CN107677831A - The method for determining the diagnosis marker for assessing schizophrenia patients - Google Patents
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention discloses a kind of method for determining the diagnosis marker for assessing schizophrenia patients, the mark can be conveniently as the diagnosis marker for assessing schizophrenia patients.A kind of method for determining the diagnosis marker for assessing schizophrenia patients, experimental method are as follows：(1) blood sample, is gathered；(2), early stage removes the high-abundance proteins in experimenter's serum, may be selected to use TCA acetone albumen precipitation methods；Or the TCA precipitation method；Or PEG precipitation of protein；Obtain the supernatant of electrophoresis test or redissolve liquid；(3) according to《Molecular Cloning:A Laboratory guide》Method and step, to supernatant or redissolve liquid carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis, obtain protein band.The present invention removes the serum of collection in early stage the interference of high-abundance proteins, improves the degree of accuracy of low-abundance protein detection, in follow-up SDS PAGE electrophoresis, separation gel is verified using 10~12%, makes electrophoretic effects good.
Description
Technical field
The present invention relates to technical field of bioengineering, is especially used for assessing the albumen that serum and schizophrenia diagnoses
Mark, it is a kind of method for determining the diagnosis marker for assessing schizophrenia patients.
Background technology
Schizophrenia is the cerebral disorders with the characteristics of being continued for a long time by a variety of severe psychiatric symptoms or constantly being aggravated.Spirit
Split disease clinical symptoms are generally divided into positive symptom and negative symptoms.Positive symptom includes：Illusion, vain hope, confusion of thinking etc.；It is cloudy
Property symptom includes：Apathy, aboulia, slow movement and social withdrawal etc..Schizophrenia incidence is high in recent years, shadow
The 1% of nearly world population is rung, accounts for global disease is always born 1%, exceedes the heart in the treatment expenditure of Chinese Spirit Split disease
Cranial vascular disease, tumour, respiratory disease and infectious disease, it is the first to arrange various Disease Spectrums.
The schizoid cause of disease is extremely complex, and still end illustrates completely at present, and this seriously inhibits its Clinics and Practices
Development.Since the 1960s, people have carried out some significant discussions to schizoid pathogenesis, successively
The hyperfunction theory of dopamine system, 5-HT theories and GABA theories are established, and develops new medicine on this basis, but
Up to the present the schizoid cause of disease of method interpretation and pathogenesis are there is no, not yet finds out the biology mark of diagnostic significance
Will.
In the past few decades, schizophrenia is concentrated mainly on the research of genome, a large amount of schizophrenia susceptibility sites
Very little is contributed to schizoid onset risk, the heredity grade explained by these susceptibility locis is significantly less than desired value.So far
There is no several experiments to distinguish similar phenotype, monitoring therapeutic process untill the present, or assess the diagnosis of disease.Due to protein
It is the final result after genetic transcription, therefore, the best route of spirit of exploration Split disease mechanism of causing a disease will likely be from protein level
Set about.
Schizoid biological indicator is diagnosed to find, domestic and foreign scholars have carried out the effort of each side for many years,
But substantial achievement is not obtained so far.Jiang Wei etc. is using analytical technique of mass spectrum to 40 serum and schizophrenias and 44
Healthy control group serum is studied, and finds there is the albumen mass-to-charge ratio peak of 15 differential expressions in serum and schizophrenia,
Wherein 6 marker proteins for having obvious differential expression, and the artificial nerve network model established, to schizoid diagnosis spirit
Sensitivity and specificity are respectively 91.7% and 93.8%.Jiang Wei etc. utilizes analytical technique of mass spectrum, the artificial neural network mould of foundation
Type, not serologic marker thing, it is difficult to applied to clinic.
Sabine etc. compares the protein spectrum of schizophreniac and Healthy People with mass-spectrometric technique, it was found that 2
The peak value of significant changes occurs.It is identified they be all the alexins of a mono-.But not further clinical research.
Liu Hui etc. has found exist in patients serum's CIC ELISA in the research to schizophrenia pathomechanism
The stronger 32kD protein bands of one specificity, a new direction is prompted for schizoid clinical research, but do not had
Benign control group is set up, also lacks the research report continued deeper into.
