Mutant proteins and related biomaterials and their applications in preparing cell models of Alzheimer's disease

Abstract

The present invention discloses a mutant protein and related biomaterials and their application in preparing an Alzheimer's disease cell model, belonging to the field of peptides. The technical problem to be solved by the present invention is to provide a mutant of human PSEN2 protein that promotes the increased production of β-amyloid peptide 42. The protein of the present invention is named T142I protein, which is as follows (a1) or (a2): (a1) the amino acid sequence of which is a protein of SEQ ID NO: 3; (a2) a fusion protein obtained by connecting the N-terminus or / and C-terminus of (a1) to a protein tag. The present invention can be used to prepare an Alzheimer's disease model.

Description

Technical Field

[0001] The present invention belongs to the field of peptides, and in particular relates to mutant proteins and related biomaterials and applications thereof in preparing Alzheimer's disease cell models. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease. The pathogenesis of AD is unknown, and it is generally believed to be caused by the combined effects of genetic and environmental factors. Typical AD patients show insidious onset and gradual progression of cognitive decline, such as memory, execution, language and visual-spatial disorders, which may be accompanied by psychiatric symptoms. About 10% of AD patients develop the disease before the age of 65, and 90% of AD patients develop the disease after the age of 65. In recent years, monoclonal antibodies targeting amyloid protein have been launched on the market, which has advanced the treatment window for AD patients. Early diagnosis is a prerequisite for early treatment, and early clinical intervention can help delay disease progression and improve the quality of life of patients.

[0003] The gold standard for AD diagnosis is autopsy pathology, but it is difficult to do it during life. AD has a large clinical variability and a high misdiagnosis rate. Imaging biomarker diagnosis such as Aβ-PET carries the risk of renal damage and radiation, and it is difficult to make an ultra-early diagnosis. In comparison, genetic diagnosis is more conducive to early and accurate diagnosis.

[0004] AD is a multi-gene genetic disease, and common pathogenic genes include APP, PSEN1 and PSEN2. The PSEN2 gene is located on chromosome 1, and the PSEN2 protein it encodes is a component of γ-secretase and is involved in the cleavage process of APP protein. PSEN2 gene mutations affect the function of γ-secretase, causing increased Aβ production and toxicity, leading to AD.

[0005] According to the Clinvar database, 39 pathogenic / suspected pathogenic variants have been found in the PSEN2 gene. Among them, 30 are copy number variants, 3 are deletion variants, and only 6 are single nucleotide missense variants. Among the 138 pathogenic / suspected pathogenic variants in the highly homologous PSEN1 gene, 115 are single nucleotide missense variants. Therefore, it is necessary to further study the PSEN2 gene variation in the population, especially single nucleotide variation, so as to understand the distribution of variation in the population and assist in the early diagnosis and treatment of AD patients. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a mutant of human PSEN2 protein that promotes the increased production of β-amyloid protein polypeptide 42 (Aβ42).

[0007] In order to solve the above technical problems, the present invention first provides a protein, named T142I protein (a mutant protein of human PSEN2 protein), which is as follows (a1) or (a2):

[0008] (a1) a protein having an amino acid sequence of SEQ ID NO: 3;

[0009] (a2) A fusion protein obtained by connecting the N-terminus or / and C-terminus of (a1) to a protein tag.

[0010] (a2) In the fusion protein, the protein tag refers to a polypeptide or protein that is fused and expressed together with the target protein (protein whose amino acid sequence is SEQ ID NO: 3) using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag and / or a His tag and / or an MBP tag and / or an HA tag and / or a myc tag and / or a GST tag and / or a SUMO tag, etc. In the fusion protein, the target protein and the tag protein are operably connected.

[0011] The present invention also provides T142I protein related biological materials.

