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Protein probe and application thereof in detecting BACE1 activity

A protein and probe technology, applied in the field of biomedicine, can solve the problems of unable to restore the physiological environment, complex probe synthesis, cumbersome data processing, etc., to avoid the interference of non-specific signals, simple and direct operation, and low detection equipment requirements. Effect

Pending Publication Date: 2022-05-13
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, most of the detection of BACE1 activity relies on chemically synthesized peptide substrates, such as the BACE1 activity detection kit sold by Sigma-Aldrich, but the kit needs to first lyse the cells and use protein lysate for detection, which cannot restore the real physiological environment
In the existing technology, the synthesis of probes for BACE1 activity detection is complex, the data processing is cumbersome, the instrument requirements are high, and the operability is low

Method used

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  • Protein probe and application thereof in detecting BACE1 activity
  • Protein probe and application thereof in detecting BACE1 activity
  • Protein probe and application thereof in detecting BACE1 activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0094] Embodiment 1 Fluorescent protein probe is constructed to pcDNA3.1

[0095] In this embodiment, fluorescent protein probes were constructed into pcDNA3.1, and the constructed probes were expressed in HEK293T cells.

[0096] Such as Figure 4 The structure diagram of the constructed plasmid is shown below, and the operation steps are described in detail below:

[0097] Use the PCR method to amplify mCherry-N and mCherry-C from the plasmid of mCherry-C1, using primers:

[0098] mCherry-N-for:5'-CTTGGTACCGAGCTCGGATCCATGGTGAGCAAGGGCGAGGAGGAT-3'(BamHI) (as shown in SEQ ID NO:1);

[0099] mCherry-N-rev: 5'-GGTGCCGCGCAGCTT-3' (as shown in SEQ ID NO: 2);

[0100] mCherry-C-for:5'-AAGCTGCGCGGCACCGAAGTGAATCTGGATGCAGAATTCCGACAGAAGAAGACCATGGGCTGGGAG-3' (as shown in SEQ ID NO:3);

[0101] mCherry-N-rev: 5'-CCACACTGGACTAGTGGATCCCTACTTGTACAGCTCGTCCATGCCGCC-3' (BamHI) (shown in SEQ ID NO: 4).

[0102] Wherein mCherry-C-for contains the base sequence (5'-GAAGTGAATCTGGATGCAGAATTCCGA-3'...

Embodiment 2

[0110] Example 2 Optimized screening of polypeptide sequences recognized by BACE1

[0111] During the construction of the probe, optimized screening was carried out for the peptide sequence recognized by BACE1:

[0112] The original 133-141: EVNLDAEFR was shortened to construct 133-140: EVNLDAEF, 134-139: VNLDAE, 134-140: VNLDAEF, 134-141: VNLDAEFR, 135-140: NLDAEF. Subsequently, a total of 6 probes were tested for fluorescence intensity and sensitivity:

[0113] The result is as Figure 9 and as shown in Table 1, Figure 9 The middle left image is the confocal image of the fluorescence intensity, Figure 9 The upper right figure shows the basic fluorescence intensities of the six probes, Figure 9 The bottom right panel shows the sensitivity of the six probes. On the whole, the sensitivity of 133-140 and 134-140 has been improved, and the fluorescence intensity has also been improved, so three kinds of peptide sequences were screened out.

[0114] Table 1

[0115]

...

Embodiment 3

[0123] Example 3 Study on the change of BACE1 activity in cell senescence

[0124] Alzheimer's disease (AD) is a neurodegenerative disease, and the occurrence and development of AD are closely related to the growth of age. BACE1 is directly related to the accumulation of Aβ, and plays an important role in the occurrence and development of AD. The purpose of this example is to use the probe of the present invention to study the change of BACE1 activity in cell senescence.

[0125] For this purpose, HEK293T cells at passage 19 and passage 69 were transfected into mCherry-BACE1-sub, and confocal imaging was performed after culture. The result is as Figure 7 As shown in Table 3, with the increase of the number of HEK293T generations, the fluorescence intensity of the probe weakened, and the activity of BACE1 continued to increase. Therefore, we can reasonably speculate that the enhanced activity of BACE1 leads to more accumulation of Aβ in brain tissue, leading to the death of...

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Abstract

The invention relates to the field of biological medicine, in particular to a protein probe and application thereof in detecting BACE1 activity. The invention provides an expression vector, the expression vector comprises a gene for coding the following protein fragments, and the protein fragments comprise an mCherry-N fragment, an mCherry-C fragment, VAMP7 and a BACE1 recognition polypeptide. The fluorescent protein probe provided by the invention is visual, dynamic, simple and convenient, has strong operability, can be implemented under physiological conditions, and can be used for detecting the activity of BACE1 in living cells.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a protein probe and its application in detecting BACE1 activity. Background technique [0002] β-secretase (BACE1), an aspartic protease mainly expressed on endosomes, plays a key role in the pathogenesis of Alzheimer's disease (AD). Vassar et al. found that BACE1 promoted 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 undergoes further cleavage by γ-secretase to form and release amyloid β (Aβ). Aβ is easy to accumulate in brain tissue, leading to the generation of aggregates and insoluble fibers, which is an important reason for the occurrence of AD. In general, BACE1 is a key enzyme in the formation of Aβ, which is closely related to the occurrence and development of AD. Therefore, BACE1 can serve as an important target for AD drug development, and the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/62C12N15/11C12N5/10C12Q1/37G01N21/64
CPCC12N15/85C07K14/43595C12Q1/37G01N21/6428C07K2319/01C12N2800/107G01N2333/96472
Inventor 仓春蕾李豪魏亚男
Owner UNIV OF SCI & TECH OF CHINA
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