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Humanization of Mouse Anti-bladder Cancer McAb by CDR3 Directed Phage Antibody Library

Author: ZhouLiJun
Tutor: WangZuo
School: PLA Military Academy of Medical Sciences
Course: Immunology
Keywords: Phage antibody library Mouse monoclonal humanized loxp-cre recombination system Bladder cancer
CLC: R392.11
Type: PhD thesis
Year: 2003
Downloads: 104
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Abstract


The subject based on antigen directional selection \First by RT-PCR method from bladder cancer monoclonal antibody secreting anti hybridoma BDI amplified mouse k chain , Fd segment gene , DNA sequence determination that belong Mκ Ⅳ , and MH3D subsets . K chain and Fd fragments cloned into the phage expression vector p3MH obtain expression in E. coli . Fd fragments of the N-terminal amino acid sequence correction by PCR-mediated site-directed mutagenesis , pro original sequence of the mouse Fab binding activity . The use of ELISA, competitive inhibition assay , immunohistochemistry experiments confirmed received Fab BDI 's Fab . Oligonucleotide primers containing the mouse CDR3 region of VH and VL combinations of the diversity of sources by overlapping PCR and recombinant DNA technology , the anti- bladder cancer Antibody BDI and CDR3 regions with human lymphocytes and then synthesized according to the sequence of mouse monoclonal build with BDI CDR3 primary human phage antibody library capacity of 2 × 10 7 < / sup> ; Recombination System loxp-cre , after cre < / sup > BS1365 bacterial intracellular positioning reorganization , increase the combination of light and heavy chain pairs , and end up with a capacity of 1 × 10 10 large capacity secondary phage antibody library . The four adsorption - elution - amplification , selection of 46 clones after the fourth round of screening by ELISA , 4 clone with EJ cells specific combination of bladder cancer cells (EJ) . Competitive inhibition experiments showed that three of the parental murine monoclonal antibody recognition epitopes . Determination of its variable region gene sequences , VH genus VH I VH II subgroup , Vk are subsets of Vk Ⅰ, Vk Ⅵ . The higher activity of clone bladder carcinoma Immunohistochemical experiments, show that the clone with cancer tissue -specific binding . In this study, mice CDR3 limited human antibody library received antibody library with a predetermined bias to favor specific antibody screening . Loxp-cre recombination system introduced phage antibody libraries , antibody libraries to build large-capacity . Without Antibody to do the template chain replace human antibody with mouse monoclonal antibody recognizing the same epitope .

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CLC: > Medicine, health > Basic Medical > Medical Immunology > Immunobiology > The biology of the immune molecules ( immunochemistry )
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