CN105087512A - Protein for detecting thyroid diseases and partial peptide of protein - Google Patents
Protein for detecting thyroid diseases and partial peptide of protein Download PDFInfo
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Abstract
The invention relates to protein, polypeptide, a detection component including the protein or the polypeptide as well as a diagnosis kit including the detection component, which are useful for the diagnosis of thyroid diseases.
Description
Technical field
The present invention relate generally to protein, this protein partial peptide, comprise they detection means and comprise the detection kit of this detection means, particularly can be used for the protein of the diagnosis of thyroid disease or somatotype, this protein partial peptide, comprise the partial peptide of this protein or this protein detection means and comprise this protein, the partial peptide of this protein or the detection kit of this detection means.
Background technology
The clinical syndrome of autoimmune thyroid disease (autoimmunethyroiddisease, AITD) to be jointly caused by h and E factor one group with abnormal thyroid function be main manifestations.This group disease belongs to organ specific autoimmune disease, and their common features are that pathology all accumulates on Tiroidina; Conforming change there is lymphocytic infiltration; All exist in serum for thyroid autoantibody; Various disease can occur together or mutually transform; There is the evidence of genetic predisposition, simultaneously this group disease shows again its respective Clinical Manifestations.
The abnormal thyroid function that various thyroid disease causes can affect the function of each system of whole body, and main manifestations is hyperthyroidism and disease of going down.The complication such as the hyperthyroidism crisis that Hyperthyroidism is sent out and hyperthyroid heart disease can jeopardize the life of patient time serious, hypothyroidism then can increase the risk of cardiovascular disorder, inborn defect greatly.In China, the investigation result display hyperthyroidism of some areas and the morbidity of disease of going down are about 2.0%, and the morbidity of subclinical hyperthyroidism and subclinical hypothyroidism reaches 3.9% and 6.1% respectively.Therefore, thyroid disease remains the important diseases affecting China's residents ' health.
Autoimmune thyroid disease is except iodine deficiency, topmost Tiroidina class disease.Mainly comprise Graves disease, Hashimoto thyroiditis, atrophic thyroiditis, subacute lymphocytic thyroiditis, latter 3 kinds are commonly referred to autoimmune thyroiditis.
Hospitals at Present for the diagnosis of autoimmune thyroid disease mainly through clinical symptom, thyroid hormones level and detect relevant TA and realize.
Summary of the invention
The object of the present invention is to provide the partial peptide of a kind of protein, this protein, comprise the detection means of this protein or its partial peptide and comprise the detection kit of this detection means, they to the diagnosis of thyroid disease or somatotype useful.This protein or its partial peptide is the object of the present invention is to provide to prepare the purposes that can be used in the diagnosis of thyroid disease or the detection kit of somatotype.
That is, the present invention comprises following technical proposals:
1. the protein be made up of the aminoacid sequence shown in SEQIDNO:1, or the following part peptide of this protein:
The polypeptide be made up of the aminoacid sequence shown in SEQIDNO:2;
The polypeptide be made up of the aminoacid sequence shown in SEQIDNO:3;
2. a detection means, it comprises:
Solid carrier, and
Be connected to the described partial peptide of the protein described in item 1 on this solid carrier or this protein.
3. the detection means according to item 2, wherein, described solid carrier is SJ modified silica-gel.
4. the detection means according to item 2 or 3, its diagnosis for thyroid disease or somatotype.
5. a detection kit, it comprises the detection means according to any one of item 2 ~ 4.
6. the detection kit described in 5, its diagnosis for thyroid disease or somatotype.
7. the protein described in 1 or the purposes of the described partial peptide of this protein in preparation detection kit.
8. the purposes according to item 7, wherein, described detection kit is used for diagnosis or the somatotype of thyroid disease.
9. according to the purposes that item 7 is stated, wherein, described detection kit comprises the detection means according to any one of item 2 ~ 4.
Apply the present invention to diagnosis or the somatotype of thyroid disease, gratifying effect can be obtained.
Accompanying drawing explanation
The schematic diagram that the making processes of Fig. 1 to SJ modified silica-gel (iPDMS film) is described;
Fig. 2 illustrates the schematic diagram of polypeptide microarrays spot sample mode;
The result photo that the spot sample mode that Fig. 3 ~ 4 display contains polypeptide LTP-71 detects different serum;
Fig. 5 ~ 6 display contains the spot sample mode of polypeptide LTP-72 to the photo of the result that different serum detects.
the embodiment of invention
protein of the present invention and partial peptide thereof
In an aspect, the invention provides a kind of diagnosis to thyroid disease or the useful protein (below also referred to as protein of the present invention) of somatotype.This protein is made up of the aminoacid sequence shown in SEQIDNO:1, it is thyroid disease autoantigen protein-thyroid peroxidase (Hollowell, J.G.etal.SerumTSH, T (4), andthyroidantibodiesintheUnitedStatespopulation (1988to1994): NationalHealthandNutritionExaminationSurvey (NHANESIII).
