A urine sugar detection reagent and kit as well as a preparation method and application thereof

Abstract

The present application relates to a urine sugar detection reagent and kit and its preparation method and application, belong to medical detection field. The urine sugar detection reagent is a recombinant binding protein, has the ability of specific binding β-D-glucose, its construction method is to replace single domain antibody CDR3 region with binding peptide segment and carry out recombination expression, the binding peptide segment has the structural characteristics similar to glucose oxidase (GOD) binding region. The present application also relates to a urine sugar detection method using the recombinant binding protein, and its related detection kit and detection chip preparation method. The detection reagent prepared by the present application is a non-enzyme recombinant protein, which has high specificity, high sensitivity, stable structure and low cost. The urine sugar detection kit or detection chip prepared by using the recombinant protein of the present application has the advantages of high sensitivity, low cost and storage resistance. The urine sugar detection method based on the above kit or detection chip using ELISA or SPR method has accurate and reliable results and high sensitivity, which can meet the rapid and accurate urine sugar detection requirements.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical detection, and particularly relates to a recombinant protein capable of specifically recognizing and combining with beta-D-glucose, a urine glucose detection reagent and kit based on the protein, and a preparation method and application thereof. The present application also relates to the application of the urine glucose detection chip or kit. BACKGROUND

[0002] Glucose is the main source of human activity, and glucose detection is of great significance in biomedicine. Among them, urine glucose detection can be used to evaluate the effectiveness and safety of new treatments for diabetes, or as a complementary indicator of treatment effectiveness, and has unique application value in the initial screening, monitoring and research of diabetes.

[0003] At present, the most important method for urine glucose detection is enzyme-based detection. This method is composed of glucose oxidase (GOD), peroxidase (POD) and a color developing system, and its principle is a continuous enzyme reaction: when urine glucose comes into contact with the detection system, GOD first catalyzes the oxidation of glucose to produce gluconic acid and hydrogen peroxide, and then POD catalyzes the color reaction of hydrogen peroxide with potassium iodide to produce free iodine, which then reacts with other substances to produce a color reaction, and the content of urine glucose is determined by color. The defects of enzyme detection are that it is greatly affected by external conditions, especially the preservation of enzymes, and if the test paper is exposed to air for a long time, it will cause false positive of urine glucose. Moreover, it is high in cost and more affected by the environment, especially when there are reducing substances (such as ascorbic acid) in the urine sample, which will interfere with the detection results and cause the detection results to be low. SUMMARY

[0004] The purpose of the present application is to solve the above problems in urine glucose detection, and to provide a rapid, convenient and low-cost urine glucose reagent and a preparation method and application thereof.

[0005] In a first aspect, the present application provides a urine glucose detection reagent, which comprises a recombinant binding protein, the recombinant binding protein can specifically bind to beta-D-glucose, when urine glucose comes into contact with the detection reagent, it can be captured by the recombinant binding protein, and the amino acid sequence of the recombinant binding protein is shown in SEQ ID NO. 46 or SEQ ID NO. 58.

[0006] In a second aspect, the present application provides a preparation method of a urine glucose detection reagent, comprising the following steps:

[0007] Step 1: Obtain the crystal structure of glucose oxidase (GOD) from the PDB database, obtain crystal structures 1GAL and 3QVR, intercept the binding region thereof, the binding region is A0 and B0, and the amino acid sequences are SEQ ID NO. 1 and SEQ ID NO. 23 respectively, and then obtain a peptide segment similar to the GOD binding region from a random peptide library;

[0008] Step 2: Calculate the structure of the above peptide segment by using the Peptide fold tool (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD4 / ), and screen out a peptide segment similar to the structure of the GOD binding region;

[0009] Step 3: Replace the CDR3 of the single-domain antibody with the peptide segment screened in step 2, replace the C of CDR1 with G, and add a His-tag at the C-terminus, and then add a cysteine at the C-terminus for coupling with a bare gold chip, to prepare a recombinant binding protein;

[0010] The amino acid sequence of the single-domain antibody is shown in SEQ ID NO. 45, and the amino acid sequence of the recombinant binding protein is shown in SEQ ID NO. 46 or SEQ ID NO. 68. The peptide segment screened in step 2 also has the ability to bind glucose and can directly prepare a urine sugar binding chip. However, the polypeptide is poor in stability, and is easily degraded when directly coupled on the chip. The single-domain antibody is stable in structure, resistant to acid and alkali, and can be expressed in prokaryotic organisms, and the preparation process is mature and low in cost. Therefore, the peptide segment in step 2 is transplanted to replace the CDR3 of the single-domain antibody to prepare a recombinant protein, so that the advantages of both can be fully utilized to achieve an ideal effect.

