Secretory Iga Chimeric Antibody Expressed In Chinese Hamster Cells

Immunoglobulin A, as the principal antibody class in the secretions that bathe the mucosal surfaces, acts as the first line of defence. In this study we used Chinese hamster ovary (CHO) cells to express an anti-H5N1 chimeric SIgA in order to provide a passive immunological agents for avian influenza prophylaxis.We have extracted total RNA from secretion of monoclonal antibodies against H5N1-avian influenza.The variable region of the heavy chain (VH) and light chain (VL) genes of an anti-H5N1 neutralizing monaclonal antibody (McAb) HA 8 were cloned by RT-PCR. Sequence analysis indicated that the Vk and VH were derived from mouse immunoglobulin genes, and The light chain variable region includes 393 base pairs, with the first 60 nucleotides encoding asignal sequence.Ⅴregion and J gene were derived from mouse antibody gene IgKVⅢand IgKJⅠrespectively.The heavy chain variable region includes 414 base pairs.VH, J gene and D-gene were derived from mouse antibody gene IgHVⅠ, IgHVⅡand IgHVⅣrespectively. Full length heavy and light chain genes of chimeric IgA were constructed by fusing VK and VH with IGHA2 and CK, respectively. The chimeric antibody expression plasmids were constructed by inserting full length heavy and light genes into the multiple cloning sites of plasmid pEF-dhfr-Neo, with antibody gene controlled by DHFR gene and NEO were used as the selectable marker, and CMV early promoter. The IgJ and pIgR expression plasmids were constructed by inserting IgJ and pIgR into the multiple cloning sites of the pcDNA3.1 (+)with CMV early promoter and NEO as the selectable marker. At the same time.The full-length chimeric light and heavy chain expressing plasmids pEF-IGK8 and pEF-IGHA8 were transfected into the CHO/dhfr- cells, and cell clones were selected with selective medium (DMEM without hypoxanthine and thymine) and methotrexate (MTX). The recombinant chimeric antibodies were purified with protein L affinity chromatography from the cell culture supernatant. The chimeric IgA antibody expression was confirmed by ELISA, SDS-PAGE and Western blot. In order to express secretory IgA molecules, the expressing plasmids pEF-IGHA8, pEF-IGK8, were transfected into the CHO/dhfr- cells, and secretory IgA in cultured cell supernatants harvested at 48-72 hours were detected by ELISA.In a word, this study successfully cloned the mouse variable region of an anti-H5N1 neutrolizing McAb, and constructed the full-length chimeric human-mouse IgA antibody gene and their expression vectors, and finally expressed the chimeric IgA and SIgA antibody in CHO cells.