CN108732355A - A kind of active detection method of measurement BACE1 digestions NRG1 and its kit - Google Patents
A kind of active detection method of measurement BACE1 digestions NRG1 and its kit Download PDFInfo
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Abstract
BACE1 can carry out digestion in the particular sequence between the regions EF and ME of 10 residues of transmembrane region of NRG1 to NRG1, present invention discover that whether the activity of BACE1 enzymes and schizoid morbidity and the course of disease and severity close association.On this basis, the application the present invention provides a kind of BACE1 digestions peptide substrate, the composition containing the substrate and kit and the substrate containing " EFME " sequence in detecting sample in the active detections of BACE1 digestions NRG1.The application can be used for schizoid diagnosis, solve the problems, such as that current schizophrenia diagnosis lacks effective molecule diagnostic criteria.
Description
Technical field
The present invention relates to medicine and field of biology, are a kind of active detection methods of external test BACE1 digestions NRG1
And its kit, this method and kit can be used for schizoid diagnosis.
Background technology
Schizophrenia (Schizophrenia) is a kind of principal characteristic mental disease lost with chronic functional, mostly in blueness
The prime of life falls ill, and crowd's risk rate is 1% (Owen M J, Craddock N, O'donovan M C.Schizophrenia:
genes at last？[J].Trends Genet,2005,21(9):518-525.).Its complicated clinical manifestation, often have thinking,
Emotion and behavior disorder and cognition social disabilitry, the course of disease are delayed more, and disability rate is high, are brought to family and society heavy
Economy and mental burden, the major issue that will be solved as public health.
Schizophrenia has polygenes pathogenic and the feature of height heterogeneity.Since its pathogenesis is indefinite, lack
Molecule diagnostic criteria is the long-term bottleneck of the medical diagnosis on disease and treatment assessment, finds the research one of schizophrenia molecular marker
It has been concerned since straight.Nature in 2014 report with schizophrenia may relevant 108 genetic locus, should to inquire into
The biomarker of disease provides important objective basis.This 108 genetic locus include Neuregulin 1 (NRG1) with
And the 4th GG hEGF receptor (ErbB4) and BACE1 (Schizophrenia Working Group of the
Psychiatric Genomics C.Biological insights from 108schizophrenia-associated
genetic loci[J].Nature,2014,511(7510):421-427.)。
BACE1 was had found by 5 independent laboratories respectively in 1999, No. 11 chromosomes was positioned at, in being widely present in
In pivot and peripheral tissues' system, wherein intracerebral content and expression activity highest.BACE1 carries out substrate cutting under acidic environment,
Play enzyme activity.The structure of BACE1 is in " bifolium ", and active site is located at N-terminal " blade " and C-terminal " blade " is formed by double leaf
In type crack, substrate is closed after two panels leaf gap is combined with active site, then while opening releases enzymolysis product.
BACE1 can selectively targeted film bound substrates, and to the cutting of substrate have sequence-specific.It is known that BACE1 can enzyme
Xie Sanshi kinds and the relevant substrate of brain function, wherein one of substrate of greatest concern is APP, which sends out in Alzheimer disease
There is critical role in interpretation of the cause, onset and process of an illness system.D in BACE192* TGS and D289* SGT is its two active regions, any one active site
Mutation can all lead to the inactivation of BACE1, influence digestion function.
NRG1 belongs to epidermal growth factor (epidermal growth factor, EGF) family, has biological activity
NRG1 spliced bodies all have EGF spline structures domain.Film is anchored NRG1 hypotypes I and III in the EF and ME apart from 10 residues of transmembrane region
Particular sequence between region can be widely distributed the BACE1 shearings of maincenter and periphery Various Tissues.Digestion products contain
EGF structural domains, referred to as NRG1-nft α and NRG1-nft β can be combined, inducing receptor dimerization, receptor with downstream ErbB receptor
The relevant cell transduction access of the development such as tyrosine phosphorylation and activation downstream PI3K-AKT, mapk kinase, it is more to play biology
Sample (Luo X, Prior M, He W, et al.Cleavage of neuregulin-1 by BACE1 or ADAM10
protein produces differential effects on myelination[J].J Biol Chem,2011,286
(27):23967-23974；Yarden Y,Sliwkowski M X.Untangling the ErbB signalling
network[J].Nat Rev Mol Cell Biol,2001,2(2):127-137.)。
Stefansson in 2002 etc. reports NRG1 for the first time by studying Iceland's schizophrenia families genome scanning
As schizophrenia susceptibility gene.It is subsequent for many years in NRG1 by Many researchers in different crowds, such as Scotsman
Group, Japanese population and India crowd, the correlating validation repeated, and have new haplotype and genetic locus successive
Report, such as new haplotype of the Chinese han population of report in 2004.The characteristics of due to schizophrenia different substantiality disease,
Being associated with not there are in all groups between NRG1 gene unconventionalities and schizophrenia, but with partial symptoms patient or certain
A little crowds are related.Bakker et al. (Bakker S C, Hoogendoorn M L, Selten J P, et al.Neuregulin
1:genetic support for schizophrenia subtypes[J].Mol Psychiatry,2004,9(12):
1061-1063；Rethelyi J M,Bakker S C,Polgar P,et al.Association study of NRG1,
DTNBP1,RGS4,G72/G30,and PIP5K2A with schizophrenia and symptom severity in a
Hungarian sample[J].Am J Med Genet B Neuropsychiatr Genet,2010,153B(3):792-
801.) it provides research for this supposition to support, it is found that NRG1 exists with the schizophrenic onset of negative symptom and be associated with.With
The concern to clinical meaning, researcher has found NRG1, and there are correlations from phenotype index in different schizophrenia, carry
Patient's prepulse inhibition PPI of NRG1 mutant is impaired, in addition it has also been found that NRG1 genetic locus, which changes, influences event-related EEG
Position ERP latent period delays.Recently, brain function research is reported, carries the crowd of NRG1 mutant for its brain structure (including ash
Matter, white matter volume and density) also have a certain impact (Mcintosh A M, Moorhead T W, Job D, et al.The
effects of a neuregulin 1 variant on white matter density and integrity[J]
.Mol Psychiatry,2008,13(11):1054-1059；Knickmeyer R C,Wang J,Zhu H,et
al.Common variants in psychiatric risk genes predict brain structure at birth
[J].Cereb Cortex,2014,24(5):1230-1246.), prompt NRG1 gene mutations are for cognitive function of patients, brain
Activation function has an impact, and provides potentially pathogenic risk.In addition, being found in schizophreniac's brain sample, NRG1/
The expression of ErbB systems is abnormal, and the wherein mRNA of I types of NRG1- is dramatically increased in brain in patients back of the body lateral prefrontal expression,
And increased degree and take before death resisting mental disease drug dosage positive correlation (Hashimoto R, Straub R E,
Weickert C S,et al.Expression analysis of neuregulin-1in the dorsolateral
prefrontal cortex in schizophrenia[J].Mol Psychiatry,2004,9(3):299-307.).This
Outside, researcher detects that NRG1 expression quantity reduces (Wang R, Wang Y, Hu R, et in Patients with Chronic Schizophrenia blood plasma
al.Decreased plasma levels of neureglin-1in drug naive patients and chronic
patients with schizophrenia[J].Neurosci Lett,2015,606:220-224)。
Invention content
The present invention is based on the active variation for finding BACE1 digestions NRG1, and schizoid morbidity specific involvement,
And a kind of active method of detection BACE1 digestions NRG1 and kit are further provided, and complete the present invention.
