Establish Of PPRV H Protein Antibody Detection Method Based On Multi-epitope Peptide And PPRV P Protein Function Research

Peste des petits ruminants virus?PPRV?which belongs to the genus Morbillivius within the family Paramyxoviridae,sheep and goat are the mainly infection host of PPRV with highly morbidity and mortality.PPRV has been listed as the type I infection disease in our country and notifiable disease by the World Organization for Animal Health?OIE?.In recently years,PPRV has caused damages serious to animal husbandry with its broken frequently in china.nevertheless,the disease is mainly preventive for treatment.hence,it is vital to PPRV by rapidly and exactly detected.Based on the choice of B cell epitopes of PPRV H protein,chemosynthesis 4 segment antigen epitopes with preferably reactionogenicity after connection as the coating antigen to establish a iELISA method for HN protein antibody detection,optimize the relevant conditions.After optimize,optimum coating concentration of multi-epitope antigen was 1.5?10^-5?g/mL,serum dilution concentration was1:100,enzyme labeled secondary antibody dilution concentration was 1:80000,serum dilution buffer and secondary antibody dilution buffer was PBST,coating buffer was bicarbonate buffer,blocking buffer was2%BSA.On the basis of positive control?0.5 and negative control?0.25,the OD450 value of serum sample<?X+2S=0.25 was negative,the OD450 value??X+3S=0.30 was positive and between 0.25 to0.30 was suspicious.The coefficient of variation of detection results at some batch and between different batch was less than 10%.it is demonstrated that this method has preferably specificity by detected serums of sheep pox virus,food and mouth disease and bluetongue virus.Its coincidence was 92.75%compare with criterion cELISA kit.P protein,as a highly phosphorylation protein,not only regulates transcription and replication of viral but also the cells cycle by interaction with N protein.Researching to function of PPRV P protein and cellular target proteins that can further clarification the concrete interaction mechanism of P protein in viral transcription and replication and virus regulation cell cycles.Construction fusion expression vector to express and purify P-his protein for the choice of cellular protein which has the potential of interaction with P protein by Pull-down/MS,results show that 60 proteins can potentially interaction with P protein and the score of 14 types proteins more than 40s and that cellular protein Vimentin can interaction with P protein after Co-IP and indirect immunofluorescence confirmed.In order to further confirm the interaction of cellular protein and P protein,to construct fusion expression vector pCMV-Flag-P and indirect immunofluorescence and Co-Immunoprecipitation assay were carried out after transfected cells,confirmed that it is surely interaction between two proteins.Exploit small interference RNA?siRNA?to disturb the expression of Vimentin protein and then infected PPRV,indirect immunofluorescence,Western Blotting and qPCR to detect the influence to PPRV replication when Vimentin protein expression downregulated.results display that the level of viral gene upregulated while N protein expression downregulated,showing that Vimentin protein can promotes viral protein expression and inhibits replication of viral genome RNA.