The Rapid Detection Methods Of Porcine Deltacoronavirus (PDCoV) And Preparation Of Its' Monoclonal Antibody

Porcine delta coronavirus disease is an enterogenous,contact and infectious disease mainly caused by porcine delta coronavirus(PDCoV).Pigs at all age stages are susceptible,mainly affecting suckling piglets aged 5?21 days.Piglets have diarrhea,vomiting and other acute clinical symptoms after infection,and the mortality rate is as high as 100%.The clinical symptoms,pathological changes and epidemiology of PDCoV infection are very similar to those of porcine epidemic diarrhea virus(PEDV),and mixed infection often occurs.It is difficult to make a diagnosis through clinical symptoms,so laboratory detection methods must be used to make a diagnosis.Nucleocapsid protein(N)is the structural protein of PDCoV,and it is highly conservative.After infection with PDCoV,the body produces the earliest antibody against N protein,lasts for a long time,and produces the largest amount of antibody.N protein is the preferred antigen for establishing immunological methods.Immunoassay based on specific monoclonal antibody plays an important role in the establishment of diagnostic kits with strong specificity,high sensitivity and good reproducibility.It is widely used in disease diagnosis,food safety detection and other fields.At present,there is no commercial reagent for the diagnosis of porcine delta coronavirus at home and abroad.Therefore,it is of great significance to establish a duplex TaqManMGB FQ-PCR method for differential detection of PEDV and PDCoV,to establish an indirect ELISA method based on recombinant N protein(rN)and to developmonoclonal antibodies against PDCoV N protein for clinical diagnosis and prevention of PDCoV.In this study,we established a duplex TaqManMGB FQ-PCR detection method for PEDV and PDCoV,expressed and purified the transformed N protein gene,and established an indirect ELISA antibody detection method based on the recombinant N protein(rN protein)The spleen cells of immunized mice were fused with SP2/0 myeloma cells by using 2000 as the fusion agent.The positive clones were obtained by the indirect ELISA antibody detection method.The monoclonal antibodies against PDCoV N protein were prepared and identified.The results are as follows1.Establishment of TaqManMGB FQ-PCR for PEDV and PDCoVAccording to the known gene sequences of PEDV and PDCoV in Genebank,the M gene of PEDV and N gene of PDCoV were compared and analyzed by Meg Align.Two pairs of primers and TaqManMGB probe pairs were designed.The optimal primer,probe concentration and reaction conditions were optimized to establish the double TaqManMGB of PEDV and PDCoV FQ-PCR detection method,and the specificity,sensitivity,repeatability and clinical experiments were carried out.The results showed that there was a good linear relationshipbetween the circulation threshold(CT)of pGEM-PEDV standard curve and the template concentration,the correlation coefficient was 0.998,and the linear relationshipbetween copy number(x)and CT value was CT=-3.374.There was a good linear relationshipbetween the cycle threshold(CT)of pGEM-PEDV standard curve and the template concentration,and the correlation coefficient was 0.998.The linear relationshipbetween copy number(x)and CT value was expressed as CT=-3.543 lgx+35.5.The sensitivity of this method for the detection of PEDV and PDCoV can reach 10~1copies/?L,indicating high sensitivity;the method can specifically detect positive samples of PEDV and PDCoV,but no specific amplification reaction for TGEV,SADS,PRo V positive samples and negative samples of PEDV and PDCoV,indicating strong specificity;the intra groupand inter grouprepeatability coefficient of variation of this method are less than 1.5%,indicating good repeatability;the method is simple,rapid and accurate.The results showed that 16cases were positive for PEDV,15 cases were positive for PDCoV,and 6 cases were double positive for PEDV and PDCoV,while 13 cases were positive for PEDV,12 cases were positive for PDCoV,and 4 cases were double positive for PEDV and PDCoV by ordinary PCR,indicating that the detection rate of FQ-PCR was higher than that of ordinary PCR.Therefore,the duplex TaqManMGB FQ-PCR assay for PEDV and PDCoV established in this study can provide a method for rapid detection and diagnosis of the above two diseases.2.Recombinant expression of PDCoV N gene and establishment of indirect ELISA based on rN proteinIn this study,PDCoV N gene was amplified and cloned into pET 32a(+)vector.After induction,expression and purification,rN protein was obtained.By optimizing the best coating conditions,serum dilution,blocking conditions and secondary antibody dilution,an indirect ELISA method based on rN protein was established.After specificity,sensitivity,repeatability and detection of clinical serum samples,the results showed that 1029 bpN gene was successfully amplified,and the expressed rN protein was about 60 k D.Western blot results showed that the rN protein had good reactivity.The results showed that the best coating concentration of rN protein was 2?g/m L,the best dilution of serum was 1:100 and the action time was 1 h,the best dilution of sheepanti porcine HRP Ig G was 1:5000 and the action time was 30 min,and the best color development time of TMB was 10 min.This method can detect PDCoV positive serum samples,but has no reaction with TGEV,PRo V,SADS,PEDV positive serum samples and PDCoV negative serum samples,indicating that the specificity is strong;the highest dilution times of five positive samples detected by the established indirect ELISA method are basically the same as those of neutralization test,indicating that this method has good sensitivity.Ninety-two clinical serum samples were selected and compared with the results of neutralization experiment,and the coincidence rate was 93.3%.Therefore,in this study,the recombinant expression of PDCoV N protein was used to establish an indirect ELISA based on rN protein,which provides a sensitive,specific and efficient tool for PDCoV monitoring and clinical diagnosis.3.Preparation of monoclonal antibody against PDCoV N proteinIn this study,inactivated and purified PDCoV cells were used as antigen to immunize pure Balb/c mice.Splenocytes of immunized mice were fused with SP2/0 cells by hybridoma cell technology.Subcloning was carried out by limited dilution method,and indirect ELISA was used to screen.At the same time,monoclonal antibody subtype was identified.Finally,one strain with Ig G subtype and stable secretion of anti PDCoV was screened The hybridoma cell line 1A4 with monoclonal antibody against N protein.Hybridoma cells were expanded and cultured,ascites were collected and purified.After stability,specificity and blocking test,the results showed that 1A4 Mc Ab had good specificity,could specifically react with PDCoV antigen,but did not cross react with PRo V,PEDV,SVA,SADS,TGEV and other antigens;it had good stability,and its titer could still reach 1:128000 after 7 consecutive passages;there was no cross reaction between 1A4Mc Ab and PRo V,PEDV,SVA,SADS,TGEV and other antigens It can be effectively blocked by specific positive serum induced by PDCoV.The preparation of monoclonal antibody against N protein of PDCoV laid a foundation for further establishment of blocking ELISA based on monoclonal antibody against rN protein and colloidal gold test stripfor rapid clinical detection.