CN114480494B - Protein probes and their use in detecting BACE1 activity - Google Patents

Abstract

The present invention relates to the field of biomedicine, and in particular to a protein probe and its application in detecting BACE1 activity. The present invention provides an expression vector, the expression vector includes a gene encoding the following protein fragments, the protein fragments include: mCherry-N fragment, mCherry-C fragment, VAMP7, BACE1 recognition polypeptide. The present invention provides a fluorescent protein probe that is intuitive, dynamic, simple, highly operable, can be implemented under physiological conditions, and can be used to detect BACE1 activity in living cells.

Description

Protein probe and its application in detecting BACE1 activity

Technical Field

The present invention relates to the field of biomedicine, and in particular to a protein probe and an application thereof in detecting BACE1 activity.

Background technique

β-Secretase (BACE1) is an aspartic protease that is mainly expressed on endosomes and plays a key role in the pathogenesis of Alzheimer's disease (AD). Vassar et al. found that BACE1 promotes the generation of a C-terminal fragment (β-CTF) containing 99 amino acid residues by mediating the cleavage of amyloid precursor protein (APP) at the β site. Subsequently, β-CTF is further cleaved by γ-secretase to form and release amyloid β (Aβ). Aβ easily accumulates in brain tissue, leading to the production of aggregates and insoluble fibers, becoming an important cause of AD. In general, BACE1 is a key enzyme in the formation of Aβ and is closely related to the occurrence and development of AD. Therefore, BACE1 can be used as an important target for AD drug development, and the development of a powerful tool with high selectivity and sensitivity for measuring BACE1 activity in living cells is crucial for the screening of AD-specific drugs.

Currently, most detection of BACE1 activity relies on chemically synthesized peptide substrates, such as the BACE1 activity detection kit sold by Sigma-Aldrich, but the kit requires cells to be lysed first and then tested using protein lysate, which cannot restore the real physiological environment. In the existing technology, the synthesis of probes for BACE1 activity detection is complex, data processing is cumbersome, instrument requirements are high, and operability is low.

Therefore, providing a protein probe and its application in detecting BACE1 activity has important practical significance.

Summary of the invention

In view of this, the present invention provides a protein probe and its application in detecting BACE1 activity. The purpose of the present invention is to develop a fluorescent probe that is intuitive, dynamic, simple, highly operable, can be implemented under physiological conditions, and can be used to detect BACE1 activity in living cells.

In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

The present invention provides an expression vector, wherein the expression vector comprises a gene encoding the following protein fragments, wherein the protein fragments comprise:

mCherry-N fragment;

mCherry-C fragment;

VAMP7;

BACE1 recognizes peptides.

In some specific embodiments of the present invention, the mCherry-N fragment is 1-132 of the mCherry amino acid sequence, and the mCherry-C fragment is 142-236 of the mCherry amino acid sequence.

In some embodiments of the present invention, 133-141, 133-140, 134-140 represent the above-mentioned BACE1 recognition polypeptides of different lengths, which include the following amino acid sequences in sequence: EVNLDAEFR, EVNLDAEF, VNLDAEF.

In some embodiments of the present invention, the above-mentioned BACE1 recognition polypeptide includes: 133-141EVNLDAEFR.

In some embodiments of the present invention, the polypeptide encoding the above 133-141 (EVNLDAEFR) recognition polypeptide has:

(16), the nucleotide sequence shown in SEQ ID NO: 16; or

(17) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (16) but is different from the nucleotide sequence shown in (16) due to the degeneracy of the genetic code; or

(18) A nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (16) or (17), and a nucleotide sequence that has the same or similar function as the nucleotide sequence shown in (16) or (17).

In some embodiments of the present invention, the above-mentioned BACE1 recognition polypeptide includes: 133-140EVNLDAEF.

In some embodiments of the present invention, the polypeptide encoding the above 133-140 (EVNLDAEF) recognition polypeptide has:

(19), the nucleotide sequence shown in SEQ ID NO: 17; or

(20) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (19) but is different from the nucleotide sequence shown in (19) due to the degeneracy of the genetic code; or

(21) A nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (19) or (20), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (19) or (20).

In some embodiments of the present invention, the above-mentioned BACE1 recognition polypeptide includes: 134-140VNLDAEF.

