Anti-vascular endothelial growth factor antibody and 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 it is difficult to guarantee the synergistic effect and the anti-tumor effect is difficult to achieve a satisfactory effect, and achieve enhanced anti-tumor effect, broaden anti-tumor methods, and reduce production costs
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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] Separation of human peripheral blood mononuclear lymphocytes: 100 healthy adults were randomly selected, and 10ml of peripheral blood was extracted from each person. Dilute 1:1 with RPMI-1640 culture medium containing 10% heparin, add to the centrifuge tube containing lymphocyte separation medium (the volume ratio of diluted venous blood to lymphocyte separation medium is 2:1), 2,000×g, Centrifuge for 17 minutes. Aspirate the milky white mononuclear cell layer at the interface of the lymphocyte separation solution, and wash twice with PBS buffer.
[0035] 1.2 Extraction of total cellular RNA
[0036] Press every 5×10 6 The ratio of cells / ml was added to Trizol reagent, 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 f...
Embodiment 2
[0077] Example 2. Construction of fusion protein
[0078] Hepcidin DNA and selected linker DNA were biosynthesized and linked to VEGF ScFv DNA using PCR technology.
[0079] PCR reaction 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; at 61°C for 30 seconds; at 72°C for 1 minute, for a total of 30 cycles, and finally at 72°C for 10 minutes. After the reaction, 5 μl of the reaction product was taken for 1% agarose gel electrophoresis analysis.
[0083] Ligation reaction reaction system:
[0084]
[0085] After the above reactants were well mixed and centrifuged to sink to the bottom of the tube, they were connected overnight at 4°C.
[0086] Transformation of the recombinant plasmid (the steps are the same as before).
[0087] Sequence analysis of DNA fragments of positive clones.
[0088] For the positive clones identified by...
Embodiment 3
[0103] Example 3. The inhibitory effect of fusion protein on the growth of different breast cancer cells
[0104] Use Transwell small chamber (type 3428, pore size 8um, US Corning-Costar Company) to measure cell invasion in vitro (perform according to the operating instructions)
[0105] Preparation of conditioned medium (as a chemokine): mouse fibroblast cell line NIH3T3, cultured with RPMI-1640 medium containing 10% calf serum at 37°C and 5% CO2, and changed to serum-free when the growth was good After culturing the culture solution for 24 hours, the supernatant was collected, sterilized by filtration, and frozen for future 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] Add 1 ml of high-sugar DMEM medium containing 0.1% BSA to the 6-well plate and hydrate the basement membrane at 37°C for 30 minutes.
[0108] Aspirate the culture medium, add 2.5ml of conditioned me...
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