The first aspect of the invention is to provide：A kind of BACE1 digestions peptide substrate, structural formula are：R1-R2-EFME-
R3-R4, wherein R2For 0-8 amino acid；R3For 0-8 amino acid；Work as R1For fluorescent emission group when, R4For fluorescent quenching group
Or fluorescent emission group；Alternatively, working as R1For fluorescent quenching group when, R4For fluorescent emission group；R1It is connected to R2Amino on, R4
It is connected to R3Carboxyl or non-alpha-amino amino on；E is glutamic acid, and F is phenylalanine, and M is methionine.
It is preferred that R2For：The 2-8 amino acid containing-GI- sequences；More preferably contain 2-5 amino acid of-GI- sequences；
Most preferably-GI-；Wherein G is glycine, and I is isoleucine.
It is preferred that R3For：The 2-8 amino acid containing-AE- sequences；More preferably contain 2-5 amino acid of-AE- sequences；
Most preferably-AEK-；Wherein A is alanine, and E is glutamic acid, and K is lysine.
It is preferred that fluorescent emission group is：Rhodamine compound, fluoresceins compound, BODIPY classes compound, EDANS
Class compound, coumarin kind compound, paramethylaminophenol class compound, cyanine compound, acridine compound, isoindoles
Compound, dansyl class compound, aminophthalic acid hydrazide kind compound, anthranilic acid compound, amino neighbour's benzene
Dicarboximide class compound, aminonaphthalimide class compound, aminobenzofur class compound, aminoquinolines
Close object or dicyano hydroquinone compound.
Fluorescent emission group is more preferably：Luminol, different luminol, rhodamine 110, rhodamine 6G, rhodamine
123, rhodamine B, carboxyl tetramethylrhodamine, fluorescein, fluorescein isothiocynate, 5-carboxyfluorescein, 6- Fluoresceincarboxylic acids,
Tetrachlorofluorescein, chlordene fluorescein, bis- chloro- 2' of carboxyl -4', 5'-, 7'- dimethoxyfluoresceins or ortho-aminobenzoic acid
(Abz).More preferably ortho-aminobenzoic acid (Abz).
It is preferred that fluorescent quenching group is：The nitrations aromatic series such as nitrobenzophenone, nitrobenzyloxycarbonyl, nitro benzoyl
Compound, indigo class compound, benzo quinones, anthraquinone analog compound, azo-compound, indenes amino benzenes compounds or
Person two or triphenylmethane compound.
Fluorescent quenching group is more preferably：2,4- dinitrophenol (Dnp), nitrobenzophenone, nitrobenzyloxycarbonyl,
Or nitro benzoyl.More preferably 2,4- dinitrophenol (Dnp).
In an embodiment of the invention, the BACE1 digestions peptide substrate is (Abz)-GIEFMEAEK-
(Dnp)-NH2、(Abz)-GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-(Abz)-NH2Or (Dnp)-
GIEFMEAEK-(Abz)-COOH。
In an embodiment of the invention, the BACE1 digestions peptide substrate is (Abz)-GIEFMEAEELK-
(Dnp)-NH2、(Abz)-GIEFMEAEELK-(Dnp)-COOH、(Dnp)-GIEFMEAEELK-(Abz)-NH2Or (Dnp)-
GIEFMEAEELK-(Abz)-COOH。
In an embodiment of the invention, the BACE1 digestions peptide substrate is (Abz)-
HLGIEFMEAEELYQK-(Dnp)-NH2、(Abz)-HLGIEFMEAEELYQK-(Dnp)-COOH、(Dnp)-
HLGIEFMEAEELYQK-(Abz)-NH2Or (Dnp)-HLGIEFMEAEELYQK- (Abz)-COOH.
The efficiency and the fluorescent emission being digested from substrate are the angles specifically brought by BACE1 digestions EFME
Degree considers that the preferably described BACE1 digestions peptide substrate is R1-GIEFMEAEK-R4, R1And R4It is as defined above.Such as it can
Think：(Abz)-GIEFMEAEK-(Dnp)-NH2、(Abz)-GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-
(Abz)-NH2Or (Dnp)-GIEFMEAEK- (Abz)-COOH.
The second aspect of the invention is to provide：A kind of composition, wherein containing above-mentioned BACE1 digestions peptide substrate.
According to the present invention, the pH value of the composition is 2.9-4.6, preferably 3.0-4.0, more preferably 3.0-3.5.
According to the present invention, the pH value of the composition can be provided by buffer solution appropriate.
According to the present invention, the buffer solution can be various buffer solutions as known in the art, including but not limited to acetic acid-
Acetate buffer, phosphoric acid-phosphate buffer, citric acid-citrate buffer solution, glycine-HCI buffer solution, phosphoric acid hydrogen
Disodium-citrate buffer solution, acetic acid-sodium chloride buffer etc..
In an embodiment of the invention, the buffer solution is acetic acid-sodium chloride buffer.It is preferred that the buffer solution
Contain 50mM acetic acid and 100mM sodium chloride.
The third aspect of the invention is to provide：A kind of kit, wherein containing above-mentioned BACE1 digestions peptide substrate.
According to the present invention, the kit further contains the buffer solution that can dissolve the BACE1 digestions peptide substrate.
According to the present invention, the kit can also further contain the lysate for being useful for cracking detected sample.
The substrate, buffer solution, lysate can be independently divided various pieces, have independent packaging, make in detection
Substrate is made into corresponding substrate reactions buffer solution by the used time with buffer solution；Can also be that substrate is dissolved in buffer solution and forms bottom
Object reaction buffer is packed as one, and lysate is independently divided for another packaging.
According to the present invention, the buffer solution provides substrate reactions buffer solution with suitable pH value, pH value 2.9-4.6, excellent
It is selected as 3.0-4.0, more preferably 3.0-3.5.
According to the present invention, the buffer solution can be various buffer solutions as known in the art, including but not limited to acetic acid-
Acetate buffer, phosphoric acid-phosphate buffer, citric acid-citrate buffer solution, glycine-HCI buffer solution, phosphoric acid hydrogen
Disodium-citrate buffer solution, acetic acid-sodium chloride buffer etc..
In an embodiment of the invention, the buffer solution is acetic acid-sodium chloride buffer.It is preferred that the buffer solution
Contain 50mM acetic acid and 100mM sodium chloride.
According to the present invention, the described lysate for cracking sample to be tested can be as known in the art various is used for
The lysate of lysate sample.In the specific embodiment of the present invention, the lysate contains：Tris-HCl,NaCl,
EDTA or its salt, EGTA or its salt, Na3VO4, glycerine and Triton X-100.It is preferred that the group of the lysate becomes：10mM
Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na3VO4, 10% glycerine and 0.5%Triton X-100.
The fourth aspect of the invention is to provide：A kind of active methods of BACE1 digestions NRG1 in detection sample, the side
Method includes the following steps：
1) sample pre-treatments；
2) by treated, the reaction containing the BACE1 digestion peptide substrates described in the first aspect of the present invention is added in sample
In system；
3) detecting step 2) gained reaction system fluorescence intensity and its rate of change.
According to the present invention, the sample is blood, urine, cerebrospinal fluid or saliva from human body；The preferably blood of human body
Liquid or cerebrospinal fluid.
When the sample is blood of human body, the sample pre-treatments of the step 1) include：A) centrifugal blood obtains blood plasma；
B) blood plasma is diluted and is handled with lysate.
According to the present invention, the lysate contains：Tris-HCl, NaCl, EDTA or its salt, EGTA or its salt, Na3VO4、
Glycerine and Triton X-100.
It is preferred that the group of the lysate becomes：10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA,
1mM Na3VO4, 10% glycerine and 0.5%Triton X-100.
According to the present invention, the reaction system in the step 2) contain BACE1 digestions peptide substrate of the present invention and
Buffer solution.
The pH value of the reaction system is 2.9-4.6, preferably 3.0-4.0, more preferably 3.0-3.5.
