【作者】 杜昆；

【导师】 余平；

【作者基本信息】 中南大学 ， 医学免疫学， 2011， 博士

【摘要】 一、研究目的1.研究CT703蛋白在沙眼衣原体感染细胞中的表达模式、持续性感染状态下CT703蛋白的表达变化。2.研究CT703蛋白对Raf/MEK/ERK信号通路的激活及抗凋亡作用。二、研究方法1.RT-PCR法扩增沙眼衣原体L2血清型CT703基因全长序列并亚克隆到原核表达质粒pGEX-6p-1中。2.IPTG诱导重组质粒pGEX-6p-1/CT703在大肠杆菌BL21中表达相应的重组融合蛋白,利用SDS-聚丙烯酰胺凝胶电泳分离并纯化重组融合蛋白,以纯化的重组融合蛋白作为免疫原免疫小鼠制备抗CT703蛋白多克隆抗体。3.采用Western Blot和免疫荧光技术,以抗CT703蛋白多克隆抗体为一抗检测CT703蛋白在沙眼衣原体急性感染状态下的表达模式。4.采用RT-PCR和Western Blot技术分别检测在干扰素-γ诱导沙眼衣原体持续性感染状态下CT703mRNA和蛋白水平表达变化。5.构建CT703基因真核表达重组质粒pcDNA4/CT703并转染HeLa细胞,Western Blot技术检测CT703蛋白是否活化Raf/MEK/ERK信号通路；并检测转染细胞在十字孢碱(Staurosporine, STS)作用下,细胞凋亡率以及Caspase-3活性变化。三、研究结果1.利用RT-PCR技术扩增沙眼衣原体L2血清型CT703基因,扩增片段长度约1.5kb,与预期理论值大小一致。重组质粒pGEX-6p-1/CT703经EcoR I和NotⅠ双酶切后,约在1.5kb处同样出现特异性DNA条带。通过DNA测序,证实插入的基因序列与GenBank公布的CT703基因序列完全一致,表明重组质粒构建成功。2.原核表达重组质粒pGEX-6p-1/CT703经IPTG在大肠杆菌BL21中诱导表达,纯化产物经考马斯亮蓝染色显示产物分子量约81kDa,由55KDa的CT703蛋白和26KDa的GST蛋白组成,与预期理论的分子量大小一致。以抗GST蛋白抗体为一抗,Western Blot技术检测纯化的重组融合蛋白,在81KDa处检测到相应的蛋白条带。Werstern Blot实验证实以重组融合蛋白免疫小鼠制备的抗血清能与CT703蛋白特异性结合；间接ELISA法检测抗血清效价,结果显示抗体滴度最高达到1：32000。3.利用Western Blot技术检测CT703蛋白在沙眼衣原体急性感染状态下的表达,感染后24小时可检测到相应的蛋白,随着感染时间的延长,蛋白表达量逐渐增多,并持续存在于整个感染过程,未感染细胞组没有检测到CT703蛋白的表达；通过免疫荧光技术检测CT703蛋白表达,最早在感染12小时即可检测到相应的蛋白。在干扰素-γ诱导沙眼衣原体持续性感染状态下,RT-PCR和Western Blot技术检测CT703 mRNA和蛋白的表达情况,结果表明持续性感染状态下CT703 mRNA和蛋白表达不呈时间依赖性,相同时间点沙眼衣原体持续性感染状态下CT703 mRNA和蛋白水平明显低于急性感染状态。4.间接免疫荧光技术对内源性的CT703蛋白进行定位,结果显示沙眼衣原体感染细胞CT703蛋白的荧光染色部位既不同于胞浆蛋白CPAF,也不同于包涵体膜蛋白CT813。5.构建的真核表达重组质粒pcDNA4/CT703经PCR、双酶切实验证实插入片段大小与CT703基因片段大小一致,测序证实插入的基因序列与GenBank公布的CT703基因序列完全一致。重组质粒pcDNA4/CT703转染到HeLa细胞后,Western Blot和免疫荧光实验均能检测到CT703蛋白的表达。6.真核表达重组质粒pcDNA4/CT703转染HeLa细胞后36小时检测磷酸化的Raf、ERK,发现二者均未被磷酸化；转染质粒36小时后,STS诱导细胞凋亡5小时,用流式细胞术检测细胞凋亡率以及用Caspase-3活性检测试剂盒检测Caspase-3活性。同时设STS诱导的HeLa细胞组、STS诱导的沙眼衣原体感染组和正常HeLa细胞组作为对照,发现4组细胞的凋亡率分别为：(93.1±2.01)%、(91.3±1.67)%、(3.21±0.87)%、(2.08±0.76)%。统计学分析,STS诱导的重组质粒转染组与正常HeLa细胞组比较有显著性差异,与STS诱导的衣原体感染组比较有显著性差异(P<0.05),与STS诱导的HeLa细胞组比较没有显著性差异(P>0.05)。Caspase-3活性检测结果与细胞凋亡率一致。四、结论1.成功构建原核表达重组质粒pGEX-6p-1/CT703以及真核表达重组质粒pcDNA4/CT703。2.沙眼衣原体急性感染状态下CT703蛋白表达呈时间依赖性增加,持续性感染状态下CT703蛋白表达不呈时间依赖性；相同时间点沙眼衣原体持续性感染状态下CT703蛋白表达明显低于急性感染状态。3.CT703蛋白不能激活Raf/MEK/ERK信号通路,不能抑制STS诱导的细胞凋亡。更多还原

