沙眼衣原体pORF5质粒蛋白单克隆抗体2H4的纯化及其免疫学特性研究  

Purification and immunological characteristics of monoclonal antibody 2H4 against Chlamydia tracho. marls pORF5 plasmid protein

作　　者：李忠玉[1] 吴移谋[1] 黄秋林[2] 粟盛梅[1] 周洲[1] 陈超群[1] 周辉[1] 钟光明[3] 

机构地区：[1]南华大学医学院微生物学与免疫学教研室,衡阳421001 [2]南华大学第一附属医院普外科 [3]美国德克萨斯大学健康科学中心

出　　处：《中华微生物学和免疫学杂志》2011年第11期1041-1045,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（30970165、81102230）;湖南省自然科学基金（09JJ3059）;湖南省高校科技创新团队资助项目（湘教通[20103212号）

摘　　要：目的 纯化抗沙眼衣原体pORF5单克隆抗体2H4并鉴定其免疫学特性.方法 大量培养pORF5单克隆抗体阳性杂交瘤细胞株并收集培养上清,采用G蛋白免疫亲和层析法纯化2H4单克隆抗体；酶联免疫吸附试验（ELISA）测定2H4效价及抗体亚类；Western blot鉴定其特异性；免疫荧光试验（IFA）检测2H4单克隆抗体的衣原体种属特异性.结果 纯化后2H4抗体的纯度高达93％；效价为1：1024,免疫球蛋白类型为IgG2a;2H4抗体不仅能特异性识别pORF5融合蛋白,而且能特异性识别Ct血清型A、D、L2、鼠农原体（MoPn）、鹦鹉热嗜农原体（6BC）质粒所编码的内源性pORF5蛋白,但不识别衣原体其他质粒蛋白和肺炎嗜衣原体（Cpn）.结论 获得了高纯度的能特异识别pORF5质粒蛋白的单克隆抗体,为进一步研究pORF5蛋白结构和功能以及沙眼衣原体诊断试剂盒的研制奠定了良好的基础.Objective To purify and characterize the monoclonal antibody （McAb） against Chlamydia trachomatis pORF5 plasmid protein.Methods The hybridoma cells stably secreting specific McAb against pORF5 were cultured in a large scale,and protein G purification by affinity chromatography was used to purify 2H4 McAb.ELISA was used to determine the antibody titer,and identify McAb isotype.Immunofluorescence assay （IFA） and Western blot were performed to detect McAb specificity.Results The purity of 2H4 antibody was 93%,the titer reached 1：1024,and 2H4 McAb was identified to belong to IgG2a isotype,2H4 McAb reacted strongly with the GST-pORF5 fusion protein and endogenous pORF5 protein expressed by Chlamydia trachomatis serovar A,D,L2,Chlamydia muridarum （ MoPn ）,Chlamydia psittaci 6BC,but not other chlamydial plasmid proteins and Chlamydia pneumoniae（Cpn） AR39 strain.Conclusion 2H4 McAb against pORF5 protein was successfully purified with a high titer and specificity which lay a foundation for further study on pORF5 protein structure and function.

关 键 词：沙眼衣原体 pORF5质粒蛋白 单克隆抗体 免疫学特性 

分 类 号：R446.6[医药卫生—诊断学]