Generation Of Monoclonal Antibody Against PRRSV HeB108 Strain And Establishment Of IFA Method

Porcine reproductive and respiratory syndrome(PRRS)is an important infectious disease that seriously endangers the pig industry.The pathogen of the disease was first isolated in China in 1996.Since 2012,a NADC30-like strain with high homology with the US PRRSV NADC30 strain has emerged in China.In recent years,this strain has gradually become widespread throughout the country and has become the main PRRSV epidemic strain in China.Prompt detection,diagnosis and control of PRRS is an important means to curb its prevalence.Monoclonal antibodies have become the first choice for the specific diagnosis and detection of NADC30-like PRRSV strains due to their high specificity and unlimited ability to produce antibodies.Therefore,it is very necessary to develop the monoclonal antibody against NADC30-like strain..In this study,ultracentrifugation and cesium chloride density gradient centrifugation were used to purify the NADC30-like PRRSV He B108 virus and use it as an antigen to immunize BALB/c mice.The mouse serum was collected 7 days after the third immunization,and the antibody titer in the serum was detected by IFA method.The mouse with the highest serum antibody titer after immunization was selected,under aseptic conditions,the splenocytes from the mouse spleen and SP2/0 were taken to cell fusion,the fusion cells were seeded in a 96-well plate,the cell supernatant was taken after 10 days of cultured,the positive cell wells were screened by the ELISA method,and the positive hybridoma cell lines were further verified by the IFA method.After three subcloning,8 positive hybridoma cell lines that stably secrete anti-PRRSV monoclonal antibodies were successfully screened,named 2D7,5B4,9G5,12F1,12G4,15G7,15D7 and 17F1,respectively.The 8 positive hybridoma cell lines obtained were identified by Western blot,and it was found that the hybridoma cell lines 9G5,12F1,2D7,12G4 and 5B4 could react with the PRRSV whole virus,and the hybridoma cell lines 15G7,15D7 and 17F1 had no obvious specific bands,suggesting that they might be conformational epitopes..Subsequently,the constructed eukaryotic expression plasmid p CMV-N targeting PRRSV N protein was used to identify the recognized protein by IFA test.The results showed that m Ab 5B4 could specifically recognize PRRSV N protein In.Then,the monoclonal antibody 5B4 cell supernatant was used as the primary antibody,and Goat anti mouse Ig G FITC fluorescent antibody was used as the secondary antibody.After optimizing the reaction conditions,the IFA detection method has good specificity and sensitivity,which is suitable for clinical detection of PRRSV.In conclusion,eight hybridoma cell lines secreting PRRSV Mc Abs were obtained by immunizing mice with purified PRRSV heb108 virus.The eight Mc Abs reacted with PRRSV with good specificity.Among them,monoclonal antibody 5b4 could recognize PRRSV N protein.An IFA detection method for PRRSV N protein was established by using this Mc Ab.This study laid the foundation for further exploring the structure and function of PRRSV,and provided technical support for clinical diagnosis and prevention of PRRSV.