家蝇Phormicin A多克隆抗体的制备及效价检测

Preparation and Titer Detection of Polyclonal Antibody Against Phormicin A of Housefly

家蝇Phormicin作为防御素家族的成员,是一类具有广普抗菌活性的抗菌肽。本研究采用原核表达法表达并纯化获得了家蝇Phormicin A蛋白。将家蝇Phormicin A原核表达蛋白与完全佐剂和不完全佐剂乳化混匀,先后免疫新西兰白兔获得其多克隆抗体。通过原核表达蛋白中和吸附实验,以及Western blot实验验证了抗体的特异性。进一步用金黄色葡萄球菌刺激家蝇三龄幼虫样品,进行内源性验证并测定抗体的效价。结果显示,该多克隆抗体既可以识别家蝇Phormicin A原核表达蛋白,也可以识别金黄色葡萄球菌刺激家蝇三龄幼虫产生的内源性Phormicin A蛋白。本研究为进一步探索Phormicin在家蝇天然免疫和防御中的机制等后续工作打下基础。

Musca domestica Phormicin,belonging to the defensin super family,is a kind of antimicrobial peptides with broad antibacterial activity.In this study,Phormicin A protein of housefly was expressed and purified by prokaryotic expression method.And it was mixed and emulsified well with complete and incomplete adjuvants.Then the polyclonal antibodies were obtained after immunizing New Zealand white rabbits.The specificity of the antibody was verified by neutralization and adsorption of prokaryotic expression protein incubated with antibody via Western blot experiments.Endogenous validation was performed and the antibody titer was also determined by using the samples of 3rd instars larvae of housefly stimulated by Staphylococcus aureus.The results showed that the polyclonal antibody could recognize not only the prokaryotic expression protein,but also the endogenous Phormicin A protein produced from 3rd instars larvae of housefly stimulated by the S.aureus.This study laid a foundation for further exploration of the mechanism of Phormicin in innate immunity and defense of housefly.