A heat-stable Aβ40 and its application

Abstract

The present invention provides an Aβ40 mutant and application thereof. The provided Aβ40 mutant comprises at least one mutation substitution or deletion at A2, F4, and S8 relative to the amino acid sequence SEQ ID NO.1 of the wild-type Aβ40, has improved thermal stability, and the thermal stability is significantly higher than that of the wild-type Aβ40 during long-term storage. The Aβ40 mutant can be used as a standard product and has greater utilization value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mutant Aβ40 with improved thermal stability and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common cause of dementia in the elderly. A key neuropathological feature of AD is extracellular amyloid plaques, including amyloid-β (Aβ) peptides of 42 and 40 amino acids in length (Aβ42 and Aβ40, respectively). Accumulation of amyloid-β (Aβ) leads to the formation of brain amyloid plaques and tangles, ultimately causing cognitive impairment (PMID: 26195256). Cerebrospinal fluid (CSF) biomarkers of Aβ are clinically used for early detection of MCI and dementia (PMID: 19622817). Wild-type (WT) Aβ40 peptide, with two hydrophilic (residues D1-K16 and E22-G29) and hydrophobic (residues L17-A21 and A30-A40) regions, aggregates rapidly in vitro and is structurally heterogeneous in aqueous solution (PMC4888851). Wild-type Aβ40 has poor thermal stability and loses activity under high temperature conditions.

[0003] Under acidic conditions close to the isoelectric point of Aβ, the absence of charge is conducive to the formation of intermolecular contacts and promotes aggregation. Methods for designing Aβ40 mutants with increased thermal stability have not been reported so far. Existing technologies mostly use stabilizers to enhance the thermal stability of Aβ40, such as: Aβ binders inhibit protein aggregation (PMC9173773); neutral pH and physiological salt concentration are conducive to slow aggregation (PMC10179685); chaperone binding proteins can form stable, highly water-soluble complexes with Aβ (PMC9700483). However, these methods have many obstacles in terms of process complexity, cost and practical application of products. Summary of the invention

[0004] The present invention aims to provide a highly thermostable Aβ40 mutant obtained by rational design and screening, and a preparation method and application thereof.

[0005] In one aspect, the present invention provides an Aβ40 mutant, whose amino acid sequence comprises at least one mutation substitution or deletion at A2, F4, S8 relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitutions or deletions at A2, F4, and S8 relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitutions A2I, F4E, and S8A relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant is as shown in SEQ ID NO.2.

[0006] In another aspect, the present invention provides one or more biological materials selected from the group consisting of:

[0007] 1) A polynucleotide molecule encoding an Aβ40 mutant;

[0008] 2) an expression vector comprising the polynucleotide molecule as described in 1);

[0009] 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

[0010] The present invention provides a polynucleotide molecule encoding the Aβ40 mutant of the present invention; wherein the polynucleotide molecule may have various modifications in the coding region, as long as the amino acid sequence of the variant of the present invention does not change with the degeneracy of the codons or does not change with the preferred codons in the organism expressing the variant. In some embodiments, the polynucleotide molecule is as shown in SEQ ID NO: 6.

[0011] The expression vector provided by the present invention comprises a polynucleotide molecule encoding the Aβ40 mutant of the present invention. In some embodiments, the expression vector further comprises one or more regulatory sequences, such regulatory sequences include but are not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequence is operably linked to the polynucleotide molecule encoding the variant. In some embodiments, the expression vector can be a linear or circular DNA molecule, which generally comprises elements such as a multiple cloning site, a resistance gene, and a replication initiation site.

[0012] In some embodiments, the expression vector of the present invention is preferably pET28a.

[0013] The host cell provided by the present invention refers to any cell that is beneficial to the expression of the variant of the present invention, that is, any cell that is susceptible to transformation, transfection or transduction with the expression vector described in the present invention, including any progeny cells that are different from the parent cell due to mutations that occur during replication.

[0014] In some embodiments, the host cell is a prokaryotic cell, which can be selected from gram-positive bacteria or gram-negative bacteria. In some embodiments, the host cell is a gram-positive bacteria, including but not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanic Bacillus, Staphylococcus, Streptococcus and Streptomyces. In some embodiments, the host cell is a gram-negative bacteria, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silvae, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21 (DE3).

