摘要
目的在利用生物信息学全面分析TgSUB3序列的基础上,制备针对该蛋白B细胞抗原表位的兔源多克隆抗体。方法采用生物信息学软件对TgSUB3蛋白的理化性质、空间结构、磷酸化/糖基化位点、B细胞抗原表位等进行分析。用原核表达、纯化的TgSUB3重组蛋白免疫新西兰大白兔,于4次加强免疫后第7 d制备兔血清,采用Protein A亲和纯化抗体蛋白,分别利用SDS-PAGE和ELISA分析抗体纯度和效价。结果生物信息学分析TgSUB3为不稳定亲水性蛋白,无信号肽,跨膜区为19-39aa,包含1个Peptidases_S8结构域(358-617aa)。二级结构中无规卷曲占51.29%,α螺旋占26.11%。TgSUB3包含65个磷酸化位点和126个糖基化位点。B细胞抗原表位区集中在45-290aa和715-937aa。SDS-PAGE显示纯化的TgSUB3多抗为单一55 ku条带,ELISA检测TgSUB3抗体滴度为1:102400。结论成功制备了兔源TgSUB3抗血清,经纯化获得高纯度高滴度的多抗。
Objective Rabbit polyclonal antibody targeting the B cell epitope of TgSUB3 was prepared based on comprehensive analysis of TgSUB3 sequence by bioinformatics method.Methods The physical and chemical properties,spatial structure,phosphorylation/glycosylation sites and B cell epitope of TgSUB3 were predicted by bioinformatics software.New Zealand white rabbits were immunized with purified TgSUB3 protein expressed in prokaryotic expression.The rabbit serum was collected on the 7th day after the fourth intensive immunization,and purified with Protein A affinity.The purity and titer of the antibody were analyzed by SDS-PAGE and ELISA,respectively.Results Bioinformatics analysis showed that TgSUB3 was an unstable hydrophilic protein with no signal peptide.The transmembrane region and Peptidases_S8 domain of TgSUB3 were 19-39aa and 358-617aa,respectively.The secondary structure mainly included random coil(51.29%)and alpha helix(26.11%).TgSUB3 including 65 phosphorylation sites and 126 glycosylation sites.The antigen region of B cell epitopes were concentrated at 45-290aa and 715-937aa.SDS-PAGE result of the purified TgSUB3 polyclonal antibody showed a specific band at around 55 ku.The ELISA result showed that the titer of TgSUB3 antibody was 1:102400.Conclusion In this study,the rabbit antiserum of TgSUB3 was successfully prepared,and the good purity and high titer polyclonal antibody was obtained after purified.
作者
李瑾
张欣
雷战
谢金晶
王龙江
尹昆
徐超
魏庆宽
孙慧
LI Jin;ZHANG Xin;LEI Zhan;XIE Jinjing;WANG Longjiang;YIN Kun;XU Chao;WEI Qingkuan;SUN Hui(Shandong Institute of Parasitic Diseases,Shandong First Medical University&Shandong Academy of Medical Sciences,Jining 272033,Shandong,China;Shandong First Medical University&Shandong Academy of Medical Sciences)
出处
《中国病原生物学杂志》
CSCD
北大核心
2024年第6期690-694,共5页
Journal of Pathogen Biology
基金
山东省自然科学基金项目(No.ZR2022MH271)
山东省医药卫生科技项目(No.202301011242)
山东省医药卫生科技发展计划项目(No.202101050153)。
