Prokaryotic Expression Of Porcine Pseudorabies Virus UL19 Antigen And Preparation Of Its Monoclonal Antibody

Porcine pseudorabies(RSEUDORABIES,PR)is caused by the pseudorabies virus(Rseudorabies virus,PRV)a variety of domestic and wild animals suffering from acute,hot infectious diseases,breathing difficulties and mental disorders as the main feature,pseudorabies virus belonging to the herpes virus family,?-herpes virus subfamily,varicella virus genus.PRV infection clinically newborn piglets nerve disorders,death,adult pigs breathing difficulties,sow reproduction disorder is characterized by serious economic losses to the aquaculture industry.Since 201 1,a high virulence of PRV appeared in many vaccinated pigs in our country,to the disease prevention and control has brought new challenges.UL19 protein is the main component of PRV capsid protein,closely integrated with the viral genome to form a ribosomal protein,and the virus transport in the host cell,while the protein assembly,the genome into the nucleus plays an irreplaceable role.Pseudorabies virus has latent infection characteristics,which can be latent dose moderate trigeminal ganglia in its natural host pigs,and does not produce lytic infection,once the host immunity is reduced,it can activate the virus to produce acute infection.In order to better study the status of PRV lurking in nerve cells,prepared u119 monoclonal antibody,it was to study the status of the virus in the cell and transfer tracer establish a good operating platform for better studing of latent infection in which the virus particles state.Meanwhile UL19 PRV involved in the host cell invasion,replication,transport and latent infection mechanisms,can analyze the distribution of the protein in the cell infection,it can also be used as viral load internal control,quantitative virus other proteins in different time periods the amount of further study of the virus in infected cells to provide a quantitative standard viral load.PRV UL19 gene fragment was amplified by PCR,and the cloned gene fragment was ligated to construct a recombinant plasmid pMD-18T-UL19,after identified,then the target gene was ligated to pcold-TF construct recombinant prokaryotic expression plasmid pCold-TF-UL19 and induced by IPTG expression and purification.In purified PRV UL19 protein antigen-immunized mice,after three free cell fusion,subcloning culture continued after screening monoclonal antibody cells.In order to establish an indirect ELISA method to screen hybridoma cell lines.Results indicated that PRV UL19 gene fragment could be effectively amplified by P CR method,its size was about 1563 bp;at the same time successfully constructed pC old-TF-UL19 expression vector,and it could be expressed by induction of IPTG by th e analysis of SDS-PAGE and Western Blot,the recombinant protein was about 110 k Da,and mainly expressed in a soluble manner.Using the established indirect ELISA method initially screened four strains of hybridoma cell lines,hybridoma cell lines cul tured by a limiting dilution method,and then the screening was performed by Western blot,finally a cell lines with the stable and highly secreting monoclonal antibody-spe cific was obtained,and named as 3D8.In this study,PRV UL19 was cloned and expressed successfully.An indirect ELISA assay was established for the detection of PRV UL19,and a monoclonal antibody cell line against PRV UL19 was prepared after immunizing mice with the recombinant protein.This study provides a good platform for the study of the status and transmission of the virus in cells,and also provides an important means for the control of the disease.