In some embodiments of the present invention, the polypeptide encoding the above 134-140 (VNLDAEF) recognition polypeptide has:

(22), the nucleotide sequence shown in SEQ ID NO: 18; or

(23) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (22) but is different from the nucleotide sequence shown in (22) due to the degeneracy of the genetic code; or

(24) A nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (22) or (23), and a nucleotide sequence that has the same or similar function as the nucleotide sequence shown in (22) or (23).

In some embodiments of the present invention, the mCherry-N-terminus in the expression vector is 1-132 of the mCherry amino acid sequence, and the mCherry-C-terminus is 142-236 of the mCherry amino acid sequence.

In some embodiments of the present invention, the nucleic acid encoding the mCherry-N fragment in the expression vector has:

(1), the nucleotide sequence shown in SEQ ID NO: 13; or

(2) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (1) but is different from the nucleotide sequence shown in (1) due to the degeneracy of the genetic code; or

(3) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (1) or (2), and having the same or similar functions as the nucleotide sequence shown in (1) or (2); and/or

The nucleic acid encoding the above mCherry-C fragment has:

(4) the nucleotide sequence shown in SEQ ID NO: 14; or

(5) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (4) but is different from the nucleotide sequence shown in (4) due to the degeneracy of the genetic code; or

(6) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (4) or (5), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (4) or (5); and/or

The nucleic acid encoding the above VAMP7 has:

(7) the nucleotide sequence shown in SEQ ID NO: 15; or

(8) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (7) but is different from the nucleotide sequence shown in (7) due to the degeneracy of the genetic code; or

(9) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (7) or (8), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (7) or (8); and/or

The above-mentioned BACE1 recognition polypeptide has:

(10), the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 11 and/or SEQ ID NO: 12; or

(11) an amino acid sequence obtained by replacing, deleting or adding one or more amino groups of the amino acid sequence shown in (10), and having the same or similar function as the amino acid sequence shown in (10); or

(12) an amino acid sequence that is at least 80% identical to the amino acid sequence of (10) or (11);

The nucleic acid encoding the BACE1 recognition polypeptide has:

(13), the nucleotide sequence shown in SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18; or

(14) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (13) but is different from the nucleotide sequence shown in (13) due to the degeneracy of the genetic code; or

(15) A nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (13) or (14), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (13) or (14).

The present invention also provides primers and combinations thereof, using the above expression vector as the target fragment to be amplified.

In some embodiments of the present invention, the above primers have:

(25) a nucleotide sequence as shown in any of SEQ ID NOs: 1 to 9;

(26) a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (25) but is different from the nucleotide sequence shown in (25) due to the degeneracy of the genetic code; or

(27) A nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (25) or (26), and a nucleotide sequence that has the same or similar function as the nucleotide sequence shown in (25) or (26).

The present invention also provides a host cell for transforming and/or transfecting the above expression vector.

The present invention also provides a protein probe, which is directly or indirectly produced via the above expression vector and/or the above host cell.

In some embodiments of the present invention, VAMP7 is used in the above protein probe to locate the probe on the endosome. VAMP7 is a protein located on the endosomal membrane. VAMP7 is amplified from the cDNA library using PCR technology, cloned into pcDNA3.1-mCherry-BACE1-sub, and placed at the front end of mCherry-BACE1-sub to express VAMP7-mCherry-BACE1-sub fusion protein. The probe is located in the endosome, thereby improving its sensitivity to BACE1 activity detection.

In some embodiments of the present invention, the above-mentioned protein probe is constructed by first connecting the localization sequence of VAMP7 to the N-terminus of the probe protein to achieve the purpose of localizing the fluorescent protein to the endosome, and VAMP7 has 187 amino acid residues on the cytoplasmic side and only 11 amino acid residues on the lumenal side, which can allow mCherry-BACE1-sub to have more spatial binding opportunities with BACE1 expressed in large quantities on the endosomal membrane, thereby increasing the efficiency of enzyme cleavage.

In some embodiments of the present invention, the above-mentioned protein probe is positioned on the endosome through VAMP7, increasing the possibility of binding to BACE1. EVNLDAEF is a polypeptide in APPswe (APPswe is a pathogenic mutant of APP, which can be more easily cleaved by BACE1) that can be recognized and cleaved by BACE1. When BACE1 recognizes EVNLDAEF, it can play an enzymatic role and cut mCherry into two protein segments, thereby losing fluorescence. This change in fluorescence can be detected by an optical system, such as a fluorescence microscope, to evaluate the activity of BACE1.

