CN103869080B - chronic neurodegenerative disease early diagnosis marker and application - Google Patents
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Abstract
The invention provides a kind of chronic neurodegenerative disease early diagnosis marker, it is β type solubility NSF attachment protein β-SNAPs, is its amino acid sequence as Seq? ID? shown in No.1, or this sequence is through replacing, lacking or add one or several amino acids formed amino acid sequence with same function.The expression of Late Cambrian β-SNAPs of the present invention and content can indicate the early stage generation of chronic neurodegenerative disease, and develop the proteomics method detecting the change of this albumen, for the early diagnosis of chronic neurodegenerative disease and early detection open new approaches, by the foundation of more deep study mechanism and loss-time curve, contribute to the loss of chronic neurodegenerative disease to drop to minimum.
Description
Technical field
The present invention relates to medical science and protein engineering field, specifically, relate to a kind of chronic neurodegenerative disease early diagnosis marker and application.
Background technology
Prion disease is the class nerve degenerative diseases occurring in mammalian central nervous system, has longer latent period, and because early treatment is for the healing important in inhibiting of this disease, the research therefore for the early diagnosis of prion disease is imperative.Research at present for nerve degenerative diseases early diagnosis mainly launches around protein science, genomics, metabolism group and the several aspect of impact observation.Protein science wherein based on two dimensional polyacrylamide gel electrophoresis is study hotspot in recent years always.Dissimilar protein in protein example can be separated by two dimensional electrophoresis.Different types of protein can be carried out high-resolution separation according to isoelectric point with molecular weight difference by two dimensional electrophoresis.2000 ~ 3000 kinds of protein can be separated by successful two dimensional electrophoresis.After electrophoresis, high sensitivity dyeing is carried out to glue, such as silver dye or fluorescent dye.If compare the similarities and differences of protein expression between two kinds of samples, the protein example both can preparing respectively under similarity condition, then carries out two dimensional electrophoresis, compares two pieces of glue after dyeing under similarity condition; Or, can by the protein example of the two respectively with different fluorochrome labels, right latter two protein example carries out the separation of two dimensional electrophoresis on one piece of glue, finally by fluorescent scanning technique analysis result.
Gel image analysis system imaging can be utilized after glue dyeing, then by analysis software, quantitative test be carried out to protein spots, and interested protein spots is positioned.By special protein spots diced system, precise cutting can be carried out in the glue region at protein spots place.Then digestions is carried out to protein in glue, enzyme cut after digestion product just can by spotting system by the surface (MALDI-TOF) of protein point sample to certain material after desalination/concentration.These protein last just can be analyzed in mass spectrometer system, thus obtain the qualitative data of protein, and these data may be used for building database or comparing analysis with existing database.
LCM-two dimensional electrophoresis-mass-spectrometric technique is the classical technology path of proteomics research, and in addition, LCM-antibody chip is also the technology path of an important proteomics research.That is, obtain interested cell type by LCM technology, prepare cell protein quality sample, protein is hybridized with antibody chip after fluorochrome label, thus can compare the similarities and differences of two kinds of sample protein matter expression.
Summary of the invention
The object of this invention is to provide a kind of chronic neurodegenerative disease early diagnosis marker.
Another object of the present invention is to provide the application of described mark in the early diagnosis of chronic neurodegenerative disease.
In order to realize the object of the invention, a kind of chronic neurodegenerative disease early diagnosis marker of the present invention, described mark is β type solubility NSF attachment protein (SolubleNSFattachmentproteins, SNAPs) β-SNAPs, its amino acid sequence is as shown in SeqIDNo.1, or this sequence is through replacing, lacking or add one or several amino acids formed amino acid sequence with same function.
The present invention also provides described mark β-SNAPs preparing the application in chronic neurodegenerative disease early diagnosis reagent.It is using β-SNAPs as immunogene, by immune animal, obtains corresponding antibodies, using the antibody of preparation as the effective constituent in diagnostic reagent or diagnostic reagent.
The present invention also provides a kind of chronic neurodegenerative disease early diagnosis reagent, and it is using β-SNAPs as immunogene, and by immune animal, the corresponding antibodies of acquisition is diagnostic reagent.
The present invention also provides a kind of chronic neurodegenerative disease early diagnosis kit, containing above-mentioned diagnostic reagent in described kit.
The chronic neurodegenerative disease related in the present invention includes but not limited to Transmissible spongiform encephalopathy (TSE) and A Cihaimo disease (AD) etc.
