中文摘要：

目的利用杆状病毒系统表达中蜂囊状幼虫病毒（chinese sacbrood virus,CSBV）结构蛋白VP1基因。方法提取感染CSBV的中蜂囊状幼虫总RNA,RT-PCR法扩增VP1基因,克隆至载体pFastBacI中,构建重组转座质粒pFastBacI-VP1,转化至感受态大肠埃希菌DHl0Bac中,获得重组杆粒rBacmid-VP1,转染至昆虫细胞sf9中,获得第1代重组杆状病毒,经透射电镜观察杆状病毒粒子;病毒连续传代3次后,RT-PCR鉴定重组杆状病毒,SDS-PAGE检测VP1蛋白的表达情况,Western blot检测表达产物的反应原性。结果重组杆粒经PCR鉴定构建正确;第1代重组杆状病毒经透射电镜观察,可见杆状病毒粒子;重组杆状病毒以M13通用引物和VP1基因特异引物进行PCR扩增,分别可见3 263和963 bp的片段,大小均与理论值一致;CSBV VP1基因能够在sf9细胞中表达,表达的重组VP1蛋白相对分子质量约31 000,可与兔抗CSBV-VP1阳性血清发生特异性反应。结论成功利用杆状病毒表达系统表达了CSBV VP1基因,为深入研究CSBV的感染机制和蜜蜂的免疫机制以及中蜂囊状幼虫病血清诊断试剂盒的研制奠定了基础。

英文摘要：

Objective To express Chinese sacbrood virus（CSBV） VP1 gene by using baculovirus system.Methods Total RNA was extracted from the bee larvae infected with CSBV,from which VP1 gene was amplified by RT-PCR and inserted into vector pFastBacI.The constructed recombinant transposition plasmid pFastBacI-VP1 was transformed to competent E.coli DH10Bac.The obtained recombinant bacmid rBacmid-VP1 was transfected to insect sf9 cells.The prepared recombinant baculovirus of passage 1 was observed for virus particles by transmission electron microscopy,then subcultured for 3 passages and identified by RT-PCR,determined for expression of VP1 protein by SDS-PAGE,and analyzed for reactogenicity of expressed product by Western blot.Results Recombinant bacmid rBacmid-VP1 was constructed correctly as proved by PCR.Baculovirus particles were observed in recombinant baculovirus of passage 1 by transmission electron microscopy.Gene fragments at lengths of 3 263 and 963 bp were amplified by PCR from recombinant baculovirus using general primer M13 and VP1 gene-specific primer respectively,which were consistent with the theoretical values.CSBV VP1 gene was expressed in sf9 cells.The expressed recombinant VP1 protein,with a relative molecular mass of about 31 000,showed specific reaction with rabbit anti-CSBV-VP1 positive serum.Conclusion CSBV VP1 gene was successfully expressed by using baculovirus expression system,which laid a foundation of further study on infection mechanism of CSBV,immune mechanism of bee and development of serological diagnostic kit for CSB.