Although these domestic and international research application two dimensional gel electrophore- sises and analytical technique of mass spectrum study schizophreniac's
Haemocyanin, and schizophrenia partial rules are disclosed, but these albumen can't be used as biomarker.
The content of the invention
, it is an object of the invention to provide a kind of method for determining the diagnosis marker for assessing schizophrenia patients
The technical problem of the solution mark is convenient as the diagnosis marker for assessing schizophrenia patients.
In order to solve the above-mentioned technical problem, a kind of diagnosis marker determined for assessing schizophrenia patients of the present invention
Method, experimental method is as follows：(1) blood sample, is gathered；(1) subject morning limosis vein blood 3ml is gathered with vacuum blood collection tube,
It is put into 30min in 4 DEG C of refrigerators and stands 1h, separates out serum；Centrifuge, rotating speed 3000r/min, centrifugation are put under (2) 4 DEG C of environment
5min, obtains experimenter's serum, dispenses 5-10 parts, -80 DEG C freeze it is standby；(2), early stage removes the high abundance in experimenter's serum
Albumen, it may be selected to use TCA acetone albumen precipitation methods；Or the TCA precipitation method；Or PEG precipitation of protein；Electrophoresis test is obtained to use
Supernatant or redissolve liquid；(3) according to《Molecular Cloning:A Laboratory guide》Method and step, to supernatant or redissolve liquid carry out ten
Dialkyl sulfonates-polyacrylamide gel electrophoresis, after electrophoresis protein band.
The present invention cuts the 150KD protein band scopes at least on 30 protein bands obtained after electrophoresis, -80 DEG C of guarantors
Deposit, concentrate and carry out mass spectral analysis.
TCA acetone albumen precipitation method of the present invention comprises the following steps：(1) 25~400ul is taken to be stored in 4-8 DEG C of ice
10%~40%TCA acetone solns of precooling in case, add in 1.5mlEP pipes, then add 50ul serum, be put into concussion and mix
Device is shaken to mixing, under 4 DEG C of environment, is placed 1 hour；Acetone soln is analysis pure acetone；10%~40%TCA solution：10
~40gTCA is added to configuration in 100ml acetone solns and obtains 10%~40%TCA acetone solns；(2) high speed refrigerated centrifuge is used
Machine, 4 DEG C of Centrifugal Environment, rotating speed 15000g centrifugation 15min, remove supernatant；(3) 0.5mlDTT acetone solns are added, are put into shake
Swing vortex mixer to shake to mixing, with high speed freezing centrifuge, 4 DEG C of Centrifugal Environment, rotating speed 15000g centrifugation 15min, retain precipitation
Thing, separation supernatant is standby, and -80 DEG C standby of supernatant freezes；DTT acetone solns：Acetone containing 20mmol/L DTT is molten
Liquid；(4) sediment is lyophilized into -70 DEG C of refrigerators powdered, the time is three days；(5) normal temperature, powdery precipitate is interior to be added
200ul rehydration solution redissolves, and obtains redissolution liquid；Rehydration solution：To contain 7mol/L urea, 2mol/L thiocarbamides, 4g/
100mlCHAPS (3- [3- (courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt), 4-two sulphur O.4g/100ml determine butanol,
The aqueous solution of 0.002g/100ml bromophenol blues, water are deionized water.
