CN104098682B - Polypeptide, the detection device comprising the polypeptide and detection kit - Google Patents

Polypeptide, the detection device comprising the polypeptide and detection kit Download PDF

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Publication number
CN104098682B
CN104098682B CN201310116801.XA CN201310116801A CN104098682B CN 104098682 B CN104098682 B CN 104098682B CN 201310116801 A CN201310116801 A CN 201310116801A CN 104098682 B CN104098682 B CN 104098682B
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polypeptide
diabetes
present
detection
detection device
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CN104098682A (en
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马雄明
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SUZHOU SIJU BIOMATERIALS CO Ltd
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SUZHOU SIJU BIOMATERIALS CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

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  • Chemical & Material Sciences (AREA)
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  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Rehabilitation Therapy (AREA)
  • Diabetes (AREA)
  • Rheumatology (AREA)
  • Toxicology (AREA)
  • Inorganic Chemistry (AREA)
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  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Detection device and detection kit the present invention relates to polypeptide, comprising the polypeptide.The polypeptide of the present invention is by SEQ ID NO：Amino acid sequence shown in 1 is formed.Polypeptide, the detection device comprising the polypeptide and the detection kit of the present invention is useful in the diagnosis of diabetes.

Description

Polypeptide, the detection device comprising the polypeptide and detection kit
Technical field
Detection device and detection kit the invention mainly relates to polypeptide, comprising the polypeptide, belong to biological technical field.
Background technology
Diabetes (Diabetes Mellitus, DM) be by h and E factor it is common caused by one group with glycometabolism Disorder is the clinical syndrome mainly showed.Insulin deficit and insulin action obstacle individually or simultaneously cause carbohydrate, fat, The metabolic disorder of protein, water and dielectric etc., clinic is using chronic hyperglycemia as principal character.Model case may occur in which diuresis, More drinks, eat more, become thin etc. and show, be i.e. " three-many-one-little " symptom.
At European diabetology meeting (European Association forthe Study of Diabetes, EASD) In meeting in 2011, IDF (International Diabetes Federation, IDF) issue latest data Display：Whole world diabetes number of patients in 2011 is up to 3.66 hundred million, 2.85 hundred million increases nearly 30% compared with 2010.Have every year 4600000 people die from diabetes, and the medical expense for diabetes is up to 465,000,000,000 dollars.Chairman IDF Jean Professor ClaudeManbaya says：" in 2011, just thering is 1 people to be beaten because of Diabetes Death, alarm bell every seven seconds ".According to China Report sound in November, 2011, diabetic's number that China has made a definite diagnosis is up to 92,400,000 people, is the first in the world.The whole nation 20 years old In adult above, diabetes morbidity is up to 9.7%.
Diabetes can be divided into type 1 diabetes, diabetes B, gestational diabetes mellitus and specific type sugar by the classification of its teiology Urine disease.
(such as type 1 diabetes are mainly in adolescence to the clinical manifestation of Hospitals at Present Main Basiss, and have typically " more than three One is few " symptom, dependent on insulin etc.) and 1 type and diabetes B are distinguished, it is as slow in being grown up to the indefinite diabetic of parting Hair style autoimmune diabetes (latent autoimmune diabetes in adults, LADA) and make a definite diagnosis 1 type glycosuria It is sick then need detect type 1 diabetes autoantibody carry out auxiliary diagnosis.
The content of the invention
It is an object of the invention to provide a kind of useful polypeptide of diagnosis to diabetes, particularly type 1 diabetes, coding The nucleic acid of the polypeptide, the expression vector comprising the nucleic acid, the host cell for having imported the expression vector, resist the polypeptide antibody, Detection device, the detection kit comprising the polypeptide or the detection device and the polypeptide comprising the polypeptide are detecting 1 type sugar The purposes of purposes, the polypeptide in the kit for being used for detecting type 1 diabetes or detection device is prepared in urine disease.
That is, the present invention includes following technical proposals：
1. a kind of polypeptide, its amino acid sequence such as SEQ ID NO：Shown in 1, i.e.,： GGTAVGGGGVAEPEQDCAVTSGEELLALPP。
2. encode the nucleic acid of the polypeptide described in claim 1.
3. include the expression vector of the nucleic acid described in claim 2.
4. the host cell of the expression vector described in claim 3 is imported.
5. one kind detection device, it includes：
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on the solid carrier.
6. detection device according to claim 5, wherein, the solid carrier is SJ modified silica-gels.
7. a kind of detection kit, it includes the polypeptide described in claim 1 or the detection described in claim 5 or 6 Device.
8. the antibody of the polypeptide described in anti-claim 1.
9. use of the polypeptide in the kit for being used for detecting type 1 diabetes or detection device is prepared described in claim 1 On the way.
The polypeptide of the present invention is applied to the diagnosis of diabetes (particularly type 1 diabetes), can be obtained gratifying Effect.
