沙眼衣原体主要外膜蛋白多肽多克隆抗体的制备与鉴定  

Preparation and identification of peptide polyclonal antibodies against major outer membrane protein of Chlamydia trachomatis

作　　者：徐青青 陈文韬 江银波 蓝银苑 潘钰莹 曾丽红 杨建江 薛耀华 郑和平 XU Qingqing;CHEN Wentao;JIANG Yinbo;LAN Yinyuan;PAN Yuying;ZENG Lihong;YANG Jianjiang;XUE Yaohua;ZHENG Heping(Guangdong Dermatology Clinical College,Anhui Medical University,Hefei 230032,China)

机构地区：[1]安徽医科大学广东皮肤病临床学院,合肥230032 [2]广东省皮肤病医院,广州510091 [3]南方医科大学皮肤病医院,广州510091

出　　处：《实用医学杂志》2022年第11期1333-1338,共6页The Journal of Practical Medicine

基　　金：国家自然科学基金项目(编号:81974307);广东省自然科学基金项目(编号:2018A030313662);广东省普通高校特色创新类项目(编号:2021KTSCX014);广东省中医药局中医药科研项目(编号:20211276)。

摘　　要：目的 沙眼衣原体主要外膜蛋白(major outer membrane protein,MOMP)在维持沙眼衣原体结构稳定性和衣原体感染宿主细胞过程中发挥重要作用,为深入研究MOMP蛋白的功能及作用机制,使用人工合成多肽制备MOMP多克隆抗体。方法 分析沙眼衣原体MOMP的抗原表位,根据15个沙眼衣原体血清型MOMP保守肽段,设计并合成MOMP特异性肽段,与血蓝蛋白偶联后免疫新西兰兔。采用免疫亲和层析纯化免疫血清,应用SDS-PAGE和ELISA评估血清抗体纯度及效价,利用Western blot和免疫荧光染色鉴定MOMP抗体特异性。结果 纯化后的血清IgG抗体纯度为89.6%,MOMP抗体效价达1∶12 800,Western blot结果显示抗体与8个血清型沙眼衣原体蛋白在40 kDa处特异性结合,免疫荧光染色证实该抗体可特异性结合沙眼衣原体。此外,MOMP抗体与进口商品化抗体明显共定位于宿主细胞内,且两种抗体荧光强度相当。结论 本研究成功制备效价高、特异性好的沙眼衣原体MOMP的兔多克隆抗体,为后续深入研究MOMP的生物学功能提供了有力工具。Objective The major outer membrane protein(MOMP)of Chlamydia trachomatis(Ct)is essential for Ct structural stability and infection of the host cell. MOMP polyclonal antibody was created using the synthetic peptide to further investigate the function and mechanism of MOMP protein. Methods Ct MOMP antigenic epitope was investigated. MOMP-specific peptide was designed and synthesized based on the conserved peptides of 15 different Ct serotype MOMPs,and New Zealand white rabbits were immunized after coupling with hemocyanin.MOMP-specific peptide was designed and synthesized based on the conserved peptides of 15 different Ct serotype MOMPs,and New Zealand white rabbits were immunized after coupling with hemocyanin.Immunoaffinity chromatography was used to separate the immune serum. SDS-PAGE was used to determine the purity of the IgG antibody,and ELISA was used to determine the titer of the MOMP antibody. Furthermore,Western blot and immunofluorescence staining were used to determine the specificity of the MOMP antibody. Results The purity of IgG antibody was 89.6%,and the titer of MOMP antibody was 1∶12800. The results of Western blot showed that the antibody specifically bound to 8 serotypes of Ct protein at 40 kDa,and indirect immunofluorescence staining showed that the antibody could specifically combined with Ct MOMP. In addition,the immunofluorescence results showed that the MOMP antibody and the imported commercial antibody were obviously co-localized in the host cells,and the fluorescence intensities of the two antibodies were comparable. Conclusions This study successfully prepared a rabbit polyclonal antibody with high titer and good specificity against Ct MOMP,providing a useful tool for future research on the biological function of MOMP.

关 键 词：沙眼衣原体 主要外膜蛋白 多肽 多克隆抗体 

分 类 号：R374.1[医药卫生—病原生物学]