Establishment Of PRRSV N Protein Double-antibody Sandwich ELISA Method And Its Effect On Toll-like Receptor-mediated Signaling Pathway

Porcine reproductive and respiratory s yndrome?PRRS?is one of the most devasting infectious disease that negatively impacts on global pig industry for three decades.It is characterized by reproductive failure in pregnant sows and respiratory s ymptoms in p iglets.It casuing pathogen,the porcine reproductive and respiratory s yndrome virus?PRRSV?,has been characterized by of instant antigen variation,a highl y restricted macrophage tropism,antibody-dependent enhancement?ADE?and persistent infection in ho st.Currentl y,vaccination of PRRSV demonstrated little success.Moreover,recent prevalence of highl y pathogenic PRRSV?HP-PRRSV?has caused huge economic losses in China.With the constant antigen variation and discovery of new cellular receptors for PRRSV,the pathogenic mechanism of the disease is still largel y unknown though tremendous efford has been made.N protein is a nucleocapsid protein encoded by the relativel y conserved PRRSV ORF7 gene,which plays an important role in protecting virus RNA,vi rus replication,transcription and virion assembl y.In earl y reports,N protein was defined as the most abandent structural protein produced during PRRSV infection,which can rapidl y induce the antibodies production in the earl y stage of infection.Meanwhi le,there were studies suggested that immunization of host by baculovirus-expressed N protein confer partial protection of host against PRRSV challenge,indicating N protein could be a protective angigen of PRRSV.Besides,other reports have confirmed that antibodies recognizing specific epitopes on N proteins can mediate the ADE effect in PAMs during PRRSV infection.Based on these previous observations,further studies were conducted in here to further explore more functions of N protein for understanding the role played by PRRSV-N protein in PRRSV pathogenesis.The main work of this study is listed as follows:1. Cloning,prokaryotic expression,purification and characterization of PRRSV ORF7.According to the nucleotide sequence of PRRSV-SD16 strain,primers were designed to amplify the ORF7 gene by PCR.The ORF7 fragment was amplified by Q5?High-Fidelity DNA Polymerase and cloned into the prokaryotic expression vector p ET28a and confirmed by DNA sequencing.After transformed into E.coli BL21?DE3?cells,protein expression was induced by IPTG.The results of SDS-PAGE showed that the N protein was mainl y expressed in the supernatant as soluble form and eas y to purify using Roche Ni Resin.Moreover,the purified N protein reacted with PRRSV positive pig ser um via Western blotting.2. Purification of N protein from PRRSV antibodies positive pig serum and establishment of PRRSV-N protein sandwich ELISA.The purified PRRSV-N protein was congjugated to CNBr-activated Sepharose^TM 4B and incubated with PRRSV antib odies positive pig serum.Polyclonal antibodies against N protein were eluted using gl ycine?p H 2.4?.The purit y of eluted antibodies was anal yzed by SDS-PAGE.Reactivit y of purified antibodes were probled using l ysate of Marc-145 infected with PRRSV-SD16strain,purified PRRSV virions?VR2332 strain?and recombinant PRRSV-N protein,as well as normal Marc-145 recombinant porcine ISG15 protein.Data suggests a successful purification of antibody against N protein from PRRSV antibodies positive pig serum.Next,the PRRSV-N protein specific monoclonal antibody?Clone No.:PP7EF11?and purified swine anti-N protein pol yclonal antibodies were used as capture antibody and detection antibody for sandwich ELISA,respectivel y.Cell culture supernatants of Marc-145,CRL-2843^{C D 1 6 3} and PAMs infected with PRRSV-JXA1 strain and uninfected cells were used as samples.Meanwhile,recombinant N protein was used as positive control.After optimizing the ELISA protocal,the results showed that there was exitence of extralcellul ar N protein in the supernatant of Marc-145,CRL-2843^{C D 1 6 3} and PAMs infected with PRRSV-JXA1 strain,indicating that there might be secretory nucleocapsid protein released from infected cell during PRRSV infection.3. Characterization of interacting parter ner of PRRSV-N protein in celllualr membrane of PAMs.Based on the principle of biotin-avidin adsorption,the biotin-conjugated PRRSV-N protein was incubated with PAMs and the interaction was visualized using APC-conjugated streptavidin.The results of flo w cytometry showed that N protein interacted with PAMs membrane protein,while there was no reaction SUMO treated PAMs or non-protein control.The purified N protein was further conjugated to CNBr-activated Sepharose^TM 4B and incubated with membrane protei n exacted from PAMs to avoding contamination of cellplasma protein.The specific interacting parter of PRRSV-N was separated by SDS-PAGE and visualized by silver staining.Gel of specific-band was further characterzied as CD169 after mass spectrometry anal ysis.4. Changes of cytokines and chemokines in PAMs treated with N protein.PAMs were pretreated with endotoxin-free PRRSV-N protein,and then stimulated by TLR3 receptor agonist Pol y?I:C?.The expression of inflammatory cytokines in PAMs was detected by real-time quantitative PCR?q PCR?.The results showed that N protein pretreatment significantl y enhanced the upregulation of IFN-?1,IFN-?,IL-6 and IL-12 induced by Pol y?I:C?,suggesting that PRRSV-N protein could sensitize PAMs.In conclusion,this study shows that secretory nucleocapsid protein is produced during PRRSV infection,and there is interaction between N protein and PAMs membrane protein CD169.Interaction between PRRSV-N and CD169could sensitize PAMs to enhance the innate response agains t secondary infection which might be the cause of cytokine storm.Our data provides new insight for understanding pathogensis of PRRSV and may benefit for future vaccine development.