Brief description of the drawings
Polypeptide is fixed on the schematic diagram on SJ modified silica-gels surface by Fig. 1 by chemical covalent.
The HPLC that Fig. 2 is confirmed to the polypeptide of the invention of chemical synthesis characterizes collection of illustrative plates.
The MS that Fig. 3 is confirmed to the polypeptide of the invention of chemical synthesis characterizes collection of illustrative plates.
The schematic diagram that Fig. 4 illustrates to the manufacturing process of SJ modified silica-gels (iPDMS films).
The figure that Fig. 5 illustrates to polypeptide microarrays chemistry fixation procedure.
Fig. 6 illustrates the schematic diagram of polypeptide microarrays spot sample mode.
Fig. 7~8 show the photo of the result detected to different serum.
The embodiment of invention
The polypeptide of the present invention
The polypeptide of the present invention is 30 peptides.30 peptides of the present invention are by SEQ ID NO：Amino acid sequence shown in 1 is formed, i.e.,： GGTAVGGGGVAEPEQDCAVTSGEELLALPP.As shown in the Examples, its serum to type 1 diabetes patient is positive instead Should, and it is negative to healthy normal person or non-type 1 diabetes patients serum.Therefore, 30 peptide the examining as type 1 diabetes Disconnected instrument is useful.
The polypeptide of the present invention can use commercially available product in the case of commercially available, further, it is also possible to suitably be changed using (1) The known method such as synthetic method or (2) enzyme reaction synthetic method is learned to obtain, wherein chemical synthesis is more easy.Closed in chemistry Into in the case of the polypeptide of the present invention, carried out by using peptide synthesizer synthesis or the semi-synthetic polypeptide.As chemical synthesis Method, it can include such as peptide solid-phase synthesis.The peptide so synthesized can use conventional meanses such as ion exchange color Spectrum, reverse phase high performance liquid chromatogram, affinity chromatography etc. are purified.Such peptide solid phase synthesis process with and subsequent peptide purification all It is well-known in the art.
In addition, in the case where producing the polypeptide of the present invention by enzyme reaction, such as International Publication pamphlet can be used Method described in No. WO2004/011653.I.e., it is possible to so produce：By the amino acid of a side or the carboxyl terminal quilt of dipeptides Amino acid or dipeptides obtained from esterification or amidatioon, amino acid (such as the carboxy protective for being in amino acid free state Amino acid) reacted in the presence of peptide synthetase, the dipeptides or tripeptides of generation.As peptide synthetase, can include：Tool There are the culture, the microbial cells separated by the culture or the thalline of the microorganism of the microorganism of the ability of generation peptide Manage thing or the microbe-derived peptide synthetase.
Moreover, in addition to above-mentioned enzyme method, chemical synthesis process, in some cases, polypeptide of the invention is also possible to It is naturally occurring (but not being separated).In the case of naturally occurring, it can also be separated.
Nucleic acid, the expression vector host cell of the present invention, and the antibody of anti-polypeptide of the invention
The invention further relates to nucleic acid (nucleic acid of the invention), expression vector (this hair comprising the nucleic acid for encoding the polypeptide Bright expression vector), imported the host cell (host cell of the invention) of the expression vector, they may be preferably used for producing The polypeptide of the present invention.The present invention nucleic acid, expression vector, host cell can using well known to a person skilled in the art method come Prepare.The invention further relates to the antibody of anti-polypeptide of the invention, and it can be used for the polypeptide of the detection present invention.The antibody of the present invention can To be prepared using well known to a person skilled in the art method.
The detection device of the present invention
The invention further relates to one kind detection device (detection device of the invention), it includes solid carrier and is connected to Polypeptide of the invention on the solid carrier.
In the present invention, solid carrier is not particularly limited, as long as solid or insoluble material, (being, for example, can With by filtering, precipitating, the material that Magnetic Isolation etc. separates from reactant mixture) carrier.
The material for forming solid carrier includes but is not limited to：Silica gel (dimethyl silicone polymer, PDMS), cellulose, Teflon It is grandTM, NC Nitroncellulose, agarose, glucan, chitosan, polystyrene, polyacrylamide, polyester, makrolon, polyamide, Polypropylene, nylon, polyvinylidene fluoride, latex, silica, glass, glass fibre, gold, platinum, silver, copper, iron, stainless steel, iron Oxysome, silicon wafer, polyethylene, polyethyleneimine, PLA, resin, polysaccharide, albumen (albumin etc.), carbon or their group Close etc..
The shape of solid carrier includes 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, can be set on the flat solid carrier such as film or plate Put pit, groove, filter membrane bottom etc..
In the present invention, magnetic bead can have the sphere diameter of about 25nm~about 1mm scopes.In preferred embodiment In, magnetic bead has the diameter of the μ m of about 50nm~about 10.The size of magnetic bead can be selected according to specific purposes.
