Construction Of Protein-protein Interaction Network Between ASFV And Host

African swine fever(ASF)is a severe infectious disease caused by African swine fever virus(ASFV)with a high fatality rate.At present,our understanding of the mechanism by which ASFV escapes host immunity is very insufficient,which limits the development of African swine fever vaccines and the screening of therapeutic drugs,which is detrimental to the prevention and control of the African swine fever epidemic.Because the ASFV genome is extremely large,it encodes nearly 200 proteins,and there are complex interactions between these proteins and host proteins.Analyzing the interaction between ASFV protein and host protein is the key to exploring the pathogenicity and immune escape mechanism of ASFV.In order to explore the pathogenicity and immune escape mechanism of ASFV from the perspective of the interaction between ASFV protein and host immune system and signal transduction protein,we used a new high-throughput yeast two-hybrid technology RLL-Y2 H,which can directly High-throughput screening of proteins between libraries is performed to obtain global information on protein-protein interactions(PPIs).We selected 1562 proteins in 28 pathways related to the porcine immune system and signal transduction,and used RLL-Y2 H yeast two-hybrid technology to screen the interaction between ASFV protein and host protein and the interaction between its own proteins,and construct ASFV and host immunity The protein interaction network of the system and signal transduction-related pathways,drawing the interaction sub-network map between the ASFV protein and the host’s different pathway proteins and the ASFV self-protein interaction network map.According to the further analysis of the interacting protein pairs,the key proteins of ASFV in the process of pathogenicity and immune escape were screened.The main contents are as follows:1.Construction of yeast library of ASFV protein,host immune system and signal transduction-related pathway proteinsA total of 1264 gene fragments in 28 pathways related to porcine immune system and signal transduction were amplified,and a host protein yeast library was constructed through yeast transformation.After self-activation detection,51 proteins with self-activation activity were removed,and finally a Prey yeast library containing1,213 host immune system and signal transduction-related proteins was obtained.At the same time,all 179 gene fragments of ASFV were amplified,and ASFV protein yeast library was constructed through yeast transformation.After self-activation detection,a Prey yeast library containing 178 ASFV proteins and a Bait yeast library containing 173 ASFV proteins were obtained.2.Drawing of global network map of interaction between ASFV protein and host proteinThrough the hybridization of the host Prey yeast library with the ASFV protein Bait yeast library and the high-throughput sequencing of positive interacting protein pairs,combined with bioinformatics analysis,we have drawn a global interaction network containing 79 ASFV proteins and 590 host proteins Atlas.3.Drawing of ASFV protein and host protein interaction sub-network map and screening of key interaction node proteinsIn order to screen the key proteins in the process of ASFV infection and immune escape,we further mapped the interaction subnetworks between ASFV protein and different host immune system and signal transduction-related pathway proteins,including: MGF-300-1L,MGF-360-9L,MGF-360-11 L interacts with the protein that regulates interferon in the host,which may be a potentially important protein that regulates the expression of host interferon;E199L interacts with the TSC2 protein that plays a GTPase activation function,and may promote the fusion of the late endosomal membrane with the ASFV inner membrane It also functions in core release;B119L(9GL)interacts with the key proteins GSDMD and CASP8 for pyroptosis regulation,and may regulate host cell pyrosis through interaction with GSDMD and CASP8proteins;E66L and host apoptosis-related proteins CARD10,MDM2 The interaction of DDIT4 and AZI2 may regulate host cell apoptosis;CP80R interacts with the host transcription factor AHR and may be involved in the initiation of ASFV gene transcription;EP153R interacts with PAP2,which may affect P53 protein by regulating the expression of PAP2 protein The activity of the host cell will eventually regulate the apoptosis of the host cell.4.Drawing of ASFV’s own protein interaction network map and screening of key interaction node proteinsIn order to understand the interaction between ASFV’s own proteins,we further screened the interactions between ASFV’s own proteins,and drew a network map containing a total of 520 interacting protein pairs among 92 ASFV proteins.Among them,as an important capsid protein of ASFV,P49 interacts between itself.From the perspective of protein interaction,it is verified that P49 protein exists in the form of multimers in the capsid.This study analyzed the interaction between ASFV’s own proteins and the host’s immune system and signal transduction proteins,and found a total of 6975 interacting protein pairs between 79 ASFV proteins and 590 host proteins,as well as ASFV itself92 A total of 520 interacting protein pairs between the two proteins provide key clues and basis for exploring the immune escape mechanism of ASFV,the development of new vaccines,and the screening of therapeutic drugs.