In some embodiments of the present invention, as shown in FIG. 3 , the replaced polypeptide in the above protein probe is located at the position where mCherry is prominent, which makes it more convenient for BACE1 to recognize and cleave the protein probe.

In some embodiments of the present invention, the above protein probe can also be constructed into a viral vector to construct a cell line that stably expresses the probe. This cell line can be used as a tool cell for screening BACE1-specific drugs and for establishing a large-scale high-throughput drug screening program.

The present invention also provides a detection product, comprising the above protein probe and an acceptable auxiliary agent.

In some embodiments of the present invention, the above-mentioned detection product does not include protein lysate.

The present invention also provides the use of the above expression vector, the above host cell, the above protein probe and/or the above detection product in preparing a kit for detecting BACE1 activity.

The present invention also provides the use of the above-mentioned expression vector, the above-mentioned host cell, the above-mentioned protein probe and/or the above-mentioned detection product in the preparation of a kit for detecting and/or diagnosing Alzheimer's disease.

The present invention also provides the use of the above expression vector, the above host cell, the above protein probe and/or the above detection product in screening drugs for preventing and/or treating Alzheimer's disease.

The present invention provides an expression vector, which includes genes encoding the following protein fragments, and the protein fragments include: mCherry-N fragment; mCherry-C fragment; VAMP7; and BACE1 recognition polypeptide.

The beneficial effects of the present invention include:

(1) Compared with existing detection reagents, the probe of the present invention does not need to use protein lysis buffer and can be detected in living cells, which can more truly reflect the activity of BACE1 in cells.

(2) Compared with some compound probes, the protein probe in the present invention can be fixedly expressed on the endosome where BACE1 is located, and can detect BACE1 activity more directly and accurately.

(3) Small chemical molecules have poor photostability and are prone to fluorescence quenching; and due to the poor pH stability of small chemical molecules, some small molecule probes are prone to hydrolysis, thereby generating nonspecific signals. The protein probe of the present invention is essentially a protein with stronger stability, thus avoiding the interference of nonspecific signals. The pKa of mCherry is <4.5, and it still has stable fluorescence in the acidic environment of the endosome (4.5 < pH < 6.5).

(4) Since the protein probe of the present invention is expressed by cells, it is conducive to conducting some dynamic and continuous experimental studies.

(5) Compared with the detection of probes using FRET fluorescence pairs, which requires a FRET fluorescence microscope, the protein probe of the present invention only requires the red fluorescence channel of mCherry and can be detected under an ordinary fluorescence microscope. It has low requirements for detection equipment, is simple and direct to operate, and is convenient for data processing.

(6) The protein probe of the present invention can be used to construct a stable expression cell line as a tool cell for BACE1-specific drug screening.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

Figure 1 shows the structure of the VAMP7-mCherry-BACE1-sub protein probe;

FIG2 shows a schematic diagram of the VAMP7-mCherry-BACE1-sub protein probe;

FIG3 is a schematic diagram showing the position of BACE1 recognition peptide EVNLDAEF replaced into mCherry;

FIG4 shows the structure of the expression plasmid of VAMP7-mCherry-BACE1-sub protein probe;

FIG5 shows the co-localization confocal image of co-transfected probes VAMP7-mCherry-BACE1-sub and Rab5a-Q79L in HEK293T cells;

FIG6 shows confocal imaging and statistical graphs of HEK293T cells transfected with probes VAMP7-mCherry-BACE1-sub-(133-141), VAMP7-mCherry-BACE1-sub-(133-140), and VAMP7-mCherry-BACE1-sub-(134-140) and then adding the BACE1 inhibitor verubecestat;

Among them: 133-141, 133-140, 134-140 represent BACE1 recognition peptides of different lengths: EV NLDAEFR, EVNLDAEF, VNLDAEF;

FIG7 shows confocal imaging and statistical graphs of probe expression in HEK293T cells of different generations;

FIG8 shows laser confocal micrographs and statistical graphs of the stably transfected cell lines containing the probe VAMP7-mCherry-BACE1-sub-(133-140) and the addition of verubecestat and overexpression of BACE1;

FIG. 9 shows laser confocal microscopy images, fluorescence intensity images and sensitivity images of HEK293T cells expressing probes 133-141, 133-140, 134-139, 134-140, 134-141 and 135-140, respectively, and then adding verubecestat.

