CN108732355B

CN108732355B - Detection method for determining activity of BACE1 enzyme-cleaved NRG1 and kit thereof

Info

Publication number: CN108732355B
Application number: CN201710277553.5A
Authority: CN
Inventors: 李人; 申勇; 张峥嵘; 高峰
Original assignee: Beijing Anding Hospital
Current assignee: Beijing Anding Hospital
Priority date: 2017-04-25
Filing date: 2017-04-25
Publication date: 2021-06-25
Anticipated expiration: 2037-04-25
Also published as: CN108732355A

Abstract

BACE1 can cut NRG 1in a specific sequence between EF and ME regions of NRG1, which are 10 residues away from a transmembrane region, and the activity of BACE1 enzyme is closely related to the onset, the course and the severity of schizophrenia. On the basis, the invention provides a BACE1 enzyme-cutting polypeptide substrate containing an EFME sequence, a composition containing the substrate, a kit containing the substrate and application of the substrate in detection of activity of BACE1 enzyme-cutting NRG 1in a detection sample. The application can be used for diagnosing schizophrenia, and solves the problem that the existing schizophrenia diagnosis lacks effective molecular diagnosis standards.

Description

Detection method for determining activity of BACE1 enzyme-cleaved NRG1 and kit thereof

Technical Field

The invention relates to the field of medicine and biology, in particular to a detection method for in vitro determination of activity of BACE1 enzyme-digested NRG1 and a kit thereof, and the method and the kit can be used for diagnosis of schizophrenia.

Background

Schizophrenia (Schizophrania) is a serious mental disease with chronic functional loss, which is mostly developed in young and middle-aged years, and the population risk is 1% (Owen M J, Craddock N, O' donovan M C. Schizophrania: genes at last [ J ]. Trends Genet,2005,21(9): 518-. The clinical manifestations of the traditional Chinese medicine are complex, the traditional Chinese medicine often has thinking, emotion and behavior disorders and cognitive social function defects, the course of disease is long, the disability rate is high, heavy economic and mental burdens are brought to families and society, and the traditional Chinese medicine becomes an important problem to be solved by public health.

Schizophrenia is characterized by a multigenic pathogenesis and a high degree of heterogeneity. Because of its unclear pathogenesis, the lack of molecular diagnostic criteria is a long-term bottleneck in the diagnosis and treatment evaluation of this disease, and research to find molecular markers of schizophrenia has been of great interest. Nature reported 108 genetic loci that may be associated with schizophrenia in 2014, providing important objective basis for exploring biomarkers of the disease. These 108 genetic loci include neuregulin 1(NRG1) and the fourth human epidermal growth factor receptor (ErbB4) and BACE1 (Schizophranilia Working Group of the Psychiatric Genomics C. biological antigens from 108 Schizophranilia-associated genetic loci [ J ] Nature 2014,511(7510): 421-.

BACE1 was discovered in 1999 by 5 independent laboratories, respectively, to map to chromosome 11, and is widely present in central and peripheral tissue systems, of which intracerebral content and expression activity are highest. BACE1 performs substrate cleavage in an acidic environment and exerts enzyme activity. The structure of BACE1 is in a double-leaf shape, the active site is located in a double-leaf-shaped crack formed by an N-end leaf and a C-end leaf, and a substrate passes through the two leavesThe sub-gap is closed after being combined with the active site, and then releases an enzymolysis product when being opened. BACE1 is capable of specifically targeting membrane bound substrates and has sequence specificity for cleavage of the substrate. BACE1 is currently known to enzymatically cleave thirty substrates related to brain function, one of the most interesting substrates being APP, a protein that plays a central role in the pathogenesis of Alzheimer's disease. D in BACE1₉₂TGS and D₂₈₉SGT is its two active regions, and mutation of either active site results in inactivation of BACE1, affecting the cleavage function.

NRG1 belongs to the Epidermal Growth Factor (EGF) family, and biologically active NRG1 splice bodies all have EGF-like domains. The specific sequence of membrane anchored NRG1 subtypes I and III, between the EF and ME regions 10 residues away from the transmembrane region, can be cleaved by BACE1, which is widely distributed in a variety of tissues, both central and peripheral. The cleavage products contain EGF domains, called NRG1-nft alpha and NRG1-nft beta, which bind to downstream ErbB receptors, induce receptor dimerization, receptor tyrosine phosphorylation and activate downstream developmental related Cell transduction pathways such as PI3K-AKT, MAPK kinase, etc., exerting biological diversity (Luo X, Prior M, He W, et al. cleavage of neuregulin-1 by BACE1 or ADAM10 protein products differential efficiencies on biology [ J ]. J. Biol Chem,2011, and (286): 23967-.

In 2002, Stefansson et al firstly report NRG1 as a schizophrenia susceptible gene through the genome scanning research of the icelandic schizophrenia family. In the following years NRG1 was repeatedly validated by many researchers in different populations, such as the scotch population, the japanese population and the indian population, and new haplotypes and genetic loci were reported one after the other, such as the new haplotypes reported in the chinese han population in 2004. Due to the heterogeneous nature of schizophrenia, the association between NRG1 gene abnormalities and schizophrenia is not present in all but is associated with partially symptomatic patients or in certain populations. Bakker et al (Bakker S C, Hoogendohorn M L, Selten J P, et al Neuredulin 1: genetic support for schizophrenic subtypes [ J ]. Mol Psychiatry,2004,9(12): 1061-1063; rethylyi J M, Bakker S C, Polgar P, et al. Association study of NRG1, DTNBP1, RGS4, G72/G30, and 5K2A with schizophrenic and symptom selectivity in a Hungarian sample [ J ]. Am J.Med Gent B neuropsyiatr Gent, 2010,153B (3):792 801.) provide a support for this finding that there is no pathological symptom of NRV 1. With the focus on clinical significance, researchers found that NRG1 correlated with different internal phenotype indicators of schizophrenia, that prepulse inhibition of PPIs in patients carrying NRG1 mutants was impaired, and that NRG1 genetic locus changes affected the event-related delay in brain ERP latency. Recently, studies on brain function reported that people carrying NRG1 mutant also had some influence on their brain structure (including gray matter, white matter volume and density) (McIntosh A M, Moorhead T W, Job D, et al. the effects of a neuroegulin 1 variant on white matter sensitivity and integration [ J ]. Mol Psychiatry,2008,13(11): 1054-1059; Knickmeyer R C, Wang J, Zhu H, et al. common variants in a psychological gene precursor tissue at biological [ J ]. Cereb Cortex 2014 24(5): 1230-6.) suggested that NRG1 gene mutation has a pathogenic effect on the cognitive function, the potential brain activation function of patients and provides a risk of disease. In addition, abnormal expression levels of the NRG1/ErbB system were found in brain specimens from schizophrenic patients, where mRNA of the NRG 1-I type was significantly increased in the dorsolateral prefrontal lobe of the brain of the patient, and the degree of the increase was positively correlated with the dose of the antipsychotic drug administered prenatally (Hashimoto R, Straub R E, Weickert C S, et al. expression analysis of neuroegulin-1 in the dorsolateral anterior cortix in schizophrenic [ J ]. Mol Psichiatry, 2004,9(3):299 and 307.). In addition, there were investigators who detected reduced expression of NRG 1in plasma of patients with chronic schizophrenia (Wang R, Wang Y, Hu R, et al, purified plasma levels of neuroeglin-1 in drug negative properties and chronic properties with schzophrenia [ J ]. Neurosci Lett,2015,606: 220-. 224).

