CN103788198B - Polypeptide, the detection means comprising this polypeptide and detection kit - Google Patents

Abstract

The present invention relates to a polypeptide, a detection device and a detection kit comprising the polypeptide. The polypeptide of the present invention consists of SEQ? ID? NO: The amino acid sequence shown in 1 constitutes. The polypeptide, detection device and detection kit comprising the polypeptide of the present invention are useful in the diagnosis of diabetes.

Description

Polypeptide, detection device and detection kit comprising the polypeptide

technical field

The invention mainly relates to a polypeptide, a detection device and a detection kit containing the polypeptide, and belongs to the field of biotechnology.

Background technique

Diabetes Mellitus ( DM ) is a group of clinical syndromes caused by genetic and environmental factors, mainly manifested by glucose metabolism disorders. Insulin deficiency and insulin action disorder alone or simultaneously cause metabolic disorders of carbohydrates, fats, proteins, water and electrolytes, and the main clinical feature is chronic hyperglycemia. In a typical case, symptoms such as polyuria, polydipsia, polyphagia, and weight loss may appear, that is, symptoms of "three excesses and one deficiency".

At the 2011 annual meeting of the European Association for the Study of Diabetes (EASD), the latest data released by the International Diabetes Federation (IDF) showed that the number of people with diabetes in the world reached 366 million in 2011, an increase of nearly 30% from 285 million in 2010. . Diabetes kills 4.6 million people each year and costs $465 billion in healthcare costs. IDF President Professor Jean-Claude Manbaya said: "In 2011, one person died of diabetes every 7 seconds, and the alarm bells are ringing". According to the Voice of China report in November 2011, the number of diagnosed diabetes patients in my country is as high as 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 according to its etiology into type 1 diabetes, type 2 diabetes, gestational diabetes and special type of diabetes.

At present, the hospital mainly distinguishes between type 1 and type 2 diabetes based on clinical manifestations (such as type 1 diabetes mostly occurs in adolescence, and has typical symptoms of "three more and one less" and relies on insulin, etc.). Adult late-onset autoimmune diabetes (latent autoimmunediabetesina adults, LADA) and the diagnosis of type 1 diabetes require the detection of type 1 diabetes autoantibodies to assist in the diagnosis.

Contents of the invention

The object of the present invention is to provide a polypeptide useful for the diagnosis of diabetes, especially type 1 diabetes, a nucleic acid encoding the polypeptide, an expression vector containing the nucleic acid, a host cell into which the expression vector is introduced, an antibody against the polypeptide, Detection device comprising the polypeptide, detection kit comprising the polypeptide or the detection device, use of the polypeptide in detecting type 1 diabetes, and use of the polypeptide in preparing a kit or detection device for detecting type 1 diabetes .

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

1. A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

2. A nucleic acid encoding the polypeptide of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A host cell into which the expression vector according to claim 3 has been introduced.

5. A detection device comprising:

solid carrier, and

The polypeptide of claim 1 linked to the solid support.

6. The detection device according to claim 5, wherein the solid support is SJ modified silica gel.

7. A detection kit comprising the polypeptide according to claim 1, or the detection device according to claim 5 or 6.

8. An antibody raised against the polypeptide of claim 1.

9. Use of the polypeptide according to claim 1 in the preparation of a kit or detection device for detecting type 1 diabetes.

Applying the polypeptide of the present invention to the diagnosis of diabetes (especially type 1 diabetes) can achieve satisfactory results.

Description of drawings

Figure 1 Schematic diagram of chemically covalently immobilizing peptides on the surface of SJ modified silica gel.

Fig. 2 is an HPLC characterization spectrum for confirming the chemically synthesized polypeptide of the present invention.

Fig. 3 is a MS characterization spectrum for confirming the chemically synthesized polypeptide of the present invention.

Figure 4 is a schematic diagram illustrating the fabrication process of SJ-modified silica gel (iPDMS thin film).

Fig. 5 is a diagram illustrating the chemical immobilization process of a polypeptide microarray.

Fig. 6 illustrates a schematic diagram of a spotting mode of a polypeptide microarray.

Figures 7-8 show photographs of the results of testing different sera.

detailed description

Polypeptides of the invention

The polypeptide of the present invention is a 30 peptide. The 30 peptide of the present invention is composed of the amino acid sequence shown in SEQ ID NO: 1, namely: LFRGGKSGELEQEEERLSKEWEDTNRWSKM. As shown in the examples, it is positive to the sera of type 1 diabetic patients and negative to the sera of healthy normal persons or non-type 1 diabetic patients. Therefore, this 30-peptide is useful as a diagnostic tool for type 1 diabetes.

