弓形蟲棒狀體蛋白的表達及相互作用蛋白的篩選
發(fā)布時間：2018-08-26 10:26
【摘要】：弓形蟲病是由剛地弓形蟲引起的一種人獸共患寄生蟲病,剛地弓形蟲隸屬于頂復(fù)門,可寄生在除紅細胞外幾乎所有有核細胞中。據(jù)估計,全世界約三分之一的人群感染弓形蟲。隨著城市的發(fā)展、寵物飼養(yǎng)隊伍不斷地擴大,弓形蟲感染的危險性有上升趨勢。棒狀體蛋白是弓形蟲分泌至宿主細胞的主要效應(yīng)因子,棒狀體蛋白定位于宿主細胞的不同部位,與宿主細胞之間存在著復(fù)雜的相互作用。該類蛋白可以通過調(diào)控膜系及骨架結(jié)構(gòu),影響宿主細胞的生物活性因子,從而阻斷其內(nèi)在的防御機制,使弓形蟲成功的完成入侵,寄生及增殖等一系列生理過程。弓形蟲棒狀體蛋白Rop28、Rop38在速殖子階段和緩殖子階段的表達存在很大差異。本研究通過反轉(zhuǎn)錄PCR成功擴增了弓形蟲Rop28、Rop38全長基因,將擴增的基因分別克隆至原核表達載體中,成功在大腸桿菌內(nèi)進行了大量表達,制備了特異性的Rop28、Rop38多克隆抗體。同時,為鑒定與Rop和Rop38相互作用的蛋白分子,構(gòu)建了Rop28和Rop38真核表達載體,在293T細胞中成功進行了表達。本研究構(gòu)建了人源成纖維細胞(HFF)和T細胞(CEM)cDNA文庫,文庫的滴度分別為,1.2×108cfu/ml和1.38×108cfu/ml。利用構(gòu)建的酵母誘餌質(zhì)粒篩選HFF文庫,獲得了與Rop2B、Rop5、Rop16、Rop17、Rop32和Rop38相互作用的宿主細胞蛋白。同時,用Rop38篩選了T細胞c DNA文庫。結(jié)果顯示Rop蛋白與宿主細胞存在著廣泛的相互作用。進一步雜交實驗證實了Rop2B和7種宿主蛋白存在相互作用;Rop5和7種蛋白存在相互作用;Rop16和14種蛋白存在相互作用;Rop17和7種蛋白存在相互作用;Rop32和12種宿主蛋白相互作用;Rop38和HFF文庫中11種宿主蛋白存在相互作用,與CEM文庫中20種蛋白存在相互作用。有文獻表明,過量表達Rop38能夠顯著抑制感染細胞的轉(zhuǎn)錄應(yīng)答。本研究利用Rop38在HFF細胞文庫和CEM文庫均篩選到PRMT7。PRMT7是一種精氨酸甲基轉(zhuǎn)移酶,是PRMTs家族的重要成員,目前已發(fā)現(xiàn)其參與調(diào)控基因表達、細胞遷移與分化個體發(fā)育等多個過程。Rop38對宿主細胞基因表達的調(diào)控很有可能是通過PRMT7通路實現(xiàn)的。本項目旨在通過篩選與弓形蟲棒狀體蛋白相互作用的宿主蛋白,更深入的進行弓形蟲病原性以及宿主相互作用的分子機制的研究,為開發(fā)抗弓形蟲病疫苗以及藥物提供理論基礎(chǔ)。
[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.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.7
本文編號：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.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.7
【參考文獻】
相關(guān)期刊論文 前1條
1 剡海闊;彭高輝;袁子國;朱興全;;抗弓形蟲核酸疫苗的研究進展[J];中國人獸共患病學報;2009年10期
，本文編號：2204576
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