Disclosure of Invention

The invention is based on the discovery that the activity change of BACE1 enzyme cutting NRG1 is associated with the pathogenesis specificity of schizophrenia, and further provides a method and a kit for detecting the activity of BACE1 enzyme cutting NRG1, thereby completing the invention.

A first aspect of the present invention provides: a BACE1 enzyme-cutting polypeptide substrate, which has a structural formula as follows: r₁-R₂-EFME-R₃-R₄Wherein R is₂Is 0-8 amino acids; r₃Is 0-8 amino acids; when R is₁When it is a fluorescence-emitting group, R₄Is a fluorescence quenching group or a fluorescence emitting group; or, when R is₁When it is a fluorescence quenching group, R₄Is a fluorescent emitting group; r₁Is connected to R₂On the amino group of (A), R₄Is connected to R₃On the carboxyl group or on the amino group of the non-alpha-amino group; e is glutamic acid, F is phenylalanine, and M is methionine.

Preferably R₂Comprises the following steps: 2-8 amino acids comprising the-GI-sequence; more preferably 2-5 amino acids comprising the-GI-sequence; most preferably-GI-; wherein G is glycine and I is isoleucine.

Preferably R₃Comprises the following steps: 2-8 amino acids comprising an-AE-sequence; more preferably 2-5 amino acids comprising the-AE-sequence; most preferably-AEK-; wherein A is alanine, E is glutamic acid, and K is lysine.

Preferred fluorescent emitting groups are: rhodamine compounds, fluorescein compounds, BODIPY compounds, EDANS compounds, coumarin compounds, p-methylamino phenol compounds, cyanine compounds, acridine compounds, isoindole compounds, dansyl compounds, aminophthalic acid hydrazide compounds, anthranilic acid compounds, aminophthalimide compounds, aminonaphthalimide compounds, aminobenzofuran compounds, aminoquinoline compounds or dicyanohydroquinone compounds.

The fluorescent emitting group is further preferably: luminol, isoluminol, rhodamine 110, rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, or anthranilic acid (Abz). More preferably anthranilic acid (Abz).

Preferred fluorescence quenching groups are: a nitrated aromatic compound such as a nitrophenyl group, a nitrobenzyloxycarbonyl group or a nitrobenzoyl group, an indigo compound, a benzoquinone compound, an anthraquinone compound, an azo compound, an indanidine compound or a di-or triphenylmethane compound.

The fluorescence quenching group is further preferably: 2, 4-dinitrophenol (Dnp), nitrophenyl, nitrobenzyloxycarbonyl, or nitrobenzoyl. More preferably 2, 4-dinitrophenol (Dnp).

In one embodiment of the present invention, the BACE1 enzyme-cleaved polypeptide substrate is (Abz) -GIEFMEAEK- (Dnp) -NH₂、(Abz)-GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-(Abz)-NH₂Or (Dnp) -GIEFMEAEK- (Abz) -COOH.

In one embodiment of the present invention, the BACE1 enzyme-cleaved polypeptide substrate is (Abz) -GIEFMEAEELK- (Dnp) -NH₂、(Abz)-GIEFMEAEELK-(Dnp)-COOH、(Dnp)-GIEFMEAEELK-(Abz)-NH₂Or (Dnp) -GIEFMEAEELK- (Abz) -COOH.

In one embodiment of the present invention, the BACE1 enzyme-cleaved polypeptide substrate is (Abz) -HLGIEFMEAEELYQK- (Dnp) -NH₂、(Abz)-HLGIEFMEAEELYQK-(Dnp)-COOH、(Dnp)-HLGIEFMEAEELYQK-(Abz)-NH₂Or (Dnp) -HLGIEFMEAEELYQK- (Abz) -COOH.

From the viewpoint of the efficiency of cleavage of the substrate, and the fact that the fluorescence emission is specifically brought about by the cleavage of EFME by BACE1, it is preferable that the BACE1 cleaves the polypeptide substrate as R₁-GIEFMEAEK-R₄，R₁And R₄Is as defined above. For example, it may be: (Abz) -GIEFMEAEK- (Dnp) -NH₂、(Abz)-GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-(Abz)-NH₂Or (Dnp) -GIEFMEAEK- (Abz) -COOH.

A second aspect of the present invention is to provide: a composition comprising a BACE1 cleavage polypeptide substrate as described above.

According to the invention, the pH of the composition is between 2.9 and 4.6, preferably between 3.0 and 4.0, more preferably between 3.0 and 3.5.

According to the invention, the pH of the composition may be provided by a suitable buffer.

According to the present invention, the buffer may be various buffers known in the art, including, but not limited to, an acetate-acetate buffer, a phosphate-phosphate buffer, a citrate-citrate buffer, a glycine-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, an acetate-sodium chloride buffer, and the like.

In one embodiment of the invention, the buffer is an acetate-sodium chloride buffer. Preferably, the buffer contains 50mM acetic acid and 100mM sodium chloride.

A third aspect of the present invention is to provide: a kit comprising a BACE1 cleavage polypeptide substrate as described above.

According to the invention, the kit further contains a buffer solution capable of dissolving the BACE1 enzyme-cleaved polypeptide substrate.

According to the invention, the kit may further comprise a lysis solution for lysing the sample to be tested.

The substrate, the buffer solution and the lysate can be respectively and independently separated parts and are independently packaged, and when the substrate, the buffer solution and the lysate are used for detection, the substrate is prepared into the corresponding substrate reaction buffer solution; alternatively, the substrate may be dissolved in a buffer to form a substrate reaction buffer as one package, and the lysate may be separately divided into another package.

According to the invention, the buffer provides the substrate reaction buffer at a suitable pH value of 2.9 to 4.6, preferably 3.0 to 4.0, more preferably 3.0 to 3.5.

According to the present invention, the lysis solution for lysing a sample to be tested may be various lysis solutions for lysing a sample known in the art. In one embodiment of the invention, the lysis solution comprises: Tris-HCl, NaCl, EDTA or its salt, EGTA or its salt, Na₃VO₄Glycerol and Triton X-100. Preferably, the composition of the lysis solution is: 10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na₃VO₄10% glycerol and 0.5% Triton X-100.

A fourth aspect of the present invention provides: a method of detecting activity of BACE1 on cleavage of NRG 1in a sample, the method comprising the steps of:

1) pretreating a sample;

2) adding the treated sample into a reaction system containing a BACE1 enzyme-cleaved polypeptide substrate in the first aspect of the invention;

3) detecting the fluorescence intensity and the change rate of the reaction system obtained in the step 2).

According to the invention, the sample is blood, urine, cerebrospinal fluid or saliva from a human; preferably human blood or cerebrospinal fluid.

When the sample is human blood, the sample pretreatment of the step 1) comprises the following steps: a) centrifuging the blood to obtain plasma; b) plasma was diluted and processed with lysis solution.

