Genetic modification of a baculovirus vector for increased expression in insect cells

Abstract

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.

Comparative Studies of Lepidopteran Baculovirus-Specific Protein FP25K: Development of a Novel Bombyx mori Nucleopolyhedrovirus-Based Vector with a Modified fp25K Gene

“…However, little genetic modification has been performed to increase the amount of foreign products or enhance the activity of the polh promoter in the BEVS. A recent study demonstrated that the deletion of five nonessential genes, v-cath, v-chiA, p26, p10, and p74, from the AcMNPV genome significantly increases the expression levels of recombinant proteins (14). Unlike most AcMNPVbased BEVSs, the BmNPV-based BEVS is particularly suitable for the mass production of foreign proteins in larvae and pupae of B. mori (36).…”

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

“…This generated virus can be used for primary expression evaluations. After the amplifi cation and titeration of the baculoviral stock, the produced high-titer seed can be used for infection of the insect cells and expression of the recombinant protein of interest in large scale (23,(27)(28)(29)(30)(31)(32)(33).…”

Genetically Engineered Dental Stem Cells for Regenerative Medicine

“…Besides, another widely used vector is baculovirus, which has low toxicity and high effi ciency in transgenic gene transfer. Another advantage of this type of vectors is that they do not get integrated into target cell genome [ 25 ]. For example, Lu et al have used baculovirus encoding TGF-β3 to modify MSCs from adipose tissue (ADSCs) to demonstrate that these cells increase chondrogenesis and stimulate cartilage formation in vitro [ 26 ].…”