Prokaryotic Expression Of The Peste Des Petits Ruminants Virus Nucleocapsid Protein And The Development Of Indirect ELISA

Peste des petits ruminants virus(PPRV)is the etiological agent of peste des petits ruminants(PPR),which is an acute and highly contagious viral disease.It mainly infects goats,sheep,antelope and other small ruminants and has a very high morbidity and mortality.PPR is a notifiable disease listed by the World Organization for Animal Health(OIE).The nucleocapsid(N)protein of PPRV is the main structural protein of the virion.It is also the most abundant viral protein in the virion and has a very high immunogenicity,thus making it an ideal target for PPRV diagnosis.In order to develop a rapid,specific and sensitive diagnostic method for PPRV,the PPRV N protein was successfully expressed using a prokaryotic expression system and then purified using Ni-NTA agarose.The purified PPRV N was used as the coating antigen,and an indirect enzyme-linked immunosorbent assay(iELISA)for the detection of PPRV-specific antisera was successfully established.In this study,a pair of primers were designed based on the N gene sequence of the PPRV China/BJ/2014 strain(GenBank accession No.KP260624).The Not ? and Sal ?restriction sites were introduced into the 5'-terminus of the forward and reverse primers,respectively.Using the artificially synthesized N gene of PPRV(China/BJ/2014 strain)as a template,PCR was utilized to amplify the full-length coding sequence of the N gene,which was then cloned into the prokaryotic expression vector pET-2 8a-c(+)to construct the recombinant plasmid pET-28a-PPRV-N.After verification using the double-enzyme cleavage method and sequence analysis,the recombinant plasmid pET-28a-PPRV-N was transformed into the competent E.coli BL21(DE3)cells which were then induced by isopropyl-p-thiogalactopyranoside(IPTG).The bacterial pellets were then analyzed by sodium dodecyl sulfate polyacrylamide gel electropheresis(SDS-PAGE)and Western-blotting analysis.The results showed that the recombinant PPRV N fusion protein was efficiently expressed in E.coli BL21(DE3)in the form of both soluble expression and inclusion bodies,and the molecular weight of His-SBV-N protein was about 80 kDa.The recombinant N protein reacted well with PPRV antisera,indicating this protein posesses good antigenicity.The recombinant PPRV N fusion protein was successfully purified using Ni-NTA affinity chromatography under native conditions.Afterwards,the purified PPRV N protein was used as the coating antigen,and an iELISA for the detection of PPRV antisera was established after optimizing the consentration of coating antigen,the serum dilution,the blocking solution,the blocking duration,the reaction duration of serum,secondary antibodies and the substrate as well as other experimental conditions.It was demonstrated that the established iELISA did not react with the antisera against Brucella,Bluetongue virus and Schmallenberg virus,which are three important diseases of goats and sheep,indicating ithas good specificity.Even the standard positive serum sample was diluted to 1:1600,it was still reactive in the iELISA,suggesting that this assay has good sensitivity.The intra-assay and inter-assay coefficient of variation values were maintained at less than 10.0%,revealing that the iELISA has good reproducibility.In addition,the diagnostic agreement between the established iELISA and a commercial ID-VET PPR Competition ELISA kit was 88%.