Interaction Verification And Protein Structure Prediction Of GPV,NGPV VP2 And Transferrin Receptor

Goose parvovirus is a member of the parvoviridae,parvovirus genus.Initially,the virus caused gosling plague in goslings and Muscovy ducks.In 1971,it caused mule ducks short beak and dwarfism syndrome(SBDS)in France.In 2015,NGPV caused duck beak atrophy and dwarfish syndrome(BADS).The host range of the virus is gradually expanding,and the phenomenon of cross-species transmission has appeared,which has posed great threats to the healthy and efficient breeding of waterfowl in China and the world.The binding of virus to cell surface receptors is the initiation link in the process of viral infection replication and is one of the decisive factors affecting the specificity of the virus host.Previous studies have shown that parvovirus capsid proteins are important for virus tropism,host range,and pathogenicity.Its VP2 can interact with receptors on the surface of the host cell,allowing the virus to enter the cell and proliferate,thereby causing effective infection.In view of this,this study takes parvovirus capsid protein VP2 as the research object,and proposes the hypothesis that transferrin receptor(TfR)is a surface receptor that the virus binds to host cells.TfR is one of the highly expressed components in the plasma membrane,it is also a powerful target for host cells infected by the virus.Through bioinformatics,molecular biology and other research methods,model the homology of virus and receptor proteins,verify the interaction between GPV-VP2,NGPV-VP2 protein and its natural host cell surface receptor TFR.The main contents of this study are as follows: 1.Alignment analysis of GPV,NGPV capsid protein VP2 and goose and duck TfR using sequence structure analysis software.2.The bait plasmid VP2-pGBKT7 was transformed into the Y2 H yeast strain,and the prey plasmid TfR-PGADT7 was transformed into the Y187 yeast strain.The two yeast strains were mixed and cultured to verify the interaction between goose TfR and GPV VP2 protein,duck TfR and NGPV VP2 protein.3.In order to prevent false positives,the GST-Pull down in vitro interaction test was performed using the VP2-GST fusion protein and the TfR-HA fusion protein.4.The location of the virus and TfR was observed with laser confocal microscope.5.Prepare a TfR polyclonal antibody with good specificity,and use it to block GEF cells and DEF cells.After blocking,respectively adsorb GPV and NGPV.Identifying viral infections using Real-time PCR.The results are as follows: Structure analysis shows that mutations of GPV VP2 and NGPV VP2 on the surface of the virus(S305N,S353 N,A379G,K430 R,H515N,D558N)are far from the area where the virus interacts with the host receptor,suggesting that the mutation is not enough to affect the binding of the virus to the receptor.The yeast hybrid system and GSTPull down test showed that goose TfR could interact with GPV VP2 protein;duck TfR could interact with NGPV VP2 protein.The results of laser confocal microscopy show that GPV and NGPV virus particles can co-localize with the surface TfR of GEF cells and DEF cells.The results of real-time quantitative PCR showed that anti-TfR antibodies reduced the efficiency of virus infection after blocking GEF cells and DEF cells.The above results prove that TfR on the surface of host cell membrane is receptor for GPV and NGPV,and it is an important protein in the process of virus adsorption to host cells and infection.This study is essential for understanding the molecular mechanism of GPV-host interactions,and developing intervention strategies for GPV infections.