Urine sugar detection reagent and kit as well as preparation method and application of urine sugar detection reagent and kit

Abstract

The invention relates to a urine sugar detection reagent and kit as well as a preparation method and application thereof, and belongs to the field of medical detection. The urine glucose detection reagent is recombinant binding protein and has the capacity of specifically binding beta-D-glucose, the construction method of the urine glucose detection reagent is that a binding peptide fragment replaces a single-domain antibody CDR3 region for recombinant expression, and the binding peptide fragment has structural characteristics similar to that of a glucose oxidase (GOD) binding region. The invention also relates to a urine sugar detection method using the recombinant binding protein, and a preparation method of a related detection kit and a detection chip thereof. The detection reagent prepared by the invention is a non-enzyme recombinant protein, and has the advantages of strong specificity, high sensitivity, stable structure and low cost; the urine glucose detection kit or detection chip prepared from the recombinant protein has the advantages of high sensitivity, low cost and storage resistance; the urine sugar detection method based on the kit or the detection chip and applying an ELISA or SPR method is accurate and reliable in result and high in sensitivity, and can meet the rapid and accurate urine sugar detection requirements.

Description

Technical Field

[0001] The present invention belongs to the field of medical testing and specifically relates to a recombinant protein that can specifically recognize and bind to β-D-glucose, as well as a urine glucose detection reagent and kit based on the protein, and their preparation and use. The present invention also relates to the use of the urine glucose detection chip or kit. Background of the Invention

[0002] Glucose is the primary source of energy for human activity, and glucose testing is of great significance in biomedicine. Urine glucose testing, due to its rapid, simple, safe, and non-invasive nature, can be used to assess the effectiveness and safety of new diabetes treatments or serve as a supplementary indicator of treatment efficacy, offering unique application value in diabetes screening, monitoring, and research.

[0003] Currently, the most prevalent method for urine glucose detection is enzymatic. This method, consisting of glucose oxidase (GOD), peroxidase (POD), and a color development system, operates on a continuous enzymatic 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. POD then catalyzes a color development reaction between hydrogen peroxide and potassium iodide. The resulting free iodine then reacts with other substances to produce color, allowing the urine glucose content to be determined by color. However, the enzymatic method is subject to significant limitations, particularly regarding enzyme storage. Prolonged exposure of the test strip to air can result in false positives for urine glucose. Furthermore, it is costly and susceptible to environmental influences. The presence of reducing substances (such as ascorbic acid) in the urine sample can interfere with the test results, resulting in a low result. Summary of the Invention

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

[0005] In a first aspect, the present invention provides a urine sugar detection reagent, which includes a recombinant binding protein that can specifically bind to β-D-glucose. When urine sugar comes into contact with the detection reagent, it can be captured by the recombinant binding protein. 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 invention provides a method for preparing a urine sugar detection reagent, comprising the following steps:

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

[0008] Step 2: Use the Peptide fold tool (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD4 / ) to calculate the structures of the above peptides and screen out peptides with structures similar to the GOD binding region;

[0009] Step 3: Replace the CDR3 of the single-domain antibody with the peptide selected in step 2, replace the C in CDR1 with G, add a His-tag to 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 to glucose and can be used to directly prepare a urine sugar binding chip. However, the polypeptide has poor stability and is easily degraded when directly coupled to the chip. The single-domain antibody has a stable structure, is acid and alkali resistant, and can be expressed in prokaryotes. The preparation process is mature and the cost is low. Therefore, the peptide segment in step 2 is transplanted to replace the CDR3 of the single-domain antibody to prepare the recombinant protein, which can fully utilize the advantages of both and achieve the desired effect.

[0011] In a third aspect, the present invention provides use of the urine sugar detection reagent in preparing a urine sugar detection product.

[0012] In a fourth aspect, the present invention provides a non-diagnostic detection method, which utilizes the urine sugar detection reagent described in the first aspect for detection, including two methods:

[0013] (1) ELISA method:

[0014] Among them, there are two methods for immobilizing binding proteins. One is to use the hydrophobic coating method to directly coat the RA series binding proteins as capture antibodies on the ELISA plate; the other is to use the biotin-avidin coating method. First, the RA series binding proteins need to be biotinylated and then coated on the avidin ELISA plate as capture antibodies.

[0015] RB series binding proteins are used as detection antibodies. The detection kit includes the following components: ELISA plate coated with capture antibody, detection antibody reagent, enzyme-labeled secondary antibody, color development solution, and stop solution.

