Screening And Preliminary Identification Of Host Cellular Proteins Interacting With VP60 Protein Of Rabbit Haemorrhagic Disease Virus Isolated From Moschus Chrysogaster

Moschus chrysogaster viral hemorrhagic disease(Mc VHD)is a kind of acute,highly lethal animal infectious diseases which caused by rabbit haemorrhagic disease virus isolated from Moschus chrysogaster.The disease is characterized by congestion,hemorrhage,and necrosis of alpine musk deer’s tissues and organs.At present,the disease has caused a serious threat to the development of artificial breeding of alpine musk deer.This virus is a strain of rabbit haemorrhagic disease virus(RHDV)which isolated and identified for the first time in the alpine musk deers.The study shows that rabbit haemorrhagic disease virus isolated from Moschus chrysogaster has the same structure as the classic RHDV genome and belongs to the RHDV G2 genotype.VP60 protein,as the structural protein of RHDV,is closely related to the pathogenicity and immunogenicity of RHDV.Therefore,the study of host cellular proteins interacting with RHDV is of great significance for exploring the infectional mechanism of RHDV isolated from Moschus chrysogaster and the prevention and control of Mc VHD.Based on this,this research carried out and completed the following tasks:(1)Screening of host cellular proteins interacting with RHDV isolated from Moschus chrysogaster.A healthy non-immune New Zealand white rabbit was used as an animal model to collect blood to separate red blood cells,and then to prepare primary rabbit liver cells,spleen cells,kidney cells,and lung cells.Using inactivated RHDV as bait protein,host cellular proteins interacting with RHDV were screened by Co-IP and LC-MS/MS.Finally,bioinformatics analysis of the differential proteins were performed.The results showed: a.A total of 88 differential proteins were screened in all test groups,of which 56 were differential proteins in the hepatocyte group.b.The bioinformatics analysis of the differential proteins of the liver cells shows that the differential proteins are mainly composed of membrane proteins,keratins,etc.They have binding capacity and various enzyme activities.Most of the differential proteins are involved in the transportation and metabolism of carbohydrates and protein modification process.In addition,there are far more differential proteins involved in metabolic pathways than those proteins involved in the other four types of signal transduction pathways.(2)Prokaryotic expression and identification of RHDV structural protein VP60 and host-related proteins.Firstly,ANXA2,EF-1α,Trx were screened as host cellular proteins that potentially interact with RHDV structural protein VP60.In addition,we constructed a double-labeled prokaryotic expression plasmid called PGEX-GST-His.On this basis,according to the corresponding gene sequences in Gen Bank,primers were designed to amplify the target genes.Then these recombinant expression plasmids called PGEX-GST-VP60-His,PGEX-GST-ANXA2,PGEX-GST-Trx,PGEX-GST-EF-1α were successsfully constructed.These correctly identified recombinant expression plasmids were respectively transformed into E.coli BL21(DE3)and induced by IPTG.Finally,all recombinant proteins were purified and identified by Western blot.The results showed that the recombinant sequence in the PGEX-GST-His plasmid was consistent with the expected sequence.The sizes of the amplified products of VP60,ANXA2,Trx,and EF-1α genes respectively were 1740 bp,1020 bp,318 bp and 1389 bp.After comparison,all amplified products had no amino acid site mutations.In additon,all recombinant proteins could be successfully expressed in E.coli BL21(DE3),and all recombinant proteins were soluble.The result of Western blot showed all recombinant proteins were correctly expressed and in line with expectations.(3)Preliminary identification of host cellular proteins interacting with RHDV isolated from Moschus chrysogaster.The His-Pull down test was used to identify host cellular proteins interacting with structural protein VP60.The results showed that VP60 protein and ANXA2 protein could interact directly in vitro,while Trx protein and EF-1α protein could respectively bind to the GST-His protein and the GST-VP60-His protein.In view of the influence of GST tag protein,whether VP60 protein directly interacts with Trx protein and EF-1α protein in vitro needs further verification.In this study,88 differential proteins were successfully screened in all test groups through Co-IP and LC-MS/MS.In addition,the 56 differential proteins screened in the hepatocyte group were mainly involved in the transportation and metabolism of carbohydrates and protein modification process.Three proteins,ANXA2,Trx,and EF-1α,were used as host celluar proteins that potentially interacted with RHDV isolated from alpine musk deer.The recombinant proteins of VP60,ANXA2,Trx,EF-1α were successfully obtained through prokaryotic expression,and all the proteins were expressed correctly and were soluble expression.On this basis,it was determined that RHDV VP60 protein and ANXA2 protein could directly bind in vitro by His-Pull down.However,the interaction between EF-1α,Trx protein and VP60 protein need further study.The results of this study laid the foundation for the in-depth study of the infectional mechanism of RHDV isolated from Moschus chrysogaster and the effective prevention and control of McVHD.