| Porcine deltacoronavirus(PDCoV)is a novel porcine intestinal coronavirus that often causes watery diarrhea,vomiting,rapid dehydration and acute death in nursing piglets aged 515 days,with a mortality rate of 50%-100%.The M protein of PDCoV is the most abundant component of the viral envelope surface and is involved in the replication and assembly process of the virus.The S protein of PDCoV is not only the main antigen that induces host production of neutralizing antibodies,but also a key protein for virus transmission across species.Therefore,this study prepared monoclonal antibodies against S and M proteins and identified their biological characteristics,in order to provide new ideas and insights for PDCoV prevention and control,vaccine research and development,immunodiagnosis and protein function research.1.Preparation and identification of PDCoV M protein monoclonal antibodiesIn this study,the highly hydrophilic region of the M protein was selected for prokaryotic expression and purification,and the recombinant protein of PDCoV M was prepared.The obtained recombinant protein was immunized in mice,and the mice that reached the antibody titer were selected for cell fusion.Through screening and subcloning,a hybridoma cell that stably secreted M protein-specific antibody was successfully obtained,which was named 24A6.Subsequent test results proved that the monoclonal antibody is suitable for IFA,Western blot,IP and other tests.The results of Western blot showed that the monoclonal antibody could specifically recognize 3 strains of PDCoV without reacting with other viruses,confirming its good specificity.The monoclonal antibody can react with PDCoV in infected LLC-PK1 cells by IP test.The monoclonal antibody prepared in this study was further used as the primary antibody to detect the expression of M protein at different time points after the virus infected the cells.The results showed that green fluorescence could be detected by IFA at 6 h,and the specific band of M protein could be detected by Western blot at 3 h,suggesting that M protein could be used as an antigen target for early detection of PDCoV-infected cells.By expressing the truncated peptide of M protein,the linear epitope recognized by the monoclonal antibody was identified for the first time,which is 103SPESRL108.Through sequence comparison analysis with 181 PDCoV strains,it was found that the amino acid sequence of this region was highly conserved.2.Preparation and identification of PDCoV S protein monoclonal antibodyIn this study,the S1 CTD(C-terminal domain)region of the S protein was selected for prokaryotic expression and purification,and the recombinant protein of PDCoV S was prepared,and a stable secretory strain of PDCoV S was successfully obtained through mouse immunization,cell fusion,screening and subcloning.The hybridoma cell with S proteinspecific antibody is named 1B3.In order to obtain a monoclonal antibody with neutralizing activity similar to the natural conformation of the PDCoV S protein,this study also immunized mice by rescuing the recombinant Sendai virus expressing the S protein.On the basis of the SeV infectious cloning plasmid constructed in our laboratory,GFP was replaced with PDCoV S protein to obtain a recombinant infectious cloning plasmid.The plasmid was further transfected into BHK-21 cells to rescue the recombinant Sendai virus.The results of IFA and Western blot showed that the recombinant Sendai virus was rescued successfully.The mice were immunized with recombinant Sendai virus,and three strains of hybridoma cells with S protein monoclonal antibodies were obtained through cell fusion,screening and subcloning,which were named 3A5,1C3 and 3D3 respectively.All four monoclonal antibodies are suitable for IFA and Western blot detection.By expressing the truncated peptide of S protein,the linear epitope recognized by 1B3 monoclonal antibody was identified for the first time,which was 534KPVLSYGPISVCSD547.The biological functions of the four monoclonal antibodies were identified.Using 24-A6 monoclonal antibody as a negative control,the neutralizing activity of 4 monoclonal antibodies was determined,among which 3A5 and 3D3 had neutralizing activity.In order to verify whether the prepared monoclonal antibody can recognize the de-N-glycosylated S protein,the cell protein infected with PDCoV was de-Nglycosylated.The results showed that after deglycosylation,it could still be detected by 1B3 strain monoclonal antibody,but the molecular weight of S protein became smaller. |
