狂犬病病毒CVS-P蛋白相互作用的宿主蛋白的筛选及初步的功能研究
发布时间：2018-09-19 17:16
【摘要】：狂犬病是由嗜神经性的狂犬病病毒（RABV）感染引起的一种重要的人兽共患传染病，一旦发病，几乎全部死亡。RABV属于弹状病毒科，狂犬病病毒属，是一种不分节段的单股负链RNA病毒，其基因组编码5种结构蛋白，分别为N、P、M、G、L蛋白。已有研究表明，RABV P蛋白是一种聚合酶辅助因子，参与了该病毒基因组的转录和复制过程。当RABV感染宿主后，，P蛋白通过和宿主蛋白STAT1、STAT2和STAT3等相互作用，抑制宿主细胞内IFN信号转导，使得RABV能够逃避宿主免疫，以利于病毒在宿主体内的传播及扩散。P蛋白还能够和LC8作用，可能介导该病毒在神经元内的逆轴浆传输，成为研究RABV致病机制的重要靶点蛋白。但到目前为止，RABV致病机制，特别是与P蛋白存在相互作用的宿主蛋白尚不明确，而对后者的深入揭示，可望为RABV致病机制的研究补充更多实验数据。 CVS作为RABV国际标准攻击毒株，对于其研究更具泛示性。酵母双杂交系统（Yeast two-hybrid system）是研究蛋白质相互作用、筛选新蛋白质的重要工具。为了寻找能够与CVS-P具有相互作用的新宿主蛋白，我们首先通过PCR扩增得到CVS-P的全长基因并构建了酵母双杂交诱饵质粒pGBKT7-CVS-P，然后将诱饵质粒转化Y2HGold酵母细胞，证实了该蛋白对酵母细胞无毒性且无自激活作用；将含有诱饵质粒的Y2HGold酵母菌和含有人脑cDNA文库的Y187酵母菌进行培养，通过营养缺陷型培养基反复多次筛选阳性克隆，获得24个阳性克隆；挑取单个酵母蓝色阳性克隆提取质粒，转化大肠杆菌并提取质粒，经PCR鉴定最终得到12个阳性克隆。将这些阳性克隆质粒测序，并与NCBI数据库进行序列比对和分析，结果显示，其中8个克隆为SNAP-associated protein (Snapin)，1个克隆为zinc finger protein350(ZNF350)，3个克隆为COP9signal some subunit5(COPS5)。经酵母回转实验验证，上述3种蛋白与P蛋白均存在相互作用，与Snapin蛋白的信号最强。 Snapin是一种与突触相关的多功能蛋白，与鹦鹉热、风湿性关节炎、帕金森、前列腺癌等及多种病毒性疾病疾病相关。Snapin是SNARE复合物的重要组分，主要分布在胞浆，参与突触前稳态的维持、BACE1等逆神经元轴浆运输、调节人类巨细胞病毒解旋酶细胞内分布、诱导钙离子的释放进而影响HIV-1在T细胞中的复制。尽管Snapin具有上述多重功能，近几年对其研究也逐渐增多，但尚未见与RABV相关的研究，所以我们将Snapin蛋白作为进一步研究的重点。. 首先通过GST pull-down技术和免疫共沉淀（Co-immunoprecipitation,Co-IP）技术证实了CVS-P和Snapin蛋白在体内和体外均存在相互作用；通过免疫荧光实验证实了二者在细胞内共定位于胞浆；应用Real-time PCR和Western blot检测，RABV感染鼠脑Snapin mRNA和蛋白水平与对照相比都呈上调趋势，当Snapin蛋白过表达时，P蛋白mRNA和蛋白水平均上调。以上实验首次表明，RABVP蛋白与宿主蛋白Snapin存在相互作用，Snapin蛋白可导致P蛋白表达上调，促进RABV复制复合物的形成，利于该病毒的转录及复制，而Snapin又是一种介导突触传递的重要蛋白，可能导致大量复制的病毒颗粒的跨突触扩散，使其致病力增强。当然以上推断还需通过后续试验进行验证，该推断一旦被证实，将为狂犬病的防治提供新的方向。
[Abstract]:Rabies is an important zoonotic infectious disease caused by rabies virus (RABV). Once it comes on, it almost all dies. RABV belongs to the family Rhabdoviridae and Rabies Virus. It is a single negative stranded RNA virus without segmenting. Its genome encodes five structural proteins, namely N, P, M, G and L proteins. These results suggest that RABV P protein is a polymerase-assisted factor involved in the process of transcription and replication of the virus genome. And diffuse. P protein can also interact with LC8, which may mediate the retroaxial transmission of RABV in neurons and become an important target protein for studying the pathogenesis of RABV. The study adds more experimental data.