According to the invention, the lysis solution contains: Tris-HCl, NaCl, EDTA or its salt, EGTA or its salt, Na₃VO₄Glycerol and Triton X-100.

Preferably, the composition of the lysis solution is: 10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na₃VO₄10% glycerol and 0.5% Triton X-100.

According to the invention, the reaction system in the step 2) contains a BACE1 enzyme-cutting polypeptide substrate and a buffer solution.

The pH of the reaction system is 2.9 to 4.6, preferably 3.0 to 4.0, more preferably 3.0 to 3.5.

According to the present invention, the buffer may be various buffers known in the art, including, but not limited to, an acetate-acetate buffer, a phosphate-phosphate buffer, a citrate-citrate buffer, a glycine-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, an acetate-sodium chloride buffer, and the like. In one embodiment of the invention, the buffer is an acetate-sodium chloride buffer. Preferably, the buffer contains 50mM acetic acid and 100mM sodium chloride.

A fifth aspect of the present invention is to provide: the use of a BACE1 enzyme-cleaved polypeptide substrate according to the first aspect of the invention, a composition according to the second aspect, a kit according to the third aspect, or a method of detection according to the fourth aspect of the invention, for the diagnosis of schizophrenia.

The study of the invention finds that the activity of BACE1 for cutting NRG1 is related to the onset or absence of schizophrenia and the course and severity of schizophrenia.

Compared with healthy people, BACE 1in schizophrenic patients has higher activity of cutting NRG1, and has significant difference. Compared with the patients with depression, the activity of BACE1 for cutting NRG 1in the patients with schizophrenia is increased, and has significant difference. The activity of BACE1 for cutting other substrates in the schizophrenic patients, for example, the activity of BACE1 for cutting APP, is not different from that of healthy people.

The increase degree of the activity of BACE1 for digesting NRG 1in a schizophrenia patient is related to the course of disease, and the shorter the course of disease or the earlier the disease occurs, the higher the increase degree of the activity of BACE1 for digesting NRG1 is; the increase of the activity of BACE1 for cutting NRG1 is gradually reduced along with the prolongation of the disease course.

The increased activity of BACE1 on NRG 1in schizophrenic patients correlates with the severity of the disease, with the higher the increased activity of BACE1 on NRG 1in patients with overall greater clinical assessments.

According to the invention, the schizophrenia is schizophrenia in the early stages of onset or in the short course of the disease.

According to the invention, the schizophrenia is severe schizophrenia.

According to the invention, said diagnosis can be used in persons suffering from schizophrenia or having a probability of developing schizophrenia. The person having a possibility of developing schizophrenia refers to a person having familial inheritance of schizophrenia.

A sixth aspect of the present invention provides: the application of the BACE1 enzyme-cutting polypeptide substrate in the first aspect of the invention, the composition in the second aspect, the kit in the third aspect and the reagent or the kit for preparing the schizophrenia diagnosis reagent.

According to the invention, the schizophrenia is severe schizophrenia.

Drawings

FIG. 1 shows that the Vmax value of the activity of BACE1 for digesting NRG1 by using sequences 1-3 as substrates in a cell over-expressing BACE1 enzyme

FIG. 2 is a graph showing the Vmean value of the activity of BACE1 for digesting NRG 1in ten minutes in cells over-expressing the BACE1 enzyme by using sequences 1-3 as substrates

FIG. 3 is a graph showing the Vmean value of the activity of BACE1 for cleaving NRG 1in cells over-expressing the BACE1 enzyme with sequences 1-3 as substrates over the entire measurement time

FIG. 4 is a graph of determining the rate of BACE1 digesting NRG 1in a cell over-expressing BACE1 enzyme by using sequences 1-3 as a substrate

FIG. 5 Vmax values of the activity of BACE1 for cleaving NRG 1in healthy human plasma using sequences 1-3 as substrates

FIG. 6 Vmean values of the activity of BACE1 for cleaving NRG 1in the first ten minutes of healthy human plasma using sequences 1-3 as substrates

FIG. 7: vmax value of activity of BACE1 enzyme-cutting NRG 1in plasma and pH value of different reaction systems by taking sequence 1 as a substrate

FIG. 8: the relation graph of the value of the average enzyme activity (Vmean) in the first ten minutes of BACE1 enzyme-cutting activity of NRG 1in plasma and the pH value of different reaction systems by taking sequence 1 as a substrate

FIG. 9: effect of two BACE1 inhibitors (LY2886721, BI) on Vmax of activity of BACE1 enzyme-cleaved NRG 1in plasma by taking sequence 1 as a substrate

FIG. 10: effect of two BACE1 inhibitors (LY2886721, BI) on the average enzyme activity (Vmean) within the first ten minutes of the enzymatic cleavage of NRG1 activity by BACE 1in plasma with sequence 1 as substrate (Vmean)

FIG. 11: effect of two BACE1 inhibitors (LY2886721, BI) on Vmax of activity of BACE1 enzyme-cleaved NRG 1in plasma by taking sequence 3 as a substrate

FIG. 12: effect of two BACE1 inhibitors (LY2886721, BI) on the average enzyme activity (Vmean) within the first ten minutes of the cleavage of NRG1 activity by BACE 1in plasma with sequence 3 as substrate

FIG. 13: the sequence 1 is taken as a substrate to detect the maximum enzyme activity of BACE1 enzymatic hydrolysis NRG 1in the plasma of schizophrenic patients and healthy people, wherein CON represents the healthy people, SCZ represents the schizophrenic patients

FIG. 14: detecting the average enzyme activity of BACE 1in the plasma of schizophrenic patients and healthy people within the first ten minutes of NRG1 enzymolysis by BACE1 by taking the sequence 1 as a substrate, wherein CON represents the healthy people, SCZ represents the schizophrenic patients

FIG. 15 a: graph of the relationship between the course of disease and the maximum enzymatic activity of BACE1 enzymatically cleaved NRG 1in plasma of schizophrenic patients, with the abscissa of the graph representing the number of months in the course of disease

FIG. 15 b: the disease course of the schizophrenic patients is within 100 months and more than 100 months, and the contrast chart of the maximum enzyme activity of BACE1 enzymatic hydrolysis NRG 1in the plasma of the schizophrenic patients

FIG. 16: graph of the relationship between CGI score of schizophrenic patients and the maximum enzymatic activity of BACE1 enzymatic NRG 1in plasma

FIG. 17: the sequence 1 is taken as a substrate to detect the maximum enzyme activity of BACE1 enzymatic hydrolysis NRG 1in the plasma of a depression patient and a healthy person, wherein CON represents the healthy person, DES represents the depression patient

FIG. 18: detecting the average enzyme activity of BACE 1in the plasma of the tristimania patient and the healthy person within the first ten minutes of NRG1 enzymolysis by using the sequence 1 as a substrate, wherein CON represents the healthy person, DES represents the tristimania patient

FIG. 19: graph of the relationship between the course of disease and the maximum enzymatic activity of BACE1 enzymatically degraded NRG 1in plasma of patients with depression, the abscissa of the graph is the number of months of the course of disease

FIG. 20: graph of relationship between CGI score of depression patients and maximum enzymatic activity of BACE1 for enzymatic NRG 1in plasma

FIG. 21: BACE1 enzyme-hydrolyzing APP maximum enzyme activity in plasma of schizophrenia patients and healthy people, wherein CON represents healthy people, SCZ represents schizophrenia patients

FIG. 22: average enzyme activity in plasma of schizophrenia patients and healthy persons within the first ten minutes of enzymatic cleavage of APP by BACE1, wherein CON represents healthy persons and SCZ represents schizophrenia patients

FIG. 23: graph of the relationship between the course of disease and the maximal enzymatic activity of BACE 1in plasma for APP cleavage in patients with schizophrenia, with the abscissa of the graph representing the number of months in the course of disease

FIG. 24: graph of the relationship between CGI score of schizophrenic patients and the maximum enzymatic activity of BACE1 for enzymatic APP in plasma

FIG. 25: western detection result graph of BACE1 enzyme over-expressed by 293T cells transfected with pKH3-HA-BACE1 plasmid

Detailed Description

The reaction principle of the invention is that BACE1 can carry out enzyme digestion on NRG 1in a specific sequence between EF and ME regions of NRG1, which are 10 residues away from a transmembrane region.

