The Research Of Dog-mouse Chimeric Antibody For Monoclonal Antibody

Canine Parvovirus(Canine parvovirus, CPV) is the member of parvovirus Parvovirus families, the viru has no virus particles enveloped and nucleocapsid is an isometric icosahedral symmetry, CPV is a strong resistance for the outside world, CPV is only a antigenic types—CPV-2, The disease has a strong infectiousness, rapid onset, severe illness and high mortality is characteristic of the disease, the spread of the disease is by direct or indirect contact between the dog. The incidence of dogs have two phenotypes: myocarditis and enteritis. The main clinical manifestations of Enteritis is severe vomiting and bloody diarrhea; The main feature of myocarditis is respiratory and cardiovascular failure. The treatment of CPV has no effects of drugs, the use of clinical treatment, symptomatic treatment and supportive therapy is a main therapy. Specific therapy is by injection hyperimmune serum and monoclonal antibody, the infection of monoclonal antibody early is a significant treatment in the CPV, however murine monoclonal antibody in clinical induce immune response in the heterologous host, reduce its effect, and even induce severe immune reactions. Therefore it is necessary to transform murine monoclonal antibody, In this study, we developed a detection of canine immunoglobulin G(Ig G) of double sandwich ELISA method, Then construct the recombinant plasmid of chimeric antibody and express in the mammalian cell, Lay the foundation for stably expressing of canine- chimeric antibody cell lines. It provided support for antibody therapy of canine parvovirus in the future.In this study, rabbit anti-dog Ig G Fc segment polyclonal antibody as coating antibody, peroxidase-labeled rabbit anti-dog Ig G(whole molecule) polyclonal antibodies for the detection of antibodies, establish a sandwich ELISA quantitative detection method, and The Ig G from canine serum purified using Protein A was used as standards for ELISA detection. The established assay was specificity for detection of canine IgG, and had no cross-reaction with other animal sera such as mouse, rabbit, horse, cow, chicken and sheep; the detection limit of the assay was around 4.8 ng/m L of serum Ig G. It aims to provide a detection method for detecting canine Ig G and easy to post-detection dogs- mouse chimeric antibody screening of expression and developing stable expression dog- mouse chimeric antibody cell lines.In this study, heavy and light chain variable region was amplified successfully from the hybridoma, amplified sequences had antibody function at the IMGT website, heavy chain and light chain constant region of canine Ig G was amplified from canine peripheral blood mononuclear cells(PBMC), and carried out by NCBI Blast analysis it is more than 99% xompared with canine Ig G heavy and light chain gene homology, and splicing chimeric antibody sequences by overlap extension PCR, it was cloned into pc DNA3.1(+) vector, the recombinant plasmid transfected CHO cells, The expression of chimeric antibody was detected by ELISA and himeric antibody was purified successfully. The study provided experimental support for dog- the expression method of canine-mouse recombinant chimeric antibody, It yet laid a material foundation for identification of antibody potency and function and the establishment of stably transfected cell lines in the future.