CN111363809B - Molecular marker for developing Alzheimer's diagnostic product - Google Patents

Abstract

The invention discloses a molecular marker for developing an Alzheimer diagnosis product, wherein the molecular markers disclosed by the invention are EEF1AKMT3 and MANEAL, and particularly relates to application of the molecular marker in preparation of the Alzheimer diagnosis product, which is carried out by detecting EEF1AKMT3 and MANEAL in a sample in vitro. The invention also discloses application of EEF1AKMT3 and MANEAL in preparation of medicaments for treating Alzheimer disease.

Description

Molecular marker for developing Alzheimer's diagnostic product

Technical Field

The invention belongs to the field of biological medicines, and relates to a molecular marker for developing an Alzheimer diagnosis product.

Background

Alzheimer's Disease (AD) is a neurodegenerative disease and The major cause of dementia (Kang L, Zhang G, Yan Y, Ke K, Wu X, Gao Y, Li J, Zhu L, Wu Q, Zhou Z (2013) The role of HSPA12B in regulating neurological apoptosis [ J ] neurohem Res 38: 311-. AD accounts for 60% -70% of the dementia syndromes in people over 65 years of age. Currently, the prevalence of AD is increasing exponentially as the population ages rapidly. AD is a huge burden on the nation, society and family of patients. However, the pathogenesis of AD is not clearly explained, and there is still a lack of effective diagnostic and therapeutic approaches. Senile Plaque (SP) formed by abnormal aggregation of amyloid beta (Abeta) outside nerve cells, neurofi-brilary tangle (NFT) formed by abnormal aggregation of Tau protein inside nerve cells and neuronal loss are the main pathological features (Lakhan SE, Kirchgessner A, Hofer M (2009) [ J ] Influmamosoma in immunochemical strain: thermal apoptosis. JTransl. Med 7: 97). There is evidence that deposition of Α β is considered to be a pathological hallmark of AD (Li J, Wang Y, Luo J, Fu Z, Ying J, Yu Y, Yu W (2012) MiR-134 inhibition therapy to sensory transduction by targeting FOXM1 in non-small cell lung cancer cells [ J ] FEBS Lett 586: 3761-.

Currently there is no accepted "gold standard" diagnostic test for Alzheimer's disease. Clinical diagnosis of alzheimer's disease is typically based on the evaluation of clinical criteria that are highly sensitive but have low specificity and are usually diagnosed only when dementia has begun in a patient. However, at this stage of the disease, it is unlikely that any treatment will be able to reverse the damage caused by the neurodegenerative process. In order to improve the management of alzheimer's disease patients, methods and tools are needed that allow diagnosis at an earlier stage of the disease.

With the development of biotechnology and the introduction of precise medical treatment, the research of gene expression profiles related to diseases is the focus at present, and the search for molecular markers that can be used for characterizing diseases is of great significance for the early diagnosis and treatment of diseases.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a molecular marker related to Alzheimer's disease, and the early diagnosis of Alzheimer's disease is realized by detecting the expression level of the molecular marker.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of a reagent for detecting a gene marker in preparation of a product for diagnosing Alzheimer's disease, wherein the gene marker is selected from one or two of EEF1AKMT3 and MANEAL.

Further, EEF1AKMT3 expression was up-regulated in Alzheimer's patients and MANEAL was down-regulated in Alzheimer's patients compared to healthy humans.

Further, the product comprises a reagent for detecting the level of the gene marker by a sequencing technology, a nucleic acid hybridization technology, a nucleic acid amplification technology and a protein immunity technology.

Further, the agent is selected from:

a probe that specifically recognizes EEF1AKMT3 or MANEAL; or

Primers for specifically amplifying EEF1AKMT3 or MANEAL; or

An antibody that specifically binds to a protein encoded by EEF1AKMT3 or MANEAL.

Furthermore, the primer sequence of the specific amplification EEF1AKMT3 is shown as SEQ ID No. 1-2, and the primer sequence of the specific amplification MANEAL is shown as SEQ ID No. 3-4.