[0011] In a third aspect, the application provides use of the urine sugar detection reagent in preparation of a urine sugar detection product.

[0012] In a fourth aspect, the application provides a detection method for non-diagnostic purposes, which utilizes the urine sugar detection reagent of the first aspect for detection, including two methods:

[0013] (i) ELISA method:

[0014] Among them, there are two methods for immobilizing the binding protein, one is to use hydrophobic coating method to directly coat the RA series binding protein as the capture antibody on the ELISA plate; the other is to use biotin-avidin coating method, which first needs to biotinylate the RA series binding protein, and then coat it as the capture antibody on the avidin ELISA plate.

[0015] The RB series binding protein serves as a detection antibody. The detection kit comprises the following components: an ELISA plate coated with a capture antibody, a detection antibody reagent, an enzyme-labeled secondary antibody, a color developing solution, and a termination solution.

[0016] The specific operation method comprises the following steps: (1) coating a capture antibody; (2) blocking; (3) washing the plate; (4) adding a sample; (5) washing the plate; (6) detecting antibody binding; (7) washing the plate; (8) adding an enzyme-labeled secondary antibody; (9) washing the plate; (10) developing color; and (11) reading.

[0017] (ii) SPR method:

[0018] SPR is a refractive index sensor, and the response value RU reflects the change in the SPR angle. The response signal depends on the concentration and temperature of the molecules on the chip surface. The response value of 1 RU is approximately equivalent to a change of 1 pg / mm in the concentration of the substances bound on the chip surface. 2 The device used in the present application is a Biacore T200, and the chips used are a bare gold chip BR100405 and an NTA chip.

[0019] (1) Chip preparation: the bare gold chip is directly coupled with the recombinant protein by relying on thiol-Au coupling, and the coupling amount is until saturation. The NTA chip relies on the His-tag at the end of the recombinant protein to bind, and the coupling amount is also until saturation.

[0020] (2) Detection: direct sample injection detection, and the glucose in the sample is combined with the recombinant protein, and eluted under the condition of pH = 2.0.

[0021] The measured binding amount is linearly related to the RU value, and the RU value can be converted.

[0022] Beneficial effects: the urine glucose detection reagent of the present application is a recombinant binding protein that can specifically bind beta-D-glucose. The recombinant binding protein has a peptide segment with the structural characteristics of a glucose oxidase (GOD) binding region screened by a random peptide library, and the CDR3 region of a single domain antibody is transplanted to construct a recombinant binding protein to enhance its stability. The detection reagent prepared by the present application is a non-enzyme recombinant protein, which has the advantages of high specificity, high sensitivity, stable structure and low cost. The urine glucose detection kit or detection chip prepared by using the recombinant binding protein of the present application also has the advantages of high sensitivity, low cost and storage resistance. The urine glucose detection method based on the above kit or detection chip and using the ELISA or SPR method has accurate and reliable results and high sensitivity. In particular, the SPR method is simple, fast, has a large processing capacity and saves manpower, and can meet the requirements of rapid and accurate urine glucose detection. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the structure of fragment 1 (A0) and fragment 2 (B0);

[0024] Figure 2 is a structure of A1-A21;

[0025] Figure 3 is a structure of B1-B21;

[0026] Figure 4 is a standard curve of ELISA for detecting urine glucose according to the present application. DETAILED DESCRIPTION

[0027] The following examples are given to illustrate the specific embodiments of the present application, but the embodiments of the present application are not limited to the following examples, and any selection and change can be made without affecting the technical effects to be achieved by the present application. The technical terms and abbreviations used in the present application have the conventional meanings known to those skilled in the art; all materials in the following examples are commercially available unless otherwise specified.

[0028] Example 1: Obtaining peptide fragments of GOD-like binding fragments from a random peptide library.

[0029] (1) Based on the published GOD protein sequence and structure, the sequence of the GOD binding fragment is obtained.

[0030] The structures 1GAL and 3QVR of GOD are obtained in the PDB database, and according to the protein structure and sequence, the glucose binding fragment 1 (A0) and fragment 2 (B0) are cut off, respectively.

