發(fā)布時間：2018-03-12 22:47

  本文選題：CT7O3　切入點：蛋白表達　出處：《中南大學(xué)》2011年博士論文　論文類型：學(xué)位論文

【摘要】：一、研究目的 1.研究CT703蛋白在沙眼衣原體感染細胞中的表達模式、持續(xù)性感染狀態(tài)下CT703蛋白的表達變化。 2.研究CT703蛋白對Raf/MEK/ERK信號通路的激活及抗凋亡作用。 二、研究方法 1.RT-PCR法擴增沙眼衣原體L2血清型CT703基因全長序列并亞克隆到原核表達質(zhì)粒pGEX-6p-1中。 2.IPTG誘導(dǎo)重組質(zhì)粒pGEX-6p-1/CT703在大腸桿菌BL21中表達相應(yīng)的重組融合蛋白,利用SDS-聚丙烯酰胺凝膠電泳分離并純化重組融合蛋白,以純化的重組融合蛋白作為免疫原免疫小鼠制備抗CT703蛋白多克隆抗體。 3.采用Western Blot和免疫熒光技術(shù),以抗CT703蛋白多克隆抗體為一抗檢測CT703蛋白在沙眼衣原體急性感染狀態(tài)下的表達模式。 4.采用RT-PCR和Western Blot技術(shù)分別檢測在干擾素-γ誘導(dǎo)沙眼衣原體持續(xù)性感染狀態(tài)下CT703mRNA和蛋白水平表達變化。 5.構(gòu)建CT703基因真核表達重組質(zhì)粒pcDNA4/CT703并轉(zhuǎn)染HeLa細胞,Western Blot技術(shù)檢測CT703蛋白是否活化Raf/MEK/ERK信號通路；并檢測轉(zhuǎn)染細胞在十字孢堿(Staurosporine, STS)作用下,細胞凋亡率以及Caspase-3活性變化。 三、研究結(jié)果 1.利用RT-PCR技術(shù)擴增沙眼衣原體L2血清型CT703基因,擴增片段長度約1.5kb,與預(yù)期理論值大小一致。重組質(zhì)粒pGEX-6p-1/CT703經(jīng)EcoR I和NotⅠ雙酶切后,約在1.5kb處同樣出現(xiàn)特異性DNA條帶。通過DNA測序,證實插入的基因序列與GenBank公布的CT703基因序列完全一致,表明重組質(zhì)粒構(gòu)建成功。 2.原核表達重組質(zhì)粒pGEX-6p-1/CT703經(jīng)IPTG在大腸桿菌BL21中誘導(dǎo)表達,純化產(chǎn)物經(jīng)考馬斯亮藍染色顯示產(chǎn)物分子量約81kDa,由55KDa的CT703蛋白和26KDa的GST蛋白組成,與預(yù)期理論的分子量大小一致。以抗GST蛋白抗體為一抗,Western Blot技術(shù)檢測純化的重組融合蛋白,在81KDa處檢測到相應(yīng)的蛋白條帶。Werstern Blot實驗證實以重組融合蛋白免疫小鼠制備的抗血清能與CT703蛋白特異性結(jié)合；間接ELISA法檢測抗血清效價,結(jié)果顯示抗體滴度最高達到1：32000。 3.利用Western Blot技術(shù)檢測CT703蛋白在沙眼衣原體急性感染狀態(tài)下的表達,感染后24小時可檢測到相應(yīng)的蛋白,隨著感染時間的延長,蛋白表達量逐漸增多,并持續(xù)存在于整個感染過程,未感染細胞組沒有檢測到CT703蛋白的表達；通過免疫熒光技術(shù)檢測CT703蛋白表達,最早在感染12小時即可檢測到相應(yīng)的蛋白。在干擾素-γ誘導(dǎo)沙眼衣原體持續(xù)性感染狀態(tài)下,RT-PCR和Western Blot技術(shù)檢測CT703 mRNA和蛋白的表達情況,結(jié)果表明持續(xù)性感染狀態(tài)下CT703 mRNA和蛋白表達不呈時間依賴性,相同時間點沙眼衣原體持續(xù)性感染狀態(tài)下CT703 mRNA和蛋白水平明顯低于急性感染狀態(tài)。 4.間接免疫熒光技術(shù)對內(nèi)源性的CT703蛋白進行定位,結(jié)果顯示沙眼衣原體感染細胞CT703蛋白的熒光染色部位既不同于胞漿蛋白CPAF,也不同于包涵體膜蛋白CT813。 5.構(gòu)建的真核表達重組質(zhì)粒pcDNA4/CT703經(jīng)PCR、雙酶切實驗證實插入片段大小與CT703基因片段大小一致,測序證實插入的基因序列與GenBank公布的CT703基因序列完全一致。重組質(zhì)粒pcDNA4/CT703轉(zhuǎn)染到HeLa細胞后,Western Blot和免疫熒光實驗均能檢測到CT703蛋白的表達。 6.真核表達重組質(zhì)粒pcDNA4/CT703轉(zhuǎn)染HeLa細胞后36小時檢測磷酸化的Raf、ERK,發(fā)現(xiàn)二者均未被磷酸化；轉(zhuǎn)染質(zhì)粒36小時后,STS誘導(dǎo)細胞凋亡5小時,用流式細胞術(shù)檢測細胞凋亡率以及用Caspase-3活性檢測試劑盒檢測Caspase-3活性。同時設(shè)STS誘導(dǎo)的HeLa細胞組、STS誘導(dǎo)的沙眼衣原體感染組和正常HeLa細胞組作為對照,發(fā)現(xiàn)4組細胞的凋亡率分別為：(93.1±2.01)%、(91.3±1.67)%、(3.21±0.87)%、(2.08±0.76)%。統(tǒng)計學(xué)分析,STS誘導(dǎo)的重組質(zhì)粒轉(zhuǎn)染組與正常HeLa細胞組比較有顯著性差異,與STS誘導(dǎo)的衣原體感染組比較有顯著性差異(P0.05),與STS誘導(dǎo)的HeLa細胞組比較沒有顯著性差異(P0.05)。Caspase-3活性檢測結(jié)果與細胞凋亡率一致。 四、結(jié)論 1.成功構(gòu)建原核表達重組質(zhì)粒pGEX-6p-1/CT703以及真核表達重組質(zhì)粒pcDNA4/CT703。 2.沙眼衣原體急性感染狀態(tài)下CT703蛋白表達呈時間依賴性增加,持續(xù)性感染狀態(tài)下CT703蛋白表達不呈時間依賴性；相同時間點沙眼衣原體持續(xù)性感染狀態(tài)下CT703蛋白表達明顯低于急性感染狀態(tài)。 3.CT703蛋白不能激活Raf/MEK/ERK信號通路,不能抑制STS誘導(dǎo)的細胞凋亡。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R374

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