SARS-CoV N蛋白与病毒mRNA相互作用的初步研究

Interaction between Nucleocapsid Protein and the Leader the Sequence of SARS-CoV RNA

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30,000 nt,为单股正链RNA。病毒基因中的1-72个核甘酸为前导序列。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。在本研究中,我们通过PCR的方法从SARS-CoV cDNA中克隆N基因,将基因克隆到大肠杆菌表达载体中,经表达纯化获得大量重组蛋白,通过亲和层析和凝胶过滤层析获得高纯度的N蛋白。同时构建前导RNA的转录模板,经体外转录得到地高辛标记的RNA。使用Northwestern分析技术,我们证实纯化的N蛋白在体外可以与RNA发生特异性的结合。N蛋白与病毒RNA的结合特性及其在病毒生活周期中的所起作用的初步研究,为下一步设计出有效的阻断病毒周期从而达到抗病毒目的的药物或疫苗奠定了基础。

SARS coronavirus （SARS-CoV） is a novel human coronavirus and was responsible for SARS outbreak in several regions of the world in 2003. The genome of SARS-CoV is about 30, 000 nucleotides in length and sequence analysis revealed that nucleotides 1-72 contain a RNA leader sequence preceding an untranslated region （UTR）. In order to confirm the interaction between the N protein and the RNA leader sequence, the N gene was cloned, sequenced and the protein was expressed in Escherichia coli as a fusion protein with His-tag. The recombination N protein was purified by Ni-NTA affinity column and G-75 column. Meanwhile, the DIG labeled RNA leader sequences were synthesized by in vitro transcription. RNA-Protein interaction was detected by Northwestern RNA blot assay. The results indicated that a specific interaction takes place between the N protein and RNA leader sequence. Our results provide some insights into the understanding of assembly of the SARS-CoV particles, which is an essential step for viral replication cycle.