[0031] (2) Obtaining similar peptide fragments from a random peptide library.

[0032] The two fragments are input into the random peptide library, the parameters are set, the similar peptide fragments are obtained, and the sequence information is shown in Table 1.

[0033] Table 1: Peptide sequence and serial number

[0034]

[0035]

[0036] Example 2: Calculation of random peptide fragment structure

[0037] The structure of the peptide fragments in Example 1 is calculated using PEP-FOLD4, and the calculation results are shown in Figures 1 to 3 , where yellow represents the N-terminal and red represents the C-terminal.

[0038] Figure 1 is the structure of fragment 1 (A0) and fragment 2 (B0), as shown in the figure, the two ends of A0 are α-helix, and the middle is a loop; the two ends of B0 form an inverse parallel β-sheet, and the middle is a loop.

[0039] Figure 2 A1-A21 are the structures of B1-B21, we select the peptide segments with similar structures to B0, respectively B3, B6, B7, B9, B12, B13, B14, B15, B17, B18, B20, a total of 11.

[0040] Figure 3 A1-A21 are the structures of B1-B21, we select the peptide segments with similar structures to B0, respectively B3, B6, B7, B9, B12, B13, B14, B15, B17, B18, B20, a total of 11.

[0041] Preparation of recombinant binding protein in Example 3

[0042] Single-domain antibody is an artificially designed antibody molecule, which has a molecular weight of only 1 / 10 of that of conventional antibodies, and the framework region is replaced by four highly conserved hydrophobic amino acids with hydrophilic amino acids, so that the single-domain antibody has high stability (heat-resistant, resistant to extreme pH), low cost (can be expressed in prokaryotes), easy to modify, etc. It is an ideal recombinant protein framework structure. The present application selects a single-domain antibody (Wang Xin-yi, Wang Xiao-qin, Wang Hong-jun, Chao Yue-hui. Screening, expression and verification of FLAG-tag nanobody [J]. Biotechnology Bulletin, 2023, 39 (10): 323-331.) reported in the literature. The 23 peptide segments selected in Example 2 are replaced with the CDR3 of the above-mentioned single-domain antibody to construct a recombinant binding protein.

[0043] The amino acid sequence of the selected single-domain antibody is shown in SEQ ID NO. 45, and the amino acid sequence is: QVQLQESGGGSVHTGGSLRLSCVASRGIYTTCSTAWYRQSPGEKERVLVASISPGGDPTYDD SVKGRFVISQDKTEKTVFTVFLQMNNLRPEDSGTYYCNSPGWVAGRCRPDFGYWGSGTQV TVSS, wherein the amino acid sequence of CDR1 is: RGIYTTCS, the amino acid sequence of CDR2 is: ISPGGDP, and the amino acid sequence of CDR3 is: NSPGWVAGRCRPDFGY.

[0044] The 23 peptide segments selected in Example 2 are replaced with the CDR3 region of the above-mentioned single-domain antibody, and the C of the original CDR1 is replaced with G to stabilize the antibody sequence, and a His-tag is added at the C-terminus for purification, and a cysteine (C) is added for coupling with a bare gold chip. The amino acid sequence of the redesigned recombinant protein is shown in SEQ ID NO. 46-SEQ ID NO. 68.

[0045] The preparation process of the recombinant binding protein is as follows:

[0046] (1) The above recombinant protein sequence is outsourced for full synthesis, and the constructed gene sequence is connected to the pET23a vector and transformed into E. coli.

[0047] (2) Recombinant bacteria culture and recombinant protein expression: The basic culture medium of the recombinant protein is TB culture medium, inoculated at a 5% inoculation amount, and cultured at 37°C for 3-5 h, and then induced with inducer galactoside (IPTG) (final concentration 0.25 mM, same below)

[0048] overnight induction; after the induction is completed, centrifugation is performed at 4000 rpm for 20 min to obtain the bacterial body containing the recombinant protein.

[0049] (3) Recombinant protein purification: The obtained bacterial body is added with lysis solution (10 mM imidazole, 500 mM NaCl, pH 7.4, 0.02 M PB) at a ratio of 1:10, and cell disruption is performed using a 700 bar high-pressure homogenizer; centrifugation is performed at 4°C and 10000 rpm for 20 min, and the supernatant is taken; the supernatant is filtered through a 0.45 μm filter, and then subjected to affinity chromatography column (GE Healthcare, US) for separation and purification of the recombinant protein, wherein the filler of the affinity chromatography column is NiSepharose High Perfomance. The purified recombinant protein is obtained.