Designing polypeptide containing 'EFME' sequence, coupling fluorescence emission group and fluorescence quenching group at two ends of polypeptide, quenching fluorescence of fluorescence emission group because fluorescence quenching group is close to fluorescence emission group in space. When the polypeptide containing the sequence of 'EFME' is cut by BACE1, a peptide fragment with a fluorescence emitting group and a peptide fragment with a fluorescence quenching group are respectively obtained by enzymolysis, the fluorescence quenching group and the fluorescence emitting group are spatially separated, and the fluorescence emitting group can emit fluorescence.

Alternatively, two or more fluorescence emitting moieties capable of dimerizing or stacking to quench fluorescence are attached to a polypeptide comprising an "EFME" sequence such that the fluorescence emitting moieties dimerize or stack to quench each other or self-quench (when the fluorescence emitting moieties are the same) the fluorescence of the fluorescence emitting moieties. When a polypeptide comprising an "EFME" sequence is cleaved by BACE1, the dimer or stacking structure of the fluorescence emitting groups attached thereto dissociates and the fluorescence emitting groups separate from each other, so that fluorescence will occur due to the high degree of fluorescence inherent in the fluorescence emitting groups.

Based on the magnitude of the fluorescence intensity emitted by the released fluorescent emitting group and the rate of change thereof, the activity of BACE1 enzyme can be qualitatively and quantitatively determined, thus reflecting the degree of activation of BACE 1.

The fluorescence intensity can be detected by various detection methods known in the art, such as a microplate reader, a flow cytometer, and the like. And a multifunctional microplate reader is preferably adopted, and the multifunctional microplate reader can sensitively acquire signals and analyze the signals, so that the detection sensitivity can be greatly improved. In addition, the instrument has high detection speed, can collect fluorescence signals of samples within 20 minutes generally, is short in time for calculation and analysis, and is suitable for detecting large-batch biological samples.

Because of high fluorescence efficiency and sensitive detection, the required sample amount is very small, and BACE1 enzyme activity detection can be carried out by taking 10ul of plasma generally; and a multi-hole plate, such as a 96-hole plate, is very suitable for simultaneously measuring a plurality of sample samples, and the operation is convenient and the effect is high.

The invention provides a method for determining the activity of BACE1 for cutting NRG 1in a schizophrenia patient. The precision RSD of the method in day and in daytime is less than 15 percent.

The diagnosis principle of the invention is that the research of the invention finds that the activity of BACE1 for cutting NRG1 is related to the onset or absence of schizophrenia and the course and severity of schizophrenia, and the relation is disease-specific and directional, namely, only patients with schizophrenia have increased activity of cutting NRG1 by BACE1, but have unchanged activity of cutting other substrates by BACE 1.

In the present invention, "fluorescence quenching" and "fluorescence quenching" have the same meaning: it means that the fluorescence intensity is decreased due to various causes or effects, such as fluorescence quenching by a fluorescence quencher, self-quenching by an excessive concentration of a fluorescent substance, mutual quenching by stacking of different fluorescent substances, and the like.

In the present invention, the "fluorescence emitting group" is a molecule or compound or group capable of emitting fluorescence, and may be a molecule or compound or group capable of emitting fluorescence known in the art, such as a compound having one aromatic ring or fused ring, an organic compound having a plurality of conjugated double bonds, including but not limited to rhodamine-based compound, fluorescein-based compound, BODIPY-based compound, EDANS-based compound, coumarin-based compound, p-methylaminophenol-based compound, cyanine-based compound, acridine-based compound, isoindole-based compound, dansyl-based compound, aminophthalide-based compound (e.g., luminol and isoluminol derivatives), anthranilic acid-based compound, aminophthalimide-based compound, aminonaphthalimide-based compound, aminobenzofuran-based compound, aminoquinoline-based compound, aminonaphthodiimide-based compound, aminobenzofuran-based compound, aminoquinoline, Dicyano hydroquinone compounds.

Of these fluorescent emitting groups, compounds having a substantially flat aromatic structure are generally capable of quenching each other or self-quenching by dimerization or stacking, including but not limited to: fluorescein compounds (e.g., fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, etc.), rhodamine compounds (e.g., rhodamine 110, rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine, etc.), cyanine compounds (e.g., hemicyanine compounds, merocyanine compounds, oxonol compounds, squaraine compounds), BODIPY compounds (e.g., 4-difluoro-4-bora) -3 a-azonia-4 a-aza-s-indane (indacene)), and the like.

"fluorescence quenching group" refers to a compound or molecule capable of reducing or no longer emitting transferred energy by accepting the energy of a fluorescence emitting group, and may be various fluorescence quenching groups known in the art, including but not limited to, nitro aromatic compounds such as nitrophenyl, nitrobenzyloxycarbonyl, nitrobenzoyl, indigo compounds, benzoquinone compounds, anthraquinone compounds, azo compounds, indene compounds, di-or triphenylmethane compounds.

"amino acid" refers to an organic compound containing an amino group and a carboxyl group, and includes 20 natural amino acids. The 20 natural amino acids are: alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), methionine (Met, M), glycine (Gly, G), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y), asparagine (Asn, N), glutamine (Gln, Q), lysine (Lys, K), arginine (Arg, R), histidine (His, H), aspartic acid (Asp, D) and glutamic acid (Glu, E).

In the present invention, the amino acid sequence is from the N-terminus to the C-terminus unless otherwise specified.

In the present invention, when the terminal amino acid at the C-terminus of the substrate polypeptide sequence carries an amino group other than an alpha amino group, for example, the terminal amino acid at the C-terminus is lysine, a fluorescence emitting group or a fluorescence quenching group may be attached to the carboxyl group of the terminal amino acid at the C-terminus or the amino group other than an alpha amino group. For convenience and clarity of illustration, the present invention is cleaved with a "-NH group at the end of the polypeptide substrate₂"represents a fluorescence emitting group or a fluorescence quenching group attached to a carboxyl group, plus" -COOH "represents a fluorescence emitting group or a fluorescence quenching group attached to an amino group other than an alpha amino group. For example: (Abz) -GIEFMEAEK- (Dnp) -NH₂Indicating that said Dnp is attached to the carboxyl group of lysine; (Abz) -GIEFMEAEK- (Dnp) -COOH, indicating that the Dnp is linked to an amino group other than the alpha amino group of lysine.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.

