微小脲原体UP3-RS02445生物信息学分析及单克隆抗体的制备  

Bioinformatics analysis of ureaplasma urealyticum UP3-RS02445 and the preparation of monoclonal antibodies

作　　者：陈恒鑫 贾晓晖[1] 李雅惠 周艳[1] 贾天军[1] 李萍[1] CHEN Hengxin;JIA Xiaohui;LI Yahui;ZHOU Yan;JIA Tianjun;LI Ping(Key Laboratory of Clinical Laboratory and Diagnostics,Hebei North University,Zhangjiakou 075000,China;Pathogenic Biology and Immunology,Hebei North University,Zhangjiakou 075000,China)

机构地区：[1]河北北方学院临床检验诊断学重点实验室,河北张家口075000 [2]河北北方学院病原生物与免疫学研究所,河北张家口075000

出　　处：《细胞与分子免疫学杂志》2024年第11期1011-1017,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：河北省高校基本科研业务费资助项目(JYT2022005)。

摘　　要：目的对微小脲原体UP3-RS02445功能进行生物信息学分析,进而构建其重组表达载体,并制备单克隆抗体(mAb)。方法利用生物信息学分析软件,对UP3-RS02445蛋白功能进行分析。构建UP3-RS02445重组表达载体,异丙基-β-D-硫代半乳糖苷(IPTG)诱导并纯化蛋白免疫雌性BALB/c小鼠,利用杂交瘤技术制备抗UP3-RS02445 mAb,分别使用Western blot法和ELISA方法检测mAb的特异性和效价。使用mAb亚型测定试纸条检测mAb的重链亚型和轻链亚型。结果生物信息学分析表明,UP3-RS02445蛋白由201个氨基酸组成,无跨膜结构域和信号肽,属于非分泌蛋白。成功构建UP3-RS02445重组表达载体并能够诱导重组蛋白的大量表达,细胞融合后经ELISA和Western blot法筛选获得两株分泌UP3-RS02445 mAb的杂交瘤细胞(A1H5和A4E2)。ELISA结果显示mAb的效价为1∶2560,Western blot法和免疫荧光技术结果均表明mAb可与UP3-RS02445蛋白特异性结合。两株mAb的重链亚型为IgG1,轻链亚型为Kappa型。结论成功制备了特异性强、效价高的UP3-RS02445 mAb,为后续UP诊断试剂的开发及蛋白功能的研究奠定了基础。Objective To make the bioinformatics analysis of Ureaplasma parvum UP3-RS02445 and prepare monoclonal antibody(mAb)against UP3-RS02445.Methods The biological characteristics of UP3-RS02445 protein were predicted by bioinformatics software.The UP3-RS02445 prokaryotic expression plasmid was constructed and the corresponding protein expression was induced by isopropyl-β-D-thiogalactoside(IPTG).Thus the expressed protein was used as immunogen to immunize female BALB/c mice.Hybridoma cell technology was used to prepare the monoclonal antibody against UP3-RS02445.The specificity and titer of monoclonal antibody were detected by Western blot and ELISA respectively.The subclass of heavy chain and subtype of light chain were identified by monoclonal antibody subtype identification test strip.Results Bioinformatics analysis showed that UP3-RS02445 protein was composed of 201 amino acids,without transmembrane domain and signal peptide,and belongs to non-secretory proteins.The recombinant prokaryotic plasmid of UP3-RS02445 was successfully constructed and the recombinant protein could be induced in large amount.After cell fusion,two hybridoma cells(A1H5 and A4E2)secreting UP3-RS02445 mAb were screened by ELISA and Western blot.The results of ELISA showed that the titers of monoclonal antibodies were 1∶2560.Western blot and Immunofluorescence technique both indicated that the antibodies could bind specifically to the UP3-RS02445 protein.The heavy chain and light chain of the two mAbs were IgG1 and kappa subtypes respectively.Conclusion We prepared the UP3-RS02445 monoclonal antibodies with well specificity and high titer which might lay foundations for the subsequent development of UP diagnostic reagents and the functional study of protein.

关 键 词：微小脲原体(UP) 生物信息学 原核表达 蛋白纯化 单克隆抗体 

分 类 号：R392[医药卫生—免疫学] R446.6[医药卫生—基础医学] R375.3