WO2014044184A1 - Protein for detecting type 1 diabetes mellitus and partial peptide thereof - Google Patents

Abstract

The present invention relates to a protein useful for the diagnosis of type 1 diabetes mellitus, a polypeptide, a detection device containing the protein or polypeptide, and a diagnostic kit containing the detecting device.

Description

 A protein that can be used to detect type 1 diabetes and a partial peptide of the protein

 This application claims the priority of the following Chinese patent applications, the entire contents of which are respectively aided? I and the description of the book: CN201210355395.8, CN2013101 16453.6, CN2013101 17129.6, CN2013101 15787.1 and 2013101 16801.X.

Technical field

 The present invention relates generally to proteins, partial peptides of the proteins, detection devices comprising the same, and detection kits comprising the same, particularly proteins useful for diagnosis or typing of type 1 diabetes, partial peptides of the protein, including the same A detection device for a protein or a partial peptide of the protein and a detection kit comprising the protein, a partial peptide of the protein or the detection device. Background technique

 Diabetes Mellitus, D) is a group of clinical syndromes characterized by disorders of glucose metabolism caused by genetic and environmental factors. Insulin deficiency and insulin dysfunction cause metabolic disorders of carbohydrates, fats, proteins, water and dielectrics alone or simultaneously, and are characterized by chronic hyperglycemia. Typical cases can appear polyuria, polydipsia, polyphagia, weight loss, etc., that is, "three more one less" disease.

 In the European Association for the Study of Diabetes (European Association for the Study of Diabetes

 At the 1st annual meeting of EASD), the International Diabetes Federation (IDF) released the latest data: In 2001, the number of patients with diabetes in the world reached 366 million, an increase of nearly 30% compared with 285 million in 2010. Every year, 4.6 million people die of diabetes, and medical expenses for diabetes are as high as $465 billion. Professor ID C. President Jean Claude Manbaya said: "In 201, one person died of diabetes every 7 seconds, and the alarm bell is ringing." According to Voice of China, in November 2011, the number of confirmed diabetes patients in China reached 92.4 million, ranking first in the world. Among adults over 20 years old in China, the incidence of diabetes is as high as 9.7%.

 Diabetes can be classified into type 1 diabetes, type 2 diabetes, gestational diabetes, and special types of diabetes according to its etiology.

At present, the hospital is mainly based on clinical manifestations (such as type 1 diabetes, which occurs mostly in adolescence, and there are Types of "three more and one less" symptoms, depending on insulin, etc.) distinguish between type 1 and type 2 diabetes, and for patients with unclear type of diabetes such as latent autoimmune diabetes in adults (LAD A), And diagnosis of type 1 diabetes requires the detection of type 1 diabetes autoantibodies to aid diagnosis. Summary of the invention

 SUMMARY OF THE INVENTION An object of the present invention is to provide a protein, a partial peptide of the protein, a detection device comprising the protein or a partial peptide thereof, and a detection kit comprising the detection device, which are useful for diagnosis or typing of type 1 diabetes. It is an object of the present invention to provide a use of the protein or a partial peptide thereof for the preparation of a test kit useful for diagnosis or typing of type 1 diabetes.

 That is, the present invention includes the following technical solutions:

 A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or a partial peptide of the protein:

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 2

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 3

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 4

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 5

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 6

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 7

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 8

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO 9

 a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10;

 a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11;

 A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12.

 2. A detection device comprising:

 Solid carrier, and

 A protein as described in item 1 attached to the solid support, or the partial peptide of the protein.

3. The detecting device according to item 2, wherein the solid carrier is SJ modified silica gel.

4. The detection device according to item 2 or 3, which is for diagnosis or typing of type 1 diabetes.

A detection kit comprising the detection device according to any one of items 2 to 4. 6. The test kit of item 5 for use in the diagnosis or typing of type 1 diabetes.