Detailed ways

The invention discloses a protein probe and application thereof in detecting BACE1 activity.

It should be understood that the expression "one or more of..." includes individually each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and/or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

In the fluorescent protein probe construction, polypeptide recognition sequence optimization and activity detection test of the present invention, the raw materials and reagents used can be purchased from the market.

The present invention will be further described below in conjunction with embodiments:

Example 1 Construction of fluorescent protein probe into pcDNA3.1

In this embodiment, a fluorescent protein probe is constructed into pcDNA3.1, and the constructed probe is expressed in HEK293T cells.

FIG4 is a diagram of the constructed plasmid structure, and the following is a detailed description of the operation steps:

mCherry-N and mCherry-C were amplified from the mCherry-C1 plasmid using PCR using primers:

mCherry-N-for: 5'-CTTGGTACCGAGCTCGGATCCATGGTGAGCAAGGGCGAGGAGGAT-3'(BamHI) (as shown in SEQ ID NO: 1);

mCherry-N-rev: 5'-GGTGCCGCGCAGCTT-3' (as shown in SEQ ID NO: 2);

mCherry-C-for: 5'-AAGCTGCGCGGCACCGAAGTGAATCTGGATGCAGAATTCCGACAGAAGAAGACCATGGGCTGGGAG-3' (as shown in SEQ ID NO: 3);

mCherry-N-rev: 5’-CCACACTGGACTAGTGGATCCCTACTTGTACAGCTCGTCCATGCCGCC-3’(BamHI) (as shown in SEQ ID NO: 4).

Among them, mCherry-C-for contains the base sequence corresponding to the polypeptide that can be recognized and cleaved by BACE1 (5'-GAAGTGAATCTGGATGCAGAATTCCGA-3') (as shown in SEQ ID NO: 5).

Subsequently, mCherry-N and mCherry-C were fused into mCherry-BACE1-sub using the PCR fusion protein program. After pcDNA3.1 was digested with BamHI, mCherry-BACE1-sub was cloned into pcDNA3.1 by homologous recombination to construct pcDNA3.1-mCherry-BACE1-sub.

Extract mRNA from HEK293T cells, reverse transcribe into cDNA, and establish a cDNA library. Use PCR to amplify VAMP7 from the cDNA library, using primers:

VAMP7-for: 5'-TTTAAACTTAAGCTTGGTACCATGGCGATTCTTTTTGCTGTTGTT-3'(KpnI) (as shown in SEQ ID NO:6);

VAMP7-rev:5’-CATGGATCCGAGCTCGGTACCTTTCTTCACACAGCTTGGCCATGT-3’(KpnI) (as shown in SEQ ID NO:7).

After single digestion of pcDNA3.1-mCherry-BACE1-sub with KpnI, VAMP7 was cloned into pcDNA3.1-mCherry-BACE1-sub by homologous recombination to construct VAMP7-mCherry-BACE1-sub-133-141. 133-141 refers to the nine amino acid sequence of the BACE1 substrate replacing the original sequence at the position of 133a.a.-141a.a. of mCherry.

Furthermore, probes with different BACE1 substrate lengths such as VAMP7-mCherry-BACE1-sub-133-140 and VAMP7-mCherry-BACE1-sub-134-140 can be constructed by point mutation.

As shown in Figure 5, the plasmids VAMP7-mCherry-BACE1-sub and Rab5a-Q79L were co-transfected into HEK293T cells. It can be clearly seen that the VAMP7-mCherry-BACE1-sub protein can be expressed in HEK293T cells and has a good co-localization with the endosome, providing a basis for the high sensitivity of the probe.

Example 2 Optimization and screening of peptide sequences recognized by BACE1

During the construction of the probe, the peptide sequence recognized by BACE1 was optimized and screened:

The original 133-141:EVNLDAEFR was shortened to construct 133-140:EVNLDAEF, 134-139:VNLDAE, 134-140:VNLDAEF, 134-141:VNLDAEFR, 135-140:NLDAEF. Then the fluorescence intensity and sensitivity of the six probes were tested:

The results are shown in Figure 9 and Table 1. The left figure in Figure 9 is a confocal image of fluorescence intensity, the upper right figure in Figure 9 is the basic fluorescence intensity of the six probes, and the lower right figure in Figure 9 is the sensitivity of the six probes. Overall, the sensitivity of 133-140 and 134-140 has improved, and the fluorescence intensity has also improved, so three peptide sequences were screened.

