METHOD FOR PRODUCING RECOMBINANT IGA2m1 ISOTYPE IMMUNOGLOBULIN IN MAMMALIAN CELLS Russian patent published in 2023 - IPC C12N15/13 C12N15/63 C12N5/10 C07K16/10 

FIELD: biotechnology.

SUBSTANCE: recombinant plasmid DNA pBiPr-ABIgA2m1P4A1-Intht is described, encoding a neutralizing monoclonal antibody P4A1 against the SARS-CoV-2 virus of the IgA2m1 isotype, containing an intronized heavy chain gene of the antibody with the nucleotide sequence SEQ ID NO:2 encoding the amino acid sequence of SEQ ID NO:1, which is under the control of the hEF1-HTLV promoter with the nucleotide sequence of SEQ ID NO:6 and the polyadenylation signal of bovine growth hormone BGH polyA with the nucleotide sequence of SEQ ID NO:7, the antibody light chain gene with the nucleotide sequence of SEQ ID NO:4 encoding the amino acid sequence of SEQ ID NO:3, which is under the control of the CMV promoter with the nucleotide sequence of SEQ ID NO:5, the polyadenylation signal of herpes virus thymidine kinase of antibody chains, and selective th marker that ensures the selection of transfected eukaryotic cells, while the hEF1-HTLV and CMV promoters are placed in a sequence that provides unidirectional transcription. A method for obtaining a eukaryotic cell line CHO DG44/pBiPr-ABIgA2m1P4A1-Intht, a producer of a neutralizing monoclonal antibody P4A1 to the SARS-CoV-2 virus of the IgA2m1 isotype by transfection of Chinese hamster ovary CHO cells with the described recombinant plasmid DNA is also described. Eukaryotic cell line CHO DG44/pBiPr-ABIgA2m1P4A1-Intht, producer of a neutralizing monoclonal antibody P4A1 to the SARS-CoV-2 virus of the IgA2m1 isotype is presented. A method for obtaining a neutralizing P4A1 monoclonal antibody to the SARS-CoV-2 virus of the IgA2m1 isotype is also presented, including: culturing the indicated cell lines in a nutrient medium, isolating the resulting target P4A1 antibody to the SARS-CoV-2 virus of the IgA2m1 isotype from the culture liquid.

EFFECT: as a result of transfection of CHO DG44 cells with the constructed recombinant plasmid pBiPr-ABIgA2m1P4A1-Intht, subsequent cultivation on a growth medium and selection of colonies resistant to methotrexate, cell cultures were obtained that stably produce neutralizing P4A1 monoclonal antibodies to the SARS-CoV-2 virus of the IgA2m1 isotype into the culture fluid with a yield of 16.6 mcg per ml of conditioned medium.

6 cl, 4 dwg, 6 tbl, 4 ex