摘要
Virus absorbing to receptors on sensitive cell surface is the first step in virus infection.Many studies have shown that human vascular endothelial cells(VECs) are the primary target cells for DV infection,but their receptors of DV have not been elucidated yet.In this study,we modified the virus overlay protein binding assay(VOPBA) to increase the screening rates,and identified proteins of 33,43,55 kDa from membrane protein extracts of ECV304 cells as the potential binding molecules for Dengue virus.Using mass spectrometry(MS),the peptide masses of a 43 kDa protein that matches to cell actin—a cytoskeleton protein in eukaryotic cells.For further identification of the 43 kDa protein and its interaction with DV-2 E protein domain III,we prepared the polyclonal antibody against the 43 kDa protein.Western blot analysis shown that the 43 kDa protein could be recognized by both immunized anti-43 kDa murine polyclonal sera and commercial mouse anti-actin polyclonal antibody.The binding specificity of DV-2 EDIII protein and the ECV304 cells 43 kDa actin protein was confirmed by competitive blocking assay and co-immunoprecipitation(co-IP) test.
Virus absorbing to receptors on sensitive cell surface is the first step in virus infection.Many studies have shown that human vascular endothelial cells(VECs) are the primary target cells for DV infection,but their receptors of DV have not been elucidated yet.In this study,we modified the virus overlay protein binding assay(VOPBA) to increase the screening rates,and identified proteins of 33,43,55 kDa from membrane protein extracts of ECV304 cells as the potential binding molecules for Dengue virus.Using mass spectrometry(MS),the peptide masses of a 43 kDa protein that matches to cell actin—a cytoskeleton protein in eukaryotic cells.For further identification of the 43 kDa protein and its interaction with DV-2 E protein domain III,we prepared the polyclonal antibody against the 43 kDa protein.Western blot analysis shown that the 43 kDa protein could be recognized by both immunized anti-43 kDa murine polyclonal sera and commercial mouse anti-actin polyclonal antibody.The binding specificity of DV-2 EDIII protein and the ECV304 cells 43 kDa actin protein was confirmed by competitive blocking assay and co-immunoprecipitation(co-IP) test.
引文