Development And Initial Application Of Indirect ELISA For Detection Of Peste Des Petits Ruminants Antibody

Peste des petits ruminants(PPR)is an acute contagious disease of sheep and goats and caused by Peste des petits ruminants virus(PPRV),which is characterized by fever,ocular and nasal discharges,necrosis and erosive stomatitis,gastroenteritis,diarrhea,dyspnea and bronchopneumonia.As morbidity and mortality is extremely high,highly contagious,PPR is regarded as Lists A disease by OIE.PPR was first noticed in Ivory Coast(i.e.Ivory Coast)in 1942,then spread to West Africa,Central Africa,the Arabian Peninsula and the South Asian subcontinent,showing a trend spread from west to east,and even spread to Europe,bringing the global animal husbandry economy a big threat.PPR is epidemic in Rutog County,Tibet Autonomous Region in China in July 2007,then the PPR epidemic throughout the country,spread fast,large span,high risk,caused serious losses to China's animal husbandry,but the origin of the epidemic is not clear.For PPR,culling and vaccination are the main means to prevent the disease,and the immune effect is an important indicator of the evaluation and control of the epidemic.Therefore,development an indirect ELISA for detection of PPR antibody to evaluate the immune control effect and achieve the purpose of immune monitoring.The PPRV is a non-segmented single-stranded and negative-stranded RNA virus and belongs to the genus Morbillivirus in the family Paramyxoviridae.PPRV genome processes 15948 nt,encoding nucleoprotein(N),Hemagglutinin protein(H),Phosphoprotein(P),fusion protein(F),matrix protein(M)and the large protein(L),and two non-structural proteins C and V.N gene is 1578 bp and highly conserved.Sequence similarities between Morbilliviruses N proteins range from 67% to 74%.N protein is a major structural protein of viral transcription and replication.This protein is the most abundant and immunogenic protein in PPRV,and its antigenicity is stability.PPRV-infected animal serum for N protein antibody accounted for high levels,but not with neutralization.Meanwhile,the protein also contains T cell epitopes,which can induce specific cellular immune response.Therefore,N protein is an ideal candidate antigen for PPRV antibody detection method,which can monitor the level of PPRV antibody in flocks and for a wide range of immunization monitoring.Using N protein as the coating antigen,an indirect ELISA method was developed to detect PPRV antibody and lay the foundation for the development of PPR immune antibody detection kit.Firstly,artificial synthesis the gene sequence of PPRV N protein to construct recombinant plasmid of pSumo-mut-N,and pSumo-mut-N was digestion EcoR I/Xho I and sequenced.The recombinant plasmid pSumo-mut-N was transformed into E.coli Arctic Express,obtained expression bacteria of the N-Arctic Express.The optimum conditions for the determination of N protein were induced by 0.5mM IPTG,and cultured at 11? for 10 h.N protein concentration was 120?g/ml.SDS-PAGE and Western blot analysis showed that the molecular weight of the recombinant protein is about 71.9kDa,and also has good immunogenicity with PPRV sheep positive serum.The N protein was purified by Ni affinity chromatography.The purified N protein was used as coating antigen,with the known PRRV negative and positive serum to optimization the detection of the conditions.The indirect ELISA method of PPRV antibody was established.The optimization of detection conditions of the indirect ELISA program was determined:(1)antigen coated: the N protein coated onto ELISA plate of 0.24ug/well with 100ul/well,were incubated at 37? for 2h;(2)blocking: 200ul/well of 5% skim milk powder and the plates incubated at 37? for 1.5h;(3)serum samples: 100ul/well of serum samples at 1:40 dilution with PBST was added to all the wells and the plates incubated at 37? for 50min;(4)HRP labeled Rabbit anti sheep IgG conjugate: 100ul/well of HRP labeled Rabbit anti sheep IgG conjugate diluted 1:5000 in PBST was added to each well and incubated at 37? for 1h;(5)color reaction: TMB was added with 100ul/well and incubated at 37? in dark for 15min;(6)Termination: the reaction was stopped with 2M H2SO4,50ul/ well,and absorbance read at 450 nm in an Microplate Reader.Collected 15 samples of goat serum from slaughterhouse and two serum samples obtained from healthy sheep immunized with PPRV,were neutralized to determine the seroprevalence of serum.According to the indirect ELISA to identify above serums,15 known serum from slaughterhouse,two serum samples obtained from healthy sheep immunized with PPRV,11 serum samples collected from sheep flock with PPRV,31 clinical samples collected from Kaifeng CDC serum(known as serum by competitive ELISA test,all was negative)were detected.Based on statistical method,the average value of negative serum OD450 X=0.470,SD=0.16594.According to the formula X ± 3SD to calculate the serum samples of negative and positive cut-off values and its criteria.Calculated X±3SDX=0.968.The standard of the indirect ELISA is that the value of OD450?0.968 is the positive,the value of OD450?0.968 is the negative sample.Evaluation of indirect ELISA by ROC curve of the sheep serum samples,the AUC=1,indicating that this indirect ELISA method has high application value,and its sensitivity and specificity for 100%.Meanwhile,the results of inter and intra batch repeatability test showed that this method has good stability and repeatability.The results of indirect ELISA showed that above 31 clinical serum samples were consistent with the results of c-ELISA,and the coincidence rate was 100%.In summary,this study constructed PPRV N protein of prokaryotic expression recombinant bacteria successfully,induced expression the N protein;the determined optimal procedure of indirect ELISA for detection of PPRV antibody by using the protein.The results of clinical serum samples showed that the ROC curve,AUC=1,indicating that its application value is high,which provides a foundation for the development of indirect ELISA for PPRV antibody test kit.