journalofClinicalEndocrinology & Metabolism 87, 489-499 (2002)) truncated protein, it can be combined by the thyroid disease autoantibody specificity in thyroid disease patients serum.Therefore, this protein is as being useful for the diagnosis of thyroid disease or the instrument of somatotype.Protein of the present invention can adopt method well-known in the art to prepare.Such as, its nucleotide sequence of coding can be inferred based on above-mentioned aminoacid sequence, and this nucleotide sequence of chemosynthesis, this nucleotide sequence of chemosynthesis is inserted in known expression vector, with this expression vector transformed host cell, cultivate this host cell and make it to express this protein, then adopting ordinary method to carry out separation and purification, thus obtain protein of the present invention.
In one aspect of the method, the invention provides to the diagnosis of thyroid disease or the useful polypeptide (below also referred to as polypeptide of the present invention) of somatotype.These polypeptide are respectively:
Polypeptide LTP-71, it is made up of the aminoacid sequence shown in SEQIDNO:2; LTRVPMDAFQVGKFPEDFES
Polypeptide LTP-72, it is made up of the aminoacid sequence shown in SEQIDNO:3; VGKFPEDFESCDSITGMNLE
As shown in the Examples, the serum of polypeptide of the present invention to thyroid disease patient is positive, and to be negative reaction to healthy normal people or non-thyroid disorders patients serum.Therefore, polypeptide of the present invention is useful as the diagnosis of thyroid disease or somatotype instrument.Protein of the present invention or polypeptide may be used for manufacturing detection means (example detection means of the present invention described as follows) or the detection kit (example detection kit of the present invention described as follows) of diagnosis or the somatotype that can be used for thyroid disease.
Polypeptide of the present invention can be suitable for adopting the known method such as (1) chemical synthesis process or (2) enzyme reaction synthetic method to obtain, but is not limited thereto, and wherein chemosynthesis is more easy.In chemosynthesis polypeptide situation of the present invention, undertaken by using peptide synthesizer synthesis or this polypeptide semi-synthetic.As chemical synthesis process, such as peptide solid-phase synthesis etc. can be listed.The peptide of such synthesis can adopt conventional means such as ion-exchange chromatography, reverse phase high performance liquid chromatogram, affinity chromatography etc. to carry out purifying.Such peptide solid phase synthesis process with and subsequent peptide purification be all well-known in the art.
In addition, when producing polypeptide of the present invention by enzyme reaction, the method such as described in No. WO2004/011653, International Publication brochure can be adopted.Namely; can produce like this: by the amino acid of a side or the C-terminal of dipeptides is esterified or amidation and the amino acid that obtains or dipeptides, the amino acid (amino acid of such as carboxy protective) that is in unbound state with amino acid react under the existence of peptide synthetase, the dipeptides of generation or tripeptides.As peptide synthetase, can list: there is the culture of microorganism of the ability generating peptide, the bacterial disposing thing of the microbial cells be separated by this culture or this microorganism or this microbe-derived peptide synthetase.
And, except above-mentioned enzyme method, chemical synthesis process, such as gene engineering method can also be adopted to produce polypeptide of the present invention.
The aminoacid sequence of polypeptide of the present invention can adopt conventional method to carry out analyzing and confirming, such as, can utilize the method such as mass spectrum, chromatogram to carry out and analyze and confirm.
detection means of the present invention
In another aspect, the present invention also provides a kind of detection means (detection means of the present invention), and it comprises solid carrier and the protein of the present invention that is connected on this solid carrier or polypeptide of the present invention.Described detection means is useful for the diagnosis of thyroid disease or somatotype.
In the present invention, solid carrier is not particularly limited, as long as the carrier of solid or insoluble material (be such as can pass through filtration, material that precipitation, magnetic resolution etc. are separated from reaction mixture).
The material forming solid carrier includes but not limited to: silica gel (polydimethylsiloxane, PDMS), Mierocrystalline cellulose, teflon
tM, Nitrocellulose, agarose, dextran, chitosan, polystyrene, polyacrylamide, polyester, polycarbonate, polymeric amide, polypropylene, nylon, poly(vinylidene fluoride), latex, silicon-dioxide, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, ferrite, silicon wafer, polyethylene, polymine, poly(lactic acid), resin, polyose, albumen (albumin etc.), carbon or their combination etc.
The shape of solid carrier comprises but is not limited to: pearl, magnetic bead, film, microcapillary, filter membrane, plate, micro plate, carbon nanotube, sensor chip etc.Just as known in the art, the solid carrier that film or plate etc. are smooth can be arranged bottom pit, groove, filter membrane etc.
