Gene of human vascular endothelial growth factor monoclonal antibody and use thereof
A technology of monoclonal antibody and vascular endothelium, applied in the direction of anti-growth factor immunoglobulin, application, genetic engineering, etc., can solve the problems of cell line instability, high cost, easy loss, etc., and achieve difficult preservation and high production cost , the effect of small molecules
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Embodiment 1
[0037] Example 1: Isolation of variable region genes of antibodies from VEGF165 monoclonal cell line
[0038] Collect the hybridoma cell line ABL-110 that grows well and can secrete VEGF165 monoclonal antibody 7 One (constructed by our laboratory, according to "Current protocol in immunology" Production of Monoclonal Antibodies), the total RNA was extracted with TRIZOL reagent.
[0039] RT-PCR catch VH gene:
[0040] With reference to the method provided by Promega (Access RT-PCRSystem and Access RT-PCRIntroductory System): one-step RT-PCR kits were used to amplify the VH gene:
[0041] The VH gene uses primer VHBack (5'-AGGTSMARCTGCAGSAGTCWGG-3') and primer VHFor (5'-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3'),
[0042] The RT-PCR reaction is a total of 50 μl system: 1 μg of total RNA, 10 μl of 5×AMV / Tf1 reaction buffer, 5 μl of primers VH Back and VH For at a concentration of 10 pmol, 1 μl of dNTP at a concentration of 500 uM, and 3 mM MgSO 4 2 μl; 1 μl of 5u AMV reverse trans...
Embodiment 2
[0052] Example 2: Assembly of scFv genes
[0053] To obtain scFv, add a flexible linker between VH and VL (G 4 S) 3 , in order to prevent the introduction of mutations during the amplification process, we use the high-fidelity enzyme Pyrobest (Dalian Bao Biology). The whole assembly schematic diagram is figure 2 , the result is as figure 1 .
[0054] The first round of PCR reaction to amplify the VH:
[0055] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; the concentration of 10 μM primer VH P 1 and VH P 2 2.5 μl: 4 μl dNTPs; 35 μl HO 2 O; High-fidelity Pyrobest enzyme 0.5 μl. Denaturation at 95°C for 5min; denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 26 cycles; extension at 72°C for 5min.
[0056] The second round of PCR reaction amplifies VL:
[0057] The 50 μl PCR reaction system is as follows: 5 μl of 10× reaction buffer; primer VL P at a concentration of 10 μM 1 and VLP 2 2.5 μl: 4...
Embodiment 3
[0060] Example 3: Enzyme digestion and connection of scFv gene
[0061] Double digestion of pET28a:
[0062] EcoRI and HindIII are Takara products. 2 μl of 10×M reaction buffer; 10 μl of vector pET28a; 1 μl each of EcoRI and HindIII; 7 μl of H 2 O. React at 37°C for 6 hours. :
[0063] The third round of amplification product in double enzyme digestion embodiment 2:
[0064] 2 μl of 10×M reaction buffer; 10 μl of the third-round PCR product; 1 μl each of EcoRI and HindIII; 7 μl of H 2 O. React at 37°C for 6 hours. 1% agarose gel electrophoresis, and the product was recovered by tapping the gel.
[0065] Link reaction:
[0066] T4 ligase is a product of Takara. 2 μl of ligation reaction buffer H 2 O; 1 μl each of the products of the last round of enzyme digestion; 16 μl H 2 O. React at 16°C for 12 hours.
[0067] Take 10 μl of the product to transform into competent DH5α. Plate, pick a single clone the next day, identify it by colony PCR, and send it to Shanghai B...
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