2006～2007年流行流感病毒减毒疫苗株的构建(英文)  

Construction of Attenuated Influenza Virus Vaccines of 2006～2007

作　　者：杨鹏辉[1] 叶艺[2] 石辛甫[1] 罗德炎[1] 张钰[1] 颜艳[1] 张鹏飞[1] 段跃强[1] 邢丽[1] 刘秀梵[3] 王希良[1] 

机构地区：[1]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071 [2]西北农林科技大学动物科技学院,杨陵712100 [3]扬州大学,农业部畜禽传染病学重点开放实验室,扬州225009

出　　处：《生物化学与生物物理进展》2008年第3期312-319,共8页Progress In Biochemistry and Biophysics

基　　金：国家高技术研究发展计划(863)资助项目(2006AA02Z450)~~

摘　　要：选择冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为骨架病毒,对其6个内部基因片段进行了全基因合成,同时人工引入5个氨基酸突变(PB1-391E,581G,661T,PB2-265S,NP-34G).HA和NA来源于2006～2007当年流行株A/New Caledonia/20/99(H1N1).8个基因片段通过与改造后的转录载体pAD3000连接,构建8个基因的拯救载体,经测序获得序列准确的拯救质粒:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS、pMDV-A-HA、pMDV-A-NA.6质粒与当年流行株的表面基因HA和NA进行"6+2"组合的病毒拯救,8个重组质粒共转染COS-1细胞,成功拯救出了具有血凝性的冷适应减毒的重组A型人流感病毒.鸡胚尿囊液中重组病毒的血凝效价为1∶29～1∶210.构建的A/AA/6/606个内部基因的病毒骨架拯救系统,为深入研究冷适应减毒人流感病毒的基因功能和新型疫苗研发奠定了基础.Six gene segments, PB1, PB2,PA, NP, M and NS, were fully synthesized which derived from the master donor virus （MDV）, cold-adapted（ca）, temperature sensitive（ts）, live attenuated influenza virus strain A/Ann Arbor/6/60（MDV-A）. Meanwhile, five amino acid substitutions （PB1-391E, 581G, 661T, PB2-265S, NP-34G） were artificially altered by human intervention. HA and NA fragments derived from the 2006-2007 circulating strain A/New Caledonia/20/99 （H1N1）. Eight fragments were ligated with modified pAD3000 for rescue plasmid construction. Eight transcription/expression plasmids were named as pMDV-A-PB2, pMDV-A-PB1, pMDV-A-PA, pMDV-A-NP, pMDV-A-M, pMDV-A-NS, pMDV-A-HA, pMDV-A-NA, respectively. The COS-1 cells were co-transfected with eight plasmids representing 6 internal viral backbone of the strain A/AA/6/60 and two plasmids containing the cDNA of the HA and NA segments of the strain A/New Caledonia/20/99 （H1N1）, the results showed that cold-adapted, attenuated reassortant influenza A virus was rescued successfully. Titers of a reassorted influenza A virus in embryonated chicken eggs ranged from 1 ： 2^9 to 1 ： 2^10. The rescue system of six internal genes used as backbone opens the way for further research on gene function and neotype vaccine candidate of cold-adapted, live attenuated human influenza virus.

关 键 词：流感病毒 反向遗传学 8质粒拯救系统 重组流感病毒 

分 类 号：R183.3[医药卫生—流行病学] R392.1[医药卫生—公共卫生与预防医学]