At present for chronic neurodegenerative disease (especially spongiform encephalopathy and A Cihaimo disease), at asymptomatic stage and early stage disease stage, also do not find effective detection means.Current detection means still rests on the histopathology after morbidity after the extraction and analysis of tissue samples and death.Owing to not curing at present the method for this kind of disease, therefore the early diagnosis of chronic neurodegenerative disease has very great meaning for the health of humans and animals.A kind of albumen N solubility second maleimide ammonia fusion attachment protein β type (β-SNAPs) existed only in brain tissue of Late Cambrian of the present invention, its expression and content can indicate the early stage generation of chronic neurodegenerative disease, and develop the proteomics method detecting the change of this albumen, for the early diagnosis of chronic neurodegenerative disease and early detection open new approaches, by the foundation of more deep study mechanism and loss-time curve, contribute to the loss of chronic neurodegenerative disease to drop to minimum.
The technology used in the present invention route is: extract the other hamster of the same age in days same sex that different time infects or do not infect Scrapie Strain 263K, the brain tissue sample of transgenosis A Cihaimo disease model mice or normal wild type mouse, carry out two dimensional electrophoresis analysis, by special software (such as, Mascot, version2.3.02, MatrixSciences company) analyze and find out the albumen that relative content has significant change, carry out MALDI-TOF/TOFMS mass spectrophotometry, the series connection spectrum obtained is searched in NCBInr database by special software, thus analyze title and the amino acid sequence of albumen.Protein-bonded biological function, finds that the albumen β-SNAPs participating in neurotransmitter regulator and vesicular traffic has important Research Significance.By Western blot (WesternBlot), quantitative test is carried out to the β-SNAPs in sample subsequently, thus determine and confirmed the early stage content of β-SNAPs in brain tissue to reduce change and gradual loss.And using WesternBlot to the effective detection method one of of the quantitative detection of β-SNAPs as early detection β-SNAPs content.
The present invention carries out two dimensional electrophoresis by the brain tissue of the mouse to prion infection, and Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis is carried out to the protein site of significant difference, find 7 potential labels that can be used as in prion disease early diagnosis altogether, WesternBlot result also demonstrate that this result simultaneously.This early diagnosis being prion disease and pathomechanism research provide new approaches.
Accompanying drawing explanation
Fig. 1 is Syria hamster brain tissue protein groups two dimensional electrophoresis glue figure in the embodiment of the present invention 1; Wherein, the albumen of arrow points mass spectrum successful identification.
Fig. 2 is the change of β-SNAPs albumen on two-dimentional gel in control group and experimental group in the embodiment of the present invention 1.
Fig. 3 is the variable quantity that in the embodiment of the present invention 1, WesternBlot detects β-SNAPs, and result meets two dimensional electrophoresis result.Use α/β-SNAPs rabbit polyclonal antibody, anti-actin monoclonal antibody, in A figure, left side three is classified as and attacks poison rear surrounding, six weeks, eight weeks β-SNAP expressions, right side three is classified as the β-SNAP expression of PBS control group in corresponding period, then carries out relative content with β-actin and compare.The analysis of B diagram data draws β-SNAPs relative variation and relative variation tendency.
Fig. 4 is 2-D standard protein reference diagram; The IPG adhesive tape of pH value 4-7 is selected in figure, a.Mw (kDa) 77pI6/6.3/6.6, b.Mw (kDa) 66.2pI5.4/5.6, c.Mw (kDa) 43pI5/5.1, d.Mw (kDa) 36pI8.3/8.5, e.Mw (kDa) 29pI5.9/6, f.Mw (kDa) 21.5pI4.6, g.Mw (kDa) 17.6pI6.8/7.3.A figure is the protein profile that standard protein carries out separately two dimensional electrophoresis, and B figure is that standard protein instructions is with reference to contrast figure.
Fig. 5 is " loss-time curve " figure of β-SNAPs.
