Methods and materials for identifying novel pesticide agents
View Patent ↗Materials and methods for identifying novel pesticide agents are disclosed herein. Specifically exemplified is a full length aminopeptidase N isolated from Manduca sexta , insect cells expressing APN, and methods of screening pesticide agents using the same. Also disclosed are methods of identifying novel APN inhibitors.
1. A transgenic cell comprising an isolated polynucleotide encoding an aminopeptidase N (APN), wherein a functional APN is produced by said cell and localized on the extracellular membrane of said cell, and said APN is able to bind a molecule selected from the group consisting of a Bacillus insect toxin protein and an APN inhibitor, wherein said polynucleotide is capable of hybridizing with a probe corresponding to the complement of the nucleic acid sequence of SEQ ID NO:2, wherein hybridization is maintained under conditions of 0.1× standard saline sodium phosphate EDTA buffer (SSPE) and 0.1% sodium dodecyl sulfate (SDS) at 65° C. for 15 minutes.
2. The transgenic cell of claim 1 wherein said APN is able to bind a Bacillus insect toxin protein.
3. The transgenic cell of claim 1 wherein said APN is able to bind an APN inhibitor.
4. The transgenic cell of claim 1 wherein said cell is an insect cell.
5. A transgenic cell comprising an isolated polynucleotide encoding an aminopeptidase N (APN), wherein a functional APN is produced by said cell and localized on the extracellular membrane of said cell, and said APN is able to bind a molecule selected from the group consisting of a Bacillus insect toxin protein and an APN inhibitor, wherein said APN has at least 95% identity with the amino acid sequence of SEQ ID NO:1.
6. A method of screening for a Bacillus insect toxin protein wherein said method comprises the steps of:
a) obtaining a transgenic cell comprising a heterologous polynucleotide encoding an aminopeptidase N (APN) wherein a functional APN is produced by said cell and localized on the extracellular membrane of said cell, and said APN is able to bind a Bacillus insect toxin protein;
b) exposing said cell to a composition suspected of comprising a Bacillus insect toxin; and
c) determining if a Bacillus insect toxin is bound to said APN,
wherein said polynucleotide is capable of hybridizing with a probe corresponding to the complement of the nucleic acid sequence of SEQ ID NO:2, wherein hybridization is maintained under conditions of 0.1× standard saline sodium phosphate EDTA buffer (SSPE) and 0.1% sodium dodecyl sulfate (SDS) at 65° C. for 15 minutes.
7. The method of claim 6 wherein said APN has at least 95% identity with the amino acid sequence of SEQ ID NO:1.
8. A method of screening for an aminopeptidase inhibitor wherein said method comprises the steps of:
a) obtaining a plurality of transgenic cells wherein each said cell comprises an isolated polynucleotide encoding an aminopeptidase N (APN), wherein a functional APN is produced by said cell and localized on the extracellular membrane of said cell, and said APN is able to bind an aminopeptidase inhibitor;
b) exposing said cells to at least one potentially APN-inhibiting compound;
c) identifying a compound that binds to said APN; and
d) assaying said compound for its ability to inhibit an aminopeptidase,
wherein said polynucleotide is capable of hybridizing with a probe corresponding to the complement of the nucleic acid sequence of SEQ ID NO:2, wherein hybridization is maintained under conditions of 0.1× standard saline sodium phosphate EDTA buffer (SSPE) and 0.1% sodium dodecyl sulfate (SDS) at 65° C. for 15 minutes.
9. The method of claim 8 wherein said compound binds to an aminopeptidase of an insect.
10. The method of claim 8 wherein said compound inhibits an aminopeptidase of an insect.
11. The method of claim 8 wherein said APN has at least 95% identity with the amino acid sequence of SEQ ID NO:1.