In the present invention, magnetic bead can have the sphere diameter of about 25nm ~ about 1mm scope.In a preferred embodiment, magnetic bead has the diameter of about 50nm ~ about 10 μm scope.The size of magnetic bead can be selected according to specific purposes.
In the present invention, the pearl be made up of the contour crosslinked spherical agarose of Sepharose has the diameter of about 24 μm ~ about 165 μm of scopes.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm ~ about 44 μm of scopes.The size of high crosslinked spherical sepharose 4B can be selected according to specific purposes.
The example with the solid carrier of water repellent surface comprises can from Polysciences, Warrington, PA or Spherotech, the polystyrene latex beads such as the goods that Liberville, IL buy.
Silicon-dioxide (SiO
2)-process or silicon-dioxide (SiO
2) example of solid carrier of base comprises the extraordinary magnetic silica pearl etc. can bought from Polysciences, Warrington, PA, it may be used for catching nucleic acid (such as DNA).Or, the M-280 etc. that can buy from DynalBiotech can also be used.
The magnetic bead with hydrophilic surface can be used for the bacterial cell of seizure proliferation period, nucleic acid and other composition.As the example of this magnetic bead, Polysciences can be listed, Warrington, the pearl (title: Biomag (registered trademark) carboxyl) that PA sells or BangsLaboratory, Inc., the name of Fishers, IN is called the pearl of MC02N/2928.Or, the M-270 etc. that DynalBiotech sells can be used.
In a preferred embodiment of the present invention, described solid carrier is SJ modified silica-gel, it is the microarray solid support material (iPDMS film, see the open CN101265329A of Chinese patent) of a kind of silicon rubber material of Suzhou Siju Biomaterials Co., Ltd.'s exploitation.This material is based on the conventional PDMS of biological study, add specific initiator composition (making this material realize surface-functionalized modification by surface initiated polymerization (SIP)) wherein, obtain through polyethylene glycol methacrylate-styrene polymer (poly (oligo (ethyleneglycol) methacrylate), pOEGMA) finishing again.SJ modified silica-gel has outstanding anti-protein non-specific adsorption (Nonspecificproteinadsorption, NPA) ability, non-specific protein absorption in complicated protein immunization can being detected controls to close to " absolute 0 " level (being near or below the limit of detection of instrument), not only can exempt the trouble closed and repeatedly clean, can also by the susceptibility using stronger amplification of signal means to improve protein microarray.And the essence of its silicon rubber imparts the stronger mechanical property of this material and good operability.Suzhou Yvonne gathers the combination assay microarray ELISA kit successfully SJ modified silica-gel being applied to 11 tumor markers compositions, achieve high-throughput and high-sensitive detection, demonstrating this material is a kind of outstanding protein microarray solid support material.Meanwhile, this material also has the adjustable characteristic of surface properties, can adjust its surface topography within the specific limits by the controlled modification reaction times.
The connection of polypeptide of the present invention and solid carrier can adopt the method for attachment that well known to a person skilled in the art polypeptide and solid carrier to carry out.Such as, for the connection on protein/polypeptide and modified silica-gel surface, 1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide [1-ethyl-3-(3-dimethylami-nopropyl) carbodiimide can be passed through, EDC] and N-hydroxy-succinamide (N-hydroxysuccinimide, NHS) reaction changes the carboxyl (-COOH) group on the macromolecular chain of modified silica-gel surface into activating group, this activating group can with protein/polypeptide on amino (-NH2) react thus realize protein/polypeptide being fixed on solid carrier surface (see such as HongweiMa, YuanziWu, XiaoliYang, XingLiu, Jian ' anHe, LongFu, JieWang, HongkeXu, YiShiandRenqianZhong,
integratedpoly (dimethysiloxane) withanintrinsicnonfoulingpropertyapproaching " Absolute " zerobackgroundinimmunoassays, anal.Chem., 82,6338 – 6342,2010).
Concentration for polypeptide of the present invention in the sampling liquid used during point sample is not particularly limited, and those skilled in the art can conventionally select, and is preferably 1 μ g ~ 1000 μ g/mL, more preferably 10 μ g ~ 500 μ g/mL.In addition, the density distributed on a solid support for polypeptide of the present invention is not particularly limited, and those skilled in the art can conventionally select, and is preferably 1 ~ 100 points/10mm2, more preferably 5 ~ 50 points/10mm2.
Detection means of the present invention is useful for the diagnosis of thyroid disease or somatotype, or it may be used for manufacturing and can be used for the diagnosis of thyroid disease or the detection kit of somatotype.
detection kit of the present invention
In one aspect of the method, the present invention also provides a kind of detection kit (detection kit of the present invention), and it comprises detection means of the present invention.This detection kit can be used for diagnosis or the somatotype of thyroid disease.