The invention provides a product for detecting the expression level of a gene marker EEF1AKMT3 or MANEAL in a sample in vitro, and the product comprises a preparation, a nucleic acid membrane strip, a chip or a kit. Where a "sample" is a substance from which nucleic acids, polypeptides, or other analytes can be obtained, including cells or cellular material.

Further, the chip includes a gene chip including specific primers or oligonucleotide probes for gene markers, and a protein chip including antibodies or ligands that specifically bind to proteins encoded by the gene markers.

Further, the kit comprises:

one or more reagents for detecting the expression level of the gene marker; and

one or more selected from the group consisting of: container, instructions for use, positive control, negative control, buffer, adjuvant or solvent.

Further, the kit comprises a reagent for detecting the expression level of the gene marker by an RT-PCR method, a qRT-PCR method, a biochip detection method, a DNA blotting method, an in situ hybridization method and an immunoblotting method.

Further, the sample is blood.

The invention provides application of a product for in vitro detection of expression levels of gene markers EEF1AKMT3 or MANEAL in a sample in preparation of a tool for diagnosing Alzheimer disease.

The invention provides application of a gene marker in preparing a pharmaceutical composition for treating Alzheimer, wherein the gene marker is EEF1AKMT3 and/or MANEAL.

Further, the pharmaceutical composition comprises an inhibitor of EEF1AKMT3 or an enhancer of MANEAL.

Further, the inhibitor is an agent that reduces the expression level of EEF1AKMT 3.

Further, the agent is a nucleic acid molecule.

Further, the nucleic acid molecule is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 7-8.

Further, the promoter is an agent that increases the expression level of MANEAL.

Furthermore, the promoter is an overexpression vector of MANEAL.

The invention provides a pharmaceutical composition for treating Alzheimer's disease, which comprises an inhibitor of EEF1AKMT3 or a promoter of MANEAL.

Further, the inhibitor is an agent that reduces the expression level of EEF1AKMT 3.

Further, the agent is a nucleic acid molecule.

Further, the nucleic acid molecule is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 7-8.

Further, the promoter is an agent that increases the expression level of MANEAL.

Furthermore, the promoter is an overexpression vector of MANEAL.

In the present invention, "marker" refers to parameters associated with one or more biomolecules (i.e., "molecular markers"), such as naturally or synthetically produced nucleic acids (i.e., individual genes, as well as coding and non-coding DNA and RNA) and proteins (e.g., peptides, polypeptides). "marker" in the context of the present invention also includes reference to a single parameter which may be calculated or otherwise obtained by taking into account expression data from two or more different markers.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. EEF1AKMT3 and MANEAL have the current International public nucleic acid databases, GeneBank, with the IDs 25895 and 149175, respectively. One skilled in the art will appreciate that in performing a sequencing analysis, the original sequencing results will be aligned to the human reference genome, and thus the genes in the screening results may contain different transcripts, so long as they can be aligned to the reference genome.

The human EEF1AKMT3 gene is located on chromosome 12, and 2 transcripts exist in EEF1AKMT3 gene in GeneBank of International public nucleic acid database. As a non-limiting example, the EEF1AKMT3 gene has the sequence NM _015433.3, NM _206914.2 of any transcript. The corresponding amino acid sequences are shown as NP-056248.2 and NP-996797.1. In the present invention, a representative EEF1AKMT3 gene sequence is shown in NM-015433.3, and an amino acid sequence is shown in NP-056248.2.

The human MANEAL gene is located on chromosome 1, and currently, 3 transcripts of MANEAL gene exist in GeneBank, the international public nucleic acid database. As a non-limiting example, the sequences of the MANEAL gene are shown in any of the transcripts NM-001031740.2, NM-001113482.1, NM-152496.2. The corresponding amino acid sequences are shown in NP-001026910.1, NP-001106954.1, and NP-689709.1. In the present invention, a representative MANEAL gene sequence is shown as NM-001031740.2, and the amino acid sequence is shown as NP-001026910.1.

The gene marker of the invention comprises genes and proteins. Such markers include DNA comprising the complete or partial sequence of the nucleic acid sequence encoding the gene marker or the complement of this sequence. Gene marker nucleic acids also include RNA containing the entire or partial sequence of any nucleic acid sequence of interest. The gene marker protein is a protein encoded by or corresponding to the DNA gene marker of the present invention. The gene marker protein comprises the complete or partial amino acid sequence of any gene marker protein or polypeptide. Fragments and variants of gene marker genes and proteins are also included within the scope of the invention.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional or translational (i.e., protein) level.

