【摘要】 应用反转录聚合酶链式反应(RT-PCR)获得水稻条纹病毒(Rice stripe virus,RSV)的4个基因NS2、NS3、CP和SP,并将它们克隆至pMD-18-T载体上.得到的重组质粒pMD-18-T-NS2、pMD-18-T-NS3、pMD-18-T-CP和pMD-18-T-SP经XbaⅠ/HindⅢ双酶切,分别与经相同方法酶切的苜蓿银纹夜蛾核型多角体病毒(Autographa california nuclear polyhedrosis virus,AcM-NPV)转移载体pFastBacHTb相连接,构建重组转移质粒pFastBacHTb-X(pFastBacHTb-NS2、pFastBacHTb-NS3、pFastBacHTb-CP和pFastBacHTb-SP).序列测定表明,目的基因准确地插入到表达载体中.重组质粒pFastBacHTb-X通过转化包含有穿梭载体的大肠杆菌(Escherichia coli)感受态细胞DH10Bac,得到重组穿梭质粒rb-X(rb-NS2、rb-NS3、rb-CP和rb-SP).rb-X侵染草地贪夜蛾(Spodoptera frugiperda)离体细胞(sf9)24-72 h后,在荧光倒置显微镜可见光200倍视野下观察到细胞增大、培养液和细胞内出现颗粒状物质、部分细胞破裂甚至裂解等一系列与正常sf9细胞形态有明显区别的现象.rb-X侵染细胞72h后,从细胞提取蛋白,电泳分析得到4个条带,大小分别为28.2、29.2、40.2和25.2 ku,与预测的4种融合蛋白大小一致.Western blotting分析分别得到4条单一条带,证明了RSV NS2、NS3、CP和SP基因在"AcMNPV-sf9昆虫细胞"真核表达体系中成功表达.更多还原

【Abstract】 The "AcMNPV-sf9 insect cells" eukaryotic expression(Autographa california nuclear polyhedrosis virus,AcMNPV;Spodoptera frugiperda,sf9) of four genes: NS2,NS3,CP and SP encoded by RSV(Rice stripe virus) was constructed in our research to get active RSV protein.The genes NS2,NS3,CP,and SP of RSV were first cloned into pMD-18-T through RT-PCR(reverse transcription-polymerase chain reaction) approach by inserting the DNA segments in the multiple cloning sites(MCS),named as pMD-18-T-X(pMD-18-T-NS2,pMD-18T-NS3,pMD-18-T-CP and pMD-18-T-SP).Then the recombinant plasmid pFastBacHTb-X(pFastBacHTb-NS2,pFastBacHTb-NS3,pFastBacHTb-CP and pFastBacHTb-SP) was constructed by double digesting in XbaⅠ/Hind Ⅲ sites of the transposing vector pFastBacHTb and recombinant plasmid pMD-18-T-X.Each plasmid was sequenced to ensure that the target gene is correctly inserted and in right reading frame.pFastBacHTb-X was introduced into the competent cells(E.coli DH10Bac) containing a shuttle vector-bacmid to produce recombinant baculovirus rb-X(rb-NS2,rb-NS3,rb-CP and rb-SP).rb-X was isolated and transfected into the sf9 cells to produce the recombinant virus named as P1-X.An increased diameter,granular appearance and cells lysis,which were much different from the morphology of normal sf9 cells,were observed under fluorescence invert microscope(×200),24-72 h after infection.Fresh insect(sf9) cells were reinfected with P1-X containing target genes to amplify viral stocks.And Pn-X was harvested,which was added at a MOI(multiplicity of infection) of 5 after 4 times of reinfection.The special bands(28.2,29.2,40.2 and 25.2 ku) were detected by SDS-PAGE analysis and western blotting showed the single band of each protein,which confirmed that the four protein encoded by RSV were correctly expressed through the "AcMNPV-sf9 insect cells" system.更多还原