Whole human anti-human interleukin-17A antibody and application thereof
A human interleukin, fully human-sourced technology, applied in the field of biomedicine, can solve problems such as inability to effectively cause CDC
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Embodiment 1
[0076] Example 1 Affinity Maturation of Antibodies
[0077] 1.1 Preparation of yeast electrotransformation competent
[0078] 1) From a fresh YPD plate, pick EBY100 yeast (Invitrogen TM ) monoclonal in 5mL YPD medium. Incubate overnight at 30°C.
[0079] 2) Inoculate the overnight culture into 50 mL of YPD medium, the initial concentration is OD600=0.1, and cultivate at 30°C until OD600=1.3-1.5, and the process takes about 6 hours.
[0080] 3) When the cells are cultured to OD600=1.3-1.5, add 500 μL of Tris-DTT buffer to the culture medium. Shake in a shaker at 30°C for 15 minutes.
[0081] 4) Centrifuge the culture at 2500 g for 3 minutes at 4°C, and gently resuspend the culture with 25 mL of ice-cold 1 M sorbitol to wash the cell pellet. Centrifuge, wash the cell pellet by gently resuspending in 1 mL of ice-cold 1M sorbitol, and centrifuge.
[0082] 5) Resuspend the cell pellet with ice-cold double-distilled water to a final volume of 300 μL, and place it on ice for la...
Embodiment 2
[0140] Example 2 Full-length antibody protein expression and purification
[0141] 1) 293FT suspension cells
[0142] Need to maintain 4×10 5 to 3×10 6 Cells / mL concentration range, usually 25mL cultured in a 125mL Erlenmeyer flask, shaker speed 120rpm, 37°C, 8% CO 2 .
[0143] 2) 24 hours before transfection cells, according to 1×10 6 cells / mL and cultured overnight, the next day the cell concentration should reach 2×10 6 cells / mL.
[0144] 3) On the day of transfection, count the cells and resuspend the cells by centrifugation to make the concentration 2.5-3.0×10 6 cells / mL.
[0145] 4) Transfer 10 mL of resuspended cells to a new 125 mL Erlenmeyer culture flask, confirm the cell concentration again, and place the cells in a cell culture incubator for culture.
[0146] 5) The gene fragments of the light chain and heavy chain of the antibody are digested by Xho1 and BamH1, and T4 enzyme is connected to the PTT5 expression vector. After the sequencing is correct, the ...
Embodiment 3
[0153] Example 3 Antibody neutralization experiment in vitro
[0154] IL-17A can induce and stimulate HT1080 (ATCC) to produce cytokine IL-6. In this experiment, the neutralizing activity of anti-IL-17A antibody was detected by IL-17A-induced HT1080 cells to produce IL-6 activity.
[0155] 1) Human fibrosarcoma cell line (HT1080) was trypsinized in a 96-well plate. Cells were counted so that there were approximately 9000 cells in each well, and cultured overnight in MEM medium containing 10% fetal bovine serum.
[0156] 2) Dilute the recombinant protein IL-17A (recombinant IL-17A antigen from PeproTech, Cat. No.: #200-17) with sterile PBS to 10 nM.
[0157] 3) The purified recombinant antibodies 9NT.NS and 9NT (Example 1) were serially diluted with sterile PBS, respectively: 1350nM, 337.5nM, 84.38nM, 21.09nM, 5.27nM, 1.32nM, 0.33nM, 0.08nM, 0.02nM And ten gradients of 0.01nM, mixed with 10nM recombinant IL-17A protein, removed HT1080 cell culture supernatant, added antibody...
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