The TCA precipitation method of the present invention comprise the following steps：(1) 12.3ul serum is taken, is added in 1.5mlEP pipes, is added
200ulPBS (phosphate buffer), the 40ul 60%TCA aqueous solution is being added, 10-20 hours are incubated in mixture of ice and water；
The 60%TCA aqueous solution:60gTCA is dissolved in the solution in 100ml water, and water is deionized water；(2) high speed freezing centrifuge is used, from
4 DEG C of thimble border, rotating speed 10000g centrifugation 30min, removes supernatant；(3) add and be stored in precooling in 4-8 DEG C of refrigerator
100ul90% aqueous acetone solutions, are incubated 15min in mixture of ice and water；90% aqueous acetone solution：Volume ratio is 90% acetone
Solution adds the solution of water, and water is deionized water, and acetone soln is analysis pure acetone；(4) high speed freezing centrifuge, centrifugal hoop are used
4 DEG C of border, rotating speed 10000g centrifugation 30min, removes supernatant, retains sediment；(5) sediment is dried in room temperature, biochemical case,
To sediment into powdered；(6) rehydration solution for adding 200ul redissolves；Rehydration solution：To contain 7mol/L urea, 2mol/L
Thiocarbamide, 4g/100mlCHAPS (3- [3- (courage acyl aminopropyl) dimethylamino] propane sulfonic acid inner salt), 4-two sulphur O.4g/100ml
Determine butanol, the aqueous solution of 0.002g/100ml bromophenol blues, water is deionized water；(7) centrifuge is used, Centrifugal Environment room temperature, is turned
Fast 10000g centrifuges 5min, takes supernatant.
PEG precipitation of protein of the present invention comprises the following steps：(1) 100ul serum is taken to add in 0.5mlEP pipes,
Add the PEG6000 aqueous solution that 100ul mass percents are 7%, 4-8 DEG C of refrigerator cold-storage 10-20 hour；The PEG6000 aqueous solution：
The solution of deionized water is dissolved in for the PEG of model 6000；(2) high speed freezing centrifuge, 4 DEG C of Centrifugal Environment, rotating speed are used
1500r/min, 20min is centrifuged, remove supernatant, obtain sediment；(3) 50ul deionized water is added in sediment, is put into shake
Swing vortex mixer to shake to mixing, obtain redissolution liquid.
The separation gel used in electrophoresis of the present invention is the gel that polyacrylamide percent by volume is 10%~12%
Solution.
The present invention verifies mass spectrometry results after 15KD protein band mass spectral analyses, using ELISA kit, uses
The original experimenter's serum sample that ZNF729 ELISA kit analysis treats without the above method, according to reagent theory
Bright book is operated.
In TCA acetone albumen precipitation method of the present invention, 50~200ul is taken to be stored in precooling in 4-8 DEG C of refrigerator
20%~30%TCA acetone solns
In TCA acetone albumen precipitation method of the present invention, 100ul is taken to be stored in the 30%TCA of precooling in 4-8 DEG C of refrigerator
Acetone soln.
The present invention according to《Molecular Cloning:A Laboratory guide》Method and step, to supernatant or redissolve liquid carry out dodecyl sulphur
Sour sodium-polyacrylamide gel electrophoresis, separation gel selects polyacrylamide percent by volume, and for 10% gel solution, electrophoresis sets
Standby initial voltage is 80V, and after dyestuff enters separation gel, voltage is increased into 120V, continues electrophoresis until dyestuff arrives at separation
Glue bottom.
The present invention removes the serum of collection in early stage the interference of high-abundance proteins, improves the standard of low-abundance protein detection
Exactness, in follow-up SDS-PAGE electrophoresis, separation gel is verified using 10~12%, makes electrophoretic effects good.
Brief description of the drawings
Fig. 1 is using redissolution liquid obtained by PEG precipitation of protein, does schizophreniac and normal healthy controls that electrophoresis obtains
The protein band contrast of person.
Fig. 2 is using redissolution liquid obtained by TCA acetone precipitations, does schizophreniac and normal healthy controls that electrophoresis obtains
The protein band contrast of person.
Fig. 3 is using supernatant obtained by the TCA precipitation method, is schizophreniac that electrophoresis obtains and normal healthy controls person
Protein band contrasts.
Fig. 4 is the mucosal adhesive path profile for including complement waterfall path and collagen ɑ -1 (II) homopolymer.
Fig. 5 is the mucosal adhesive path for including thrombospondin, cDNA FLJ61587 and the albumen of hyaluronic acid -1
Figure.
Fig. 6 be include complement factor H (sp | 08603), cDNA FLJ75416 (tr | A8K5T0) and complement factor B (tr |
B4E1Z4 the complement waterfall path profile including).
Embodiment
Specific embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.