When the polypeptide of the present invention is commercially available, commercially available products can be used. In addition, known methods such as (1) chemical synthesis method or (2) enzymatic reaction synthesis method can also be suitably used to obtain, wherein chemical synthesis is more easy. In the case of chemically synthesizing the polypeptide of the present invention, it is carried out by synthesizing or semi-synthesizing the polypeptide using a peptide synthesizer. Examples of chemical synthesis methods include peptide solid phase synthesis and the like. The peptide thus synthesized can be purified by conventional means such as ion exchange chromatography, reversed-phase high performance liquid chromatography, affinity chromatography and the like. Such solid-phase peptide synthesis methods and subsequent peptide purification are well known in the art.

In addition, when the polypeptide of the present invention is produced by an enzymatic reaction, for example, the method described in International Publication No. WO2004/011653 can be used. That is, it is possible to produce an amino acid or dipeptide obtained by esterifying or amidating the carboxyl terminus of one amino acid or dipeptide, and an amino acid in a free state with the amino acid (for example, a carboxyl-protected amino acid) in the peptide synthetase. In the presence of the reaction, the generated dipeptide or tripeptide. Examples of the peptide synthetase include a culture of a microorganism capable of producing peptides, a microbial cell isolated from the culture, a processed product of the microbial cell, or a peptide synthase derived from the microorganism.

Moreover, in addition to the above-mentioned enzymatic method and chemical synthesis method, in some cases, the polypeptide of the present invention may also be naturally occurring (but not isolated). Where they occur naturally, they can also be isolated.

Nucleic acid of the present invention, expression vector host cell, and antibody against the polypeptide of the present invention

The present invention also relates to a nucleic acid encoding the polypeptide (the nucleic acid of the present invention), an expression vector comprising the nucleic acid (the expression vector of the present invention), and a host cell into which the expression vector has been introduced (the host cell of the present invention), which are preferably used for Polypeptides of the invention are produced. The nucleic acid, expression vector, and host cell of the present invention can be prepared by methods known to those skilled in the art. The invention also relates to antibodies against the polypeptides of the invention, which can be used to detect the antibodies of the invention. Antibodies of the present invention can be prepared by methods known to those skilled in the art.

Detection device of the present invention

The present invention also relates to a detection device (the detection device of the present invention), which includes a solid support and the polypeptide of the present invention linked to the solid support.

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

Materials constituting solid supports include, but are not limited to: silica gel (polydimethylsiloxane, PDMS), cellulose, Teflon ^™ , nitrocellulose, agarose, dextran, chitosan, polystyrene , polyacrylamide, polyester, polycarbonate, polyamide, polypropylene, nylon, polyvinylidene fluoride, latex, silica, glass, fiberglass, gold, platinum, silver, copper, iron, stainless steel, iron Oxygen, silicon wafers, polyethylene, polyethyleneimine, polylactic acid, resins, polysaccharides, proteins (albumin, etc.), carbon, or combinations thereof.

The shapes of solid supports include, but are not limited to: beads, magnetic beads, thin films, microtubes, filter membranes, plates, microplates, carbon nanotubes, sensor chips, etc. As is well known in the art, flat solid supports such as films or plates can be provided with pits, grooves, bottoms of filter membranes, and the like.

In the present invention, the magnetic beads may have a spherical diameter ranging from about 25 nm to about 1 mm. In a preferred embodiment, the magnetic beads have a diameter in the range of about 50 nm to about 10 μm. The size of the magnetic beads can be selected according to the specific application.

In the present invention, beads made of highly cross-linked spherical agarose such as Sepharose have a diameter ranging from about 24 μm to about 165 μm. Preferably, the highly cross-linked spherical agarose beads have a diameter in the range of about 24 μm to about 44 μm. The size of the highly cross-linked spherical agarose beads can be selected according to the specific application.

Examples of solid supports with hydrophobic surfaces include polystyrene latex beads such as those available from Polysciences, Warrington, PA or Spherotech, Liberville, IL.

Examples of silica (SiO ₂ )-treated or silica (SiO ₂ )-based solid supports include supermagnetic silica beads, etc., available from Polysciences, Warrington, PA, which can be used to capture nucleic acids such as DNA ). Alternatively, M-280, which is commercially available from DynalBiotech, etc. can also be used.

Magnetic beads with a hydrophilic surface can be used to capture bacterial cells, nucleic acids, and other components during the proliferating phase. Examples of such magnetic beads include beads sold by Polysciences, Warrington, PA (name: Biomag (registered trademark) carboxyl), or beads with the name MC02N/2928 available from BangsLaboratory, Inc., Fishers, IN. Alternatively, M-270 sold by DynalBiotech or the like can be used.