[0050] rpm for 20 min, and the supernatant is taken; the supernatant is filtered through a 0.45 μm filter, and then subjected to affinity chromatography column (GE Healthcare, US) for separation and purification of the recombinant protein, wherein the filler of the affinity chromatography column is NiSepharose High Perfomance. The purified recombinant protein is obtained.

[0051] Example 4 Binding protein screening

[0052] An ELISA plate coated with glycogen is used for screening.

[0053] (1) Binding: The recombinant protein is diluted to 1 μg / mL, and 100 μL / well is added to the enzyme-labeled hole, and 37°C is combined for 1 h. The negative control hole is not added.

[0054] (2) Washing plate: Wash the plate 5 times with 0.05% PBST.

[0055] (3) Detecting antibody binding: Anti-HRP / Anti-6xHis is diluted by 1:5000 with 1xPBS, and 100 μL / well is added to the enzyme-labeled hole, and 37°C is combined for 1 h.

[0056] (4) Washing plate: Wash the plate 5 times with 0.05% PBST.

[0057] (5) Color development: 100 μL / well of TMB working solution is added, and 37°C is reacted for 6 min in the dark. After the color development reaction is completed, 50 μL / well of 2M H2SO4 is added to terminate the reaction.

[0058] (6) Reading: OD was read by microplate reader 630 and OD 450 at 450 nm.

[0059] The positive judgment criterion was that the ratio was greater than 1.0, and the results of 23 samples were all positive. It was shown that the structure screening method used in the application could screen out recombinant proteins with similar structures and similar functions. In order to save cost and improve efficiency, according to the order from large to small, the recombinant binding proteins with the highest positive value, RA1 (the sequence is shown as SEQ ID NO. 46) and RB3 (the sequence is shown as SEQ ID NO. 58), were selected for the next experiment.

[0060] Example 5 Kit preparation

[0061] (I) ELISA detection method

[0062] The first embodiment of the method is to directly coat the binding protein RA1 as the capture antibody on the ELISA plate by hydrophobic coating method, and the binding protein RB3 as the detection antibody. The detection kit includes the following components: ELISA plate coated with capture antibody, detection antibody reagent, enzyme-labeled secondary antibody, color developing solution and termination solution.

[0063] Specific operation method:

[0064] (1) Coating capture antibody: dilute RA1 to 1 μg / mL, add 300 μL / well to the blank enzyme-labeled plate, and react overnight at 4°C.

[0065] (2) Blocking: remove excess liquid, wash the plate with PBS for 3 times, and then block with 0.5% BSA.

[0066] (3) Washing plate: wash the plate with 0.05% PBST for 5 times.

[0067] (4) Sample addition: add the sample to be tested and different concentration gradient glucose standard, and react for 1 h at 37°C.

[0068] (5) Washing plate: wash the plate with 0.05% PBST for 5 times.

[0069] (6) Detection antibody binding: dilute RB3 to 1 μg / mL with 1×PBS, and add 100 μL / well to the enzyme-labeled hole, and combine for 1 h at 37°C.

[0070] (7) Washing plate: wash the plate with 0.05% PBST for 5 times.

[0071] Add secondary antibody: dilute Anti-HRP / Anti-6×His to 1:5000 with 1×PBS, and add 100 μL / well to the enzyme-labeled hole, and combine for 1 h at 37°C.

[0072] (8) Washing: wash the plate with 0.05% PBST for 5 times.

[0073] (9) Color development: add 100 μL / well of TMB working solution, and react at 37°C for 6 min in the dark. After the color development reaction is completed, add 50 μL / well of 2M H2SO4 to terminate the reaction.

[0074] (10) Reading: read OD 630 and OD 450 at 450 nm.

[0075] The second embodiment of the method is to use a biotin-avidin coating method. First, the binding protein RA1 is biotinylated, and is coated on an avidin ELISA plate as a capture antibody, and the binding protein RB3 is used as a detection antibody. The detection kit comprises the following components: an ELISA plate coated with a capture antibody, a detection antibody reagent, an enzyme-labeled secondary antibody, a color developing solution, and a termination solution. The present application uses a Thermo Scientific EZ-Link NHS-biotin reagent, and the biotinylation process is as follows: dilute RA1 to 2 mg / mL, add 3 μL of biotin reagent to 1 mL of protein solution, and react at room temperature for 30 min.

