Method for preparing homogeneous amyloid fiber

Abstract

The method comprises the following steps: dissolving beta-amyloid protein powder by using a sodium hydroxide solution, centrifuging, adding a proper amount of phosphate buffer solution into supernate, and centrifuging to prepare a protein monomer solution; oscillating and incubating at 37 + / -2 DEG C for 3-5 days, and observing that the protein is aggregated to form primary amyloid fibers; taking the primary amyloid fiber sample, carrying out ultrasonic treatment in a water bath, uniformly blowing, adding into a freshly prepared protein monomer solution, uniformly mixing, and carrying out oscillation incubation in a constant-temperature shaking table at 37 + / -2 DEG C for 3-5 days to prepare a second-generation amyloid fiber sample; according to the method, amyloid fiber samples from the third generation to the twelfth generation are prepared. And taking the prepared 12th-generation amyloid fiber sample as a seed, carrying out water bath ultrasonic treatment, uniformly blowing, adding into a freshly prepared amyloid protein monomer solution, and carrying out oscillation incubation in a constant-temperature shaking table at 37 + / -2 DEG C for 2-4 days to obtain the amyloid protein. The amyloid fiber prepared by the method has the characteristics of uniform particle size, high thermal stability and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of fiber preparation, and in particular relates to a method for preparing uniform amyloid fibers. Background Art

[0002] Amyloid fibrils are highly ordered fibrous aggregates formed by self-assembly of polypeptides or protein molecules, and they show great potential for application in the biomedical field. They are not only pathological markers of many diseases, but also key factors in the occurrence and development of diseases, and have a significant impact on the functions of tissues and organs. They have broad application prospects in the early diagnosis and intervention treatment of diseases, the design and precise delivery of targeted drugs, and the construction of disease models and mechanism analysis. For example, the FDA-approved drugs aducanumab (J. Sevigny, P. Chiao, T. Bussière, PH Weinreb, L. Williams, M. Maier, R. Dunstan, S. Salloway, T. Chen, Y. Ling, J. O'Gorman, F. Qian, M. Arastu, M. Li, S. Chollate, MS Brennan, O. Quintero-Monzon, RH Scannevin, HM Arnold, T. Engber, K. Rhodes, J. Ferrero, Y. Hang, A. Mikulskis, J. Grimm, C. Hock, RM Nitsch, A. Sandrock, Nature, 2016, 537, 50-56) and lencanizumab (CH van Dyck, CJ Swanson, P. Aisen, RJ Bateman, C. Chen, M. Gee, M. Kanekiyo, D. Li, L. Reyderman, S. Cohen, L. Froelich, S. Katayama, M. Sabbagh, B. Vellas, D. Watson, S. Dhadda, M.Irizarry, LD Kramer, T. Iwatsubo, N. Engl. J. Med., 2023, 388, 9-21), can selectively bind to amyloid deposits in the brains of patients with Alzheimer's disease and clear the deposited proteins in the brain by activating the immune system. Therefore, it shows significant efficacy in patients with early Alzheimer's disease. Aducanumab has been shown to reduce amyloid plaque deposition in the brain and has shown a protective effect on cognitive function in some studies. During an 18-month treatment cycle, lencanerumab slowed the rate of cognitive decline in patients with early Alzheimer's disease by 27%.

[0003] However, the self-assembly process of proteins is a complex and delicate dynamic balance, which is regulated by a variety of environmental factors. This process produces a variety of different aggregates, and these aggregates show a wide range of differences in morphology, structure, and function. These aggregates provide both rich information and considerable challenges in biomedical research. Aducanumab and Lencanerumab also face similar problems. Since it can target and recognize a variety of different forms of aggregates, there are differences in its specificity for binding to aggregates of different forms, resulting in its ability to target and recognize highly toxic amyloid aggregates that is not optimal. This difference in binding will not only weaken the actual efficacy of the drug in the body, making it difficult to fully exert its expected therapeutic effect, but may also pose a potential threat to the safety of the drug. Therefore, in the development and application of such targeted drugs, how to optimize their binding specificity and improve the accuracy of targeted binding to reduce side effects and enhance drug efficacy has become a key issue that needs to be solved urgently. Through in vitro regulation and condition optimization, amyloid fiber samples with high uniformity and stability are screened out, and then highly specific targeted drugs are designed based on the amyloid fibers. This can effectively avoid potential side effects caused by nonspecific binding, effectively slow down the rate of cognitive decline, and provide patients with new treatment options.