Fig. 6 is the β-SNAPs of MALDL-TOF Mass Spectrometric Identification Trypsin Induced, and mark peptide section meets β-SNAPs peptide section mass theory value.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The discovery of embodiment 1 chronic neurodegenerative disease early diagnosis marker β-SNAPs
1. the preparation of brain tissue sample
The Scrapie Strain 263K that proterties is stablized, performance is determined is injected to Syria hamster brain tissue.The brain homogenate 50 μ l infecting the hamster of itch is thus injected in weanling some Syria hamster brains.With the PBS of same dosage inject with in other hamster brain of the age in days same sex in contrast.Difference the 4th, 6,8 week after the implantation, selects hamster and the control group hamster of inorganization morphological change, carries out euthanasia, extract its brain tissue and be kept in-80 DEG C of refrigerators it.Female A Cihaimo disease transgenic mice (TheJacksonLaboratory) [B6C3-Tg (APPSWE at 12 monthly ages, PSEN1dE9) 85Dbo/Mmjaxmice] and the brain tissue of suitable wild type normal control mice, be kept at equally after extraction in-80 DEG C of refrigerators.
2. sample preparation
With lysis buffer [the 7M urea containing full constituent protease inhibitor cocktail (Roche company), 2M sulphur is urinated, 2%(w/v) CHAPS, 0.2%(v/v) pharmalyte, 65mM dithiothreitol (DTT) (DTT)] brain tissue sample of hamster is suspended also, and homogenate is even.Brain homogenate is carried out to ultrasonic (25% amplitude) break process (each circulation: ultrasonic 1s stops 5s) of 12 circulations at 4 DEG C.After placing 30s on ice, the centrifugal 1h(4 DEG C of 25000 × g).With 2-D quantification kit (GEHealthcare company), accurate quantitative analysis is carried out to the protein content in centrifugal rear supernatant.To contain 450 μ g albumen in every increment product for standard, packing is kept in-80 DEG C, uses in order to two-dimensional gel electrophoresis and WesternBlot.
3. two-dimensional gel electrophoresis
Rehydration is carried out to the brain tissue protein sample in above-mentioned steps.Rehydration buffer [7M urea, 2M thiocarbamide, 2% (w/v) CHAPS, 0.5% (v/v) pharmalyte, 3% (w/v) DTT, 0.002% (v/v) bromophenol blue] adds in protein sample and makes cumulative volume be 450 μ l.Join in long Stationary pH 4-7 (non-linear) gradient strip of 24cm (GEHealthcare company), place 18h in room temperature.To be undertaken etc. by following steps that point focusing (IEF): 250V2h, 1000V2h, 5000V2h, 3h are progressive increases to 10000V(HoeferIEF100 system).Sample strip is balance 18min in 0.5mMPH6.8Tris-HCl damping fluid (6M urea, 30% glycerine, 2%SDS, 2%DTT), then, balances 15min again after changing buffer components DTT into 2.5% iodoacetamide.Sample strip is transferred to (HoeferSE900 system) in 12.5%SDS-PAGE gel and carries out second to electrophoretic separation, 1 watt/gel electrophoresis 5h, 2 watts/gel electrophoresis 30h.Taking-up gel after completing, fix 1h in immobile liquid (50% methyl alcohol, 5% acetic acid) after, stained over night in coomassie brilliant blue staining liquid (20% methyl alcohol, 10% phosphoric acid, 10% ammonium sulfate, 0.125% coomassie G-250).
4. graphical analysis
After dyeing, gel scanner (UMAXPowerLook2100XL-USB) scans under 500dpi.Image is analyzed by Dymensionsoftware dedicated analysis software (Syngene company).T inspection is carried out at least 3 separate test findings.Selective light strength difference at the protein site (p<0.05) of 1.5 times or more, in order to carry out next step mass spectrophotometry, comprising β-SNAPs albumen.
5. the discovery of spectrometer analysis and β-SNAPs
Cut by the protein site selected from gel, be placed in the Eppendorf pipe of 1.5mL, tri-distilled water rinses twice, rinses for several times with 100mM ammonium bicarbonate, then with 100% pure acetonitrile dehydration, is placed in the freeze drying of vacuum freezedrying pump.Add the 100mM ammonium bicarbonate of 50 μ L containing 10MmDTT in each sample hose, 57 DEG C of reducing action 1h, after room temperature cooling, remove excess liq, add rapidly subsequently with the freshly prepared 55mMIAA of 100mM ammonium bicarbonate, alkylation 30min is placed in room temperature dark place.React rear 100mM ammonium bicarbonate and rinsed 2 times, then add the freeze-drying of isopyknic 100mM ammonium bicarbonate with after pure acetonitrile dehydration.Often effective 25 μ L trypsase digest, and 37 DEG C are spent the night.By 5% trifluoroacetic acid (TFA)/50% acetonitrile (ACN) extracting twice, each 15min, combining extraction liquid freeze-drying.0.8 μ L matrix CHCA (α-cyano-4-hydroxy-cinnamicacid) solution (0.5g/L, solvent 0.1%TFA/50%ACN) is added, upper MALDL target plate, at room temperature natural drying in the sample of freeze-drying after extraction.In addition, put 0.5 μ L and do not add the 0.5g/LCHCA solution of sample as blank.Analyze with MALDI-TOF/TOF high resolving power tandem mass spectrometer.MS terminate rear direct selection and the PMF contrasting matrix scheme variant, mass range 700-3200Da and minimum signal to noise ratio (S/N ratio) be 20 peptide section carry out MS/MS analysis.Series connection spectrum Mascot, version2.3.02 (MatrixSciences company) software searches for NCBInr database title and the amino acid sequence of determining albumen, and the scoring of MSCOT to albumen is not less than 59 points and is considered as identifying successful albumen.If a protein site detects multiple albumen, estimate its molecular mass in conjunction with 2-D standard protein (precious bioengineering company limited) (Fig. 4) and carry out pI inspection, selecting the albumen that mark is higher.