The method for determining the diagnosis marker for assessing schizophrenia patients, experimental method are as follows：
(1) blood sample, is gathered
1st, gathered to be put into 4 DEG C of refrigerators in subject's morning limosis vein blood 3ml, 30min with vacuum blood collection tube and stand 1h, analysis
Go out serum.
2nd, centrifuge is put under 4 DEG C of environment, rotating speed 3000r/min, centrifuges 5min, obtains experimenter's serum, dispenses 5-10
Part, -80 DEG C freeze it is standby.
(2), early stage first removes the high-abundance proteins in experimenter's serum, to make low-abundance protein be easy to detect
Come, make 150KD protein bands be easy to detect, the method that following three kinds of early stages remove high-abundance proteins.
Dispense patient and normal human serum and indicate numbering.
First, TCA (trichloroacetic acid) acetone albumen precipitation method
1st, take 25~400ul to be stored in 10%~40%TCA acetone solns of precooling in 4-8 DEG C of refrigerator, add 1.5mlEP
In pipe, 50ul serum is then added, concussion vortex mixer is put into and shakes to mixing, under 4 DEG C of environment, place 1 hour.
Acetone soln is analysis pure acetone.
10%~40%TCA solution：10~40gTCA is added to configuration in 100ml acetone solns and obtains 10%~40%
TCA acetone solns.
2nd, with high speed freezing centrifuge, 4 DEG C of Centrifugal Environment, rotating speed 15000g centrifugation 15min, supernatant is removed.
3rd, 0.5mlDTT acetone solns are added, concussion vortex mixer is put into and shakes to mixing, with high speed freezing centrifuge, centrifugation
4 DEG C of environment, rotating speed 15000g centrifugation 15min, retains sediment, separation supernatant is standby, and -80 DEG C standby of supernatant freezes.
DTT acetone solns：Acetone soln containing 20mmol/L DTT (dithiothreitol (DTT)).
4th, sediment is lyophilized into -70 DEG C of refrigerators powdered.
5th, normal temperature, the interior rehydration solution for adding 200ul of powdery precipitate redissolve, must redissolve liquid and do electrophoresis.
Rehydration solution：To contain 7mol/L urea, 2mol/L thiocarbamides, 4g/100mlCHAPS (3- [3- (courage acyl aminopropyl)
Dimethylamino] propane sulfonic acid inner salt), 4-two sulphur O.4g/100ml determine butanol, the aqueous solution of 0.002g/100ml bromophenol blues, water
For deionized water.
The effect of standby supernatant：It is for examining this TCA acetone albumen precipitations method to remove high abundance egg early stage
Whether white step succeeds.Redissolution liquid and supernatant after sediment is redissolved while after doing electrophoresis, if 150kD albumen is simultaneously
Occur, illustrate that albumen treatment effect early stage is bad；If redissolve has in liquid, do not have in supernatant, it is high to illustrate that this time removes early stage
The step of abundance protein, is successful.
Embodiment 1
Early stage, with TCA (trichloroacetic acid) acetone albumen precipitation method, the high-abundance proteins in experimenter's serum were removed, tool
The desirable 200ul10%TCA solution of body.
Embodiment 2
With embodiment 1,400ul20%TCA solution specifically can use.
Embodiment 3
With embodiment 1,100ul30%TCA solution specifically can use.
Embodiment 4
With embodiment 1,100ul40%TCA solution specifically can use.
Embodiment 5
With embodiment 1,50ul20%TCA acetone solns are specifically can use.
Embodiment 6
With embodiment 1,25ul40%TCA acetone solns are specifically can use.
Embodiment 7
With embodiment 1,100ul20%TCA acetone solns are specifically can use.
Embodiment 8
With embodiment 1,200ul30%TCA acetone solns are specifically can use.
Embodiment 9
With embodiment 1,400ul10%TCA acetone solns are specifically can use.
2nd, the TCA precipitation method
1st, 12.3ul serum is taken, is added in 1.5mlEP pipes, adds 200ulPBS (phosphate buffer), is adding 40ul
The 60%TCA aqueous solution, in mixture of ice and water be incubated 10-20 hours.
The 60%TCA aqueous solution:60gTCA is dissolved in the solution in 100ml water, and water is deionized water.