In the present invention, the pearl made of the high crosslinked spherical agarose such as Sepharose has about 24 μm~about 165 μm The diameter of scope.Preferably, high crosslinked spherical sepharose 4B has the diameter of about 24 μm~about 44 μ ms.High crosslinked spherical fine jade The size of lipolysaccharide pearl can be selected according to specific purposes.
The example of solid carrier with hydrophobic surface include can from Polysciences, Warrington, PA or The polystyrene latex beads such as the product of Spherotech, Liberville, IL purchase.
Silica (SiO2)-processing or silica (SiO2) include can be from for the example of solid carrier of base Extraordinary magnetic silica pearl etc. of Polysciences, Warrington, PA purchase, its can be used for catching nucleic acid (such as DNA).Or the M-280 etc. that can be bought from Dynal Biotech can also be used.
Magnetic bead with hydrophilic surface can be used for bacterial cell, nucleic acid and the other compositions for catching proliferation period.As The example of the magnetic bead, Polysciences, the pearl (title of Warrington, PA sale can be included：Biomag (registrations Trade mark) carboxyl) or Bangs Laboratory, Inc., Fishers, IN entitled MC02N/2928 pearl.Or M-270 that Dynal Biotech are sold etc. can be used.
In a preferred embodiment of the present invention, the solid carrier is SJ modified silica-gels.Suzhou Yvonne consor thing materials Expect a kind of microarray solid support material (the iPDMS films, referring to Chinese patent of silicone rubber material of Co., Ltd's exploitation CN101265329A).This material be by biological study commonly use PDMS based on, add wherein specific initiator into Part (making the material surface-functionalized modification can be realized by surface initiated polymerization (SIP)), then by polyethylene glycol methyl What acrylate (poly (oligo (ethylene glycol) methacrylate), pOEGMA) surface modification obtained.SJ changes Property silica gel has outstanding anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) ability, Non-specific protein absorption control during can complicated protein immunization be detected is to close to " absolute 0 " level (is near or below The detectable limit of instrument), it can not only exempt closing and the trouble being cleaned multiple times, can also be by using stronger amplification of signal Means improve the sensitivity of protein microarray.And the essence of its silicon rubber impart the stronger mechanical performance of the material and Good operability.SJ modified silica-gels are successfully applied to the more of 11 tumor markerses composition by the poly- companies of Suzhou Yvonne Index joint inspection microarray ELISA kit, realize high flux and highly sensitive detection, it was demonstrated that this material is a kind of outstanding Protein microarray solid support material.Meanwhile this material also has the adjustable characteristic of surface nature, can pass through control The modification reaction time adjusts its surface topography within the specific limits.
The connection of the polypeptide and solid carrier of the present invention can use well known to a person skilled in the art polypeptide and solid to carry The connection method of body is carried out.For example, for connection of the protein/polypeptide with modified silica-gel surface, 1- second can be passed through Base -3- (3- dimethyl aminopropyls)-carbodiimides [1-ethyl-3- (3-dimethyl ami-nopropyl) Carbodiimide, EDC] and n-hydroxysuccinimide (N-hydroxysuccinimide, NHS) reaction by modified silica-gel Carboxyl (- COOH) group on the macromolecular chain of surface is changed to activated group, and the activated group can be with the ammonia of institute's band on protein/polypeptide Protein/polypeptide is fixed on solid carrier surface (referring to Fig. 1) by base (- NH2) reaction so as to realize.
It is not particularly limited for the concentration of polypeptide of the invention in the sampling liquid that is used during point sample, those skilled in the art Can be according to conventional selection, the μ g/mL of preferably 1 μ g~1000 μ g/mL, more preferably 10 μ g~500.In addition, for the more of the present invention The density that peptide is distributed on a solid support is not particularly limited, those skilled in the art can according to conventional selection, preferably 1~ 100 points/10mm2, more preferably 5~50 points/10mm2。
The detection device of the present invention can be used for detecting diabetes (particularly type 1 diabetes) or prepare for detecting The kit of diabetes (particularly type 1 diabetes).
The detection kit of the present invention
The invention further relates to a kind of detection kit (detection kit of the invention), and it includes the polypeptide of the present invention or inspection Survey device.The detection kit is preferred for detecting diabetes (particularly type 1 diabetes), is more preferably used for detecting tuberculosis simultaneously Send out type 1 diabetes.
The detection device of the present invention or the polypeptide of the present invention are the important documents of the detection kit of the present invention.The detection of the present invention Kit can also include：
1. the serum dilution or serum dilution component solution that prepare：Serum dilution, such as there is Beijing to match biology of speeding Sample dilution (production code member 070021-S2), the sample-adding of Zhengzhou Bo Weijia bio tech ltd of Science and Technology Ltd. Change colour Sample dilution (production code member bwj010103) etc..The serum dilution is used for dilute serum, the serum of kit detection Dilute suitable multiple, such as 2~200 times, preferably 10~100 times.
The detection kit of the present invention can also include：