[0004] Based on this, this application was developed. Summary of the invention

[0005] The present invention aims to overcome the defects of the prior art and provide a method for preparing highly uniform amyloid fibers. The method screens and prepares amyloid fiber samples with high uniformity and stability through in vitro regulation and condition optimization. The prepared amyloid fibers have the characteristics of uniform particle size and high thermal stability.

[0006] To achieve the above object, the present invention adopts the following technical solution: A method for preparing uniform amyloid fibers comprises the following steps: 1) Dissolve the freeze-dried β-amyloid protein powder in sodium hydroxide solution, centrifuge at 4°C, 10000-14000 rpm for 3-5 minutes, and separate the supernatant; add an appropriate amount of phosphate buffer to the supernatant, transfer to a centrifugal filter, centrifuge at 4°C, 3000-6000 rpm for 3-5 minutes, and the filtrate is the protein monomer solution; 2) Place the prepared protein monomer solution in a constant temperature shaker at 37±2°C and 160–300 rpm for 3–5 days to observe that the protein has aggregated to form primary amyloid fibers; 3) Take the above-mentioned primary amyloid fiber sample, ultrasonicate it in a water bath for 3-5 minutes at room temperature, blow it evenly and mix it with the freshly prepared protein monomer solution, then incubate it at 37±2°C and 160-300 rpm with constant temperature shaking, and prepare the second generation amyloid fiber sample after 3-5 days; 4) Pretreating the second generation amyloid fiber sample according to the method in step 3) above, and then preparing the third to twelfth generation amyloid fiber samples; wherein, for the third to sixth generations, each generation of amyloid fiber samples is incubated under shaking conditions at 37±2°C and 160–300rpm for 1–2 days, and for the seventh to twelfth generations, each generation of amyloid fiber samples is incubated under shaking conditions at 37±2°C and 160–300rpm for 4–8 hours; the twelfth generation fiber sample obtained by transmission electron microscopy is an amyloid fiber with uniform morphology, and the twelfth generation amyloid fiber sample is retained for later use; 5) The prepared 12th generation amyloid fiber sample was used as a seed, and ultrasonicated in a water bath for 3-5 minutes at room temperature. After being blown evenly, it was mixed with the freshly prepared protein monomer solution, and then incubated at 37±2℃ and 160-300 rpm for 2-4 days to obtain highly uniform amyloid fibers.

[0007] Specifically, in step 1), sodium hydroxide solution can be added to the freeze-dried β-amyloid protein powder, and the final concentration after the protein is dissolved is 15-25 mg / mL.

[0008] Further preferably, 50-100 mM sodium hydroxide solution can be added to 1 mg of freeze-dried β-amyloid protein powder, and the final concentration after protein dissolution is 18-22 mg / mL.

[0009] Specifically, in step 1), an appropriate amount of 10–50 mM phosphate buffer can be added to the supernatant to make the final protein concentration 50–200 μM.

[0010] Further preferably, in step 1), the pH of the phosphate buffer is 7.2-7.5, and contains 0.01-0.02% sodium azide.

[0011] Specifically, in step 3), the primary amyloid fiber sample can be added to the freshly prepared protein monomer solution at a volume percentage of 2-10% after being blown uniformly.

[0012] Specifically, in step 5), the 12th generation amyloid fiber sample can be added to the freshly prepared protein monomer solution at a volume percentage of 2-10% after being blown uniformly.

[0013] As a preferred embodiment, the method for preparing highly uniform amyloid fibers comprises the following steps: 1) Add 50 mM sodium hydroxide solution to 1 mg of freeze-dried β-amyloid protein powder. The final concentration of the protein after dissolution is 20 mg / mL. Centrifuge at 13000 rpm for 3 minutes at 4°C to separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide) to the supernatant to make the final protein concentration 100 μM. Transfer the above solution to a centrifugal filter and centrifuge at 6000 rpm for 3 minutes at 4°C. The filtrate is the protein monomer solution; 2) The prepared protein solution was transferred to a vial, placed in a constant temperature shaker, and incubated at 37°C and 200 rpm for 4 days. Transmission electron microscopy showed that the protein had aggregated to form primary amyloid fibers. 3) Take the above-mentioned first-generation amyloid fiber sample, ultrasonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). After mixing the two, transfer them to a vial and incubate them in a 37°C constant temperature shaker at 200 rpm. After 4 days, the second-generation amyloid fiber sample was prepared; 4) According to the above fiber pretreatment method, the second generation fiber sample was subjected to water bath ultrasonic treatment to prepare the third to twelfth generation amyloid fiber samples. Among them, for the third to sixth generations, each generation of samples was incubated at 37°C, 200 rpm for 1 day. For the seventh to twelfth generations, each generation of samples was incubated at 37°C, 200 rpm for 6 hours. The twelfth generation fiber sample obtained by transmission electron microscopy was an amyloid fiber with uniform morphology, and the twelfth generation fiber sample was retained for later use; 5) The prepared 12th generation amyloid fiber sample was used as a seed, ultrasonicated in a water bath for 5 minutes at room temperature, and then added to the freshly prepared protein monomer solution at a ratio of 2-10% (v / v) after being blown evenly. The solution was incubated in a constant temperature shaker at 37°C for 2-4 days to prepare highly uniform amyloid fibers.

