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| Fusion Expression of Peptide Sequences Bound to Midgut Receptors of Empoasca vitis and the Interaction with Brash Border Membrane Vesicle (BBMV) |
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Abstract Empoasca vitis (Gothe), one of the most serious tea plant (Camellia sinensis) insects, occurs throughout the Chinese tea growing areas and causes significant losses in both production and quality of tea. It is of important significance to solve pest problems by biological pesticide and initially explore the intoxication mechanisms of Bacillus thuringiensis Cry toxins to E. vitis midgut cells. Using specific primers, peptide sequence bound to midgut receptors by screening phage library was amplified. The 750 bp PCR product was purified and cloned into the cloning vector pMD-18T, then the recombinant plamid DNA was used for EcoRⅠ/XhoⅠdigested identification and gene sequencing. The targeted fragment was subcloned into expression vector, and then the strain with recombinant plasmid of pT-egfp-32a was induced and expressed. Moreover, the fusion protein was purified by His affinity chromatography and its binding activity to E. vitis midgut was verified by Western blot. Results indicated that the relative molecular weight of T-EGFP was about 46 kD in supernatant after purification step. After that, it also achieved good effect that Western blot had been identified its binding activity to E. vitis midgut brash border membrane vesicle (BBMV). Successful projects for expression, purification and verification of T-EGFP bound to midgut receptors of E. vitis would provide basic data for further research on understanding the interaction mechanism between Cry toxins and E. vitis midgut cells, and also contribute to further study of directed modification for Bt Cry toxin against E. vitis.
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Received: 29 June 2015
Published: 05 October 2015
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