[0012] The T142I protein-related biological material provided by the present invention is any one of the following (b1) to (b7):

[0013] (b1) a nucleic acid molecule encoding the T142I protein;

[0014] (b2) an expression cassette containing the nucleic acid molecule described in (b1);

[0015] (b3) a recombinant vector containing the nucleic acid molecule described in (b1) or a recombinant vector containing the expression cassette described in (b2);

[0016] (b4) a recombinant microorganism containing the nucleic acid molecule described in (b1), a recombinant microorganism containing the expression cassette described in (b2), or a recombinant microorganism containing the recombinant vector described in (b3);

[0017] (b5) a transgenic animal cell containing the nucleic acid molecule described in (b1) or a transgenic animal cell containing the expression cassette described in (b2);

[0018] (b6) transgenic animal tissue containing the nucleic acid molecule described in (b1) or transgenic animal tissue containing the expression cassette described in (b2);

[0019] (b7) An organ of a transgenic animal containing the nucleic acid molecule described in (b1) or an organ of a transgenic animal containing the expression cassette described in (b2).

[0020] In the above biological material, the nucleic acid molecule (b1) may be a DNA molecule (such as a gene encoding T142I protein) or an RNA molecule. The DNA molecule may be genomic DNA or cDNA. The nucleic acid molecule may be a genomic gene or a cDNA gene of the protein. The cDNA gene is a cDNA molecule that includes a coding sequence (CDS) of the protein. The coding sequence may be SEQ ID NO: 4.

[0021] The gene encoding the T142I protein may specifically be as follows (c1) or (c2):

[0022] (c1) a DNA molecule whose coding sequence is SEQ ID NO: 4;

[0023] (c2) A DNA molecule derived from a mammal that has more than 75% identity with (b1) and encodes the T142I protein.

[0024] Alignment for determining the percentage of sequence identity can be achieved in a variety of known ways, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms required to achieve maximum alignment over the entire length of the compared sequences. However, for the purposes of this article, sequence comparison computer program ALIGN-2 is used to generate sequence identity percentage values. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington, D.C., 20559, along with user documentation, and its registration number in the U.S. Copyright Office is TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California), or can be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and will not change.

[0025] Specifically, the 75% or more identity is 80% or more identity. Specifically, the 75% or more identity is 85% or more identity. Specifically, the 75% or more identity is 90% or more identity. Specifically, the 75% or more identity is 91% or more identity, 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, 96% or more identity, 97% or more identity, 98% or more identity or 99% or more identity. More specifically, the 75% or more identity may be at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0026] In the above biological materials, the expression cassette described in (b2) refers to a DNA capable of expressing the protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing the nucleic acid molecule of the above protein. The regulatory sequence can guide the coding sequence to express the above protein in a suitable host cell under compatible conditions. The regulatory sequence includes, but is not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence and a transcription terminator. At a minimum, the regulatory sequence should include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites into the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a linker may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that has transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of mRNA that is important for translation of the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a signal peptide coding region that encodes an amino acid sequence attached to the amino terminus of the protein that can guide the encoded protein into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein into the secretory pathway of the host cell used may be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory sequences are systems that can respond to chemical or physical stimuli (including in the presence of regulatory compounds) to open or close gene expression. Other examples of regulatory sequences are those that can amplify genes. In these cases, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequences.

[0027] In the above biological materials, the recombinant vector (b3) can be a cloning vector or an expression vector. When preparing the expression vector, the nucleic acid molecule encoding the above protein can be located in the vector so as to be operably linked to the appropriate expression regulatory sequence. The recombinant expression vector can be any vector (such as a plasmid or virus) that is convenient for recombinant DNA operations and expression of nucleic acid sequences. The choice of vector usually depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be a linear or closed plasmid. The vector can be an autonomously replicating vector (i.e., a complete structure that exists outside the chromosome and can replicate independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any mechanism to ensure self-replication. Alternatively, the vector is a vector that can be integrated into the chromosome and replicated together with the integrated chromosome when introduced into the host cell. The vector contains one or more selection markers that facilitate the selection of transformed cells. The selection marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers prototrophy to auxotrophs, etc. Examples of bacterial selection markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline. The vector contains elements that enable the vector to be stably integrated into the host cell genome, or that ensure that the vector replicates autonomously in the cell independently of the cell genome. In the case of autonomous replication, the vector may also contain an origin of replication that enables the vector to replicate autonomously in the target host cell. The origin of replication may carry a mutation that makes it temperature-sensitive in the host cell (see, for example, fEhrlich, 1978, Proceedings of the National Academy of Sciences of the United States of America 75: 1433). More than one copy of the nucleic acid molecule encoding the above protein may be inserted into the host cell to increase the yield of the gene product. The number of copies of the nucleic acid molecule may be increased by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or inserting an amplifiable selection marker together with the nucleic acid molecule, and selecting cells containing amplified copies of the selectable marker gene and thus additional copies of the nucleic acid molecule by culturing the cells in the presence of a suitable selection agent. The operations for connecting the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0028] In the above biological materials, the microorganism (b4) may be bacteria, yeast, algae or fungi.

