用8质粒病毒拯救系统产生冷适应减毒的重组A型人流感病毒的研究  

Generation of cold-adapted, attenuated reassortant human influenza A virus with eight-plasmid system

作　　者：杨鹏辉[1] 石辛甫[1] 颜艳[1] 罗德炎[1] 张钰[1] 邢丽[1] 龙进学[2] 刘秀梵[2] 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]扬州大学农业部畜禽传染病学重点开放实验室

出　　处：《中华微生物学和免疫学杂志》2008年第2期139-143,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家863项目（2006AA022450）

摘　　要：目的利用流感病毒8质粒病毒拯救系统，产生冷适应减毒的重组A型人流感病毒，建立以冷适应流感病毒株为拯救骨架的反向遗传学技术平台。方法以冷适应、温度敏感、减毒的A／Ann Arbor／6／60（H2N2）流感病毒株作为拯救病毒的骨架，人工合成了该病毒株的6个内部基因片段，即PB2、PB1、PA、NP、M和NS，同时引入5个氨基酸突变作为标签。6个基因片段通过与改造后的转录载体pAD3000连接，构建6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。结果6质粒与PR8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活性的冷适应减毒的重组A型人流感病毒，命名为rMDV-A。鸡胚尿囊液中重组病毒的血凝（HA）效价为1：2^9～1：2^10。结论构建的A／Ann Arbor／6／60的6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提供了试验材料。Objective To set up a technical platform of reverse genetics based on the 8 plasmid. virus rescue system of cold-adapted influenza virus strain. Methods The cold-adapted, temperature sensitive, live attenuated influenza virus strain A/AnnArbor/6/60 （H2N2） was chosen as the master donor virus （MDV） for rescue research, and its six internal gene fragments PB2 ,PB1 ,PA ,NP,M and NS were artificially synthesized. Meanwhile, five amino acid mutations have been introduced as tags. Six fragments were ligated with modified pAD3000 for the construction of rescue plasmid. Six transcription/expression plasmids （pMDV-A-PB2, pMDV-A-PB1, pMDV-A-PA, pMDV-A-NP, pMDV-A-M, and pMDV-A-NS） were obtained, and their sequences were accurate. Results The reassorted virus named as rMDV-A contains HA and NA gene segments derived from PR8 strain along with six gene segments, PB2, PB1, PA, NP, M and NS, from MDV. The COS-1 cells were co-transfected with eight recombinant plasmids. The results showed that a cold-adapted, attenuated reassortant influenza A virus with hemagglutination activity was rescued successfully by ＂6 ＋2＂ combination of MDV and PR8, and the allanotoic fluid of the injected eggs gave a positive hemagglutinin （HA） fiter ranging from 1 ： 2^9 to 1 ： 2^10. Conclusion The rescue system of six internal genes of A/AA/6/60 used as backbone has provided experimental materials for further research on the gene function and novel vaccine candidate of cold-adapted, 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：R686[医药卫生—骨科学]