In a preferred embodiment of the present invention, the solid support is SJ modified silica gel. A silicon rubber microarray solid support material (iPDMS film, see Chinese patent CN101265329A) developed by Suzhou Siju Biomaterials Co., Ltd. This material is based on PDMS, which is commonly used in biological research, and specific initiator components are added to it (so that the material can be modified by surface-initiated polymerization (SIP) to achieve surface functional modification), and then polyethylene glycol methyl Acrylate (poly (oligo (ethyleneglycol) methacrylate), pOEGMA) surface modification obtained. SJ modified silica gel has excellent anti-protein non-specific adsorption (Nonspecific protein adsorption, NPA) ability, which can control non-specific protein adsorption in complex protein immunoassay to close to "absolute 0" level (close to or lower than the detection limit of the instrument), Not only can the trouble of blocking and multiple washings be eliminated, but also the sensitivity of the protein microarray can be improved by using stronger signal amplification means. Moreover, the nature of its silicone rubber endows the material with strong mechanical properties and good operability. Suzhou Siju Company has successfully applied SJ modified silica gel to a multi-index joint detection microarray ELISA kit composed of 11 tumor markers, achieving high-throughput and high-sensitivity detection, proving that this material is an excellent solid support material for protein microarrays. At the same time, this material also has the characteristics of adjustable surface properties, and its surface morphology can be adjusted within a certain range by controlling the modification reaction time.

The connection of the polypeptide of the present invention to a solid support can be carried out using methods known to those skilled in the art for linking a polypeptide to a solid support. For example, for the attachment of proteins/polypeptides to modified silica surfaces, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide [1-ethyl-3-(3- The reaction of dimethylami-nopropyl)carbodiimide, EDC] and N-hydroxysuccinimide (N-hydroxysuccinimide, NHS) changes the carboxyl (-COOH) group on the polymer chain on the surface of the modified silica gel to an activation group, and the activation The group can react with the amino group (-NH2) on the protein/polypeptide to immobilize the protein/polypeptide on the surface of the solid support (see Figure 1).

There is no special limitation on the concentration of the polypeptide of the present invention in the spotting solution used for spotting, and those skilled in the art can choose according to routine, preferably 1 μg-1000 μg/mL, more preferably 10 μg-500 μg/mL. In addition, there is no special limitation on the distribution density of the polypeptide of the present invention on the solid support, which can be selected by those skilled in the art according to routine, preferably 1-100 dots/10mm ² , more preferably 5-50 dots/10mm ² .

The detection device of the present invention can be used to detect diabetes (especially type 1 diabetes), or prepare a kit for detecting diabetes (especially type 1 diabetes).

Detection kit of the present invention

The present invention also relates to a detection kit (the detection kit of the present invention), which includes the polypeptide or the detection device of the present invention. The detection kit is preferably used for detecting diabetes (especially type 1 diabetes).

The detection device of the present invention or the polypeptide of the present invention is a requirement of the detection kit of the present invention. The detection kit of the present invention may also include:

1. Prepared serum diluent or serum diluent component solution: serum diluent, such as sample diluent (product number 070021-S2) from Beijing Saichi Biotechnology Co., Ltd., Zhengzhou Boweijia Biotechnology Co., Ltd. Add color-changing sample diluent (product number bwj010103), etc. The serum diluent is used to dilute the serum, and the serum detected by the kit should be diluted by an appropriate multiple, such as 2-200 times, preferably 10-100 times.

The detection kit of the present invention may also include:

2. Concentrated lotion: After incubating the serum and enzyme-labeled secondary antibody on the surface of the solid carrier, wash off the unbound antibody and enzyme-labeled secondary antibody on the surface of the solid carrier with a lotion. The concentrated lotion is, for example, 1% Tween 20 aqueous solution, which needs to be diluted 2-40 times, preferably 5-20 times when used.

The detection kit of the present invention may also include:

3. Enzyme-labeled secondary antibody solution: Type 1 diabetes autoantibodies in the serum of patients with type 1 diabetes can bind to the polypeptide of the present invention on a solid carrier (such as SJ modified silica gel), the secondary antibody can bind to the antibody, and the secondary antibody on the The label reacts with a luminescent substrate to emit detectable light. The enzyme-labeled secondary antibody can be, for example, horseradish peroxidase-labeled goat anti-human IgG. As the enzyme-labeled secondary 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. There is no special limitation on the concentration of the enzyme-labeled secondary antibody in the enzyme-labeled secondary antibody solution, which may be, for example, 1 ng-1000 ng/mL.