[0029] Specifically, the bacteria is any one of the following (d1) to (d3):

[0030] (d1) Gram-negative bacteria;

[0031] (d2) Escherichia bacteria;

[0032] (d3) Escherichia coli.

[0033] In the above biological materials, the animal cells in (b5) may be isolated mammalian cells. The mammals include humans. The mammalian cells may not include animal germ cells, animal fertilized eggs and animal embryonic stem cells, and may be somatic cells or cell lines.

[0034] In the above biological materials, the animal organ (b7) may be an isolated animal organ, and may not include an embryo.

[0035] Exemplarily, the recombinant vector is obtained by inserting the gene into a vector. Exemplarily, the recombinant vector is obtained by inserting the gene into an expression vector. Exemplarily, the recombinant vector is obtained by inserting the gene into a mammalian expression vector. As an example, the mammalian expression vector is a pcDNA™3.1 (-) mammalian expression vector.

[0036] Exemplarily, the transgenic animal cell is obtained by introducing the gene into an animal cell as a recipient cell. Exemplarily, the transgenic animal cell is obtained by introducing the recombinant vector into an animal cell as a recipient cell. Specifically, the recipient cell is a mammalian cell. Specifically, the mammalian cell is a human cell. As an example, the mammalian cell is a HEK293 cell.

[0037] The present invention also protects the use of T142I protein in preparing an Alzheimer's disease model.

[0038] The present invention also protects the use of T142I protein-related biological materials in preparing Alzheimer's disease models.

[0039] The Alzheimer's disease model is an Alzheimer's disease cell model or an Alzheimer's disease animal model.

[0040] The present invention also protects a method for preparing an Alzheimer's disease cell model, comprising the following steps: introducing a gene encoding T142I protein into a receptor cell to obtain a recombinant cell, wherein the recombinant cell is an Alzheimer's disease cell model.

[0041] Specifically, the gene is introduced into the recipient cell via a recombinant vector; the recombinant vector contains the gene. Exemplarily, the recombinant vector is obtained by inserting the gene into a vector. Exemplarily, the recombinant vector is obtained by inserting the gene into an expression vector. Exemplarily, the recombinant vector is obtained by inserting the gene into a mammalian expression vector. As an example, the mammalian expression vector is a pcDNA™3.1 (-) mammalian expression vector.

[0042] In the method, the recipient cell may be an isolated mammalian cell. The mammal includes a human. The mammalian cell may not include animal germ cells, animal fertilized eggs and animal embryonic stem cells, such as somatic cells or cell lines.

[0043] In the method, the recombinant cell has an increased ability to produce β-amyloid polypeptide 42 compared to the recipient cell. In the method, the comparison is performed under comparable conditions.

[0044] The present invention can prepare an Alzheimer's disease model, which can be used for mechanism research, drug screening, etc. of Alzheimer's disease, and has great application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the result of the functional verification in Example 2. DETAILED DESCRIPTION

[0046] Herein, unless otherwise defined herein, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Examples of resources describing many of the terms used herein related to molecular biology can be found in: Alberts et al., Molecular Biology of The Cell, 5th edition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th edition, Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007.

[0047] Unless otherwise indicated, the terms "nucleic acid," "nucleotide," and "polynucleotide" encompass both DNA and RNA.

[0048] The term "vector" refers to a nucleic acid molecule that allows the insertion of foreign nucleic acid without destroying the ability of the vector to replicate and / or integrate in the host cell. The vector may include a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. The vector may also include one or more selectable marker genes and other genetic elements. An integration vector is capable of integrating itself into the host nucleic acid. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of the inserted gene. The vector may be a plasmid vector or a viral vector.