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

[0017] (2) SPR method:

[0018] SPR is a refractive index sensor. Its 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. A response value of 1RU is roughly equivalent to a change of 1pg / mm2 in the concentration of the bound substance on the chip surface. 2 The equipment used in the present invention is Biacore T200, and the chips used are bare gold chip BR100405 and NTA chip.

[0019] (1) Chip preparation: Bare gold chips are directly coupled to recombinant proteins via thiol-Au coupling until the coupling amount is saturated. NTA chips are coupled via the His-tag at the end of the recombinant protein, and the coupling amount is also saturated.

[0020] (2) Detection: Direct injection detection, glucose in the sample binds to the recombinant protein and is eluted at pH = 2.0.

[0021] The measured binding amount is linearly correlated with the RU value and can be converted based on the RU value.

[0022] Beneficial effects: The urine sugar detection reagent of the present invention is a recombinant binding protein that can specifically bind to β-D-glucose. The recombinant binding protein has a peptide segment with structural characteristics similar to the glucose oxidase (GOD) binding region screened by a random peptide library, and then transplanted to replace the CDR3 region of the single-domain antibody to construct a recombinant binding protein to enhance its stability. The detection reagent prepared by the present invention is a non-enzymatic recombinant protein with strong specificity, high sensitivity, stable structure and low cost; the urine sugar detection kit or detection chip prepared using the recombinant binding protein of the present invention also has the advantages of high sensitivity, low cost and storage resistance; the urine sugar detection method based on the above-mentioned kit or detection chip using ELISA or SPR method has accurate and reliable results and high sensitivity, especially the SPR method is simple and fast to operate, has a large processing volume, saves manpower, and can meet the needs of fast and accurate urine sugar detection. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 It is the structure of A1~A21;

[0025] Figure 3 It is the structure of B1 to B21;

[0026] Figure 4 This is the standard curve for detecting urine sugar by ELISA of the present invention. DETAILED DESCRIPTION

[0027] The following examples are given to illustrate the specific embodiments of the present invention, but the embodiments of the present invention are not limited to the following examples. Any selection and modification can be made within the scope that does not affect the technical effects to be achieved by the present invention. The technical terms and abbreviations used in the present invention have the conventional meanings known to those skilled in the art; unless otherwise specified, all materials in the following examples were purchased from commercial channels.

[0028] Example 1: Obtaining peptide segments similar to GOD 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 was obtained.

[0030] The structures 1GAL and 3QVR of GOD were obtained from the PDB database, and the glucose binding fragment 1 (A0) and fragment 2 (B0) were respectively intercepted based on their protein structure and sequence.

[0031] (2) Obtain similar peptides from the random peptide library.

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

[0033] Table 1 Peptide sequences and serial numbers

[0034]

[0035]

[0036] Example 2 Calculation of random peptide structures

[0037] PEP-FOLD4 was used to calculate the peptide structure in Example 1. The calculation results are as follows: Figures 1 to 3 As shown, yellow represents the N-terminus and red represents the C-terminus.

[0038] Figure 1 This is the structure of fragment 1 (A0) and fragment 2 (B0). As shown in the figure, A0 has α-helix at both ends and a loop in the middle; B0 forms an antiparallel β-sheet at both ends and a loop in the middle.

[0039] Figure 2 It is the structure of A1 to A21. We selected peptide segments with similar structures to A0, namely A1, A3, A4, A7, A9, A12, A15, A16, A18, A19, A20, and A21, a total of 12.

[0040] Figure 3 It is the structure of B1 to B21. We selected peptide segments with similar structures to B0, namely B3, B6, B7, B9, B12, B13, B14, B15, B17, B18, and B20, a total of 11.

[0041] Example 3 Preparation of recombinant binding protein

[0042] A single-domain antibody is an artificially designed antibody molecule with a molecular weight of only 1 / 10 of that of a conventional antibody. Four highly conserved hydrophobic amino acids in the framework region are replaced with hydrophilic amino acids. Therefore, single-domain antibodies have the characteristics of high stability (heat resistance, extreme pH resistance), low cost (can be expressed in prokaryotes), and easy modification, making them an ideal recombinant protein framework structure. The present invention selected a single-domain antibody that binds to a FLAG-tag as reported in the literature (Wang Xinyi, Wang Xiaoqian, Wang Hongjun, Chao Yuehui. Screening, expression and verification of FLAG-tagged nanoantibodies [J]. Biotechnology Bulletin, 2023, 39(10): 323-331.), and replaced the CDR3 of the single-domain antibody with the 23 peptide segments selected in Example 2 to construct a recombinant binding protein.