2nd, with high speed freezing centrifuge, 4 DEG C of Centrifugal Environment, rotating speed 10000g centrifugation 30min, supernatant is removed；
3rd, the 100ul90% aqueous acetone solutions for being stored in precooling in 4-8 DEG C of refrigerator are added, are incubated in mixture of ice and water
15min。
90% aqueous acetone solution：The acetone soln that volume ratio is 90% adds the solution of water, and water is deionized water, acetone soln
To analyze pure acetone.
4th, using high speed freezing centrifuge, 4 DEG C of Centrifugal Environment, rotating speed 10000g centrifugation 30min, supernatant is removed, is retained
Sediment.
5th, sediment is dried in room temperature, biochemical case, to sediment into powdered.
6th, the rehydration solution for adding 200ul redissolves.
Rehydration solution：To contain 7mol/L urea, 2mol/L thiocarbamides, 4g/100mlCHAPS (3- [3- (courage acyl aminopropyl)
Dimethylamino] propane sulfonic acid inner salt), 4-two sulphur O.4g/100ml determine butanol, the aqueous solution of 0.002g/100ml bromophenol blues.
7th, using centrifuge, Centrifugal Environment room temperature, rotating speed 10000g centrifugation 5min, supernatant is taken to do electrophoresis.
3rd, PEG (polyethylene glycol) precipitation of protein
1st, take 100ul serum to add in 0.5mlEP pipes, it is water-soluble to add the PEG6000 that 100ul mass percents are 7%
Liquid, 4-8 DEG C of refrigerator cold-storage 10-20 hour.
The 7%PEG6000 aqueous solution：Mass percent is that the PEG of 7% model 6000 is dissolved in the molten of deionized water
Liquid.
3rd, with high speed freezing centrifuge, 4 DEG C, rotating speed 1500r/min of Centrifugal Environment, 20min is centrifuged, removes supernatant, obtain
Sediment.The sediment is immune complex.
4th, 50ul deionized water is added in sediment, concussion vortex mixer is put into and shakes to mixing, obtain redissolution liquid, redissolve liquid
Do electrophoresis.
The redissolution liquid obtained using PEG (polyethylene glycol) precipitation of protein, it is best to do electrophoretic effects.
(3) electrophoresis
According to《Molecular Cloning:A Laboratory guide》Method and step, to above-mentioned supernatant or redissolve liquid carry out dodecyl sodium sulfonate
Sodium-polyacrylamide gel electrophoresis (SDS-PAGE), separation gel is selected, the separation gel used in the electrophoresis is polyacrylamide
Percent by volume is 10~12% gel solution., initial voltage 80V, after dyestuff enters separation gel, voltage is increased to
120V, continue electrophoresis until dyestuff arrives at separation gel bottom.It is specifically good from 10% separation gel effect.Walk glue and complete post analysis
Electrophoretogram.
As shown in Figure 1 to 4, Fig. 1 is to use above-mentioned PEG precipitation of protein, and separation gel is coagulated using 10% polyacrylamide
The electrophoretogram of glue, wherein 25,26,27 be the protein band of schizophreniac, N120, N121 are the albumen of normal healthy controls person
Band, confirm that difference of the 150kD protein bands between schizophreniac and Healthy People is obvious with reference to albumen marker.
Fig. 2 is to use above-mentioned TCA acetone precipitations, and separation gel uses the electrophoretogram of 10% polyacrylamide gel, wherein
05th, 06,07,08 be schizophreniac protein band, N1, N2 be normal healthy controls person protein band.
Fig. 3 is to use the above-mentioned TCA precipitation method, and separation gel uses the electrophoretogram of 10% polyacrylamide gel, wherein 01,
02nd, 03,04,05 be schizophreniac protein band, N3, N4 be normal healthy controls person protein band.
Protein band obtained by counting experiments organizes the positive rate with control group in patient, as shown in table 1.
Table 1
Table 1 is the serum of 30 schizophreniacs of collection, 30 manic or depressive patients and 30 normal persons, is passed through
The 150KD protein band positive reaction numerical tabulars that the above method measures.The wherein 150KD protein bands of schizophreniac
Strong positive reaction reaches 87%, and normal artificial 0.The expression quantity of the protein band of the 150KD in serum and schizophrenia
Linear combination can significantly distinguish schizophrenia and normal population, therefore this protein band can be used as and comment schizophreniac
Early warning factor, make clinical treatment more personalized.