[0049] The term "plasmid" refers to a structure composed of genetic material designed to direct the transformation of target cells. A "plasmid" includes a plasmid backbone. A "plasmid backbone" contains multiple genetic elements that are positioned and oriented in sequence with other necessary genetic elements so that the nucleic acid in the nucleic acid cassette can be transcribed and, if necessary, translated in the transfected cells. A "plasmid backbone" may contain one or more unique nucleic acid restriction sites. A "plasmid" is capable of autonomous replication in a host or organism, thereby replicating the cloned sequence. A "plasmid" can confer certain phenotypes on the host organism that are selectable or easily detectable. A "plasmid" or "plasmid backbone" may have a linear or circular configuration. The components of a "plasmid" may include, but are not limited to, a DNA molecule comprising: (1) DNA; (2) a plasmid backbone; (3) a sequence encoding the gene of interest; and (4) regulatory elements responsible for transcription, translation, RNA stability, and replication.

[0050] The term "nucleic acid restriction site" or "restriction site" refers to a deoxyribonucleic acid sequence at which specific restriction endonucleases cleave the molecule.

[0051] The term "transfection" refers to the operation of introducing exogenous DNA into cultured cells by exposing the cultured cells to the exogenous DNA. Transfection methods include, but are not limited to, microinjection, electroporation, calcium phosphate precipitation, liposome fusion (e.g., liposome transfection), or gene gun. As an example, the transfection is by means of Lip2000 transfection. Transformation can occur by various mechanisms, such as transfection, electroporation, or particle bombardment.

[0052] The term "operably linked" refers to the operable connection of a nucleic acid sequence or an amino acid sequence, which are in a functional relationship with each other. When used to refer to nucleic acids, it refers to the functional connection between a promoter or other regulatory element and a gene-related transcribable DNA sequence or a gene's coding sequence (coding sequence, CDS), so that regulatory elements such as promoters play a role in starting, assisting in causing and / or promoting the transcription and expression of the relevant transcribable DNA sequence or coding sequence in at least certain cells, tissues, developmental stages and / or diseases. For example, operably linked promoters, enhancer elements, coding sequences (CDS), open reading frames (ORFs), 5' and 3' UTRs, and terminator sequences cause the accurate production of nucleic acid molecules (e.g., RNA). In some instances, operably linked nucleic acid elements lead to the transcription of open reading frames and ultimately to the production of polypeptides (i.e., the expression of open reading frames). In other instances, operably linked peptides are peptides in which functional domains are placed at appropriate distances from each other to confer the intended function of each domain.

[0053] The term "transcribable DNA" refers to DNA that can be transcribed into an RNA molecule.

[0054] The term "promoter" may generally refer to a DNA that contains an RNA polymerase binding site, a transcription start site, and / or a TATA box and assists or promotes transcription of a transcribable DNA. A promoter may be produced by artificial synthesis or may be derived from a known or naturally occurring promoter. A promoter may also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter may be a constitutively active promoter (i.e., a promoter that is continuously active / "on"), an inducible promoter (i.e., a promoter whose state (active / "on" or inactive / "off") is controlled by an external stimulus (e.g., the presence of a specific temperature, compound, or protein), a spatially restricted promoter (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), or may be a temporally restricted promoter (i.e., a promoter that is "on" or "off" at a specific stage of embryonic development or a specific stage of a biological process). Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-β-D-thiogalactoside (IPTG)-regulated promoter, lactose-induced promoter, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducers of inducible promoters include, but are not limited to, regulation by molecules such as doxycycline, RNA polymerase (e.g., T7 RNA polymerase), estrogen receptor, estrogen receptor fusion protein, etc.

[0055] The term "transcription terminator sequence" (also known as "transcription terminator element", "transcription terminator" or "terminator" or "terminator sequence") refers to a nucleotide sequence that terminates transcription. In the present invention, the sequence is located in the 3' linker region of the multicloning sequence, but can also be located at other sites of the plasmid. In one embodiment, the terminator is derived from the Escherichia coli rrnB operon. These sequences ensure that the transcription of the target nucleic acid sequence will not read into other functional regions of the plasmid.

[0056] The term "transcription" or "transcription" refers to the process of forming RNA molecules on a DNA template through complementary base pairing. This process is mediated by RNA polymerase.

