Preparation Of Monoclonal Antibodies Against Feline Parvovirus By Single B Cell Antibody Technique

Feline parvovirus(FPV),also known as feline panleukopenia virus,belongs to the family Parvovirus and is a member of Parvovirus.Cats infected by FPV show symptoms such as high fever,vomiting,hemorrhagic enteritis,diarrhea,and severe leukopenia.Cats are one of the most common companion animals and play an important role in providing people with emotional support.At present,the main treatment methods for feline parvovirus disease at home and abroad are symptomatic treatment and specific treatment based on mouse monoclonal antibodies,while mouse monoclonal antibodies are highly immunogenic and prone to immune rejection.Therefore,it is necessary to develop safe and effective monoclonal antibodies against FPV,to reduce mortality and losses,and to provide new genetically engineered antibodies for the clinical treatment of feline parvovirus disease.This study mainly includes the following aspects:1.Expression,labeling and identification of FPV VP2 proteinSf9 cells were infected with baculovirus of generation r Bac-FPV-VP2 P2 preserved by National Veterinary Medicine Technology Research Center,and the expression of FPV VP2 protein was confirmed by Western-blot and SDS-PAGE.The FPV VP2 protein was purified by 8% PEG 6000,and then purified by molecular sieve.Finally,the high-purity FPV VP2 protein(0.33mg/m L)was successfully purified,and the hemagglutination titer was 1: 217.Virus-like particles(VLPs)with a diameter of about 24 nm were observed under the electron microscope,which was similar to the morphology of natural FPV,and the VP2 protein was successfully labeled with biotin.2.Selection of cat single B cells secreting FPV antibody and determination of antibody sequence from catsThe experimental cats were immunized with FPV inactivated vaccine once every21 days,for a total of 3 times.Serum was collected every 7 days from the 14 th day after the first immunization,and the humoral immune response level of the experimental cats was detected by indirect ELISA,HI and virus neutralization test.During the immune cycle,the HIghest binding titer of ELISA serum was 1: 1024000,the highest hi titer was 1: 8192,and the highest neutralization titer was 1: 22727.3. Venous blood was collected from cats with high humoral immune response at 28 days after the third immunization,and peripheral blood mononuclear cells(PBMCs)were isolated.With the biotin-labeled FPV VP2 protein as capture antigen,120 CD21+/CD27+/ Ig G+/ FPV VP2+ memory B lymphocytes were sorted by flow cytometry.The heavy chain and light chain variable region genes of cat Ig G were amplified from a single B cell by single cell PCR and sequenced,and finally 53 pairs of light and heavy chain variable region gene sequences were obtained.3.Expression and identification of whole cat antibodyThe constant region sequence of cat antibody was searched on NCBI and IMGT official website,and finally the constant region sequence of cat antibody heavy chain Ig G1 a and light chain λ was selected.Seventeen genes of heavy chain variable region and light chain variable region were selected and spliced with the constant region sequences of cat antibody heavy chain Ig G1 a and light chain λ,respectively,and were entrusted to Nanjingjinsirui Science & Technology Biology Corp to construct expression plasmids.The heavy chain and light chain expression plasmids were co-transfected into Chinese hamster ovary cells(CHO)to express and purify the antibody.The reactivity of the antibody was verified by indirect ELISA,indirect immunofluorescence test(IFA)and Western-blot,and the neutralization activity of the antibody was verified by virus neutralization test.Finally,all the 17 antibodies were successfully expressed,and 10 monoclonal antibodies with FPV specificity were screened out.Among them,8 antibodies can specifically bind to FPV VP2 protein by Western-blot,and 4 antibodies can recognize FPV by IFA.Only E4 and G4 antibodies have neutralization activity,and the neutralization titer is 1:10 and 1:11.2 respectively.In summary,this study first established a platform for preparing all-cat monoclonal antibodies based on flow sorting of single B cells.By using this technique,two strains of neutralizing antibodies against FPV were successfully screened,both of which can recognize FPV VP2 protein by Western-blot.The study laid a foundation for the subsequent development of FPV neutralizing antibody,and also provided a tool for the subsequent development of all-cat antibody.