Subparagraph Sars Coronavirus Pumc02 Strains Of Genome-wide Cdna Clone And The N Protein Function

The SARS-associated coronavirus is a new coronavirus, which caused a novel acute epidemic disease called severe acute respiratory syndrome(SARS). It is a single-stranded, positive-sense RNA virus, and belongs to the coronaviridae family. The genome of the SARS-CoV is 29.7kb in length, and the genomic organization is typical of coronaviruses, possessing the characteristic gene order: 5'-replicase, spike(S), envelope(E), membrane(M) and nucleocapsid(N)-3', and the short untranslated regions at both termini. The replicase gene which comprises approximately two-thirds of the whole genome undergoes cotranslational proteolytic processing, and release several non-structural proteins, including 3C-like proteinase(3CL^PRO), RNA-dependent RNA polymerase (RdRp), adenosine triphosphatase/ helicase (Hel/NTPase) and mRNA cap-1 methyltransferase (MTase). There are four open reading frames downstream of replicase gene , which are predicted to encode the structural proteins S, E, M and N. All of these proteins are essential in the life cycle of the SARS-CoV.Understanding the genome and exploring the function of the essential proteins of the SARS-CoV may aid us in elucidating the pathogenesis of the virus, and are the premise to search for the effective drugs and completely conquer the disease. The thesis are divided into two part, 1) twelve cDNA fragments covering the whole genome of the PUMC02 strain of SARS-CoV were obtained , and 2) the function of the important structure protein N was studied.In the first part, according to the sequences of the SARS-CoV genomes published in NCBI as well as the analysis of the restriction ribonuclease map, twelve pairs of primers were designed and the whole genome was divided into twelve fragments of about 3kb. The twelve cDNA fragments were amplified by RT-PCR with the template of the total RNA extracted from the Vero E6 cells infected by the SARS-CoV PUMC02 strain , and cloned...