Screening And Identification Of Interaction Protein Between F1L Protein Of OrfV And Host Cells

Orf is a contact and epithelial zoonotic infectious disease caused by OrfVirus（OrfV）.OrfV mainly infects sheep and goats,and the incidence of lambs is as high as90%～100%.The main characteristic lesions are proliferative damage in lips and nose,which is an important infectious disease endangering the sheep industry.OrfV is a member of the genus Parapoxvirus,family Poxviridae and is an enveloped linear double-stranded DNA virus.F1L protein is a surface envelope protein of virus.It has been found that F1L protein has heparin binding activity,which is related to virus adsorption and invasion of host cells and plays an important role in the early stage of virus infection.Lamb testicular（LT）cells are sensitive cells for OrfV proliferation and there must be interacting proteins on the surface or inside of the cell membrane.Therefore,the screening and identification of OrfV F1L proteins interacting with host cells are of great significance for the analysis of viral pathogenesis.In this study,the yeast two-hybrid c DNA library of lamb testicular cells and OrfV F1L protein bait strain were constructed.The host cell proteins interacting with OrfV F1L protein were screened by yeast two-hybrid technology,and further verified by reverse hybridization,Co-IP and His-Pull down,colocalization of F1L protein interacting with host cells protein by confocal laser scanning microscopy.Lay the foundation for exploring the pathogenic mechanism of OrfV.1.Construction and identification of yeast two-hybrid c DNA library of lamb testicular cellsThe aim of this study was to construct a yeast two-hybrid c DNA library of lamb testicular cells and evaluate the quality of the library.The lamb testicular（LT）was collected to prepare LT cells.After stable passage,the total RNA of LT cells was extracted by TRIzol method,and the concentration and purity of total RNA were detected.The full-length c DNA was synthesized by SMART technology,and the p GADT7-Rec vector and c DNA were co-transformed into Y187 yeast competent cells by homologous recombination.Positive clones were screened by nutrient-deficient selective medium（SD/-Leu）to construct c DNA libraries,and the library titer,total capacity,recombination rate and insert were tested.The results showed that LT cells were successfully prepared and the yeast two-hybrid c DNA library of LT cells was constructed.The library titer was 2.33×10⁸ CFU·m L^-1,the total capacity of the library was 3.5×10⁹ CFU,the library recombination rate was 100%,and the length of the inserted fragment was mainly concentrated in 250-2 000 bp.The results indicated that the constructed c DNA library of LT cells was of good quality and could be used for subsequent yeast two-hybrid screening.2.Construction and identification of OrfV F1L bait strainThis experiment aimed to construct OrfV F1L protein bait strain.The DNA of Orf disease material was extracted,the F1L gene was amplified by PCR,and it was cloned into p MD18-T vector.After identification by PCR,double restriction digestion and sequencing to obtain p MD18-T-F1L clone plasmid.p MD18-T-F1L cloning plasmid and p GBKT7 vector were digested with restriction endonucleases Eco R I and Nco I at the same time,ligated and transformed into DH5αcompetent cells to construct bait plasmid p GBKT7-F1L.Transformation of p GBKT7-F1L decoy plasmid into Y2H Gold yeast competent cells using PEG/Li Ac method.Western-Blot was used to identify whether the bait protein was correctly expressed in yeast cells,and then SD/-Trp/-Leu（DDO）,SD/-Trp/-Leu/-His/-Ade（QDO）selective medium was used to detect its self-activation activity,and at the same time,the OD_{600 nm} of the bait strain and the empty vector strain at different time periods was determined,and the difference in the growth curves of the two strains was compared to detect whether the bait protein had cytotoxicity.The results showed that the amplified F1L gene was 1 029 bp,and the p GBKT7-F1L bait plasmid and the bait strain p GBKT7-F1L/Y2H Gold were successfully constructed.And the size of the bait protein verified by Western-Blot was about 61 k Da.At the same time,the bait protein had no self-activation activity detected by DDO and QDO selective media.The growth curve of the bait strain determined by OD_{600 nm} showed that the bait protein had no toxic effect.The results indicated that the OrfV F1L protein bait strain was successfully constructed in this experiment,which met the conditions of subsequent two-hybrid screening.3.Screening and identification of OrfV F1L protein and host cell interaction proteinThis assay aims to screen and identify cellular proteins that interact with OrfV F1L protein on LT cells.Hybridization of LT cell c DNA library with p GBKT7-F1L/Y2H Gold bait strain.Screening 3 times on selective medium of QDO and SD/-Trp/-Leu/-His/-Ade/X-α-gal/Ab A（QDO/X/A）,and then verified by revertant hybridization,Co-IP,His-Pull down and laser confocal for interactions.The results showed that 57 host cell proteins preliminarily screened by yeast two-hybrid system interacted with F1L protein.Furthermore,five host cell proteins interacting with F1L protein were identified by revertant hybridization,namely eukaryotic translation initiation factor 3 subunit I（e IF3i）,alpha actinin 4（ACTN4）,fibronectin 1（FN1）,matrix metallopeptidase 2（MMP2）and LGALS1.LGALS1 and F1L proteins were selected from the five proteins for Co-IP and His-Pull down tests,which confirmed that LGALS1 protein and F1L protein interacted in and out of cells.Colocalization of F1L protein and LGALS1protein in cytoplasm by laser confocal for interactions,and colocalization of F1L protein and LGALS1 protein in cytoplasm by confocal laser scanning microscopy.The results indicated that F1L protein of OrfV interacted with LGALS1 in LT cells.