Take proper range carefully to cut 150KD protein bands, -80 DEG C of preservations, concentrate and carry out mass spectral analysis.
(4) mass spectral analysis
The composition of 150kD protein bands does mass spectral analysis.
Mass spectral analysis is completed by Hua Da Science and Technology Ltd..Because the object of mass spectral analysis is typically small molecule peptide fragment, because
This forms small peptide fragment first using trypsase by proteolysis, and caused peptide fragment after enzymolysis is freezed, then with various concentrations
Formic acid (FA) and the mixing of acetonitrile (ACN) matrix, and point sample is to mass spectrum point template, in Shimadzu Corporation LC-20AD model nanoliter liquid
In chromatography, separated by first mass spectrometric and second order mses, and carry out molecular weight determination, obtain the albumen shown in table 2
Content table reaches spirogram table.
Table 2
Table 2 is the albumen table that the obvious 11 kinds of albumen of comparison in difference that mass spectral analysis obtains is carried out to 150KD protein bands
Up to amount data, it is 1. the expressing quantity of schizophreniac, is 2. manic or depressive patient expressing quantity, is 3. strong
The expressing quantity of health collator, albumen coding is sp wherein in analytical data of mass spectrum storehouse | A6NN14, corresponding albumen is Zinc
Finger protein 729 (zinc finger protein 72 9) albumen, its P value 1. 3. compareed have reached 0.050, can be by the Zinc
Early warning factor of the albumen of finger protein 729 as evaluation and test schizophreniac, makes clinical treatment more personalized.
P values (P value) are exactly that the sample view result resulting when null hypothesis is true or more extreme result occur
Probability.If P value very littles, the probability very little for the occurrence of illustrating null hypothesis, and in the event of, according to small probability principle,
The reasons why we just have reason to refuse null hypothesis, and P values are smaller, and we refuse null hypothesis is more abundant.In a word, P values are smaller, show to tie
Fruit is more notable, and above content is explanation of the Baidupedia to P values.Medically think P value≤0.05, conspicuousness is obvious, Ke Yizuo
For diagnosis marker.
(5) using ELISA kit checking mass spectrometry results.
In order to verify the accuracy of mass spectral results, using 4 kinds of albumen in the 11 kinds of albumen listed in mass spectral analysis, i.e.,
CFH, CFB, ZNF729 and THBS-1 ELISA (enzyme linked immunosorbent assay (ELISA)) kit, analysis application is without the above method
Treated original experimenter's serum sample, is operated according to reagent specification.The numerical value of table 3 is obtained after statistical analysis.
The detection of zinc finger protein 72 9 in original serum sample, preferably carried out multiple dilution factor conditions under detection.
Table 3
Table 3 is the schizophrenia treated using the ELISA kit analysis of four kinds of albumen without (two) step method
Statistical analysis, list are carried out in the original serum of disease patient, manic or depressive patient and normal person after the concentration of four kinds of albumen
It is compared.Four kinds of albumen are：(Recombinant Human Thrombospondin-1 recombination human platelets are anti-by THBS1
Answer albumen), CFH (complement factor H complement factor Hs), CFB (complement factor B complement factor Bs)；
ZNF729 (zinc finger protein 72s 9 of zinc finger protein 729).Conclusion is that ZNF729 P values are less than 0.05, it was demonstrated that mass spectrum
It is correct to analyze the high conclusion of conspicuousnesses of the obtained ZNF729 in the serum of schizophreniac and normal person.
(6) analyzed using Mascot2.3.02 softwares (protein identification software).
