兔轮状病毒VP7基因在重组杆状病毒中的表达  

Expression of VP7 Gene of Lapine Rotavirus in Recombinant Baculovirus

作　　者：周洁[1,2,3] 王娱[4] 胡建华[1,2,3] 高诚[1,2,3] 

机构地区：[1]上海实验动物研究中心 [2]上海市计划生育科学研究所 [3]上海市实验动物质量监督检验站,上海200032 [4]扬州大学兽医学院,扬州225009

出　　处：《实验动物与比较医学》2009年第4期215-219,共5页Laboratory Animal and Comparative Medicine

基　　金：上海市科委基金项目（064909008）

摘　　要：目的利用Bac—to-Bac杆状病毒表达系统表达兔轮状病毒VP7基因。方法根据Gen Bank发表的LaRV308／01VP7基因序列（AF5028201），设计一对特异性引物，RT—PCR扩增获得VP7基因，将目的片段克隆入pFastBacHTa中，构建重组转移载体pFastBHa-VP7。将重组转移载体转化DH10Bac感受态细菌，与Bacmid发生位点特异性转座作用，获得重组穿梭载体Bacmid-VP7，通过脂质体将其转染昆虫细胞Sf9。结果获得了重组杆状病毒Bacmid—VP7，Western blot和IFA分析表明VP7蛋白在昆虫细胞中获得正确表达且表达产物具有抗原性。结论本研究利用Bac—to-Bac杆状病毒表达系统成功表达兔轮状病毒VP7蛋白，为进一步建立诊断学方法奠定了实验基础。Objective To express the LaRV VP7 protein gene with Bac-to-Bac baculovirus expression system. Method According to LaRV VP7 gene sequence in Genbank, a pair of specific primers was designed and synthesized. The VP70RF gene was amplified by RT-PCR., and then inserted into the transfer vehicle pFastbacHTa to obtain the recombinant transfer vectors pFastBHa-VP7, which was used to transform into E.coli DH10Bac, producing specific transposition with Bacmid to yield the recombinant shuttle vehicles, Bacmid-VPT. Then the Bacmid-VP7 was further used to transfect Sf9 insect cells by lipofectin to generate recombinant baculovirus expressing VP7 recombination protein. Results The recombinant baculovirus and Bacmid-VP7 was obtained. The IFA and Western blotting detection confirmed that the gene had been expressed in sf9 cells, and the expression product could specially react to the sera of LaRV. Conclusion The LaRV VPTprotein is expressed successfully by Bac-to-Bac baeulovirus expression system, which may lay foundation for development of diagnostic technique.

关 键 词：兔轮状病毒 VP7基因 杆状病毒表达 

分 类 号：S852.65[农业科学—基础兽医学]