| Immunoglobulin A (IgA), as the principal antibody class in the secretions that bathe the mucosal surfaces, acts as the first line of defence. So far there is no attempt using secretory IgA against H5N1 virus in animal respiratory tract or digestive tract. 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.In order to clone the genes of SIgA subunits with"Genomic DNA splicing"technique established in our lab, highly efficient exon primers were designed to amplify the exons directly from genomic DNA extract. An overlapping PCR was then performed with manually designed overlapping primers to join adjacent exons together to form a full-length coding sequence. The full-length PCR products were purified and ligated with pGEM-T Easy Vector. After transformation, clones were screened and positive clones were subjected to sequencing. Sequence analysis confirmed that the cloned sequences were identical to the relative entries of the GenBank database.The variable region of the heavy chain (VH) and light chain (VL ) genes of an anti-H5N1 neutralizing monaclonal antibody (McAb) HA9 were cloned by RT-PCR. Sequence analysis indicated that the Vk and VH were derived from mouse immunoglobulin genes, and the heavy chain variable region includes 414 base pairs, with the first 57 nucleotides encoding a signal sequence. VH, D-gene and J gene were derived from mouse antibody gene IgHVâ… ,IgHVâ…£and IgHVâ…£respectively. The light chain variable region includes 393 base pairs, with the first 60 nucleotides encoding asignal sequence. V region and J gene were derived from mouse antibody gene IgKVâ…¢and IgKJâ… respectively.Full length heavy and light chain genes of chimeric IgA were constructed by fusing VH and VK 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 CMV early promoter, and DHFR gene and NEO were used as the selectable marker. 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-IGHA9 and pEF-IGK9 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-IGHA9, pEF-IGK9, pcDNA3.1(+)-IgJ and pcDNA3.1(+)-SC were transfected into the CHO/dhfr- cells, and secretory IgA in cultured cell supernatants harvested at 48-72 hours were detected by ELISA.In Conclution, this study successfully cloned human secretory IgA subunit genes IGHA2, IgJ, pIgR/SC and 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. |
