Modified polypeptide and use thereof

Abstract

The present invention belongs to the technical field of detection reagents, and relates to a modified polypeptide and the use thereof. The modified β amyloid protein polypeptide comprises peptide fragment I, peptide fragment II, and peptide fragment III located between the peptide fragment I and the peptide fragment II. The peptide fragment I contains a sequence as shown in SEQ ID NO: 9, the peptide fragment II contains a sequence as shown in SEQ ID NO: 18, and the peptide fragment III is composed of non-hydrophobic amino acids. Linker compounds are added between the peptide fragment I and the peptide fragment III, as well as between the peptide fragment II and the peptide fragment III. Compared with the original polypeptide, the modified polypeptide is reduced in terms of synthesis difficulty and cost, and improved in terms of stability. In terms of reaction activity with antibodies, the modified form is consistent with the original polypeptide, is a better raw material form for a calibrator of a diagnostic reagent, and is beneficial to development and wide application of a detection kit.

Description

A polypeptide modification and its application Technical Field

[0001] The present invention belongs to the technical field of detection reagents and relates to a polypeptide modification and application thereof. Background Art

[0002] Alzheimer's disease (AD), the most common form of dementia, is a common neurodegenerative disorder. With the aging population and increased life expectancy, AD has become one of the most serious threats to human health, following cancer, heart disease, and diabetes. Its continued development will pose a serious social problem. In recent years, the amyloid cascade hypothesis has gained increasing recognition among researchers. This hypothesis posits that excessive production of amyloid-β (Aβ) in the brain is the primary cause of AD. Aβ is formed by cleavage of the amyloid precursor protein (APP) by β-secretase and γ-secretase. It consists of 39-42 amino acid residues, is highly hydrophobic, and has the ability to self-aggregate. Studies have shown that Aβ1-40 (Aβ40) is present at high levels in the brains of AD patients, while Aβ1-42 (Aβ42) aggregates are highly toxic. Furthermore, changes in markers such as Aβ40 or Aβ42 / Aβ40 can be detected in the blood before AD patients develop clinical symptoms. As a continuously progressive disease, better treatment effects depend on earlier diagnosis. Early diagnosis and design and development of targeting Aβ are of great significance for the treatment of AD.

[0003] The development of detection reagents for Aβ42 and Aβ42 / Aβ40 markers not only faces the difficulty of preparing high-affinity and high-specificity antibodies, but also presents stability issues due to the high hydrophobicity and self-aggregation ability of the Aβ1-40 or Aβ1-42 calibrator peptides. Therefore, the amino acid sequence of the calibrator peptides is designed and modified. Adjusting the amino acid sequence without affecting the reactivity with the antibody while improving its stability will facilitate the wider application of the detection reagents.

[0004] Summary of the Invention

[0005] To optimize the stability of calibrator polypeptides for β-amyloid proteins such as Aβ1-40 and Aβ1-42, the present invention provides modified calibrator polypeptides. The resulting modified polypeptides can remain stable for a long time and their reactivity with antibodies is consistent with that of the original polypeptides. Therefore, they can be used to prepare AD diagnostic reagent calibrators or AD diagnostic kits.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention first provides a modified β-amyloid protein polypeptide, which includes peptide segment 1, peptide segment 2, and peptide segment 3 located between peptide segment 1 and peptide segment 2. Peptide segment 1 contains DAEFR (SEQ ID NO: 9), peptide segment 2 contains VGGVV (SEQ ID NO: 18), and peptide segment 3 is composed of non-hydrophobic amino acids. A connecting compound is added between peptide segment 1 and peptide segment 3, and between peptide segment 2 and peptide segment 3.

[0008] In a further embodiment, the amino acids in the modified β-amyloid protein polypeptide are all D-amino acids.

