Expression And Function Of SARS-CoV N And Agkistin-s

Section one: expression and function of SARS-CoV N protein. A novel member of coronavirus, the severe acute respiratory syndrome coronavirus (SARS-CoV), was identified as the causative agent of SARS. SARS-CoV is an enveloped virus containing a single stranded, positive-sense RNA virus. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, we expressed the recombinant SARS-CoV N protein using prokaryotic and eukaryotic expression system. Recombinant N protein was purified to near homogeneity by Ni~2+-NTA affinity chromatography. We used the purified recombinant protein expressed from prokaryotic cells to immunize the New Zealand whit rabbit and achieve the high titer and superiority antisera.The SARS-CoV N protein contains a putative nuclear localization signal (KKDKKKK, 370-376aa). Previous studies confirmed that nucleolar localization of the N protein is a common feature of the coronaviurs family. We examined the subcellular localization of the N protein by confocal microscopy in Trichoplusia ni BT1 Tn5Bl-4 and Hela cells. The proteins were shown to localize either to the cytoplasm alone or the nucleolus. This feature is possibly of functional significance. It has been reported that apoptosis plays an important role in the pathophysiology of SARS. Apoptosis is a general phenomenon in SARS and apoptotic cells increased significantly in the spleen, lung and lymph nodes of SARS patients as compared with normal tissues. The apoptotic cells included pneumocytes, lymphocytes and monocytes. In the case of virus -infected cells, there is increasing evidence that many viruses encode proteins, which interact intimately with the biochemical pathways regulating apoptosis. In our studies, we show that the expression of the SARS-CoV N gene is sufficient to induce apoptosis in Hela cells. Further studies showed that N protein induced apoptosis by capsase pathway.Section two: expression and function of agkistin-s. Disintegrins are a family of small proteins mainly derived from snake venom. Most of the disintegrin containthe RGD or the Lys-Gly-Asp sequence, which is the structural motif act as potent antagonists of several integrins. There are several reports demonstrating that disintegrins containing the RGD sequence inhibit tumor metastasis by blocking tumor cell adhesion to ECMs and by inhibit angiogenesis. Disintegrin inhibit tumor angiogenesis by blocking endothelial cells adhesion to the ECM to induce endothelial cell apoptosis, suppress integrin-signaling pathways.Previous work in our laboratory has shown agkistin, a snake venom, metalloproteases (SVMPs) from the venom of Agkistrodon halys, possesses anti-platelet aggregation activity. In this study we further examined the anti-angiogenic activity of agkistin-s, the disintegrin domain of agkistin. Recombinant agkistin-s was produced in Escherichia coli by subcloning its cDNA into pET28a vector, and the effect of purified agkisin-s was evaluated. At concentration of 0.5-1.5 uM, the recombinant agkistin-s exhibited inhibitory activities on the bovine aortic endothelial cells (BAECs) migration and proliferation in a dose-dependent manner. In addition, it exhibited an effective anti-angiogenic effect when assayed by using the 10-day-old embryo chick CAM model. Furthermore, it potently induced BAECs apoptosis as examined by electrophoretic and flow cytometric assays. General caspase inhibitors B-D-fmk inhibited the cleavage of DNA fragmentation.