Development Of Blocking ELISA Antibody Detection Kits Of Peste Des Petits Ruminants Virus

The Peste des Petits Ruminants Virus(PPRV)is a pathogenic source of infectious diseases such as sheep and other small ruminants.The protein is PPRV nucleoprotein,which has good immunogenicity.In this research,the prokaryotic expression plasmid pET-28a-N was constructed and the prokaryotic expression protein of PPRV was purified.The monoclonal antibody against PPRV N protein was prepared and the Bac-PPRV-N expressing PPRV N protein was constructed.On this basis,a blocking ELISA method for detecting PPRV antibody was established,which can provide an effective tool for clinical diagnosis of PPRV.The specific research contents are as follows:1.Preparation and Identification of PPRV N Monoclonal AntibodiesThe protein N of PPRV was expressed using a prokaryotic expression system.The mice were immunized with purified protein.Six monoclonal antibodies secreting antiPPRV N protein were screened by cell fusion and subcloning.Respectively,named as 1H5,2H5,1A7,1G5,1A6 and 2F5.Six monoclonal antibodies were identified as IgG,the subtypes were IgG2 b,and the light chains were Kappa chains.Through the IFA and Western Blot analysis,the six monoclonal antibodies were able to specifically recognize the N protein expressed by PPRV.By truncating the N gene,the epitopes of monoclonal antibody 1H5,2H5,1G5,2F5 were found in the base region of 112 aa to 144 aa of N gene by Western Blot,the epitopes of monoclonal antibody 1A7,1A6 are in the base region of 144 aa to 240 aa of N gene.The antibody was screened for detection of the blocking activity by PPRV positive reference serum and negative reference serum,The blocking rates of the selected monoclonal antibody 1H5,2H5,1A7,1G5,1A6 and 2F5 were 79.28%,71.74%,93.32%,84.29%,73.82% and 74.49% respectively.2.Construction of PPRV N Protein Baculovirus Expression PlasmidThe shuttle plasmid Bac-PPRV-N was constructed using the PfastBac HTB vector in the baculovirus expression system.The constructed recombinant plasmid was transfected into sf9 cells to rescue the recombinant baculovirus Bac-PPRV-N.By IFA and Western Blot analysis,the constructed Bac-PPRV-N expressed PPRV N protein can react with the monoclonal antibody against the N protein.3.The development of PPRV N protein blocking Antibody Detection ELISA kitThe purified prokaryotic expression of protein N as coating antigen,monoclonal antibody 1A7 with blocking activity was subjected to HRP labeling as enzyme secondary antibody.A total of 262 clinical serum samples were detected by comparison with the PPRV antibody test kit manufactured by ID.VET in France.The total coincidence rate was 94.2%.The blocking ELISA kits developed by Qingdao Riel Biology,A total 241 clinical samples were detected,the total coincidence rate was 97.9%.During the intermittent repeat test and the intra-batch repeat test,the variation coefficient of the kit was within 10%,indicating that the kit had good reproducibility.Through the experiment of sensitivity,specificity experiment and shelf lifes showed that the blocking ELISA established in this experiment has the advantages high sensitivity and specificity,and is suitable for clinical detection of PPRV antibody.