弓形虫棒状体蛋白的表达及相互作用蛋白的筛选
发布时间：2018-08-26 10:26
【摘要】：弓形虫病是由刚地弓形虫引起的一种人兽共患寄生虫病,刚地弓形虫隶属于顶复门,可寄生在除红细胞外几乎所有有核细胞中。据估计,全世界约三分之一的人群感染弓形虫。随着城市的发展、宠物饲养队伍不断地扩大,弓形虫感染的危险性有上升趋势。棒状体蛋白是弓形虫分泌至宿主细胞的主要效应因子,棒状体蛋白定位于宿主细胞的不同部位,与宿主细胞之间存在着复杂的相互作用。该类蛋白可以通过调控膜系及骨架结构,影响宿主细胞的生物活性因子,从而阻断其内在的防御机制,使弓形虫成功的完成入侵,寄生及增殖等一系列生理过程。弓形虫棒状体蛋白Rop28、Rop38在速殖子阶段和缓殖子阶段的表达存在很大差异。本研究通过反转录PCR成功扩增了弓形虫Rop28、Rop38全长基因,将扩增的基因分别克隆至原核表达载体中,成功在大肠杆菌内进行了大量表达,制备了特异性的Rop28、Rop38多克隆抗体。同时,为鉴定与Rop和Rop38相互作用的蛋白分子,构建了Rop28和Rop38真核表达载体,在293T细胞中成功进行了表达。本研究构建了人源成纤维细胞(HFF)和T细胞(CEM)cDNA文库,文库的滴度分别为,1.2×108cfu/ml和1.38×108cfu/ml。利用构建的酵母诱饵质粒筛选HFF文库,获得了与Rop2B、Rop5、Rop16、Rop17、Rop32和Rop38相互作用的宿主细胞蛋白。同时,用Rop38筛选了T细胞c DNA文库。结果显示Rop蛋白与宿主细胞存在着广泛的相互作用。进一步杂交实验证实了Rop2B和7种宿主蛋白存在相互作用;Rop5和7种蛋白存在相互作用;Rop16和14种蛋白存在相互作用;Rop17和7种蛋白存在相互作用;Rop32和12种宿主蛋白相互作用;Rop38和HFF文库中11种宿主蛋白存在相互作用,与CEM文库中20种蛋白存在相互作用。有文献表明,过量表达Rop38能够显著抑制感染细胞的转录应答。本研究利用Rop38在HFF细胞文库和CEM文库均筛选到PRMT7。PRMT7是一种精氨酸甲基转移酶,是PRMTs家族的重要成员,目前已发现其参与调控基因表达、细胞迁移与分化个体发育等多个过程。Rop38对宿主细胞基因表达的调控很有可能是通过PRMT7通路实现的。本项目旨在通过筛选与弓形虫棒状体蛋白相互作用的宿主蛋白,更深入的进行弓形虫病原性以及宿主相互作用的分子机制的研究,为开发抗弓形虫病疫苗以及药物提供理论基础。
[Abstract]:Toxoplasma gondii is a zoonotic parasitic disease caused by Toxoplasma gondii. Toxoplasma gondii belongs to the apical phylum and can be parasitized in almost all nucleated cells except red blood cells. It is estimated that about 1/3 people worldwide are infected with Toxoplasma gondii. With the development of the city, the pet keeping team is expanding and the risk of Toxoplasma gondii infection is on the rise. Coryloid protein is the main effector factor secreting from Toxoplasma gondii to host cells. The rodlike proteins are located in different parts of host cells and have complex interactions with host cells. This kind of protein can affect the biological activity factors of host cells by regulating the membrane system and skeleton structure, thus blocking its internal defense mechanism, and making Toxoplasma gondii successfully complete a series of physiological processes, such as invasion, parasitism and proliferation. The expression of Toxoplasma gondii Coryloid protein (Rop28,Rop38) was different from that of Toxoplasma gondii (Toxoplasma gondii) in tachyzoite stage and bradymeric stage. In this study, the full-length Rop28,Rop38 gene of Toxoplasma gondii was successfully amplified by reverse transcription PCR. The amplified gene was cloned into prokaryotic expression vector and successfully expressed in Escherichia coli, and the specific polyclonal antibody of Rop28,Rop38 was prepared. In order to identify the protein molecules interacting with Rop and Rop38, the eukaryotic expression vectors of Rop28 and Rop38 were constructed and successfully expressed in 293T cells. In this study, the human fibroblast (HFF) and T cell (CEM) cDNA libraries were constructed. The titers of the libraries were 1.2 脳 108cfu/ml and 1.38 脳 108cfur / ml, respectively. The recombinant yeast bait plasmid was used to screen the HFF library and the host cell proteins interacting with Rop2B,Rop5,Rop16,Rop17,Rop32 and Rop38 were obtained. At the same time, T cell c DNA library was screened by Rop38. The results showed that Rop protein interacted extensively with host cells. Further hybridization experiments confirmed the interaction between Rop2B and 7 host proteins, rop5 and 7 proteins, rop16 and 14 proteins, rop17 and 7 proteins, respectively, and the interaction between Rop32 and 12 host proteins. Interaction between Rop38 and 11 host proteins in HFF library, It interacts with 20 proteins in CEM library. It has been shown that overexpression of Rop38 can significantly inhibit the transcriptional response of infected cells. In this study, Rop38 was used to screen out that PRMT7.PRMT7 is an arginine methyltransferase and an important member of PRMTs family in HFF cell library and CEM library. It has been found that PRMT7.PRMT7 is involved in the regulation of gene expression. The regulation of gene expression in host cells by Rop38 during cell migration and differentiation and ontogenesis may be mediated by PRMT7 pathway. This project aims to further study the pathogenicity of Toxoplasma gondii and the molecular mechanism of host interaction by screening host proteins that interact with the rodlike proteins of Toxoplasma gondii. To provide a theoretical basis for the development of Toxoplasma gondii vaccine and drugs.