 7. The use of the protein of Item 1 or the partial peptide of the protein in the preparation of a test kit.

 8. The use according to item 7, wherein the test kit is for diagnosis or typing of type 1 diabetes.

 9. The use according to any one of items 7 to 8, wherein the detection kit comprises the detection device according to any one of items 2 to 4.

 The application of the present invention to the diagnosis or typing of type 1 diabetes can achieve satisfactory results. DRAWINGS

 Fig. 1 is a schematic view showing the process of producing SJ modified silica gel (iPDMS film). Figure 2 illustrates a schematic of the peptide microarray spotting mode.

 Figures 3 to 4 show photographs of the results of testing for different sera. DETAILED DESCRIPTION OF THE INVENTION

 Protein of the invention and partial peptide thereof

In one aspect, the present invention provides a protein (hereinafter also referred to as a protein of the present invention) useful for diagnosis or typing of type 1 diabetes. This protein consists of the amino acid sequence shown in SEQ ID NO: 1, which is a type 1 diabetes autoantigen PDX-1 protein (Shi-Wu Li, Vijay Koya, YiLi, William Donelan, Peng Lin, Westley H Reeves and Li- Jim Yang, Pancreatic duodenal homeobox 1 protein is a novel b-cell-specific autoantigen for type I diabetes, Laboratory Investigation (2010) 90, 31 - 39 ) truncated protein, which can be associated with type 1 diabetes in patients with type 1 diabetes Autoantibodies specifically bind. Therefore, this protein is useful as a tool for diagnosis or typing of type 1 diabetes. The proteins of the invention can be prepared by methods well known in the art. For example, a nucleotide sequence encoding the same can be inferred based on the above amino acid sequence, and the nucleotide sequence can be chemically synthesized, and the chemically synthesized nucleotide sequence can be inserted into a known expression vector, and the expression vector can be used to transform the host. Cells, cultured and expressed by the host cell, and then isolated and purified by conventional methods to obtain the protein of the present invention. In another aspect, the present invention provides a polypeptide useful for diagnosis or typing of type 1 diabetes. Gold ^^ 翱

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L£L£80/£10Z 13/I3d In the case of a polypeptide, it is carried out by synthesizing or semi-synthesizing the polypeptide using a peptide synthesizer. Examples of the chemical synthesis method include a peptide solid phase synthesis method and the like. The peptide thus synthesized can be purified by a conventional means such as ion exchange chromatography, reversed phase high performance liquid chromatography, affinity chromatography or the like. Such peptide solid phase synthesis methods and subsequent peptide purification are well known in the art.

 Further, in the case of producing the polypeptide of the present invention by an enzymatic reaction, for example, the method described in International Publication No. WO2004/011653 can be employed. That is, it can be produced by: an amino acid or a dipeptide obtained by esterifying or amidating a carboxyl group of one amino acid or a dipeptide, and an amino acid having a free amino acid (for example, a carboxyl-protected amino acid) in a peptide synthetase. The dipeptide or tripeptide formed in the presence of the reaction. Examples of the peptide synthetase include a culture of a microorganism having the ability to produce a peptide, a microbial cell isolated from the culture, a bacterial cell treated product of the microorganism, or a peptide synthetase derived from the microorganism.

 Further, in addition to the above-described enzymatic method or chemical synthesis method, for example, a genetic engineering method can be employed to produce the polypeptide of the present invention.

 The amino acid sequence of the polypeptide of the present invention can be analyzed and confirmed by a conventional method, and can be analyzed and confirmed by, for example, mass spectrometry, chromatography or the like. Detection device of the invention

 In another aspect, the invention also provides a detection device (detection device of the invention) comprising a solid support, and a protein of the invention or a polypeptide of the invention linked to the solid support. The detection device is useful for the diagnosis or typing of type 1 diabetes.

 In the present invention, the carrier is not particularly limited as long as it is a carrier which is a solid or insoluble material (e.g., a material which can be separated from the reaction mixture by filtration, precipitation, magnetic separation, etc.).

