Cxcl16 Interact With SARS-CoV N Protein In and Out Cell / 中国病毒学·英文版

Our study investigated the host cell protein which can interact with SARS-CoV N protein, and explored the functional connections. The eukaryotic expression vectors pEGFP-N1/SARS-CoVN and pdsRed2-N1/ CXCL16 were constructed and used to co-transfect HEK293FT cells by the calcium phosphate method. The HIS-tagged fusion protein SARS-CoVN-GFP was then built and purified for the binding assay in vitro. The co-localization of SARS-CoVN and CXCL16 in the cytoplasm of HEK293FT cells was also shown using confocal laser scanning microscopy. It is suggested that their interaction might be through direct combination. Under a fluorescence microscope, it was observed that the purified fusion protein SARS-CoVN-GFP was attached to the cell membrane of CXCL16-transfected cells, indicating that SARS-CoVN and CXCL16 can be mutually combined.

Screen and Identification of The Protein-protein Interactors in The Host Cell With The SARS Coronavirus Nucleocapsid Protein / 生物化学与生物物理进展

15 SARS-CoV N Protein Interacting Protein (NPIP) were selected from host cells using Yeast Two-hybrid system (Y2H). These are Angiogenin, acyglycerol kinase, cytochrome oxydase subunit I, CXC chemokine ligand 16, epidermal growth factor receptor pathway substrate 15, glutathione S-transferase kappa 1,integrin beta 1, jun oncogene, NIMA (never in mitosis gene a)-related kinase 10, protein tyrosine kinase 2 beta,homo sapiens SH3KBP1 binding protein 1 and ubiquitin specific peptidase 53. With the aid of immunological co-precipitation (CO-IP), it was confirmed that chemokine CXCL16 was the interactor with SARS-CoV N protein in host cells.

Cloning and expression of MxA gene from chickens in E.coli and identification of biological activity of MxA / 中国免疫学杂志

Objective:To clone chickens MxA gene, construct its recombinant expression plasmid and induce the expression of fusion protein using a prokaryotic expression system.Methods:The MxA gene fragment was amplified by RT-PCR from CEF cells and subcloned into the pMD18-T vector, filtrated the positive clone and reclaimed the MxA.Subcloned the MxA into the prokaryotic expression plasmid pGEX-6p-1. After recombinant plasmid was induced by IPTG, the expressed proteins were analyzed by SDS-PAGE, the NDV intervence experiment and VSV-CEF restrain experiment.Results:The sequence of MxA gene amplified by RT-PCR was the same as the sequence in gene map of Genebank; SDS-PAGE, the NDV intervence experiment and VSV-CEF restrain experiment showed that a protein was expressed, the molecular weight of this protein was 45 000, which was the same as the fusion protein GST-MxA.Conclusion:The MxA is cloned and its recombinant expression plasmid is constructed successfully.The fusion protein GST-MxA is successfully expressed in the prokaryotic expression system E.coli DH5? induced by IPTG. This research lay a foundation for further studying on ints antiviral effects and exploring new way of antiviral medication.