A thermally stable NFL and its application
By designing amino acid mutations at specific sites, a high thermal stability NF-L mutant was prepared, which solved the problem of insufficient thermal stability of NF-L in the prior art, and achieved stability and reactivity under 37°C for 7 days.
Patent Information
- Application Number
- CN202411673836.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-21
- Publication Date
- 2025-04-04
AI Technical Summary
The prior art is difficult to design a highly thermostable phosphorylated neurofilament protein NF-L, especially in the diagnosis of Alzheimer's disease, with a need for an NF-L mutant capable of maintaining stability at 37°C for 7 days.
By performing amino acid mutation or deletion at sites such as F4, A45, R107, E110, A203, S308, T360, and T398, a high thermal stability NFL mutant was designed, and its preparation and expression were achieved through the combination of polynucleotide molecules, expression vectors and host cells.
The high thermal stability of the NFL mutant was achieved, and the reactive CV was able to maintain less than 10% at 37°C and 7 days, significantly improving its stability relative to the wild type.
Smart Images
Figure CN119751628A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a mutant NFL with improved thermal stability and a preparation method and application thereof. Background Art
[0002] Alzheimer's disease (AD) Neurofilament proteins are well-established hallmarks of acute and chronic neuronal damage and are increased in multiple neurodegenerative diseases. Brain tissue contains primarily full-length neurofilaments and a fragment with a C-terminal tail domain. Neurofilament concentrations are increased in AD patients compared to healthy cohorts, with some neurofilament species being more differentially expressed than others. There are at least three major truncations of NF-L in the cerebrospinal fluid that are increased to varying degrees in AD. These fragments include NF-L165 (in Coil 1B), NF-L324 (in Coil 2B), and NF-L530 (in the C-terminal tail domain) (PMID: 35415607).
[0003] Most neurofilament proteins in cerebrospinal fluid exist in the form of domains. This also explains that the half-life of full-length NF-L outside the brain tissue is short and it is easily hydrolyzed into multiple domains. Methods for designing neurofilament protein NF-L mutants with increased thermal stability have not yet been reported. Faced with the hot demand for global AD diagnosis, as an important marker, phosphorylated neurofilament protein NF-L with high thermal stability has extremely high value. Summary of the invention
[0004] The present invention aims to provide an NFL mutant with high thermal stability obtained through rational design and screening, and a preparation method and application thereof.
[0005] In one aspect, the present invention provides an NFL mutant, whose amino acid sequence comprises at least one mutation substitution or deletion at F4, A45, R107, E110, A203, S308, T360, T398 relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitutions or deletions at F4, A45, R107, E110, A203, S308, T360, and T398 relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitutions of F4Y, A45P, R107 K, E110F, A203Y, S308I, T360W, and T398C 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 NFL 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 NFL 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: 4.
[0011] The expression vector provided by the present invention comprises a polynucleotide molecule encoding the NFL 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 connected 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 NFL 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 NFL mutant of the present invention.
[0019] In another aspect, the present invention provides a kit comprising the NFL mutant of the present invention.
[0020] In another aspect, the present invention provides the use of the NFL mutant in preparing a standard product. In some embodiments, the standard product is used in conjunction with an NFL detection kit or is included in an NFL detection kit.
[0021] In another aspect, the present invention provides use of the NFL mutant in preparing NFL antigen.
[0022] In another aspect, the present invention provides use of the NFL mutant in preparing NFL antibodies.
[0023] The NFL mutant provided by the present invention has significantly improved stability compared to the wild-type NFL, 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-NFL recombinant plasmid
[0025] Figure 2 This is the SDS-PAGE electrophoresis detection result of NFL mutant and wild-type NFL. 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 NFL
[0029] According to existing reports, the thermal stability of NFL is closely related to its recognition antibody binding. By analyzing the protein structure of NFL, searching for proteolysis-related segments, and improving stability parameters, the present invention selects sites away from diagnostic antibody recognition sites to avoid affecting its recognition antibody binding. These sites include F4, R107, E110, S308, T360, and T398. In addition, the present invention selects hydrophobic core sites in some non-core regions, hoping to improve the stability of protein values by improving internal hydrophobic interactions. These sites include A45 and A203.
