摘要
目的以鸡胚高度适应株A/PR/8/34株为重组流感病毒骨架,利用反向遗传技术拯救冷适应致弱的重组A型人流感病毒。方法对冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株的PB2基因片段,进行了全基因序列合成,同时人工引入PB2265(N265S)氨基酸的突变。PB2基因片段通过与改造后的转录/表达载体pAD3000连接,构建PB2基因的拯救载体。该重组质粒与PR8进行“7+1”组合的病毒拯救,共转染COS-1细胞。结果经测序获得序列准确的拯救质粒pMDV-A-PB2,利用反向遗传技术成功拯救出了具有血凝性(1×25)的冷适应的重组A型流感病毒。结论利用反向遗传技术成功拯救冷适应致弱的重组A型人流感病毒,该系统为深入研究甲型人流感病毒的基因功能和新型疫苗研发奠定了基础。
Objective To generate cold-adapted reassortant influenza A virus by reverse genetics with A/PR/8/34 strain as a backbone. Methods PB2 fragments of cold-adapted （ca）, temperature sensitive （ts）, and live attenuated influenza virus A/Ann Arber/6/60 （H2N2） strain were synthesized and PB^265 （N265S） amino acid mutation was introduced for rescue research. The PB2 fragment was ligated with modified pAD3000 for rescue plasmid construction. Recombinant transcription/expression plasmid was obtained and designed as pMDV-A- PB2. COS-1 cell was cotransfected combination of one pMDV-A-PB2 and seven PR8 fragments. Results The recombinant virus can be rescued successfully and the virus titers of a recombinant influenza A virus in embryonated chicken eggs was 2^5 . Conclusion The rescue system is a base for farther research on gene function and novel vaccine candidate of human influenza A virus.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第3期335-338,共4页
Immunological Journal
基金
国家科技攻关项目(2004BA519A70)资助
