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Published on: August 23, 2018
Expression of functional Anopheles merus alpha-amylase in the baculovirus/Spodoptera frugiperda system
P Chimoy Effio1, A V Folgueras-Flatschart, W R Montor
1Instituto de Quimica, Universidade de São Paulo, São Paulo, Brazil.
Abstract:
The Anopheles merus (Diptera, Nematocera, Culicoidea) alpha-amylase gene (AmerAmy, GenBank Accession Number U01210) was amplified with its own or with the Zabrotes subfasciatusalpha-amylase signal peptide (ZsAmerAmy, GenBank Accession Number AY270183) by PCR, using designed primers. The AmerAmy gene was sequenced from its promotor to the TGA codon. As a positive control, the Z. subfasciatusalpha-amylase gene with its own signal peptide (ZsAmy, GenBank Accession Number AF255722) was also amplified by PCR. These three sequences were inserted into the baculovirus genome using the Bac-to-Bac trade mark system. Recombinant baculovirus preparations were used to infect Sf9 Spodoptera frugiperda insect cells. The A. merusalpha-amylase was successfully expressed as an active enzyme detected mainly in cell culture supernatants.
Insights
Anopheles merus alpha-amylase was successfully expressed in insect cells. This active enzyme was detected in cell culture supernatants, paving the way for further research into its function.
Area of Science:
- Molecular Biology
- Biochemistry
- Entomology
Background:
- Alpha-amylase is a crucial enzyme in carbohydrate metabolism.
- Understanding Anopheles merus alpha-amylase can provide insights into insect physiology and potential pest control strategies.
Purpose of the Study:
- To express the Anopheles merus alpha-amylase gene in a heterologous system.
- To investigate the activity and localization of the expressed enzyme.
Main Methods:
- PCR amplification of the Anopheles merus alpha-amylase gene and its variants.
- Gene sequencing from promoter to TGA codon.
- Insertion of sequences into the baculovirus genome using the Bac-to-Bac system.
- Infection of Sf9 insect cells with recombinant baculovirus.
Main Results:
- Successful amplification and sequencing of the Anopheles merus alpha-amylase gene.
- Expression of active Anopheles merus alpha-amylase in Sf9 cells.
- The enzyme was primarily detected in cell culture supernatants.
Conclusions:
- The Anopheles merus alpha-amylase gene can be successfully expressed in Sf9 insect cells.
- The expressed enzyme is active and secreted into the culture medium.
- This expression system provides a valuable tool for studying Anopheles merus alpha-amylase.