[0015] In another aspect, the present invention provides a method for preparing the Aβ40 mutant, comprising: (1) culturing the host cell of the present invention under conditions suitable for mutant expression; and (2) recovering the mutant.

[0016] In some embodiments, the method for recovering the mutant can be a method well known in the art, such as centrifugation, filtration, treatment with a crystal protein precipitant (salting out method), extraction, ultrasonic fragmentation, ultrafiltration, dialysis, various chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc., HPLC, and combinations of the above methods.

[0017] In some embodiments, the preparation method also includes a step of purifying the variant, and the purification step can be a method well known in the art, such as chromatography (such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), isoelectric focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0018] In another aspect, the present invention provides a composition comprising the Aβ40 mutant of the present invention.

[0019] In another aspect, the present invention provides a kit comprising the Aβ40 mutant of the present invention.

[0020] In another aspect, the present invention provides the use of the Aβ40 mutant in preparing a standard product. In some embodiments, the standard product is used in conjunction with an Aβ40 detection kit or is included in an Aβ40 detection kit.

[0021] In another aspect, the present invention provides use of the Aβ40 mutant in preparing Aβ40 antigen.

[0022] In another aspect, the present invention provides use of the Aβ40 mutant in preparing Aβ40 antibodies.

[0023] The Aβ40 mutant provided by the present invention has significantly improved stability compared to the wild-type Aβ40, and its heat resistance reaches 37°C. Under the condition of 7 days, the reactivity CV on the chemiluminescence platform is less than 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the construction of pET28a-Aβ40 recombinant plasmid;

[0025] Figure 2 This is the result of SDS-PAGE electrophoresis detection of Aβ40 mutant and wild-type Aβ40. DETAILED DESCRIPTION

[0026] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0027] The technical scheme of the present invention is further described below in conjunction with specific examples. However, the following examples are merely examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims. In the following examples, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the field, and the experimental methods and technical means used are conventional methods and means in the field.

[0028] Example 1: Preparation of mutant thermostable Aβ40

[0029] According to existing reports, the thermal stability of Aβ40 is closely related to its recognition antibody binding. The present invention analyzes the protein structure of Aβ40, searches for segments related to protein self-aggregation, and improves stability parameters. Therefore, the present invention selects sites away from the diagnostic antibody recognition site to avoid affecting its recognition antibody binding. These sites include F4 and S8. In addition, the present invention selects hydrophobic core sites in some non-catalytic core regions, hoping to improve the stability of the protein value by improving internal hydrophobic interactions. These sites include A2.

[0030] The gene sequence of wild-type Aβ40 is shown in SEQ ID NO.5, and the gene sequence of mutant is shown in SEQ ID NO.6 (A2I, F4E, S8A), SEQ ID NO.7 (A2I), and SEQ ID NO.8 (F4E). After DNA sequence synthesis, PCR amplification was performed, and then the genes were introduced into the BamHI and XhoI restriction sites of the expression vector pET28a to obtain recombinant expression vectors, generating 4 expression plasmids, namely pET28a-Aβ40 plasmid (wild-type), (A2I), (F4E) and (A2I, F4E, S8A) ( Figure 1 ), the specific synthetic sequence includes the purification tag 6×His sequence added at the 5' end and the TAA stop codon at the 3' end. This plasmid was transformed into Escherichia coli BL21 competent cells for expression host construction, and cloned strains were obtained after antibiotic resistance plate coating screening; after successful recombination, the obtained strains were activated overnight in LB medium at 37°C and added to the fermentation broth (LB medium), cultured to OD600 value of 0.6-0.8, and IPTG with a final concentration of 0.5 mol was added to continue culturing for 4-6 hours, and then centrifuged at 12000rmp and 4-8°C to collect the strains, and the collected strains were washed with 0.2M PBS buffer with a pI value of 7.0 to obtain the bacteria: after high-pressure crushing, affinity chromatography and ion exchange chromatography were performed to purify Aβ40 wild type, mutants (A2I), (F4E) and (A2I, F4E, S8A). After protein purification, the storage buffer was 20 mM Tris, pH 8.0, 150 mM NaCl, 5% glycerol, stored at -20°C. The concentration was determined by absorbance at 280 nm.

