Construction Of Complementary System For The Deletion Of IE180 Gene Of Pseudorabies Virus

Pseudorabies virus(PRV)is the causative agent of pseudorabies in pigs.The virus can infect most mammals,while pigs are the natural hosts of PRV.PRV is a neuro invasive pathogen that can establish life-long latency in peripheral nervous system(PNS),which brings great difficulty to eradication of PRV.The virus,like other members of the alpha herpesvirus family,it can limited express their viral genes,resulting the production of latency-associated transcripts in PNS.The IE180 is the only expression product of immediate-early gene of PRV,which expressed immediately after infection.The IE180 is the switch of the PRV transcriptional cascade;several studies demonstrated the PRV recombinant clone plasmid of deleting two copies of IE180 completely lost the ability to infect host or cell lines,demonstrating IE180 is necessary for PRV infection.At present,the biological function of IE180 is still controversial,in order to further study of IE180,we built a recombination clone plasmid of deleting the IE180 promoter,this study use the pBAC-JS2012 infectious clone plasmid,which has been constructed by the researchers in our laboratory,and the galk selection system to construct the IE180 promoter double deletion clone plasmid.This study provides a tool for studying the gene function of IE180,the preparation process comprises the following steps:1.Preparation of polyclonal antibody against IE180.Three segments sequences of IE180 gene were cloned into pcold-TF vector to construct recombinant prokaryotic expression plasmid pcold-TF-IE180.After purification and immunization,we obtained the polyclonal antibody against IE180,it shows great immunore activity to IE180 protein.2.Construction of partial deletion clone plasmid of IE180 gene.Using the infectious clone plasmid,the mutant screening strain SW102-PRVBAC and the galk selection system,the IE180 promoter double deletion clone plasmid was constructed.In this process,IE180 TATA-box gene and its surrounding bases(left 297bp,right 172bp)were replaced with galk gene.After identification,we use the same method to replace another TATA-box gene and its surrounding bases(left 134bp,right 309bp)with Kan~+marker gene.The results of PCR and western blot demonstrate that IE180 gene has been deleted completely.Hence,we successfully screened the IE180 promoter double deletion clone plasmid pPRV-DIE180-BAC.3.Construction of the complementary deletion system of IE180 gene.In order to compensate for the defective expression of IE180 gene in pPRV-DIE180-BAC,we constructed the eukaryotic expression plasmid pCCAGGS-IE180.The results show that the IE180 protein was expressed after co-transinfected the p CCAGGS-IE180 and pPRV-DIE180-BAC plasmids into cell lines,which demonstrates the p CCAGGS-IE180 plasmid is complementary to the p PRV-DIE180-BAC plasmid.This tool can realize PRV replication successfully when the IE180 gene was deleted.