Protein for detecting adamts13 activity and use thereof

Abstract

The present application relates to the field of protein, in particular to a kind of protein for detecting the activity of ADAMTS13 and its application.The protein for detecting the activity of ADAMTS13 described in the present application at least includes A1) nanobody as shown in SEQ ID NO.1;A2) vWF (D1596-R1668) variant as shown in SEQ ID NO.2;A3) positive chain polypeptide as shown in SEQ ID NO.3;A4) negative chain polypeptide as shown in SEQ ID NO.4;A5) circularly permuted GFP (cpGFP) as shown in SEQ ID NO.6.The protein for detecting the activity of ADAMTS13 described in the present application can directly determine the ADAMTS13 enzyme activity in blood sample, biological sample or chemical sample, and is suitable for clinical point-of-care testing (POCT), biological sample screening and drug development scenarios.

Description

Technical Field

[0001] This invention relates to the field of proteins, and more specifically to a protein for detecting ADAMTS13 activity and its applications. Background Technology

[0002] Thrombotic thrombocytopenic purpura (TTP) is a highly fatal disseminated thrombotic microangiopathy, with ADAMTS13 activity deficiency as its etiological basis. ADAMTS13 is a zinc metalloproteinase that, under physiological conditions, can specifically cleave von Willebrand factor (vWF). Under pathological activity deficiency, vWF accumulates into large molecular clusters, activating panmicrothrombi via the platelet pathway.

[0003] The clinical manifestations of TTP are very similar to those of HUS (hemolytic uremic syndrome) and DIC (disseminated intravascular coagulation), but the clinical management is quite different. Therefore, a series of diagnostic tests around ADAMTS13 have become a key point for the differential diagnosis and clinical treatment of TTP. However, due to its complex reaction mechanism, monopoly of gold standard raw materials, and expensive testing instruments, no relevant testing research or registered products have been launched in China to date, resulting in the consumption and loss of social and medical resources.

[0004] Currently, there are several methods for detecting ADAMTS13 activity, including: (1) Immunoradioassay and collagen binding method: The substrate is a full-fragment vWF polymer, which requires the addition of a denaturing agent for unfolding. However, there is no denaturing agent in the human body, which does not meet physiological conditions, and the reaction time is relatively long, around 16-48 hours; (2) Fluorescence resonance energy transfer method: The chemically synthesized substrate FRETS-vWF73 will reduce the fluorescence quenching effect after the substrate is specifically cleaved. That is, ADAMTS13 in normal human plasma will enhance fluorescence after action, while patients with insufficient activity will have no effect or weakened fluorescence. However, this detection method is developed by several domestic companies. Foreign companies have a technological monopoly, and the synthesis of substrates with 73 peptides modified with fluorescent and quenching groups is extremely expensive; (3) Enzyme-linked immunosorbent assay: based on double-antibody sandwich ELISA technology, but the molecular weight of ADAMTS13 recombinant protein is large, the testing process is complex and time-consuming, making it difficult to meet the needs of large-scale TTP screening, and it is not suitable for the clinical needs of emergency diagnosis and treatment of patients in the emergency room; (4) Magnetic microparticle chemiluminescence detection method: using the abundance of His after the GST-vWF73-His substrate is cleaved for chemiluminescence detection (or other tags), but its instruments and reagents are expensive, and the cost of use is extremely high. Based on the shortcomings of the above detection methods, there is an urgent need for a series of detection methods for ADAMTS13 that are efficient, fast and accurate. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to provide a novel protein based on green fluorescent protein spatial conformation recombination technology for detecting the activity of ADAMTS13, thereby enabling rapid and accurate detection of ADAMTS13 activity.

[0006] In a first aspect, the present invention provides a protein for detecting the activity of ADAMTS13, said protein comprising at least the following components:

[0007] A1) Circular arrangement of GFP (cpGFP) as shown in SEQ ID NO.1;

[0008] A2) V Part 1 of the vWF (D1596-R1668) variant as shown in SEQ ID NO.2;

[0009] A3) V Part 2 of the vWF (D1596-R1668) variant as shown in SEQ ID NO.3;

[0010] A4) Positive-chain polypeptides as shown in SEQ ID NO.4;

[0011] A5) Negative-chain polypeptides as shown in SEQ ID NO.5;

[0012] A6) Nanobody as shown in SEQ ID NO.6, wherein the positive chain polypeptide and the negative chain polypeptide are adjacent, and V Part1 of the vWF(D1596-R1668) variant and V Part2 of the vWF(D1596-R1668) variant are located at the two ends of the positive chain polypeptide and the negative chain polypeptide, respectively.

