沙眼衣原体CT703蛋白在感染细胞中的表达及功能初探

发布时间：2018-03-12 22:47

本文选题：CT7O3　切入点：蛋白表达　出处：《中南大学》2011年博士论文　论文类型：学位论文

【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(P0.05),与STS诱导的HeLa细胞组比较没有显著性差异(P0.05)。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。
[Abstract]:First, the purpose of the study
1. the expression pattern of CT703 protein in Chlamydia trachomatis infected cells and the change of the expression of CT703 protein in the persistent infection state.
2. study the activation and anti apoptosis effect of CT703 protein on Raf/MEK/ERK signaling pathway.
Two, research methods
The full-length sequence of serotype CT703 gene of Chlamydia trachomatis L2 was amplified by 1.RT-PCR and subcloned into the prokaryotic expression plasmid pGEX-6p-1.
The expression of the corresponding recombinant fusion protein in Escherichia coli BL21 recombinant plasmid pGEX-6p-1/CT703 2.IPTG induced by SDS- polyacrylamide gel electrophoresis for separation and purification of recombinant fusion protein, the purified recombinant fusion protein as immunogen to prepare mouse polyclonal antibody against CT703 protein.
3., we used Western Blot and immunofluorescence technology to detect the expression pattern of CT703 protein in Chlamydia trachomatis under the condition of acute infection by anti CT703 protein polyclonal antibody.
4. RT-PCR and Western Blot were used to detect the changes in the expression of CT703mRNA and protein in the continuous infection of Chlamydia trachomatis induced by interferon - gamma.
5. construction of CT703 gene recombinant eukaryotic expression plasmid pcDNA4/CT703 and transfected into HeLa cells, the detection of CT703 protein Western Blot whether activation of Raf/MEK/ERK signaling pathway; and transfected cells in staurosporine (Staurosporine, STS) under the action of apoptosis rate and Caspase-3 activity.
Three, the results of the study
1. by RT-PCR amplification of Chlamydia trachomatis serotype L2 CT703 gene, amplified fragment length of about 1.5kb, the size is consistent with the expected theoretical values. The recombinant plasmid pGEX-6p-1/CT703 by EcoR I and Not I digested the same specific DNA bands about 1.5kb. Through DNA sequencing, confirmed identical gene sequences of CT703 gene sequences with the insertion of GenBank released, showed that the recombinant plasmid was successfully constructed.
2. the prokaryotic expression recombinant plasmid pGEX-6p-1/CT703 expression induced by IPTG in Escherichia coli BL21, purified by Coomassie brilliant blue staining showed that the molecular weight of about 81kDa, consisting of 55KDa CT703 protein and 26KDa GST protein, consistent with the expected molecular weight. A theory with anti GST antibody for the detection of Western, Blot technology the purified recombinant fusion protein, the protein bands of.Werstern Blot to the experimental detection at 81KDa confirmed that the recombinant fusion protein in mice immunized with antiserum prepared to specifically bind to CT703 protein; indirect ELISA detection results showed that the anti serum titer, antibody titer reached 1:32000.
The expression of CT703 protein was detected using 3. Western Blot technology in Chlamydia trachomatis infection condition, 24 hours after infection can be detected in the corresponding protein, with prolonged infection, the expression gradually increased, and continued to exist in the whole process of infection, the non infected cells was not detected the expression of CT703 protein was detected by CT703; protein immunofluorescence expression, the earliest in 12 hours of infection can be detected. The corresponding protein in interferon gamma induced persistent chlamydial infection condition, the expression of RT-PCR and Western Blot to detect CT703 mRNA and protein, the results show that persistent expression in a time-dependent manner under the condition of CT703 protein and mRNA infection, at the same time persistent chlamydial infection under the condition of CT703 mRNA and protein levels were significantly lower than the acute infection.
4., indirect immunofluorescence technique was used to locate the endogenous CT703 protein. The results showed that the fluorescent staining sites of CT703 protein in Chlamydia trachomatis infected cells were different from cytoplasmic protein CPAF and inclusion body membrane protein CT813..
The recombinant eukaryotic expression plasmid pcDNA4/CT703 constructed by PCR 5., double enzyme digestion experiments confirmed that the inserted fragments and CT703 gene fragments, identical gene sequences of CT703 gene sequences and GenBank sequencing confirmed that the insertion released. The recombinant plasmid pcDNA4/CT703 was transfected into HeLa cells, Western Blot and immunofluorescence assay were detected the expression of CT703 protein.
6. recombinant eukaryotic expression plasmid pcDNA4/CT703 was transfected into HeLa cells after 36 hours to detect the phosphorylation of Raf, ERK, found that two were not to be phosphorylated; transfection 36 hours after 5 hours, cell apoptosis induced by STS, flow cytometry was used to detect the apoptosis rate and Caspase-3 activity detection kit to detect the activity of Caspase-3. HeLa cells were induced by STS and STS induced by Chlamydia trachomatis infection group and normal HeLa cells as control group, apoptosis of cells in 4 groups respectively: (93.1 + 2.01)% and (91.3 + 1.67)% and (3.21 + 0.87)% and (2.08 + 0.76)%. Statistical analysis, STS induction of the recombinant plasmid was transfected into HeLa cell group and the normal group with significant difference, there was significant difference between infection group and STS induced by Chlamydia trachomatis (P0.05), and HeLa cells induced by STS. There was no significant difference between (P0.05).Caspase-3 activity and apoptosis rate of detection results Agreement.
Four. Conclusion
1. the prokaryotic expression recombinant plasmid pGEX-6p-1/CT703 and eukaryotic expression recombinant plasmid pcDNA4/CT703. were successfully constructed
2. the expression of CT703 protein increased in a time dependent manner under the condition of Chlamydia trachomatis acute infection. The expression of CT703 protein was not time-dependent in persistent infection state. At the same time, the expression of CT703 protein in Chlamydia trachomatis persistent infection state was significantly lower than that in acute infection state.
3.CT703 protein does not activate the Raf/MEK/ERK signaling pathway and does not inhibit the apoptosis induced by STS.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R374

【参考文献】