Detection kit of the present invention must comprise detection means of the present invention.Detection kit of the present invention can also comprise:
1. the serum sample diluent prepared or serum sample diluent component solution: Sample dilution, such as, have the Sample dilution (production code member 070021-S2) of Sai Chi bio tech ltd, Beijing, the application of sample variable color Sample dilution (production code member bwj010103) etc. of Bo Weijia bio tech ltd, Zhengzhou.Such Sample dilution is the PBS solution containing the composition such as BSA, sucrose, and containing sanitas, clarified liq, can directly use.This Sample dilution is used for dilute serum, and the serum that test kit detects will dilute suitable multiple, such as 2 ~ 200 times, preferably 10 ~ 100 times.
Detection kit of the present invention can also comprise:
2. concentrated washing lotion and washing lotion: after solid carrier surface hatches serum and enzyme labelled antibody, the unconjugated antibody of solid carrier surface and enzyme labelled antibody need be washed by washing lotion.Concentrated washing lotion is such as the polysorbas20 aqueous solution of 1%, need dilute 2 ~ 40 times, preferably 5 ~ 20 times during use.Can dilute by 1:9 by concentrating washing lotion (10 ×) purified water or distilled water, configure washing lotion.Such as, add 50mL in 450mL purified water or distilled water and concentrate washing lotion (10 ×), fully mix.The washing lotion do not used is placed on 2 ~ 8 DEG C of preservations, can preserve 3 months.
Detection kit of the present invention can also comprise:
3. enzyme labelled antibody solution: the thyroid disease autoantibody in thyroid disease patients serum can protein of the present invention on solid carrier (such as SJ modified silica-gel) or polypeptide be combined, enzyme labelled antibody can with antibodies, and the marker on enzyme labelled antibody can react with luminous substrate, thus send detectable light.Enzyme labelled antibody can be the goat anti-human igg of such as horseradish peroxidase-labeled.As enzyme labelled antibody solution, the Goat anti human IgG (H+L) of the horseradish peroxidase-labeled that Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge produces can be listed, production code member ZB-2304.Being not particularly limited the concentration of enzyme labelled antibody in enzyme labelled antibody solution, can be such as 1ng ~ 1000ng/mL.
Detection kit of the present invention can also comprise:
4. luminous substrate mixing solutions: luminous substrate mixing solutions can react with the horseradish peroxidase that marks on enzyme labelled antibody, make to react and send the chemical light luminous substrate mixing solutions that instrument can detect and comprise luminous substrate liquid A-superoxol, and luminous substrate liquid B-luminol (luminol,3-aminophthalic acid cyclic hydrazide) solution.Adopt two kinds of luminous substrate liquid to mix with equal-volume during use in advance, obtain luminous substrate mixing solutions (using in 45 minutes).Luminol only has crosses just meeting luminescence by oxidizer treatment.The mixed aqueous solution of usual use hydrogen peroxide and a kind of hydroxide bases is as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen G&W:
2H
2O
2→O
2+2H
2O
Luminol,3-aminophthalic acid cyclic hydrazide and oxyhydroxide generate a pairs of anion when reacting, the dioxygen oxidation that it can be gone out by peroxide decomposition, and product is an organo-peroxide.This superoxide is very unstable, decomposites nitrogen immediately, generates the 3-aminophthalic acid of excited state.During excited state to ground state transforms, the energy of release exists with the form of photon, and wavelength is positioned at the blue light components of visible ray.The SuperSignal ELISAFemtoMaximumSensitivitySubstrate of the example such as ThermoSeientific company of luminous substrate mixing solutions, article No. 37074.
Detection kit of the present invention can also comprise:
5. one or more reaction cavity (such as Chinese patent Granted publication CN202054829U).
Detection kit of the present invention can also comprise:
6. other are for detecting the detection molecules (such as polypeptide, protein, nucleic acid etc.) of diabetes (particularly thyroid disease).
Detection kit of the present invention can also comprise:
7. working instructions, wherein describe diagnosis or somatotype that this detection kit may be used for thyroid disease.
Reaction member and reaction kit can such as adopt gel imaging instrument to carry out chemiluminescence imaging.As gel imaging instrument, such as GELAS4000mini type or sky energy 5500 type Microarray image instrument can be adopted.
Embodiment
Below, by embodiment, more specific description is carried out to the present invention, but be not the restriction to the technology of the present invention scope.By the record of this specification sheets, those skilled in the art can be easy to modify the present invention/change, and these are included in technical scope of the present invention.Scope of the present invention is determined by claims.
1. polypeptide preparation and confirmation
The each polypeptide used in embodiment by the synthesis of gill biochemical (Shanghai) Co., Ltd., and confirms described polypeptide by mass spectrum.The sign of polypeptide confirms by hydrogen spectrum and mass spectrum:
LTP-71: liquid phase chromatography detects purity: 96.55% (area normalization method): mass spectroscopy detects: calculate molecular weight: 2314.63, test value 2313.88;
LTP-72: liquid phase chromatography detects purity: 95.03% (area normalization method): mass spectroscopy detects: calculate molecular weight: 2218.45, test value 2218.48.