[0013] Further, the order of the protein parts used to detect ADAMTS13 activity from N-terminus to C-terminus is as follows: circularly arranged GFP (cpGFP), V Part1 of the vWF (D1596-R1668) variant, negative-chain polypeptide, positive-chain polypeptide, V Part2 of the vWF (D1596-R1668) variant, and nanobody. Optionally, the protein used to detect ADAMTS13 activity may further include an N-terminal or C-terminal tag sequence.

[0014] The sequences of each part are as follows:

[0015] SEQ ID NO: 1:

[0016] NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVN GHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY

[0017] SEQ ID NO: 2:

[0018] DREQAPNVVYMVTGNPTSDEIKRLPGDI

[0019] SEQ ID NO: 3:

[0020] KELQLVPIGVAPNANVQELKRIGWPNAPILIQDFETLPREAPDLVLQR

[0021] SEQ ID NO: 4:

[0022] AQLKKKIAALKKKNAQLKWKIAALKKKLA

[0023] SEQ ID NO: 5:

[0024] SQLEKEIAALEKENAQLEWEIAALEKELA

[0025] SEQ ID NO: 6:

[0026] QVQLVESGGGLVRPGGSLRLSCVDSERTSYPMGWFRRAPGKEREFVASITWSGIDPTYADSVADRFTTSRDVANNTLYLQMNSLKHEDTAVYYCAAKAPVGNSSSPYDFDYWGQGTQVTVS

[0027] Furthermore, the various parts can be directly connected or connected through flexible connectors (GGGS)n, where n is an integer ≥1, representing the number of repetitions of the GGGS sequence, preferably 1≤n≤10.

[0028] In a specific embodiment of the present invention, the various parts are directly connected.

[0029] Furthermore, the amino acid sequence of the protein used to detect ADAMTS13 activity includes at least one of the following:

[0030] B1) The amino acid sequence includes the amino acid sequence shown in SEQ ID NO.7;

[0031] SEQ ID NO.7:

[0032] NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVS GEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYDREQAPNVVYMVTGN PTSDEIKRLPGDIGGGSAQLKKKIAALKKKNAQLKWKIAALKKKLAGGGSGGGSQLEKEIAALEKENAQLEWEIAALEAKELQLVPIGVAPNANVQELKRIGWPNAPILIQDFETLPREAPDLVLQRG GGSGGGSQVQLVESGGGLVRPGGSLRLSCVDSERTSYPMGWFRRAPGKEREFVASITWSGIDPTYADSVADRFTTSRDVANNTLYLQMNSLKHEDTAVYYCAAKAPVGNSSSPYDFDYWGQGTQVTVS

[0033] B2) An amino acid sequence of a fusion protein having the same function, obtained by linking a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in B1). Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO. 8.

[0034] SEQ ID NO.8

[0035] MASWSHPQFEKNVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDV NGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYDREQAPNVVY MVTGNPTSDEIKRLPGDIGGGSAQLKKKIAALKKKNAQLKWKIAALKKKLAGGGSGGGSQLEKEIAALEKENAQLEWEIAALEAKELQLVPIGVAPNANVQELKRIGWPNAPILIQDFETLPREAPDLVL QRGGGSGGGSQVQLVESGGGLVRPGGSLRLSCVDSERTSYPMGWFRRAPGKEREFVASITWSGIDPTYADSVADRFTTSRDVANNTLYLQMNSLKHEDTAVYYCAAKAPVGNSSSPYDFDYWGQGTQVTVS

[0036] Furthermore, the protein used to detect the activity of ADAMTS13 can form a hairpin-shaped structure through positive-chain polypeptides and negative-chain polypeptides.

