Protein antigen combination for detecting premature Alzheimer's disease and application

Abstract

The invention belongs to the field of biological detection, and particularly relates to a protein antigen combination for detecting premature Alzheimer's disease. According to the specific technical scheme, the protein antigen combination for detecting the early-onset Alzheimer's disease at least comprises ERBB2IP and / or LSP1. The invention provides a novel protein antigen and a combination thereof, which can be used for detecting or diagnosing the early screening Alzheimer's disease, and is particularly suitable for risk assessment and prediction before the Alzheimer's disease is attacked; meanwhile, the Alzheimer's disease and other types of dementia can be identified; and related reagents or kits can be further prepared according to needs.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a protein antigen combination and application for early-onset Alzheimer's disease detection. Background Art

[0002] Alzheimer's disease is one of the most common types of dementia worldwide. According to the age of onset, we divide AD into two categories, EOAD and LOAD. The former mainly refers to AD patients who develop symptoms before the age of 65, accounting for only 5% of the total number of AD, while the latter mainly refers to patients who develop symptoms after the age of 65. Compared with LOAD patients, EOAD patients have significant differences in clinical symptoms, genetics, imaging, and pathophysiological changes.

[0003] Protein co-expression analysis is a special bioinformatics analysis method that can be used to explore changes in biological networks, signaling pathways, and cell types in human tissues. Modules of co-expressed proteins can be associated with clinical feature information to clarify the relationship between them. In addition, the most closely related proteins in these co-expression modules may contain key drivers of disease onset. Therefore, this method has been widely used to discover potential biomarkers or therapeutic targets.

[0004] At present, some studies have explored the application of blood markers in the early diagnosis of Alzheimer's disease. However, these markers often have problems such as insufficient specificity, low sensitivity or poor stability, making them difficult to be widely used in clinical practice. In addition, a single biomarker is often difficult to fully reflect the pathological process of AD, so it is necessary to develop a combination of multiple biomarkers to improve the accuracy and reliability of detection. In view of the shortcomings of the prior art, the present invention aims to provide a protein antigen combination for the detection of early-onset Alzheimer's disease and its application. Summary of the invention

[0005] The purpose of the present invention is to provide a protein antigen combination and application for the detection of early-onset Alzheimer's disease. The protein antigen combination contains two protein antigens that are closely related to the pathological process of AD, which can achieve early and accurate detection of AD and provide strong support for clinical intervention and treatment.

[0006] To achieve the above-mentioned object of the invention, on the one hand, the present invention provides a protein antigen combination for detecting early-onset Alzheimer's disease, wherein the antigen combination at least includes ERBB2IP and / or LSP1.

[0007] Furthermore, the amino acid sequence of the ERBB2IP is shown in SEQ ID NO: 1; and the amino acid sequence of the LSP1 is shown in SEQ ID NO: 2.

[0008] On the other hand, the present invention also provides the use of the antigen combination in the preparation of a product for detecting / identifying early-onset Alzheimer's disease.

[0009] Furthermore, the product includes a reagent or a kit.

[0010] On the other hand, the present invention also provides a kit for detecting Alzheimer's disease prepared by using a combination of antigens.

[0011] Furthermore, the sample used in the kit is whole blood or serum.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention provides two new protein antigens, ERBB2IP and LSP1, and their combination, which can be used for the detection or diagnosis of early-onset Alzheimer's disease, especially for risk assessment and prediction before the onset of Alzheimer's disease, and can significantly enhance the recognition ability of early-onset Alzheimer's disease. These combined markers can more accurately distinguish AD patients from healthy people or patients with other neurodegenerative diseases, reduce the misdiagnosis and missed diagnosis rates; and can be further prepared into related reagents or kits as needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 This is a box plot of key proteins in EOAD brain tissue;

[0016] Figure 2 The expression of ERBB2IP and LSP1 in 3-month-old, 6-month-old, and 10-month-old 5×FAD mice;

[0017] Figure 3 Box plots of 3-month-old, 6-month-old, and 10-month-old 5×FAD mice;

[0018] Figure 4 This is the cellular localization map of ERBB2IP in the brain of 10-month-old 5×FAD mice;

[0019] Figure 5 is the expression of ERBB2IP in primary neurons. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0022] The present invention firstly finds that the two proteins ERBB2IP and LSP1 are closely related to the diagnosis of early-onset Alzheimer's disease. The amino acid sequence of ERBB2IP is shown in SEQ ID NO: 1; the amino acid sequence of LSP1 is shown in SEQ ID NO: 2.