The material constituting the body carriers include, but are not limited to: silicone (polydimethylsiloxane, the PDMS), cellulose, Teflon ^TM Yu nitro cellulose, agarose, dextran, chitosan, polystyrene , polyacrylamide, polyester, polyacrylate, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, silica, glass, fiberglass, gold, platinum, silver, copper, iron, stainless steel , ferrite, silicon wafer, polyethylene, polyethyleneimine, polylactic acid, resin, polysaccharide, protein (albumin, etc.), a combination thereof, and the like.

The shape of the solid carrier includes, but is not limited to, beads, magnetic beads, membranes, microtubes, membranes, Plates, microplates, carbon nanotubes, sensor chips, etc. As is well known in the art, pits, grooves, filter bottoms, etc. can be placed on a flat solid support such as a film or sheet.

 In the present invention, the magnetic beads may have a sphere diameter ranging from about 25 nm to about 1 mm. In a preferred embodiment, the magnetic beads have a diameter ranging from about 50 ηηη to about ΙΟμηϊ. The size of the beads can be selected according to the specific application.

 In the present invention, beads made of highly crosslinked spherical agarose such as Sejpharose have a diameter ranging from about 24 μί to about 165 μm. Preferably, the highly crosslinked spherical agarose beads have a diameter ranging from about 24 μηη to about 44 μΐϊΐ. The size of the highly crosslinked spherical blush beads can be selected according to the specific application.

 Examples of solid carriers having a hydrophobic surface include those available from Polysciences, W arringtoii,

PA or Spheroteeh, Libemlle, [L purchase of products such as polyphenylene film rubber beads.

The silicon dioxide (S) ₂ ) - treated or disilicon (Si0 ₂ ) based solid carrier inverted includes ultramagnetic dimethoxy beads available from Polysciences, Warrington, PA, etc., which can be used to capture nucleic acids. (eg DNA) „ or; M-280, etc., which can be purchased from Dynal Biotech.

 Magnetic beads with a hydrophilic surface can be used to capture bacterial cells, nucleic acids, and other growth during the proliferative phase.

'Minute. Examples of the magnetic beads include, for example, beads sold by Polysciences, Warrington, PA (name: Biomag (registered two standard) L base), or Bangs Laboratory, inc.. Fishers, IN under the name MC02N/2928 bead. Alternatively, you can use M-270, etc., sold by Dynal Biotech.

In a preferred embodiment of the present invention, the solid carrier is SJ modified silica gel, which is a silicone rubber microarray solid support material developed by Suzhou Yuju Biomaterial Co., Ltd. (iPDMS film, see Chinese patent publication) CN101265329A). This material is based on PDMS commonly used in biological research, in which a specific initiator component is added (so that the material can be surface functionalized by surface initiated polymerization (SIP)), followed by polyethylene glycol sulfhydryl Acrylate (poly(oligo(ethylene glycol) methacrylate), pOEGMA) surface modification obtained. SJ modified silica gel has excellent anti-protein non-specific adsorption (NPA) ability, which can control the adsorption of non-specific proteins in complex protein immunoassay to near the "absolute 0" level (close to or below the detection limit of the instrument) ), not only can avoid the trouble of blocking and multiple cleaning, but also can improve the sensitivity of protein microarray by using stronger signal amplification means. Moreover, the nature of its silicone rubber gives the material strong mechanical properties and good operability. Suzhou Jingju has successfully applied SJ modified silica gel to a multi-index combined microarray ELISA kit consisting of 11 tumor markers, which has achieved high-throughput and high-sensitivity detection, which proves that this material is an excellent one. Protein microarray solid support material. At the same time, this material also has the property of adjustable surface properties, which can be adjusted within a certain range by controlling the modification reaction time.