2. the preparation of detection means
Detection chip be with SJ modified silica-gel (iPDMS film) for solid support material, be prepared from by point sample immobilized polypeptide solution thereon.Modified silica-gel be add in traditional polydimethyl siloxane material band olefin-terminal, the initiator of surface initiated polymerization, and be fixed in the three-dimensional structure of polydimethylsiloxane by heat cross-linking (si-h bond bonding), obtain SJ modified silica-gel, its making processes as shown in Figure 1.
A and B is wherein two components of polydimethylsiloxane, polydimethylsiloxane (Poly (dimethylsiloxane), Sylgard184) buy from Dow corning (DowCorning) company, comprise liquid composition A (composition is metallic platinum catalyst and the diformazan siloxanes polymer precursor mixture being with vinyl) and crosslinking agent B (composition is the dimethyl siloxane precursor with vinyl and Si-H group) two kinds of compositions.C is the initiator of end strips vinyl, is purchased from Hangzhou Dong Wei company.Polymer on finally modifying is that oligomeric ethylene glycol methacrylate monomer (Oligo (ethyleneglycol) methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) is bought in Aldrich.Polydimethylsiloxane precursor A and crosslinking agent B are fully mixed with A:B:C=10:1:0.5 ratio with the initiator C of band vinyl end.Make transparent elastic silicone rubber by curing reaction, then carry out finishing by sip technique and can obtain SJ modified silica-gel.Experiment shows, the surface of SJ modified silica-gel have enough highdensity, by the initiator of covalently immobolization, it can pass through surface initiated polymerization (SIP), and to realize surface macromolecule modified.The surface that reaction acquisition polyoxyethylene glycol (PolyethyleneGlycol, PEG) is modified is carried out in use poly (OEGMA) (polyethylene glycol methacrylate-styrene polymer), realizes the ability of stronger anti-albumen non-specific adsorption.
The SJ modified silica-gel film made need be kept in 4 DEG C of refrigerators.
Adopt brilliant core PersonalArrayerTM16 people's point sample instrument to prepare polypeptide microarrays on modified silica-gel, process is:
1) pre-treatment
By SJ modified silica-gel thin slice (15 × 15mm
2) be immersed in activation solution, take out with deionized water drip washing 3 times after 30min, dry up with nitrogen, be used for point sample at once.
2) point sample
Sampling liquid is diluted and gets well and transfer in the corresponding micropore of 384 orifice plate, 384 orifice plates of band sample are placed on point sample instrument base station, pretreated modified silica-gel thin slice are placed on the base station of point sample instrument simultaneously, carry out point sample at once.Point sample envrionment conditions is room temperature (25 DEG C), and humidity set is 50%.On the polypeptide microarrays made, the point sample amount of each point is about 0.6nL, and sampling point radius is 200 μm.
3) chemistry is fixing
The polypeptide microarrays just made will be placed on fixing at least 6h in climatic chamber (26 DEG C, 60% humidity).Chemistry fixation procedure is as follows:
First by point sample instrument by include catch peptide molecule damping fluid point on modified silica-gel film, then damping fluid starts evaporation, catch peptide molecule and the surface intimate contact interacting of SJ modified silica-gel, by Chemical bond, the high molecular end-COOH of ploy (OEGMA) on the modified silica-gel surface and-NH of peptide molecule
2formed and stablize covalent linkage, and then chemically active peptide molecule will be had to be fixed on SJ modified silica-gel surface.
4) assemble
The polypeptide microarrays of fixing 6h must assemble in two days.First by gum, SJ modified silica-gel thin slice is attached on special reaction column, covers reaction cavity.A reactor is made up of two reaction columns and a reaction cavity.
5) preserve
The polypeptide microarrays assembled, needs to vacuumize sealing, is kept in the refrigerator of 4 DEG C, for subsequent use.
3. detect by detection means
Checking procedure
1, before starting to detect, concentrated washing lotion is added purified water in the ratio of 1:10 or distilled water dilutes, must washing lotion after dilute, direct use, use liquid-transfering gun that 2mL washing lotion is added to chip surface, soak chip 3 minutes, ensure that chip surface is fully wet out.
2, test serum sample Sample dilution is mixed according to 1:40 dilution.
3, discard the washing lotion of soaking chip, under the state that chip surface is completely moistening, the serum after each serum sample draws 200 μ L dilutions joins in chip reactor.
4, chip reactor is put into chip permanent seat, be put on shaking table, open shaking table, frequency 150 revs/min, incubated at room 30 minutes.