[0023] Based on this new discovery, the present invention provides the use of the above proteins in preparing products (such as kits) for detecting and diagnosing Alzheimer's disease. The above proteins can be used alone or in combination to form a protein composition.

[0024] Example

[0025] The results of proteomic analysis of brain tissue are as follows Figure 1 As shown, Figure 1 The box plots in the middle show the ERBB2IP and LSP1 protein levels in the control group and the EOAD group detected in brain tissue. The thick line in the middle of the box plot, and the line segments above and below represent the median, the third quartile, and the first quartile, respectively. The asterisk represents the significance level of the Student's t test: ****p≤0.0001.

[0026] The results showed that compared with the control group, the expression of ERBB2IP and LSP1 in the EOAD group was significantly increased. Figure 1 shown.

[0027] Mouse model validation experiment: The present invention selected 5×FAD mice, which were transferred with human APP and PSEN1 genes, including 5 AD-related mutations, and AD neuropathological changes and symptoms appeared at a relatively early stage. In this example, 3-month-old, 6-month-old and 10-month-old 5×FAD mice and WT mice of the same litter were selected, and their brain tissues were analyzed by immunoblotting.

[0028] Figure 2 Middle: Western blotting experiment to detect the ERBB2IP and LSP1 protein levels in the brains of 3-month-old, 6-month-old, and 10-month-old 5×FAD mice and their littermate WT mice. β-actin and GAPDH were used as controls.

[0029] Figure 3 Box plots showing comparative analysis of ERBB2IP and LSP1 protein levels between 3-month-old, 6-month-old, and 10-month-old 5×FAD mice and littermate WT mice (n=6 for each month of age for 5×FAD mice and n=6 for WT mice). The bold line in the middle of the box plot, the line segments above and below represent the median, the third quartile, and the first quartile, respectively. Asterisks represent the significance level of the Student's t test: *p≤0.05, **p≤0.01, and ns represents p>0.05.

[0030] The results showed that compared with WT mice, the expression of ERBB2IP and LSP1 in 3-month-old and 6-month-old 5×FAD mice did not change significantly, while the expression of ERBB2IP and LSP1 in 10-month-old 5×FAD mice increased significantly (Figure 2A). Figure 2 and Figure 3 shown).

[0031] In summary, ERBB2IP and LSP1 may have an impact on the late progression of EOAD.

[0032] Immunofluorescence was used to explore the cellular localization of ERBB2IP and LSP1. Neurons were labeled with MAP2 and nuclei were labeled with DAPI in frozen brain tissue sections of 10-month-old 5×FAD mice. Figure 4 The cellular localization of ERBB2IP in the cerebral cortex of 10-month-old 5×FAD mice. The red part is the ERBB2IP protein labeled with the ERBB2IP antibody, the green part is the MAP2 protein labeled with the MAP2 (neuronal marker) antibody, and the blue part is the cell nucleus labeled with the DAPI antibody. The scale bar is 20um.

[0033] The results showed that ERBB2IP and MAP2 showed obvious co-localization in neurons of frozen brain tissue sections.

[0034] Isolation and culture of primary neurons. Figure 5 As shown, the red part is the ERBB2IP protein labeled by the ERBB2IP antibody, the green part is the MAP2 protein labeled by the MAP2 (neuronal marker) antibody, and the blue part is the cell nucleus labeled by the DAPI antibody. The scale bar is 20um.

[0035] The results showed that after using MAP2 to label neurons, the results were similar to those presented in frozen brain tissue sections ( Figure 5 ), indicating that ERBB2IP is expressed in neurons.

[0036] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A protein antigen combination for early-onset Alzheimer's disease detection, It is characterized in that The antigen combination includes at least ERBB2IP and / or LSP1.

2. The protein antigen combination for detecting early-onset Alzheimer's disease according to claim 1, It is characterized in that The amino acid sequence of the ERBB2IP is shown in SEQ ID NO: 1; the amino acid sequence of the LSP1 is shown in SEQ ID NO:

2.

3. Use of the antigen combination according to any one of claims 1 to 2 in the preparation of a product for detecting / identifying early-onset Alzheimer's disease.

4. The use according to claim 3, It is characterized in that The product includes a reagent or a kit.

5. A kit for detecting Alzheimer's disease prepared using the antigen combination according to any one of claims 1 or 2.

6. The kit according to claim 5, It is characterized in that The sample used in the kit is whole blood or serum.

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