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2006 Progress Report: Collaborative Research: Cost-Effective Production of Baculovirus Insecticides (TSE03-D)
EPA Grant Number: R831421Title: Collaborative Research: Cost-Effective Production of Baculovirus Insecticides (TSE03-D)
Investigators: Murhammer, David W. , Bonning, Bryony C. , Feiss, Michael G.
Institution: University of Iowa , Iowa State University
EPA Project Officer: Richards, April
Project Period: January 1, 2004 through December 31, 2006 (Extended to December 31, 2007)
Project Period Covered by this Report: January 1, 2006 through December 31, 2007
Project Amount: $320,000
RFA: Technology for a Sustainable Environment (2003)
Research Category: Pollution Prevention/Sustainable Development
Description:
Objective:The long-term goal of this research project is to develop a more cost-effective method for mass producing baculovirus insecticides. The specific objective of this research project is to develop methods to overcome the accumulation of FP mutants that would otherwise occur on repeated baculovirus passage in cell culture. This includes two approaches. First, the base sequence of the fp25k gene (whose mutation leads to FP mutants) is being modified. This research is being conducted in the Bonning laboratory (Iowa State University). Second, the FP25K protein is being expressed from the host cell genome. This research is being conducted in the Murhammer laboratory (University of Iowa).
Background
Biopesticides are useful in overcoming the significant disadvantages of chemical pesticides, such as detrimental effects on nontarget organisms, higher animals, and soil fertility. The current production cost of potential biopesticides, such as the baculovirus (which kills only targeted insects and is environmentally benign), however, is significantly higher than that of chemical pesticides. Costs could be significantly reduced by using a continuous production process. Unfortunately, traditional continuous processes are not feasible because of accumulation of few polyhedra (FP) and defective interfering particle (DIP) mutants that render the product useless as a biopesticide. In this context, our primary goal is to overcome the FP mutation, resulting, generally, from the insertion of host cell DNA sequences, known as transposons, into the baculovirus fp25k gene. In addition, we will examine whether FP mutants are a necessary precursor of the DIP mutants. Depending on the outcome of our investigation, further reactor design may be needed to overcome the DIP mutants.
Progress Summary:Expression of Viral fp25k Protein in Sf-9 Cells
We have developed a recombinant insect cell line containing the fp25k gene under the control of the late baculovirus p6.9 promoter (p6.9-FP-Sf-9 cell line). Further, we have expressed the MBP-FP25K fusion protein in Escherichia coli and purified it to obtain the antibody for the FP25K protein (conducted in University of Iowa, Dr. Murhammer’s laboratory and Dr. Feiss’ laboratory).
Experimental Steps
1. Construction of the pIB vector (Invitrogen) having the p6.9-fp25k cassette.
- PCR amplification of:
- The p6.9 promoter from the pAcMLF9 vector.
- The fp25k gene from pUC19-fp25k vector.
- The p6.9 promoter and the fp25k gene to obtain the p6.9-fp25k cassette.
- Putting the p6.9-fp25k cassette into the pIB/V5-His vector by ligation.
2. Transfection of the Sf-9 cells with the p6.9-fp25k-pIB vector and selection of stable p6.9-fp25k-Sf-9 clones using blasticidin.
3. Checking the presence of the p6.9-fp25k cassette in Sf-9 cells by diagnostic PCR.
4. Synthesis of MBP-FP25K fusion protein.
- Construction of the pMAL vector having the fp25k gene.
- Expression of the MBP-FP25K fusion protein in E. coli.
- Purification of the fusion protein using amylase resin column.
- Optimization of the antibody and antigen concentration for Western blot.
- Testing of the antibody for MBP-FP25K protein by Western blot.
5. Checking the expression of protein in p6.9-fp25k-Sf-9 cells.
- By Western blot.
- By immunofluorescent assay.
Passaging of the Stabilized AcMNPV from Bonning’s Laboratory
A passaging experiment was conducted to check the potential of the stabilized fp25k gene in the recombinant AcMNPV at the shaker flask level. The baculovirus stock was transferred every 48 hours to infect the insect cells to start a new passage. It has been found that the number of polyhedra/cell is greater in case of the stabilized virus than the wild type cells at several passages. Also the number of cells having more polyhedra is higher in case of the stabilized AcMNPV.
Conclusions
The stabilized baculovirus has shown potential in preliminary studies, but additional studies are needed to determine if this baculovirus will overcome FP mutant accumulation during long-term cell culture. Studies are in progress to determine the effectiveness of expressing the fp25k protein from the host insect cell in overcoming FP mutant accumulation.
Future Activities:Testing of the Modified Virus and the Cell in Bioreactor System
The stabilized virus (with no TTAA transposons sites) and the recombinant insect cell (expressing FP25K protein) will be tested in continuous bioreactor system by examining the characteristics of FP and DIP mutation and polyhedra productivity. Specifically, the following will be monitored:
- Cell growth and cell viability using our recently purchased Vi-Cell instrument from Beckman-Coulter.
- Infectivity of the baculovirus using end point dilution assay/enzyme-linked immunosorbent assay using the gp64 antibody.
- Counting polyhedra using Transmission Electron Microscopy (TEM).
- Identification of FP mutation by PCR/Southern blot/Western blot/Immunofluorescence.
- Identification of DIP mutation using TEM.
We shall use two different reactor systems:
- A two-stage continuous bioreactor system connected in series.
- A three stage reactor system with two continuous stirred tank reactors and a multiple airlift bioreactor (simulating plug flow system).
No journal articles submitted with this report: View all 4 publications for this project
Supplemental Keywords:biopesticide, continuous production, baculovirus,
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TREATMENT/CONTROL, Sustainable Industry/Business, Scientific Discipline, RFA, Technology for Sustainable Environment, Sustainable Environment, Technology, Biochemistry, Environmental Chemistry, Ecology and Ecosystems, Economics and Business, baculovirus, biotechnology, bioengineering, insecticide production, agriculture, bioinsecticides, innovative technology
Progress and Final Reports:
2004 Progress Report
2005 Progress Report
Original Abstract
2007 Progress Report