The gene markers of the present invention are detected using a variety of nucleic acid and protein techniques known to those of ordinary skill in the art, including but not limited to: nucleic acid sequencing, nucleic acid hybridization, nucleic acid amplification technology and protein immunization technology.

The nucleic acid amplification technique of the invention is selected from the group consisting of Polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA) and Nucleic Acid Sequence Based Amplification (NASBA). Among them, PCR requires reverse transcription of RNA into DNA before amplification (RT-PCR), TMA and NASBA to directly amplify RNA.

Protein immunization techniques include sandwich immunoassays, such as sandwich ELISA, in which detection of a biomarker is performed using two antibodies that recognize different epitopes on the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunization can be carried out, for example, in the form of microtiter plates or strips.

In some embodiments, the expression level of the biomarker is detected at the transcriptional level. Various methods for specific DNA and RNA measurements using nucleic acid hybridization techniques are known to those skilled in the art. Some methods involve electrophoretic separation (e.g., Southern blots for detecting DNA and Northern blots for detecting RNA), but measurements of DNA and RNA can also be made without electrophoretic separation (e.g., by dot blotting). Southern blots of genomic DNA (e.g., from humans) can be used to screen for Restriction Fragment Length Polymorphisms (RFLPs) to detect the presence of a genetic disorder affecting a polypeptide of the invention. All forms of RNA can be detected, including but not limited to messenger RNA (mRNA), micro RNA (miRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).

The choice of the nucleic acid hybridization format is not critical. Various nucleic acid hybridization formats include, but are not limited to, sandwich assays and competitive or alternative assays. Detection of the hybridization complex may require binding of the signal-producing complex to the duplex of the target and probe polynucleotide or nucleic acid. Typically, this binding occurs through a ligand-anti-ligand interaction, such as an interaction between a ligand-coupled probe and a signal-coupled anti-ligand. The binding of the signal-generating complex is also readily accelerated by exposure to ultrasonic energy.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The term "chip", also known as "array", refers to a solid support comprising attached nucleic acid or peptide probes. Arrays typically comprise a plurality of different nucleic acid or peptide probes attached to the surface of a substrate at different known locations. These arrays, also known as "microarrays," can generally be produced using either mechanosynthesis methods or light-guided synthesis methods that incorporate a combination of photolithography and solid-phase synthesis methods. The array may comprise a flat surface, or may be nucleic acids or peptides on beads, gels, polymer surfaces, fibers such as optical fibers, glass, or any other suitable substrate. The array may be packaged in a manner that allows for diagnostic or other manipulation of the fully functional device.

A "microarray" is an ordered array of hybridization array elements, such as polynucleotide probes (e.g., oligonucleotides) or binding agents (e.g., antibodies), on a substrate. The matrix may be a solid matrix, for example, a glass or silica slide, beads, a fiber optic binder, or a semi-solid matrix, for example, a nitrocellulose membrane. The nucleotide sequence may be DNA, RNA or any permutation thereof.

Various probe arrays have been described in the literature and can be used in the context of the present invention to detect markers that may be associated with the phenotypes described herein. For example, a DNA probe array chip or a larger DNA probe array wafer (otherwise, individual chips may be obtained by breaking the wafer) is used in one embodiment of the present invention. The DNA probe array wafer generally comprises a glass wafer on which a high-density array of DNA probes (short DNA fragments) is placed. Each of these wafers may hold, for example, about 6000 million DNA probes for identifying longer sample DNA sequences (e.g., from an individual or population, e.g., containing a marker of interest). The identification of sample DNA by the DNA probe set on the glass wafer was performed by DNA hybridization. When a DNA sample is hybridized to an array of DNA probes, the sample binds to those probes whose sample DNA sequences are complementary. By assessing which probes the DNA of an individual sample hybridizes more strongly to, it is possible to determine whether a known nucleic acid sequence is present in the sample, and thus whether a marker found in the nucleic acid is present. This can also be used to allow discrimination of single nucleotides by controlling the hybridization conditions. Arrays provide a convenient embodiment for the simultaneous (or tandem) detection of multiple polymorphic markers.

