E型沙眼衣原体主要外膜蛋白多克隆抗体的制备及鉴定  

Preparation and identification of polyclonal antibody against major outer membrane protein of Chlamydia trachomatis serovar E

作　　者：李明洋 魏荷婷 陈天枫 胡瑜 沈玉婷 董海艳[1] 朱珊丽[1] 张丽芳[1] LI Ming-yang;WEI He-ting;CHEN Tian-feng;HU Yu;SHEN Yu-ting;DONG Hai-yan;ZHU Shan-li;ZHANG Li-fang(Institute of Molecular Viruses and Immunology,Department of Microbiology and Immunology,Wenzhou Medical University,Wenzhou 325035,Zhejiang Province,China;不详)

机构地区：[1]温州医科大学分子病毒与免疫研究所微生物学与免疫学教研室,浙江温州325035 [2]温州医科大学精神医学学院,浙江温州325035 [3]温州医科大学仁济学院,浙江温州325035

出　　处：《中国生物制品学杂志》2020年第9期1014-1018,1022,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31700160);浙江省自然科学基金(LY16H190008);温州市科技局资助项目(Y20170011);温州医科大学科研发展基金(RTJ17015)。

摘　　要：目的制备E型沙眼衣原体(Chlamydia trachomatis,Ct)主要外膜蛋白(major outer membrane protein,MOMP)多克隆抗体,并在体外检测其生物学特性。方法通过分子克隆方法构建重组表达质粒pET21a(+)/MOMP,转化E.coli BL21(DE3),IPTG诱导表达重组MOMP蛋白,经镍螯合亲和层析胶纯化后,经皮下多点注射免疫日本大耳白兔,制备兔多克隆抗体,分别采用ELISA和Western blot法检测抗体的效价和特异性。在HeLa细胞中培养E血清型Ct,通过卢戈和吉姆萨染色法观察细胞中Ct包涵体的形成情况,间接免疫荧光法检测制备的MOMP多克隆抗体对Ct天然MOMP的结合特异性。结果Ct MOMP可在原核表达系统中高效表达;制备的兔抗MOMP多克隆抗体ELISA效价可达1∶256000,可特异性结合、识别融合及天然的Ct MOMP。结论制备的兔抗Ct MOMP多克隆抗体效价高、特异性强,为后续进一步研究Ct的致病机制及开发新的免疫诊断制剂奠定了基础。Objective To prepare the polyclonal antibody against major outer membrane protein(MOMP)of Chlamydia trachomatis(Ct)and determine its biological characteristics in vitro.Methods Recombinant plasmid pET21 a(+)/MOMP was constructed by molecular cloning method and transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed recombinant MOMP was purified by nickel ion affinity chromatography and injected s.c.into Japanese rabbits in several sites to prepare polyclonal antibody.The prepared antibody was determined for titer and specificity by ELISA and Western blot respectively.Ct serovar E was propagated in HeLa cells and observed for formation of inclusion body by Lugo and Giemsa staining.The binding specificity of prepared polyclonal antibody against MOMP to natural MOMP of Ct was tested by indirect immunofluorescence assay(IFA).Results Ct MOMP was highly expressed in E.coli BL21(DE3).The prepared polyclonal antibody against MOMP reached an ELISA titer of 1∶256000,which bound and recognized fusion and natural Ct MOMP specifically.Conclusion The prepared rabbit polyclonal antibody against Ct MOMP showed high titer and specificity,which laid a foundation of further study on the pathogenic mechanism of Ct and development of novel immunodiagnostic kit.

关 键 词：沙眼衣原体 主要外膜蛋白 多克隆抗体 

分 类 号：R374[医药卫生—病原生物学]