猫泛白细胞减少症病毒VP2基因在昆虫细胞中的表达  

Expression of FPV VP2 gene in insect cells

作　　者：杜莹[1] 冯昊[1] 张仁舟[1] 杨松涛[1] 夏咸柱[1] 

机构地区：[1]解放军军事医学科学院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130062

出　　处：《中国兽医学报》2011年第4期461-464,共4页Chinese Journal of Veterinary Science

摘　　要：根据猫泛白细胞减少症病毒(Feline panleukopenia virus,FPV)GT-2株VP2蛋白全长基因序列设计1对特异性引物,以GT-2毒株为模板,PCR扩增VP2基因片段(去除终止密码子);与转座载体pFastBacI连接,构建重组质粒pFastBac-VP2,并进行测序鉴定;鉴定正确后,转化到含有辅助质粒的大肠杆菌DH10Bac中,将目的基因定向插入到Bacmid质粒中;经蓝白斑筛选后获得重组Bacmid-VP2,转染Sf9昆虫细胞。电镜负染结果表明:重组Bacmid-VP2在昆虫细胞中包装,形成了大量的杆状病毒粒子;VP2蛋白得到表达并形成了FPV病毒样颗粒。直接免疫荧光试验证明,FPV VP2蛋白在重组杆状病毒中获得了表达。利用双抗体夹心ELISA法检测病毒样颗粒的表达量,表明目的基因在昆虫细胞中获得了较高水平的表达。将表达蛋白作抗原免疫小鼠,经抗体测定证明表达的重组蛋白能诱导机体产生特异性免疫应答。On the basis of the entire gene sequence of the FPV VP2,a pair of specified primer were designed.Taking FPV GT-2 as its template and excluding the stop codon,and connected it with pFastBacI transposon vector,so as to construct and identify recombinant plasmid pFastBac-VP2.After correct identification,we transferred it into Escherichia coli DH10Bac containing assistant plasmid,inserting the targeted gene into Bacmid plasmid.After white-blue plaque selection,the reconstructed Bacmid-VP2 came into being,then transfected Sf9 insect cells.The result of negative staining electron microscope indicated that reconstructed Bacmid-VP2 being packed in insect cells gave birth to a considerable number of baculovirus particles;VP2 protein was expressed and gave birth to FPV virus-like particles.FPV VP2 protein was successfully expressed in reconstructed baculovirus particles by direct immnofluorescence experiment.The detection of the expression level of virus-like particles through double antibody sandwich ELISA technique indicated that targeted gene gained high-level expression in insects cells.Antibody detection in a mouse taking the expressed protein as its antigen proved that expressed reconstructed protein was able to generate a specific immune response.

关 键 词：猫泛白细胞减少症病毒 VP2基因 杆状病毒表达系统 昆虫细胞 

分 类 号：S852.65[农业科学—基础兽医学]