[0009] In a further embodiment, in the above-mentioned modified β-amyloid protein polypeptide, the amino acid sequence of peptide segment one may be DAEFR (SEQ ID NO: 9) or DAEFRH (SEQ ID NO: 10), the amino acid sequence of peptide segment two may be VGGVVIA (SEQ ID NO: 11), MVGGVVIA (SEQ ID NO: 12), GLMVGGVV (SEQ ID NO: 13) or IGLMVGGVV (SEQ ID NO: 14), peptide segment three contains D and E, and the amino acid sequence of peptide segment three may specifically be DEDEDEDEDEDE (SEQ ID NO: 15) or DDDEDDDEDDDE (SEQ ID NO: 16), and the non-hydrophobic linking compound may be (PEG4)3, (CH2)6 or GGGS (SEQ ID NO: 17).

[0010] In a further embodiment, the number of amino acid residues in the modified polypeptide is less than that in the original sequence of the β-amyloid protein polypeptide. The structure of the modified polypeptide is a straight chain.

[0011] The present invention further provides modified Aβ1-42 polypeptides. The modified Aβ1-42 polypeptides include Aβ42-V1, Aβ42-V2, and Aβ42-V3. The modified Aβ42 polypeptides Aβ42-V1, Aβ42-V2, and Aβ42-V3 are modified from the original Aβ42 polypeptide having the amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (shown in SEQ ID NO: 1).

[0012] Furthermore, the amino acid sequence of the modified Aβ42 polypeptide Aβ42-V1 is DAEFR-(PEG4)3-DEDEDEDEDEDE-(PEG4)3-VGGVVIA (shown in SEQ ID NO: 3), and the amino acid molecular weight is 2832.

[0013] Furthermore, the amino acid sequence of the modified Aβ42 polypeptide Aβ42-V2 is DAEFRH-(CH2)6-DDDEDDDEDDDE-(CH2)6-MVGGVVIA (shown in SEQ ID NO: 4), and the amino acid molecular weight is 3117.

[0014] Furthermore, the amino acid sequence of the modified Aβ42 polypeptide Aβ42-V3 is DAEFRH-GGGS-DEDEDEDEDEDE-GGGS-MVGGVVIA (shown in SEQ ID NO: 5), and the amino acid molecular weight is 3705.

[0015] Furthermore, the preparation method of the modified Aβ42 polypeptides Aβ42-V1, Aβ42-V2, and Aβ42-V3 comprises the following steps: chemically synthesizing the amino acid sequences of the modified Aβ42 polypeptides using a peptide solid-phase synthesis method to obtain the complete sequences; and then desalting the resulting products using HPLC reverse-phase column chromatography to obtain the modified Aβ42 polypeptides.

[0016] The present invention further provides modified Aβ1-40 polypeptides. The modified Aβ1-40 polypeptides include Aβ40-V1, Aβ40-V2, and Aβ40-V3. The modified Aβ40 polypeptides, Aβ40-V1, Aβ40-V2, and Aβ40-V3, are modified from the original Aβ40 polypeptide having the amino acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (shown in SEQ ID NO: 2).

[0017] Furthermore, the amino acid sequence of the modified Aβ40 polypeptide Aβ40-V1 is DAEFR-(PEG4)3-DEDEDEDEDEDE-(PEG4)3-GLMVGGVV (shown in SEQ ID NO: 6), and the amino acid molecular weight is 3001.

[0018] Furthermore, the amino acid sequence of the modified Aβ40 polypeptide Aβ40-V2 is DAEFRH-(CH2)6-DDDEDDDEDDDE-(CH2)6-IGLMVGGVV (shown in SEQ ID NO: 7), and the amino acid molecular weight is 3222.

[0019] Furthermore, the amino acid sequence of the modified Aβ40 polypeptide Aβ40-V3 is DAEFRH-GGGS-DEDEDEDEDEDE-GGGS-IGLMVGGVV (shown in SEQ ID NO: 8), and the amino acid molecular weight is 3810.

[0020] Furthermore, the preparation method of the modified Aβ40 polypeptides Aβ40-V1, Aβ40-V2, and Aβ40-V3 comprises the following steps: chemically synthesizing the amino acid sequences of the modified Aβ40 polypeptides using a peptide solid-phase synthesis method to obtain the complete sequences; and then desalting the resulting sequences using HPLC reverse-phase column chromatography to obtain the modified Aβ40 polypeptides.

