酶联免疫分析法探测Cry1Ab蛋白在不同介质中的构象变化  

Probing the Conformation Change of Cry1Ab Toxin in Different Medium by Enzyme-linked Immunosorbent Assay

作　　者：邬建敏[1] 奚凤娜[1] 叶庆富[2] 刘维屏[3] 

机构地区：[1]浙江大学理学院化学系,杭州310027 [2]浙江大学原子核农业科学研究所,杭州310029 [3]浙江大学环境科学研究所,杭州310029

出　　处：《分析化学》2005年第8期1105-1108,共4页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金资助项目(No.20277031和No.20177021)

摘　　要：采用酶联免疫分析(ELISA)方法,根据构象变化后的蛋白与抗体结合能力下降从而导致ELISA测定值降低的原理,探测了转cry1Ab基因水稻表达的Cry1Ab蛋白在不同溶液介质中的热致构象变化行为,以及不同有机溶剂及溶液pH值对该蛋白构象变化的影响程度.实验表明,CrylAb蛋白在不同条件下的构象变化程度可以灵敏地通过ELISA方法检测.在不同的介质中,Cry1Ab蛋白的热致构象变化程度不同.在Na2SO4介质中,该蛋白具有较高的热稳定性;SDS的存在,可以促进该蛋白的构象变化.常温下,25%(V/V)的有机溶剂乙腈、异丙醇、甲醇、乙醇均能使该蛋白的构象发生转变,其中以乙腈最为显著.醇类溶剂对Cry1Ab蛋白的构象影响程度随疏水性增大而增大;溶液pH值也对该蛋白的构象变化产生影响.pH在 8～10之间,该蛋白构象能保持稳定;酸或过碱性的溶液均能使蛋白构象偏离原始状态,从而引起ELISA测定值的降低.另外,腐殖酸能在一定时间内保持Cry1Ab蛋白构象的稳定性.Enzyme-linked immunoadsorbent assay （ELISA） was utilized to probe the conformation change of Cry1Ab protein expressed by transgenic Cry1Ab rice based on the change of its binding capacity with antibody resulting from conformation transition. Stability of Cry1Ab protein in different temperature, solvent composition, pH and different organic solvents were measured. Results showed that the antibody on the micro-well plate of ELISA kits could sensitively recognized conformation transition of Cry1Ab protein without need of highly purified protein. The conformation change induced by temperature was depended on the solvent composition. Relatively high stability could be achieved in sodium sulfate （Na2SO4） solution. In contrast, sodium dodecyl sulfate （SDS） accelerated its temperature-induced conformation transition. At room temperature,25% （V/V） organic solvent such as methanol, ethanol, 2-propanol and acetonitrile could significantly cause Cry1Ab protein conformation change. Among all the solvents tested, the acetonitrile worked strongest. The extent of the transition in alcohols was highly correlation with the hydrophobicity of the solvents, pH also had an effect on the conformation transition of Cry1Ab protein. The native structure of Cry1Ab protein was stable in pH 8 - 10. Too acidic or alkaline media destabilized the protein as demonstrated by the decrease of ELISA results. Moreover, humic acids from soil could remarkably stabilize the conformational of Cry1Ab protein even in acidic condition.

关 键 词：酶联免疫分析法 Cry1Ab蛋白 介质 温度 构象变化 苏云金杆菌 

分 类 号：Q51[生物学—生物化学]