The probe has a base sequence complementary to a specific base sequence of the target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA, RNA, or a polynucleotide obtained by substituting a part or all of nucleotides with an artificial nucleic acid such as PNA, LNA, ENA, GNA, TNA, or the like.

The antibodies against the proteins encoded by the above-mentioned genes used in the present invention are used in the broadest sense and specifically encompass, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, multispecific antibodies and antibody fragments. Such antibodies can be chimeric, humanized, human and synthetic.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, such variants typically being present in minor amounts, except for possible variants that may arise during the course of production of the monoclonal antibody. Such monoclonal antibodies typically include an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select unique clones from a collection of multiple clones, such as hybridoma clones, phage clones, or recombinant DNA clones. It will be appreciated that the selected target binding sequence may be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are generally uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be generated by a variety of techniques, including: hybridoma methods, recombinant DNA methods, phage display techniques, and techniques for generating human or human-like antibodies in animals that have partially or wholly human immunoglobulin loci or genes encoding human immunoglobulin sequences.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remaining portion of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

Non-human"humanized" forms of (e.g., murine) antibodies refer to chimeric immunoglobulins, immunoglobulin chains or fragments thereof such as Fv, Fab ', F (ab') ² Or other antigen binding subsequences of antibodies.

An "antibody fragment" comprises a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab') ² And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for the heavy and light chains) are shed from the folded structure of these two domains, contributing to the amino acid residues that bind antigen and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) can have the ability to recognize and bind antigen, with only a lower affinity than the entire binding site.

In the present invention, the nucleic acid membrane strip comprises a substrate and oligonucleotide probes immobilized on the substrate; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

Statistical method

In the present invention, the experiment is repeated at least 3 times, the result data are expressed in the form of mean value ± standard deviation, and the difference between the two is determined by t test, and it is considered to have statistical significance when P is less than 0.05.

The invention has the advantages and beneficial effects that:

the invention discovers molecular markers related to Alzheimer for the first time, wherein the molecular markers are EEF1AKMT3 and MANEAL, EEF1AKMT3 and MANEAL show differential expression in Alzheimer patients, and whether a subject suffers from Alzheimer can be judged by detecting the expression level of the molecular markers, so that early diagnosis of Alzheimer is realized.

Drawings

FIG. 1 is a graph showing the expression of the gene marker Alzheimer, wherein A is a graph showing the expression of EEF1AKMT3, and B is a graph showing the expression of MANEAL.

FIG. 2 is a graph showing the effect of EEF1AKMT3 and MANEAL on nerve cell proliferation.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers.

Example 1 screening of Gene markers associated with Alzheimer's disease

1. Sample collection

4 samples of healthy human blood and blood from Alzheimer's patients were collected, mixed with anticoagulants, and subjected to high throughput sequencing.

2. Preparation and Mass analysis of RNA samples

RNA was extracted using a blood extraction kit from Takara, the procedure was as follows:

1) transferring 0.25ml of Blood sample into a centrifuge tube, adding 0.75ml of RNAlso Blood, and repeatedly sucking up and down by using a pipette gun until the cells are completely lysed;

2) adding chloroform (1/5 volume amount of sample solution + RNAlso Blood volume amount), covering the centrifugal tube cover tightly, mixing until the solution is milky white, and standing at room temperature for 5 min;

3) centrifuging at 4 deg.C for 15min at 12,000 Xg, sucking supernatant, and transferring to new centrifuge tube;

4) adding isopropanol with equal volume into the supernatant, turning the centrifuge tube upside down, mixing, and standing at room temperature for 10 min;

5) centrifuging at 4 deg.C for 10min at 12,000 Xg;

6) discarding the supernatant, adding equivalent 75% ethanol, washing the precipitate with Vortex oscillation, centrifuging at 7,500 Xg and 4 deg.C for 5min, and discarding the supernatant;

7) drying the precipitate at room temperature, adding a proper amount of RNase-free water to dissolve the precipitate after drying the precipitate;

8) and detecting the concentration of the RNA, and identifying the yield and purity of the RNA.

