滑液囊支原体DnaK蛋白单克隆抗体的制备及抗原表位的初步鉴定  

Preparation of Monoclonal Antibody Against DnaK Protein of Mycoplasma synoviae and Preliminary Identification of Its Epitopes

作　　者：刘凯 赵云海 何肖肖 马海云 王青 刘玉东 包世俊[1] LIU Kai;ZHAO Yun-Hai;HE Xiao-Xiao;MA Hai-Yun;WANG Qing;LIU Yu-Dong;BAO Shi-Jun(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070

出　　处：《农业生物技术学报》2025年第3期689-696,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32072863)。

摘　　要：热休克蛋白(DnaK)是生物在受不利因素影响下产生的具有高度保守性的应激蛋白,具有载体和佐剂双重作用,然而目前对滑液囊支原体(Mycoplasma synoviae, MS) DnaK蛋白研究较少。本研究为制备MS DnaK蛋白的单克隆抗体(monoclonal antibody, MAb)并对其抗原表位进行初步鉴定,构建重组质粒进行重组蛋白表达,并纯化出滑液囊支原体重组蛋白DnaK (rMSDnaK蛋白),以rMSDnaK蛋白免疫BALB/c小鼠(Mus musculus)。选择抗体水平高的小鼠,分离其脾脏,将脾细胞与骨髓瘤细胞融合,通过有限稀释法和间接ELISA法筛选出状态良好的阳性杂交瘤细胞,将其注射至昆明鼠腹腔后收集并纯化腹水。经SDS-PAGE鉴定,rMSDnaK成功表达。采用间接ELISA法筛选出1株可分泌抗DnaK抗体且稳定性良好的杂交瘤细胞株,命名为6G6。对MAb 6G6亚型进行鉴定,结果为IgG3亚类。纯化后的MAb经间接ELISA法测定效价为1∶102 400。Western blot结果显示,MAb 6G6仅与rMSDnaK蛋白发生反应。间接免疫荧光结果显示,MAb 6G6仅与MS发生荧光反应。Western blot结果证明,制备的截短体MS DnaKΔ3-3能够与rMSDnaK蛋白反应,证明单克隆抗体识别的抗原表位区域是C端506~596位氨基酸。本研究成功制备出抗MS DnaK单克隆抗体并对其进行抗原表位鉴定,为后续建立检测MS的双抗夹心ELISA法提供生物材料。Heat shock protein(DnaK)is a highly conserved stress protein produced by organisms under the influence of unfavorable factors,it has dual roles of carrier and adjuvant,however,the DnaK protein of Mycoplasma synoviae(MS)is less studied.In this study,in order to prepare a monoclonal antibody(MAb)of MS DnaK protein and preliminarily characterize its antigenic epitope,the recombinant plasmid was constructed and the recombinant protein was expressed.The recombinant protein of Mycoplasma synoviae,DnaK(rMSDnaK protein)was purified.BALB/c mice(Mus musculus)were immunized with rMSDnaK protein.Mice with high antibody levels were selected,their spleens were isolated,and the collected splenocytes were fused with myeloma cells(SP2/0 cells),after which positive hybridoma cells in good condition were screened by limited dilution and indirect ELISA,and ascites were collected and purified after injection into the peritoneal cavity of quinacrine mice.The rMSDnaK was successfully expressed identified by SDS-PAGE.Indirect ELISA was used to screen one hybridoma cell line,named 6G6,which could secrete anti-DnaK antibody and had good stability.The MAb 6G6 subtype was identified as IgG3 subclass.The potency of the purified MAb was determined by indirect ELISA to be 1∶102400.Western blot results showed that MAb 6G6 reacted only with rMSDnaK protein.Immunofluorescence assay(IFA)results showed that MAb 6G6 reacted fluorescently only with MS.Western blot results demonstrated that the prepared truncated MS DnaKΔ3-3 was able to react with rMSDnaK protein,which proved that the antigenic epitope region recognized by the monoclonal antibody was amino acids 506~596 at the C-terminus.In this study,the anti-MS DnaK MAb was successfully prepared and its antigenic epitope was identified,which provides biological materials for the subsequent establishment of MS detection by double-antibody sandwich ELISA for MS detection.

关 键 词：滑液囊支原体(MS) DnaK蛋白 单克隆抗体 抗原表位鉴定 

分 类 号：S852.62[农业科学—基础兽医学]