Preparation Of Neutralizing Antibody Against GP3 Protein Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome(PRRS)is one of the diseases currently endangering the global pig breeding industry,caused by PRRS virus(PRRSV)infection.Symptoms include anorexia and fever in sows,respiratory disorders,miscarriage,stillbirth and mummified fetuses in late pregnancy,and respiratory diseases and mass deaths in piglets.The disease caused huge losses to my country's pig industry.Due to the lack of effective drugs at present,the immune effect of existing vaccines is not ideal,especially the recombination of vaccine strains and wild viruses to form new mutant strains,which makes the prevention and control of the disease extremely difficult.Neutralizing antibody,as a kind of antibody that kills virus accurately and efficiently,plays an important role in diagnosis and treatment in veterinary clinic.There are many viral proteins in PRRSV that can induce the body to produce neutralizing antibodies.Among them,the structural protein GP3 contains neutralizing antibody epitopes,and GP3 is considered to be related to the body's protective immunity.In this study,by preparing GP3 monoclonal antibody,studying its virus neutralizing activity,and expressing a single-chain antibody with neutralizing activity,it provides a favorable tool for further research on PRRSV,has important application value,and lays the foundation for the prevention and control of PRRS.This research is divided into three parts:(1)Prokaryotic expression,purification and renaturation of PRRSV GP3 proteinFirst,the GP3 gene of PRRSV TA-12 strain was cloned into the pET-32a empty vector using the method of double digestion and ligation.After transforming DH5?competent cells,the PCR-positive plasmid of the bacterial liquid was extracted and verified by double digestion,pET-32a-GP3 recombinant plasmid.After identification by sequencing,it was transformed into BL21 competent cells and induced to express.After verification and analysis by SDS-PAGE gel electrophoresis and protein gel Coomassie brilliant blue staining,the PRRSV GP3 recombinant protein was insoluble and expressed,with a size of about 35 kDa and then passed through Ni-The GP3 recombinant protein was purified by NTA affinity chromatography,and the purified PRRSV GP3 recombinant protein was renatured by urea gradient dialysis to detect the protein concentration.An immunogen for immunizing mice is thus obtained.(2)PRRSV GP3 protein monoclonal antibody preparation and virus neutralizationThe obtained PRRSV GP3 recombinant protein was renatured by gradient dialysis,and the renatured GP3 protein was verified by SDS-PAGE gel electrophoresis and protein gel Coomassie brilliant blue staining.The protein concentration was detected by BCA method.Balb/C mice were immunized.After three immunizations,blood was collected from the tail vein of the mice.The serum was collected to detect the serum antibody level of the mice by indirect ELISA test.After the mouse serum reached the cell fusion condition,the mice were boosted.Mouse splenocytes and cultured SP2/0 mouse myeloma cells were fused by chemically adding fusion agent polyethylene glycol(PEG).After 14 days of cell fusion,the expressed GP3 protein was used as the antigen,and the ELISA enzyme-labeled wells were coated.The supernatant of the fusion cells was detected by indirect ELISA test,and the positive hybridoma cells that could secrete antibodies were screened.After three subcloning,both cells were obtained.Two hybridoma cells that can secrete antibodies and can be passaged indefinitely were named GP3-8B7 and GP3-7B3.The specificity of these two GP3monoclonal antibodies was verified by indirect immunofluorescence assay(IFA)and Western Blot assay.Two hybridoma cells of GP3 antibody were injected into Balb/C mice according to the corresponding number of cells to prepare GP3 monoclonal antibody.The obtained two GP3 monoclonal antibodies were stored in an ultra-low temperature environment of-80?.The amount of virus per cell well was fixed at 0.1 MOI.The GP3 monoclonal antibody was diluted 2 times,the virus was mixed with antibodies of different dilutions in a ratio of 1:1,and the mixture was incubated at 37? for 1 h.The cells were incubated at 37? for 2 h,At the same time,set up controls(the negative serum stored before immunization of mice was diluted by 2 times,mixed with virus to infect cells)and(positive serum stored before fusion of mice was diluted by 2 times,mixed with virus to infect cells).After 2 hours,the maintenance solution was replaced,and the cytopathic changes were observed day by day.After about 3 days,the cell state was stable.The fluorescence was observed by IFA test to detect the effect of GP3 monoclonal antibody on PRRS virus neutralization.The results showed that the negative serum did not neutralize the virus.,GP3 mouse positive serum has the effect of neutralizing the virus with a titer of 2~3.The virus neutralization titers of the two monoclonal antibodies of GP3-8B7 and GP3-7B3 are both 2~5.(3)Preparation of GP3 single-chain antibody and virus neutralization of antibodyAntibodies are minimized in order to make them more readily available in large quantities.By amplifying the variable region light chain and heavy chain in the GP3monoclonal antibody,artificially adding a linker(Linker)to connect the light chain variable region(L)and heavy chain variable region(H)of the GP3 monoclonal antibody with pET-28a-sumo empty vector was constructed into a recombinant gene.After double-enzyme digestion verification and sequencing results comparison,the GP3-8B7 and GP3-7B3 single-chain antibody genes were finally successfully constructed.The protein was obtained by prokaryotic expression.The molecular weight of the two GP3 single-chain antibody proteins was 54 kDa.SDS-PAGE gel electrophoresis confirmed that it was an insoluble protein,and it was renatured by gradient dialysis method,and its protein concentration was detected by BCA method.The obtained two GP3 single antibody proteins were purified and renatured,verified by SDS-PAGE gel electrophoresis,and their protein concentrations were detected by BCA method.Using a GFP-tagged PRRSV strain,the amount of virus per cell well was fixed.The protein was mixed with PRRS virus in equal proportion after 2-fold dilution,and the same operation was performed as the virus neutralization test for detecting monoclonal antibody.The cells gradually stabilized at about 3 d,and were directly observed under an inverted fluorescence microscope.The results showed that the virus neutralization titer of GP3-8B7single chain antibody was 2~3,and the virus neutralization titer of GP3-7B3 single chain antibody was 2~4.In this study,two neutralizing antibodies against PRRSV GP3 protein were obtained,and the single-chain antibodies of these two monoclonal antibodies were expressed in prokaryotic cells.The prepared single-chain antibodies had good neutralizing activity,laying the foundation for the prevention and control of PRRS.