Preparation And Application Of Polyclonal Antibody Against RNA Dependent RNA Polymerase Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV)belongs to the coronavirus family,α-Coronavirus.PEDV infection leads to porcine epidemic diarrhea(PED),which mainly causes severe diarrhea,vomiting and dehydration of pigs,resulting in extremely high mortality of suckling piglets,causing huge economic losses to the global pig industry.The coronavirus genome encodes a total of 16 nonstructural proteins.RNA dependent RNA polymerase(RdRp)is an important nonstructural protein encoded by nonstructural protein12 and plays a central role in RNA replication and transcription of viral RNA.It is the core protein of replication complex and is used to express genes downstream of replicase genes.In this study,the active RdRp protein was prepared by prokaryotic expression.And its activity was measured by FITC labeling method.A rabbit polyclonal antibody against RdRp was prepared with purified RdRp protein as antigen.Its serum titer was determined by ELISA,and its effectiveness and specificity were determined by Western Blot and immunofluorescence.The half-life of RdRp protein was measured by Western Blot method with rabbit polyclonal antibody against RdRp as the primary antibody.The results of the experimental study are as follows.1 Preparation and validation of polyclonal antibody to RdRp In this experiment,the Rd RP gene was successfully cloned from PEDV CV777 strain by using E.coli prokaryotic expression technology,and the recombinant expression vector p ET28a(+)-RdRp was constructed and sequenced for validation.Then,it was transferred into the recombinant Escherichia coli strain BL21,and recombinant expression was performed through IPTG induction to obtain and purify the His-tag RdRp fusion protein.The obtained protein was detected by SDS-PAGE method.After concentration testing,the purified RdRp protein was emulsified with an equivalent volume of Freund’s adjuvant,and then was injected subcutaneously at multiple points on the back to inoculate 6-week-old female white rabbits.Heart blood was collected 10 days after the fourth immunization.After purification of the serum,anti-RdRp rabbit polyclonal antibodies were obtained.After purification of the polyclonal antibody,the titer of the RdRp polyclonal antibody was detected using indirect ELISA technology,while the specificity of the RdRp polyclonal antibody was analyzed using Western Blot and indirect immunofluorescence technology.The results showed that the sequencing results of the recombinant vector were consistent with the RdRp gene,indicating that the recombinant vector was successfully constructed in this experiment.The SDS-PAGE results showed that the recombinant strain BL21 successfully expressed the target protein and only had a single band after purification,indicating that the RdRp protein was successfully prepared and purified in this experiment.The rabbit polyclonal antibody prepared in this experiment has a high titer of 1:100000,and can specifically bind to RdRp in PEDV and RdRp expressed in eukaryotic expression vectors,proving its good specificity.2 Application of RdRp in the study of AOFP3 anti PEDV In this experiment,purified RdRp was taken as sample,FITC-labeled ploy A as substrate,purified RdRp as polymerase,and the activity of RdRp was calculated by measuring the absorbance and fluorescence value of the reactant.At the same time,AOFP3 with different concentrations was added to the reaction system to determine the effect of AOFP3 on the activity of RdRp.Then,the eukaryotic vector of RdRp was transfected into vero cells.After 72 hours,actinomycin was added to stop the expression of RdRp.From now on,the cells after 0hours,2 hours,4 hours,6 hours,10 hours,and 14 hours were taken as samples respectively.The anti-RdRp rabbit polyclonal antibody was used as the first antibody,and the half-life was detected by Western Blot method.AOFP3 was also added in the detection process to determine the effect of AOFP3 on the activity of RdRp.The result shows that the activity of RdRp is 2 pmol/μg/h,and AOFP3 can reduce its activity.With the increase of AOFP3 concentration,the reduction effect is enhanced.The half-life of RdRp is about 5.47 hours,and AOFP3 has no effect on the half-life of RdRp.To sum up,the prokaryotic expression recombinant plasmid p ET28a(+)-RdRp his was constructed in this experiment,and the rabbit polyclonal antibody against RdRp with good specificity was further prepared and identified by various means,providing experimental materials for the follow-up study of PEDV replication mechanism and the development of new antiviral drugs or vaccine adjuvants based on this.In addition,the activity and half-life of RdRp were detected in this experiment,which provides an important molecular basis for further study of PEDV RdRp and an important raw material for screening RdRp antagonists,which is of great clinical significance for the prevention and treatment of PEDV.