According to the present invention, the buffer solution can be various buffer solutions as known in the art, including but not limited to acetic acid-
Acetate buffer, phosphoric acid-phosphate buffer, citric acid-citrate buffer solution, glycine-HCI buffer solution, phosphoric acid hydrogen
Disodium-citrate buffer solution, acetic acid-sodium chloride buffer etc..In an embodiment of the invention, the buffer solution is vinegar
Acid-sodium chloride buffer.It is preferred that the buffer solution contains 50mM acetic acid and 100mM sodium chloride.
The fifth aspect of the invention is to provide：BACE1 digestions peptide substrate described in the first aspect of the present invention, second
The detection method described in kit or the 4th aspect described in terms of composition, third described in a aspect, is diagnosing
Application in schizophrenia.
The present invention the study found that whether the active height of BACE1 digestions NRG1 is with schizoid morbidity, and
The schizoid course of disease and severity are relevant.
Compared with Healthy People, the activity of the BACE1 digestions NRG1 in schizophreniac's body increases, and has conspicuousness
Difference.Compared with patients with depression, the activity of the BACE1 digestions NRG1 in schizophreniac's body increases, and with notable
Sex differernce.And in schizophreniac's body other substrates of BACE1 digestions activity, such as BACE1 digestions APP activity, with
Healthy People is compared, without difference.
The active degree that increases of BACE1 digestions NRG1 in schizophreniac's body is related to the course of disease, and the course of disease is shorter
Or morbidity is got over early stage, to increase degree higher for BACE1 digestions NRG1 active；With the extension of the course of disease, BACE1 digestions NRG1's
The active degree that increases continuously decreases.
The active of BACE1 digestions NRG1 in schizophreniac's body increases degree and the severity of disease phase
It closes, to increase degree higher for the clinical total more serious patient BACE1 digestions NRG1 of evaluation active.
According to the present invention, the schizophrenia be fall ill early stage schizophrenia or the short schizophrenia of the course of disease
Disease.
According to the present invention, the schizophrenia is severe schizophrenia.
According to the present invention, the diagnosis can be used for schizophrenia or with generation schizophrenia possibility
People.It is described have occur schizophrenia possibility people refer to its there are the genetic people of schizophrenic families.
The sixth aspect of the invention is to provide：BACE1 digestions peptide substrate described in the first aspect of the present invention, second
Kit described in terms of composition, third described in a aspect diagnoses schizoid reagent or kit preparing
In application.
According to the present invention, the schizophrenia be fall ill early stage schizophrenia or the short schizophrenia of the course of disease
Disease.
According to the present invention, the schizophrenia is severe schizophrenia.
According to the present invention, the diagnosis can be used for schizophrenia or with generation schizophrenia possibility
People.It is described have occur schizophrenia possibility people refer to its there are the genetic people of schizophrenic families.
Description of the drawings
Fig. 1 measures the active of BACE1 digestions NRG1 using sequence 1-3 as substrate in the cell for being overexpressed BACE1 enzymes
Vmax numerical value
Fig. 2 using sequence 1-3 as substrate, measured in the cell for being overexpressed BACE1 enzymes BACE1 digestions NRG1 it is active before
Vmean numerical value in ten minutes
Fig. 3 measures the active complete of BACE1 digestions NRG1 using sequence 1-3 as substrate in the cell for being overexpressed BACE1 enzymes
Vmean numerical value in portion's time of measuring
Fig. 4 measures the rate diagram of BACE1 digestions NRG1 using sequence 1-3 as substrate in the cell for being overexpressed BACE1 enzymes
Fig. 5 measures the active Vmax numerical value of BACE1 digestions NRG1 using sequence 1-3 as substrate in human normal plasma
Fig. 6 is using sequence 1-3 as substrate, in human normal plasma in active preceding ten minutes of measurement BACE1 digestions NRG1
Vmean numerical value
Fig. 7：It is substrate with sequence 1, the active Vmax numerical value of BACE1 digestions NRG1 and different reaction systems in blood plasma
PH value relational graph
Fig. 8：It is substrate with sequence 1, the activity of BACE1 digestions NRG1, the average enzymatic activity in preceding ten minutes in blood plasma
(Vmean) relational graph of numerical value and the pH value of different reaction systems
Fig. 9：It is substrate with sequence 1, two kinds of BACE1 inhibitor (LY2886721, BI) are to BACE1 digestions NRG1 in blood plasma
The influence of active Vmax
Figure 10：It is substrate with sequence 1, two kinds of BACE1 inhibitor (LY2886721, BI) are to BACE1 digestions NRG1 in blood plasma
The influence of average enzymatic activity (Vmean) in active preceding ten minutes
Figure 11：It is substrate with sequence 3, two kinds of BACE1 inhibitor (LY2886721, BI) are to BACE1 digestions NRG1 in blood plasma
The influence of active Vmax
Figure 12：It is substrate with sequence 3, two kinds of BACE1 inhibitor (LY2886721, BI) are to BACE1 digestions NRG1 in blood plasma
The influence of average enzymatic activity (Vmean) in active preceding ten minutes
Figure 13：It is substrate with sequence 1, detects BACE1 enzymolysis NRG1 in the blood plasma of schizophreniac and Healthy People
Maximum enzyme activity, CON represents Healthy People in figure, and SCZ represents schizophreniac
Figure 14：It is substrate with sequence 1, detects BACE1 enzymolysis NRG1 in the blood plasma of schizophreniac and Healthy People
Average enzyme activity in preceding ten minutes, CON represents Healthy People in figure, and SCZ represents schizophreniac
Figure 15 a：BACE1 digests the relationship between the maximum enzyme activity of NRG1 in the course of disease and blood plasma of schizophreniac
Scheme, abscissa is course of disease months in figure
Figure 15 b：The course of disease of schizophreniac is within 100 months and 100 months or more, BACE1 enzymes in blood plasma
Solve the comparison diagram of the maximum enzyme activity of NRG1
Figure 16：BACE1 digests the relationship between the maximum enzyme activity of NRG1 in schizophreniac CGI score values and blood plasma
Figure
Figure 17：It is substrate with sequence 1, detects the maximum of BACE1 enzymolysis NRG1 in the blood plasma of patients with depression and Healthy People
Enzyme activity, CON represents Healthy People in figure, and DES represents patients with depression
Figure 18：It is substrate with sequence 1, detects preceding ten of BACE1 enzymolysis NRG1 in the blood plasma of patients with depression and Healthy People
Average enzyme activity in minute, CON represents Healthy People in figure, and DES represents patients with depression
Figure 19：BACE1 digests the relational graph between the maximum enzyme activity of NRG1, figure in the course of disease and blood plasma of patients with depression
Middle abscissa is course of disease months
Figure 20：BACE1 digests the relational graph between the maximum enzyme activity of NRG1 in patients with depression CGI score values and blood plasma
Figure 21：BACE1 digests the maximum enzyme activity of APP in the blood plasma of schizophreniac and Healthy People, CON generations in figure
Table Healthy People, SCZ represent schizophreniac
Figure 22：BACE1 digests the average enzyme activity in preceding ten minutes of APP in the blood plasma of schizophreniac and Healthy People
Power, CON represents Healthy People in figure, and SCZ represents schizophreniac
Figure 23：BACE1 digests the relationship between the maximum enzyme activity of APP in the course of disease and blood plasma of schizophreniac
Scheme, abscissa is course of disease months in figure
Figure 24：BACE1 digests the relationship between the maximum enzyme activity of APP in schizophreniac CGI score values and blood plasma
Figure
Figure 25：The 293T cells for having transfected pKH3-HA-BACE1 plasmids are overexpressed the western testing results of BACE1 enzymes
Figure
Specific implementation mode
The present invention reaction principle be, BACE1 can NRG1 apart from the regions EF and ME of 10 residues of transmembrane region it
Between particular sequence, to NRG1 carry out digestion.
