Study On Monoclonal Antibody Preparation And Tissue Profile Of Porcine Integrin αvβ6 As FMDV Receptor

Food-and-mouth disease (FMD) is a highly contagious disease of artiodactyl, which is caused by foot-and-mouth disease virus (FMDV). FMDV belongs to the aphthovirus of Picornaviridae and seven different serotypes of FMDV, namely O, A, C, SAT1, SAT2, SAT3 and Asia1. The first step of FMDV infects cells is to bind surface receptor of cellular membrane, it has been demonstrated that there are at least five receptors for FMDV, include integrinαvβ1,αvβ3,αvβ6,αvβ8 and heparan sulfate(HS).Recombinant plasmidporcine B612p was transformed into BL21(DE3) host cells, which contained gene of porcine integrinβ6 subunit (ligand-binding domain, LBD) as FMDV receptor, Expression of the recombinant plasmid was induced with IPTG, and resulted in a high level of protein expression.The monoclonal antibody(McAb) C4C7, E7C7, B8C9, C2D8 and B7B6 against porcine integrinβ6 subunit LBD as FMDV receptor were produced by fusing Sp2/0 myeloma cells with spleen cells of BALB/c mice immunied four times with purified porcine integrinβ6 subunit LBD recombinat fusion protein. Masculine hybridoma cell was cloned and sieved by limiting dilution assay and indirect Enzyme-linked immunsorbent assay (ELISA), McAb especially reacted with integrinβ6 subunit LBD recombinat fusion protein as determined by SDS-PAGE electrophoresis assay, indirect Enzyme linked immunsor-bent assay (ELISA), Western blot analysis and indirect immunofluoresence assay (IFA). Five hybridoma cell strains against porcineβ6 subunit LBD McAb with high affinity and specificity was prepared successfully, McAbs were secreted respectively as IgG2a (C4C7, E7C7, B8C9, B7B6) and IgG2b (C2D8)subtypes.Which will lay a foundation for further using of the McAb to detect expressing disposition of integrinαvβ6 in tissue.CHOK1 cell was stablly transfected by pcDNA3.1(+)-β6p eucaryon expression vector, expressing disposition of integrinβ6 subunit was detected by indirect immuno- fluoresence assay (IFA). Tissue profile of porcine integrinαvβ6 in porcine tongue, lip, trachea and so on, was detected by indirect immunohistochemistry assay(IHA) and IFA. The monoclonal antibody against porcine integrinβ6 subunit LBD as FMDV receptor as first antibody, tow IgG antibodies of goat against mice are respectively labelled by fluorescein isothiocyanate(FITC) and horseradish peroxidase(HRP) as secondary antibody. Results: Viridis fluorescence was appeared in cell membrane and cytoplasm of CHOK1 cell stablly transfected by pcDNA3.1(+)-β6p eucaryon expression vector, idio-positive reaction was appeared in cell membrane and cytoplasm of porcine tongue, lip, trachea and so on, which indicated integrinαvβ6 receptor was generally expressed in porcine tissue cell, which will lay a foundation for further understanding of the roles of integrinαvβ6 in FMDV infection.