弓形虫肌动蛋白抗原表位抗体的制备及应用
Preparation and Application of Epitope Antibodies of Toxoplasma Actin
庄宝灿 1朱进进 1余莉1
作者信息
- 1. 安徽医科大学病原生物学教研室病原生物学安徽省重点实验室人畜共患病安徽高校省级重点实验室,合肥 230032
- 折叠
摘要
制备弓形虫肌动蛋白(TgActin)多肽表位抗体并探讨其应用价值.BepiPred 1.0 Server在线分析设计TgActin抗原表位,并与多种属Actin进行同源比对,选取并合成高特异性多肽片段序列免疫新西兰兔.末次免疫后收集兔血清并使用Protein G纯化抗体,SDS-PAGE检测抗体纯度,ELISA测定抗体效价,Western blot分析抗体的特异性及TgActin表位抗体作为内参抗体的可能性,免疫荧光观察胞内速殖子形态.结果显示,ELISA测定抗体效价为1∶128 000,Western blot结果显示抗体特异性识别弓形虫Actin,免疫荧光染色显示TgActin表位抗体可以标记纳虫泡内速殖子.结果表明,成功制备了TgActin多肽表位抗体,可用做弓形虫蛋白检测的内参抗体及其在细胞内的免疫荧光检测,为后续弓形虫研究提供了便利条件.
Abstract
To prepare the antibodies against the epitopes of Toxoplasma actin(TgActin),the structure of TgActin were analyzed by BepiPred 1.0 Server and a specific polypeptide with a length of 12 amino acids was selected and synthesized for immunization of New Zealand rabbits.One week after the last immunization,serum samples were collected from the immunized rabbits and antibodies were purified by Protein G.The purity of antibodies was examined by SDS-PAGE and the titer was determined by ELISA.Moreover,the specificity of antibodies was analyzed by Western blot.Intracellular tachyzoites were detected by immunofluorescence using TgActin antibodies as the primary antibody.The results showed that the antibody titer was 1:128 000 in ELISA and the antibodies recognized TgActin in Western blotImmunofluorescence staining showed that the antibodies against TgActin bound to tachyzoites in parasitophorous vacuoles.In conclusion,the antibodies against the epitopes of TgActin were successfully prepared,and could be used as the internal reference antibody for the detection of T.gondii protein and immunofluorescence detection in cells.
关键词
弓形虫/肌动蛋白/表位抗体/内参抗体/免疫荧光Key words
Toxoplasma gondii/actin/epitope antibody/loading control antibody/immunofluorescence引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据