Design includes the polypeptide of " EFME " sequence, and fluorescent emission group and fluorescent quenching are coupled respectively at the both ends of polypeptide
Group at this time because fluorescent quenching group and fluorescent emission group spatially approach, and quenches the fluorescence of fluorescent emission group.Work as packet
Polypeptide containing " EFME " sequence digests the peptide fragment for being respectively provided with fluorescent emission group and carries fluorescence after BACE1 digestions
The peptide fragment of quencher, fluorescent quenching group and fluorescent emission group are spatially separated, and fluorescent emission group can send out glimmering
Light.
Alternatively, by two or more dimerization or accumulation can occur and the fluorescent emission group of fluorescent quenching is made to be connected to
Including on the polypeptide of " EFME " sequence so that dimerization or accumulation occur for fluorescent emission group, to mutually be quenched or itself quenches
Go out the fluorescence of (when fluorescent emission group is identical) fluorescent emission group.When the polypeptide for including " EFME " sequence, by BACE1 digestions
Afterwards, the dimer or packed structures of the fluorescent emission group connected thereon dissociate, and fluorescent emission group is separated from each other, to
Due in fluorescent emission group high fluorescent, it will there is fluorescence.
It, can be qualitative and quantitative according to the size and its rate of change of the fluoresced intensity of fluorescent emission group released
Measurement BACE1 enzymatic activitys, to react the degree that BACE1 is activated.
Various detection methods as known in the art, such as microplate reader, fluidic cell may be used in the detection of fluorescence intensity
Instrument etc..It is preferred that using multi-function microplate reader, which can sensitively acquire signal, and be analyzed, and detection is greatly improved
Sensitivity.In addition, the detection speed of the instrument is fast, the fluorescence signal of sample can be generally acquired in 20 minutes, carried out
Analysis is calculated, the time is short, is suitble to large batch of biology sample detection.
Due to fluorescence efficiency height, detection is sensitive, and required sample size very little generally takes 10ul blood plasma that can carry out BACE1 enzymes
Activity determination；And it is particularly suitable for using porous plate, such as 96 orifice plates are carried out at the same time multiple sample sample measures, easy to operate,
Effect is high.
The present invention provides method to measure the activity of BACE1 digestions NRG1 in schizophreniac's body.The method
In a few days, day to day precision RSD be respectively less than 15%.
The diagnosis principle of the present invention is that present invention research finds the active height and spirit point of BACE1 digestions NRG1
Whether splitting the morbidity of disease and the schizoid course of disease and severity it is relevant, and this association is special with disease
Directive property, that is, only schizophreniac, the activity of BACE1 digestions NRG1 increases, and BACE1 digestions other substrates
Activity do not change.
In the present invention, " fluorescent quenching ", " Ying Guang temper go out " it is identical with the meaning of " fluorescent quenching "：Refer to fluorescence intensity because
A variety of causes or effect and reduce, such as fluorescence quencher leads to fluorescent quenching, self-quenching caused by fluorescent material concentration is excessive,
Different fluorescent materials are quenched mutually caused by stacking.
In the present invention, " fluorescent emission group " is the molecule or compound or group that can send out fluorescence, can be this
The known molecule that can send out fluorescence or compound or group in field, such as have the compound there are one aromatic ring or condensed ring,
Organic compound with multiple conjugated double bonds, including but not limited to rhodamine compound, fluoresceins compound, BODIPY
Class compound, EDANS classes compound, coumarin kind compound, paramethylaminophenol class compound, cyanine compound, acridine
Compound, isoindoles compound, dansyl class compound, aminophthalic acid hydrazide kind compound (such as luminol and different
Derivative of Luminol), anthranilic acid compound, aminophthalimide class compound, amino naphthalenes two formyls it is sub-
Aminated compounds, aminobenzofur class compound, aminoquinolines, dicyano hydroquinone compound.
In these fluorescent emission groups, there are one the compounds of substantially flat aromatic structure generally can be transferred through two for tool
Dimerization or accumulation and occur mutually to be quenched or itself is quenched, the compound includes but not limited to：Fluoresceins compound is (such as
Fluorescein, fluorescein isothiocynate, 5-carboxyfluorescein, 6- Fluoresceincarboxylic acids, tetrachlorofluorescein, chlordene fluorescein, carboxyl-
Bis- chloro- 2' of 4', 5'-, 7'- dimethoxyfluoresceins etc.), rhodamine compound (such as rhodamine 110, rhodamine 6G, rhodamine
123, rhodamine B, carboxyl tetramethylrhodamine etc.), flower cyanines class is (as half flower cyanines class, portion spend cyanines class, oxonols class, the sour cyanines in side
Class), BODIPY classes compound (such as miscellaneous (the borata) -3a- nitrogen -4a- azepine-s- dihydroindene of bis- fluoro- 4- boron of 4,4-
(indacene)) etc..
" fluorescent quenching group " is the energy referred to by receiving fluorescent emission group, and reduces or no longer emit and turn
The compound or molecule for moving energy, can be various fluorescent quenching groups as known in the art, including but not limited to nitrobenzene
The nitrations such as base, nitrobenzyloxycarbonyl, nitro benzoyl aromatic compound, indigo class compound, benzo quinones chemical combination
Object, anthraquinone analog compound, azo-compound, indenes amino benzenes compounds, two or triphenylmethane compound.
" amino acid " refers to the organic compound containing amino and carboxyl, including 20 kinds of natural amino acids.20 kinds of natural ammonia
Base acid is：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), Soviet Union
Propylhomoserin (Thr, T), tyrosine (Tyr, Y), asparagine (Asn, N), glutamine (Gln, Q), relies cysteine (Cys, C)
Propylhomoserin (Lys, K), arginine (Arg, R), histidine (His, H), aspartic acid (Asp, D) and glutamic acid (Glu, E).
In the present invention, unless otherwise specified, amino acid sequence is from N-terminal to C-terminal.
In the present invention, when the end amino acid of the C-terminal of substrate polypeptide sequence carries the amino of non-α amino, such as C-terminal
End amino acid be lysine, fluorescent emission group or fluorescent quenching group may be coupled to the end amino acid of the C-terminal
On carboxyl or on the amino of non-α amino.For convenience of and clearly demonstrate, digestion peptide substrate of the present invention end plus "-
NH2" represent fluorescent emission group or fluorescent quenching group is connected on carboxyl, in addition "-COOH " represent fluorescent emission group or
Fluorescent quenching group is connected on the amino of non-α amino.Such as：(Abz)-GIEFMEAEK-(Dnp)-NH2, indicate the Dnp
It is connected on the carboxyl of lysine；(Abz)-GIEFMEAEK- (Dnp)-COOH indicates that the Dnp is connected to the non-alpha of lysine
On the amino of amino.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Furthermore, it is to be understood that after having read recorded content of the invention, this field skill
Art personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within limited range of the present invention.
The method of the present invention is illustrated by specific embodiment below in conjunction with the accompanying drawings, but the invention is not limited in
This.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The processing method of plasma sample：
Blood vessel overturning will be taken after blood drawing for several times, with the drawn blood liquid of mixing.It is centrifuged after heparin tube is stood half an hour
(3500rpm × 10 minute) isolate leucocyte, haemocyte and blood plasma.Take 10 μ L blood plasma lysates (10mM Tris-HCl,
PH 7.4,150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na3VO4, 10% glycerine and 0.5%Triton X-100) into
5 times of dilutions of row, are shaken, treated, and plasma sample is spare later.