 The attachment of the polypeptide of the present invention to a solid support can be carried out by a method of attaching a polypeptide to a solid support well known to those skilled in the art. For example, for the attachment of a protein/polypeptide to a surface of a modified silica gel, it can be passed through 1-ethyl-3-(3-diamidinopropyl)-carbodiimide [l-ethyl-3-(3- The reaction of dimethyl ami-nopropyl)carbodiimide, EDC] with N-hydroxysuccinimide (NHS) changes the carboxyl (-COOH) group on the polymer chain of the modified silica gel to an activating group. The activating group can react with the amino group (-NH2) carried on the protein/polypeptide to immobilize the protein/polypeptide on the surface of the solid support (see, for example, Hongwei Ma, Yuanzi Wu, Xiaoli Yang, Xing Liu, Jian'an He, Long Fu, Jie Wang, Hongke Xu, Yi Shi and Renqian Zhong, Integrated poly(dimethysiloxane ) with an intrinsic nonfouling property approaching "Absolute " zero background in immunoassays, Anal. Chem., 82, 6338-6342, 2010 ).

The concentration of the polypeptide of the present invention in the spotting solution used for spotting is not particularly limited, and those skilled in the art can select it as usual, preferably lg~100 (^g/mL, more preferably l(^g~50(^) Further, the density of the polypeptide of the present invention distributed on a solid carrier is not particularly limited, and can be selected by a person skilled in the art, preferably from 1 to 100 points/10 mm ² , more preferably from 5 to 50 points/10 mm ^{2 .} .

 The detecting device of the present invention is useful for diagnosis or typing of type 1 diabetes, or it can be used for manufacturing a test kit which can be used for diagnosis or typing of type 1 diabetes. Detection kit of the invention

 In another aspect, the present invention also provides a detection kit (detection kit of the present invention) comprising the detection device of the present invention. The test kit can be used for the diagnosis or typing of type 1 diabetes.

 The detection kit of the present invention must contain the detection device of the present invention. The detection kit of the present invention may further comprise:

1 · With a good serum sample dilution or serum sample dilution solution: Sample dilution, such as sample dilution of Beijing Saichi Biotechnology Co., Ltd. (product number 070021-S2), Zhengzhou Boweijia Biotechnology Co., Ltd. The company's sample color change sample dilution (product number bwj010103) and so on. This type of sample dilution is a PBS solution containing BSA, sucrose and other ingredients, containing preservatives, clear liquid, and can be used directly. The sample diluent is used to dilute the serum, and the serum detected by the kit is diluted by an appropriate multiple, for example, 2 to 200 times, preferably 10 to 100 times.

 The detection kit of the present invention may further comprise:

 2. Concentrated lotion and lotion: After incubating the serum and the enzyme-labeled antibody on the surface of the solid carrier, the unbound antibody and the enzyme-labeled antibody on the surface of the solid carrier are washed away with a washing solution. The concentrated washing solution is, for example, a 1% Tween 20 aqueous solution, which is diluted 2-40 times, preferably 5-20 times when used. The lotion can be configured by diluting the concentrated washing solution (10x) with purified water or distilled water at 1:9. For example, add 450 mL of concentrated washing solution (10Ox) to 450 mL of purified water or distilled water and mix well. The unused lotion is stored at 2~8 °C and can be stored for 3 months.

 The detection kit of the present invention may further comprise:

 3. Enzyme-labeled antibody solution: The type 1 diabetes autoantibody in the serum of type 1 diabetic patients can be bound to the protein or polypeptide of the present invention on a solid carrier (for example, SJ modified silica gel), and the enzyme-labeled antibody can bind to the antibody, and the enzyme The label on the target antibody can react with the luminescent substrate to emit detectable light. The enzyme-labeled antibody may be, for example, horseradish peroxidase-labeled goat anti-human IgG. As the enzyme-labeled antibody solution, horseradish peroxidase-labeled goat anti-human IgG (H+L) produced by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., product number ZB-2304, can be cited. The concentration of the enzyme-labeled antibody in the enzyme-labeled antibody solution is not particularly limited and may be, for example, from 1 ng to 1000 ng/mL.