[0021] In addition, the present invention also provides for the use of the aforementioned modified β-amyloid protein polypeptides for preparing a calibrator for an Alzheimer's disease diagnostic reagent. In specific applications, the Alzheimer's disease diagnostic reagent calibrator may comprise only a modified Aβ42 polypeptide or only a modified Aβ40 polypeptide, or the Alzheimer's disease diagnostic reagent calibrator may comprise both a modified Aβ42 polypeptide and a modified Aβ40 polypeptide. When using a modified Aβ42 polypeptide as a calibrator for an Alzheimer's disease diagnostic reagent, at least one of Aβ42-V1, Aβ42-V2, and Aβ42-V3 may be used. When using a modified Aβ40 polypeptide as a calibrator for an Alzheimer's disease diagnostic reagent, at least one of Aβ40-V1, Aβ40-V2, and Aβ40-V3 may be used.

[0022] Beneficial effects of the present invention:

[0023] Based on the original peptide sequences of amyloid β peptides, such as Aβ1-42 and Aβ1-40, this invention modifies the original amino acid sequence to obtain peptide variants with lower synthesis difficulty and cost, as well as improved stability. Modification methods include amino acid substitution and the addition of hydrophilic polymers, resulting in multiple peptide variants. Compared to the original peptides, the modified variants have lower synthesis difficulty and cost, while improving stability. Furthermore, their reactivity with antibodies is consistent with that of the original peptides, making them superior raw materials for diagnostic reagent calibrators, facilitating the development and widespread application of detection kits.

[0024] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0026] FIG1 is a calibration curve of Aβ42-original as a calibrator.

[0027] FIG2 is a calibration curve of Aβ42-V1 as a calibrator.

[0028] FIG3 is a calibration curve using Aβ42-V2 as a calibrator.

[0029] FIG4 is a calibration curve using Aβ42-V3 as a calibrator.

[0030] FIG5 is a calibration curve of Aβ40-original as a calibrator.

[0031] FIG6 is a calibration curve using Aβ40-V1 as a calibrator.

[0032] FIG7 shows the calibration curve of Aβ40-V2 as a calibrator.

[0033] FIG8 is a calibration curve using Aβ40-V3 as a calibrator. DETAILED DESCRIPTION

[0034] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] The SP2 / 0 mouse myeloma cells used in the examples were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, and BALB / c mice were purchased from Suzhou Hengxinchen Biopharmaceutical Co., Ltd.

[0036] Example 1 Preparation of Aβ42 polypeptide variants

[0037] Human beta-amyloid protein Aβ42 polypeptide contains 42 amino acid residues, and the original amino acid sequence is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA.

[0038] Based on the original amino acid sequence of the Aβ42 peptide, three variants of the Aβ42 peptide were designed using molecular engineering methods. The amino acid sequence of the variant Aβ42-V1 is DAEFR-(PEG4)3-DEDEDEDEDEDE-(PEG4)3-VGGVVIA, containing 24 amino acid residues; the amino acid sequence of the variant Aβ42-V2 is DAEFRH-(CH2)6-DDDEDDDEDDDE-(CH2)6-MVGGVVIA, containing 26 amino acid residues; and the amino acid sequence of the variant Aβ42-V3 is DAEFRH-GGGS-DEDEDEDEDEDE-GGGS-MVGGVVIA, containing 34 amino acid residues. All amino acids are D-amino acids.

[0039] The preparation of the above three Aβ42 polypeptide modifications is specifically as follows:

[0040] (1) Based on the amino acid sequence of the modified Aβ42 polypeptide, the entire sequence was synthesized using an automatic peptide synthesizer and desalted using HPLC reverse phase column chromatography;

[0041] (2) then determining the molecular weight of the peptide obtained in (1) using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry;

[0042] (3) The purity of the polypeptide purified in (1) was then identified using high performance liquid chromatography (HPLC).

[0043] The measurement results are:

[0044] The modified Aβ42 peptide, Aβ42-V1, has a molecular weight of 2832 amino acids and is a small linear peptide with a purity of >98%.