3. construction and sequencing of cDNA libraries

The construction and sequencing of the cDNA library are completed by the Huada gene, and the steps are as follows:

1) total RNA DNase I digestion: digesting DNA fragments existing in a Total RNA sample by using DNase I, purifying and recovering reaction products by using magnetic beads, and finally dissolving the reaction products in DEPC water;

2) removal of rRNA: taking a digested Total RNA sample, removing rRNA by using a Ribo-Zero kit of Epicentre, detecting Agilent 2100 after removing the rRNA, and verifying the rRNA removing effect;

3) RNA disruption: taking the sample in the previous step, adding a breaking Buffer, and placing the sample in a PCR instrument for thermal breaking till 140-;

4) reverse transcription one-strand synthesis: adding a proper amount of primers into the broken sample, fully and uniformly mixing, reacting for a certain time at a proper temperature of a Thermomixer to open a secondary structure and combine with the primers, adding a one-chain synthesis reaction system Mix prepared in advance, and synthesizing one-chain cDNA on a PCR instrument according to a corresponding procedure;

5) synthesis of reverse transcription two-chain: preparing a double-chain synthesis reaction system, reacting on a Thermomixer at a proper temperature for a certain time to synthesize double-chain cDNA with dUTP, and purifying and recovering reaction products by using magnetic beads;

6) and (3) repairing the tail end: preparing a tail end repairing reaction system, reacting in a Thermomixer at a proper temperature for a certain time, repairing the viscous tail end of a cDNA double-chain obtained by reverse transcription under the action of enzyme, purifying and recovering a tail end repairing product by using magnetic beads, and finally dissolving a sample in EB Solution;

7) 3' end of cDNA plus "A": preparing an A reaction system, reacting in a Thermomixer at a proper temperature for a certain time, and adding A basic groups to the 3' end of a product cDNA with repaired end under the action of enzyme;

8) ligation of cDNA 5' adapter: preparing a joint connection reaction system, reacting in a Thermomixer at a proper temperature for a certain time, connecting a joint with the A base under the action of enzyme, and purifying and recovering a product by using magnetic beads;

9) UNG digested cDNA double strand: preparing a UNG digestion reaction system, digesting two strands in double-stranded DNA by UNG enzyme, and purifying and recovering a product by using magnetic beads;

10) PCR reaction and product recovery: preparing a PCR reaction system, selecting a proper PCR reaction program, amplifying the product obtained in the previous step, carrying out magnetic bead purification and recovery on the PCR product, dissolving the recovered product in EB solution, and labeling;

11) and (3) detecting the quality of the library: the library quality was checked using Agilent 2100Bioanalyzer and ABI StepOneplus Real-Time PCR System;

12) and (3) machine sequencing: and (4) detecting a qualified library, adding NaOH to denature the library into a single chain, and diluting the single chain to a certain computer-loading concentration according to the expected computer-loading data quantity. Adding the library after denaturation and dilution into the FlowCell, hybridizing with a linker on the FlowCell, completing bridge PCR amplification on a cBot, and finally sequencing by using an Illumina Hiseq x-ten platform.

4. Bioinformatics analysis

RNA-seq read mapping was performed using TopHat v1.3.1, RNA-seq fragment numbers were normalized by Cufflinks v1.0.3 to calculate relative abundance of transcripts, differential expression was detected using cuffdiff, p values were pooled using invert normal method in meta assay, genes were considered significantly differentially expressed when FDR value < 0.01.

5. Results

Sequencing results show that compared with healthy people, EEF1AKMT3 and MANEAL show a significant difference in Alzheimer patients, wherein EEF1AKMT3 is up-regulated in the Alzheimer patients, and MANEAL is down-regulated in the Alzheimer patients.

Example 2 QPCR sequencing verified differential expression of genes

1. Blood samples from 31 alzheimer patients and 24 healthy persons were collected for QPCR validation in the manner as described in example 1.

2. RNA extraction

RNA was extracted from blood using Takara RNA extraction kit, see example 1 for specific procedures.

3、QPCR

1) Design of primers

Primers were designed based on the gene sequences of EEF1AKMT3, MANEAL and GADPH, and the primer sequences are shown below.