2. concentrate washing lotion：After solid carrier surface is incubated serum and ELIAS secondary antibody, solid carrier table need to be washed with washing lotion Face uncombined antibody and ELIAS secondary antibody.Concentration washing lotion is, for example, the 1% polysorbas20 aqueous solution, need to dilute during use 2~40 times, It is preferred that 5~20 times.
The detection kit of the present invention can also include：
3. ELIAS secondary antibody solution：Type 1 diabetes autoantibody in type 1 diabetes patients serum can be with solid carrier (example Such as SJ modified silica-gels) on polypeptide of the invention combine, secondary antibody can be with antibody binding, and the label on secondary antibody can be with luminous bottom Thing reacts, so as to send detectable light.ELIAS secondary antibody can be the goat anti-human igg of such as horseradish peroxidase-labeled.Make For ELIAS secondary antibody solution, the horseradish peroxidase-labeled that Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge produces can be included Goat anti human IgG (H+L), production code member ZB-2304.There is no special limit to concentration of the ELIAS secondary antibody in ELIAS secondary antibody solution System, can be such as 1ng~1000ng/mL.
The detection kit of the present invention can also include：
4. luminescent solution component solution：Luminescent solution can react with the horseradish peroxidase marked on secondary antibody so that reaction hair Go out the detectable chemical light of instrument.Luminescent solution is mixed by two kinds of solution, is A liquid-hydrogenperoxide steam generator respectively, and B Liquid-luminous ammonia solution.Luminol (luminol) is only crossed with oxidizer treatment and can just lighted.Usually using hydrogen peroxide and one kind The mixed aqueous solution of hydroxide bases is as exciting agent.Under horseradish peroxidase enzyme catalytic, decomposing hydrogen dioxide solution is oxygen and water：
2H2O2→O2+2H2O
Luminol and hydroxide generate a pairs of anion when reacting, and it can be by oxygen oxygen that hydrogen peroxide decomposites Change, product is an organic peroxide.The peroxide is very unstable, decomposites nitrogen immediately, generates the 3- ammonia of excitation state Base phthalic acid.During excitation state converts to ground state, the energy of release exists in the form of photon, and wavelength is located at the indigo plant of visible ray Light part.The example of luminescent solution component solution such as Thermo Seientific companiesELISA Femto Maximum Sensitivity Substrate, article No. 37074.
The detection kit of the present invention can also include：
5. one or more reaction cavity (such as Chinese patent Granted publication CN202054829U).
The detection kit of the present invention can also include：
6. other be used to detecting diabetes (particularly type 1 diabetes) detection molecules (such as polypeptide,
Protein, nucleic acid etc.).
The detection kit of the present invention can also include：
7. operation instructions.
Embodiment
Hereinafter, more specific description is carried out to the present invention by embodiment, but is not the limit to the technology of the present invention scope It is fixed.By the record of this specification, those skilled in the art readily to the present invention can modify/change, and these are included In the technical scope of the present invention.
The preparation and confirmation of 1.30 peptides
30 peptides used in embodiment have SEQ ID NO：Amino acid sequence shown in 1, it is limited by Shanghai gill biochemistry Company synthesizes, and the sign collection of illustrative plates of the polypeptide is shown in Fig. 2~3, can confirm that and synthesized the polypeptide.
2. detect the preparation of device
Detection chip is for solid support material with SJ modified silica-gels (iPDMS films), is fixed thereon by point sample more Peptide solution is prepared.Modified silica-gel is to add with olefin-terminal, surface in traditional polydimethyl siloxane material to draw The initiator of polymerisation is sent out, and is fixed to by heat cross-linking (si-h bond bonding) in the three-dimensional structure of dimethyl silicone polymer, Obtain a kind of new material i.e. SJ modified silica-gels.Its manufacturing process is as shown in Figure 4.
A and B therein be dimethyl silicone polymer two components, dimethyl silicone polymer (Poly (dimethylsiloxane), Sylgard 184) buy from Dow corning (DowCorning) company, include liquid composition A (composition is band for (composition is metallic platinum catalyst and the diformazan siloxanes macromolecule precursor mixture with vinyl) and crosslinking agent B Have the dimethyl siloxane precursor of vinyl and Si -- H) two kinds of compositions.C is initiator of the end with vinyl, is purchased from Hangzhoupro Zhou Dongwei companies.It is oligomeric ethylene glycol methacrylate monomers to carry out the macromolecule in surface modification finally by sip technique (Oligo (ethylene glycol) methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) buy in Aldrich.By polydimethylsiloxane precursor A and crosslinking agent B with the initiator C with vinyl end with A:B:C=10:1: 0.5 ratio is sufficiently mixed.Transparent elastic silicone rubber is made up of curing reaction, surface modification is then carried out by sip technique It can obtain SJ modified silica-gels.Experiment shows that there is highdensity enough, drawing by covalent bond fixation on the surface of SJ modified silica-gels Agent is sent out, it can realize that surface is macromolecule modified by surface initiated polymerization (SIP).Use poly (OEGMA) (poly- second two Alcohol methacrylate) carry out reaction obtain polyethylene glycol (Polyethylene Glycol, PEG) modification surface, realize compared with The ability of strong anti-albumen non-specific adsorption.
The SJ modified silica-gels film made need to be stored in 4 DEG C of refrigerators.
