SARS冠状病毒N蛋白与病毒前导RNA相互作用的初步研究

发布时间：2018-03-14 01:21

本文选题：SARS冠状病毒　切入点：N蛋白　出处：《武汉大学》2005年硕士论文　论文类型：学位论文

【摘要】：SARS冠状病毒(SARS—CoV)是一种新型的冠状病毒,其基因组大小约为30,000nt,为单股正链RNA。病毒基因中的1-72个核苷酸为前导序列。核衣壳(Nucleocapsid,N)蛋白是冠状病毒的主要结构蛋白,它在病毒基因转录、翻译以及病毒颗粒包装中起重要作用。以前对鼠肝炎病毒(Mouse hepatitis virus,MHV)的研究表明,N蛋白在发挥重要作用时,都离不开与病毒RNA的相互作用。N蛋白的中的丝氨酸和精氨酸富集区(SR-Rich region)可以和病毒RNA 5’端的前导序列(RNA leader sequence)发生特异性的结合。其中前导序列中的UCUAA保守序列对结合起关键作用。对比MHV和SARS-CoV的序列,在SARS-CoV中,N蛋白也存在一段SR-Rich区域,前导序列中也有UCUAA保守序列。因此,我们预测这两个区域为病毒N蛋白和RNA结合的关键序列,他们的结合可能在病毒颗粒的组装中发挥重要的作用。本研究中,我们通过PCR的方法从SARS-CoV的cDNA中克隆出N基因,将N基因及其片断的基因克隆到大肠杆菌表达载体中经诱导表达获得大量的重组蛋白,通过亲和层析和凝胶过滤层析得到高纯度的蛋白;同时构建前导RNA的转录模板,经体外转录得到地高辛(digoxin)标记的RNA。通过重组蛋白在体外与RNA进行结合分析,结果表明,SARS病毒N蛋白在体外可以与病毒前导RNA发生特异性的结合,并且N蛋白中的丝氨酸、精氨酸富集区为结合的主
[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.
【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

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