[0030] (1) Strain construction: The gene sequence of the wild type NFL is shown in SEQ ID NO.3, and the gene sequence of the mutant is shown in SEQ ID NO.4. After DNA sequence synthesis, PCR amplification was performed and then introduced into the BamHI and XhoI restriction sites of the expression vector pET28a to obtain a recombinant expression vector, generating a pET28a-NFL plasmid ( 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. The plasmid was transformed into E. 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 liquid (LB medium), cultured until the OD600 value was 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.
[0031] (2) Protein purification: The collected strains were washed with 0.2M PBS buffer with a pI value of 7.0 to obtain the bacterial cells: after high-pressure crushing, affinity chromatography and ion exchange chromatography were performed to purify the NFL mutants. After protein purification, the storage buffer formula was 20mM Tris, pH 8.0, 300mM NaCl, 5% glycerol, and stored at -20°C. The concentration was determined by absorbance at 280nm and compared with the wild-type protein by SDS-Page gel analysis ( Figure 2 ).
[0032] Example 2: Reactivity assay
[0033] The synthesized NFL mutants and wild-type NFL proteins were diluted to 1 mg / mL using phosphate buffer. The samples were diluted to gradients at a ratio of 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:512000, 1:1024000, 1:2048000, 1:4096000, 1:8192000, 1:16384000, 1:32768000, and 1:65536000, and the reactivity of the synthesized NFL mutants and wild-type NFL proteins was evaluated using the NFL in vitro diagnostic kit (Hunan Novi Medical Technology Co., Ltd., M6701CB). The above samples were transferred to Hitachi cups and tested using a chemical immunoassay analyzer (Shenzhen Yingkai Biotechnology Co., Ltd., fully automatic chemiluminescence immunoassay analyzer Shine i2910). The test results are shown in Table 1.
[0034] Table 1:
[0035] Antigen dilution gradient NFL Mutants Wild-type NFL 1:4000 22409528 22758579 1:8000 12285852 11979363 1:16000 6057041 6116276 1:32000 2944513 2964506 1:64000 1389024 1460674 1:128000 718284 715422 1:256000 351062 353048 1:512000 174526 172559 1:1024000 86273 86386 1:2048000 44000 44530 1:4096000 21634 21730 1:8192000 11636 11601 1:16384000 6628 6575 1:32768000 4080 3915 1:65536000 2681 2672 blank 1518 1781
[0036] The results showed that the NFL mutant was reactive with anti-NFL antibodies, and the intensity of the reaction was nearly consistent with that of the wild-type NFL protein, indicating that the NFL mutant had the same antigenicity as the wild-type NFL protein.
[0037] Example 3: Thermal stability determination
[0038] The stability of the above-mentioned NFL mutants and wild-type NFL proteins was tested using the NFL in vitro diagnostic kit (Hunan Novi Medical Technology Co., Ltd., M6701CB). The concentration of the NFL protein was adjusted to 700 pg / mL with a calibrator diluent (PBS + 2% BSA), and the aliquots were placed in a 37°C incubator for 7 days, and the endpoint method was used for detection. The samples were tested using a fully automatic chemiluminescence instrument (Shenzhen Yingkai Biotechnology Co., Ltd., fully automatic chemiluminescence immunoassay Shine i2910), and the test results are shown in Table 2.
[0039] Table 2:
[0040]
[0041] After 7 days of heat damage, the reaction value of the NFL mutant decreased less than that of the wild-type NFL, and its stability was significantly improved.
Claims
1. An NFL mutant, whose amino acid sequence comprises at least one mutation substitution or deletion at F4, A45, R107, E110, A203, S308, T360, T398 relative to SEQ ID NO.
1.
2. The mutant according to claim 1, wherein the amino acid sequence comprises F4Y, A45P, R107 K, E110F, A203Y, S308I, T360W and T398C 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 NFL 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 NFL 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 NFL mutant according to claim 1. A kit comprising the NFL mutant according to claim 1.
9. Use of the NFL mutant according to claim 1 in preparing a standard product.
10. Use of the NFL mutant according to claim 1 in preparing NFL antigen or NFL antibody.