质粒蛋白pORF5在沙眼衣原体感染细胞中的定位及免疫原性研究(英文)

Immunogenicity of plasmid protein pORF5 and its localization in Chlamydia Trachomatis infected cells

目的分析沙眼衣原体隐蔽性质粒编码的质粒蛋白pORF5在感染细胞中的定位并初步探讨免疫原性特征。方法 PCR扩增pORF5质粒蛋白编码基因,构建原核表达重组体pGEX-6p/pORF5,重组体转化大肠杆菌XL1-blue中诱导表达融合蛋白;融合蛋白经Glutathione Sepharose亲和层析纯化后免疫小鼠,制备单克隆抗体和多克隆抗体,间接免疫荧光技术及免疫印迹鉴定抗体的特异性,并分析pORF5质粒蛋白在感染细胞中的分布特征。采用ELISA方法分析pORF5质粒蛋白的免疫原性。结果 pORF5原核表达重组体成功构建,融合蛋白在大肠杆菌中可溶性表达;pORF5主要分布于宿主细胞胞浆,但也少量分布在EB、RB上;pORF5能与衣原体患者血清及鼠免疫血清发生强烈地免疫反应。结论 pORF5为衣原体分泌蛋白,并具有很强的免疫原性。

We aimed to localize the plasmid protein pORF5 in chlamydia infected cells and investigate the immunogenicity of pORF5.Firstly,the open reading frame encoding for pORF5 protein from the Chlamydia trachomatis plasmid was amplified and cloned into the pGEX-6p vector.Then the recombinant plasmid pGEX-6p/pORF5 was transformed into XL1-blue E.coli with glutathione-s-transferase（GST） to express fusion protein.After purified with Glutathione Sepharose 4B beads,the pORF5 fusion protein was used to immunize mice to make monoclonal antibody and polyclonal antibody,which were subsequently used to localize the endogenous pORF5 protein by indirect immunofluorescence assay（IFA）.At the same time,ELISA was used to determine the immunogenicity of pORF5 plasmid protein through recognizing the pool sera of patients infected with Chlamydia trachomatis and the pool sera of mice immunized with pORF5 fusion protein.pORF5 was dominantly expressed in the cytosol of the Chlamydia-infected cells,and also appeared in the RBs and EBs in small quantity;pORF5 protein could strongly reacted with antiserum.In conclusion,pORF5 plasmid protein is identified as a secreted protein with good immunogenicity,which lay the foundation for studying the biological activities and the development of the Chlamydia trachomatis vaccine against this pathogen.