中文摘要：

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.

英文摘要：

The ＂AcMNPV-sD insect cells＂ eukaryotic expression （Autographa california nuclear polyhedrosis virus, AcMNPV ; Spo- dopterafrugiperda, sD） 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 Xba I / 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, pFastBacHTo-X was introduced into the competent ceils （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 sD cells to produce the recombinant virus named as P1-X. An increased diameter, granular ap- pearance and ceils lysis, which were much different from the morphology of normal sD cells, were observed under fluorescence invert microscope （ x 200）, 24 -72 h after infection. Fresh insect （sD） 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-sD insect cells＂ system.