[0043] The amino acid sequence of the selected single-domain antibody is shown in SEQ ID NO.45, which has the following amino acid sequence: 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 CDR3 region of the single-domain antibody was replaced with each of the 23 peptides selected in Example 2. The C in the original CDR1 was replaced with a G to stabilize the antibody sequence. A His-tag was added to the C-terminus for purification, and a cysteine (C) was added for coupling to a bare gold chip. The amino acid sequences of the redesigned recombinant proteins are shown in SEQ ID NOs. 46 to 68.

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

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

[0047] (2) Recombinant bacterial culture and recombinant protein expression: The basal culture medium for recombinant protein was TB medium, inoculated at 5% inoculum, cultured at 37°C for 3-5 h, and the inducer IPTG (final concentration 0.25 mM, the same below) was added.

[0048] The cells were induced overnight. After induction, the cells were centrifuged at 4000 rpm for 20 min to obtain the recombinant protein-containing bacteria.

[0049] (3) Recombinant protein purification: lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.4, 0.02 M PB) was added to the obtained bacteria at a ratio of 1:10, and the cells were broken using a 700 bar high-pressure homogenizer; the cells were homogenized at 4°C and 10,000

[0050] The mixture was centrifuged at 370 nm for 20 min at 400 nm and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter and then purified by affinity chromatography using NiSepharose High Performance (GE Healthcare, US) to obtain the purified recombinant protein.

[0051] Example 4 Binding Protein Screening

[0052] Screening was performed using glycogen-coated ELISA plates.

[0053] (1) Binding: Dilute the recombinant protein to 1 μg / mL and add 100 μL / well to the enzyme-labeled wells. Bind at 37°C for 1 h. Do not add the protein to the negative control wells.

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

[0055] (3) Detection of antibody binding: dilute anti-HRP / Anti-6×His at 1:5000 with 1×PBS and add 100 μL / well to the enzyme-labeled wells for binding at 37°C for 1 h.

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

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

[0058] (6) Reading: Read OD using a microplate reader 630 and OD 450 Absorbance value.

[0059] The criterion for positive identification was a ratio greater than 1.0, and all 23 samples were positive. This demonstrates that the structural screening method used in the present invention can screen for recombinant proteins with similar structures and functions. To save costs and improve efficiency, the recombinant binding proteins with the highest positive values, RA1 (sequence shown in SEQ ID NO. 46) and RB3 (sequence shown in SEQ ID NO. 58), were selected in descending order for the next step of the experiment.

[0060] Example 5 Kit Preparation

[0061] (1) ELISA test method

[0062] In the first embodiment of this method, binding protein RA1 is directly coated onto an ELISA plate using a hydrophobic coating method as the capture antibody, and binding protein RB3 is used as the detection antibody. The detection kit includes the following components: an ELISA plate coated with the capture antibody, a detection antibody reagent, an enzyme-labeled secondary antibody, a developer, and a stop solution.

[0063] Specific operation method:

[0064] (1) Coating capture antibody: Dilute RA1 to 1 μg / mL, add 300 μL / well to a blank ELISA plate, and react at 4°C overnight.

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

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

[0067] (4) Sample addition: Add the sample to be tested and glucose standards with different concentration gradients respectively, and react at 37°C for 1 h.

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

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

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

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

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

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

[0074] (10) Reading: Read OD using a microplate reader 630 and OD 450 Absorbance value.

[0075] The second implementation of this method uses a biotin-avidin coating method. First, the binding protein RA1 is biotinylated and coated on an avidin ELISA plate as the capture antibody, with the binding protein RB3 serving as the detection antibody. The detection kit includes the following components: an ELISA plate coated with the capture antibody, a detection antibody reagent, an enzyme-labeled secondary antibody, a developer, and a stop solution. The present invention uses the Thermo Scientific EZ-Link NHS-biotin reagent. The biotinylation process is as follows: RA1 is diluted to 2 mg / mL, 3 μL of the biotin reagent is added to 1 mL of the protein solution, and the reaction is allowed to proceed at room temperature for 30 minutes.

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

[0077] The test results of the two implementation methods are as follows Figure 4 As shown, the urine sugar concentration in the sample to be tested can be calculated based on the absorbance value and the standard curve, and the optimal detection range is 0.1 to 1.0 mmol / L.

[0078] ELISA standard curve R prepared by two implementation methods 2 All of them are greater than 0.99, with high fit and high reliability. Among them, the direct hydrophobic coating method has some influence on the activity of the binding protein, so the overall signal value is lower, but the ratio is not much different, which is almost consistent with the measurement results of the biotin-coated plate.