[0057] The term "introduction" refers to an operation of transferring the gene or a recombinant vector having the gene into a cell so that the gene can be expressed in the cell.

[0058] The expression may be transient expression, sustained expression or stable expression.

[0059] The term "transient expression" means that the introduced genetic material is not integrated into the host cell genome or replicated and can therefore be degraded or translocated to other compartments over a period of time.

[0060] The term "constant expression" refers to the introduction of a gene of interest into a cell together with genetic elements that enable the genetic material to replicate and / or be maintained episomally (i.e., extrachromosomally) in the cell. This can result in apparently stable transformation of the cell without the need for integration of the new genetic material into the host cell's chromosomes.

[0061] The term "stable expression" refers to the introduction of genetic material into the chromosomes of a target cell, where it integrates and becomes a permanent part of the genetic material of that cell. Gene expression following stable introduction can permanently alter the properties of the cell and its progeny through replication, resulting in stable transformation.

[0062] The term "comparable conditions" refers to the same or similar environmental conditions. Environmental conditions include in vitro culture conditions such as culture temperature, culture medium, and gas environment.

[0063] Aβ: β-amyloid protein.

[0064] Aβ40: amyloid β-peptide 40.

[0065] Aβ42: amyloid β-peptide 42.

[0066] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0067] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0068] Serum-free Opti-MEM I medium (Opti-MEM™ I reduced serum medium, phenol red-free): Brand Gibco TM , ThermoFisher Company, product catalog number is 11058021. Lip2000 (full name Lipofectamine™ 2000 transfection reagent): brand Invitrogen TM , ThermoFisher, catalog number 11668500.

[0069] Example 1. Discovery of mutant proteins

[0070] The PSEN2 gene in the human genome is shown in NCBI Reference Sequence: NG_007381.2 (12-DEC-2024).

[0071] A SNP mutation in the PSEN2 gene was found in high-throughput sequencing of Alzheimer's patients. The SNP mutation caused a mutation in an amino acid residue of the encoded protein. The protein before the mutation was named PSEN2 protein. The protein after the mutation was named T142I protein. The PSEN2 protein is shown in SEQ ID NO: 1. The T142I protein is shown in SEQ ID NO: 3. The difference between the PSEN2 protein and the T142I protein is only in the 142nd amino acid residue, which is threonine (T) in the PSEN2 protein and isoleucine (I) in the T142I protein.

[0072] Example 2: Functional verification of mutant proteins

[0073] 1. Construction of recombinant plasmid

[0074] The double-stranded DNA molecule shown in SEQ ID NO: 2 was used to replace the pcDNA™3.1(-) vector. XOt Ⅰ and EcoR The small fragment between the restriction sites I was cut, and other sequences were kept unchanged to obtain the recombinant plasmid, which was named as the recombinant plasmid pcDNA3.1-PSEN2.

[0075] The double-stranded DNA molecule shown in SEQ ID NO: 4 was used to replace the pcDNA™3.1(-) vector. XOt Ⅰ and EcoR The small fragment between the restriction sites of I and II was cut, and other sequences were kept unchanged to obtain the recombinant plasmid, which was named as the recombinant plasmid pcDNA3.1-T142I.

[0076] pcDNA™3.1(-) vector, full name pcDNA™3.1 (-) mammalian expression vector, Invitrogen, product catalog number V79520.

[0077] 2. Functional verification of mutant proteins

[0078] 1. Cell culture

[0079] Take a 12-well plate and inoculate HEK293 cell suspension (2×10 5 cells / well) and cultured in serum-free Opti-MEM I medium until the cell density reached 90%.

[0080] 2. Preparation of transfection complexes

[0081] Dilute 1000ng of recombinant plasmid pcDNA3.1-PSEN2 in 50μl serum-free Opti-MEM I medium and mix gently to obtain solution 1. Dilute 5μl of Lip2000 in 50μl serum-free Opti-MEM I medium and let stand at room temperature for 5 minutes to obtain solution 2. Mix solution 1 and solution 2 and let stand at room temperature for 20 minutes to obtain a 100μl WT group transfection complex (this is the transfection dose for 1 well).