5, the serum sample in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
6, after having cleaned, each chip reactor adds 200 μ L enzyme labelled antibody solution respectively, chip reactor is put into chip permanent seat, is put on shaking table, opens shaking table, frequency 150 revs/min, incubated at room 30 minutes.
7, the enzyme labelled antibody solution in chip reactor is discarded, with 15mL washing lotion cleaning reaction cavity and chip surface 3 times.
8, in advance luminous substrate liquid A and luminous substrate liquid B can be mixed with equal-volume, obtain luminous substrate mixing solutions (using in 45 minutes).To be cleaned complete after, take off reaction cavity, each chip surface adds 15 μ L luminous substrate mixing solutionss respectively, makes luminous substrate mixing solutions can be laid on chip surface uniformly.
9, the chip adding luminescent solution is placed in gel imaging instrument chemiluminescence imaging, and sentence read result.
Healthy normal human serum, thyroid disease patients serum and other disease patient's serum samples are provided by chain hospital, and disease criterion all through Clinical Laboratory confirmation, all obtains the Informed Consent Form of supplier.
Negative control has PBS damping fluid (namely to hatch without test serum in the 3rd step, and hatch by PBS solution, all the other steps are identical) contrast, the contrast of serum dilution, and the contrast of negative serum (referring to the serum of Healthy People and non-thyroid disorders patient).
The spot sample mode of polypeptide microarrays is as shown in Figure 2: wherein, the sample of leg-of-mutton 20 points is human IgG, as locating point; The sample of foursquare 4 points is PB sampling liquid, as blank; The sample of circular point is other thyroid disease autoantigen protein polypeptide, as Testing index (these polypeptide have response to illustrate in detected serum have thyroid disease autoantibody); For polypeptide of the present invention (polypeptide that the aminoacid sequence shown in SEQIDNO:2 is formed), it is thyroid disease autoantigen protein polypeptide to the sample polypeptides of star point, can produce response to Patients With Various Thyroid Disorders serum.
Use this polypeptide microarrays to detect thyroid disease patients serum and negative control according to above-mentioned detecting step, response modes as shown in Figure 3-4: wherein, Fig. 3 shows the detected result of negative control, only has the sample of the point shown in trilateral to have response.Fig. 4 shows the detected result of thyroid disease patients serum, and the sample of trilateral, circle and star point has response.It should be noted that, instrument records signal value from low to high, and corresponding signaling point color is by black-red-Bai gradual change.
With the polypeptide that the aminoacid sequence shown in another polypeptide SEQIDNO:3 of the present invention is formed, point sample has been carried out by above-mentioned pattern, obtain different polypeptide microarrays, these polypeptide microarrays are used to detect thyroid disease patients serum and negative control respectively according to above-mentioned detecting step, result all obtains the response modes identical with the above-mentioned response modes using the above-mentioned polypeptide microarrays comprising sp-1 to obtain, specifically as seen in figs. 5-6: wherein, Fig. 5 shows the detected result of negative control, only has the sample of the point shown in trilateral to have response.Fig. 6 shows the detected result of thyroid disease patients serum, and the sample of trilateral, circle and star point has response.It should be noted that, instrument records signal value from low to high, and corresponding signaling point color is by black-red-Bai gradual change.
The result obtained for any one the detection thyroid disease patients serum used in these 2 polypeptide is analyzed, and namely gets the signal value (S of each luminous point with genepix software
d), and the background (S of whole result figure
b), according to SNR=(S
d-S
b)/S
bobtain the snr value (SNR) of each luminous point, the results are shown in Table 1.
