Recombinant antibody and preparation method thereof
A technology for recombining antibodies and heavy chains, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antibodies, etc., can solve the problems of affecting drug efficacy, different immunogenicity of antibodies, and easy development of drug resistance in patients. The effect of significantly inhibiting the effect
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Embodiment 1
[0025] Example 1 Preparation of recombinant antibody
[0026] To express the recombinant antibody of the present invention, refer to the method of Wood et al., J Immunol. 145:3011 (1990), etc., in CHO cells, a monoclonal antibody that specifically binds to the extracellular region of CD20. The expression vector containing the antibody gene was constructed by conventional molecular biology methods (optiCHO antibody express system was purchased from Invitrogen Company). The host cell was a cell line derived from CHO-k1 cells (ATCC CCL61). To construct a high-yielding stable cell line, the steps are as follows: host cells were grown in suspension in CD-CHO medium (Gbico, CA), the host cells in the logarithmic growth phase were centrifuged, and the suspension was re-growth in fresh CD-CHO medium , count, and adjust the cell density until 1.43 × 10 8 cells / ml, take 400 μl of the above cell suspension into the electric shock cup, then add 20 μg of the linearized plasmid, and pipet...
Embodiment 2
[0027] Example 2 Recombinant antibody anti-tumor experiment
[0028] The anti-tumor effect of the recombinant antibody was evaluated by the growth inhibitory effect of human A431 tumor cells in nude mice. A brief description is as follows: A431 cells (ATCC, CRL-7907) were grown in DMEM medium containing 13% fetal bovine serum and supplemented with 3mM glutamine. Collect A431 cells and resuspend in PBS so that 100 μl volume contains 4×10 8 Cells, 5-6 weeks old female nude mice were injected into the right axilla with 100 μl of the above cell suspension. When the tumor volume reaches 80-150mm 3 At the beginning of group administration, 20 rats in each group. The administration volume of the buffer solution was 100 μl, once every three days for a total of 8 administrations, and the administration method was intraperitoneal injection. The recombinant antibody was administered once every three days for a total of 8 times at a dose of 5 mg antibody per kilogram of body weight. ...
Embodiment 3
[0029] Example 3 Experiment of Recombinant Antibody Inhibiting Endothelial Cell Proliferation in Vitro
[0030] Inhibition of endothelial cells by recombinant antibody was used to evaluate its anti-tumor effect. The specific operation is as follows: Add growth factors (BulletKit, Lonza) and 12% fetal bovine serum (purchased from Sigma) to endothelial cell basic medium (DMEM, Sigma) to prepare endothelial cell growth medium. When human umbilical vein endothelial cells (HUVEC, ATCC, Cat No: CRL-1730) grew to a dense monolayer, 5×10 4 The density of cells / well was seeded in a 96-well plate, and no growth factors were added to the assay medium at this time. The positive control antibody was Avastin, and the recombinant antibody was added in a certain dilution gradient. After incubation for 1 hour, the recombinant antibody was added to make the final concentration 10ng / mL. After 4 days of cultivation in a 37°C, 5% CO2 incubator. 10 μL of MTT (Invitrogen) was added to each well a...
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