Anti-human c-met human-mouse chimeric monoclonal antibody and its application
A monoclonal antibody, expression vector technology, applied in application, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. Problems such as the weakening of anti-drug efficacy
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[0055] In the present invention, the preparation method of the anti-human c-Met human-mouse chimeric monoclonal antibody preferably comprises the following steps:
[0056] 1) The 1D20 heavy chain variable region, the 3C36 heavy chain variable region and the 3D63 heavy chain variable region are double-digested with BamHI and XbaI, respectively, to obtain the 1D20 heavy chain variable region and the 3C36 heavy chain variable region. Variable region and cleaved 3D63 heavy chain variable region;
[0057] The plasmid vector pcDNA3.1(+) was double digested with BamHI and XbaI to obtain the linearized plasmid vector pcDNA3.1(+);
[0058] Ligate the enzyme-cut 1D20 heavy chain variable region, enzyme-cut 3C36 heavy chain variable region and enzyme-cut 3D63 heavy chain variable region into the linearized plasmid vector pcDNA3.1(+) to obtain the recombinant plasmid pcDNA3.1- H;
[0059] 2) The 1D20 light chain variable region, the 3C36 light chain variable region and the 3D63 light ch...
Embodiment 1
[0070] Mouse immunity and titer detection
[0071] Immunize 5 female Balb / c mice for 6-8 weeks, and each mouse is immunized with human c-met extracellular antigen (NP_000236) (Met 1-Thr 932) labeled with HIS tag each time, the dose is 50 μg, and the first immunization will The immunogen and an equal volume of complete Freund's adjuvant were used to make an emulsifier, and the abdomen was injected subcutaneously in multiple points; the same dose of immunogen was taken at intervals of 2 to 3 weeks to make an emulsifier with an equal volume of incomplete Freund's adjuvant, and the abdomen was subcutaneously injected in multiple points injection. One week after the three immunizations, blood was taken to determine the serum titer. Mice with qualified titers were given a booster immunization. Three days after the booster immunization, the spleens of the mice were frozen and stored in liquid nitrogen for later use.
[0072] One week after the last immunization, 50-60 μL of blood wa...
Embodiment 2
[0074] Biopanning variable region sequences
[0075] Total RNA was extracted from the spleen of mouse A immunized with c-Met extracellular antigen by the trizol method, and the murine antibody variable region gene was amplified with the murine antibody scFv gene amplification kit (Cat. No.: P001Z), and a segment composed of multiple The peptide linker Linker (SEQ ID No.13: SSGGGGSGGGGGGSSRSS) composed of glycine (Gly) and serine (Ser) connects the heavy chain and light chain variable regions of the antibody to form a single-chain antibody gene fragment scFv, and the scFv single-chain antibody gene fragment Cloned into the phagemid vector pCANTAB5E to construct the scFv phage display library, so that the single-chain antibody scFv could be displayed and expressed in the phage display library, figure 2 Schematic diagram of vector construction for phage exhibition. After plasmid extraction, the phage plasmids were electrotransformed to build a library with a storage capacity of...
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