Isolation And Identification Of Porcine Delta Coronavirus And Preparation Of Monoclonal Antibody Against N Protein

Porcine diarrhoeal disease has always been one of the main threats to the healthy development of pig industry.In 2012,porcine delta coronavirus(PDCoV)HKU 15first landed in Hong Kong,China,and then spread in many regions of China.PDCoV is susceptible to pigs of different ages,especially the most serious harm to suckling piglets.Vomiting,dehydration,severe diarrhea are the main clinical characteristics,and the incidence rate and mortality rate are higher.At present,our research on pdcov is still unknown,and there is no commercial vaccine to prevent and control the disease.So far,pdcov has been prevalent in many countries and regions in the world,such as China,the United States,Korea and so on.Therefore,it is imperative to carry out scientific research on pdcov epidemiological investigation,virus isolation and identification,and establishment of detection and diagnosis methods.In this study,intestinal tract and intestinal contents of piglet were collected and tested in large-scale pig farms in Hebei province.PDCoV positive samples were inoculated into LLC-PK1 cells,and then passed to the sixth generation.Typical cytopathic lesions were found.The pdcov positive virus was successfully isolated by RT-PCR detection,observation of cytopathy,genome sequencing and pHylogenetic tree analysis Strain CHN-TS1-2019(GenBank No.MT663769).According to the pdcov gene sequence published in GenBank,a pair of specific PCR primers were designed to amplify the N gene of CHN-TS1-2019 strain.The N gene was connected with pet-30a(+)expression vector to construct pET-30a-PDCoV-N recombinant plasmid.The recombinant plasmid was transformed into E.coli BL21 expression strain,and pdcov was induced by IPTG BALB/c mice were immunized with purified N protein and Freund’s adjuvant as antigen.Spleen cells of immunized mice were fused with SP2/0 tumor cells.The positive hybridoma cell lines were screened by indirect ELISA.The specific reaction of monoclonal antibody(mAb)was verified by indirect immunofluorescence and Western blot.After screening and identification,three hybridoma cell lines(3F8,8B7,9D3)secreting stable antibody were successfully obtained.The antibody titers were 100×2~8,100×2~7and 100×2~9,respectively.The heavy chain and light chain of 3F8 and8B7 McAbs were IgG1 subtype andκsubtype respectively;the heavy chain of 9D3strain was IgG2b subtype and the light chain wasλsubtype.The successful development of monoclonal antibodies against pdcov N protein provides valuable materials for the control of the disease,the study of protein function and the establishment of serological detection methods.