[0031] Example 2: Reactivity assay

[0032] The synthesized Aβ40 mutant and wild-type Aβ40 protein were diluted to 1 mg / mL using phosphate buffer. The samples were diluted to gradients at a ratio of 1:100000, 1:200000, 1:400000, 1:800000, 1:1600000, 1:3200000, and 1:6400000, and the reactivity of the synthesized Aβ40 mutant and wild-type Aβ40 protein was evaluated using the Aβ1-40 in vitro diagnostic kit (Hunan Novogene Medical Technology Co., Ltd., M1701CB). The samples were transferred to a Hitachi cup and tested using a fully automatic chemiluminescence immunoassay analyzer (Shenzhen Yingkai Biotechnology Co., Ltd., Shine i2910). The test results are shown in Table 1.

[0033] Table 1:

[0034] Antigen dilution gradient Aβ40 (A2I, F4E, S8A) Aβ40 (A2I) Aβ40(F4E) Aβ40 (wild type) 1:100000 8612568 9039816 8912649 9825631 1:200000 4354832 4663950 4465538 5068863 1:400000 2207327 2664030 2491270 2660681 1:800000 1219410 1416591 1316435 1397179 1:1600000 651350 862171 700708 749015 1:3200000 349188 431136 361935 386049 1:6400000 201528 219080 198706 213090 blank 1854 1816 1756 1858

[0035] The results showed that the Aβ40 mutant reacted to the in vitro diagnostic kit, and the reaction intensity was almost consistent with that of the wild-type Aβ40 protein, indicating that the Aβ40 mutant had the same antigenicity as the wild-type Aβ40 protein.

[0036] Example 3: Thermal stability determination

[0037] The stability of the above Aβ40 mutants and wild-type Aβ40 proteins was tested using an Aβ40 in vitro diagnostic kit (Hunan Novi Medical Technology Co., Ltd., M1701CB). The concentration of Aβ40 protein was adjusted to 10ug / mL intermediate stock solution (the above 1mg / mL Aβ40 protein (0.1mL) was diluted 100 times with phosphate buffer to obtain 10mL intermediate stock solution), and the intermediate stock solution was divided (1ml / tube) and placed in a 37°C incubator for 1 / 2 / 3 / 4 / 5 / 6 / 7 / 10 / 14 days, and the control was placed at 2-8°C for storage. The 37°C culture was placed in reverse order, and the sample was taken out and diluted to 10ng / mL with phosphate buffer at the time. The sample was tested using a fully automatic chemiluminescence immunoassay analyzer (Shenzhen Yingkai Biotechnology Co., Ltd., Shine i2910) as a sample. The test results are shown in Table 2.

[0038] Table 2:

[0039]

[0040] After 7 days of heat damage, the signal reduction of Aβ40 mutants (A2I, F4E, S8A) and mutant Aβ40 (A2I) was less than 10%, which was relatively stable and significantly better than the wild-type Aβ40 protein (45.8%). After 10 / 14 days, the wild-type protein decreased by more than half (54.9%, 67.5%), and the Aβ40 mutants (A2I, F4E, S8A) still decreased by less than 10%, which was more stable than the wild-type protein.

Claims

1. An Aβ40 mutant, whose amino acid sequence comprises at least one mutation substitution or deletion at A2, F4, S8 relative to SEQ ID NO.

1.

2. The mutant according to claim 1, wherein the amino acid sequence comprises A2I, F4E and S8A substitutions relative to SEQ ID NO.

1.

3. The mutant according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

4. One or more biological materials selected from the following: 1) A polynucleotide molecule encoding the Aβ40 mutant according to claim 1; 2) an expression vector comprising the polynucleotide molecule as described in 1); 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

5. A polynucleotide molecule encoding the mutant according to claim 3, wherein the nucleotide sequence thereof is as shown in SEQ ID NO.

4.

6. A method for preparing an Aβ40 mutant, comprising: (1) culturing the host cell according to claim 4 under conditions suitable for mutant expression; and (2) recovery of mutants. 7 . A composition comprising the Aβ40 mutant according to claim 1 . A kit comprising the Aβ40 mutant according to claim 1.

9. Use of the Aβ40 mutant according to claim 1 in preparing a standard product. 10 . Use of the Aβ40 mutant according to claim 1 in preparing Aβ40 antigen or Aβ40 antibody.

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