The method according to the invention is described below by way of specific examples with reference to the accompanying drawings, to which, however, the invention is not restricted.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Plasma sample processing method:

after blood drawing, the blood vessel is turned over for several times to mix the drawn blood. The blood collection tube was left to stand for half an hour and then centrifuged (3500 rpm. times.10 minutes), thereby separating leukocytes, blood cells, and plasma. mu.L of plasma lysate (10mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na)₃

VO

₄10% glycerol and 0.5% Triton X-100) were diluted 5-fold before shaking and the treated plasma samples were ready for use.

Detecting enzyme activity by using a multifunctional microplate reader:

adding the processed plasma sample into a 96-hole black light-shielding plate, adding a substrate reaction buffer solution according to the volume ratio of 1: 1in the light-shielding environment, recording the fluorescence signal intensity within 1 hour under the conditions of 37 ℃ of temperature, setting parameters of a Biotek multifunctional microplate reader (Synergy H1), 320nm of excitation wavelength, 420nm of emission wavelength and 60-75 of gain value, and finishing data acquisition within 20 min.

Two indexes of maximum enzyme activity Vmax and average enzyme activity Vmean are selected to measure the enzyme activity.

Determination of total protein concentration in plasma:

reagent a, 1L: BCA (1%), Na were weighed separately₂CO₃.H₂O(2％)，Na₂C₄H₄O₆.2H₂O(0.16％)，NaOH(0.4％)，NaHCO₃(0.95%) water was added to 1L and the pH was adjusted to 11.25.

Reagent B, 50 ml: taking CuSO₄.5H₂O (4%), distilled water was added to 50 ml.

BCA working solution: 100ml of A is taken and mixed evenly with 2ml of reagent B.

Standard protein solution: bovine serum albumin was weighed and prepared as a 5mg/ml solution.

Protein concentration was determined and a standard curve was drawn according to the BCA protein concentration assay.

And (3) measuring the absorbance value of the sample plasma, searching a standard curve, and calculating the protein concentration (g/L) in the plasma to be measured. And calculating the maximum enzyme activity Vmax/ug and the average enzyme activity Vmean/ug of unit mass according to the protein concentration of the sample.

The data statistical method comprises the following steps:

using spss18. statistical analysis software to manage and statistically analyze data, wherein all statistical analyses adopt double-sided test, and P <0.05 is taken as significant statistical significance (except for special description); the quantitative index is described by using the mean +/-standard deviation, the minimum and the maximum, and the qualitative index is described by using frequency and percentage.

Comparison between groups: according to the data distribution type, the comparison among the quantitative index groups is carried out by using a parameter (t test, variance analysis and the like) test or a non-parameter test (rank sum test, CMH test and the like), and the comparison among the qualitative index groups is carried out by using a chi-Square test or a Fisher exact probability test.

Quantitative indices pairwise comparisons between groups type i error expansion was controlled using the Bonferroni method.

And (3) correlation analysis: pearson's method was used depending on the data type.

Study case and healthy population collection:

the clinical data of the Beijing mental disease and a sample resource library platform are used for bringing patients with schizophrenia, patients with depression and healthy people.

Grouping objects: a compact International Neuropsychiatric Interview (Mini-International clinical Interview for Neuropsychiatric Interview, MINI) was used as a diagnostic tool. Patients with schizophrenia: the diagnosis is carried out according to DSM-IV paranoid schizophrenia diagnosis standard, and the patients are diagnosed with schizophrenia; ② patients with depression: the diagnostic standard of DSM-IV depression is met, and the depression is confirmed to be diagnosed. ③ healthy subjects: healthy individuals without a family history of psychosis.

Grouping standard: firstly, all the objects to be grouped must be diagnosed by at least one doctor with the main treating and qualification above; the Han nationality, the age of which is 18-65 years old, is unlimited in nature, is right-handed and has enough audio-visual power to complete the research; the primary school and the cultural degree (at least 6 years of education) are normal in intelligence (IQ is more than or equal to 70); laboratory and functional examination (including conventional liver and kidney function electrocardiogram and electroencephalogram) have no meaningful abnormality.

Exclusion criteria: there are organic diseases of brain or body diseases accompanied by severe instability at present or before; history of abuse of alcohol, psychoactive substances and drugs; ② eliminating secondary mental symptoms (somatic diseases, drugs or other mental diseases); ③ pregnant or lactating women; fourthly, the authors are extremely excited, impulsive, suicide or violence out of order; patients receiving electric convulsion or magnetic stimulation within 6 months; sixthly, the consciousness loss exceeds 1 hour after head trauma; seventhly, the group with the recurrent schizophrenia needs to eliminate depression after the schizophrenia; the tristimania group needs to exclude the people accompanied with the psychotic symptoms.

The rejection standard is that the test scheme is violated; the final diagnosis of the person who does not meet the group entry standard; thirdly, the final data is not complete and the curative effect can not be judged.

All subjects signed informed consent.

Psychiatric disease severity classification:

evaluated by investigator, clinical Global impression Scale (Global expressions Scale CGI): is suitable for evaluating the state of illness and observing the curative effect of various mental diseases. Comprises the severity of illness, the total evaluation of curative effect and the curative effect index, and adopts an 8-grade scoring method of 0-7 points. ② Positive and negative symptoms scale (Positive and negative syndrome scale PANSS): mainly used for assessing the existence of mental symptoms and the severity of each symptom. Including positive symptoms scale, negative symptoms scale, general psychopathological symptoms scale, following the entry definition and the "scoring criteria". ③ Hamilton Depression Scale (HAMD-17): the method is mainly used for clinically evaluating the depression state. The project adopts a grade 5 grading method of 0-4, and the general demarcation scores are respectively 24, 17 and 7.

Example 1

Preparation of BACE1 enzyme-cleaved polypeptide substrate:

sequence 1: (Abz) -GIEFMEAEK- (Dnp) -NH₂

Sequence 2: (Abz) -GIEFMEAEELK- (Dnp) -NH₂

And (3) sequence: (Abz) -HLGIEFMEAEELYQK- (Dnp) -NH₂

Synthesized and supplied by gill biochemical (shanghai) ltd.

The prepared sequences and their numbering are used in the examples below.

Example 2

Respectively adopting the polypeptides of the sequences 1-3 as substrates to detect the activity of BACE1 for cutting NRG1

1. Assays Using cell systems overexpressing BACE1 protein

293T cells were cultured, and 1. mu.g of plasmid was transfected into cells in a 10cm dish by using transfection reagent to overexpress pKH3-HA-BACE1 plasmid (wherein the gene sequence of BACE1 protein is NC-000011.10, inserted between HindIII and Sal I cleavage sites of pKH3-HA plasmid, pKH3-HA plasmid was purchased from Youbaobao). After transfection at 37 ℃ with 5% CO₂Under the conditions of (1) for 36 hours. The transfected cells were shown to overexpress BACE1 protein by western assay (see FIG. 25).