【Abstract】 Objective:1. To investigate the expression pattern of CT703 protein in Chlamydia trachomatis-infected cells by Western Blot and immunofluorescence assay and investigate the expression change of CT703 protein under Chlamydia trachomatis persistent infection.2. To investigate whether CT703 protein can activate Raf/MEK/ERK signaling pathway and investigate the antiapoptotic avtivity of CT703 protein.Methods:1. The full length of CT703 gene of Chlamydia trachomatis was amplified by RT-PCR, and was cloned into the prokaryotic expression vector pGEX-6p-1.2. The fusion protein GST-CT703 was expressed in the E. coli BL21 strain induced with IPTG, and then was separated and purified with SDS-PAGE gel, purified fusion protein was used to raise antigen-specific antibodies.3. To detection the expression pattern of CT703 protein by Western Blot and immunofluorescence assaies under chlamydia trachomatis acute infection.4. To examine the expression change of CT703 mRNA by RT-PCR and that of protein by Western Blot assay under Chlamydia trachomatis persistent infection induced with IFN-y.5. To construct the recombinant eukaryotic expression plasmid of pcDNA4/CT703. To investigate whether CT703 protein can activate Raf/MEK/ERK signaling pathway and examine the apoptotic rate and caspase-3 activity induced by STS in HeLa cells transfected transiently with pcDNA4/CT703.Results:1. About 1.5kb DNA band amplified by RT-PCR was seen in the 1% agarose gel, which was consistent with the entire CT703 gene fragment. Digested with the restriction endonuclease EcoRⅠand No tⅠ, about 1.5kp DNA fragment was cleaved off from the recombinant plasmid. Sequence analysis revealed that the fragment inserted into the pGEX-6p-1 vectors was 100% homologous to the published nucleotide sequence in GeneBank.2. The recombinant expression plasmid pGEX-6p-1/CT703 was overproductive in E. coli BL21 induced with IPTG. The product was an 81-kDa fusion protein composed of the 26-kDa pGEX-6p-1 vector expression protein and the 55-kDa CT703 protein, which was consistent with prediction Molecular Weight. The purified fusion protein showed one clear band on SDS-PAGE and was detected with anti-GST antibody by Western Blot. The mouse antiserum could bind to CT703 protein specially. The result showed the antibody titer examined by ELISA reached to 1:32000.3. The expression of CT703 protein in Chlamydia trachomatis-infected cells was examined by Western Blot assay; the result showed that CT703 protein was first detected at 24 h after infection, and the protein levels increased with progression of infection, but not in the parallel uninfected HeLa cells. In IF A, the CT703 protein was monitored as early as 12 h after infection. The expression of CT703 mRNA and protein was not time-dependent under persistent infection induced with IFN-γ. Both the CT703 mRNA and protein production greatly decreased at the same time points under persistent infection, as compared with under acute infection.4. Localization of CT703 protein in Chlamydia trachomatis-infected cells by indirect immunofluorescence assay. The result showed that the localization of CT703 protein was different from CPAF and CT813 protein which were known plasmosin and Inc protein, respectively.5. Eukaryotic expression plasmid pcDNA4/CT703 was constructed successfully, which was demonstrated by PCR, restriction enzyme digestion and sequence analysis. The expression of CT703 protein in HeLa cells after the plasmid was transfected was demonstrated by Immunofluorescence assay and Western Blot.6. The eukaryotic expression plasmid pcDNA4/CT703 was transfected transiently into cells, and then cultured 36 h, the pRaf and pERK were detected by Western Blot assay, and the result demonstrated CT703 protein can not activate Raf and MEK. The apoptotic rate and caspase-3 activity induced by STS for 5h was examined in HeLa cells transfected transiently with pcDNA4/CT703 for 36 h. The HeLa cell induced with STS, Chlamydia trachomatis-infected HeLa cells induced with STS and alone HeLa cells were as the control groups. The apoptotic rates were (93.1±2.01)%、(91.3±1.67)%、(3.21±0.87)%、(2.08±0.76)% respectively. The apoptotic rate of STS-induced cells transfected transiently with pcDNA4/CT703 was significant difference when compared with HeLa cells group and Chlamydia trachomatis-infected cells group induced with STS (P<0.05), but no significant difference when compared with HeLa cells group induced with STS (P>0.05). The result of caspase-3 activity was consistent with that of apoptotic rate. Conclusions:1. The prokaryotic expression plasmid pGEX-6p-1/CT703 and eukaryotic expression plasmid pcDNA4/CT703 was constructed successfully.2. The expression of CT703 protein was time-dependent increase under Chlamydia trachomatis acute infection, but was not time-dependent under persistent infection. CT703 protein production greatly decreased at the same time points under Chlamydia trachomatis persistent infection, as compared with under Chlamydia trachomatis acute infection.3. CT703 protein can not activate Raf/MEK/ERK signaling pathway, and can not inhibit cell apoptosis induced with STS.更多还原