The theoretical mass spectra data progress that tandem mass spectrum data and SwissProt/UniProt database simulations are obtained
Match somebody with somebody, so as to obtain protein coverage rate and protein classes result, and carry out gene ontology (gene ontology, GO), albumen phase
Cluster (cluster of orthologous groups of proteins, COG), capital of a country gene and the genome hundred of adjacent class
The functional annotation of section's pandect (Kyoto encyclopedia of genesand genomes, KEGG) path.Using IPA (eggs
White matter software for analyzing spectrum) in schizophreniac's blood plasma paraprotein (including zinc finger protein 72 9) that mass spectral analysis is obtained
11 kinds of albumen bring into mucosal adhesive functional network.This network includes complement waterfall, ECM (extracellular matrix)-acceptor
Effect and infection of staphylococcus aureus.Biological function molecular network figure is obtained, as shown in Figures 4 to 6.Further illustrate
These band mass spectrum compositions have been primarily involved in molecule adhesion approach function.
This research finds another Abnormal Eggss when 32kD protein bands are carried out and further studied on the past Research foundation
Informal voucher band, molecular weight 150kD, and carried out mass spectral analysis.Found in this research in depression, mania it has also been found that such egg
In vain, and positive rate is close with schizophrenia.And studied in the past and do not carry out such research.Pre-experiment stage is by constantly entering
Row adjustment, optimize each experimental procedure and experiment condition, include the adjustment of serum applied sample amount, the preparation of sample-loading buffer is constantly excellent
Change the intermediate processing of albumen, the application repeatedly of the separation gel of various concentrations, it is found that the redissolution liquid that the 7%PEG precipitation method obtain is done
PAGE gel electrophoresis pattern effect is good, and the experimental result drawn on this basis is stable, and reproducible, protein band is clear
It is clear.
Claims (10)
1. a kind of method for determining the diagnosis marker for assessing schizophrenia patients, experimental method are as follows：
(1) blood sample, is gathered；
(1) gathered to be put into 4 DEG C of refrigerators in subject's morning limosis vein blood 3ml, 30min with vacuum blood collection tube and stand 1h, separated out
Serum；
It is put into centrifuge under (2) 4 DEG C of environment, rotating speed 3000r/min, centrifuges 5min, obtain experimenter's serum, dispenses 5-10 parts, -80
DEG C freeze standby；
(2), early stage removes the high-abundance proteins in experimenter's serum, may be selected to use TCA acetone albumen precipitation methods；Or TCA
The precipitation method；Or PEG precipitation of protein；Obtain the supernatant of electrophoresis test or redissolve liquid；
(3) according to《Molecular Cloning:A Laboratory guide》Method and step, to supernatant or redissolve liquid carry out dodecyl sodium sulfate-
Polyacrylamide gel electrophoresis, after electrophoresis protein band.
2. the method according to claim 1 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：150KD protein band scopes at least on 30 protein bands are cut, -80 DEG C of preservations, concentrates and carries out mass spectral analysis.
3. the method according to claim 1 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：The TCA acetone albumen precipitation method comprises the following steps：
(1) take 25~400ul to be stored in 10%~40%TCA acetone solns of precooling in 4-8 DEG C of refrigerator, add 1.5mlEP pipes
It is interior, 50ul serum is then added, concussion vortex mixer is put into and shakes to mixing, under 4 DEG C of environment, place 1 hour；
Acetone soln is analysis pure acetone；
10%~40%TCA solution：10~40gTCA is added to configuration in 100ml acetone solns and obtains 10%~40%TCA third
Ketone solution；
(2) high speed freezing centrifuge is used, 4 DEG C of Centrifugal Environment, rotating speed 15000g centrifugation 15min, removes supernatant；
(3) 0.5mlDTT acetone solns are added, concussion vortex mixer is put into and shakes to mixing, with high speed freezing centrifuge, centrifugal hoop
4 DEG C of border, rotating speed 15000g centrifugation 15min, retains sediment, separation supernatant is standby, and -80 DEG C standby of supernatant freezes；
DTT acetone solns：Acetone soln containing 20mmol/L DTT；
(4) sediment is lyophilized into -70 DEG C of refrigerators powdered, the time is three days；
(5) normal temperature, the interior rehydration solution for adding 200ul of powdery precipitate redissolve, and obtain redissolution liquid；
Rehydration solution：To contain 7mol/L urea, 2mol/L thiocarbamides, 4g/100mlCHAPS (3- [3- (courage acyl aminopropyl) diformazans
Amino] propane sulfonic acid inner salt), 4-two sulphur O.4g/100ml determine butanol, the aqueous solution of 0.002g/100ml bromophenol blues, and water is to go
Ionized water.
