Preparation Of Monoclonal Antibody Against GP4 Of PRRSV And Development Of Candidate Strains For Replication-deficient Vaccine

Porcine Reproductive and Respiratory Syndrome(PRRS)serious threat to the swine industry.The development of an effective new vaccine as soon as possible is the focus of preventing this disease.Among them,replication-deficient vaccine have certain research value because of their safety.To rescue ORF4 replication-deficient PRRSV,this study was conducted in three parts:(1)Preparation of monoclonal antibody against GP4 of PRRSV:recombinant Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)Glycoprotein 4(GP4)was truncated expression and purified.GP4 monoclonal antibody was prepared by immunizing mice with recombinant GP4.The results of SDS-PAGE indicated that the recombinant GP4 was successfully expressed in the supernatant form.The results of WB and IFA indicated that the monoclonal antibody Anti-A-GP4-70 was successfully prepared and specifically bound to the antigen.(2)Construction of Marc-145 cell line stably expressing the ORF4 gene of PRRSV: the recombinant lentiviral plasmid p LV-EF1a-EGFP-2A-ORF4 was constructed using the full-length c DNA infectious cloning plasmid p JXwn06 as a template.After transfection in HEK293 FT cells with Lipofectamine,the lentivirus was harvested and transduced into Marc-145 cells.The cell line Marc-145-GP4 was screened by puromycin and limiting dilution,and verified with the prepared monoclonal antibody.The results of PCR and RT-PCR indicated that the ORF4 gene was successfully integrated into the cell chromosome and could perform normal transcription and translation.The results of WB and IFA indicated that the ORF4 gene was successfully expressed on Marc-145 cells.(3)Rescue ORF4 replication-deficient PRRSV using trans-complementation: the recombinant c DNA infectious clone deletion plasmid p JXwn06-△ORF4 was constructed by Overlap PCR using the full-length c DNA infectious cloning plasmid p JXwn06 as a template.The replication-deficient virus r JXwn06-△ORF4was rescued by trans-complementation of cell line Marc-145-GP4 using in vitro transcription and electrotransfection.The results of PCR and IFA indicated that the replication-deficient virus was successfully rescued and could only grow and reproduce in the Marc-145 cell line expressing the ORF4 gene.This study lays the foundation for the rapid detection of PRRS and the functional study of GP4 and provides candidate strains for a new replication-deficient vaccine of PRRS.