Using brilliant core16 people's point sample instruments prepare polypeptide microarrays, mistake on modified silica-gel Cheng Wei：
1) pre-process
By SJ modified silica-gel thin slices (15 × 15mm2) be immersed in activating solution, taking-up deionized water elution 3 after 30min It is secondary, dried up with nitrogen, be immediately used to point sample.
2) point sample
Sampling liquid is diluted good and is transferred in the corresponding micropore of 384 orifice plates, 384 orifice plates with sample are placed in point sample instrument On base station, while the modified silica-gel thin slice of pretreatment is placed on the base station of point sample instrument, carries out point sample at once.Point sample environmental condition For room temperature (25 DEG C), humidity set 50%.The point sample amount each put on manufactured polypeptide microarrays is about 0.6nL, and sampling point is partly Footpath is 200 μm.
3) chemistry is fixed
The polypeptide microarrays just made will be placed in climatic chamber (26 DEG C, 60% humidity) fixed at least 6h.Chemistry is solid It is as shown in Figure 5 to determine process.
The buffer solution point for capturing peptide molecule will be included on modified silica-gel film by point sample instrument first, then buffered Liquid starts to evaporate, and capture peptide molecule and the surface intimate contact of SJ modified silica-gels simultaneously interacts, and passes through chemical bond, modified silicon High molecular end-the COOH of ploy (OEGMA) on glue surface and peptide molecule-NH2Stable covalent bond is formed, and then will be had Chemically active peptide molecule is fixed on SJ modified silica-gels surface.
5) assemble
Fixed 6h polypeptide microarrays must assemble in two days.SJ modified silica-gel thin slices are attached to by gum first On special reaction column, reaction cavity is covered.One reactor is made up of two reaction columns and a reaction cavity.
6) preserve
The polypeptide microarrays that assemble are stored in 4 DEG C of refrigerator, it is necessary to vacuumize sealing, standby.
3. detected with detection device
Checking procedure
1st, before starting detection, concentrated cleaning solutions are pressed 1:10 ratio adds purified water or distilled water is diluted, and dilutes After the completion of directly use.2mL cleaning fluids are added to chip surface using liquid-transfering gun, immersion chip 3 minutes, ensure chip surface quilt Complete wetting.
2nd, by test serum sample Sample dilution according to 1:40 dilutions mix.
3rd, the cleaning fluid of immersion chip is discarded, in the state of chip surface moistens completely, each serum sample draws 200 Serum after μ L dilutions is added in chip reactor.
4th, chip reactor is put into chip fixed seat, be put on shaking table, open shaking table, 150 revs/min of frequency, room temperature It is incubated 30 minutes.
5th, the serum sample in chip reactor is discarded, reaction cavity and chip surface 3 times are cleaned with 15mL washing lotions.
6th, after the completion of cleaning, each chip reactor is separately added into 200 μ L enzyme labelled antibody solution, and chip reactor is put into Chip fixed seat, is put on shaking table, opens shaking table, 150 revs/min of frequency, is incubated at room temperature 30 minutes.
7th, the enzyme labelled antibody solution in chip reactor is discarded, reaction cavity and chip surface 3 times are cleaned with 15mL washing lotions.
8th, after the completion of cleaning, reaction cavity is removed, each chip surface is separately added into 15 μ L luminous substrate liquid, makes luminescent solution Chip surface can be uniformly laid on.
9th, the chip for adding luminescent solution is placed in chemiluminescence imaging in gel imager, and sentence read result.
Type 1 diabetes patients serum and other disease patient's serum samples are provided by chain hospital.Serum is by related personnel Transported with parcels such as ice cube/dry ice or be handed to laboratory soon.
Negative control have PBS (do not have to test serum i.e. in the 3rd step to be incubated, and be incubated with PBS solution, remaining Step is identical) control, the control of serum dilution, and patients with negative (referring to Healthy People and non-type 1 diabetes patient) serum Control.
The spot sample mode of polypeptide microarrays is as shown in Figure 6.Wherein, the sample of 20 points of triangle is human IgG, as reality The anchor point tested；The sample of 4 points of square is PB sampling liquids, the blank control as experiment；The sample polypeptides of star point As polypeptide SEQ ID NO of the invention：1, it is type 1 diabetes autoantigen protein polypeptide, can be to type 1 diabetes patient's blood It is clear to produce response；The sample of circular dot is other type 1 diabetes autoantigen protein polypeptides, the Testing index as experiment (these polypeptides have in response explanation detection serum and have type 1 diabetes autoantibody).
Experimental result is as shown in Fig. 7~8.Wherein, Fig. 7 shows the testing result of negative control, only shown in triangle The sample of point have response.Fig. 8 shows the testing result of type 1 diabetes patients serum, triangle, star point and/or circle Sample have response.It should be noted that instrument measures signal value from low to high, corresponding signaling point color by it is black-white gradually Become.
<110>Suzhou Siju Biomaterials Co., Ltd.
<120>Polypeptide, the detection device comprising the polypeptide and detection kit
<130> Sp-15
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 30
<212> PRT
<213>Artificial sequence
<220>
<223>Type 1 diabetes antigenic polypeptide
<400> 1
GGTAVGGGGVAEPEQDCAVTSGEELLALPP 30

