(2017) Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells. Iranian Journal of Biotechnology. pp. 172-178. ISSN 1728-3043
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Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells.pdf Download (318kB) |
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 DH(10)Bac 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/M-13 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.
Item Type: | Article |
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Keywords: | Bacmid Baculovirus Canine Parvovirus GFP VP2 canine parvovirus strains vp2 gene-sequences host-range capsid structure evolution vectors dogs baculoviruses proteins culture Biotechnology & Applied Microbiology |
Divisions: | |
Page Range: | pp. 172-178 |
Journal or Publication Title: | Iranian Journal of Biotechnology |
Journal Index: | ISI |
Volume: | 15 |
Number: | 3 |
Identification Number: | https://doi.org/10.15171/ijb.1558 |
ISSN: | 1728-3043 |
Depositing User: | مهندس مهدی شریفی |
URI: | http://eprints.bmsu.ac.ir/id/eprint/4377 |
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