Multi-function microplate reader detects enzymatic activity：
96 hole black are added in plasma sample after processing to be protected from light in plate, under light protected environment, according to 1:Bottom is added in 1 volume ratio
Object reaction buffer, in 37 DEG C of temperature, Biotek multi-function microplate readers (Synergy H1) parameter setting, excitation wavelength 320nm,
Under the conditions of launch wavelength 420nm, yield value 60-75, fluorescence signal intensity in record 1 hour, completion data are adopted in 20min
Collection.
Maximum enzyme activity Vmax and average two kinds of indexs of enzyme activity Vmean are selected to weigh enzyme activity size.
Measure the total protein concentration of blood plasma：
Reagent A, 1L：BCA (1%), Na are weighed respectively2CO3.H2O (2%), Na2C4H4O6.2H2O (0.16%), NaOH
(0.4%), NaHCO3(0.95%), 1L is added water to, adjusts pH value to 11.25.
Reagent B, 50ml：Take CuSO4.5H2O (4%), adds distilled water to 50ml.
BCA working solutions：A 100ml are taken to be uniformly mixed with reagent B 2ml.
Standard protein solution：Bovine serum albumin(BSA) is weighed, the solution of 5mg/ml is made.
According to BCA determination of protein concentration methods, measures albumen concentration and draw standard curve.
The absorbance value of determination sample blood plasma searches standard curve, finds out protein concentration in test plasma (g/L).And
According to sample protein concentration, the maximum enzyme activity Vmax/ug of unit of account quality and average enzyme activity Vmean/ug.
The method of data statistics：
The management and statistical analysis of data are carried out using spss18. statistical analysis softwares, all statistical analyses are all made of double
Side is examined, with P<0.05 is to be statistically significant (except specified otherwise)；Quantitative target uses mean ± standard deviation, most
Small value, maximum value description, qualitative index are described using frequency, percentage.
Comparison among groups：According to data distribution type, quantitative target comparison among groups use parameter (t inspections/variance analysis etc.)
It examines or non-parametric test (rank sum test/CMH inspections etc.) is compared, qualitative index comparison among groups are examined using chi-Square
It tests or Fisher exact methods is examined.
Multigroup of quantitative target is compared two-by-two accidentally to be expanded using Bonferroni methods control class Ⅰ malocclusion.
Correlation analysis：Pearson methods are used according to data type.
Study the acquisition of case and healthy population：
By Beijing mental disease clinical data and sample resource pool platform, it is included in schizophreniac, depression is suffered from
Person and Healthy People.
Enter group objects：Utilize concise international psychoneural interview (Mini-International clinical
Interview for Neuropsychiatric Interview, MINI) it is used as diagnostic tool.1. schizophreniac：
Meet DSM-IV schizophrenia paranoid type diagnostic criteria, is diagnosed as schizophrenia crowd；2. patients with depression：Meet DSM-
IV criteria for depression, is diagnosed as depression.3. health volunteer：The healthy individuals of free-event survival family history.
Inclusion criteria：1. it is all enter group objects must be subject to by least one doctor with attending physician and the above qualification
It makes a definite diagnosis；2. Han nationality, the age, male or female, dextro manuality had enough audiovisual power to complete research between 18-65 Sui；3. primary school and
The above schooling (at least receiving an education 6 years), normal intelligence (IQ >=70)；4. laboratory and functional check (including blood routine liver
Renal function electrocardiogram electroencephalogram etc.) without significant exception.
Exclusion criteria：1. previously or at present there is brain organic disease or with serious unstable physical disease；Alcohol, essence
Refreshing active material and drug abuse history；2. excluding secondary mental symptom (physical disease, drug or other mental diseases)；3. pregnant
It is pregnent or women breast-feeding their children；4. being on wires, getting excited, committing suiside or violence loner；Electricity twitch or magnetic thorn have been received 5. being inscribed for 6 months
Swash curer；6. head injury, the loss of consciousness is more than 1 hour person；7. Recurrent schizophrenia group need to exclude post-schizophrenia
Depression；8. depression group need to be excluded with psychotic symptoms person.
Rejecting standard:1. violating testing program；2. last diagnostic does not meet inclusion criteria person；3. final data is not completely without method
Judge curative effect person.
All research objects sign informed consent form.
Mental disease severity classification：
By researcher's assessment tool, 1. clinical global impression scale (Global Impressions Scale CGI)：It is suitable
State of an illness appraisal for various mental diseases and observation of curative effect.Including coincident with severity degree of condition, curative effect general comment, therapeutic index, using 0
~7 points of 8 grades of point-scores.2. Positive and Negative Symptoms (Positive and negative syndrome scale
PANSS)：It is mainly used for the severity for evaluating the presence or absence of mental symptom and every symptom.Including Positive Symptom Scale, negative disease
Shape scale, general spirit pathological state scale, it then follows entry define and《Standards of grading》.3. Hamilton depressive scale
(HAMD-17)：It is mainly used for clinically evaluating depressive state.Project uses 0~4 point of 5 grades of point systems, general Cut-off score,
Respectively 24 points, 17 points and 7 points.
Embodiment 1
The preparation of BACE1 digestion peptide substrates：
Sequence 1：(Abz)-GIEFMEAEK-(Dnp)-NH2
Sequence 2：(Abz)-GIEFMEAEELK-(Dnp)-NH2
Sequence 3：(Abz)-HLGIEFMEAEELYQK-(Dnp)-NH2
It is synthesized and is provided by gill biochemistry (Shanghai) Co., Ltd..
The sequence and its number of the preparation are along in following embodiment.
Embodiment 2
The polypeptide that sequence 1-3 is respectively adopted is substrate, the activity of detection BACE1 digestions NRG1
1, it is tested using the cell system for being overexpressed BACE1 albumen
293T cells are cultivated, pKH3-HA-BACE1 plasmid (the wherein genes of BACE1 albumen are overexpressed using transfection reagent
Sequence is NC_000011.10, is inserted between Hind III and Sal the I restriction enzyme sites of pKH3-HA plasmids, the purchase of pKH3-HA plasmids
From excellent precious biology), transfection amount is the 1 μ g plasmids of cell transfecting of 10cm culture dishes.In 37 DEG C, 5%CO after transfection2Under conditions of train
It supports 36 hours.(see attached drawing 25) is detected through western, it was demonstrated that the cell after transfection has been overexpressed BACE1 albumen.
Cell culture medium is removed, is washed 2 times with the PBS of precooling, the cell 4ml lysates for being 80% for 10cm density
(10mM Tris-HCl, pH 7.4,150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na3VO4, 10% glycerine, and
0.5%Triton X-100) cell is cracked, supernatant is collected in 4 DEG C of 12000g centrifugations 10 minutes, that is, obtains BACE1 mistakes
Express the cell pyrolysis liquid of albumen.
Acetic acid-sodium chloride buffer is prepared, wherein containing 50mmol/L acetic acid and 100mmol/L NaCl, uses sodium hydroxide
It is tuned into pH4.5.Sequence 1-3 is dissolved in DMSO respectively, is configured to 2mM original solutions, the acetic acid-sodium chloride for adding pH4.5 is slow
Fliud flushing is mixed, and is respectively prepared the substrate reactions buffer solution of sequence 1-3, final concentration of 40 μM of substrate sequence 1-3.
The cell pyrolysis liquid 96 hole black of addition that the BACE1 of 50ul is overexpressed to albumen are protected from light in plate, under light protected environment, are pressed
According to 1:1 volume ratio is separately added into the substrate reactions buffer solution containing sequence 1-3, at 37 DEG C of temperature, Biotek multifunctional enzymes
Mark instrument (Synergy H1) parameter setting, excitation wavelength 320nm, launch wavelength 420nm, under the conditions of yield value 60-75, record 1
Fluorescence signal intensity in hour.