Find 7 potential labels (comprising β-SNAPs albumen) that can be used as in prion disease early diagnosis altogether, table 1 is depicted as the Protein Information of successful identification.
The different albumen that table 1 goes out to express by MALDI-TOF/TOFMS Analysis and Identification
Biological action analysis is carried out to the determined albumen of mass spectrophotometry, in conjunction with the light-intensity difference of two-dimensional gel electrophoresis image, can find that β-SNAPs albumen has great Research Significance.Just there is remarkable minimizing at asymptomatic stage in β-SNAPs, and along with the prolongation of infection time, the content of β-SNAPs is fewer and feweri in spongiform encephalopathy and A Cihaimo disease infected animal brain.β-SNAPs participates in neurotransmitter regulator and vesicular traffic, has been that indispensable albumen is transmitted in cynapse.Its content reduces, directly cause the reduction of memory, locomitivity, in conjunction with the research of domestic and foreign literature to this albumen, can infer that β-SNAPs and chronic neurodegenerative disease have direct relation, and because it is in the marked change of the early stage content of disease, meet the feature of chronic neurodegenerative disease biological mark.
6.WesternBlot verifies
Take out the protein sample extracted in step 2, carry out WesternBlot analysis (BIO-RAD company) with the sample containing 50 μ g albumen.Carry out WesternBlot analysis in pairs in groups, 263K infection hamster, A Cihaimo disease transgenic mice respectively its corresponding control group are analyzed.Analyze and all ensure to repeat to ensure real result for 3 times.Step is as follows:
Polyacrylamide gel electrophoresis: prepare 12%SDS-PAGE gel according to a conventional method, every hole application of sample 10 μ l, deposition condition is concentrated glue 90V25min, separation gel 120V90min.
Electricity turns: 80V4h.
Close: 5% skimmed milk power, 37 DEG C of 1h.
Primary antibodie is hatched: α/β-SNAPs rabbit polyclonal antibody (SantaCruzBiotechnology company), 100 times of dilutions, and 4 DEG C of overnight incubation are washed 3 times, each 5min.
Two anti-hatch: the goat anti-rabbit antibodies selecting horseradish peroxidase-labeled, hatch 1h for 37 DEG C, wash 3 times, each 5min.
Colour developing: develop the color with the chemical luminescence for liquid of BIO-RAD, ChemiDoc gel imaging system carries out image acquisition.
De-antibody and β-actin are hatched again: wash away the antibody developed the color, resist by anti-actin monoclonal antibody and corresponding two and β-actin is hatched, carry out relative content with β-SNAPs after exposure to compare, to eliminate the impact that in loading sample, protein content is different.
WesternBlot analyzes and further determined that namely β-SNAPs has occurred remarkable reduction in early days in Transmissible spongiform encephalopathy and the asymptomatic of A Cihaimo disease really, and along with the prolongation of infection time in Transmissible spongiform encephalopathy, β-SNAPs have also appeared gradual loss (Fig. 5).Prove the direct relation of β-SNAPs and chronic neurodegenerative disease development thus, thus prove the feasibility of β-SNAPs as chronic neurodegenerative disease biological mark.And WesternBlot analytical approach is the effective method for measuring β-SNAPs content in the early stage process of disease.
Fig. 1 is Syria hamster brain tissue protein groups two dimensional electrophoresis glue figure; Wherein, the albumen (see table 1) of arrow points mass spectrum successful identification.IPG adhesive tape pH scope 4-7, right side molecular weight is determined according to 2-D standard protein.