EEF1AKMT3 gene:

SEQ ID NO.1 (forward primer): 5'-AGATCCCGAATCTGAGTC-3'

SEQ ID NO.2 (reverse primer): 5'-GGCTCTTCTCCGAGTAAG-3', respectively;

MANEAL gene:

SEQ ID NO.3 (forward primer): 5'-CACATCCAACCCTACAAG-3'

SEQ ID NO.4 (reverse primer): 5'-TTCCAGTTGTGTCAATGAT-3', respectively;

GAPDH gene:

SEQ ID No.5 (forward primer): 5'-AATCCCATCACCATCTTCCAG-3'

SEQ ID NO.6 (reverse primer): 5'-GAGCCCCAGCCTTCTCCAT-3'

2) Real-time quantitative PCR

TaKaRa One Step TB Green ^TM Prime Script ^TM The RT-PCR kit (Code No. RR066A) was used for PCR reaction, and the reaction system and reaction conditions are shown in Table 1.

TABLE 1QPCR reaction System and reaction conditions

In the Thermal Cycler

PCR amplification is carried out on the Time System amplification instrument, after the reaction is finished, the amplification curve and the dissolution curve of Real Time PCR are confirmed, and relative quantification is carried out by the delta CT method.

4. ROC analysis

The variables EEF1AKMT3 and MANEAL were analyzed by SPSS for ROC analysis to determine the diagnostic potency, sensitivity and specificity of the genes.

5. Results

The results are shown in fig. 1, compared with healthy people, the expression of EEF1AKMT3 is up-regulated by about 5.7 times in Alzheimer patients, and the expression of MANEAL is down-regulated by about 3.9 times in Alzheimer patients, and the difference has statistical significance (P <0.05), which suggests that EEF1AKMT3 and MANEAL can be used as molecular markers for Alzheimer diagnosis.

The ROC analysis results are shown in table 2, and the AUC value with EEF1AKMT3 as the detection variable is 0.927; the AUC value with MANEAL as the test variable was 0.895. The specificity and the sensitivity of EEF1AKMT3 and MANEAL used as detection variables are high, and the diagnosis efficiency is higher.

TABLE 2ROC analysis results

a. Under the non-parametric assumption

b. Zero hypothesis: real area is 0.5

Example 3 functional verification of Gene markers

1. Cell culture

PC12 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ under 5% CO ₂ Culturing in an incubator with the relative humidity of 90 percent, carrying out passage at the ratio of 1:3, changing the culture solution after 24 hours when the cells enter a logarithmic growth phase, and carrying out different interventions according to experimental requirements.

2. Transfection

2.1 treatment of cells before transfection

One day before transfection, 5X 10 wells were plated in 6-well plates ⁵ Cells/well were cultured in antibiotic-free medium for one day at a cell density of 60% at transfection, and replaced with serum-free medium before transfection.

2.2 Gene overexpression vectors and design and Synthesis of interfering RNAs

Interfering RNA aiming at EEF1AKMT3 is designed and synthesized by Shanghai Ji code pharmaceutical technology Limited company: siRNA-EEF1AKMT3, control was general siRNA-NC. The sequence of siRNA-EEF1AKMT3 is shown in SEQ ID NO. 7-8.

An overexpression vector of MANEAL is designed and synthesized by the Shanghai Jikai gene, a specific PCR amplification primer is synthesized according to the sequence of MANEAL, and restriction enzyme cutting sites are added to a 5 'end primer and a 3' end primer. cDNA extracted and reverse transcribed from Alzheimer patients is used as an amplification template, the cDNA sequence is inserted into a eukaryotic cell expression vector after double restriction enzyme digestion by restriction enzyme, and the obtained recombinant vector is connected for subsequent experiments.

The experiment was divided into three groups: blank control, negative control: transfecting no-load, and transfecting siRNA-NC; experimental groups: siRNA-EEF1AKMT3 was transfected, and an overexpression vector of MANEAL was transfected. Transfection was carried out using lipofectamine 2000 from Invitrogen, the detailed procedure was performed according to the instructions.

