Mohammad Sadegh Hashemzadeh1,2, Seyed Jafar Mousavy1, Ruhollah Dorostkar2, Fatemeh Fotouhi3, Firouz Ebrahimi1. 1. Department of Biology, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran. 2. Applied Virology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran. 3. Department of Influenza and other Respiratory viruses, Pasteur Institute of Iran, Tehran, Iran.
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.
Background: The importance of viral protein-2 (VP2) of canine parvovirus (CPV) in binding to humancancer 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.
Authors: Richard B Hitchman; Robert D Possee; Andrew T Crombie; Adam Chambers; Kim Ho; Evangelia Siaterli; Olga Lissina; Heather Sternard; Robert Novy; Kathryn Loomis; Louise E Bird; Raymond J Owens; Linda A King Journal: Cell Biol Toxicol Date: 2009-08-05 Impact factor: 6.691
Authors: Zhiyuan Wen; Ling Ye; Yulong Gao; Lei Pan; Ke Dong; Zhigao Bu; Richard W Compans; Chinglai Yang Journal: Antiviral Res Date: 2009-09-20 Impact factor: 5.970
Authors: Banenat B Dogonyaro; Anna-Mari Bosman; Kgomotso P Sibeko; Estelle H Venter; Moritz van Vuuren Journal: Vet Microbiol Date: 2013-04-30 Impact factor: 3.293
Authors: Karsten Hueffer; John S L Parker; Wendy S Weichert; Rachel E Geisel; Jean-Yves Sgro; Colin R Parrish Journal: J Virol Date: 2003-02 Impact factor: 5.103