[0045] The modified Aβ42 peptide, Aβ42-V2, has a molecular weight of 3117 amino acids and is a small linear peptide with a purity of >98%.

[0046] The modified Aβ42 polypeptide Aβ42-V3 has an amino acid molecular weight of 3705, is a small linear peptide, and has a purity of >98%.

[0047] Example 2 Preparation of Aβ40 polypeptide variants

[0048] Human beta-amyloid protein Aβ40 polypeptide contains 40 amino acid residues, and the original amino acid sequence is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV.

[0049] Based on the original amino acid sequence of the Aβ40 peptide, three variants of the Aβ40 peptide were designed using molecular engineering methods. The amino acid sequence of the variant Aβ40-V1 is DAEFR-(PEG4)3-DEDEDEDEDEDE-(PEG4)3-GLMVGGVV, containing 25 amino acid residues; the amino acid sequence of the variant Aβ40-V2 is DAEFRH-(CH2)6-DDDEDDDEDDDE-(CH2)6-IGLMVGGVV, containing 27 amino acid residues; and the amino acid sequence of the variant Aβ40-V3 is DAEFRH-GGGS-DEDEDEDEDEDE-GGGS-IGLMVGGVV, containing 35 amino acid residues. All amino acids are D-amino acids.

[0050] The preparation of the above three Aβ40 polypeptide modifications is specifically as follows:

[0051] (1) Based on the amino acid sequence of the modified Aβ40 polypeptide, the entire sequence was synthesized using an automatic peptide synthesizer and desalted using HPLC reverse phase column chromatography;

[0052] (2) then determining the molecular weight of the peptide obtained in (1) using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry;

[0053] (3) The purity of the purified polypeptide obtained in (1) was then identified using high performance liquid chromatography (HPLC).

[0054] The measurement results are:

[0055] The Aβ40 peptide variant Aβ40-V1 has a molecular weight of 3001 amino acids and is a small linear peptide with a purity of >98%.

[0056] The Aβ40 peptide variant Aβ40-V2 has a molecular weight of 3222 amino acids and is a small linear peptide with a purity of >98%.

[0057] The Aβ40 polypeptide modified product Aβ40-V3 has an amino acid molecular weight of 3810, is a small linear peptide, and has a purity of >98%.

[0058] Example 3 Verification of the Reactivity between Modified Aβ42 Peptides and Antibodies

[0059] Three batches of BALB / c mice were immunized with Aβ(37-42) synthetic peptide (sequence: GGVVIA) and Aβ(1-5) synthetic peptide (sequence: DAEFR), three mice each time. Each mouse was immunized with 50 μg of antigen each time, for a total of three immunizations. After the booster immunization, the spleen cells of the immunized mice were aseptically taken and fused with SP2 / 0 mouse myeloma cells. The positive hybridoma cell lines were subcloned and screened three times to obtain two hybridoma cell lines that secreted specific monoclonal antibodies. Hybridoma cell line A specifically recognized the peptide Aβ(1-5) sequence (as an antibody for detecting Aβ40 and Aβ42), and hybridoma cell line B specifically recognized the peptide Aβ(37-42) sequence (as an antibody for capturing Aβ42). After one week of adaptive feeding, 10-12 week old BALB / c mice were intraperitoneally injected with the immunosuppressant liquid paraffin, 0.5 mL / mouse. Seven days later, hybridoma cells that stably secreted antibodies and were in good condition were inoculated in the abdomen, about 1×10 6 ~2×10 6 After 7-10 days, the mice were allowed to swell, and ascites were extracted and the supernatants were pooled. Protein G was used to purify the ascites from both mice, and the purified antibodies were subjected to SDS-PAGE for purity verification. The results showed bands at 50 kD and 25 kD for both antibodies, with grayscale analysis showing purities exceeding 90%. Titer verification of both antibodies revealed a titer of 1:128,000.

