RESUMO
Measles virus is an enveloped virus with a non-segmented negative-sense RNA genome. Two envelope glycoproteins on the viral surface, namely hemagglutinin (H) and membrane fusion protein (F), are responsible for the virus entry into susceptible host cells. The specific interaction between H and its cellular receptors is a key step in successful virus infection, determining the infectivity and tissue tropism of the measles virus. Thus far, three H receptors have been identified, including the complement regulatory molecule CD46, the signaling lymphocyte activation molecule (SLAM) and the cell adhesion molecule Nectin-4. Here, we reviewed our molecular understanding on the recognition mechanism of these receptors by the viral H protein, aiming to promote future studies on antiviral drug design and measles virus-based oncolytic therapy.
Assuntos
Animais , Humanos , Antígenos CD , Metabolismo , Moléculas de Adesão Celular , Metabolismo , Hemaglutininas Virais , Metabolismo , Vírus do Sarampo , Virulência , Fisiologia , Proteína Cofatora de Membrana , Metabolismo , Fusão de Membrana , Proteínas de Fusão de Membrana , Metabolismo , Receptores de Superfície Celular , Metabolismo , Receptores Virais , Metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
<p><b>OBJECTIVE</b>To obtain streptavidin-tagged human granulocyte-macrophage colony-stimulating factor (SA/hGM-CSF) fusion protein and evaluate its bioactivity .</p><p><b>METHODS</b>PET24a-6His-SA-L-hGM-CSF and PET24a-hGM-CSF-L-SA-6His plasmids were constructed and expressed in Rosetta (DE3) host bacteria to generate the fusion proteins. The two fusion proteins were refolded by gradient dialysis after Ni-NTA affinity chromatography and finally purified using DEAE-sepharose FF anion exchange chromatography. MTT method was used to evaluate the effect of SA/hGM-CSF fusion proteins in inducing the proliferation of human erythroleukemia cells (TF-1). The efficiency of the fusion proteins for surface modification of biotinylated MB49 tumor cells was evaluated by flow cytometry.</p><p><b>RESULTS</b>The recombinant fusion proteins SA-hGM-CSF and hGM-CSF-SA were highly expressed in Rosetta (DE3) at about 20% of the total bacterial proteins, with a purity of about 96% after purification. The two fusion proteins exhibited bifunctional activities, namely the pro-proliferation effect on human erythroleukemia cells (TF-1) and SA-mediated high-affinity binding to biotinylated cell surfaces (with an anchoring modified rate of about 99%).</p><p><b>CONCLUSION</b>SA/hGM-CSF bi-fusion proteins obtained in this study lays the groundwork for the development of cancer cell vaccines with surface modification by hGM-CSF.</p>
Assuntos
Humanos , Biomarcadores , Vacinas Anticâncer , Linhagem Celular Tumoral , Diterpenos , Farmacologia , Escherichia coli , Metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Proteínas de Fusão de Membrana , Plasmídeos , Estreptavidina , QuímicaRESUMO
<p><b>OBJECTIVE</b>To construct the expression plasmid of S2 extracellular domain (S2ED) of SARS-coronavirus (SARS- Cov) spike protein (S protein) and enhanced green fluorescent protein (EGFP) to obtain the fusion protein expressed in prokaryotic cells.</p><p><b>METHODS</b>S2ED based on bioinformatics prediction and EGFP sequence were amplified by PCR and inserted into pET-14b plasmid. The recombinant protein His-S2ED-EGFP was expressed in E. coli by IPTG induction. After purification by Ni-NTA agarose beads, the soluble fractions of the fusion protein were collected and identified by SDS-PAGE and Western blotting. The fusion of S2ED with Hela cell membranes was observed with fluorescent microscope.</p><p><b>RESULTS</b>The pET-14b-S2ED-EGFP plasmid was correctly constructed and highly expressed in BL21 (DE3). When incubated with Hela cells, the purified protein could not internalize through membrane fusion.</p><p><b>CONCLUSIONS</b>The expression plasmid containing S2ED of SARS-Cov S protein and EGFP sequence is constructed successfully. Although the recombinant protein obtained has not shown the expected fusion effect with Hela cell membrane, this work may enrich the understanding of the process of membrane fusion mediated by S2 protein and lay the foundation for future study of targeting cell transport system based on cell-specific binding peptide.</p>
Assuntos
Humanos , Escherichia coli , Genética , Metabolismo , Proteínas de Fluorescência Verde , Genética , Metabolismo , Células HeLa , Fusão de Membrana , Proteínas de Fusão de Membrana , Glicoproteínas de Membrana , Genética , Proteínas Recombinantes de Fusão , Genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Genética , Glicoproteína da Espícula de Coronavírus , Proteínas do Envelope Viral , GenéticaRESUMO
The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or deltaLNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and deltaLNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of deltaLNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated deltaLNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.