PDF(Sci-hub)
Abstract：
Background: The importance of viral protein-2 (VP2) of canine parvovirus (CPV) in binding to human cancer cells, production of veterinary vaccines and diagnostic kits has motivated several researches on producing this protein. Objectives: Our purpose was to construct recombinant bacmid shuttle vectors expressing VP2 of CPV using Bac-to-Bac baculoviral expression system. Materials and Methods: Mini-Tn7 transposones engineered in pFastBac1 donor vectors were used to construct expression cassettes of GFP and CPV-VP2. The plasmids were transferred into E. coli DH10Bac competent cells. Site-specific transposition of the genes into bacmid was accomplished using helper plasmid. Occurrence of Transposition was confirmed via PCR using specific primers and PUC/M13 universal primers. The recombinant bacmid DNAs were transfected into Sf9 cells using cationic lipids to generate new recombinant baculoviruses expressing GFP and CPV-VP2. GFP and VP2 expressions were evaluated by fluorescence microscopy and western analysis, respectively. Results: Cloning, subcloning and recombination processes of both GFP and VP2 were accomplished and verified. Accuracy of transfection process was confirmed by GFP fluorescence microscopy.VP2 expression was verified by SDS-PAGE and western analysis. Conclusions: Two Bac-to-Bac expression systems were designed to produce recombinant VP2 and GFP in insect cells.
摘要：
背景：犬细小病毒（CPV）的病毒蛋白2（VP2）在与人类癌细胞结合中的重要性，兽用疫苗和诊断试剂盒的生产推动了一些生产这种蛋白质的研究。目的：我们的目的是使用Bac-to-Bac杆状病毒表达系统构建表达CPVVP2的重组bacmid穿梭载体。材料和方法:在pFastBac1供体载体中工程改造的Mini-Tn7转座子用于构建GFP和CPV-VP2的表达盒。将质粒转移到大肠杆菌DH10Bac感受态细胞中。使用辅助质粒完成基因到bacmid中的位点特异性转座。使用特异性引物和PUC/M13通用引物通过PCR确认转座的发生。使用阳离子脂质将重组杆状DNA转染到Sf9细胞中以产生表达GFP和CPV-VP2的新重组杆状病毒。GFP和VP2表达通过荧光显微镜和蛋白质分析进行评估，分别。结果:克隆,完成并验证了GFP和VP2的亚克隆和重组过程。转染过程的准确性通过GFP荧光显微镜确认。通过SDS-PAGE和western分析验证VP2表达。结论:设计两个Bac-to-Bac表达系统以在昆虫细胞中产生重组VP2和GFP。
