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| Expression of Membrane-bound APN from Brown Planthopper (Nilaparvata lugens) Midgut Epithelial Membrane in Sf9 Cell Line |
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Abstract Abstract The brown planthopper (BPH)(Nilaparvata lugens) is one of the insect pests significantly damage the rice production in China. Transgenic crops, carrying Bacillus thuringsis (Bt) genes, is widely utilized in the control of agricultural insect pests, including lepidopteran and coleopteran. However, BPH is not susceptible to most of Bt toxins. Thus, management of BPH will become an urgent problem. Binding of Cry toxins with aminopeptidase N (APN) anchored on midgut brush boarder membrane has been reported as a key step in the intoxication-mechanism of Cry toxins. In this study, full-length sequence of a putative membrane-bound APN, which was previously identified from midgut transcriptome of BPH, was cloned into pDEST8 vector, and then transformed into DH10Bac competent cells to produce recombinant Bacmid-bphAPN plasmid. Through baculovirus expression vector system, bphAPN was eukaryotically expressed in a Spodoptera frugiperda (Sf9) cell line. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that compared to control Sf9 cells, a specific circa 110 kD band was detected in the Sf9 cells transfected by Bacmid-bphAPN plasmid. The successfully preparation of bphAPN in Sf9 was then verified by western blot analysis using anti-bphAPN primary antibody and LC-MS/MS analysis. Immunofluorescent localization showed that strong fluorescence was observed in the cells expressed bphAPN, while weak fluorescence was observed from the control cells (without over expression of bphAPN). Cytotoxicity assays showed that compared to the control cells, over expression of bphAPN in Sf9 cells did not significantly increase the cell mortality after exposure to Cry1Ac toxin. Results of this work indicated that the bphAPN, which was highly expressed in the midgut of BPH nymphs could be successfully prepared in vitro by eukaryotic expression. These results will be helpful to analyze the characters of BPH midgut APN and may provide information for innovation of novel pore-forming toxins, such as Bt Cry toxins, against BPH.
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Received: 22 December 2017
Published: 02 May 2018
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