Expression of Virally Encoded Fibroblast Growth Factors in the Baculovirus Expression Vector System
Presenter(s)
Hailey Palmer
Abstract
The goal of this project is to determine whether baculovirus vFGFs are mitogenic, meaning whether they can stimulate mitosis and cell division. Baculoviruses constitute a family of long, double stranded DNA viruses that primarily infect insects. Within the viral genome is a gene that produces viral fibroblast growth factors (vFGFs), which studies have shown induce chemotaxis in host cells. In addition to chemotaxis, FGF genes normally induce cell proliferation during development and wound healing, however, there are no published studies that examine the mitogenic activity of vFGFs. Previous studies at Winona State suggested that bacterially produced FGF may have reduced protein function, potentially due to a large 42 kDa purification tag affecting its activity. As a result, the activity of vFGF observed in earlier studies may not have accurately represented its true potential in a viral expression system. This provided reasoning for the current experiment, in which the vFGF gene without the purification tag will be produced to allow accurate evaluation of its effects on cell proliferation and mitogenic activity. The vFGF will be produced in the baculovirus expression vector system (BEVS) using the transfer vector pVL1393, which contains the extremely powerful polyhedrin promoter used to drive high levels of gene expression. Once produced, the recombinant vFGF will be tested for mitogenic activity. pAcFGF-3 and pCfFGF-3 are plasmid DNA constructs that carry the full length viral FGF genes from Autographa californica multiply embedded nucleopolyhedrovirus and Choristoneura fumiferana multiply embedded nucleopolyhedrovirus. These plasmids were used as the donor source of the FGF insert for cloning into pVL1393. All plasmids (vector and inserts) were amplified in E. coli, which served as a host for plasmid replication, allowing for production of large quantities of DNA needed for cloning. Ampicillin selection was used to ensure that only the bacterial cells carrying plasmids with the gene AmpR, an ampicillin-resistance marker, survived and grew. The DNA was then purified by maxiprep. Following plasmid amplification and purification, restriction digests were performed to isolate and cut the plasmids at the appropriate sites and generate DNA fragments for cloning. Ligation reactions were then performed to insert the vFGF gene into the pVL1393 vector. The ligation products were transformed into E. coli, and then transformed cells were plated on media to identify the potential recombinant colonies. These transformants will be screened to confirm the successful cloning of the vFGF insert into the transfer vector. Once the transfer vector is confirmed to have the vFGF gene, it will be introduced into insect cells, causing a crossing over recombination event to make two new recombinant viruses expressing the vFGF genes under the control of the polh promoter. Studies are ongoing to discover whether baculovirus vFGFs are mitogenic, whether the recombinant construct has been successfully generated, and whether expression of vFGF in the baculovirus system produces stronger biological activity in cell proliferation assays.
College
College of Science & Engineering
Department
Biology
Campus
Winona
First Advisor/Mentor
Casey Finnerty
Location
Kryzsko Great River Ballroom, Winona, Minnesota; United States
Start Date
4-23-2026 1:00 PM
End Date
4-23-2026 2:00 PM
Presentation Type
Poster Session
Format of Presentation or Performance
In-Person
Session
2a=1pm-2pm
Poster Number
49
Expression of Virally Encoded Fibroblast Growth Factors in the Baculovirus Expression Vector System
Kryzsko Great River Ballroom, Winona, Minnesota; United States
The goal of this project is to determine whether baculovirus vFGFs are mitogenic, meaning whether they can stimulate mitosis and cell division. Baculoviruses constitute a family of long, double stranded DNA viruses that primarily infect insects. Within the viral genome is a gene that produces viral fibroblast growth factors (vFGFs), which studies have shown induce chemotaxis in host cells. In addition to chemotaxis, FGF genes normally induce cell proliferation during development and wound healing, however, there are no published studies that examine the mitogenic activity of vFGFs. Previous studies at Winona State suggested that bacterially produced FGF may have reduced protein function, potentially due to a large 42 kDa purification tag affecting its activity. As a result, the activity of vFGF observed in earlier studies may not have accurately represented its true potential in a viral expression system. This provided reasoning for the current experiment, in which the vFGF gene without the purification tag will be produced to allow accurate evaluation of its effects on cell proliferation and mitogenic activity. The vFGF will be produced in the baculovirus expression vector system (BEVS) using the transfer vector pVL1393, which contains the extremely powerful polyhedrin promoter used to drive high levels of gene expression. Once produced, the recombinant vFGF will be tested for mitogenic activity. pAcFGF-3 and pCfFGF-3 are plasmid DNA constructs that carry the full length viral FGF genes from Autographa californica multiply embedded nucleopolyhedrovirus and Choristoneura fumiferana multiply embedded nucleopolyhedrovirus. These plasmids were used as the donor source of the FGF insert for cloning into pVL1393. All plasmids (vector and inserts) were amplified in E. coli, which served as a host for plasmid replication, allowing for production of large quantities of DNA needed for cloning. Ampicillin selection was used to ensure that only the bacterial cells carrying plasmids with the gene AmpR, an ampicillin-resistance marker, survived and grew. The DNA was then purified by maxiprep. Following plasmid amplification and purification, restriction digests were performed to isolate and cut the plasmids at the appropriate sites and generate DNA fragments for cloning. Ligation reactions were then performed to insert the vFGF gene into the pVL1393 vector. The ligation products were transformed into E. coli, and then transformed cells were plated on media to identify the potential recombinant colonies. These transformants will be screened to confirm the successful cloning of the vFGF insert into the transfer vector. Once the transfer vector is confirmed to have the vFGF gene, it will be introduced into insect cells, causing a crossing over recombination event to make two new recombinant viruses expressing the vFGF genes under the control of the polh promoter. Studies are ongoing to discover whether baculovirus vFGFs are mitogenic, whether the recombinant construct has been successfully generated, and whether expression of vFGF in the baculovirus system produces stronger biological activity in cell proliferation assays.
Comments
Palmer, Hailey