447n

Towards Continuous Biopesticide Production in Insect Cell Culture: Overcoming Mutations in Fp25k Baculovirus Gene

Lopamudra Giri1, David W. Murhammer1, Bryony Bonning2, and Mike Feiss3. (1) Chemical and Biochemical Engineering, University of Iowa, 302 Finkbine Lane, # 6, Iowa City, IA 52246, (2) Entomology, Iowa State University, (3) Microbiology, University of Iowa

Use of chemical pesticides has several undesirable consequences, including the development of insect resistance, reduction of soil fertility and adverse health effects on non-target organisms. In contrast, baculoviruses can be used as a potential bioinsecticide since they do not affect beneficial insects and are environmentally benign. However, the main drawbacks of such bioinsecticide are its sluggish action and high production cost when compared to prevailing chemical pesticides. Of these drawbacks, the production cost can be considerably reduced by producing the baculovirus in continuous cell culture. In such production mode, however, few polyhedra (FP) and defective interfering particle (DIP) mutants tend to accumulate, rendering the bioinsecticide ineffective. The goal of this research is to overcome such mutations in continuous cell culture. The FP mutants generally result from the insertion of host cell DNA sequences known as transposons into the fp25k gene present in the baculovirus genome. The current research is based on the hypothesis that FP mutant accumulation can be either eliminated by expressing FP25K protein in host cell genome or by removing the transposon target sites (TTAA) from the wild type baculovirus genome. Towards this, a recombinant insect cell line (Spodoptera frugiperda) has been developed having the fp25k gene under a late viral promoter. In addition to this, a recombinant virus (Autographa californica) has been developed with the removed transposon sites (performed at Iowa State University, Prof Bonning's Laboratory). Also, MBP-FP25K fusion protein has been expressed in E. coli and the protein is purified through amylose resin column in our laboratory to obtain the antibody binding to FP25K protein. In future, the abovementioned recombinant insect cell and virus will be tested in a two-stage continuous bioreactor system to ascertain whether they overcome the FP mutation. Further, phenotypic and genotypic analysis of the resulting viruses from the bioreactor will be performed in order to gather insights into the relationship between FP and DIP mutations. More specifically, the hypothesis that FP mutants are a necessary precursor of DIP mutants will be tested. In case the above hypothesis fails, i.e., the stabilized cell or the recombinant insect cell overcomes FP mutation but not DIP mutation, then design and testing of a plug flow bioreactor will be undertaken in order to eliminate DIP mutation.