Preparation Of Polyclonal Antibodies Against PRRSV Nonstructural Protein Nsp8 And Analysis Of The Interaction Between Nsp8 And Nsp9

Porcine reproductive and respiratory syndrome(PRRS)is an economically devastating viral disease affecting the swine industry worldwide.The etiological agent,PRRS virus(PRRSV),possesses a positive-strand RNA viral genome with nine open reading frames(ORFs).The ORF1 a and ORF1 b encode the polyproteins pp1 a and pp1 ab,which are processed at least fourteen non-structural proteins(nsps)through the processing of proteases.After reading the relevant literature on non-structural proteins of PRRSV,we found that the structure and function of PRRSV non-structural proteins nsp8 still remain unknown.A preliminary study of nsp8 protein was conducted in this thesis.First,we used online databases to analyze the nsp8 protein and searched for data related to nsp8 protein structure and function.Then we constructed prokaryotic expression plasmids to express nsp8 and purified the nsp8 protein,which was used to immunize rabbits to prepare nsp8 polyclonal antibody,and the polyclonal antibody binding specificity and potency were tested by dot blot and Western blot.Finally,we investigated the interaction of nsp8 with other nsps and identified the interaction between nsp8 and nsp9 proteins by yeast two-hybrid.To verify the nsp8-nsp9 interaction,we further performed pull-down and bimolecular fluorescence complementation(Bi FC)assays.In this study,we obtained a rabbit antiserum against PRRSV nsp8 protein and found that the polyclonal antibody had obvious nsp8 binding capacity.However,Western blot results also showed that there were some non-specific antibodies in the serum.In the interaction analysis of nsp8 and nsp9 proteins,yeast two-hybrid and BiFC assays showed that nsp8 protein interacted with nsp9 protein.Furthermore,in this study we found that the nsp8 protein expressed by bacterial cells was unstable and could extremely easily be degraded.Whether the eukaryotic expression system can improve the stability of nsp8,and the nsp8 and nsp9 protein interaction plays a role in the virus replication,these questions remain to be studied.