Separate The Binding Protein Of Periplocoside E On Midgutbrush Boder Membrane Vesicle Of Mythimna Separata By Affinity Chromatography

Affinity chromatography is a powerful proteomic tool. This separation method is based on the specific interaction between immobilized ligands and their target proteins. Affinity chromatography offers high selectivity, high resolution, and intermediate to high capacity. At present, this technology is widely used in the field of protein research and preparation. In this study, periplcoside E was used as an affinity ligand for research the binding protein of periplcosides in the BBMV of Mythimna separata. It is conducive for lay the foundation of study the periplcosides’ s target protein in BBMV. The main findings are as follows:In this experiment, Periplcoside E, as a ligand, was used to produced affinity column.The periplcoside E-hemisuccinates was synthesized by combining periplocoside E with succinic anhydride, and it was identified by HPLC, MS and NMR. The results showed that the periplcoside E-hemisuccinates was synthesized successful and the periplcoside E-hemisuccinates was coupled into EAH-Sephorase4 B by Carbodiimide method. The conjugation rate achieved 4.58mg/ml by spectrometry.Which is comply with the affinity chromatography requirements.The synthetical affinity column was applied to purified binding protein of periplcoside E from BBMV in Mythimna separata. The SDS-PAGE results showed that there are five specific binding protein bands, 240 KDa, 130 KDa, 110 KDa,80KDa, 50 KDa. After in-gel trypsin digestion, followed by peptide extraction and LC-MS/MS analysis. There are 18 kinds of proteins were identified from the five specific bands ultimately, includes Cry1 A toxin receptor A, NADH dehydrogenase subunit 5, actin 2OG-Fe(2) oxygenase superfamily protein and so on.