[0079] (2) SPR method

[0080] SPR is a refractive index sensor. Its 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. A response value of 1RU is roughly equivalent to a change of 1pg / mm2 in the concentration of the bound substance on the chip surface. 2 The equipment used in the present invention is Biacore T200, and the chips used are bare gold chip BR100405 and NTA chip.

[0081] (1) Chip preparation: Bare gold chips were respectively coated with recombinant binding proteins RA1 (SEQ ID NO. 46) and RB3 (SEQ ID

[0082] No. 58) relies directly on thiol-Au coupling, and the coupling amount is increased to saturation. NTA chips rely on the His-tag at the end of the recombinant protein, and the coupling amount is also increased to saturation.

[0083] (2) Detection: Direct injection detection, glucose in the sample binds to the recombinant protein and is eluted at pH 2.0.

[0084] The measured binding amount is linearly correlated with the RU value and can be converted based on the RU value.

[0085] The SPR method has high sensitivity and can process large sample volumes. It can automatically process 384 samples during unattended operation, making it suitable for large-scale sample processing. The detection chip prepared in this invention is coupled to the recombinant binding proteins RA1 and RB3, respectively, and has a detection limit as low as 0.01 mmol / L, making it particularly suitable for detecting low-concentration urine glucose.

[0086] In summary, the detection reagent prepared by the present invention is a non-enzymatic recombinant protein with strong specificity, high sensitivity, stable structure and low cost; the urine sugar detection kit or detection chip prepared using the recombinant protein of the present invention also has the advantages of high sensitivity, low cost and storage resistance; the urine sugar detection method based on the above-mentioned kit or detection chip using ELISA or SPR method has accurate and reliable results with high sensitivity, especially the SPR method is simple and fast to operate, has a large processing volume, saves manpower, and provides a strong guarantee for large-scale detection.

[0087] Obviously, those skilled in the art may make various changes and modifications to this application without departing from the spirit and scope of this application. Thus, if these modifications and variations of this application fall within the scope of the claims of this application and their equivalents, this application is intended to include these modifications and variations.

Claims

1. A urine sugar detection reagent, characterized in that: The detection reagent includes a recombinant binding protein, which can specifically bind to β-D-glucose. 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 sugar detection reagent according to claim 1, characterized in that: The steps include: Step 1: Obtain the crystal structure of glucose oxidase GOD from the PDB database, intercept its binding region, and then obtain peptides from the random peptide library; Step 2: Peptide screening; Step 3: The amino acid sequence of the donor single domain antibody is shown in SEQ ID NO.

45. The peptide segment screened in step 2 is used to replace the CDR3 of the donor single domain antibody, and the C of CDR1 is replaced with G. A His-tag is added to the C-terminus, and a cysteine is added to the C-terminus for coupling to a bare gold chip to prepare a recombinant binding protein with an amino acid sequence shown in SEQ ID NO. 46 or SEQ ID NO.

58.

3. Use of the urine sugar detection reagent according to claim 1 in the preparation of urine sugar detection products.

4. A urine sugar detection kit, characterized in that: The kit comprises the urine sugar detection reagent according to claim 1, and also comprises an ELISA plate coated with a capture antibody, a detection antibody reagent, an enzyme-labeled secondary antibody, a color development solution and a stop solution.

5. A detection method for non-diagnostic purposes, characterized in that: The urine sugar detection reagent according to claim 1 is used for detection, and the detection method is an ELISA method. The recombinant binding protein according to claim 1 is immobilized on an ELISA plate by hydrophobic coating. After sample addition, incubation, plate washing, addition of capture antibody, plate washing, addition of color developing antibody, plate washing, color development, termination and reading, the urine sugar concentration in the sample to be tested is calculated by the standard curve.

6. The detection method according to claim 5, characterized in that The recombinant binding protein is first biotinylated and then coated on an avidin ELISA plate through biotin. After adding samples, incubating, washing the plate, adding capture antibodies, washing the plate, adding color developing antibodies, washing the plate, developing, stopping and reading, the urine sugar concentration in the sample to be tested is calculated using a standard curve.

7. A detection method for non-diagnostic purposes, characterized in that: The urine sugar detection reagent according to claim 1 is used for detection, and the detection method is the SPR method. The recombinant binding protein according to claim 1 is immobilized on a bare gold chip through the terminal thiol group, and the immobilization amount is saturated. Then, the sample is added for detection, and the urine sugar concentration in the sample to be tested is calculated by the change in the RU value.

8. The detection method according to claim 7, characterized in that The recombinant binding protein is immobilized on the NTA chip via the terminal His-tag, and the immobilization amount is saturated. Then, the sample is added for detection, and the urine sugar concentration in the sample to be tested is calculated based on the change in RU value.

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