Signal to noise ratio when table 1 uses 2 polypeptide to detect thyroid disease human serum
| Polypeptide | LTP-71 | LTP-72 |
| Signal to noise ratio | 8.9 | 7.2 |
SEQUENCELISTING
<110> Suzhou biomaterial company limited of Yvonne office
<120> can be used for detecting the protein of thyroid disease and the partial peptide of this protein
<130>LTP-71(2)
<160>3
<170>PatentInversion3.5
<210>1
<211>770
<212>PRT
<213> artificial sequence
<400>1
MetArgAlaLeuAlaValLeuSerValThrLeuValMetAlaCysThr
151015
GluAlaPhePheProPheIleSerArgGlyLysGluLeuLeuTrpGly
202530
LysProGluGluSerArgValSerSerValLeuGluGluSerLysArg
354045
LeuValAspThrAlaMetTyrAlaThrMetGlnArgAsnLeuLysLys
505560
ArgGlyIleLeuSerProAlaGlnLeuLeuSerPheSerLysLeuPro
65707580
GluProThrSerGlyValIleAlaArgAlaAlaGluIleMetGluThr
859095
SerIleGlnAlaMetLysArgLysValAsnLeuLysThrGlnGlnSer
100105110
GlnHisProThrAspAlaLeuSerGluAspLeuLeuSerIleIleAla
115120125
AsnMetSerGlyCysLeuProTyrMetLeuProProLysCysProAsn
130135140
ThrCysLeuAlaAsnLysTyrArgProIleThrGlyAlaCysAsnAsn
145150155160
ArgAspHisProArgTrpGlyAlaSerAsnThrAlaLeuAlaArgTrp
165170175
LeuProProValTyrGluAspGlyPheSerGlnProArgGlyTrpAsn
180185190
ProGlyPheLeuTyrAsnGlyPheProLeuProProValArgGluVal
195200205
ThrArgHisValIleGlnValSerAsnGluValValThrAspAspAsp
210215220
ArgTyrSerAspLeuLeuMetAlaTrpGlyGlnTyrIleAspHisAsp
225230235240
IleAlaPheThrProGlnSerThrSerLysAlaAlaPheGlyGlyGly
245250255
AlaAspCysGlnMetThrCysGluAsnGlnAsnProCysPheProIle
260265270
GlnLeuProGluGluAlaArgProAlaAlaGlyThrAlaCysLeuPro
275280285
PheTyrArgSerSerAlaAlaCysGlyThrGlyAspGlnGlyAlaLeu
290295300
PheGlyAsnLeuSerThrAlaAsnProArgGlnGlnMetAsnGlyLeu
305310315320
ThrSerPheLeuAspAlaSerThrValTyrGlySerSerProAlaLeu
325330335
GluArgGlnLeuArgAsnTrpThrSerAlaGluGlyLeuLeuArgVal
340345350
HisAlaArgLeuArgAspSerGlyArgAlaTyrLeuProPheValPro
355360365
ProArgAlaProAlaAlaCysAlaProGluProGlyIleProGlyGlu
370375380
ThrArgGlyProCysPheLeuAlaGlyAspGlyArgAlaSerGluVal
385390395400
ProSerLeuThrAlaLeuHisThrLeuTrpLeuArgGluHisAsnArg
405410415
LeuAlaAlaAlaLeuLysAlaLeuAsnAlaHisTrpSerAlaAspAla
420425430
ValTyrGlnGluAlaArgLysValValGlyAlaLeuHisGlnIleIle
435440445
ThrLeuArgAspTyrIleProArgIleLeuGlyProGluAlaPheGln
450455460
GlnTyrValGlyProTyrGluGlyTyrAspSerThrAlaAsnProThr
465470475480
ValSerAsnValPheSerThrAlaAlaPheArgPheGlyHisAlaThr
485490495
IleHisProLeuValArgArgLeuAspAlaSerPheGlnGluHisPro
500505510
AspLeuProGlyLeuTrpLeuHisGlnAlaPhePheSerProTrpThr
515520525
LeuLeuArgGlyGlyGlyLeuAspProLeuIleArgGlyLeuLeuAla
530535540
ArgProAlaLysLeuGlnValGlnAspGlnLeuMetAsnGluGluLeu
545550555560
ThrGluArgLeuPheValLeuSerAsnSerSerThrLeuAspLeuAla
565570575
SerIleAsnLeuGlnArgGlyArgAspHisGlyLeuProGlyTyrAsn
580585590
GluTrpArgGluPheCysGlyLeuProArgLeuGluThrProAlaAsp
595600605
LeuSerThrAlaIleAlaSerArgSerValAlaAspLysIleLeuAsp
610615620
LeuTyrLysHisProAspAsnIleAspValTrpLeuGlyGlyLeuAla
625630635640
GluAsnPheLeuProArgAlaArgThrGlyProLeuPheAlaCysLeu
645650655
IleGlyLysGlnMetLysAlaLeuArgAspGlyAspTrpPheTrpTrp
660665670
GluAsnSerHisValPheThrAspAlaGlnArgArgGluLeuGluLys
675680685
HisSerLeuSerArgValIleCysAspAsnThrGlyLeuThrArgVal
690695700
ProMetAspAlaPheGlnValGlyLysPheProGluAspPheGluSer
705710715720
CysAspSerIleThrGlyMetAsnLeuGluAlaTrpArgGluThrPhe
725730735
ProGlnAspAspLysCysGlyPheProGluSerValGluAsnGlyAsp
740745750
PheValHisCysGluGluSerGlyArgArgValLeuValTyrSerCys
755760765
ArgHis
770
<210>2
<211>20
<212>PRT
<213> artificial sequence
<400>2
LeuThrArgValProMetAspAlaPheGlnValGlyLysPheProGlu
151015
AspPheGluSer
20
<210>3
<211>20
<212>PRT
<213> artificial sequence
<400>3
ValGlyLysPheProGluAspPheGluSerCysAspSerIleThrGly
151015
MetAsnLeuGlu
20
Claims (9)
1. the protein be made up of the aminoacid sequence shown in SEQIDNO:1, or the following part peptide of this protein:
The polypeptide be made up of the aminoacid sequence shown in SEQIDNO:2;
The polypeptide be made up of the aminoacid sequence shown in SEQIDNO:3.
