Detection method for measuring BACE1 enzyme digestion NRG1 activity and kit thereof

A technology of kits and diagnostic reagents, which is applied in the fields of medicine and biology, and can solve problems such as impaired prepulse inhibition of PPI, delayed brain potential ERP latency, etc.

Active Publication Date: 2018-11-02
BEIJING ANDING HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the focus on clinical significance, researchers have found that NRG1 is associated with different endophenotype indicators of schizophrenia, patients with NRG1 mutants have impaired prepulse inhibition of PPI, and also found that NRG1 genetic locus changes affect event correlation. ERP latency delay

Method used

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  • Detection method for measuring BACE1 enzyme digestion NRG1 activity and kit thereof
  • Detection method for measuring BACE1 enzyme digestion NRG1 activity and kit thereof
  • Detection method for measuring BACE1 enzyme digestion NRG1 activity and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Preparation of BACE1 restriction peptide substrate:

[0128] Sequence 1: (Abz)-GIEFMEAEK-(Dnp)-NH 2

[0129] Sequence 2: (Abz)-GIEFMEAEELK-(Dnp)-NH 2

[0130] Sequence 3: (Abz)-HLGIEFMEAEELYQK-(Dnp)-NH 2

[0131] Synthesized and provided by Gil Biochemical (Shanghai) Co., Ltd.

[0132] The prepared sequence and its numbering are used in the following examples.

Embodiment 2

[0134] The peptides of sequence 1-3 were used as substrates to detect the activity of BACE1 digestion of NRG1

[0135] 1. Experiment with a cell system overexpressing BACE1 protein

[0136] Cultivate 293T cells, use transfection reagent to overexpress pKH3-HA-BACE1 plasmid (where the gene sequence of BACE1 protein is NC_000011.10, insert between HindⅢ and Sal I restriction site of pKH3-HA plasmid, pKH3-HA plasmid (Purchased from Ubao Bio), cells with a transfection volume of 10 cm culture dish were transfected with 1 μg plasmid. After transfection at 37℃, 5% CO 2 Cultivate for 36 hours under the same conditions. Tested by western (see attached Figure 25 ), which proves that the transfected cells overexpress the BACE1 protein.

[0137] Remove the cell culture medium, wash twice with pre-cooled PBS, and use 4ml lysis buffer (10mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na 3 VO 4 , 10% glycerol, and 0.5% Triton X-100) to lyse the cells, and centrifuge at 12000g at 4°C f...

Embodiment 3

[0149] Detection of the activity of NRG1 digested by BACE1 under different pH values

[0150] Prepare different buffer systems: prepare acetic acid-sodium chloride buffer solution, which contains 50mmol / L acetic acid and 100mmol / L NaCl, and adjust to pH3.0, pH3.5, pH4.0, and pH4.5 with sodium hydroxide.

[0151] Dissolve Sequence 1 in DMSO, prepare 2mM original solution, and mix with pH3.0, pH3.5, pH4.0, and pH4.5 acetic acid-sodium chloride buffer to make the substrate reaction buffer. The final concentration of the substance is 40 μM.

[0152] Collect the blood of healthy people and process the plasma sample. Add the processed plasma sample to a 96-well black light-proof plate. In a dark environment, add the substrate reaction buffers of different pH values ​​of sequence 1 in sequence, and perform fluorescence at 37°C. Test, see the result Figure 7-8 .

[0153] by Figure 7 It can be seen that under different pH values, the measured value of the maximum enzyme activity of BACE1 di...

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Abstract

BACE1 can perform enzyme digestion on NRG1 in specific sequences among EF and ME areas in the 10-residue distance to a transmembrane region of NRG1, so that the invention finds that activity of BACE1enzyme is closely related to schizophrenia morbidity, disease courses and severity degrees. On the basis, the invention provides a BACE1 enzyme digestion polypeptide substrate containing an EFME sequence, compound containing the substrate, a kit and application of the substrate in detecting BACE1 enzyme digestion NRG1 activity of a detected sample. The application can be used for diagnosing schizophrenia, and the problem that existing schizophrenia diagnosis lacks an effective molecule diagnosis standard is solved.

Description

Technical field [0001] The invention relates to the fields of medicine and biology, and is an in vitro detection method and kit for determining the activity of BACE1 digestion of NRG1. The method and kit can be used for the diagnosis of schizophrenia. Background technique [0002] Schizophrenia (Schizophrenia) is a severe mental illness accompanied by chronic functional loss. It usually occurs in young adults and the population risk rate is 1% (Owen MJ, Craddock N, O'donovan M C. Schizophrenia: genes at last?[J].Trends Genet,2005,21(9):518-525.). Its clinical manifestations are complex, often with thinking, emotional and behavioral disorders, and cognitive and social functional deficits. The course of the disease is prolonged and the disability rate is high. It brings a heavy economic and spiritual burden to the family and society, and will become an important public health solution. problem. [0003] Schizophrenia has the characteristics of polygenic disease and high heterogenei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/573
CPCG01N33/573G01N33/6896
Inventor 李人申勇张峥嵘高峰
Owner BEIJING ANDING HOSPITAL CAPITAL MEDICAL UNIV
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