Table 1

It was found that the following three polypeptide sequences of different lengths, 133-141:EVNLDAEFR, 133-140:EVNLDAEF, and 134-140:VNLDAEF, can all be used as substrates for BACE1 cleavage and participate in probe construction.

As shown in Figure 6 and Table 2, 10 μM of the BACE1 inhibitor verubecestat was used to detect the three probes VAMP7-mCherry-BACE1-sub-(133-141), VAMP7-mCherry-BACE1-sub-(133-140) and VAMP7-mCherry-BACE1-sub-(134-140). The right side of Figure 6 shows the average fluorescence intensity of each endosome, the average fluorescence intensity of the endosome in each cell, the total fluorescence intensity of each endosome and the total fluorescence intensity of all endosomes in each cell. When the BACE1 inhibitor verubecestat was added, the BACE1 activity was weakened and the probe could not be cleaved, so the fluorescence intensity was enhanced.

This demonstrates that the three probes constructed based on VAMP7-mCherry-BACE1-sub are practical in detecting BACE1 activity and may be helpful in screening BACE1 drugs with different binding sites due to their different sequences.

Table 2

ImageJ was used to analyze the fluorescence intensity of the laser confocal microscopy results, and the average fluorescence intensity and total fluorescence intensity of the endosomes (dots in the picture) and the average fluorescence intensity and total fluorescence intensity of the endosomes in each cell were recorded. The values are reported as mean + standard error of the mean (SEM). Statistical significance was calculated using the Student's t-test, and p<0.05 was considered statistically significant, indicated by * in the following form: *p<0.05, **p<0.01, ***p<0.001. The scale bar in the figure is 10μm.

Example 3 Study on the changes of BACE1 activity during cell senescence

Alzheimer's disease (AD) is a neurodegenerative disease, and the development of AD is closely related to aging. BACE1 is directly related to the accumulation of Aβ and plays an important role in the development of AD. The purpose of this example is to use the probe of the present invention to study the changes in BACE1 activity during cell aging.

For this purpose, mCherry-BACE1-sub was transfected into HEK293T cells at the 19th and 69th generations, and confocal imaging was performed after culture. The results are shown in Figure 7 and Table 3. As the number of HEK293T generations increases, the fluorescence intensity of the probe decreases, and the activity of BACE1 continues to increase. Therefore, we can reasonably speculate that the enhanced activity of BACE1 leads to more Aβ accumulation in brain tissue, leading to the death of normal neurons, and then inducing AD symptoms. This is consistent with the results obtained by previous researchers in mouse and human brain tissues, further proving the practicality of the probe.

table 3

mean SEM P19 1 0.05306 P69 0.84174 ^* 0.03685

ImageJ was used to analyze the fluorescence intensity of the laser confocal microscopy results, and the average fluorescence intensity of each cell was calculated. The values were reported as mean + standard error of the mean (SEM). Statistical significance was calculated using the Student's t-test, and p<0.05 was considered statistically significant, indicated by * in the following form: *p<0.05, **p<0.01, ***p<0.001. The scale bar in the figure is 10 μm.

Example 4

The gene sequence of VAMP7-mCherry-BACE1-sub-(133-140) was cloned into the pSin-EF2-EGFP-Pur lentiviral vector by homologous recombination. The primers used were:

VAMP7-mCherry-BACE1-sub-133-140-Sin-for: 5'-TTCTTCCATTTCAGGTGTCGTGAGGAATTCATGGCGATTCTTTTT-3'(EcoRI) (as shown in SEQ ID NO: 8)

VAMP7-mCherry-BACE1-sub-133-140-Sin-rev: 5’-GAATTGGCCGCCCTAGATGCATGCGGATCCCTACTTGTACAGCTC-3’ (BamHI) (as shown in SEQ ID NO: 9), and obtained the lentiviral backbone plasmid.

Subsequently, the plasmid pSin-EF2-VAMP7-mCherry-BACE1-sub-pur and the packaging plasmids PAX2 and PMD2.G were transfected into HEK293T cells using LentiFit at a mass ratio of 2:2:1 to produce lentivirus. HEK293T cells were infected with lentivirus and screened with puromycin to obtain a cell line that stably expressed the VAMP7-mCherry-BACE1-sub-133-140 probe.

