【摘要】：狂犬病是由嗜神經(jīng)性的狂犬病病毒（RABV）感染引起的一種重要的人獸共患傳染病，一旦發(fā)病，幾乎全部死亡。RABV屬于彈狀病毒科，狂犬病病毒屬，是一種不分節(jié)段的單股負鏈RNA病毒，其基因組編碼5種結構蛋白，分別為N、P、M、G、L蛋白。已有研究表明，RABV P蛋白是一種聚合酶輔助因子，參與了該病毒基因組的轉錄和復制過程。當RABV感染宿主后，，P蛋白通過和宿主蛋白STAT1、STAT2和STAT3等相互作用，抑制宿主細胞內IFN信號轉導，使得RABV能夠逃避宿主免疫，以利于病毒在宿主體內的傳播及擴散。P蛋白還能夠和LC8作用，可能介導該病毒在神經(jīng)元內的逆軸漿傳輸，成為研究RABV致病機制的重要靶點蛋白。但到目前為止，RABV致病機制，特別是與P蛋白存在相互作用的宿主蛋白尚不明確，而對后者的深入揭示，可望為RABV致病機制的研究補充更多實驗數(shù)據(jù)。 CVS作為RABV國際標準攻擊毒株，對于其研究更具泛示性。酵母雙雜交系統(tǒng)（Yeast two-hybrid system）是研究蛋白質相互作用、篩選新蛋白質的重要工具。為了尋找能夠與CVS-P具有相互作用的新宿主蛋白，我們首先通過PCR擴增得到CVS-P的全長基因并構建了酵母雙雜交誘餌質粒pGBKT7-CVS-P，然后將誘餌質粒轉化Y2HGold酵母細胞，證實了該蛋白對酵母細胞無毒性且無自激活作用；將含有誘餌質粒的Y2HGold酵母菌和含有人腦cDNA文庫的Y187酵母菌進行培養(yǎng)，通過營養(yǎng)缺陷型培養(yǎng)基反復多次篩選陽性克隆，獲得24個陽性克隆；挑取單個酵母藍色陽性克隆提取質粒，轉化大腸桿菌并提取質粒，經(jīng)PCR鑒定最終得到12個陽性克隆。將這些陽性克隆質粒測序，并與NCBI數(shù)據(jù)庫進行序列比對和分析，結果顯示，其中8個克隆為SNAP-associated protein (Snapin)，1個克隆為zinc finger protein350(ZNF350)，3個克隆為COP9signal some subunit5(COPS5)。經(jīng)酵母回轉實驗驗證，上述3種蛋白與P蛋白均存在相互作用，與Snapin蛋白的信號最強。 Snapin是一種與突觸相關的多功能蛋白，與鸚鵡熱、風濕性關節(jié)炎、帕金森、前列腺癌等及多種病毒性疾病疾病相關。Snapin是SNARE復合物的重要組分，主要分布在胞漿，參與突觸前穩(wěn)態(tài)的維持、BACE1等逆神經(jīng)元軸漿運輸、調節(jié)人類巨細胞病毒解旋酶細胞內分布、誘導鈣離子的釋放進而影響HIV-1在T細胞中的復制。盡管Snapin具有上述多重功能，近幾年對其研究也逐漸增多，但尚未見與RABV相關的研究，所以我們將Snapin蛋白作為進一步研究的重點。. 首先通過GST pull-down技術和免疫共沉淀（Co-immunoprecipitation,Co-IP）技術證實了CVS-P和Snapin蛋白在體內和體外均存在相互作用；通過免疫熒光實驗證實了二者在細胞內共定位于胞漿；應用Real-time PCR和Western blot檢測，RABV感染鼠腦Snapin mRNA和蛋白水平與對照相比都呈上調趨勢，當Snapin蛋白過表達時，P蛋白mRNA和蛋白水平均上調。以上實驗首次表明，RABVP蛋白與宿主蛋白Snapin存在相互作用，Snapin蛋白可導致P蛋白表達上調，促進RABV復制復合物的形成，利于該病毒的轉錄及復制，而Snapin又是一種介導突觸傳遞的重要蛋白，可能導致大量復制的病毒顆粒的跨突觸擴散，使其致病力增強。當然以上推斷還需通過后續(xù)試驗進行驗證，該推斷一旦被證實，將為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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

【參考文獻】

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1 楊齊衡,李林;酵母雙雜交技術及其在蛋白質組研究中的應用[J];生物化學與生物物理學報;1999年03期

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