[0014] The invention provides uniform amyloid fibers prepared by the method.

[0015] The method of the present invention is applicable to the preparation of common amyloid fibers, such as α-synuclein fibers, β-amyloid protein fibers and islet amyloid protein fibers. This paper takes the preparation of amyloid fibers from β-amyloid protein, a key protein of Alzheimer's disease, as an example to prove that the method is practical and efficient. Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The present invention prepares an amyloid fiber sample with high uniformity and stability through in vitro regulation and condition optimization. The prepared amyloid fiber has the characteristics of uniform particle size and high thermal stability. These characteristics endow the amyloid fiber prepared by this method with the characteristics of further application, greatly expanding its application range in the fields of biochemistry and chemistry.

[0016] 2. The present invention provides a new method for preparing amyloid fibers, which enriches the understanding of the structure of amyloid fiber protein, while ensuring its stability, and expanding the application prospects of amyloid protein in biochemistry, pharmacy, brain science and other fields.

[0017] 3. The present invention fibrillates β-amyloid protein, a key protein in Alzheimer's disease, which is an effective and promising protein modification strategy to change its morphological characteristics. It is expected to develop a detection method for such pathological protein fibers, providing a new idea for the clinical diagnosis of the corresponding diseases.

[0018] 4. The present invention prepares an amyloid fiber with uniform particle size and high stability after iterative incubation treatment of β-amyloid protein, a key protein of Alzheimer's disease, thereby improving the comprehensive utilization value of β-amyloid protein and having great application potential in the fields of pharmacy and diagnostic medicine.

[0019] 5. The present invention has low production cost, simple operation, and the raw materials used are cheap and easy to obtain. The present invention modifies the protein to a high value, greatly improving the application value of amyloid fibers in the fields of pharmacy and diagnostic medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The dynamic light scattering data of the amyloid fibers prepared according to the present invention show that the particle size of the amyloid fibers prepared by this method is uniform, which is 1101 nm; Figure 2 The circular dichroism spectrum data of the amyloid fiber prepared according to the present invention, the left figure is the circular dichroism spectrum of the amyloid fiber at 20°C (the lowest temperature during the measurement process) and 90°C (the highest temperature during the measurement process), and the right figure depicts the concentration changes of species that contribute to the secondary structure of the amyloid fiber under different temperature conditions. It was determined that the melting point of the amyloid fiber was 72.2±0.3°C, indicating that it has high thermal stability; Figure 3 This is a picture of the microscopic structure characteristics of the amyloid fiber prepared according to the present invention, taken through a transmission electron microscope. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0022] Example 1 A method for preparing highly uniform amyloid fibers comprises the following steps: 1) Add an appropriate amount of 50 mM sodium hydroxide solution to 1 mg of freeze-dried β-amyloid protein to make the final concentration of the protein after dissolution to 20 mg / mL. Centrifuge at 4°C, 13000 rpm for 3 minutes to separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide, mass percentage concentration) to the supernatant to make the final protein concentration 100 μM. Transfer the above solution to a centrifugal filter and centrifuge at 4°C, 6000 rpm for 3 minutes. The filtrate is the protein monomer solution.

[0023] 2) The prepared protein monomer solution was transferred to a vial, placed in a constant temperature shaker, and incubated at 37°C, 200 rpm for 4 days. Transmission electron microscopy showed that the protein had aggregated to form primary amyloid fibers.

[0024] 3) Take the above-mentioned primary amyloid fiber sample, ultrasonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). After mixing the two, transfer them to a vial and incubate them in a constant temperature shaker at 37°C and 200 rpm. After 4 days, the second generation amyloid fiber sample is prepared.

