Designing Two Individual AcMNPV Polyhedrin-Plus Bac-to-Bac Expression System in order to Express GFP and CPV-VP2 in Insect Cells

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-specifi c transposition of the genes into bacmid was accomplished using helper plasmid. Occurrence of Transposition was confi rmed via PCR using specifi c 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 fl uorescence microscopy and western analysis, respectively. Results: Cloning, subcloning and recombination processes of both GFP and VP2 were accomplished and verifi ed. Accuracy of transfection process was confi rmed by GFP fl uorescence microscopy.VP2 expression was verifi ed by SDS-PAGE and western analysis. Conclusions: Two Bac-to-Bac expression systems were designed to produce recombinant VP2 and GFP in insect cells.

Biosynthesis of a VLP-type nanocarrier specific to cancer cells using the BEVS expression system for targeted drug delivery

“…As demonstrated in the previous studies [ 12 – 15 ], first we generated two baculoviruses expressing VP2 and EGFP proteins. The expression of recombinant EGFP protein in the cells infected with recombinant baculoviruses encoding the EGFP gene was observed by an inverted fluorescent microscope.…”

“…The EGFP gene was coupled with the VP2 gene in all steps parallelly, as a control [ 13 , 14 ]. The constructed recombinant bacmids including VP2 and EGFP genes were analyzed by PCR panel, using designed primers and PUC/M13 universal primers and then transfected into Sf9 insect cells using cationic lipids to generate two new recombinant baculoviruses expressing EGFP and CPV-VP2 (Ethic NO.IR.BMSU.REC.1400.037) [ 15 ].…”

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles

“…The baculovirus expression system is widely used in protein expression, especially for the expression of large molecular weight proteins and their complexes, such as CSP (148.5 kDa) [18]. The traditional baculovirus expression system often relies on the transfection of recombinant baculovirus DNA isolated from bacteria [31, 32]. In contrast, the Ac-MultiBac system used in this study does not need transfection but employs DAP-deficient bacteria that infect insect cells to produce recombinant baculovirus () [13, 20, 23–25, 33].…”

Study of the immunogenicity of the VP2 protein of canine parvovirus produced using an improved Baculovirus expression system

“…Previous studies reported that VP2 protein could be produced in E. coli and insect expression system [15,16,[18][19][20][21]. Although E. coli expression system has been used widely for recombinant proteins production in laboratory and industrial scale due to its simplicity and economy, the solubility of target proteins could sometimes be low [22][23][24].…”