CT358蛋白在沙眼衣原体感染细胞中的定位及特性分析

确定沙眼衣原体CT358蛋白在衣原体感染细胞中的位置并初步鉴定其生物学功能．采用PCR方法从D型沙眼衣原体的基因组中扩增CT358基因，并克隆入pGEX和pDSRedC1表达载体中．将重组质粒pGEX-CT358转化到XL1-blue宿主菌，并诱导表达融合蛋白GST-CT358．纯化后的CT358融合蛋白免疫小鼠制备抗体，应用间接免疫荧光技术对CT358蛋白在衣原体感染细胞内的定位及表达模式进行分析．同时，pDSRedC1-CT358重组质粒瞬时转染HeLa细胞，观察CT358蛋白对衣原体感染的影响．实验结果证明CT358蛋白为沙眼衣原体包涵体膜蛋白．该蛋白质在衣原体感染12 h后就表达定位于包涵体膜上，直至持续到整个感染周期，转基因在胞浆表达的CT358融合蛋白不影响其后的衣原体感染．该研究为深入研究衣原体与宿主细胞间相互作用提供了新的线索，并可为衣原体性的治疗、预防提供新方向．

Localization and Characterization of Hypothetical Protein CT358 in The Chlamydia trachomatis-Infected Cells

To localize and characterize the hypothetical protein CT358 in the chlamydial infected cells. CT358 gene from the Chlamydia trachomatis(C. trachomatis) serovar D genome was amplified and cloned into the pGEX and pDSRedC1 vectors. The recombinant plasmid pGEX-CT358 was constructed and expressed as GST fusion proteins. The GST-CT358 fusion protein was used to immunize mice to raise the antibodies, which specifically recognized CT358 without cross-reacting with other unrelated proteins. The antibodies were then used to localize the endogenous CT358 protein and determine the expression pattern in Chlamydial infected cells using an indirect immunofluorescence assay (IFA). Meanwhile, pDSRedC1-CT358 recombinant plasmid was transfected to HeLa cells to evaluate the effect of CT358 expression on the subsequent chlamydial infection. The hypothetical protein CT358 was identified in the inclusion membrane of C. trachomatis-infected cells for the first time,and it was detected as early as 12 h after C. trachomatis infection and remained in the inclusion membrane throughout the rest of the infection cycle. Cytosolic expression of CT358 via a transgene failed to affect the subsequent chlamydial infection. These observations together have demonstrated that CT358 is a newly identified chlamydial inclusion membrane protein, giving the potentially importance for further understanding the mechanisms of chlamydial intracellular parasitism.