The result of the active Vmax and Vmean of sequence 1-3 detection BACE1 digestions NRG1 is as shown in Figs 1-4.As seen from Figure 1,
Use sequence 1 for substrate, the active Vmax values of the BACE1 digestions NRG1 of detection are higher than sequence 3 and sequence 2.It can by Fig. 2 and 3
See, use sequence 1 for substrate, in active preceding ten minutes of the BACE1 digestions NRG1 of detection and in whole detection durations
Vmean values are above sequence 3 and sequence 2.Fluorescence intensity level and time are mapped, are presented as that digestion rate is denoted as Fig. 4, by Fig. 4
As it can be seen that the digestion rate of sequence 1 is higher than sequence 3 and sequence 2.
It is preliminary to judge that sequence 1 is used as substrate, reaction effect according to the experimental result for the cell system for being overexpressed BACE1 albumen
Rate is higher, can be conducive to shorten detection time in future is detected, improve detection efficiency.
2, it is tested using human normal plasma
Sequence 1-3 is dissolved in DMSO respectively, is configured to 2mM original solutions, with acetic acid-sodium chloride buffer of pH3.1 into
Row mixing, is respectively prepared the substrate reactions buffer solution of sequence 1-3, final concentration of 40 μM of substrate sequence 1-3.Acetic acid-sodium chloride
Buffer solution includes 50mmol/L acetic acid and 100mmol/L NaCl.
Healthy human blood is acquired, plasma sample processing is carried out, 96 hole black, which are added, in plasma sample after processing is protected from light in plate,
Under light protected environment, the substrate reactions buffer solution of sequence 1-3 is sequentially added, in 37 DEG C of temperature, fluoroscopic examination is carried out, as a result sees Fig. 5-
6。
As seen from Figure 5, use sequence 1 for substrate, the active Vmax values for detecting the BACE1 digestions NRG1 in blood plasma are higher than
Sequence 3 and sequence 2.As seen from Figure 6, it uses sequence 1 for substrate, detects active preceding ten of the BACE1 digestions NRG1 in blood plasma
Vmean values in minute are less than sequence 3, are higher than sequence 2.
Since the BACE1 enzyme contents in human normal plasma are less than the cell system for being overexpressed BACE1, sequence 1 is used as substrate,
Reaction efficiency is higher, and the detection reaction that sequence 1 is substrate is caused to be quickly completed, and the Vmean values in preceding ten minutes is made to be less than sequence
Row 3.
The Vmax and Vmean of comprehensive health human plasma detection are as a result, sequence 1 and 3 can be better than sequence 2 as substrate.
Embodiment 3
The activity of BACE1 digestions NRG1 is detected under different pH value
Prepare different buffer systems：Prepare acetic acid-sodium chloride buffer, wherein containing 50mmol/L acetic acid and
100mmol/L NaCl are tuned into pH3.0, pH3.5, pH4.0, pH4.5 respectively with sodium hydroxide.
Sequence 1 is dissolved in DMSO, is configured to 2mM original solutions, respectively with the vinegar of pH3.0, pH3.5, pH4.0, pH4.5
Acid-sodium chloride buffer is mixed, and is made substrate reactions buffer solution, final concentration of 40 μM of substrate.
Healthy human blood is acquired, plasma sample processing is carried out, 96 hole black, which are added, in plasma sample after processing is protected from light in plate,
Under light protected environment, the substrate reactions buffer solution of the different pH value of sequence 1 is sequentially added, in 37 DEG C of temperature, carries out fluoroscopic examination, knot
Fruit sees Fig. 7-8.
As seen from Figure 7：Under different pH value, the numerical value of the maximum enzyme activity of BACE1 digestions NRG1 in the blood plasma measured, with
The pH value of reaction system is increased and is reduced, and the numerical value measured in pH3.0 is maximum.
As seen from Figure 8：Under different pH value, the activity of BACE1 digestions NRG1 in the blood plasma measured is flat in preceding ten minutes
The numerical value of equal enzymatic activity, as the pH value of reaction system increases and reduces, and the numerical value measured in pH3.0 is maximum.
Illustrate that the pH value of reaction system can have an impact the activity of BACE1 digestions NRG1 in blood plasma, is examined for detection sensitivity
Consider, preferably the pH value of reaction system be 3.0-3.5, most preferably 3.0 ± 0.1.
Embodiment 4
The specific assay of detection method
According to BACE1 design features, two kinds of BACE1 inhibitor are chosen：LY2886721, BI detect the inhibitor pair
The active influences of BACE1 digestions NRG1.
Acquire healthy human blood, carry out plasma sample processing, will treated blood plasma lysate respectively with two kinds of inhibitor
Incubation at room temperature 1 hour, final concentration of each inhibitor in incubation system are respectively 10 μM and 50 μM, and it is molten that blank is added in control group
Agent DMSO.
96 hole black are added in plasma sample after incubation to be protected from light in plate, the substrate reactions buffering of the pH3.0 of sequence 1 is added
Liquid carries out fluoroscopic examination, as a result sees attached drawing 9-10 in 37 DEG C of temperature.
As seen from Figure 9：Compared with the control group, BACE1 digestions NRG1 is most after LY2886721 being added, in the blood plasma measured
Big enzymatic activity reduces, and 50 μM of LY2886721 is higher than the inhibiting effect of maximum enzyme activity 10 μM of LY2886721, exists
Dose-effect relationship；50 μM of BI also has inhibiting effect to the maximum enzyme activity of BACE1 digestions NRG1 in blood plasma；
As seen from Figure 10：Compared with the control group, after LY2886721 being added, the work of BACE1 digestions NRG1 in the blood plasma measured
Property, the average enzymatic activity in preceding ten minutes reduces, and 50 μM of LY2886721 is higher than 10 to the inhibiting effect of average enzymatic activity
μM LY2886721, there are dose-effect relationships；50 μM of BI also has inhibition to the average enzymatic activity of BACE1 digestions NRG1 in blood plasma
Effect.
Since both inhibitor are the specific inhibitors of BACE1 enzymes, after both inhibitor are added, fluorescence intensity quilt
Inhibit, illustrates to detect fluorescent effect, i.e. digestion effect, be generated by BACE1 in blood plasma rather than other protease cutting sequences 1
's.
According to the experimental result of sequence 1,50 μM of inhibitor concentration is chosen, using same experiment condition and method, inspection
The specificity of row 3, the result is shown in Figure 1 1-12 is sequenced.
By Figure 11 and 12 as it can be seen that compared with the control group, after 50 μM of LY2886721 and BI is added, in the blood plasma measured
The maximum enzyme activity of BACE1 digestions NRG1 and average enzymatic activity are not all substantially reduced.Since LY2886721, BI are BACE1
Specific inhibitor is that substrate remains to show higher fluorescence with sequence 3, prompts the fluorescence after the inhibitor is added
Generation may be from other protease cutting sequences 3 in blood plasma.Thus illustrate, from the specificity of detection, sequence 1 is excellent
In sequence 3.
Embodiment 5
The comparison of schizophreniac and BACE1 cuttings NRG1 enzyme activities in human normal plasma
The blood plasma for acquiring schizophreniac (38) and Healthy People (37) respectively, is substrate with sequence 1, using reality
The detection method of plasma sample in example 2 is applied, the enzyme activity of BACE1 cuttings NRG1 in blood plasma is detected.
As a result see attached drawing 13-14.
As seen from Figure 13, the maximum enzyme activity of BACE1 enzymolysis NRG1 is higher than Healthy People in the blood plasma of schizophreniac,
Difference is significant.
As seen from Figure 14, BACE1 digests the average enzyme activity in preceding ten minutes of NRG1 in schizophreniac's blood plasma
Slightly above Healthy People.