The yeast two-hybrid system is an important tool for studying protein interactions and screening new proteins. In order to find new host proteins that can interact with CVS-P, we first amplified the full-length gene of CVS-P by PCR and then amplified it. A yeast two-hybrid bait plasmid pGBKT7-CVS-P was constructed and transformed into Y2HGold yeast cells. It was confirmed that Y2HGold yeast containing bait plasmid and Y187 yeast containing human brain cDNA library were cultured repeatedly in a nutrient-deficient medium. Twenty-four positive clones were obtained by screening positive clones, and one yeast blue positive clone was selected to extract plasmid, transformed into E. coli and extracted plasmid. Twelve positive clones were identified by PCR. The positive clones were sequenced and compared with NCBI database. The results showed that eight of them were SNAP-associated. Protein (Snapin), one clone was zinc finger protein 350 (ZNF350), and three clones were COP9 signal some Subunit 5 (COPS5).
Snapin is a synaptic-related multifunctional protein associated with parrot fever, rheumatoid arthritis, Parkinson's disease, prostate cancer, and many other viral diseases. Snapin is an important component of SNARE complex, mainly distributed in the cytoplasm, involved in the maintenance of presynaptic homeostasis, BACE1 and other antineuronal axoplasmic transport, regulating the decomposition of human cytomegalovirus. Although Snapin has the multiple functions mentioned above, the research on it has been increasing gradually in recent years, but there is no research related to RABV, so we take Snapin protein as the focus of further research.
The interaction between CVS-P and Snapin was confirmed by GST pull-down technique and Co-immunoprecipitation (Co-IP) technique in vivo and in vitro; the co-localization of CVS-P and Snapin in the cytoplasm was confirmed by immunofluorescence assay; the Snapin mRNA and Snapin mRNA in the brain of RABV infected rats were detected by Real-time PCR and Western blot. When Snapin protein was overexpressed, the mRNA and protein levels of P protein were up-regulated. These results showed for the first time that RABVP protein interacted with Snapin, and Snapin protein could up-regulate the expression of P protein, promote the formation of RABV replication complex, and facilitate the transcription and replication of the virus. Snapin is also an important protein that mediates synaptic transmission, which may lead to the transsynaptic diffusion of a large number of replicated viral particles and enhance their pathogenicity.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
本文编号：2250794
[Abstract]:Rabies is an important zoonotic infectious disease caused by rabies virus (RABV). Once it comes on, it almost all dies. RABV belongs to the family Rhabdoviridae and Rabies Virus. It is a single negative stranded RNA virus without segmenting. Its genome encodes five structural proteins, namely N, P, M, G and L proteins. These results suggest that RABV P protein is a polymerase-assisted factor involved in the process of transcription and replication of the virus genome. And diffuse. P protein can also interact with LC8, which may mediate the retroaxial transmission of RABV in neurons and become an important target protein for studying the pathogenesis of RABV. The study adds more experimental data.
The yeast two-hybrid system is an important tool for studying protein interactions and screening new proteins. In order to find new host proteins that can interact with CVS-P, we first amplified the full-length gene of CVS-P by PCR and then amplified it. A yeast two-hybrid bait plasmid pGBKT7-CVS-P was constructed and transformed into Y2HGold yeast cells. It was confirmed that Y2HGold yeast containing bait plasmid and Y187 yeast containing human brain cDNA library were cultured repeatedly in a nutrient-deficient medium. Twenty-four positive clones were obtained by screening positive clones, and one yeast blue positive clone was selected to extract plasmid, transformed into E. coli and extracted plasmid. Twelve positive clones were identified by PCR. The positive clones were sequenced and compared with NCBI database. The results showed that eight of them were SNAP-associated. Protein (Snapin), one clone was zinc finger protein 350 (ZNF350), and three clones were COP9 signal some Subunit 5 (COPS5).
Snapin is a synaptic-related multifunctional protein associated with parrot fever, rheumatoid arthritis, Parkinson's disease, prostate cancer, and many other viral diseases. Snapin is an important component of SNARE complex, mainly distributed in the cytoplasm, involved in the maintenance of presynaptic homeostasis, BACE1 and other antineuronal axoplasmic transport, regulating the decomposition of human cytomegalovirus. Although Snapin has the multiple functions mentioned above, the research on it has been increasing gradually in recent years, but there is no research related to RABV, so we take Snapin protein as the focus of further research.
The interaction between CVS-P and Snapin was confirmed by GST pull-down technique and Co-immunoprecipitation (Co-IP) technique in vivo and in vitro; the co-localization of CVS-P and Snapin in the cytoplasm was confirmed by immunofluorescence assay; the Snapin mRNA and Snapin mRNA in the brain of RABV infected rats were detected by Real-time PCR and Western blot. When Snapin protein was overexpressed, the mRNA and protein levels of P protein were up-regulated. These results showed for the first time that RABVP protein interacted with Snapin, and Snapin protein could up-regulate the expression of P protein, promote the formation of RABV replication complex, and facilitate the transcription and replication of the virus. Snapin is also an important protein that mediates synaptic transmission, which may lead to the transsynaptic diffusion of a large number of replicated viral particles and enhance their pathogenicity.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65
【参考文献】
相关期刊论文 前1条
1 杨齐衡,李林;酵母双杂交技术及其在蛋白质组研究中的应用[J];生物化学与生物物理学报;1999年03期
本文编号：2250794
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