【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：2204576
[Abstract]:Toxoplasma gondii is a zoonotic parasitic disease caused by Toxoplasma gondii. Toxoplasma gondii belongs to the apical phylum and can be parasitized in almost all nucleated cells except red blood cells. It is estimated that about 1/3 people worldwide are infected with Toxoplasma gondii. With the development of the city, the pet keeping team is expanding and the risk of Toxoplasma gondii infection is on the rise. Coryloid protein is the main effector factor secreting from Toxoplasma gondii to host cells. The rodlike proteins are located in different parts of host cells and have complex interactions with host cells. This kind of protein can affect the biological activity factors of host cells by regulating the membrane system and skeleton structure, thus blocking its internal defense mechanism, and making Toxoplasma gondii successfully complete a series of physiological processes, such as invasion, parasitism and proliferation. The expression of Toxoplasma gondii Coryloid protein (Rop28,Rop38) was different from that of Toxoplasma gondii (Toxoplasma gondii) in tachyzoite stage and bradymeric stage. In this study, the full-length Rop28,Rop38 gene of Toxoplasma gondii was successfully amplified by reverse transcription PCR. The amplified gene was cloned into prokaryotic expression vector and successfully expressed in Escherichia coli, and the specific polyclonal antibody of Rop28,Rop38 was prepared. In order to identify the protein molecules interacting with Rop and Rop38, the eukaryotic expression vectors of Rop28 and Rop38 were constructed and successfully expressed in 293T cells. In this study, the human fibroblast (HFF) and T cell (CEM) cDNA libraries were constructed. The titers of the libraries were 1.2 脳 108cfu/ml and 1.38 脳 108cfur / ml, respectively. The recombinant yeast bait plasmid was used to screen the HFF library and the host cell proteins interacting with Rop2B,Rop5,Rop16,Rop17,Rop32 and Rop38 were obtained. At the same time, T cell c DNA library was screened by Rop38. The results showed that Rop protein interacted extensively with host cells. Further hybridization experiments confirmed the interaction between Rop2B and 7 host proteins, rop5 and 7 proteins, rop16 and 14 proteins, rop17 and 7 proteins, respectively, and the interaction between Rop32 and 12 host proteins. Interaction between Rop38 and 11 host proteins in HFF library, It interacts with 20 proteins in CEM library. It has been shown that overexpression of Rop38 can significantly inhibit the transcriptional response of infected cells. In this study, Rop38 was used to screen out that PRMT7.PRMT7 is an arginine methyltransferase and an important member of PRMTs family in HFF cell library and CEM library. It has been found that PRMT7.PRMT7 is involved in the regulation of gene expression. The regulation of gene expression in host cells by Rop38 during cell migration and differentiation and ontogenesis may be mediated by PRMT7 pathway. This project aims to further study the pathogenicity of Toxoplasma gondii and the molecular mechanism of host interaction by screening host proteins that interact with the rodlike proteins of Toxoplasma gondii. To provide a theoretical basis for the development of Toxoplasma gondii vaccine and drugs.
【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
【参考文献】
相关期刊论文 前1条
1 剡海阔;彭高辉;袁子国;朱兴全;;抗弓形虫核酸疫苗的研究进展[J];中国人兽共患病学报;2009年10期
，本文编号：2204576
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