黄鳍鲷骨骼肌α-辅肌动蛋白的分离纯化及抗体制备

Separation,Purification and Antibody Preparation of α-Actinin from Skeletal Muscle of Sparus Latus

目的:黄鳍鲷骨骼肌α-辅肌动蛋白的分离纯化及多克隆抗体制备。方法:采用低温抽提、硫酸铵分级沉淀及两次DEAE-Sepharose离子交换等方法获得α-辅肌动蛋白,并通过免疫大鼠制备了抗α-辅肌动蛋白多克隆抗体。结果:经SDS-PAGE检测得到高纯度的α-辅肌动蛋白,分子质量约为100kDa。Westernblot检测结果显示,制备的多克隆抗体具有较高的效价和良好的特异性。结论:从海水黄鳍鲷骨骼肌中得到高度纯化的α-辅肌动蛋白,并制备了滴度高、特异性好的多克隆抗体,为研究鱼类保鲜储藏过程中α-辅肌动蛋白的分解变化提供了重要的手段。

Objective：To purify and prepare polyclonal antibody of α-actinin from the skeletal muscle of sea bream （Sparus latus）. Methods：α-actinin was extracted at low temperature and purified by ammonium sulfate fractionation and repeated column chromatographies on DEAE-Sepharose. Polyclonal antibody was prepared by immunizing rats with purified α-actinin. Results：The molecular weight of purified α-actinin determined by SDS-PAGE was approximately 100 kDa. Western blot analysis revealed that the antibody holds higher titer and specificity in reaction with purified α-actinin and α-actinin in sea bream myofibrils. Conclusion：α-actinin was purified from the skeletal muscle of sea bream（Sparus latus）and specific polyclonal antibody was prepared,which will be helpful in the study of the degradation of α-actinin during cold storage of fish.