Single-chain antibody of fully-human anti-human interleukin-21 receptor and application thereof
A single-chain antibody and human interleukin technology, applied in the field of bioengineering, can solve the problems of incomplete elimination of immunogenicity and reduction of therapeutic value
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Embodiment 1
[0035] Example 1 Screening of fully human anti-IL-21R single-chain antibody
[0036] Dilute the IL-21R extracellular region protein (purchased from Beijing Sino Biological Technology Co., Ltd.) to 100 μg / ml with coating buffer (100 mM Tris, NaCl 150 mM, pH 9.0), take 4 ml and add it to the immunotube, and wrap it at room temperature. overnight; the next day, discard the supernatant, and quickly wash the tube 3 times with PBS; fill the immunotube with 2% MPBS (PBS containing 2% skimmed milk), and block at 37°C for 2 hours; discard the blocking solution, and quickly wash with PBS tube 3 times; the phage antibody library (10 12 ~10 13 p.f.u) (Dana library, donated by Harvard Medical School) was suspended in 4ml 2% MPBS and added to the immunotube. After repeated inversion at room temperature for 30 minutes, it was allowed to stand at room temperature for more than 90 minutes; the supernatant was discarded and replaced with PBS containing 0.1% Tween-20 Wash the tube 10 times, th...
Embodiment 2
[0037] Example 2 Soluble expression and separation and purification of anti-IL-21R single-chain antibody
[0038] Select the correctly sequenced TG1 strain, collect phagemids, infect and express Escherichia coli HB2151 according to routine operations (antibody drug engineering, P51), culture to OD600nm≈0.6, add IPTG at a final concentration of 0.1mM, induce overnight at 20°C, 12% SDS -PAGE detection of induced expression results.
[0039] Centrifuge at 6000rpm at 4°C for 5min to collect the cells; resuspend the cells in PBS, add 1mmol / L phenylmethylsulfonyl fluoride, and ultrasonically break (ultrasonic 2s, interval 2s, 10min in total); centrifuge at 12000rpm at 4°C for 20min, collect clear; supernatant nickel affinity chromatography column (purchased from GE), with different concentrations of imidazole elution; 12% SDS-PAGE detection of purified results (see Figure 4 ), the target protein was stored at -20°C, named C2, its amino acid sequence was SEQ ID NO: 1 and SEQ ID NO:...
Embodiment 3
[0040] Western Blot identification of embodiment 3 anti-IL-21R single chain antibody
[0041] Purified C2 was subjected to denaturing SDS-PAGE electrophoresis with a separation gel concentration of 12%; 4°C, 100mA constant current transfer for 2 hours, and the protein was transferred to a PVDF membrane (purchased from Millipore); after the transfer, the membrane was transferred at 5% Block overnight at 4°C in MTBS (TBS containing 5% skimmed milk); dilute anti-His mouse antibody (purchased from Millipore) with 5% MTBS at 1:2000, incubate at 37°C for 1.5h, wash with TBS 3 times, each time for 5min; Dilute HRP-anti-Mouse secondary antibody (purchased from Lianke Biotech) with 5% MTBS at 1:5000, incubate at 37°C for 1.5h, wash with TBS 3 times, 5min each time; use DAB to develop color. (See Figure 5 )
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