Construction Of A CDNA Library Of Chicken Embryo Fibroblast And Preparation Of The Monoclonal Antibody That Can Inhibit Infectious Bursal Disease Virus Infection

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease, a highly contagious immunosuppressive disease which causes an acute disease characterized by high morbidity and mortality to 3 to 6 week chickens. IBDV replicates specifically in developing B lymphocytes(preB lymphocytes)in the bursal of Fabricius (BF). During replication, viral proteins induce apoptosis, resulting in a rapid depletion of B lymphocytes and the lesions of the BF as the necrosis of the BF with subsequent inflammation and atrophy of that organ. Surviving chickens as well as younger chickens that are infected with IBDV generally show no clinical signs, but may be immunosuppressed during their remaining lifetimes. As a result it causes significant losses to the poultry industry.Generally, field isolates (virulent strains) of IBDV propagates only in chicken lymphocytes but not in chicken embryo fibroblast (CEF). With successive blind passages in CEF and vero cells the virus becomes progressively adapted to growing in both cells. Adaptation of wild-type IBDV, however, always seems to correlate with attenuation. So there may be different molecules act as the receptor of IBDV or corelative elements with IBDV infection existing on CEF and Vero cells and lymphocytes which is associated with the adapted IBDV and very virulent IBDV infection, respectively. They play the crucial role in the course of IBDV infection by mediating the virus binding and penetration.In order to do further research on IBDV receptor and relative elements mediating IBDV infection, we have done series of works they are as follows: First, we constructed a cDNA eukaryotic expression library of CEF and established a simple method to prepare high efficient electrocompetent cells of E. coli strain DH10B. Then on the basis of immunocytochemistry we set up a method that is named as the number of infection positive cell reduction test in which the biotin–streptavidin systerm is applied. It is used to screen the monoclonal antibody (mAb) that can blocks IBDV infection. With the mathod we selected three strains of mAb that can inhibate IBDV infection.At last we identified some charactors of the mAb. As a result the protective ratio of the best one is up to 84.5%. It also has the character of does dependent in infection inhibation. By flow cytometry we definated that the molecular what the mAb is against is on the surface of CEF. It indicated that the molecular plays an important role in the course of IBDV infection. Maby it is one of the IBDV receptors or at least it is a relative element in IBDV infection. Because the mAb can not inhibated IBDV infection completely, we speculate that another receptors or molculars also exsist that play an important role in IBDV infecion. What we have done have settled the basis to diclose the gene of IBDV receptor on the surface of CEF or to reveal the mechanism of IBDV infection.