2.3 detection of transfection Effect

The expression levels of EEF1AKMT3 and MANEAL in the cells were measured using QPCR and real-time quantitative PCR assays were performed as described in example 2.

3. CCK-8 assays for the Effect of EEF1AKMT3 and MANEAL on AD cells

Cells were collected in log phase and adjusted to 5X 10 cell concentration ⁴ Perml, 100. mu.L of the cell suspension was inoculated into a 96-well plate and placed at 37 ℃ in 5% CO ₂ Incubate overnight, and set five replicate wells per group. The experiments were divided into three groups, blank control group, model group, experimental group. Control group: the cells were not subjected to any treatment; negative control group: cells transfected with empty or siRNA-NC but no drug treatment; model group: cells were subjected to A.beta.only _1-42 Processing; experimental groups: cell transfection of overexpression vector or siRNA-EEF1AKMT3 and A beta _1-42 And (6) processing. After 48h of drug treatment, 10 μ LCCK-8 solution was added to each well, incubated at 37 ℃ for 4h, and the absorbance value (OD value) of each well was measured at a wavelength of 450nm in a microplate reader

4. Statistical analysis

All data were processed using graphpad software, data are expressed as mean ± standard deviation (mean ± SD), differences between groups were analyzed using paired sample t-test, and p <0.05 indicates statistical differences.

5. Results

The expression level of EEF1AKMT3 after transfection of siRNA-EEF1AKMT3 (0.24 ± 0.0755) was significantly lower than that of the blank and negative control groups (blank vs experiment group, P value 0.0033, ×, negative vs experiment group, P value 0.007, ×) and no significant change in the expression level of EEF1AKMT 2 (0.963 ± 0.0306) of the negative control group transfected siRNA-NC (blank vs negative control group, P value 0.173, ns).

The expression level of MANEAL in the blank control group is taken as a reference value and is determined as 1, and the transfection result shows that the expression level (4.18 +/-0.676) of MANEAL in the experimental group after the vector for over-expressing MANEAL is transfected is obviously higher than that in the control group and the negative control group (the blank control group vs experiment group, the P value is 0.0147, the negative control group vs experiment group, the P value is 0.016, and the MANEAL in the negative control group without transfection load (0.953 +/-0.0513) is not obviously changed compared with the blank control group (the blank control group vs negative control group, the P value is 0.256, ns)

CCK-8 assay results showed that the OD values of the experimental group (transfected MANEAL over-expression vector and siRNA-EEF1AKMT3) were significantly increased compared to the model group (FIG. 2), indicating that MANEAL and EEF1AKMT3 affected cell proliferation.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

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<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cacatccaac cctacaag 18

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttccagttgt gtcaatgat 19

<210> 5

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatcccatca ccatcttcca g 21

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gagccccagc cttctccat 19

<210> 7

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

agauguugac auuuucaucc u 21

<210> 8

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gaugaaaaug ucaacaucua u 21

Claims (8)

1. Application of a reagent for detecting mRNA expression level of a gene marker in preparation of a product for diagnosing Alzheimer's disease is characterized in that the gene marker is EEF1AKMT 3.

2. The use according to claim 1, wherein the product comprises reagents for detecting the level of expression of mRNA of a gene marker by sequencing, nucleic acid hybridization or nucleic acid amplification techniques.

3. Use according to claim 1 or 2, characterized in that the agent is selected from:

a probe that specifically recognizes EEF1AKMT 3; or

Primers for specifically amplifying EEF1AKMT 3.

4. The use of claim 3, wherein the primer sequence for specific amplification of EEF1AKMT3 is shown in SEQ ID NO. 1-2.

5. The use of claim 1, wherein the product comprises a nucleic acid membrane strip, chip or kit.

6. The use of claim 5, wherein the chip comprises a gene chip comprising oligonucleotide probes specific for gene markers.

7. The use according to claim 5, wherein the kit comprises:

one or more reagents for detecting the level of expression of the gene marker mRNA; and

one or more selected from the group consisting of: container, instructions for use, positive controls, negative controls, buffer.

8. The use of claim 7, wherein the kit comprises reagents for detecting the expression level of mRNA of a gene marker by RT-PCR, qRT-PCR, biochip detection, southern blotting or in situ hybridization.

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