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

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.