An anti-vascular endothelial growth factor antibody and its fusion protein with hepcidin
A technology of endothelial growth factor and fusion protein, which is applied in the field of single-chain antibody and fusion protein with hepcidin, can solve the problems that the anti-tumor effect is difficult to achieve satisfactory results, and it is difficult to ensure synergistic effect, so as to achieve enhanced anti-tumor role, broaden the effect of anti-tumor
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Embodiment 1
[0032] Example 1 Preparation of anti-VEGF bispecific antibody
[0033] 1.1 Establishment of high-capacity natural antibody library.
[0034] Isolation of human peripheral blood mononuclear lymphocytes: 100 healthy adults were randomly selected, and 10ml of peripheral blood was drawn from each person. Dilute 1:1 with RPMI-1640 medium containing 10% heparin, add it to a centrifuge tube containing lymphocyte separation medium (the volume ratio of diluted venous blood and lymphocyte separation medium is 2:1), 2,000 × g, Centrifuge for 17 minutes. The milky white mononuclear cell layer at the interface of lymphocyte separation medium was aspirated and washed twice with PBS buffer.
[0035] 1.2 Extraction of total cell RNA
[0036] per 5 x 10 6 Trizol reagent was added at the ratio of cells / ml, and the cells were lysed by pipetting. Incubate at room temperature for 5 minutes, transfer to a DEPC-treated EP tube, add 1 / 5 volume of chloroform, shake vigorously for 15 seconds, and ...
Embodiment 2
[0077] Example 2. Construction of fusion protein
[0078] Hepcidin DNA and selected linker DNA were biosynthesized and ligated to VEGF ScFv DNA using PCR techniques.
[0079] PCR reaction system:
[0080]
[0081] Add deionized water to a final volume of 50 μl.
[0082] PCR parameters: After denaturation at 94°C for 3 minutes, PCR was performed at 94°C for 30 seconds; 61°C for 30 seconds; After the reaction, 5 µl of the reaction product was taken for analysis by 1% agarose gel electrophoresis.
[0083] Ligation reaction reaction system:
[0084]
[0085] The above-mentioned reactants were thoroughly mixed and centrifuged to sink to the bottom of the tube, and then ligated at 4°C overnight.
[0086] Transformation of recombinant plasmids (the same steps as before).
[0087] Sequence analysis of positive cloned DNA fragments.
[0088] The positive clones were identified by bacterial liquid PCR, and 1 ml of bacterial liquid was taken to complete the sequencing by Nosei ...
Embodiment 3
[0103] Example 3. Inhibitory effect of fusion protein on the growth of different breast cancer cells
[0104] Use Transwell chamber (type 3428, pore size 8um, Corning-Costar, USA) to measure cell invasiveness in vitro (according to the operating instructions)
[0105] Preparation of conditioned medium (as chemokine): mouse fibroblast cell line NIH3T3, cultured with RPMI-1640 medium containing 10% calf serum at 37°C, 5% CO2, and replaced with serum-free when the growth is good After culturing the culture solution for 24 hours, the supernatant was collected, sterilized by filtration, and frozen for later use.
[0106] 200ul / well of extracellular matrix was coated on the bottom of the Transwell chamber and polymerized in a 37°C incubator for 1 hour.
[0107] 1 ml of high-glucose DMEM medium containing 0.1% BSA was added to the 6-well plate to hydrate the basement membrane at 37°C for 30 minutes.
[0108] Aspirate the culture medium and add 2.5ml of 1:1 conditioned medium and co...
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