Full Genomic Analysis Of Porcine Rotavirus GD-01-2015 And Establishment Of A Sandwich ELISA For PoRV Detection

Rotavirus(RV)is one of the important pathogens of zoonotic diarrhea,in which porcine rotavirus(Porcine Rotavirus,PoRV)is one of the important causes of piglet diarrhea.The prevalence of the disease is widespread,the incidence is higher.Death occurs in some cases due to severe dehydration,acid-base balance and secondary infection,especially while complicated by porcine transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PEDV)co-infection,mortality rate can be extremely high.The disease is widely distributed in the world,causes serious damage in livestock industry.In this study,we studied the full genomic analysis of porcine rotavirus GD-01-2015,the dewelopment of monoclonal antibody against PoRV and the establishment of sandwich ELISA for detection of PoRV's antigen,it is of great significance to study the development and pathogenesis of PoRV vaccine and lay the foundation for the clinical diagnosis of PoRV.1.Full genomic analysis of porcine rotavirus GD-01-2015An porcine rotavirus strain,GD-01-2015,was successfully isolated from fecal samples of a diarrheic piglet,which was identified as porcine rotavirus infection by RT-PCR.This strain was passaged in a monolayer of Vero including the initial adaptation;Reference to published group A of rotavirus CC0912-1 and designed 11 pairs of primers,than the full gene of GD-01-2015 was cloned and sequenced,and the 11 gene fragments were analyzed by RotaC software,genetic variation analysis and bioinformatics analysis.The results showed that the complete genotype of GD-01-2015 was G5-P[7]-I5-R1-C1-M1-A1-N1-T1-E1-H1;The homology of GD-01-2015 to Korean K5 was 99%;Bioinformatics analysis predicted that the protein has a good antigenicity,VP1?VP3?VP4?VP7?NSP1?NSP2 and NSP4 belong to the stable protein,VP7 and NSP4 contain two and one transmembrane domain structure respectively,and VP7 belongs to hydrophobic prptein.2 The dewelopment of monoclonal antibody against PoRVFirst,Vero cells were infected with PoRV-GD-01-2015,and then the concentrated cells containing PoRV-GD-01-2015 as immunogen and immunized Balb/c mice,after two cell fusions,according to the results of indirect immunofluorescence(IFA),we successfully obtained two monoclonal antibodies against PoRV,and named PoRV-2F10 and PoRV-6F5.Western-Blot confirmed that the resulting monoclonal antibody identified only viral antigens.Identified by commercialization kit,the two types of monoclonal antibodies were IgG,Kappa chain.The two monoclonal antibodies were purified from mice ascites with Protein G column,and it laid the foundation for the subsequent establishment of the detection method.3 Establishment of sandwich ELISA for detection of PoRV's antigenAdding ELISA results showed that two monoclonal antibodies were identified different antigenic sites on PoRV protein.After grouping test,PoRV-6F5 was set up as the capture antibody while PoRV-2F10 was set up as the HRP-labeled antibody to establish the sandwich-ELISA.We found that the working concentration of capture antibody and HRP-labeled antibody were identified as 3.5?g/mL(PoRV-6F5)and 0.25?g/mL(PoRV-2F10),incubation time of antigen and HRP-labeled antibody was 60min,the best coloring time was 20min;The result of critical value determination test showed that positive in this sandwich-ELISA was value OD450>0.165.Using the sandwich-ELISA to detect PoRV,it was found the minimum detectable level was 1.3 ×10 ? TCID50;The sandwich-ELISA was specific to PoRV and showed no cross-reactivity with PEDV?TGEV and negative Vero cells;The coefficient of variation within batch or between batches was less than 10%,show that the method has good repeatability.