[0076] The subsequent kit preparation and detection process are the same as above.

[0077] The detection results of the two embodiments are shown in Table 1. Figure 4 As shown in Table 1, the concentration of urine glucose in the sample to be detected can be calculated according to the absorbance value and the standard curve, and the optimal detection range is 0.1-1.0 mmol / L.

[0078] The R 2 of the ELISA standard curve prepared by the two embodiments is greater than 0.99, the fitting degree is high, and the reliability is high. Among them, the direct hydrophobic coating method has a slight impact on the activity of the binding protein, so the overall signal value is slightly low, but the ratio is not much different from the measurement results of the biotin-coated well plate.

[0079] (II) SPR method

[0080] SPR is a refractive index sensor, and the response value RU reflects the change in SPR angle. The response signal depends on the concentration and temperature of the molecules on the chip surface, and 1 RU of the response value is roughly equivalent to a change of 1 pg / mm 2 in the concentration of the substances on the chip surface. The equipment used in the present application is Biacore T200, and the chips used are bare gold chip BR100405 and NTA chip.

[0081] (1) Chip preparation: respectively coat the bare gold chip with the recombinant binding proteins RA1 (SEQ ID NO. 46) and RB3 (SEQ ID

[0082] NO.58) Directly rely on sulfydryl-Au coupling, coupling amount is up to saturation. NTA chip relies on the end His-tag binding of recombinant protein, coupling amount is also up to saturation.

[0083] (2) Detection: Direct injection detection, glucose in the sample is combined with recombinant protein, and eluted under the condition of pH2.0.

[0084] The measured binding amount is linearly related to the RU value, and can be converted according to the RU value.

[0085] The SPR method has high sensitivity, large sample processing capacity, and can automatically process 384 samples when unattended operation, and is suitable for large-scale sample processing. The detection chip prepared in the application is coupled with recombinant binding proteins RA1 and RB3, respectively, and the detection limit can be as low as 0.01 mmol / L, and is especially suitable for low-concentration urine sugar detection.

[0086] In summary, the detection reagent prepared in the application is a non-enzyme recombinant protein, which has high specificity, high sensitivity, stable structure and low cost; the urine sugar detection kit or detection chip prepared by using the recombinant protein of the application also has the advantages of high sensitivity, low cost and storage resistance; the urine sugar detection method based on the above kit or detection chip and using ELISA or SPR method has accurate and reliable results and high sensitivity, and the SPR method is simple and fast in operation, has large processing capacity and saves manpower, which provides a strong guarantee for large-scale detection.

[0087] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Therefore, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A urine glucose detection reagent, characterized in that, The detection reagent includes a recombinant binding protein that specifically binds to β-D-glucose, and the amino acid sequence of the recombinant binding protein is shown in SEQ ID NO.46 or SEQ ID NO.

58.

2. A method for preparing the urine glucose detection reagent as described in claim 1, characterized in that, Includes the following steps: Step 1: Obtain the crystal structure of glucose oxidase GOD from the PDB database, extract its binding regions as shown in SEQ ID NO.1 (A0) and SEQ ID NO.23 (B0), and then obtain peptides from the random peptide library; Step 2: The Peptide Fold tool is used to calculate the structure of the above peptides and screen out peptides with similar GOD binding region structures. The peptide sequences are as follows: SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.26, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.32, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.

43. Step 3: The amino acid sequence of the donor single-domain antibody is shown in SEQ ID NO.

45. The peptide selected in Step 2 is used to replace CDR3 of the donor single-domain antibody, C in CDR1 is replaced with G, a His-tag is added to the C-terminus, and a cysteine ​​residue is added to the C-terminus for coupling with the bare gold chip. The recombinant binding protein with the amino acid sequence shown in SEQ ID NO.46 or SEQ ID NO.58 is obtained by screening.

3. The application of the urine glucose detection reagent as described in claim 1 in the preparation of urine glucose detection products.

4. A urine glucose detection kit, characterized in that, The kit includes the urine glucose detection reagent as described in claim 1, and also includes an ELISA plate, enzyme-labeled secondary antibody, chromogenic solution and stop solution.

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