Establishment Of Blocking ELISA Method Based On Nanobody Against Porcine Epidemic Diarrhea Virus S Protein

Porcine epidemic diarrhea virus(PEDV),a member of the coronavirus.By the end of2010,a serious epidemic of porcine epidemic diarrhea(PED)occurred in China,causing huge economic losses.The onset of pigs showed vomiting,watery diarrhea,dehydration and other clinical symptoms,resulting in slow growth or even death of pigs.The mortality rate of piglets infected by PEDV was higher,and atrophic enteritis was seen in the pathological examination of the pigs.The clinical symptoms and histopathological features of PED are similar to those of transmissible gastroenteritis(TGE).Therefore,the differential diagnosis of these two diseases mainly depends on molecular and serological tests.PEDV contains four structural proteins and 17 non-structural proteins,in which the spike protein(S)is located on the surface of the virus in the form of trimer.It mainly mediates the basic biological functions of the virus,such as cell membrane fusion and receptor binding,and contains many neutral epitopes,can induce neutralizing antibody production in host.Therefore,the antibody level of PEDV S protein in pig serum is more representative of neutralizing antibody level in pigs.In this study,the purified recombinant protein of truncated PEDV S was subcutaneously injected into Bactrian camels,and six nanobodies with different amino acid sequences were screened by phage display technology.After that,we carried out a biotin labelling of a nanobody and initially established a blocking ELISA method for detecting antibodies against PEDV S protein in pig serum.The main contents and results are as follows:1.The purified recombinant protein of truncated PEDV S was injected subcutaneously to immunize Bactrian camels.After four immunizations,the titer of specific antibodies in the serum of Bactrian camels reached 1:256000.Peripheral blood lymphocyte separation of Bactrian camel,extracting lymphocyte RNA,reverse transcription cDNA,VHH fragment amplified by nested PCR,constructed into pCANTAB-5E vector and transformed into TG1competent cells.The VHH phage antibody display library was obtained.After identification,the diversity of the library was good,the capacity of the library was 2.1 x 10~7pfu/mL,and the positive rate was 85%.After three rounds of screening and enrichment of the display library,six nanobodies with different amino acid sequences were screened out.All of them have good binding ability and specificity for truncated PEDV S recombinant protein,named Nb1-Nb6.2.Select nanobody Nb3,amplify its VHH fragment and fuse the Avitag sequence at the3'end,construct it into pET-21b vector,transform it into BL21(DE3)competent cells,and use IPTG to induce its expression.Finally,the fusion protein of Nb3-Avitag was successfully obtained by prokaryotic expression.And the purity is high after purification.After that,the fusion protein of Nb3-Avitag was labeled with biotin.It was identified that the biotinylated Nb3 still had good binding ability to the truncated PEDV S recombinant protein.3.Using biotinylated nanobody Nb3(Nb3-Biotin),a method of blocking ELISA for detecting PEDV S protein in pig serum was established.By exploring and optimizing the reaction conditions,the reaction time(total reaction time 3.5 hours)was shortened as far as possible,and the highest sensitivity was guaranteed.It has been proved that this method does not cross-react with TGEV,PRRSV,PCV,PRV,PPV and JEV antibody positive sera,and has good repeatability.In clinical sample detection,the overall coincidence rate of this method with commercial diagnostic kits is 92.59%.In summary,in this study,phage display technology was used to screen 6 specific nanobodies targeting truncated PEDV S protein.Based on biotin streptavidin system,a blocking ELISA method for detection of PEDV S protein antibody in pig serum was established,which provided a tool for the study of PEDV and monitoring of clinical neutralizing antibodies.