4. the method according to claim 1 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：The TCA precipitation method comprise the following steps：
(1) 12.3ul serum is taken, is added in 1.5mlEP pipes, adds 200ulPBS (phosphate buffer), is adding 40ul's
The 60%TCA aqueous solution, 10-20 hours are incubated in mixture of ice and water；
The 60%TCA aqueous solution:60gTCA is dissolved in the solution in 100ml water, and water is deionized water；
(2) high speed freezing centrifuge is used, 4 DEG C of Centrifugal Environment, rotating speed 10000g centrifugation 30min, removes supernatant；
(3) the 100ul90% aqueous acetone solutions for being stored in precooling in 4-8 DEG C of refrigerator are added, are incubated in mixture of ice and water
15min；
90% aqueous acetone solution：Volume ratio is that 90% acetone soln adds the solution of water, and water is deionized water, and acetone soln is point
Analyse pure acetone；
(4) high speed freezing centrifuge is used, 4 DEG C of Centrifugal Environment, rotating speed 10000g centrifugation 30min, supernatant is removed, retains precipitation
Thing；
(5) sediment is dried in room temperature, biochemical case, to sediment into powdered；
(6) rehydration solution for adding 200ul redissolves；
Rehydration solution：To contain 7mol/L urea, 2mol/L thiocarbamides, 4g/100mlCHAPS (3- [3- (courage acyl aminopropyl) diformazans
Amino] propane sulfonic acid inner salt), 4-two sulphur O.4g/100ml determine butanol, the aqueous solution of 0.002g/100ml bromophenol blues, and water is to go
Ionized water；
(7) centrifuge is used, Centrifugal Environment room temperature, rotating speed 10000g centrifugation 5min, takes supernatant.
5. the method according to claim 1 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：The PEG precipitation of protein comprises the following steps：
(1) take 100ul serum to add in 0.5mlEP pipes, add the PEG6000 aqueous solution that 100ul mass percents are 7%,
4-8 DEG C of refrigerator cold-storage 10-20 hour；
The PEG6000 aqueous solution：The solution of deionized water is dissolved in for the PEG of model 6000；
(2) high speed freezing centrifuge is used, 4 DEG C, rotating speed 1500r/min of Centrifugal Environment, centrifuges 20min, supernatant is removed, must precipitate
Thing；
(3) 50ul deionized water is added in sediment, concussion vortex mixer is put into and shakes to mixing, obtain redissolution liquid.
6. the method according to claim 1 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：The separation gel used in the electrophoresis is the gel solution that polyacrylamide percent by volume is 10%~12%.
7. the method according to claim 2 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：After mass spectral analysis, mass spectrometry results are verified using ELISA kit, are analyzed not using ZNF729 ELISA kit
The original experimenter's serum sample treated by the above method, is operated according to reagent specification.
8. the method according to claim 3 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：In the TCA acetone albumen precipitation method, 50~200ul is taken to be stored in 20%~30% of precooling in 4-8 DEG C of refrigerator
TCA acetone solns.
9. the method according to claim 8 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：In the TCA acetone albumen precipitation method, 100ul is taken to be stored in the 30%TCA acetone solns of precooling in 4-8 DEG C of refrigerator.
10. the method according to claim 6 for determining the diagnosis marker for assessing schizophrenia patients, its feature
It is：According to《Molecular Cloning:A Laboratory guide》Method and step, to supernatant or redissolve liquid carry out dodecyl sodium sulfate-poly- third
Acrylamide gel electrophoresis, separation gel select polyacrylamide percent by volume for 10% gel solution, electrophoresis equipment it is initial
Voltage is 80V, and after dyestuff enters separation gel, voltage is increased into 120V, continues electrophoresis until dyestuff arrives at separation gel bottom.
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| CN112037854A (en) * | 2020-10-15 | 2020-12-04 | 深圳市龙岗中心医院 | Method and system for acquiring tumor methylation marker based on methylation chip data |
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