Bt毒素蛋白的纯化 - yzxzcom.PPT

Bt毒素与BBMVs的相互作用的研究 BtA毒素与BBMVs的相互作用的研究 Bt、BtA与BBMV相互作用的研究 Bt与BBMV的解离常数（KD)和解离速率（kids） BtA与BBMV的解离常数（KD)和解离速率（kids） BtA的KD值仅 为Bt的29% BtA的kids值仅 为Bt的44% Bt和BtA杀虫效果的比较 以小菜蛾为供试虫研究Bt与BtA的杀虫毒力 ? 回归线方程 置信区间 LD50(μg/ml) 毒力倍数 Bt Y=6.2334+2.9499X 0.2905--0.5019 0.3818 1 BtA Y=15.6362+9.1049X 0.0019--2.4381 0.0679 5.6 技术展望 BtA藕合效率的鉴定 Octet RED System 荧光光谱法 高效液相色谱法 SDS法 BtA对靶标害虫抗药性延缓的机理 BtA在靶标害虫中肠的定位 BtA与靶标害虫中肠受体的结合 RT-PCR研究BtA对受体表达影响 蛋白质组研究BtA对受体表达作用 * 尊敬的各位领导，各位专家，我今天汇报的项目是“藕合型生物毒素BtA杀虫作用机理的研究”。 * 首先介绍一下立项依据 * * * * * The Octet will monitor change in wavelength shift over time. This real-time binding measurement can be used to calculate on and off rates, and ultimately concentration by plotting rates against concentration. The same protein side chains (the amine on exposed lysine residues) are involved in the biosensor immobilization for either biotin-NHS + streptavidin biosensors OR direct coupling to AR biosensors. In general it is easier to make certain the protein is not over-modified/immobilized using the biotinylation procedure. During biotinylation, the protein is coupled to the biotin in solution phase at neutral pH. This allows control of the ratio of biotin to protein in solution and thus the extent of biotinylation. In direct coupling using amine reactive biosurfaces, the surface is heavily activated and then the protein exposed to the activated surface at low pH (pH 5 typically). It is difficult to control this type of reaction and insure the protein is not overly pinned down to the surface. If the protein is immobilized by too many lysines, it may denature. Addtionally, with biotinylation methods there are different length linkers available to prevent steric hindrance if needed. The Fortebio website has several technical notes on rapid and gentle biotinylation protocols. * Using strepavidin coated biosensors: Step 1 is Baseline- buffer only- want nice flat line, showing zero NSB Step 2 load B-Ab- want gradual increase that eventually flattens out. St