 The detection kit of the present invention may further comprise:

 4. Luminescent substrate mixed solution: The luminescent substrate mixed solution can be reacted with the horseradish peroxidase labeled on the enzyme-labeled antibody, so that the reaction emitting device can detect the chemical photoluminescent substrate mixed solution including the luminescent substrate liquid A. - a hydrogen peroxide solution, and a luminescent substrate solution B - a luminescent ammonia (luminol) solution. In use, two kinds of luminescent substrate liquids are previously mixed in an equal volume to obtain a luminescent substrate mixed solution (used within 45 minutes). Luminescent ammonia emits light only when it is treated with an oxidizing agent. A mixed aqueous solution of hydrogen peroxide and a hydroxide base is usually used as an activator. Under the catalysis of horseradish peroxidase, the hydrogen peroxide is decomposed into oxygen and water:

2 H ₂ 0 ₂ →0 ₂ + 2 3⁄40

When luminol reacts with hydroxide, it forms a double negative ion, which is oxidized by oxygen decomposed by hydrogen peroxide, and the product is an organic peroxide. The peroxide is unstable and immediately decomposes nitrogen to form an excited state of 3-aminophthalic acid. Energy released from the excited state to the ground state In the form of photons, the wavelength is in the blue portion of visible light. Example of a luminescent substrate mixed solution. HyperSignal® ELISA Femto Maximum from Thermo Seientific

Sensitivity Substrate, Catalog No. 37074.

 The detection kit of the present invention may further comprise:

 5. One or more reaction chambers (for example, Chinese Patent Authorization Announcement CN202054829U). The detection kit of the present invention may further comprise:

 6. Other test molecules (eg, peptides, proteins, nucleic acids, etc.) used to detect diabetes (especially type 1 diabetes).

 The detection kit of the present invention may further comprise:

 7. Instruction manual. Preferably, it is stated that the test kit can be used for diagnosis or typing of type 1 diabetes.

 The reaction device and the reaction kit can be subjected to chemiluminescence imaging, for example, using a gel imager. As the gel imager, for example, a GE LAS4000mini type or a Tianneng 5500 array imager can be used.

Example

 Hereinafter, the present invention will be more specifically described by the examples, but the scope of the invention is not limited. Modifications/modifications of the present invention can be easily made by those skilled in the art from the description of the present specification, and these are included in the technical scope of the present invention. The scope of the invention is defined by the claims.

1. Preparation and confirmation of peptides

 Each of the polypeptides used in the examples was synthesized by Jill Biochemical (Shanghai) Co., Ltd., and the polypeptide was confirmed by mass spectrometry.

2. Preparation of detection device

The detection chip is prepared by using SJ modified silica gel (iPDMS film) as a solid support material, and the polypeptide solution is fixed by spotting. The modified silica gel is an initiator for surface-initiated polymerization with an olefin end added to a conventional polydisiloxane siloxane material, and is fixed to polydidecyl siloxane by thermal crosslinking (silicon hydrogen bonding). In the three-dimensional structure of the alkane, SJ modified silica gel is obtained, and the production process thereof is shown in FIG. 1 . Among them, A and B are two components of polydidecylsiloxane, and poly(dimethylsiloxane) (Sylgard 184) is purchased from Dow Corning, USA, and contains liquid component A ( The composition is a mixture of a metal platinum catalyst and a vinyl disiloxane polymer precursor) and a crosslinking agent B (the composition is a dimercaptosiloxane precursor having an ethyl group and a Si-H group) Two ingredients. C is an initiator with a vinyl group at the end and was purchased from Hangzhou Dongwei Company. The final modified polymer was oligoethylene glycol methacrylate monomer (01igo (ethylene glycol) methacrylate, the following tube was called OEGMA, molecular weight Mw = 526) was purchased from Aldrich. The polydithiosiloxane precursor A and crosslinker B were thoroughly mixed with the vinyl terminated initiator C in a ratio of A:B:C=10:1:0.5. A transparent elastic silicone rubber is prepared by a curing reaction, and then surface-modified by a SIP technique to obtain an SJ-modified silica gel. Experiments have shown that the surface of SJ modified silica gel has a sufficiently high density of initiators fixed by covalent bonds, which can achieve surface polymer modification by surface initiated polymerization (SIP). Poly(OEGMA) (polyethylene glycol methacrylate) was used to obtain a polyethylene glycol (PEG) modified surface to achieve strong anti-protein non-specific adsorption.