[0060] Reactivity was determined using ELISA. Specifically, microplates were coated with 0.1 μg / mL of the original Aβ42 peptide (Aβ42-original), the modified Aβ42-V1, Aβ42-V2, and Aβ42-V3 in carbonate buffer (pH 9.5) overnight at 4°C. Two-fold serial dilutions of the homemade Aβ42 capture antibody and Aβ42 detection antibody were then added to the microplates and reacted at 37°C for 1 hour. After washing, goat anti-mouse IgG-HRP (50 ng / mL) was added. After a 1-hour reaction, the plates were washed three times and the colorimetric readings were obtained. As shown in Table 1, the activities of the modified Aβ42-V1, Aβ42-V2, and Aβ42-V3 were consistent with those of the original Aβ42 peptide in the reaction with the Aβ42 capture antibody or detection antibody.

[0061] Table 1 Reactivity of Aβ42 polypeptide modifications with Aβ42 capture antibodies or Aβ42 detection antibodies

[0062] Example 4 Verification of the Reactivity between Modified Aβ40 Peptides and Antibodies

[0063] Three batches of BALB / c mice were immunized with Aβ(35-40) synthetic peptide (sequence: MVGGVV), 3 mice each time. Each mouse was immunized with 50 μg of antigen each time, for a total of 3 immunizations. After the booster immunization, the spleen cells of the immunized mice were aseptically taken and fused with SP2 / 0 mouse myeloma cells. The positive hybridoma cell lines were subcloned and screened 3 times to obtain a hybridoma cell line C that secreted a specific monoclonal antibody that specifically recognized the Aβ(35-40) sequence (as an Aβ40 capture antibody). After one week of adaptive feeding, 10-12 week old BALB / c mice were intraperitoneally injected with the immunosuppressant liquid paraffin, 0.5 mL / mouse. Seven days later, hybridoma cells that stably secreted antibodies and were in good condition were inoculated in the abdomen, about 1×10 6 ~2×10 6 After 7-10 days, the mice were allowed to swell, and ascites were extracted and the supernatants were pooled. Protein G was used to purify the ascites, and the purified antibodies were subjected to SDS-PAGE for purity verification. The results showed bands at 50 kD and 25 kD, with grayscale analysis showing purities exceeding 90%. Antibody titers were also confirmed at 1:128,000 for both antibodies.

[0064] Reactivity was determined using ELISA. Specifically, microplates were coated with 100 μL of 0.1 μg / mL of the original Aβ40 peptide (Aβ40-original), the modified Aβ40-V1, Aβ40-V2, and Aβ40-V3 in carbonate buffer (pH 9.5) at 4°C overnight. Two-fold serial dilutions of the homemade Aβ40 capture antibody and Aβ40 detection antibody were then added to the microplates and reacted for 1 hour at 37°C. After washing, goat anti-mouse IgG-HRP (50 ng / mL) was added, and the reaction was continued for 1 hour, followed by washing three times and colorimetric reading. As shown in Table 2, the modified Aβ40-V1, Aβ40-V2, and Aβ40-V3 exhibited the same activity as the original Aβ40 peptide in reactions with the Aβ40 capture and detection antibodies.

[0065] Table 2 Reactivity of Aβ40 polypeptide modifications with Aβ40 capture antibodies or Aβ40 detection antibodies

[0066] Example 5: Validation of polypeptide modifications as calibrator samples

[0067] Aβ42 detection system: The methodological mode is the double antibody sandwich method, and the detection instrument is the Cosma magnetic particle chemiluminescence analyzer. Specifically, 50 μL of biotin-labeled Aβ42 capture antibody, 50 μL of sample (calibrator or test sample of different concentrations), and 50 μL of Aβ42 detection antibody are added to the instrument in sequence. After reacting for 20 minutes, wash three times. The instrument sends the reaction mixture into the dark room, and adds 100 μL of chemiluminescence pre-excitation solution and 100 μL of chemiluminescence excitation solution in sequence for luminescence reaction, and finally records the luminescence intensity. Draw a calibration curve based on the concentration and luminescence intensity of the calibrator (as shown in Table 3). The Aβ42 content of the test sample is calculated based on the calibration curve and the luminescence intensity of the test sample. The linear range of the calibration curve is 1 to 1024 pg / mL. Figures 1-4 are the calibration curves of Aβ42-original, Aβ42-V1, Aβ42-V2, and Aβ42-V3 as calibrators, respectively, where the Y-axis represents the logarithm of the luminescence value and the X-axis represents the logarithm of the concentration of the Aβ42 calibrator.

