本文選題：SARS冠狀病毒　切入點：N蛋白　出處：《武漢大學》2005年碩士論文　論文類型：學位論文

【摘要】：SARS冠狀病毒(SARS—CoV)是一種新型的冠狀病毒,其基因組大小約為30,000nt,為單股正鏈RNA。病毒基因中的1-72個核苷酸為前導序列。核衣殼(Nucleocapsid,N)蛋白是冠狀病毒的主要結(jié)構(gòu)蛋白,它在病毒基因轉(zhuǎn)錄、翻譯以及病毒顆粒包裝中起重要作用。以前對鼠肝炎病毒(Mouse hepatitis virus,MHV)的研究表明,N蛋白在發(fā)揮重要作用時,都離不開與病毒RNA的相互作用。N蛋白的中的絲氨酸和精氨酸富集區(qū)(SR-Rich region)可以和病毒RNA 5’端的前導序列(RNA leader sequence)發(fā)生特異性的結(jié)合。其中前導序列中的UCUAA保守序列對結(jié)合起關(guān)鍵作用。對比MHV和SARS-CoV的序列,在SARS-CoV中,N蛋白也存在一段SR-Rich區(qū)域,前導序列中也有UCUAA保守序列。因此,我們預測這兩個區(qū)域為病毒N蛋白和RNA結(jié)合的關(guān)鍵序列,他們的結(jié)合可能在病毒顆粒的組裝中發(fā)揮重要的作用。 本研究中,我們通過PCR的方法從SARS-CoV的cDNA中克隆出N基因,將N基因及其片斷的基因克隆到大腸桿菌表達載體中經(jīng)誘導表達獲得大量的重組蛋白,通過親和層析和凝膠過濾層析得到高純度的蛋白;同時構(gòu)建前導RNA的轉(zhuǎn)錄模板,經(jīng)體外轉(zhuǎn)錄得到地高辛(digoxin)標記的RNA。通過重組蛋白在體外與RNA進行結(jié)合分析,結(jié)果表明,SARS病毒N蛋白在體外可以與病毒前導RNA發(fā)生特異性的結(jié)合,并且N蛋白中的絲氨酸、精氨酸富集區(qū)為結(jié)合的主
[Abstract]:SARS coronavirus (SARS-CoV) is a new type of coronavirus. Its genome size is about 30 ~ 000nt.The nucleotide sequence of 1-72 nucleotides in the gene of SARS coronavirus is a leading sequence. The nucleocapsidn protein is the main structural protein of coronavirus, which is transcribed in viral gene. Translation and packaging of virus particles play an important role. Previous studies on mouse hepatitis virus (MHVV) have shown that the N protein plays an important role. The serine and arginine rich region in the interaction. N protein of the virus can bind to the leader sequence of the 5 'terminal of the virus. The conserved UCUAA sequence of the leading sequence is the conserved pair of the UCUAA sequence in the leading sequence of the virus RNA 5' terminal, and the serine and arginine rich region in the serine and arginine rich region can bind to the leader sequence of the 5 'terminal of the virus. The combination plays a key role in comparing the sequences of MHV and SARS-CoV, There is also a SR-Rich region in the N protein in SARS-CoV and a conserved UCUAA sequence in the leading sequence. Therefore, we predict that these two regions are the key sequences of viral N protein and RNA binding. Their binding may play an important role in the assembly of viral particles. In this study, the N gene was cloned from SARS-CoV cDNA by PCR, and the N gene and its fragments were cloned into E. coli expression vector to obtain a large number of recombinant proteins. High purity protein was obtained by affinity chromatography and gel filtration chromatography. At the same time, the transcription template of leading RNA was constructed, and digoxin labeled RNA was obtained by in vitro transcription. The recombinant protein was analyzed by binding to RNA in vitro. The results showed that the N protein of SARS-CoV could bind specifically to the virus precursor RNA in vitro, and serine and arginine rich region of the N protein were the main binding regions.
【學位授予單位】：武漢大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R373

【參考文獻】

相關(guān)期刊論文 前1條

1 祝慶余 ,秦鄂德,于曼,司炳銀,楊保安,劉洪,呂富雙,常國輝,彭文明,范寶昌,鄧永強,韓偉國,石玉玲,李林海,張泮河,趙秋敏,曹務春;新分離的冠狀病毒與嚴重急性呼吸綜合征病原關(guān)系的研究[J];解放軍醫(yī)學雜志;2003年06期

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