环形泰勒虫TaSDP基因的表达与功能的初步研究
发布时间：2018-10-18 17:01
【摘要】：环形泰勒虫是一种蜱传血液原虫,能引起牛的热带泰勒虫病。环形泰勒虫和小泰勒虫裂殖体感染的牛淋巴细胞在体外表现具有无限增值(永生化)的能力,即其具有转化宿主淋巴细胞的特性,因此被称为可转化淋巴细胞泰勒虫。泰勒属的寄生虫是截止目前已发现的可使哺乳动物细胞发生永生化的唯一一类真核生物,为寄生虫与宿主相互作用研究提供了理想模型。截至目前,人们已经揭示了可转化淋巴细胞泰勒虫参与调控宿主细胞的部分机制,涉及到的分子有Tash AT(Theileria annulata macroschizont-specific AT hook-containing protein)、SVSP(subtelomere-encodedvariable secreted protein)等,并证明与NF-κB、ISG15、TGF-β、PI-3K、E2F等分子及凋亡调控路径有关。目前,人们已经证明了小泰勒虫Tp SCOP(T.parva schizont-derived cytoskeleton-bindingprotein)蛋白能够激活宿主细胞的NF-κB通路,进而实现对宿主细胞的调控。由于环形泰勒虫Ta SDP(T.annulata schizont-derived protein)蛋白与已知的的小泰勒虫Tp SCOP蛋白为同源蛋白。本研究对Ta SDP基因进行了原核表达,利用了原核表达获得的重组蛋白制备了兔源多克隆抗体并对该蛋白在环形泰勒虫感染细胞上进行了定位,同时运用双荧光素酶报告基因系统就该蛋白对HEK293T细胞NF-κB信号通路的影响做了初步的探索,确定了电穿孔转染环形泰勒虫感染细胞的最佳电转缓冲液和电压,为研究环形泰勒虫Ta SDP蛋白在转化的宿主细胞中的作用奠定基础。主要研究结果如下:1.Ta SDP基因的表达及Ta SDP蛋白多克隆抗体的制备。利用PCR技术从环形泰勒虫c DNA中扩增Ta SDP基因,构建原核表达载体进行蛋白的表达,r Ta SDP蛋白以包涵体的形式存在;将纯化后的融合蛋白免疫家兔,制备多克隆抗体。制备的抗体不仅可与重组表达的His-Ta SDP发生反应,而且可以识别天然的虫体蛋白,为开展其在细胞调控方面的研究准备了材料。2.Ta SDP蛋白在环形泰勒虫感染细胞中的定位。为探寻Ta SDP蛋白在环形泰勒虫感染细胞内的位置,以纯化的抗r Ta SDP蛋白免疫血清为一抗,运用间接免疫荧光实验,并借助于激光共聚焦显微镜对该蛋白进行细胞定位。结果表明,Ta SDP蛋白定位于环形泰勒虫裂殖体上。该蛋白可以作为一个标记分子用于后续裂殖体相关的研究。3.Ta SDP蛋白对HEK293T细胞内NF-κB转录活性的影响。为确定环形泰勒虫Ta SDP蛋白对HEK293T细胞内NF-κB转录活性的影响,我们运用了双荧光素酶报告基因系统。结果表明:Ta SDP蛋白对HEK293T细胞内NF-κB通路具有显著激活作用,活性升高大约1.7倍。实验中对环形泰勒虫感染细胞的C5细胞系的电转染条件进行了筛选,确定了最佳的电转缓冲液及相应的最适电压。为将双荧光素酶报告基因系统用于环形泰勒虫感染细胞的研究奠定了基础。
[Abstract]:Taylor ring is a tick-borne blood protozoa that causes tropical Taylor's disease in cattle. The bovine lymphocytes infected by Taylor's ring and small Taylor's lobes have the ability of infinite increment (immortalization) in vitro, that is, they have the characteristics of transforming host lymphocytes, so they are called transforming lymphocyte Taylor's. The parasites of the genus Taylor are the only eukaryotes found to immortalize mammalian cells, which provide an ideal model for the study of the interaction between parasites and hosts. Up to now, it has been revealed that Taylor's can participate in the regulation of host cells, involving Tash AT (Theileria annulata macroschizont-specific AT hook-containing protein), SVSP (subtelomere-encodedvariable secreted protein) and other molecules, and NF- 魏 B, ISG15 TGF- 尾, PI-3K,E2F and other molecules and apoptosis regulation pathway related to the role of TGF- 尾, PI-3K,E2F and so on. At present, it has been proved that the Tp SCOP (T.parva schizont-derived cytoskeleton-bindingprotein) protein can activate the NF- 魏 B pathway of host cells and thus regulate the host cells. Because the Ta SDP (T.annulata schizont-derived protein) protein is homologous to the known Tp SCOP protein. In this study, Ta SDP gene was expressed in prokaryotic cells and rabbit polyclonal antibody was prepared by using the recombinant protein expressed in prokaryotic expression. The effects of the protein on the NF- 魏 B signaling pathway in HEK293T cells were studied by using double luciferase reporter gene system. The optimal electroporation buffer and voltage were determined. The results provide a basis for the study of the role of Ta SDP protein in transformed host cells. The main results are as follows: expression of 1.Ta SDP gene and preparation of polyclonal antibody against Ta SDP protein. The Ta SDP gene was amplified from c DNA of Taylor's ring by PCR technique, and the prokaryotic expression vector was constructed to express, r Ta SDP protein in the form of inclusion body. The purified fusion protein was immunized with rabbit to prepare polyclonal antibody. The prepared antibody can not only react with recombinant His-Ta SDP, but also recognize natural insect proteins, which provides a material for the study of cell regulation. The localization of 2.Ta SDP protein in the cells infected with Taylor's ring. In order to find out the position of Ta SDP protein in the cells infected with Taylor's ring, the purified anti-r Ta SDP protein immunized serum was used as the first antibody. The cell localization of the protein was carried out by means of indirect immunofluorescence assay and laser confocal microscopy. The results showed that the, Ta SDP protein was located on the merozoite of Taylor's ring. This protein can be used as a marker molecule for further studies on splitosome. The effect of 3.Ta SDP protein on the transcriptional activity of NF- 魏 B in HEK293T cells. In order to determine the effect of Ta SDP on the transcriptional activity of NF- 魏 B in HEK293T cells, a double luciferase reporter gene system was used. The results showed that: Ta SDP protein significantly activated the NF- 魏 B pathway in HEK293T cells, and the activity increased about 1.7 times. In the experiment, the electrotransfection conditions of C5 cell line infected by Taylor's ring were screened, and the optimal electrotransposable buffer and the corresponding optimum voltage were determined. The results laid a foundation for the application of double luciferase reporter gene system in the study of Taylor ring infection cells.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
