Micromolecule antibody affinity peptide and applications thereof
A small molecule antibody and affinity peptide technology, applied in the field of molecular biology, can solve the problems of protein A being unstable, unusable, and increasing operational complexity.
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Embodiment 1
[0031] Example 1 Polypeptide Synthesis
[0032] 1) Activation of the resin: Weigh 1000mg of FMOC Cys-wang Resin, add 10-15mL of DMF and soak for 30min (soak all the resin), to make it fully swell.
[0033] 2) Deprotection: Press filtration to remove the DMF soaked in the resin, add 10mL of DMF solution containing 20% piperidine, blow and boil with nitrogen for 15min, then press filter to remove the DMF solution containing 20% piperidine, and remove the FMOC group that protects the amino group Wash the resin three times with 10mL isopropanol and three times with 10mL DMF, and then use the ninhydrin method to detect that the resin should turn black or purple.
[0034] 3) Condensation reaction: connect the first amino acid R 5 , the amount of FMOC-amino acid (His or Lys) weighed is 1.4mmol / g resin, 910mg O-benzotriazole-N,N,N′,N′-tetramethyluronium tetrafluoroboric acid (TBTU), Add 10mL DMF and 0.45g 1-hydroxybenzotriazole (HOBt) and mix well, then add 0.52mL DIEA to form a...
Embodiment 2
[0039] Example 2 Determination of IgG affinity characteristics
[0040] 1) Prepare 10mmol / L Hepes buffer, pH 7.4, NaCl content 150mmol / L.
[0041] 2) Use the liquid prepared in 1) as the mobile phase, and run the BiacoreT200 analyzer.
[0042] 3) Insert the detection chip CM5 and set the flow rate to 30 μL / min.
[0043] 4) Inject 100 μL each of EDC and NHS in sequence to activate the chip.
[0044] 5) Dissolve IgG in sodium acetate buffer, pH 4.5, IgG 100μg / mL.
[0045] 6) Inject 100 μL of the IgG solution in 5), and immobilize IgG on the surface of the chip.
[0046]7) Small molecule antibody affinity peptide 1 (amino acid sequence shown in SEQ ID No.11), small molecule antibody affinity peptide 2 (amino acid sequence shown in SEQ ID No.34) and natural ligand (ProG and ProA), dissolved in 1) to prepare liquid, content 100μg / mL.
[0047] 8) Sequentially inject different concentrations of peptides and natural ligands (concentrations from high to low are 500 μm, 250 μm, 125...
Embodiment 3
[0053] Example 3 Comparison of adsorption properties of small molecule antibody affinity peptides with IgG and HAS
[0054] 1) Prepare 10mmol / L Hepes buffer, pH 7.4, NaCl content 150mmol / L.
[0055] 2) Use the liquid prepared in 1) as the mobile phase, and run the BiacoreT200 analyzer.
[0056] 3) Insert the detection chip CM5 and set the flow rate to 30 μL / min.
[0057] 4) Inject 100 μL each of EDC and NHS in sequence to activate the chip.
[0058] 5) Dissolve IgG in sodium acetate buffer, pH 4.5, IgG 100μg / mL. Also dissolve HSA in sodium acetate buffer, pH 4.5, HSA 100 μg / mL.
[0059] 6) Inject 100 μL of the IgG solution in 5), and immobilize IgG on the surface of the chip.
[0060] 7) Inject 100 μL of the HSA solution in 5), and immobilize HSA on the surface of another chip.
[0061] 8) Dissolve small molecule antibody affinity peptide 3 (amino acid sequence shown in SEQ ID No.1) and small molecule antibody affinity peptide 4 (amino acid sequence shown in SEQ ID No.4) ...
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