Fig. 2 is the change of β-SNAPs albumen on two-dimentional gel in control group and experimental group.After attacking poison, surrounding, six weeks, eight weeks β-SNAP express change; Wherein, arrow points SNAP albumen, result is by Mass Spectrometric Identification gained, and the visible albumen of naked eyes reduces surrounding gradually from after attacking poison.
Fig. 3 is the variable quantity that WesternBlot detects β-SNAPs, and result meets the result of two dimensional electrophoresis.After attacking poison, reducing appears in surrounding, and six to eight weeks reduced more obvious.Use α/β-SNAPs rabbit polyclonal antibody, anti-actin monoclonal antibody, in A figure, left side three is classified as and attacks poison rear surrounding, six weeks, eight weeks β-SNAP expressions, right side three is classified as the β-SNAP expression of PBS control group in corresponding period, then carries out relative content with β-actin and compare.The analysis of B diagram data draws β-SNAPs relative variation and relative variation tendency.
Fig. 4 is the distribution of 2-D standard protein on 2-DE gel (2-D standard protein reference diagram).The IPG adhesive tape of pH value 4-7 is selected in the present invention, a.Mw (kDa) 77pI6/6.3/6.6 in figure, b.Mw (kDa) 66.2pI5.4/5.6, c.Mw (kDa) 43pI5/5.1, d.Mw (kDa) 36pI8.3/8.5, e.Mw (kDa) 29pI5.9/6, f.Mw (kDa) 21.5pI4.6, g.Mw (kDa) 17.6pI6.8/7.3.A figure is the protein profile that standard protein carries out separately two dimensional electrophoresis, and B figure is that standard protein instructions is with reference to contrast figure.
Fig. 6 is the β-SNAPs of MALDL-TOF Mass Spectrometric Identification Trypsin Induced, and mark peptide section meets β-SNAPs peptide section mass theory value.
The application of embodiment 2 chronic neurodegenerative disease early diagnosis marker β-SNAPs
The present invention also provides described mark β-SNAPs preparing the application in chronic neurodegenerative disease early diagnosis reagent.Be specially: using β-SNAPs as immunogene, by immune animal, obtain corresponding antibodies, using the antibody of preparation as the effective constituent in diagnostic reagent or diagnostic reagent.
Utilize above-mentioned diagnostic reagent to prepare chronic neurodegenerative disease early diagnosis kit, thus provide possibility for the early diagnosis of disease.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
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Claims (3)
1. chronic neurodegenerative disease early diagnosis marker is preparing the application in chronic neurodegenerative disease early diagnosis reagent, wherein, described chronic neurodegenerative disease early diagnosis marker is β type solubility NSF attachment protein β-SNAPs, and its amino acid sequence is as shown in SeqIDNo.1.
2. application according to claim 1, is characterized in that, using β-SNAPs as immunogene, by immune animal, obtains corresponding antibodies, using the antibody of preparation as the effective constituent in diagnostic reagent or diagnostic reagent.
3. application according to claim 1, is characterized in that, described chronic neurodegenerative disease comprises Transmissible spongiform encephalopathy and A Cihaimo disease.
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| EP3242134A1 (en) * | 2016-05-04 | 2017-11-08 | Euroimmun Medizinische Labordiagnostika AG | Assay for the diagnosis of a neurological disease |
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| CN1733932A (en) * | 2005-07-18 | 2006-02-15 | 山东省医药生物技术研究中心 | Adenocarcinoma marker and its uses |
| CN102373773A (en) * | 2010-08-16 | 2012-03-14 | 苏州富通电器塑业有限公司 | Polymer wall tile and preparation method thereof |
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| CN1733932A (en) * | 2005-07-18 | 2006-02-15 | 山东省医药生物技术研究中心 | Adenocarcinoma marker and its uses |
| CN102373773A (en) * | 2010-08-16 | 2012-03-14 | 苏州富通电器塑业有限公司 | Polymer wall tile and preparation method thereof |
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| beta-soluble NSF attachment protein[Bos taurus]，NCBI Reference Sequence:NP_001039698;NCBI;《NCBI Protein》;20140215;第1页 * |
| PREDICTED: beta-soluble NSF attachment protein-like [Cricetulus griseus],NCBI Reference Sequence: XP_003509919.1;NCBI;《NCBI Protein》;20111026;第1页 * |
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