CAJ | 학술논문

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.
목적 이용류감병독8질립병독증구계통,산생랭괄응감독적중조A형인류감병독,건립이랭괄응류감병독주위증구골가적반향유전학기술평태.방법 이랭괄응、온도민감、감독적A/Ann Arbor/6/60(H2N2)류감병독주작위증구병독적골가,인공합성료해병독주적6개내부기인편단,즉PB2、PB1、PA、NP、M화NS,동시인입5개안기산돌변작위표첨.6개기인편단통과여개조후적전록재체pAD3000련접,구건6개기인적증구재체,경측서획득서렬준학적증구질립:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS.결과 6질립여PR8적표면기인HA화NA진행"6+2"조합적병독증구,8개중조질립공전염COS-1세포,성공증구출료구유혈응활성적랭괄응감독적중조A형인류감병독,명명위rMDV-A.계배뇨낭액중중조병독적혈응(HA)효개위1∶2 9～1∶2 10.결론 구건적A/Ann Arbor/6/60적6개내부기인적병독골가증구계통,위심입연구랭괄응감독인류감병독적기인공능화신형역묘연발제공료시험재료.
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 successfullv bv"6+2" combination of MDV and PR8, and the allanotoic fluid of the injected eggs gave a posigenes 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.