The cell culture medium was removed, washed 2 times with pre-cooled PBS, and 4ml of lysis buffer (10mM Tris-HCl, pH 7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na) for 10cm of 80% density cells₃VO₄10% glycerol, and 0.5% Triton X-100), and centrifuging at 12000g and 4 ℃ for 10 minutes to collect supernatant, namely obtaining a cell lysate of BACE1 over-expressed protein.

An acetic acid-sodium chloride buffer containing 50mmol/L acetic acid and 100mmol/L NaCl was prepared, and adjusted to pH4.5 with sodium hydroxide. The sequences 1-3 are respectively dissolved in DMSO to prepare 2mM original solution, then acetic acid-sodium chloride buffer solution with pH4.5 is added for mixing to respectively prepare substrate reaction buffer solutions of the sequences 1-3, and the final concentration of the substrate sequences 1-3 is 40 mu M.

Adding 50ul of BACE1 overexpression protein cell lysate into a 96-hole black light-shielding plate, respectively adding substrate reaction buffer containing sequences 1-3 according to the volume ratio of 1: 1in a light-shielding environment, setting parameters of a Biotek multifunctional enzyme labeling instrument (Synergy H1) at the temperature of 37 ℃, and recording the fluorescence signal intensity within 1 hour under the conditions of excitation wavelength of 320nm, emission wavelength of 420nm and gain value of 60-75.

Sequences 1-3 the results of BACE1 for detecting Vmax and Vmean of NRG1 activity are shown in FIGS. 1-4. As can be seen from FIG. 1, the Vmax value of the activity of detecting BACE1 for digesting NRG1 is higher than that of sequence 3 and sequence 2 by using sequence 1 as a substrate. As can be seen from FIGS. 2 and 3, the Vmean values of the activity of BACE1 for digesting NRG1 detected in the first ten minutes and the entire duration of detection are higher than those of sequences 3 and 2 by using sequence 1 as a substrate. The fluorescence intensity values are plotted against time, as the cleavage rate is shown in FIG. 4, and it can be seen from FIG. 4 that the cleavage rate of SEQ ID No. 1 is higher than that of SEQ ID Nos. 3 and 2.

According to the experimental result of a cell system over expressing BACE1 protein, the sequence 1 is preliminarily judged to be used as a substrate, the reaction efficiency is higher, and the detection time can be favorably shortened and the detection efficiency can be improved in future detection.

2. Testing with healthy human plasma

The sequences 1-3 were dissolved in DMSO to prepare 2mM stock solutions, which were mixed with acetic acid-sodium chloride buffer solution of pH3.1 to prepare substrate reaction buffers of the sequences 1-3, respectively, with a final concentration of 40. mu.M for the substrate sequences 1-3. The acetic acid-sodium chloride buffer solution comprises 50mmol/L acetic acid and 100mmol/L NaCl.

Collecting blood of healthy people, processing the blood plasma sample, adding the processed blood plasma sample into a 96-hole black light-shielding plate, sequentially adding substrate reaction buffer solutions with sequences 1-3 in the light-shielding environment, and performing fluorescence detection at the temperature of 37 ℃, wherein the result is shown in figures 5-6.

As can be seen from FIG. 5, the Vmax value for detecting the activity of BACE 1in plasma for digesting NRG1 is higher than that of the sequences 3 and 2 by using the sequence 1 as a substrate. As can be seen from FIG. 6, the Vmean value within the first ten minutes of detecting the activity of BACE 1in plasma for cleaving NRG1 is lower than that of sequence 3 and higher than that of sequence 2 by using sequence 1 as a substrate.

As the BACE1 enzyme content in the plasma of healthy people is lower than that of a cell system over expressing BACE1, the reaction efficiency is higher by taking the sequence 1 as a substrate, so that the detection reaction taking the sequence 1 as the substrate is quickly completed, and the Vmean value in the first ten minutes is lower than that of the sequence 3.

Combining the results of Vmax and Vmean of healthy human plasma assays, sequences 1 and 3 will outperform sequence 2 as substrates.

Example 3

Detecting the activity of BACE1 for cutting NRG1 at different pH values

Different buffer systems were prepared: an acetic acid-sodium chloride buffer solution containing 50mmol/L acetic acid and 100mmol/L NaCl was prepared, and adjusted to pH3.0, pH3.5, pH4.0, and pH4.5 with sodium hydroxide, respectively.

The sequence 1 was dissolved in DMSO to prepare a 2mM stock solution, which was mixed with acetic acid-sodium chloride buffers of pH3.0, pH3.5, pH4.0, and pH4.5, respectively, to prepare a substrate reaction buffer solution, the final concentration of the substrate being 40. mu.M.

Collecting blood of healthy people, processing the blood plasma sample, adding the processed blood plasma sample into a 96-hole black light-shielding plate, sequentially adding substrate reaction buffer solutions with different pH values of the sequence 1in a light-shielding environment, and carrying out fluorescence detection at the temperature of 37 ℃, wherein the result is shown in figures 7-8.

As can be seen from fig. 7: the maximum enzyme activity of BACE1 for digesting NRG 1in plasma was measured at different pH values, and decreased with the increase of pH value of the reaction system, and the maximum value was measured at pH 3.0.

As can be seen from fig. 8: at different pH values, the activity of BACE 1in plasma for cutting NRG1 is measured, and the value of the average enzyme activity in the first ten minutes is reduced along with the increase of the pH value of the reaction system, and the value measured at the pH value of 3.0 is the maximum.

The pH value of the reaction system has an influence on the activity of BACE 1in digesting NRG 1in plasma, and the pH value of the reaction system is preferably 3.0-3.5, and most preferably 3.0 +/-0.1 for the detection sensitivity.

Example 4

Specificity test of detection method

According to the structural characteristics of BACE1, two BACE1 inhibitors are selected: LY2886721, BI, and detecting the influence of the inhibitor on the activity of BACE1 for cutting NRG 1.

Collecting blood of healthy human, processing a plasma sample, respectively incubating the processed plasma lysate with two inhibitors at room temperature for 1 hour, wherein the final concentration of each inhibitor in an incubation system is 10 mu M and 50 mu M respectively, and adding a blank solvent DMSO into a control group.

Adding the incubated plasma sample into 96-well black light shielding plate, adding substrate reaction buffer solution with pH3.0 of sequence 1, and performing fluorescence detection at 37 deg.C, the result is shown in figure 9-10.

As can be seen from fig. 9: compared with a control group, after LY2886721 is added, the maximum enzyme activity of BACE1 enzyme-digested NRG 1in the plasma is measured to be reduced, and the inhibition effect of 50 mu M LY2886721 on the maximum enzyme activity is higher than that of 10 mu M LY2886721, so that an amount-effect relationship exists; the BI with the concentration of 50 mu M also has an inhibiting effect on the maximum enzyme activity of BACE1 for cutting NRG 1in plasma;

as can be seen from fig. 10: compared with a control group, after LY2886721 is added, the measured activity of BACE1 enzyme-cutting NRG 1in plasma is reduced, the average enzyme activity in the first ten minutes is reduced, and the inhibition effect of 50 mu M LY2886721 on the average enzyme activity is higher than that of 10 mu M LY2886721, and an amount-effect relationship exists; BI at 50. mu.M also inhibited the average enzymatic activity of BACE1 enzyme on NRG 1in plasma.