[0025] 4) Pre-treat the second generation amyloid fiber sample according to the method in step 3) above, and then prepare the third to twelfth generation amyloid fiber samples. Among them, for the third to sixth generations, each generation of amyloid fiber samples was shaken and incubated at 37°C and 200 rpm for 1 day. For the seventh to twelfth generations, each generation of amyloid fiber samples was shaken and incubated at 37°C and 200 rpm for 6 hours. The twelfth generation fiber sample obtained by transmission electron microscopy was an amyloid fiber with uniform morphology, and the twelfth generation amyloid fiber sample was retained for later use.

[0026] 5) The prepared 12th generation amyloid fiber sample was used as a seed, and ultrasonicated in a water bath for 5 minutes at room temperature. After being blown evenly, it was added to the freshly prepared protein monomer solution at a ratio of 10% (v / v), mixed, and then incubated in a constant temperature shaker at 37°C at 200 rpm for 2 days to prepare highly uniform amyloid fibers.

[0027] Figure 1 The dynamic light scattering data of the prepared amyloid fibers are given, indicating that the particle size of the amyloid fibers prepared by this method is uniform, which is 1101 nm.

[0028] Figure 2The circular dichroism spectrum data of the prepared amyloid fibers are given. The melting point of the amyloid fibers is measured to be 72.2 ± 0.3℃, indicating that they have high thermal stability.

[0029] Figure 3 A transmission electron micrograph of the prepared amyloid fibers is given, which shows the microstructural characteristics of the amyloid fibers, whose diameter is in the nanometer scale and whose length can extend to the micrometer scale range.

[0030] Example 2 A method for preparing highly uniform amyloid fibers comprises the following steps: 1) Add an appropriate amount of 50 mM sodium hydroxide solution to 1 mg of freeze-dried β-amyloid protein to make the final concentration of the protein after dissolution to 20 mg / mL. Centrifuge at 4°C, 13000 rpm for 3 minutes to separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide, mass percentage concentration) to the supernatant to make the final protein concentration 100 μM. Transfer the above solution to a centrifugal filter and centrifuge at 4°C, 6000 rpm for 3 minutes. The filtrate is the protein monomer solution.

[0031] 2) The prepared protein monomer solution was transferred to a vial, placed in a constant temperature shaker, and incubated at 37°C, 200 rpm for 4 days. Transmission electron microscopy showed that the protein had aggregated to form primary amyloid fibers.

[0032] 3) Take the above-mentioned primary amyloid fiber sample, ultrasonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). After mixing the two, transfer them to a vial and incubate them in a constant temperature shaker at 37°C and 200 rpm. After 4 days, the second generation amyloid fiber sample is prepared.

[0033] 4) The second generation amyloid fiber sample was treated according to the method in step 3) above to prepare the third to twelfth generation amyloid fiber samples. Among them, for the third to sixth generations, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm for 1 day. For the seventh to twelfth generations, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm for 6 hours. The twelfth generation fiber sample obtained by transmission electron microscopy was an amyloid fiber with uniform morphology, and the twelfth generation amyloid fiber sample was retained for later use.

[0034] 5) The prepared 12th generation amyloid fiber sample was used as a seed, and ultrasonicated in a water bath for 5 minutes at room temperature. After being blown evenly, it was added to the freshly prepared protein monomer solution at a ratio of 2% (v / v), mixed, and then incubated in a constant temperature shaker at 37°C at 200 rpm for 4 days to prepare highly uniform amyloid fibers.

[0035] Example 3 A method for preparing highly uniform amyloid fibers comprises the following steps: 1) Add an appropriate amount of 50 mM sodium hydroxide solution to 1 mg of freeze-dried β-amyloid protein to make the final concentration of the protein after dissolution to 20 mg / mL. Centrifuge at 4°C, 13000 rpm for 3 minutes to separate the supernatant. Add an appropriate amount of 50 mM phosphate buffer (pH 7.4, containing 0.01% sodium azide, mass percentage concentration) to the supernatant to make the final protein concentration 100 μM. Transfer the above solution to a centrifugal filter and centrifuge at 4°C, 6000 rpm for 3 minutes. The filtrate is the protein monomer solution.

[0036] 2) The prepared protein monomer solution was transferred to a vial, placed in a constant temperature shaker, and incubated at 37°C, 200 rpm for 4 days. Transmission electron microscopy showed that the protein had aggregated to form primary amyloid fibers.