Claims (8)

1. a kind of polypeptide, its amino acid sequence such as SEQ ID NO：Shown in 1, GGTAVGGGGVAEPEQDCAVTSGEELLALPP.
2. encode the nucleic acid of the polypeptide described in claim 1.
3. include the expression vector of the nucleic acid described in claim 2.
4. the host cell of the expression vector described in claim 3 is imported.
5. one kind detection device, it includes：
Solid carrier, and
It is connected to the polypeptide described in the claim 1 on the solid carrier.
6. detection device according to claim 5, wherein, the solid carrier is SJ modified silica-gels.
7. a kind of detection kit, it includes the polypeptide described in claim 1 or the detector described in claim 5 or 6 Part.
8. purposes of the polypeptide in the kit for being used for detecting type 1 diabetes or detection device is prepared described in claim 1.
CN201310116801.XA 2012-09-21 2013-04-03 Polypeptide, the detection device comprising the polypeptide and detection kit Expired - Fee Related CN104098682B (en)

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PCT/CN2013/083737 WO2014044184A1 (en) 2012-09-21 2013-09-18 Protein for detecting type 1 diabetes mellitus and partial peptide thereof

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102307992A (en) * 2008-11-20 2012-01-04 成血管细胞系统公司 Method for treating or preventing a pancreatic dysfunction

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102307992A (en) * 2008-11-20 2012-01-04 成血管细胞系统公司 Method for treating or preventing a pancreatic dysfunction

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Pancreas duodenum homeobox-1 regulates pancreas development during embryogenesis and islet cell function in adulthood;Hongxiang Hui et al.;《European Journal of Endocrinology》;20021231;第146卷;摘要，第130页表1 *

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