Since these two inhibitors are specific inhibitors of the BACE1 enzyme, the fluorescence intensity was inhibited after addition of these two inhibitors, indicating that a fluorescent effect, i.e., a cleavage effect, was detected, resulting from cleavage of sequence 1 by BACE 1in plasma, but not by other proteases.

Based on the sequence 1 experiment results, 50 μ M inhibitor concentration was selected, and the specificity of sequence 3 was determined by the same experiment conditions and method, and the results are shown in FIGS. 11-12.

As can be seen from FIGS. 11 and 12, neither the maximum nor the average enzymatic activity of BACE1 for digesting NRG1 was significantly reduced in plasma when compared to the control after addition of 50. mu.M LY2886721 and BI. Since LY2886721, BI is a specific inhibitor of BACE1, the sequence 3 substrate still showed higher fluorescence after the inhibitor was added, suggesting that the fluorescence may result from cleavage of sequence 3 by other proteases in plasma. This indicates that sequence 1 is superior to sequence 3 in terms of specificity of detection.

Example 5

Comparison of BACE1 cleavage NRG1 enzymatic Activity in schizophrenia patients and healthy human plasma

Plasma of schizophrenia patients (38 cases) and healthy persons (37 cases) were collected, and the enzymatic activity of BACE1 cutting NRG 1in the plasma was measured by the method for measuring plasma samples in example 2 using sequence 1 as a substrate.

The results are shown in FIGS. 13-14.

As can be seen from FIG. 13, the maximum enzyme activity of BACE1 enzymatic hydrolysis NRG 1in the plasma of schizophrenia patients is higher than that of healthy people, and the difference is significant.

As can be seen in FIG. 14, the average enzyme activity of BACE 1in the plasma of schizophrenic patients within the first ten minutes of enzymatic cleavage of NRG1 was slightly higher than that of healthy persons.

Example 6

Comparison of BACE1 cleavage NRG1 Activity in plasma of schizophrenia patients of varying severity

The plasma of the schizophrenia patient in example 5 is also used, the sequence 1 is used as a substrate, and the enzyme activity of BACE1 cutting NRG 1in the plasma is detected by adopting the plasma sample detection method in example 2.

The maximum enzyme activity of BACE1 to cut NRG 1in the plasma of the tested patient is plotted with the disease course of the patient, and the result is shown in figure 15a, according to a Person method fitting, the length of the disease course of the patient is related to the maximum enzyme activity of BACE1 to cut NRG1, and the statistical significance is achieved, and the maximum enzyme activity of BACE1 to cut NRG 1in the plasma shows a descending trend along with the prolongation of the disease course, namely, the enzyme activity of BACE1 to cut NRG 1in the plasma of the patient with a short disease course is higher, and conversely, the activity of BACE 1in the plasma of the patient with a long disease course is lower. Furthermore, according to the disease course within 100 months and the disease course above 100 months, the duration of the disease course of the patients is differentiated, the maximum enzyme activity of BACE1 cutting NRG 1in the plasma of the patients is counted, and the patients with the disease course within 100 months (21 cases) are found, the maximum enzyme activity of BACE1 cutting NRG 1in the plasma of the patients with the disease course above 100 months (17 cases), and the difference is significant, and the result is shown in figure 15 b.

The patients were divided into the group of patients with low CGI (14 cases), the group of patients with CGI (8 cases) and the group of patients with high CGI (16 cases) according to the CGI score, and the maximum enzyme activity of BACE1 to cut NRG 1in the plasma of the three groups of patients was counted, and the result is shown in fig. 16, as the CGI score is increased, the maximum enzyme activity of BACE1 to cut NRG 1in the plasma is increased, wherein the maximum enzyme activity of the group of patients with low CGI is lower than that of the group of patients with high CGI, and the significant difference indicates that the activity of BACE1 to cut NRG 1in the plasma of the patients with schizophrenia with more severe clinical overall assessment is higher than that of the patients with.

Example 7

Activity of BACE 1in plasma of depression patients to cleave NRG1

The plasma of depression patients (19 cases) and healthy persons (18 cases) were collected respectively, and the enzyme activity of BACE1 cutting NRG 1in the plasma was tested by the method for testing the plasma sample in example 2 with the sequence 1 as the substrate.

The results are shown in FIGS. 17-20.

As can be seen from fig. 17, the maximum enzymatic activity of BACE1 enzyme cleaving NRG 1in the plasma of depression patients was comparable to that of healthy people with no statistically significant difference (P ═ 0.9082).

As can be seen from fig. 18, the average enzyme activity of BACE 1in the plasma of depressed patients within the first ten minutes of digesting NRG1 was comparable to that of healthy people with no statistically significant difference (P ═ 0.7965).

The maximum enzyme activity of BACE1 for cutting NRG 1in the plasma of the tested depression patients is plotted with the disease course of the patients, and the result is shown in figure 19, according to a Person method fitting, the length of the disease course of the patients is not related to the maximum enzyme activity of BACE1 for cutting NRG1, and the maximum enzyme activity of BACE1 for cutting NRG 1in the plasma is not changed along with the prolongation of the disease course, namely, the activity of BACE1 for cutting NRG 1in the plasma of the depression patients is not related to the disease course of the patients.

The depression patients are divided into patient groups with low CGI (8 cases), medium CGI (8 cases) and high CGI (3 cases) according to the CGI scores, the maximum enzyme activity of BACE1 cutting NRG 1in the plasma of the three groups of patients is counted, the result is shown in figure 20, the maximum enzyme activity of the patient group in the CGI is higher than that of the patient group with low CGI and high CGI, but the difference of two-two comparison between the three groups is not significant.

Example 8

Activity of BACE 1in plasma of schizophrenia patients and healthy persons to cut APP

The enzymatic activity of BACE 1in cleaving APP in plasma was measured by collecting plasma from patients with schizophrenia (35 cases) and healthy persons (35 cases) according to the method described in the literature (Deletion of vascular nerve factor receptor inhibitor amplification β generation and preservation and memory details in Alzheimer's micro journal of Experimental medicine 2007,178(5): 829. 841).

The results are shown in FIGS. 21-24.

As can be seen from fig. 21, the maximum enzymatic activity of BACE1 enzyme cleaving APP in the plasma of schizophrenic patients is not significantly different from that of healthy persons.

As can be seen in FIG. 22, the average enzyme activity of BACE 1in the plasma of schizophrenic patients during the first ten minutes of cleavage of APP by BACE1 was not significantly different from that of healthy persons.

The maximum enzyme activity of BACE1 cutting APP in the plasma of the tested patient is plotted with the disease course of the patient for statistics, the result is shown in figure 23, according to the Person fitting method, the disease course length of the patient is not related to the maximum enzyme activity of BACE1 cutting APP, and the maximum enzyme activity of BACE1 cutting APP in the plasma is not changed along with the prolongation of the disease course, namely, the enzyme activity of BACE1 cutting APP in the plasma of the patient is not related to the disease course of the patient.

