生物化学与生物物理进展
生物化學與生物物理進展
생물화학여생물물리진전
PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS
2008年
3期
312-319
,共8页
杨鹏辉%叶艺%石辛甫%罗德炎%张钰%颜艳%张鹏飞%段跃强%邢丽%刘秀梵%王希良
楊鵬輝%葉藝%石辛甫%囉德炎%張鈺%顏豔%張鵬飛%段躍彊%邢麗%劉秀梵%王希良
양붕휘%협예%석신보%라덕염%장옥%안염%장붕비%단약강%형려%류수범%왕희량
流感病毒%反向遗传学%8质粒拯救系统%重组流感病毒
流感病毒%反嚮遺傳學%8質粒拯救繫統%重組流感病毒
류감병독%반향유전학%8질립증구계통%중조류감병독
influenza virus%reverse genetics%eight-plasmid system%reassorted influenza virus
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l:279-1:210.构建的A/AA/6/60 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奠定了基础.
選擇冷適應、溫度敏感、減毒的A/Ann Arbor/6/60(H2N2)流感病毒株作為骨架病毒,對其6箇內部基因片段進行瞭全基因閤成,同時人工引入5箇氨基痠突變(PB1-391E,581G,661T,PB2-265S,NP-34G).HA和NA來源于2006-2007噹年流行株A/New Caledonia/20/99(H1N1).8箇基因片段通過與改造後的轉錄載體pAD3000連接,構建8箇基因的拯救載體,經測序穫得序列準確的拯救質粒: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質粒與噹年流行株的錶麵基因HA和NA進行"6+2"組閤的病毒拯救,8箇重組質粒共轉染COS-1細胞,成功拯救齣瞭具有血凝性的冷適應減毒的重組A型人流感病毒.鷄胚尿囊液中重組病毒的血凝效價為l:279-1:210.構建的A/AA/6/60 6箇內部基因的病毒骨架拯救繫統,為深入研究冷適應減毒人流感病毒的基因功能和新型疫苗研髮奠定瞭基礎.
선택랭괄응、온도민감、감독적A/Ann Arbor/6/60(H2N2)류감병독주작위골가병독,대기6개내부기인편단진행료전기인합성,동시인공인입5개안기산돌변(PB1-391E,581G,661T,PB2-265S,NP-34G).HA화NA래원우2006-2007당년류행주A/New Caledonia/20/99(H1N1).8개기인편단통과여개조후적전록재체pAD3000련접,구건8개기인적증구재체,경측서획득서렬준학적증구질립:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS、pMDV-A-HA、pMDV-A-NA.6질립여당년류행주적표면기인HA화NA진행"6+2"조합적병독증구,8개중조질립공전염COS-1세포,성공증구출료구유혈응성적랭괄응감독적중조A형인류감병독.계배뇨낭액중중조병독적혈응효개위l:279-1:210.구건적A/AA/6/60 6개내부기인적병독골가증구계통,위심입연구랭괄응감독인류감병독적기인공능화신형역묘연발전정료기출.
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,58lG,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.Eiight 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-l 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 mnged from 1:29to l:210.The rescue system of six intemal 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.