本文编号：2279777
[Abstract]:Taylor ring is a tick-borne blood protozoa that causes tropical Taylor's disease in cattle. The bovine lymphocytes infected by Taylor's ring and small Taylor's lobes have the ability of infinite increment (immortalization) in vitro, that is, they have the characteristics of transforming host lymphocytes, so they are called transforming lymphocyte Taylor's. The parasites of the genus Taylor are the only eukaryotes found to immortalize mammalian cells, which provide an ideal model for the study of the interaction between parasites and hosts. Up to now, it has been revealed that Taylor's can participate in the regulation of host cells, involving Tash AT (Theileria annulata macroschizont-specific AT hook-containing protein), SVSP (subtelomere-encodedvariable secreted protein) and other molecules, and NF- 魏 B, ISG15 TGF- 尾, PI-3K,E2F and other molecules and apoptosis regulation pathway related to the role of TGF- 尾, PI-3K,E2F and so on. At present, it has been proved that the Tp SCOP (T.parva schizont-derived cytoskeleton-bindingprotein) protein can activate the NF- 魏 B pathway of host cells and thus regulate the host cells. Because the Ta SDP (T.annulata schizont-derived protein) protein is homologous to the known Tp SCOP protein. In this study, Ta SDP gene was expressed in prokaryotic cells and rabbit polyclonal antibody was prepared by using the recombinant protein expressed in prokaryotic expression. The effects of the protein on the NF- 魏 B signaling pathway in HEK293T cells were studied by using double luciferase reporter gene system. The optimal electroporation buffer and voltage were determined. The results provide a basis for the study of the role of Ta SDP protein in transformed host cells. The main results are as follows: expression of 1.Ta SDP gene and preparation of polyclonal antibody against Ta SDP protein. The Ta SDP gene was amplified from c DNA of Taylor's ring by PCR technique, and the prokaryotic expression vector was constructed to express, r Ta SDP protein in the form of inclusion body. The purified fusion protein was immunized with rabbit to prepare polyclonal antibody. The prepared antibody can not only react with recombinant His-Ta SDP, but also recognize natural insect proteins, which provides a material for the study of cell regulation. The localization of 2.Ta SDP protein in the cells infected with Taylor's ring. In order to find out the position of Ta SDP protein in the cells infected with Taylor's ring, the purified anti-r Ta SDP protein immunized serum was used as the first antibody. The cell localization of the protein was carried out by means of indirect immunofluorescence assay and laser confocal microscopy. The results showed that the, Ta SDP protein was located on the merozoite of Taylor's ring. This protein can be used as a marker molecule for further studies on splitosome. The effect of 3.Ta SDP protein on the transcriptional activity of NF- 魏 B in HEK293T cells. In order to determine the effect of Ta SDP on the transcriptional activity of NF- 魏 B in HEK293T cells, a double luciferase reporter gene system was used. The results showed that: Ta SDP protein significantly activated the NF- 魏 B pathway in HEK293T cells, and the activity increased about 1.7 times. In the experiment, the electrotransfection conditions of C5 cell line infected by Taylor's ring were screened, and the optimal electrotransposable buffer and the corresponding optimum voltage were determined. The results laid a foundation for the application of double luciferase reporter gene system in the study of Taylor ring infection cells.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7
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