[0068] Table 3

[0069] Aβ40 detection system: The methodological model is a double antibody sandwich method. Specifically, 50 μL of biotin-labeled Aβ40 capture antibody, 50 μL of sample (calibrator or test sample of different concentrations), and 50 μL of Aβ40 detection antibody are added to the instrument in sequence. After reacting for 20 minutes, the reaction is washed three times. The instrument sends the reaction mixture into a dark room, and 100 μL of chemiluminescent pre-excitation solution and 100 μL of chemiluminescent excitation solution are added in sequence for luminescence reaction. Finally, the luminescence value is recorded. A calibration curve is drawn based on the logarithmic value of the calibrator concentration and the logarithmic value of the luminescence value (as shown in Table 4). The Aβ40 content of the test sample is calculated based on the calibration curve and the luminescence intensity value of the test sample. The linear range of the calibration curve is 10-2430 pg / mL. Figures 5-8 are the calibration curves of Aβ40-original, Aβ40-V1, Aβ40-V2, and Aβ40-V3 as calibrators, respectively, where the Y-axis represents the logarithm of the luminescence value and the X-axis represents the logarithm of the concentration of the Aβ40 calibrator.

[0070] Table 4

[0071] Sample Validation: 50 samples from confirmed Alzheimer's disease (AD), 50 samples from patients with cognitive impairment (MCI), 50 samples from patients with normal cognition (CU), and 50 samples from other dementias (non-AD) were collected. Different peptide forms were used as calibrators to evaluate the consistency of sample test results. The results showed that the modified peptides, when used as calibrators, exhibited high consistency in sample detection performance compared to the original peptides, as shown in Tables 5 and 6.

[0072] Table 5

[0073] Table 6

[0074] Example 6 Stability Verification of Aβ42 Polypeptide Modifications as Calibrators

[0075] The original Aβ42 peptide and its modified peptide calibrants were stored at 2-8°C for 14 months, and their quality changes were assessed over the 14-month period. Calibrators were tested at 0, 1, 3, 6, 10, and 14 months, and the luminescence values ​​at each concentration were compared to the control (0 month). Stability was considered acceptable if the relative deviation was <15%. Table 7 shows that the original Aβ42 peptide began to show instability in the first month, with luminescence values ​​deviating from the control by -18.1 to -15.8%. Tables 8, 9, and 10 show that the three modified Aβ42 peptides met the stability criteria. After 14 months of storage, the luminescence values ​​of Aβ42-V1 and the control deviated by -0.3 to 0.9%, less than 15%. After 14 months of storage, the luminescence intensity values ​​of Aβ42-V2 and the control deviated by -1.0 to 0.8%, less than 15%. After 14 months of storage, the luminescence values ​​of Aβ42-V3 and the control deviated by -0.8 to 0.5%, less than 15%.

[0076] Table 7

[0077] Table 8

[0078] Table 9

[0079] Table 10

[0080] Example 7 Stability Verification of Aβ40 Polypeptide Modifications as Calibrators

[0081] The original Aβ40 peptide and its modified peptide calibrants were stored at 2-8°C for 14 months, and their quality changes were assessed over the 14-month period. Calibrators were tested at 0, 1, 3, 6, 10, and 14 months, and the luminescence values ​​at each concentration were compared to the control (0 month). Stability was considered acceptable if the relative deviation was <15%. The results in Table 11 indicate that the original Aβ40 peptide began to show instability in the first month, with luminescence values ​​deviating from the control by -16.0 to -17.0%. As shown in Tables 12, 13, and 14, the three modified Aβ40 peptides met the stability criteria. After 14 months of storage, the luminescence values ​​of Aβ40-V1 and the control deviated by -0.2 to 1.2%, less than 15%; after 14 months of storage, the luminescence values ​​of Aβ40-V2 and the control deviated by -0.1 to 0.8%, less than 15%; and after 14 months of storage, the luminescence values ​​of Aβ40-V3 and the control deviated by -0.5 to 0.1%, less than 15%.