Patients are divided into patient groups with low CGI (13 cases), medium CGI (7 cases) and high CGI (15 cases) according to CGI scores, and the maximum enzyme activity of BACE1 for cutting APP in the plasma of the three groups of patients is counted, so that the maximum enzyme activity of the BACE 1in the patient group with medium CGI is lower than that of the patient group with low CGI and high CGI as shown in figure 24, but no significant difference exists between the three groups, and the severity of the schizophrenia patients is not related to the activity of the BACE1 for cutting APP in the plasma.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

The application of a BACE1 enzyme-cleaved polypeptide substrate in preparing a schizophrenia diagnostic reagent or a kit, wherein the BACE1 enzyme-cleaved polypeptide substrate has a structural formula as follows: r₁-GI-EFME-AEK-R₄When R is₁When it is a fluorescence-emitting group, R₄Is a fluorescence quenching group or a fluorescence emitting group; or, when R is₁When it is a fluorescence quenching group, R₄Is a fluorescent emitting group; e is glutamic acid, F is phenylalanine, M is methionine, G is glycine, I is isoleucine, A is alanine, and K is lysine.
2. The use of claim 1, wherein the fluorescence emitting group is: rhodamine compounds, fluorescein compounds, BODIPY compounds, EDANS compounds, coumarin compounds, p-methylamino phenol compounds, cyanine compounds, acridine compounds, isoindole compounds, dansyl compounds, aminophthalic acid hydrazide compounds, anthranilic acid compounds, aminophthalimide compounds, aminonaphthalimide compounds, aminobenzofuran compounds, aminoquinoline compounds or dicyanohydroquinone compounds;

the fluorescence quenching group is: a nitrated aromatic compound such as a nitrophenyl group, a nitrobenzyloxycarbonyl group or a nitrobenzoyl group, an indigo compound, a benzoquinone compound, an anthraquinone compound, an azo compound, an indanidine compound or a di-or triphenylmethane compound.
3. The use of claim 2, wherein said fluorescence emitting group is luminol, isoluminol, rhodamine 110, rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, or anthranilic acid.
4. The use of claim 2, wherein the fluorescence quenching group is: 2, 4-dinitrophenol, nitrophenyl, nitrobenzyloxycarbonyl, or nitrobenzoyl.
5. The use of claim 1, wherein the BACE1 enzyme-cleaved polypeptide substrate is (Abz) -GIEFMEAEK- (Dnp) -NH₂、(Abz)-GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-(Abz)-NH₂Or (Dnp) -GIEFMEAEK- (Abz) -COOH.
6. The use as claimed in any one of claims 1 to 5, wherein the schizophrenia is schizophrenia in early onset or severe schizophrenia.
7. The use according to any one of claims 1 to 5, wherein the diagnosis of schizophrenia is performed in a person suffering from schizophrenia or having a likelihood of developing schizophrenia; the person having a possibility of developing schizophrenia refers to a person having familial inheritance of schizophrenia.
8. The use according to any one of claims 1 to 5, wherein the diagnostic reagent or diagnostic kit further comprises a buffer.
9. The use of claim 8, wherein the buffer has a pH of 2.9 to 4.6.
10. The use of claim 9, wherein the buffer has a pH of 2.9 to 4.0.
11. The use of claim 10, wherein the buffer has a pH of 2.9 to 3.5.
12. The use of claim 8, wherein the buffer is an acetate-acetate buffer, a phosphate-phosphate buffer, a citrate-citrate buffer, a glycine-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, or an acetate-sodium chloride buffer.
13. The use of claim 8, wherein the buffer is an acetate-sodium chloride buffer comprising 50mM acetic acid and 100mM sodium chloride.
14. The use according to any one of claims 1 to 5, wherein the diagnostic reagent or diagnostic kit further comprises a sample lysate comprising: Tris-HCl, NaCl, EDTA or its salt, EGTA or its salt, Na₃VO₄Glycerol and Triton X-100.
15. The use of claim 14, wherein the composition of the lysis solution is: 10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na₃VO₄10% glycerol and 0.5% Triton X-100.
16. A BACE1 enzyme-cutting polypeptide substrate, which has a structural formula as follows: r₁-GI-EFME-AEK-R₄When R is₁When it is a fluorescence-emitting group, R₄Is a fluorescence quenching group or a fluorescence emitting group; or, when R is₁When it is a fluorescence quenching group, R₄Is a fluorescent emitting group; e is glutamic acid, F is phenylalanine, M is methionine, G is glycine, I is isoleucine, A is alanine, and K is lysine.
17. The BACE1 cleaved polypeptide substrate of claim 16, wherein the fluorescence emitting group is: rhodamine compounds, fluorescein compounds, BODIPY compounds, EDANS compounds, coumarin compounds, p-methylamino phenol compounds, cyanine compounds, acridine compounds, isoindole compounds, dansyl compounds, aminophthalic acid hydrazide compounds, anthranilic acid compounds, aminophthalimide compounds, aminonaphthalimide compounds, aminobenzofuran compounds, aminoquinoline compounds or dicyanohydroquinone compounds;

the fluorescence quenching group is: a nitrated aromatic compound such as a nitrophenyl group, a nitrobenzyloxycarbonyl group or a nitrobenzoyl group, an indigo compound, a benzoquinone compound, an anthraquinone compound, an azo compound, an indanidine compound or a di-or triphenylmethane compound.
18. The BACE1 cleaved polypeptide substrate of claim 17, wherein the fluorescence emitting group is luminol, isoluminol, rhodamine 110, rhodamine 6G, rhodamine 123, rhodamine B, carboxytetramethylrhodamine, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein, or anthranilic acid.
19. The BACE1 cleaved polypeptide substrate of claim 17, wherein the fluorescence quenching group is: 2, 4-dinitrophenol, nitrophenyl, nitrobenzyloxycarbonyl, or nitrobenzoyl.
20. The BACE1 cleaved polypeptide substrate of claim 16, which is (Abz) -GIEFMEAEK- (Dnp) -NH₂、(Abz)-GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-(Abz)-NH₂Or (Dnp) -GIEFMEAEK- (Abz) -COOH.
21. A composition comprising a BACE1 cleaved polypeptide substrate according to any one of claims 16-20.
22. The composition of claim 21, wherein the composition has a pH of 2.9 to 4.6.
23. The composition of claim 22, wherein the composition has a pH of 2.9 to 4.0.
24. The composition of claim 23, wherein the composition has a pH of 2.9 to 3.5.
25. The composition of any one of claims 21-24, wherein the pH of the composition is provided by a buffer that is an acetate-acetate buffer, a phosphate-phosphate buffer, a citrate-citrate buffer, a glycine-hydrochloric acid buffer, a disodium hydrogen phosphate-citric acid buffer, or an acetate-sodium chloride buffer.
26. The composition of claim 25, wherein the buffer is an acetate-sodium chloride buffer comprising 50mM acetic acid and 100mM sodium chloride.
27. A kit comprising a BACE1 enzyme-cleaved polypeptide substrate of any one of claims 16-20 or a composition of any one of claims 21-26.
28. The kit of claim 27, further comprising a sample lysate comprising: Tris-HCl, NaCl, EDTA or its salt, EGTA or its salt, Na₃VO₄Glycerol and Triton X-100.
29. The kit of claim 28, wherein the lysate consists of: 10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na₃VO₄10% glycerol and 0.5% Triton X-100.