Characterization Of The Interaction Between Newcastle Disease Virus M Protein And Chicken Importin ?1 Protein

Newcastle disease virus(NDV)is an enveloped virus with a non-segmented,single-stranded,negative-sense RNA genome that encodes at least six viral proteins.Of all these structural proteins,the M protein is the most abundant protein in virions and forms an outer protein shell around the nucleocapsid,constituting the bridge between the viral envelope and the nucleocapsid.Like the M proteins of other paramyxoviruses,the NDV M protein is demonstrated to be a multifunctional and nucleocytoplasmic shuttling protein.In our previous studies,the fluorescent co-localization indicated that co-expression of M and the importin ?1deletant disrupted the nuclear localization of M,and the results of Co-IP revealed that M protein could interact with importin ?1,which demonstrated that importin ?1 might participate the nuclear localization of NDV M protein.However,Co-IP assay did not reflect the direct interaction between M protein and importin ?1 protein.Therefore,additional experiments were needed to verify their interaction.At the present,pull-down assay is widely used to examine the in vivo protein-protein interaction.In this study,the recombinant plasmids expressing the NDV M protein and chicken importin ?1 protein were constructed and GST pull-down assay was used to verify the interaction between M protein and importin?1 protein.1 Cloning and bioinformatics analysis of chicken importin ?1 geneSpecific primers were designed and used to amplify the CDS region of chicken importin?1 gene from DF-1 cells.The physicochemical properties,secondary structure and phylogenetic tree of chicken importin ?1 were predicted by the bioinformatic tools.[Results]The full-length CDS region of chicken importin ?1 gene was 2268 bp and encoded 755 amino acids.The molecular formula of chicken importin ?1 protein was C3712H5901N979O1152S46 with a relative molecular mass about 84.15 k Da and p I 4.61.Protein secondary structure prediction showed that chicken importin ?1 protein containedabundant secondary structure and were mainly alpha helix(64.90%).Subcellular localization predictions revealed that the protein was localized in the cytoplasm.The results of gene homology and phylogenetic tree revealed that chicken importin ?1 gene had a close relationship with quail.2 Construction of prokaryotic expression vector of chicken importin ?1 gene and its expression in Escherichia coliIn this study,the recombinant plasmid p CR2.1-importin ?1 was used as a template to carry out PCR amplification using specific primers for the chicken importin ?1 gene.The target gene was recovered by gel and subcloned into the prokaryotic expression vector p ET-32a(+).Construction of a recombinant prokaryotic expression vector p ET-32a-importin?1.The correct recombinant prokaryotic expression vector p ET-32a-importin ?1 was transformed into E.coli BL21(DE3),and the IPTG concentration,induction temperature and induction time were optimized to determine the optimal expression conditions of the recombinant protein.The results showed that the recombinant prokaryotic expression vector p ET-32a-importin ?1 was successfully constructed and induced by IPTG and its conditions were optimized.When the final concentration of IPTG was 0.1 m M,the temperature was37°C,and the induction time was 4 h,the expression of the target protein was the highest.The molecular weight of the protein is approximately 97 k Da,which is consistent with the expected size.The His-importin ?1 recombinant protein existed as soluble and inclusion bodies.3 Construction of prokaryotic expression vector of NDV M Gene and its expression in Escherichia coliIn this study,the recombinant plasmid p CMV-HA-M stored in our laboratory was used as a template to perform PCR amplification using NDV M gene-specific primers.After the gel was recovered,the target gene was double-digested and then digested with the same enzymes.The prokaryotic expression vector p GEX-6p-1 was ligated and transformed into E.coli.Plasmids were extracted and identified by enzyme digestion.The recombinant prokaryotic expression vector p GEX-6p-M was constructed.The correct recombinant prokaryoticexpression vector was transformed into E.coli BL21(DE3)strain,and the IPTG concentration,induction temperature and induction time were optimized to determine the optimal expression conditions.The results showed that the recombinant prokaryotic expression vector p GEX-6p-M was successfully constructed and induced by IPTG.The results showed that when the final concentration of IPTG was 0.1 m M,the temperature was37°C,and the induction time was 5 h,the expression of the target protein was the highest in Escherichia coli.The target protein has a molecular weight of approximately 68 k Da,which is consistent with the expected size.The results of the expression pattern of the target protein revealed that the GST-M recombinant protein was mainly expressed as an inclusion body.4 Characterization of the interaction between NDV M protein and chicken importin ?1protein by pull-down assayIn this study,the inclusion body weight histones of GST-M were dealt with through denaturation and renaturation to recover activity.The interaction between GST-M(used as bait protein)and His-importin ?1(used as prey protein)was verified by GST pull-down assay.The results showed that the activated GST-M recombinant protein was recovered by protein refolding kit.When GST-M was used as bait protein,it could capture the His-importin ?1protein,while GST alone could not.These results demonstrated that the in vivo interaction between NDV M protein and chicken importin ?1 protein was verified by GST pull-down assay,which will provide foundation for further studying the role of importin ?1 in the nuclear localization of NDV M protein and the replication and pathogenicity of NDV.