2. a detection means, it comprises:
Solid carrier, and
Be connected to the described partial peptide of protein according to claim 1 on this solid carrier or this protein.
3. detection means according to claim 2, wherein, described solid carrier is SJ modified silica-gel.
4. detection means according to claim 2, its diagnosis for thyroid disease or somatotype.
5. a detection kit, it comprises the detection means according to any one of claim 2 ~ 4.
6. detection kit according to claim 5, its diagnosis for thyroid disease or somatotype.
7. the purposes of described partial peptide in preparation detection kit of protein according to claim 1 or this protein.
8. purposes according to claim 7, wherein, described detection kit is used for diagnosis or the somatotype of thyroid disease.
9. purposes according to claim 7, wherein, described detection kit comprises the detection means according to any one of claim 2 ~ 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410199407.1A CN105087512A (en) | 2014-05-13 | 2014-05-13 | Protein for detecting thyroid diseases and partial peptide of protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410199407.1A CN105087512A (en) | 2014-05-13 | 2014-05-13 | Protein for detecting thyroid diseases and partial peptide of protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105087512A true CN105087512A (en) | 2015-11-25 |
Family
ID=54568847
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410199407.1A Pending CN105087512A (en) | 2014-05-13 | 2014-05-13 | Protein for detecting thyroid diseases and partial peptide of protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105087512A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112312921A (en) * | 2018-07-02 | 2021-02-02 | 美国西门子医学诊断股份有限公司 | Novel immunoassay for thyroid peroxidase autoantibodies |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993003146A1 (en) * | 1991-07-30 | 1993-02-18 | Basil Rapoport | Disease associated b-cell epitopes of tpo and use thereof |
| WO1993005072A1 (en) * | 1991-08-28 | 1993-03-18 | Basil Rapoport | Disease associated human autoantibodies specific for human thyroid peroxidase |
| FR2848566A1 (en) * | 2002-12-11 | 2004-06-18 | Centre Nat Rech Scient | New peptides recognized by autoantibodies against human thyroperoxidase, useful for diagnosis and treatment of autoimmune diseases and thyoid cancer, also antibodies specific for them |
| CN102532322A (en) * | 2011-12-29 | 2012-07-04 | 浙江大学 | Preparation and application of rabbit anti-swine TPO polyclonal antibody |
| CN103665138A (en) * | 2012-09-21 | 2014-03-26 | 苏州偲聚生物材料有限公司 | Polypeptide, detecting apparatus including same and detecting kit |
-
2014
- 2014-05-13 CN CN201410199407.1A patent/CN105087512A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993003146A1 (en) * | 1991-07-30 | 1993-02-18 | Basil Rapoport | Disease associated b-cell epitopes of tpo and use thereof |
| WO1993005072A1 (en) * | 1991-08-28 | 1993-03-18 | Basil Rapoport | Disease associated human autoantibodies specific for human thyroid peroxidase |
| FR2848566A1 (en) * | 2002-12-11 | 2004-06-18 | Centre Nat Rech Scient | New peptides recognized by autoantibodies against human thyroperoxidase, useful for diagnosis and treatment of autoimmune diseases and thyoid cancer, also antibodies specific for them |
| CN102532322A (en) * | 2011-12-29 | 2012-07-04 | 浙江大学 | Preparation and application of rabbit anti-swine TPO polyclonal antibody |
| CN103665138A (en) * | 2012-09-21 | 2014-03-26 | 苏州偲聚生物材料有限公司 | Polypeptide, detecting apparatus including same and detecting kit |
Non-Patent Citations (3)
| Title |
|---|
| GUO J.等: "Search for the autoantibody immunodominant region on thyroid peroxidase: epitopic footprinting with a human monoclonal autoantibody locates a facet on the native antigen containing a highly conformational epitope", 《THE JOURNAL OF IMMUNOLOGY》 * |
| P. HOBBY等: "Identification of an immunodominant region recognized by human autoantibodies in a three-dimensional model of thyroid peroxidase", 《ENDOCRINOLOGY》 * |
| ***等: "抗甲状腺球蛋白抗体与抗甲状腺过氧化物酶抗体在甲状腺功能异常诊断中的应用", 《临床研究》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112312921A (en) * | 2018-07-02 | 2021-02-02 | 美国西门子医学诊断股份有限公司 | Novel immunoassay for thyroid peroxidase autoantibodies |
| US11327074B2 (en) | 2018-07-02 | 2022-05-10 | Siemens Healthcare Diagnostics Inc. | Thyroid peroxidase autoantibody immunoassay |
| US11835518B2 (en) | 2018-07-02 | 2023-12-05 | Siemens Healthcare Diagnostics Inc. | Thyroid peroxidase autoantibody immunoassay |
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