Embodiment 6
The active comparisons of BACE1 digestions NRG1 in the different schizophreniac's blood plasma of severity
It is substrate with sequence 1 equally with the blood plasma of schizophreniac in embodiment 5, using blood plasma sample in embodiment 2
The detection method of product detects the enzyme activity of BACE1 cuttings NRG1 in blood plasma.
BACE1 in the blood plasma of surveyed patient is cut into the maximum enzyme activity of NRG1 and the course of disease of patient carries out mapping statistics,
As a result it as shown in attached drawing 15a, is fitted according to Person methods, finds the maximum enzyme activity of patient's course of disease length and BACE1 cuttings NRG1
Power is related, has statistical significance, and with the extension of the course of disease, BACE1 cuts the maximum enzyme activity presentation decline of NRG1 in blood plasma
Trend, that is, BACE1 cuts NRG1 enzyme activity highers in the shorter patients blood plasma of the course of disease, conversely, in the longer patients blood plasma of the course of disease
BACE1 activity is lower.Further, according to the course of disease be within 100 months and the course of disease be 100 months or more, distinguish the course of disease of patient
Duration counts the maximum enzyme activity that BACE1 in its blood plasma cuts NRG1, finds patient (21) of the course of disease within 100 months,
The maximum enzyme activity of BACE1 cuttings NRG1 is more than patient (17) of the course of disease at 100 months or more in its blood plasma, and difference has aobvious
Work property, the result is shown in Figure 1 5b.
Patient is divided into according to CGI score values to patient group (8) and the CGI high in the low patient groups of CGI (14), CGI
Patient group (16), the maximum enzyme activity that BACE1 in the blood plasma of this three groups of patients cuts NRG1 is counted, as a result such as 16 institute of attached drawing
Show, with the increase of CGI score values, the maximum enzyme activity of BACE1 cuttings NRG1 increases in blood plasma, wherein patient group low CGI
Maximum enzyme activity is less than the patient group of CGI high, and significant difference, illustrates clinical total more serious schizophrenia of evaluation
The activity of the BACE1 cuttings NRG1 patient lower than evaluation score is high in patients blood plasma.
Embodiment 7
The activity of BACE1 digestions NRG1 in patients with depression blood plasma
The blood plasma for acquiring patients with depression (19) and Healthy People (18) respectively, is substrate with sequence 1, using embodiment
The detection method of plasma sample in 2 detects the enzyme activity of BACE1 cuttings NRG1 in blood plasma.
As a result see attached drawing 17-20.
As seen from Figure 17, the maximum enzyme activity of BACE1 digestions NRG1 is suitable with Healthy People in the blood plasma of patients with depression,
Statistically there was no significant difference (P=0.9082).
As seen from Figure 18, the average enzyme activity in patients with depression blood plasma in preceding ten minutes of BACE1 digestions NRG1 with it is strong
Health people's is suitable, and statistically there was no significant difference (P=0.7965).
BACE1 in the blood plasma of surveyed patients with depression is cut into the maximum enzyme activity of NRG1 and the course of disease of patient is mapped
As a result as shown in Fig. 19 statistics is fitted according to Person methods, find the maximum of patient's course of disease length and BACE1 cuttings NRG1
Enzyme activity is uncorrelated, and with the extension of the course of disease, the maximum enzyme activity of BACE1 cuttings NRG1 does not change in blood plasma, that is, suppression
BACE1 digestion NRG1 activity is not associated with patient's course of disease in strongly fragrant disease patients blood plasma.
Patients with depression is divided into the patient of (8) and CGI high (3) in CGI low (8), CGI according to CGI score values
Group counts the maximum enzyme activity that BACE1 in the blood plasma of this three groups of patients cuts NRG1, as a result as shown in Fig. 20, the trouble in CGI
The maximum enzyme activity of person's group higher than CGI is low and CGI high patient group, but between three groups two-by-two comparing difference without notable
Property.
Embodiment 8
The activity of schizophreniac and BACE1 cuttings APP in human normal plasma
The blood plasma for acquiring schizophreniac (35) and Healthy People (35), according to document (Deletion of
tumor necrosis factor death receptor inhibits amyloidβgeneration and prevents
learning and memory deficits in Alzheimer’s mice.Journal of Experimental
Medicine.2007,178(5):829-841) the method recorded detects the enzyme activity of BACE1 cuttings APP in blood plasma.
As a result see attached drawing 21-24.
As seen from Figure 21, in the blood plasma of schizophreniac the maximum enzyme activity and Healthy People of BACE1 digestions APP phase
Than difference does not have conspicuousness.
As seen from Figure 22, the average enzyme activity in schizophreniac's blood plasma in preceding ten minutes of BACE1 digestions APP with
Healthy People is compared, and difference does not have conspicuousness.
BACE1 in the blood plasma of surveyed patient is cut into the maximum enzyme activity of APP and the course of disease of patient carries out mapping statistics, is tied
Fruit is as shown in Fig. 23, is fitted according to Person methods, it is found that patient's course of disease length cuts the maximum enzyme activity of APP not with BACE1
Correlation, with the extension of the course of disease, the maximum enzyme activity of BACE1 cuttings APP does not change in blood plasma, that is, BACE1 in patients blood plasma
Cut the course of disease onrelevant of the enzyme activity and patient of APP.
Patient is divided into the patient group of (7) and CGI high (15) in CGI low (13), CGI according to CGI score values, is united
Count the maximum enzyme activity that BACE1 in the blood plasma of this three groups of patients cuts APP, as a result as shown in Fig. 24, patient group medium CGI
Maximum enzyme activity less than CGI is low and the patient group of CGI high, but illustrate schizophrenia without significant difference between three
BACE1 cuts the active onrelevant of APP in the severity and blood plasma of disease patient.
More than, embodiments of the present invention are illustrated.But the present invention is not limited to the above embodiments.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in the guarantor of the present invention
Within the scope of shield.
Claims (10)
- Application of the 1.BACE1 digestions peptide substrate in preparing schizophrenia diagnosis reagent or kit, the BACE1 digestions The structural formula of peptide substrate is：R1-R2-EFME-R3-R4, wherein R2For 0-8 amino acid；R3For 0-8 amino acid；Work as R1For When fluorescent emission group, R4For fluorescent quenching group or fluorescent emission group；Alternatively, working as R1For fluorescent quenching group when, R4It is glimmering Light emitting group；R1It is connected to R2Amino on, R4It is connected to R3Carboxyl or non-alpha-amino amino on；E is glutamic acid, and F is Phenylalanine, M are methionine；It is preferred that R2For：The 2-8 amino acid containing-GI- sequences；More preferably contain 2-5 amino acid of-GI- sequences；It is optimal It is selected as-GI-；Wherein G is glycine, and I is isoleucine；It is preferred that R3For：The 2-8 amino acid containing-AE- sequences；More preferably contain 2-5 amino acid of-AE- sequences；It is optimal It is selected as-AEK-；Wherein A is alanine, and E is glutamic acid, and K is lysine；It is preferred that the fluorescent emission group is：Rhodamine compound, fluoresceins compound, BODIPY classes compound, EDANS Class compound, coumarin kind compound, paramethylaminophenol class compound, cyanine compound, acridine compound, isoindoles Compound, dansyl class compound, aminophthalic acid hydrazide kind compound, anthranilic acid compound, amino neighbour's benzene Dicarboximide class compound, aminonaphthalimide class compound, aminobenzofur class compound, aminoquinolines Close object or dicyano hydroquinone compound；Preferably luminol, different luminol, rhodamine 110, rhodamine 6G, Rhodamine 123, Rhodamine B, carboxyl tetramethylrhodamine, fluorescein, fluorescein isothiocynate, 5-carboxyfluorescein, 6- Fluoresceincarboxylic acids, tetrachloro Fluorescein, chlordene fluorescein, bis- chloro- 2' of carboxyl -4', 5'-, 7'- dimethoxyfluoresceins or ortho-aminobenzoic acid (Abz)；It is preferred that the fluorescent quenching group is：The nitrations aromatic series such as nitrobenzophenone, nitrobenzyloxycarbonyl, nitro benzoyl Compound, indigo class compound, benzo quinones, anthraquinone analog compound, azo-compound, indenes amino benzenes compounds or Person two or triphenylmethane compound；Preferably：2,4- dinitrophenol (Dnp), nitrobenzophenone, nitrobenzyloxycarbonyl, Or nitro benzoyl.