The detection kit of the present invention may also include:

4. Luminescent solution component solution: The luminescent solution can react with the horseradish peroxidase labeled on the secondary antibody, so that the reaction emits chemical light that can be detected by the instrument. The luminous liquid is made by mixing two solutions, which are liquid A—hydrogen peroxide solution, and liquid B—luminescent ammonia solution. Luminol (luminol) glows only when treated with an oxidizing agent. A mixed aqueous solution of hydrogen peroxide and a hydroxide base is usually used as the activator. Under the catalysis of horseradish peroxidase, hydrogen peroxide is decomposed into oxygen and water:

2H ₂ O ₂ →O ₂ +2H ₂ O

When luminol reacts with hydroxide, a double negative ion is generated, which can be oxidized by oxygen decomposed by hydrogen peroxide, and the product is an organic peroxide. The peroxide is very unstable and immediately decomposes nitrogen to generate excited 3-aminophthalic acid. During the transition from the excited state to the ground state, the released energy exists in the form of photons, and the wavelength is in the blue part of visible light. An example of the luminescent liquid component solution is, for example, SuperSignal® ELISA FemtoMaximum Sensitivity Substrate from ThermoSeientific Company, product number 37074.

The detection kit of the present invention may also include:

5. One or more reaction chambers (for example, Chinese patent authorization announcement CN202054829U).

The detection kit of the present invention may also include:

6. Other detection molecules (such as polypeptides, proteins, nucleic acids, etc.) for the detection of diabetes (especially type 1 diabetes).

The detection kit of the present invention may also include:

7. Instructions for use.

Example

Hereinafter, the present invention will be described more specifically through examples, but the technical scope of the present invention is not limited. Through the description of this specification, those skilled in the art can easily modify/change the present invention, and these are included in the technical scope of the present invention.

1.30 Preparation and Confirmation of Peptides

The 30 peptide used in the examples has the amino acid sequence shown in SEQ ID NO: 1, and was synthesized by Shanghai Tricapeptide Biotechnology Co., Ltd. The characterization maps of the peptide are shown in Figure 2 and Figure 3, which confirms that the peptide was synthesized.

2. Fabrication of Detection Devices

The detection chip is made of SJ modified silica gel (iPDMS film) as the solid support material, on which the polypeptide solution is immobilized by spotting. Modified silica gel is added to the traditional polydimethylsiloxane material with an olefin-terminated, surface-initiated polymerization initiator, and fixed to polydimethylsiloxane by thermal crosslinking (silicon hydrogen bonding). In the three-dimensional structure of alkanes, a new material, SJ modified silica gel, is obtained. Its production process is shown in Figure 4.

A and B are two components of polydimethylsiloxane, and polydimethylsiloxane (Poly(dimethylsiloxane), Sylgard184) is purchased from Dow Corning (DowCorning), USA, and contains liquid component A (component It is a mixture of metal platinum catalyst and dimethyl siloxane polymer precursor with vinyl group) and crosslinking agent B (the composition is a dimethyl siloxane precursor with vinyl and Si-H groups) Element. C is an initiator with a vinyl group at the end, purchased from Hangzhou Dongwei Company. The final modified polymer is oligo(ethyleneglycol)methacrylate (Oligo(ethyleneglycol)methacrylate, hereinafter referred to as OEGMA, molecular weight Mw=526) purchased from Aldrich. Thoroughly mix polydimethylsiloxane precursor A and crosslinker B with vinyl-terminated initiator C at a ratio of A:B:C=10:1:0.5. The transparent elastic silicone rubber is made by curing reaction, and then the surface is modified by SIP technology to obtain SJ modified silicone rubber. Experiments show that the surface of SJ-modified silica gel has a sufficiently high density of initiators immobilized by covalent bonds, which can realize surface polymer modification by surface-initiated polymerization (SIP). Poly(OEGMA) (polyethylene glycol methacrylate) was used to react to obtain a polyethylene glycol (Polyethylene Glycol, PEG) modified surface to achieve a strong ability to resist non-specific protein adsorption.

The prepared SJ modified silica gel film needs to be stored in a refrigerator at 4°C.

The crystal core ^? PersonalArrayerTM16 personal spotting instrument is used to prepare peptide microarrays on modified silica gel. The process is as follows:

1) Preprocessing

Soak the SJ modified silica gel sheet (15×15mm ² ) in the activation solution, take it out after 30 minutes, rinse it with deionized water three times, dry it with nitrogen, and use it for spotting immediately.

