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

作　　者：王兵[1,2,3] 覃海 赵杰琼 姚杨 韦鹏威 宋朝蓉 李道远 张其柱 朱贵明[1] 吴建伟[6] Wang Bing;Qin Hai;Zhao Jieqiong;Yao Yang;Wei Pengwei;Song Chaorong;Li Daoyuan;Zhang Qizhu;Zhu Guiming;Wu Jianwei(Key Laboratory of Biology and Medical Engineering,Guizhou Medical University,Guiyang,550025;Engineering Research Center of Medical and Pharmacy Biotechnology,Guizhou Medical University,Guiyang,550025;Department of Biotechnology,School of Biology and Engineering,Guizhou Medical University,Guiyang,550025;Department of Cardiovascular Medicine,The Tang Du hospital of Air Force Medical University,Xi'an,710038;The First Affiliated Hospital of Guizhou Medical University,Guiyang,550025;Department of Human parasites,School of Basic Medical Sciences,Guizhou Medical University,Guiyang,550025)

机构地区：[1]贵州医科大学生物与医学工程重点实验室,贵阳550025 [2]贵州医科大学医药生物技术工程研究中心,贵阳550025 [3]贵州医科大学生物与工程学院,生物技术教研室,贵阳550025 [4]空军军医大学唐都医院,心血管内科,西安710038 [5]贵州医科大学第一附属医院,贵阳550025 [6]贵州医科大学基础医学院,病原生物学教研室,贵阳550025

出　　处：《基因组学与应用生物学》2021年第3期1040-1046,共7页Genomics and Applied Biology

基　　金：国家自然科学基金(31760250);贵州省科技计划(黔科合基础〔2019〕1273号);贵州医科大学博士启动基金(Academy-PHD-J-2014-018);贵阳市科技计划(筑科合同〔20151001〕社15号);贵州省卫健委科学计划(gzwjkj2015-1-028);贵阳市政府-贵州医科大学联合基金(筑科合同〔20161001〕023)和(筑科合同〔2017〕5-26号)共同资助。

摘　　要：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.

关 键 词：Phormicin 多克隆抗体 家蝇 抗菌肽 

分 类 号：Q939.91[生物学—微生物学]