 The prepared SJ modified silica film should be stored in a 4 ° C refrigerator.

 The peptide microarray was prepared on modified silica gel using a Crystal Core® PersonalArrayerTM 16 Personal Spotter. The process was:

 1) Pretreatment

The SJ modified silica gel sheet (15 x 15 mm ² ) was immersed in the activation solution, and after 30 minutes, it was taken out and rinsed with deionized water three times, and dried with nitrogen, and immediately used for spotting.

 2) Dotting

 Dilute the spotting solution and transfer it to the corresponding microwell of the 384-well plate. Place the 384-well plate with the sample on the abutment of the spotter and place the pre-treated modified silica gel on the base of the spotter. On the stage, I will do the same immediately. The ambient conditions were room temperature (25 ° C) and humidity set to 50%. The spotted amount of each dot on the prepared polypeptide microarray was about 0.6 nL, and the spot radius was 200 μm.

 3) Chemical fixation

 The prepared peptide microarray should be fixed in a constant temperature and humidity chamber (26 ° C, 60% humidity) for at least 6 h. The chemical fixation process is as follows:

First, the buffer containing the capture polypeptide molecule is spotted on the modified silica gel membrane by the spotter, and then the buffer begins to evaporate, and the capture polypeptide molecules are in intimate contact with the surface of the SJ modified silica gel and interact with each other. By chemical bonding, the terminal-COOH of the purified (OEGMA) polymer on the surface of the modified silica gel forms a stable covalent bond with the -NH ₂ of the polypeptide molecule, thereby immobilizing the chemically active polypeptide molecule on the surface of the SJ modified silica gel. .

 4) Assembly

 The peptide microarray fixed for 6 h must be assembled within two days. First, the SJ modified silicone rubber sheet is pasted on a special reaction column by a backing, and the reaction chamber is covered. One reactor consists of two reaction columns and one reaction chamber.

 5) Save

 The assembled peptide microarray needs to be vacuum sealed and stored in a refrigerator at 4 ° C for use.

3. Detecting with a detecting device

 Inspection procedure

 1. Before starting the test, add the concentrated washing solution to the purified water or distilled water for dilution in the ratio of 1:10. After the dilution is completed, the washing liquid is used. Directly use, add 2 mL of the washing solution to the surface of the chip with a pipetting gun, soak the chip 3 Minutes to ensure that the surface of the chip is completely wetted.

 2. Dilute the serum sample to be tested with a dilution of 1:40.

 3. Discard the washing solution of the soaking chip. After the surface of the chip is completely wet, 200 μl of diluted serum is taken from each serum sample and added to the chip reactor.

 4. Place the chip reactor in the chip holder, place it on the shaker, turn on the shaker at a frequency of 150 rpm, and incubate for 30 minutes at room temperature.

 5. Discard the serum sample in the chip reactor and clean the reaction chamber and the surface of the chip 3 times with 15 mL of the wash solution.

 6. After the cleaning is completed, add 200 μM of enzyme-labeled antibody solution to each chip reactor, place the chip reactor in the chip holder, place on the shaker, turn on the shaker, and rotate at 150 rpm for 30 minutes at room temperature.

 7. Discard the enzyme-labeled antibody solution in the chip reactor and wash the reaction chamber and chip surface 3 times with 15 mL of the washing solution.

8. The luminescent substrate liquid A and the luminescent substrate liquid B may be mixed in an equal volume in advance to obtain a luminescent substrate mixed solution (used within 45 minutes). After the cleaning is completed, the reaction chamber is removed, and 15 L of the luminescent substrate mixed solution is added to each chip surface to uniformly spread the luminescent substrate mixed solution. On the surface of the chip.

