These 8 recombinant plasmids were co-transfected into 293T cells and then treated with TPCK trypsin at 48h post-transfection.The supernatant and cells were inoculated into 9-day-old specific pathogen free(SPF)chicken embryonated eggs and then the rescued viruses were confirmed by hemagglutination assay.The rescued SH1(rSH1)was recovered and the sequences of 8 genes were identical to their parental virus SH1.These results indicated that the reverse genetics system of H9N2 subtype avian influenza virus was established successfully,providing a technological platform for further research on the molecular mechanisms of pathogenicity and transmission.