Monclonal Antibody Preparation Of Porcine Sapelovirus VP1 Protein And Establishment Of Blocking ELISA Method

Porcine sapelovirus(PSV)is a pathogen of swine disease that has attracted increasing attention in recent years.It can cause diarrhea,respiratory distress,and severe reproductive and nervous system diseases in pigs.As early as the 60 s of last century,PSV pathogens have been isolated and reported,and PSV was not isolated for the first time in China until 2011,and subsequent epidemiological investigations showed that PSV has been widely prevalent in many regions of China.Therefore,in-depth exploration of VP1 protein can establish effective diagnostic methods,and is also an important direction for the preparation of effective antiviral drugs and vaccines,so as to achieve disease prevention and control measures.In conclusion,in this study,the recombinant His-VP1 protein was successfully expressed through the prokaryotic expression system,and two monoclonal antibodies against the PSV VP1 protein were prepared and identified.Based on the antibodies and the VP1 protein,a PSV blocking ELISA antibody detection method was established.The specific test contents are as follows:Cloning and prokaryotic expression of the PSV VP1 gene.In order to obtain the immunogenic PSV VP1 protein,the VP1 gene was first amplified by PCR,and VP1 was cloned onto the pET-32 a expression vector by restriction enzymes and ligases;Subsequently,the recombinant plasmid was transformed into DH5α competent cells to obtain the correct recombinant plasmid.Finally,it was transferred to E.coli Rosetta,IPTG was added for induction,and the expression was identified by SDS-PAGE electrophoresis.The results showed that the His-VP1 recombinant protein was successfully expressed by the prokaryotic expression system,and the purified recombinant VP1 protein could react with clinically positive serum and had good immunogenicity.Preparation of monoclonal antibodies to PSV VP1 protein.In order to prepare monoclonal antibody against PSV VP1 protein,BALB/c mice were immunized with purified VP1 protein.After high level of antibody was produced in mice,the spleen cells were fused with SP2/0 cells.Indirect enzyme-linked immunosorbent assay(ELISA)was used to screen positive hybridoma cell lines,and the reactivity of monoclonal antibody and VP1 protein was identified by indirect immunofluorescence assay(IFA);The specificity of the monoclonal antibody was identified by indirect ELISA and WB test;Finally,the two Mabs were subtyped by mouse monoclonal antibody isotype ELISA kit.The results showed that two monoclonal antibodies against PSV VP1 protein were successfully obtained,named Mab 9F10 and Mab 15E4.The Mab 9F10 belonging to a subclass Ig G1/κ-type and the Mab 15E4 belonging to a subclass Ig G2b/κ-type were successfully identified,and the light chain subtype was kappa.Identification and analysis of epitopes recognized by monoclonal antibodies to PSV VP1 protein.In order to explore the epitopes of PSV VP1 protein recognized by these two monoclonal antibodies,we first used software to predict the distribution of dominant epitopes of PSV VP1 protein,and according to the predicted results,the dominant epitopes were expressed in vitro.The reactivity of the monoclonal antibodies with the truncated VP1 protein was tested by WB.The minimal epitope recognized by the antibody was identified.The homology of the identified epitopes in porcine sapelo virus was further analyzed,and the three-dimensional structure prediction of PSV VP1 protein was carried out,and the distribution of the identified epitopes on VP1 protein was analyzed.The results showed that the epitope recognized by Mab 15E4 was 203YDGDG207,which was conserved in different PSV genotypes.However,the epitope recognized by Mab 9F10 was 8QAIVNRT14,which was significantly different among different PSV strains.Structural modeling analysis showed that the two novel B cell epitopes were linear and located on the surface of VP1 protein.Establishment of PSV blocking ELISA antibody detection method.In order to establish an efficient and accurate method for the detection of PSV antibody,the purified VP1 protein and monoclonal antibody 15E4 were used as the coating antigen and detection antibody,respectively.The conditions of the method were optimized,and the specificity and repeatability of the method were evaluated;Subsequently,the coincidence rate between this method and neutralization test was analyzed by detecting clinical sera.The results showed that: In this blocking method,the VP1 protein was coated with 0.25 μg/m L and detected by HRP-Mab-15E4 diluted 2000 times.The positive rate was defined as S/N value ≤0.5017.The results of the assay were negative for positive sera of porcine Delta coronavirus and porcine epidemic diarrhea virus,indicating that the assay had good specificity.The method has good repeatability,stable detection results of the same batch and different batches,and the coincidence rate with virus neutralization test is up to 85%.In some areas of Henan province,PSV antibody was detected positive in pig serum,indicating that PSV had spread in Henan province.In summary,PSV VP1 protein with good immunogenicity was successfully expressed and purified.Two monoclonal antibodies 9F10 and 15E4 against VP1 protein were successfully selected and the epitopes recognized by the antibodies were further identified.Finally,a PSV-blocking ELISA antibody detection method was preliminarily established based on Mab 15E4 and VP1 proteins.This study provides support for the study of the function of VP1 protein and provides experimental basis for the development of a commercial ELISA kit for PSV antibody detection.