A heat-stable GFAP and its application

Abstract

The present invention provides a GFAP mutant and application thereof. The provided GFAP mutant comprises at least one mutation substitution or deletion at K107, A216, A227, N375 and G402 relative to the amino acid sequence SEQ ID NO.1 of the wild-type GFAP, has improved thermal stability, and is significantly higher than the wild-type GFAP in thermal stability during long-term storage. The GFAP mutant can be used as a standard product and has greater utilization value.

Description

Technical Field

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

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease with clinical manifestations of cognitive decline and behavioral disruption. AD is expected to affect more than 152 million people by 2050 and is now considered a common senile dementia with major pathological features (PMID: 30488277). Blood biomarkers are considered as tools for diagnosis, prognosis and monitoring of Alzheimer's disease (AD). Although amyloid beta peptide (Aβ) and Tau are the main blood biomarkers, recent studies have identified other reliable candidates that can serve as measurable indicators of the pathological condition. One such candidate is glial fibrillary acidic protein GFAP, an astrocyte cytoskeletal protein that can be detected in blood samples. There is increasing evidence that blood GFAP levels can be used to detect early AD (PMID: 37174709).

[0003] GFAP is mainly found in astrocytes, a type of glial cell in the brain (PMID: 37174709). GFAP is expressed at low levels in healthy people, but its expression level is significantly increased in the brains of AD patients. Methods for designing glial fibrillary acidic protein GFAP mutants with increased thermal stability have not yet been reported. Faced with the hot demand for AD diagnosis worldwide, as an important marker, the highly thermally stable glial fibrillary acidic protein GFAP has extremely high value. Summary of the invention

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

[0005] In one aspect, the present invention provides a GFAP mutant, whose amino acid sequence comprises at least one mutation substitution or deletion at K107Q, A216Q, A227H, N375K, G402R relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitution or deletion at K107, A216, A227, N375 and G402 relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitutions at K107, A216, A227, N375 and G402 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 a GFAP 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 GFAP 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 GFAP 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 GFAP 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 GFAP mutant of the present invention.

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

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

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

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

[0023] The GFAP mutant provided by the present invention has significantly improved stability compared with the wild-type GFAP, 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-GFAP recombinant plasmid;

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

[0029] According to existing reports, the thermal stability of GFAP is closely related to its recognition antibody binding. By analyzing the protein structure of GFAP, searching for segments related to protein breakage, and improving stability parameters, the present invention selects sites away from the diagnostic antibody recognition site to avoid affecting its recognition antibody binding. These sites include K107, N375, and G402. In addition, the present invention selects hydrophobic core sites in some non-core regions, hoping to improve the stability of the protein value by improving internal hydrophobic interactions. These sites include A216 and A227.

[0030] (1) Strain construction: The gene sequence of wild-type GFAP is shown in SEQ ID NO.3, and the gene sequence of GFAP 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 expression vector pET28a to obtain a recombinant expression vector, generating pET28a-GFAP 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 GFAP 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 GFAP mutants and wild-type GFAP proteins were diluted to 1 mg / mL using phosphate buffer. The samples were diluted to gradient ratios 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 synthetic GFAP mutants and wild-type GFAP proteins was evaluated using the GFAP in vitro diagnostic kit (Hunan Novi Medical Technology Co., Ltd., M5701CB). 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 GFAP mutant wild-type GFAP 1:4000 26260477 27892868 1:8000 14291731 15444358 1:16000 7227019 7908867 1:32000 3573884 3879999 1:64000 1778316 1912613 1:128000 838303 983049 1:256000 420251 477052 1:512000 207298 234490 1:1024000 96514 116546 1:2048000 47769 58237 1:4096000 24229 29473 1:8192000 12963 14937 1:16384000 6967 8035 1:32768000 4208 4561 1:65536000 2722 2943 blank 2128 2222

[0036] The results showed that the GFAP mutant was reactive with anti-GFAP antibodies, and the intensity of the reaction was nearly consistent with that of the wild-type GFAP protein, indicating that the GFAP mutant had the same antigenicity as the wild-type GFAP protein.

[0037] Example 3: Thermal stability determination

[0038] The stability of the above GFAP mutants and wild-type GFAP proteins was tested using the GFAP in vitro diagnostic kit (Hunan Novi Medical Technology Co., Ltd., M5701CB). The concentration of GFAP protein was adjusted to 700 pg / mL with a calibrator diluent (PBS + 2% BSA), and the samples were placed in a 37°C incubator for 7 days, and the endpoint method was used for detection. The samples were tested using an automatic chemiluminescence analyzer (Shenzhen Yingkai Biotechnology Co., Ltd., automatic chemiluminescence immunoassay analyzer Shine i2910), and the test results are shown in Table 2.

[0039] Table 2:

[0040]

[0041] After 7 days of heat damage, the response value of the GFAP mutant decreased less than that of the wild-type GFAP, and its stability was significantly improved.

Claims

1. A GFAP mutant, the amino acid sequence of which comprises at least one mutation substitution or deletion at K107, A216, A227, N375, G402 relative to SEQ ID NO.

1.

2. The mutant according to claim 1, wherein the amino acid sequence comprises substitutions of K107, A216, A227, N375 and G402 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 GFAP 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 a GFAP 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 GFAP mutant according to claim 1 . A kit comprising the GFAP mutant according to claim 1.

9. Use of the GFAP mutant according to claim 1 in preparing a standard product.

10. Use of the GFAP mutant according to claim 1 in preparing GFAP antigen or GFAP antibody.

Images