 9. The chip with the luminescent liquid is placed in a gel imager for chemiluminescence imaging, and the results are interpreted.

 Serum samples from healthy normal human serum, serum of patients with type 1 diabetes, and other disease patients were provided by the cooperative hospital. The disease standards were confirmed by clinical tests, and the informed consent of the provider was obtained.

 Negative controls were PBS buffer (ie, incubated with the serum to be tested in step 3, but incubated with PBS, the rest of the steps were the same), serum dilution control, and negative serum (referring to healthy people and non-type 1 diabetes) Control of the patient's serum).

 The spotting pattern of the polypeptide microarray is shown in Figure 2: wherein the sample of the 20 points of the triangle is human IgG as the anchor point; the sample of the 4 points of the square is the PB spotting solution, as a blank control; The sample is another type 1 diabetes autoantigen protein polypeptide, which is used as a detection index (these peptides have responses indicating that the detected serum has type 1 diabetes autoantibodies); the star point sample polypeptide is the polypeptide sp-Ι of the present invention, It is a type 1 diabetes autoantigen protein peptide that responds to serum in patients with type 1 diabetes.

 The polypeptide microarray was used to detect the serum and negative control of type 1 diabetic patients according to the above detection steps. The response patterns are shown in Figures 3 to 4: wherein, Figure 3 shows the results of the negative control, only the points indicated by the triangles. The sample is responsive. Figure 4 shows the results of serum testing for patients with type 1 diabetes, with samples from triangles, circles, and star points responding. It should be noted that the signal value measured by the instrument is from low to high, and the corresponding signal point color is changed from black to red and white.

 Any of the other polypeptides of the invention (sp-6, sp-8, sp-14, sp-15, sp-1, l-1-2, sp-1-3, sp-1) 4, sp-1-5 and sp-1-6) instead of the above-mentioned polypeptide sp-1, were spotted according to the above pattern, and different polypeptide microarrays were obtained, and the polypeptide microarrays were used according to the above detection steps for type 1 respectively. The serum of the diabetic patient and the negative control were tested, and as a result, the same response pattern as that obtained using the above-described sp-1-containing polypeptide microarray was obtained.

For sp-1, sp-6, sp-8, sp-14, sp-15, sp-l - l, sp-1 -2, sp-1 -3 , sp-1 -4, sp-1- 5 and the results of any of the 11 peptides of sp-1-6 for detecting the serum of patients with type 1 diabetes were analyzed, that is, the signal value (S _D ) of each light-emitting point was taken by genepix software, and the whole result was obtained. The background (S _B ) of the graph obtains the signal-to-noise ratio (SNR) of each luminous point according to SNR = (S _D - S _B ) / S _B . The results are shown in Table 1.

Table 1 Signal-to-noise ratio when using 11 peptides to detect human serum of type 1 diabetes

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Claims

 Claim

 A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, or a partial peptide of the protein:

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2;

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3;

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4;

 a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 5;

 a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6;

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7;

 a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 8;

 a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9;

 a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 10;

 a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11;

 A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12.

 2. A detection device comprising:

 Solid carrier, and

 The protein of claim 1, or the partial peptide of the protein, attached to the solid support.

 The detecting device according to claim 2, wherein the solid carrier is an SJ-modified silicone gel.

 4. A detection device according to claim 2 for use in the diagnosis or typing of type 1 diabetes.

A detection kit comprising the detection device according to any one of claims 2 to 4.

6. A test kit according to claim 5 for use in the diagnosis or typing of type 1 diabetes.

The use of the protein of claim 1 or the partial peptide of the protein in the preparation of a test kit

 8. Use according to claim 7, wherein the test kit is for the diagnosis or typing of type 1 diabetes.

 The use according to claim 7, wherein the detection kit comprises the detection device according to any one of claims 2-4