Amyloid protein oligomer conformation type epitope polypeptide and application thereof
A technology of amyloid protein and antigenic epitope, applied in the direction of peptides, metabolic diseases, nervous system diseases, etc., can solve the problems of patient death, achieve good immunogenicity, and eliminate side effects
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Embodiment 1
[0023] A polypeptide (PEOZ for short), whose amino acid sequence is: Cys-Ser-His-Ala-Leu-His-Asn-Asn-Cys, or one or more amino acids are substituted, deleted, or added to the above-mentioned amino acid sequence, And it has the function of inhibiting the aggregation of amyloid protein. The invention can effectively stimulate the body to produce a specific antibody for recognizing the amyloid oligomer, and the antibody can inhibit the aggregation and cytotoxicity of the amyloid protein.
[0024]The N-terminus of the polypeptide was modified with PEG2000 (sequence: PEG-Cys-Ser-His-Ala-Leu-His-Asn-Asn-Cys), and alanine was deleted (sequence: Cys-Ser-His-Leu -His-Asn-Asn-Cys), replacement of serine with alanine (sequence: Cys-Ala-His-Ala-Leu-His-Asn-Asn-Cys) or insertion of alanine (sequence: Cys-Ser -His-Ala-Leu-His-Asn-Asn-Cys), the test shows that the PEOZ polypeptide can still retain the ability to bind to A11 after certain modifications, a certain amino acid is replaced, dele...
Embodiment 2
[0034] Example 2: Binding of PEOZ to polyclonal antibody A11
[0035] Coat 100 μl of PEOZ (1 mg / well) on a 96-well ELISA microplate (with no PEOZ-coated wells as a control), place at 37°C for 2 h, block with 3% BSA at 37°C for 2 h, 200 μL / well. Wash the plate 3 times with PBS. Add 100 μl A11 (1:500) to each well, incubate at room temperature for 1 h, and wash 3 times with PBS containing 0.1% Tween-20. After incubation at room temperature for 1 h, the cells were washed 3 times with PBS containing 0.1% Tween-20. Add 100 μl diluted goat anti-rabbit HRP enzyme-labeled secondary antibody (1:5000) to each well, incubate at room temperature for 1 h, and wash 3 times with PBS containing 0.1% Tween-20. Add 100 μL of substrate solution TMB to each well, place at 37°C for 20 min, then add 50 μl of 1 mmol / L sulfuric acid to each well to stop the reaction, and measure the OD value (wavelength 450 nm) on an enzyme-linked immunosorbent detector. figure 1 The indicated PEOZ can be specifi...
Embodiment 3
[0036] Example 3: PEOZ with substituted, deleted or inserted amino acids still binds to A11
[0037] The N-terminus of PEOZ was modified with PEG2000 (referred to as PEG-PEOZ) (sequence: PEG-Cys-Ser-His-Ala-Leu-His-Asn-Asn-Cys), and alanine was deleted (referred to as: PEOZ- ?A) (sequence: Cys-Ser-His-Leu-His-Asn-Asn-Cys), serine replaced by alanine (referred to as PEOZ-S / A) (sequence: Cys-Ala-His-Ala -Leu-His-Asn-Asn-Cys) or inserted alanine (abbreviation: PEOZ-G) (sequence: Cys-Ser-His-Ala-Leu-His-Asn-Asn-Cys). In order to detect the binding of the modified polypeptides to A11, these polypeptides were combined with the unmodified PEOZ with a histidine tag to compete with A11, and then ELISA was used to detect the binding of the latter to A11. Add 100 ml of the mixture of 1 mg unmodified tagged PEOZ and 10 mg modified PEOZ into the wells coated with A11, then use ELISA to detect the combination of tagged PEOZ and A11, and measure the OD value, see results figure 2 . It s...
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