[0082] Dilute 1000ng of recombinant plasmid pcDNA3.1-T142I in 50μl serum-free Opti-MEM I medium and mix gently to obtain solution 3. Dilute 5μl of Lip2000 in 50μl serum-free Opti-MEM I medium and let stand at room temperature for 5 minutes to obtain solution 2. Mix solution 3 and solution 2 and let stand at room temperature for 20 minutes to obtain a 100μl T142I transfection complex (this is the transfection dose for 1 well).

[0083] 3. Transfect cells and culture

[0084] Take the 12-well plate that has completed step 1, and divide it into three groups of 4 wells each. Add the WT group transfection complex to the 4 wells in the first group, add the T142I group transfection complex to the 4 wells in the second group, and add serum-free Opti-MEM I medium (100 μl / well) to the 4 wells in the third group; then, shake gently to mix; then, place in a cell culture incubator (37°C, CO2) and culture for 48 hours; then, collect the supernatant.

[0085] The supernatant collected from the first group of wells was named WT supernatant.

[0086] The supernatant collected from the second group of wells was named T142I supernatant.

[0087] The supernatant collected from the third group of wells was named NC supernatant.

[0088] 4. Detection

[0089] The samples tested were: WT supernatant (4 samples) or T142I supernatant (4 samples) or NC supernatant (4 samples).

[0090] Take the test sample and detect the content of Aβ40 and Aβ42. The content of Aβ40 was detected by Human Amyloid beta 40ELISA Kit according to the instructions. The content of Aβ42 was detected by Human Aβ42 Ultrasensitive ELISA Kit according to the instructions. Human Amyloid beta 40 ELISA Kit: Brand Invitrogen, ThermoFisher, product catalog number KHB3481. Human Aβ42 Ultrasensitive ELISA Kit: Brand Invitrogen, ThermoFisher, product catalog number KHB3544.

[0091] The experiment was repeated three times, and the results were expressed as mean ± standard deviation. The Mann-Whiteny test was used for comparison among the groups.

[0092] Neither Aβ40 nor Aβ42 was detected in the NC supernatant. Compared with the WT supernatant, the Aβ42 content of the T142I supernatant was increased (86.6±4.5 vs. 59.8±5.8, P=0.0079). Compared with the WT supernatant, there was no significant difference in the Aβ40 content of the T142I supernatant (2182.0±118.0 vs. 2211.9±66.3, P=0.5476). Compared with the WT supernatant, the Aβ42 / Aβ40 ratio of the T142I supernatant was increased (0.040±0.002 vs. 0.027±0.002, P=0.0079). The results are shown in Figure 1 (ns indicates not significant, ** indicates P < 0.01). The results showed that the mutation of amino acid residue 142 increased the production of Aβ42, thus leading to the occurrence of AD.

[0093] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A protein, designated as T142I protein, characterized in that: It is as follows (a1) or (a2): (a1) a protein having an amino acid sequence of SEQ ID NO: 3; (a2) A fusion protein obtained by connecting the N-terminus or / and C-terminus of (a1) to a protein tag.

2. A nucleic acid molecule encoding the T142I protein according to claim 1.

3. An expression cassette comprising the nucleic acid molecule of claim 2.

4. A recombinant vector containing the nucleic acid molecule of claim 2.

5. A recombinant vector containing the expression cassette of claim 3.

6. A recombinant microorganism containing the nucleic acid molecule of claim 2.

7. A recombinant microorganism comprising the expression cassette of claim 3.

8. A recombinant microorganism containing the recombinant vector according to claim 4.

9. A transgenic animal cell containing the nucleic acid molecule of claim 2; the cell is a somatic cell or a cell Tie.

10. A transgenic animal cell containing the expression cassette of claim 3; the cell is a somatic cell or a cell line.

11. A transgenic animal tissue containing the nucleic acid molecule of claim 2; said tissue does not include an embryo.

12. A transgenic animal tissue containing the expression cassette of claim 3; said tissue excluding embryos.

13. An organ of a transgenic animal containing the nucleic acid molecule of claim 2; said organ excluding embryos.

14. An organ of a transgenic animal comprising the expression cassette of claim 3; said organ excluding an embryo.

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