2) Spotting

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 base of the spotting instrument, and place the pretreated modified silica gel sheet on the base of the spotting instrument On the stage, sample immediately. The ambient conditions for spotting samples were room temperature (25°C), and the humidity was set at 50%. The sample volume of each point on the prepared polypeptide microarray is about 0.6nL, and the sample point radius is 200 μm.

3) chemical fixation

The newly prepared peptide microarray should be fixed in a constant temperature and humidity chamber (26°C, 60% humidity) for at least 6 hours. The chemical fixation process is shown in Figure 5.

Firstly, the buffer solution containing the capture polypeptide molecules is spotted on the modified silica gel film through the spotting instrument, and then the buffer solution begins to evaporate, and the capture polypeptide molecules are in close contact with the surface of SJ modified silica gel and interact, through chemical combination, the modified silica gel The terminal -COOH of the poly(OEGMA) polymer on the surface forms a stable covalent bond with the -NH ₂ of the polypeptide molecule, and then the chemically active polypeptide molecule is fixed on the surface of SJ modified silica gel.

5) Assembly

Peptide microarrays fixed for 6 hours must be assembled within two days. First, paste the SJ modified silica gel sheet on the special reaction column through the back glue, and cover the reaction chamber. A reactor consists of two reaction columns and a reaction chamber.

6) save

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

3. Detection with detection device

Inspection steps

1. Before starting the test, dilute the concentrated cleaning solution with purified water or distilled water at a ratio of 1:10, and use it directly after dilution. Use a pipette gun to add 2mL of cleaning solution to the chip surface and soak the chip for 3 minutes to ensure that the chip surface is completely wetted.

2. Dilute the serum sample to be tested with the sample diluent at a ratio of 1:40 and mix well.

3. Discard the cleaning solution soaking the chip, and when the surface of the chip is completely wet, absorb 200 μL of diluted serum for each serum sample and add it to the chip reactor.

4. Put the chip reactor into the chip holder, put it on the shaker, turn on the shaker, the frequency is 150 rpm, and incubate at room temperature for 30 minutes.

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

6. After cleaning, add 200 μL of enzyme-labeled antibody solution to each chip reactor, put the chip reactor into the chip holder, put it on the shaker, turn on the shaker, frequency 150 rpm, and incubate at room temperature for 30 minutes.

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

8. After cleaning, remove the reaction chamber, and add 15 μL of luminescent substrate solution to each chip surface, so that the luminescent solution can be evenly spread on the chip surface.

9. Place the chip added with luminescent liquid in a gel imager for chemiluminescence imaging, and interpret the results.

Serum samples from patients with type 1 diabetes and other diseases were provided by cooperative hospitals. Serum is delivered or couriered to the laboratory by relevant personnel in packages such as ice cubes/dry ice.

Negative controls include PBS buffer (that is, incubate with PBS solution instead of the serum to be tested in step 3, and the rest of the steps are the same), serum dilution control, and negative patients (referring to healthy people and non-type 1 diabetics) patient) serum controls.

The spotting pattern of the peptide microarray is shown in Figure 6. Among them, the sample of 20 points in the triangle is human IgG, as the positioning point of the experiment; the sample of 12 points in the square is the PB spotting solution, as the blank control of the experiment; the sample polypeptide of the star point is the polypeptide SEQIDNO of the present invention : 1, it is the type 1 diabetes autoantigen protein polypeptide, which will respond to the serum of type 1 diabetes patients; the samples in the circle point are other type 1 diabetes autoantigen protein polypeptides, which are used as the detection indicators of the experiment (these polypeptides respond It shows that there are type 1 diabetes autoantibodies in the serum).

The experimental results are shown in Figures 7-8. Among them, FIG. 7 shows the detection results of the negative control, and only the samples at the points indicated by the triangles respond. Fig. 8 shows the test results of sera from patients with type 1 diabetes, and samples with triangles, stars and/or circles responded. It should be noted that when the signal value measured by the instrument changes from low to high, the color of the corresponding signal point gradually changes from black to white.

Claims (8)

1. the polypeptide be made up of the aminoacid sequence shown in SEQIDNO:1.

2. the nucleic acid of coding polypeptide according to claim 1.

3. comprise the expression vector of nucleic acid according to claim 2.

4. imported the host cell of expression vector according to claim 3.

5. a detection means, it comprises:

Solid carrier, and

Be connected to the polypeptide according to claim 1 on this solid carrier.

6. detection means according to claim 5, wherein, described solid carrier is iPDMS film.

7. a detection kit, it comprises the detection means described in polypeptide according to claim 1 or claim 5 or 6.

8. polypeptide according to claim 1 is for the preparation of the purposes detected in the test kit of type 1 diabetes or detection means.