- 2. application as described in claim 1, the BACE1 digestion peptide substrates are (Abz)-GIEFMEAEK- (Dnp)- NH2、(Abz)-GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-(Abz)-NH2、(Dnp)-GIEFMEAEK- (Abz)-COOH、(Abz)-GIEFMEAEELK-(Dnp)-NH2、(Abz)-GIEFMEAEELK-(Dnp)-COOH、(Dnp)- GIEFMEAEELK-(Abz)-NH2、(Dnp)-GIEFMEAEELK-(Abz)-COOH、(Abz)-HLGIEFMEAEELYQK- (Dnp)-NH2、(Abz)-HLGIEFMEAEELYQK-(Dnp)-COOH、(Dnp)-HLGIEFMEAEELYQK-(Abz)-NH2Or (Dnp)-HLGIEFMEAEELYQK-(Abz)-COOH。
- 3. application as claimed in claim 1 or 2, the schizophrenia is the schizophrenia or severe of morbidity early stage Schizophrenia.
- 4. application as described in any one of claims 1-3, the schizoid diagnosis is for schizophrenia Or with people's expansion that schizophrenia possibility occurs；The people with generation schizophrenia possibility refers to that it is deposited In the genetic people of schizophrenic families.
- 5. application according to any one of claims 1-4, buffering is further included in the diagnostic reagent or diagnostic kit Liquid；It is preferred that the pH of the buffer solution is 2.9-4.6, preferably 3.0-4.0, more preferably 3.0-3.5；It is preferred that the buffer solution is Acetic acid-acetate buffer, phosphoric acid-phosphate buffer, citric acid-citrate buffer solution, glycine-HCI buffer solution, Disodium hydrogen phosphate-citrate buffer solution or acetic acid-sodium chloride buffer；It is preferred that the buffer solution is acetic acid-sodium chloride buffer Liquid contains 50mM acetic acid and 100mM sodium chloride.
- 6. application as described in any one in claim 1-5, further contain sample in the diagnostic reagent or diagnostic kit Lysate；It is preferred that the lysate contains：Tris-HCl, NaCl, EDTA or its salt, EGTA or its salt, Na3VO4, glycerine and Triton X-100；The group of the more preferable lysate becomes：10mMTris-HCl, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM Na3VO4, 10% glycerine and 0.5%Triton X-100.
- 7. a kind of BACE1 digestions peptide substrate, structural formula are：R1-R2-EFME-R3-R4, wherein R2For 0-8 amino acid；R3 For 0-8 amino acid；Work as R1For fluorescent emission group when, R4For fluorescent quenching group or fluorescent emission group；Alternatively, working as R1For When fluorescent quenching group, R4For fluorescent emission group；R1It is connected to R2Amino on, R4It is connected to R3Carboxyl or non-alpha-amino On amino；E is glutamic acid, and F is phenylalanine, and M is methionine；It is preferred that R2For：The 2-8 amino acid containing-GI- sequences；More preferably contain 2-5 amino acid of-GI- sequences；It is optimal It is selected as-GI-；Wherein G is glycine, and I is isoleucine；It is preferred that R3For：The 2-8 amino acid containing-AE- sequences；More preferably contain 2-5 amino acid of-AE- sequences；It is optimal It is selected as-AEK-；Wherein A is alanine, and E is glutamic acid, and K is lysine；It is preferred that the fluorescent emission group is：Rhodamine compound, fluoresceins compound, BODIPY classes compound, EDANS Class compound, coumarin kind compound, paramethylaminophenol class compound, cyanine compound, acridine compound, isoindoles Compound, dansyl class compound, aminophthalic acid hydrazide kind compound, anthranilic acid compound, amino neighbour's benzene Dicarboximide class compound, aminonaphthalimide class compound, aminobenzofur class compound, aminoquinolines Close object or dicyano hydroquinone compound；Preferably luminol, different luminol, rhodamine 110, rhodamine 6G, Rhodamine 123, Rhodamine B, carboxyl tetramethylrhodamine, fluorescein, fluorescein isothiocynate, 5-carboxyfluorescein, 6- Fluoresceincarboxylic acids, tetrachloro Fluorescein, chlordene fluorescein, bis- chloro- 2' of carboxyl -4', 5'-, 7'- dimethoxyfluoresceins or ortho-aminobenzoic acid (Abz)；It is preferred that the fluorescent quenching group is：The nitrations aromatic series such as nitrobenzophenone, nitrobenzyloxycarbonyl, nitro benzoyl Compound, indigo class compound, benzo quinones, anthraquinone analog compound, azo-compound, indenes amino benzenes compounds or Person two or triphenylmethane compound；Preferably：2,4- dinitrophenol (Dnp), nitrobenzophenone, nitrobenzyloxycarbonyl, Or nitro benzoyl.
- 8. BACE1 digestions peptide substrate as claimed in claim 7 is (Abz)-GIEFMEAEK- (Dnp)-NH2、(Abz)- GIEFMEAEK-(Dnp)-COOH、(Dnp)-GIEFMEAEK-(Abz)-NH2、(Dnp)-GIEFMEAEK-(Abz)-COOH、 (Abz)-GIEFMEAEELK-(Dnp)-NH2、(Abz)-GIEFMEAEELK-(Dnp)-COOH、(Dnp)-GIEFMEAEELK- (Abz)-NH2、(Dnp)-GIEFMEAEELK-(Abz)-COOH、(Abz)-HLGIEFMEAEELYQK-(Dnp)-NH2、(Abz)- HLGIEFMEAEELYQK-(Dnp)-COOH、(Dnp)-HLGIEFMEAEELYQK-(Abz)-NH2Or (Dnp)- HLGIEFMEAEELYQK-(Abz)-COOH。
- 9. a kind of composition contains the BACE1 digestion peptide substrates described in claim 7 or 8；It is preferred that the pH value of the composition For 2.9-4.6, preferably 3.0-4.0, more preferably 3.0-3.5；It is preferred that the pH value is provided by buffer solution；It is preferred that the buffering Liquid is acetic acid-acetate buffer, phosphoric acid-phosphate buffer, citric acid-citrate buffer solution, glycine-HCI buffering Liquid, disodium hydrogen phosphate-citrate buffer solution or acetic acid-sodium chloride buffer；It is preferred that the buffer solution is acetic acid-sodium chloride Buffer solution contains 50mM acetic acid and 100mM sodium chloride.
- 10. a kind of kit, containing described in claim 7 or 8 BACE1 digestions peptide substrate or claim 9 described in Composition；It is preferred that further containing sample dissociation liquid in the kit；It is preferred that the lysate contains：Tris-HCl,NaCl, EDTA or its salt, EGTA or its salt, Na3VO4, glycerine and Triton X-100；The group of the more preferable lysate becomes：10mM Tris-HCl, 150mM NaCl, 1mM EDTA, 1mMEGTA, 1mM Na3VO4, 10% glycerine and 0.5%Triton X-100.
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| CN117965529A (en) * | 2024-02-01 | 2024-05-03 | 伊莱瑞特(武汉)生物技术有限公司 | Cell lysate, enzyme activity detection kit in cells and application |
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