[0082] Table 11

[0083] Table 12

[0084] Table 13

[0085] Table 14

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. A modified amyloid β protein polypeptide, characterized in that: The polypeptide modification includes peptide segment 1, peptide segment 2, and peptide segment 3 located between peptide segment 1 and peptide segment 2, wherein peptide segment 1 contains the sequence shown in SEQ ID NO: 9, peptide segment 2 contains the sequence shown in SEQ ID NO: 18, and peptide segment 3 is composed of non-hydrophobic amino acids. Connecting compounds are added between peptide segment 1 and peptide segment 3, and between peptide segment 2 and peptide segment 3.

2. The modified β-amyloid polypeptide according to claim 1, characterized in that The amino acids in the polypeptide modification are all D-type amino acids.

3. The modified β-amyloid protein polypeptide according to claim 1, characterized in that The amino acid sequence of the peptide segment 1 is shown in SEQ ID NO: 9 or SEQ ID NO:

10.

4. The modified β-amyloid polypeptide according to claim 1, characterized in that The amino acid sequence of the second peptide segment is shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:

14.

5. The modified β-amyloid polypeptide according to claim 1, characterized in that The peptide segment three contains amino acids D and E.

6. The modified β-amyloid polypeptide according to claim 5, characterized in that The amino acid sequence of peptide segment three is shown in SEQ ID NO: 15 or SEQ ID NO:

16.

7. The modified β-amyloid polypeptide according to claim 1, characterized in that The linking compound is (PEG4)3, (CH2)6 or as shown in SEQ ID NO:

17.

8. The modified β-amyloid polypeptide according to claim 1, characterized in that The number of amino acid residues in the polypeptide modification is less than the number of amino acid residues in the original sequence of the β-amyloid protein polypeptide.

9. The modified β-amyloid polypeptide according to claim 1, characterized in that The structure of the polypeptide modification is a straight chain.

10. The modified β-amyloid polypeptide according to claim 1, characterized in that The amyloid β polypeptide is the Aβ1-42 polypeptide shown in SEQ ID NO: 1, or the Aβ1-40 polypeptide shown in SEQ ID NO:

2.

11. The modified β-amyloid polypeptide according to claim 10, characterized in that: The amino acid sequence of the modified Aβ42 polypeptide is shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

12. The modified β-amyloid polypeptide according to claim 10, characterized in that: The amino acid sequence of the modified Aβ40 polypeptide is shown in SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

13. The modified β-amyloid polypeptide according to any one of claims 1 to 12, characterized in that: The preparation method of the modified β-amyloid protein polypeptide comprises: According to the amino acid sequence of the modified β-amyloid protein polypeptide, the polypeptide solid phase synthesis method is used for chemical synthesis to obtain the full sequence, and then HPLC reverse phase column chromatography is used for desalting to obtain the modified β-amyloid protein polypeptide.

14. Use of the modified amyloid β polypeptide according to any one of claims 1 to 12 in the preparation of a calibrator for an Alzheimer's disease diagnostic reagent.

15. An Alzheimer's disease diagnostic reagent calibrator, characterized in that: Prepared from the modified β-amyloid protein polypeptide according to any one of claims 1 to 12.

16. Use of the modified β-amyloid protein polypeptide according to any one of claims 1 to 12 in the preparation of a diagnostic kit for Alzheimer's disease.

17. A diagnostic kit for Alzheimer's disease, characterized in that: The invention comprises the modified β-amyloid polypeptide according to any one of claims 1 to 12.

18. A method for detecting the content of β-amyloid protein, characterized in that: When detecting the content of β-amyloid protein, the β-amyloid protein polypeptide modification according to any one of claims 1 to 12 is used as a calibrator.

19. A method for diagnosing Alzheimer's disease, characterized in that: First, the method according to claim 18 is used to detect the content of β-amyloid protein, and then auxiliary diagnosis of Alzheimer's disease is performed based on the content of β-amyloid protein.

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