Verubecestat was added to the cell line, and on the other hand, the cell line was overexpressed with BACE1 by transient transfection. Laser confocal microscopy was performed 24 hours later, and the fluorescence intensity was calculated using imageJ. The results are shown in Table 4 and Figure 8. Similar to Figure 6, the addition of verubecestat will increase the fluorescence intensity of the probe; and after overexpressing BACE1, the BACE1 activity in the cell line is enhanced, causing the probe to be further cleaved, resulting in a decrease in fluorescence intensity. In short, Figure 8 verifies the sensitivity and practicality of the cell line to BACE1 activity by inhibiting and enhancing BACE1 activity, providing a good tool for subsequent screening of BACE1 drugs and further research on the relationship between BACE1 and AD.

Table 4

mean SEM DMSO 1 0.04614 verubecestat 2.45406 ^*** 0.18026 Control 1 0.07164 BACE1 0.73837 ^** 0.04053

ImageJ was used to analyze the fluorescence intensity of the laser confocal microscopy results, and the total fluorescence intensity of the endosomes (dots in the figure) was counted. Values are reported as mean + standard error of the mean (SEM). Statistical significance was calculated using the Student's t-test, and p<0.05 was considered statistically significant, indicated by * in the following form: *p<0.05, **p<0.01, ***p<0.001. The scale bar in the figure is 10 μm.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

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gtatatcttt gtatcactga tgatgatttt gaacgttccc gagcctttaa ttttctgaat 240

gagataaaga agaggttcca gactacttac ggttcaagag cacagacagc acttccatat 300

gccatgaata gcgagttctc aagtgtctta gctgcacagc tgaagcatca ctctgagaat 360

aagggcctag acaaagtgat ggagactcaa gcccaagtgg atgaactgaa aggaatcatg 420

gtcagaaaca tagatctggt agctcagcga ggagaaagat tggaattatt gattgacaaa 480

acagaaaatc ttgtggattc ttctgtcacc ttcaaaacta ccagcagaaa tcttgctcga 540

gccatgtgta tgaagaacct caagctcact attatcatca tcatcgtatc aattgtgttc 600

atctatatca ttgtttcacc tctctgtggt ggatttacat ggccaagctg tgtgaagaaa 660

<210> 16

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 16

gaagtgaatc tggatgcaga attccga 27

<210> 17

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 17

gaagtgaatc tggatgcaga attc 24

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 18

gtgaatctgg atgcagaatt c 21

Claims (9)

1. An expression vector, characterized in that,

The sequence of the expression vector elements is VAMP7-mCherry-N fragment-BACE 1 recognition polypeptide-mCherry-C fragment;

the amino acid sequence of the BACE1 recognition polypeptide is shown as SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12;

The nucleotide sequence of the nucleic acid encoding the mCherry-N fragment is shown as SEQ ID NO. 13;

the nucleotide sequence of the nucleic acid encoding the mCherry-C fragment is shown in SEQ ID NO. 14.

2. The expression vector of claim 1, wherein:

the nucleotide sequence of the nucleic acid for encoding the VAMP7 is shown as SEQ ID NO. 15;

The nucleotide sequence of the nucleic acid encoding the BACE1 recognition polypeptide is shown as SEQ ID NO. 16, SEQ ID NO. 17 or SEQ ID NO. 18.

3. A host cell, characterized in that it is transformed and/or transfected with an expression vector according to claim 1 or claim 2.

4. A protein probe produced directly or indirectly by an expression vector according to claim 1 or claim 2, or a host cell according to claim 3.

5. An assay product comprising the protein probe of claim 4 and an acceptable adjuvant.

6. The test product of claim 5, wherein the test product does not include a protein lysate.

7. Use of an expression vector according to claim 1 or claim 2, a host cell according to claim 3, a protein probe according to claim 4, a detection product according to claim 5 or claim 6 for the preparation of a kit for detecting BACE1 activity.

8. Use of an expression vector according to claim 1 or claim 2, a host cell according to claim 3, a protein probe according to claim 4, a detection product according to claim 5 or claim 6 for the preparation of a kit for the detection or diagnosis of alzheimer's disease.

9. Use of an expression vector according to claim 1 or claim 2, a host cell according to claim 3, a protein probe according to claim 4, a test product according to claim 5 or claim 6 for screening for a medicament for preventing or treating alzheimer's disease.