The Structured And Expressed Of The Porcined Antibody As Well As The Detection Of The Activity Of The Recombinant Protein

Haemophilus parasuis (H. parasuis) that belongs to the Pasteurellaceae family is a Gram-negative,nonhemolytic, NAD-dependent bacterium. The organism is an important upper-respiratory-tract pathogen inswine and is the etiological agent of Glasser’s disease. This diesease is characterized by fibrinouspolyserositis, poly-arthritis, meningitis, arthritis syndrome, acute pneumonia without polyserositis, and acutesepticemia. In recent years, H. parasuis infection has been increasingly implicated as a major cause of nurserymortality in commercial swine herds, especially in specific-pathogen-free herds. PCR assay is the mainmethod to detect the disease.Currently, the vaccination and the antibiotics treatment were two main methods to prevent and controlGlasser’s disease. To date,15serotypes of this pathogen have been identified, with serotypes4and5beingprevalent among field isolates in many countries, but more than20%of strains are untypeable by currentserotyping methods. The currently available H. parasuis commercial inactivated vaccine provide protectiononly against challenge with a homologous serotype but not with a heterologous serotype.The potential of monoclonal antibodies (mAbs) as drug treatments in human and animals has beenrecognizsd. Administering murine antibodies to humans or other animals is inadvisable as they may berecognized as foreign by immune system, resulting in an anti-antibody response which may lead toanaphylactic shock and/or immune compled disease. Therefore, the murine mAbs have limitations when theyare used in the clinic therapy. The use of chimeric human/mouse antibodies or humanized murine antibodiesor even fully human antibodies is a better prospect for antibody therapy.In our study, we have built on our previous studies with the murine mAb1D8that provided thecross-protections among15serotypes of H. parasuis to construct the chimeric antibodies in the baculovirusexpression system.. Each of the genes encoding VH and VL of MAb1D8was amplified from the cDNA ofhybridoma cells that produce MAb1D8. The genes of swine IgG-2b constant regions (CH and CL) wereseparately amplified from the cDNA of lymphocytes that were isolated from swine PMBC. The variableregions from murine1D8MAb and constant regions from swine IgG-2b were spliced into the whole H and Lchains of chimeric antibody using fusion PCR. The chimeric H and L chain genes were constructed and werecloned into pFastBac-dual vector that has two promoters and cloning sites. The chimeric antibody wasexpressed in the baculovirus system. After mixing and incubating the antibody protein that expressed withHPS, we evenly coat them with the TSA medium. Finding that antibody protein has a very good antibacterialeffect in vitro and the effect is of up to80-90%. In the process of antibacterial test in vivo, the negativecontrol mice all died, the positive control and the recombinant protein mice are50%survival rate.Thechimeric antibody was expressed and showed the ability of inhibiting bacterial growth. This study provided anew alternative strategy for infectious disease control in domestic pigs.