Preparation Of Monoclonal Antibodies Against Porcine Rotavirus VP6 And Establishment Of A Double Antibody Sandwich ELISA Assay

Porcine Rotavirus（PoRV）is one of the common pathogens that cause viral diarrhea in piglets.When piglets are infected with PoRV,they will have symptoms such as vomiting,loss of appetite,diarrhea and even dehydration,and are prone to secondary bacterial infection,resulting in growth retardation or decreased survival rate of piglets,which brings serious pecuniary losses to the pig farming industry.The PoRV genome consists of 11segments of double-stranded RNA,which encodes 6 structural proteins and 6 non-structural proteins.The structural protein VP6,which accounts for about 51%of the total viral protein,is the most abundant protein in the virus,and its nucleotide sequence is highly conserved among different G genotype strains,and is usually used as an antigen for laboratory diagnosis.In this study,porcine rotavirus VP6 protein was expressed by swine pox virus vector,and anti-VP6 monoclonal antibodies were prepared.Based on this,a double antibody sandwich ELISA method was established for the detection of porcine rotavirus samples.1.Expression of porcine rotavirus VP6 protein by swine pox virus vectorThe porcine rotavirus VP6 protein is the most abundant structural protein in the virus,its nucleotide sequence is highly conserved,and it has superior antigenicity,making it a commonly used detection antigen in experimental diagnosis.In this study,vaccinia virus promoter P28 was used as VP6 promoter,G3 type PoRV nucleic acid was used as template to amplify VP6 gene,and the gene fragment P28-VP6-his was cloned into p SWE178 basic plasmid to construct recombinant plasmid p SWE178-P28-VP6-his.Recombinant swine pox virus r SWE178-P28-VP6-his expressing VP6 protein was obtained by homologous recombination and plaque purification techniques.SDS-PAGE results showed that the recombinant swine pox virus successfully expressed VP6 protein,the size of which was about 45k Da,and the expression mode was soluble.2.Preparation of monoclonal antibodies against porcine rotavirus VP6proteinIn this study,the inactivated porcine rotavirus（strain PoRV-WN）was used as immunogen to immunize BALB/c mice.The spleen cells of mice No.3 were fused with SP2/0 cells by hybridoma technique.Using VP6-his protein expressed by recombinant swine pox virus as detection antigen,three hybridoma cell lines secreting anti-VP6 monoclonal antibodies were screened by indirect ELISA,which were named 4G7,4E12 and 11B8,respectively.The reaction characteristics of the three Mc Abs were determined:（1）Western blot was performed using VP6-his protein and PoRV as samples,the results showed that11B8 reacted with VP6-his protein and PoRV,but 4G7 and 4E12 did not react with them.It can be inferred that 11B8 targets the linear epitope of VP6 protein,while 4G7 and 4E12target the conformational epitope of VP6 protein.（2）Indirect immunofluorescence assay was carried out between the three Mc Abs and MA104 cells infected with G3,G5 and G9type PoRV,the results showed that the reaction characteristics of the three Mc Abs were different:4G7 reacted with G3,G5 and G9 PoRV,while 4E12 and 11B8 only reacted with G3 and G9 PoRV,but not with G5 PoRV.3.Establishment of a double antibody sandwich ELISA methodThe rabbit was immunized with the VP6-his protein expressed by recombinant swine pox virus as immunogen.After five times of immunization,the rabbit serum antibody titer was 1.024×10⁶.The rabbit anti-VP6 polyclonal antibody was purified by ammonium sulfate precipitation method.The purified rabbit polyclonal antibody titer was 5.12×10⁵ and the concentration of protein was 11 mg/m L.The purified rabbit polyantibody was used as capture antibody to coat the enzyme plate,and the mouse anti-VP6 monoclonal antibody4G7（ascites titer was 5.12×10⁵）was used as detection antibody to establish a double antibody sandwich ELISA detection method.Through square titration,the optimum coating concentration of rabbit anti-VP6 polyclonal antibody was determined to be 2.5μg/m L,the best working condition of monoclonal antibody 4G7 was diluted at 8000,the best incubation time for antigen to be detected was 1h,and the working conditions of enzyme-labeled second antibody（goat anti-mouse Ig G-HRP）were diluted for 1:6000 and reacted for 1h.The standard curve was established by using the standard protein VP6 as the detection sample,the linear range of detection was 25～200 ng/m L,and the regression equation was y=1.361ln（x）-3.6745（R²=0.9922）.The standard curve was established by using PoRV with known virus titer as the detection sample,the linear range of detection was 1.25×10⁴～2×10⁵TCID₅₀,and the regression equation was y=1.171ln（x）-10.227（R²～0.9965）.53 clinical pig fecal samples were collected and PoRV was detected by the established double antibody sandwich ELISA method and real-time fluorescence quantitative PCR method respectively.The results showed that 15 positive fecal samples were detected by double antibody sandwich ELISA method,with a positive rate of 28.3%;20 positive fecal samples were detected by fluorescence quantitative method,with a positive rate of 37.7%;and the coincidence rate of the two methods was 83%.