CH2 fragment mutant of human antibody IgG and application thereof
A technology for human antibodies and mutants, applied in applications, antibody mimics/scaffolds, biochemical equipment and methods, etc., can solve problems such as poor stability, weak anti-aggregation ability, and reduced drug efficacy
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0018] Embodiment 1: candidate clone screening
[0019] Before screening candidate clones, it is necessary to construct a C-terminal phage display library of CH2. The construction method is the same as the existing Chinese patent application document (201810525234.6), and the sequence of CH2 is also disclosed in this patent.
[0020] 1. Panning experiment
[0021] 1) Connect 5 tubes of TG1 bacteria, 2 mL each. Shake to OD in about 4 hours 600 = 0.6.
[0022] 2) The phage library was first heated at 80°C for 10 minutes, and then left at room temperature for 20 minutes.
[0023] 3) Closed Panning orifice plate
[0024] a) Aspirate the coated protein and wash the well plate once with PBS;
[0025] b) Add phage to BSA wells, 5 wells with 10 wells in total 12 1, the final concentration of milk was 2%, 100 μL per well. Add 3% milk to anti-CH2 wells, 200 μL per well;
[0026] c) Block for 1 h at 37°C.
[0027] 4) combine
[0028] BSA well phages were transferred to anti-CH2...
Embodiment 2
[0056]Example 2: CS-20-1, CS-20-2 molecular conformation and existing form (monomer, dimer, etc.)
[0057] AKTA analysis of CS-20-1 and CS-20-2 existing forms: the purified CS-20-1 and CS-20-2 proteins were concentrated to 1 mg / mL, PBS (pH7.4) was used as the elution buffer, Pass through Column Superdex75 Increase 10 / 300GL, wherein the flow rate is 1mL / min, and then detect its existing form.
[0058] The result is as figure 2 As shown, the protein concentration of CS-20-1, CS-20-2 and CH2 is 1mg / mL, analyzed by ColumnSuperdex75 Increase 10 / 300GL (GE Company), and compared with the standard curve, it can be found that the protein molecular weight is about 14kDa, the result indicating that they exist as monomers.
[0059] CD detection of protein molecular conformation: Dilute CS-20-1 and CS-20-2 proteins to 0.3mg / mL, PBS (pH7.4) as a control, and use different wavelength conditions at the near ultraviolet end (wavelength λ=190nm~260nm) Detect its circular dichroism, and then...
Embodiment 3
[0061] Example 3: CH2 and CS-20-1, CS-20-2 stability and anti-aggregation comparison
[0062] Anti-aggregation analysis: adjust the final concentration of CH2 and CS-20-1, CS-20-2 protein to 1mg / mL, treat in a water bath at 50°C, take samples at regular intervals, detect the absorbance at λ=320nm and record data.
[0063] Compared with the anti-aggregation strength of CH2 protein, from Figure 4 It can be found that the signals of CS-20-1 and CS-20-2 rise slowly, indicating that their anti-aggregation ability is better.
PUM
| Property | Measurement | Unit |
|---|---|---|
| Concentration | aaaaa | aaaaa |
| Molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More