[0037] 3) Take the above-mentioned primary amyloid fiber sample, ultrasonicate it in a water bath for 5 minutes at room temperature, blow it evenly, and add it to the freshly prepared protein monomer solution at a ratio of 10% (v / v). After mixing the two, transfer them to a vial and incubate them in a constant temperature shaker at 37°C and 200 rpm. After 4 days, the second generation amyloid fiber sample is prepared.

[0038] 4) The second generation amyloid fiber sample was treated according to the method in step 3) above to prepare the third to twelfth generation amyloid fiber samples. Among them, for the third to sixth generations, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm for 1 day. For the seventh to twelfth generations, each generation of amyloid fiber samples was incubated at 37°C and 200 rpm for 6 hours. The twelfth generation fiber sample obtained by transmission electron microscopy was an amyloid fiber with uniform morphology, and the twelfth generation amyloid fiber sample was retained for later use.

[0039] 5) The prepared 12th generation amyloid fiber sample was used as a seed, and ultrasonicated in a water bath for 5 minutes at room temperature. After being blown evenly, it was added to the freshly prepared protein monomer solution at a ratio of 5% (v / v), mixed, and then incubated in a constant temperature shaker at 37°C at 200 rpm for 3 days to prepare highly uniform amyloid fibers.

[0040] In summary, the method of the present invention screens and prepares amyloid fiber samples with high uniformity and stability through in vitro regulation and condition optimization, and the prepared amyloid fibers have the characteristics of uniform particle size and high thermal stability.

Claims

1. A method for preparing uniform amyloid fibers, characterized in that: The steps include: 1) Dissolve the freeze-dried β-amyloid protein powder in sodium hydroxide solution, centrifuge, and separate the supernatant; add an appropriate amount of phosphate buffer to the supernatant, centrifuge, and the filtrate is the protein monomer solution; 2) Place the prepared protein monomer solution in a constant temperature shaker and incubate at 37±2°C for 3–5 days to observe that the protein has aggregated to form primary amyloid fibers; 3) Take the above-mentioned first-generation amyloid fiber sample, ultrasonicate it in a water bath at room temperature, blow it evenly and mix it with the freshly prepared protein monomer solution, then incubate it at a constant temperature of 37±2℃ with shaking, and prepare the second-generation amyloid fiber sample after 3-5 days; 4) Pretreating the second generation amyloid fiber sample according to the method in step 3) above, and then preparing the third to twelfth generation amyloid fiber samples; wherein, for the third to sixth generations, each generation of amyloid fiber samples is incubated at 37±2°C with shaking for 1-2 days, and for the seventh to twelfth generations, each generation of amyloid fiber samples is incubated at 37±2°C with shaking for 4-8 hours; 5) The prepared 12th generation amyloid fiber sample is used as a seed, ultrasonicated in a water bath, blown evenly and mixed with the freshly prepared protein monomer solution, and then incubated at a constant temperature of 37±2℃ with shaking for 2-4 days.

2. The method for preparing uniform amyloid fibers according to claim 1, characterized in that: In step 1), sodium hydroxide solution is added to the freeze-dried β-amyloid protein powder, and the final concentration after protein dissolution is 15-25 mg / mL.

3. The method for preparing uniform amyloid fibers according to claim 2, characterized in that: Add 50–100 mM sodium hydroxide solution to 1 mg of lyophilized β-amyloid protein powder to a final concentration of 18–22 mg / mL after protein solubilization.

4. The method for preparing uniform amyloid fibers according to claim 3, characterized in that: In step 1), add an appropriate amount of 10–50 mM phosphate buffer to the supernatant to make the final protein concentration 50–200 μM.

5. The method for preparing uniform amyloid fibers according to claim 4, characterized in that: In step 1), the pH of the phosphate buffer is 7.2-7.5 and contains 0.01-0.02% sodium azide.

6. The method for preparing uniform amyloid fibers according to claim 1, characterized in that: In step 3), the primary amyloid fiber sample is blown evenly and then added into the freshly prepared protein monomer solution at a volume percentage of 2-10%.

7. The method for preparing uniform amyloid fibers according to claim 1, characterized in that: In step 5), the 12th generation amyloid fiber sample is blown uniformly and added into the freshly prepared protein monomer solution at a volume percentage of 2-10%.

8. Uniform amyloid fibers prepared by the method according to any one of claims 1 to 7.

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