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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12h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.
To determine the position of Chlamydia trachomatis CT358 protein in chlamydial infected cells and preliminary identification of its biological function.C CT358 gene was amplified from the genome of Chlamydia trachomatis by PCR and cloned into pGEX and pDSRedC1 expression vector.The recombinant plasmid pGEX -CT358 was transformed into XL1-blue host strain to induce the expression of fusion protein GST-CT358. The purified CT358 fusion protein was used to immunize mice and the expression of CT358 protein in chlamydial infected cells was detected by indirect immunofluorescence The recombinant plasmid pDSRedC1-CT358 was transiently transfected into HeLa cells to observe the effect of CT358 protein on the infection of Chlamydia.It was proved that the CT358 protein was the inclusion body membrane protein of Chlamydia trachomatis.The protein was expressed in inclusion bodies 12h after chlamydia infection Membrane, until the entire infection cycle, the CT358 fusion protein expressed in the cytoplasm of transgenics did not affect the subsequent infection of chlamydia.The study provides new clues for further study of the interaction between chlamydia and host cells, and for chlamydial The treatment, prevention provides a new direction.