[0037] In a second aspect, the present invention provides a biomaterial selected from at least one of the following:

[0038] C1) A nucleic acid molecule containing the protein encoding the activity of ADAMTS13 described above;

[0039] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0040] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0041] C4) Recombinant microorganisms containing the nucleic acid molecule of C1), or recombinant microorganisms containing the expression cassette of C2), or recombinant microorganisms containing the recombinant vector of C3);

[0042] C5) Recombinant cells, wherein the recombinant cells contain recombinant cells of C1) nucleic acid molecules, or recombinant cells of C2) the expression cassette of the recombinant vector, or recombinant cells of C3) the recombinant vector, or recombinant cells of C4) recombinant microorganisms.

[0043] Furthermore, the nucleotide sequence of the nucleic acid molecule described in C1) is shown in SEQ ID NO.9.

[0044] SEQ ID NO.9:

[0045]

[0046] Furthermore, the recombinant microorganism described in C4) or the recombinant cell described in C5) includes at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0047] It should be noted that this invention does not specifically limit the types and sources of recombinant microorganisms and recombinant cells. Any commercially available recombinant microorganisms or recombinant cells capable of expressing exogenous proteins are protected under this invention. Optionally, the Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis are all commercially available products.

[0048] In a third aspect, the present invention provides a kit comprising at least the protein or the biological material described above for detecting the activity of ADAMTS13.

[0049] Furthermore, the kit also includes a solvent for diluting the protein used to detect ADAMTS13 activity. Optionally, the solvent is at least one of HEPES buffer, MES buffer, PBS buffer, Bis-Tris buffer, and Tris-HCl buffer, with a pH range of at least one in the range of 5.0-9.0.

[0050] In one specific embodiment of the present invention, the solvent is HEPES buffer solution. Optionally, the HEPES buffer solution comprises 25-250 mM HEPES, pH 5.0-7.8, 50-300 mM NaCl, 2-25 mM CaCl2, and 0.01%-0.50% BSA.

[0051] Furthermore, the kit is used to detect the activity of ADAMTS13 in blood samples, biological samples, or chemical samples. Optionally, any blood sample collected from the subject is acceptable; preferably, the blood sample is selected from at least one of whole blood, plasma, and serum; more preferably, the blood sample is serum.

[0052] Furthermore, the kit also includes a coagulation inhibitor, preferably, the coagulation inhibitor comprising at least one of heparin and sodium citrate.

[0053] The detection principle of this kit is as follows: ADAMTS13 acts as a cleavage enzyme of von Willebrand factor (vWF), specifically cleaving the peptide bond between Y1605 and M1606 in the A2 domain of vWF. The minimal functional substrate for ADAMTS13 is the 73-amino acid domain composed of D1596-R1668 in vWF. Further research revealed that in addition to cleaving the conserved sequence A1600-G1609, three other regions, D1614-P1622, I1642-I1651, and E1600-L1666, play an important role in the localization and cleavage efficiency activation of ADAMTS13.

[0054] When Nb, the anti-cpGFP protein, is in close proximity to cpGFP, molecular interactions prevent cpGFP from forming a functional fluorescent group, resulting in the entire fusion protein being almost non-fluorescent. This spatial proximity can be achieved through protein fusion via genetic engineering or by fusing structures with opposite charges. However, as the spatial distance increases, Nb detaches from the opening in the cpGFP tube, and cpGFP resumes luminescence.

[0055] By fusing with strongly charged fragments, Nb and cpGFP can be pulled close to each other by the electrostatic attraction of the strongly charged fragments, resulting in merging. Introducing ADAMTS13 cleavage sites between Nb and strongly charged fragments, or between cpGFP and strongly charged fragments, removes the pull of the strongly charged fragments on Nb or cpGFP when ADAMTS13 is present, causing Nb and cpGFP to separate and cpGFP to resume luminescence.

[0056] This change can be detected and quantified using a known fluorescence intensity meter, including the fluorescence intensity, the amount of change, and the rate of change, thereby calculating the activity of ADAMTS13 in the sample. The excitation wavelength of cpGFP is 488±10 nm, and the detection emission wavelength is 510±10 nm. The detection reaction time can be 5-60 minutes. The fluorescence intensity can be detected at the end of the reaction or at equal time intervals during the reaction, and the rate of change of fluorescence intensity can be calculated. Both methods can reflect the activity of ADAMTS13 in the sample.

[0057] In a fourth aspect, the present invention provides a method for preparing the protein for detecting the activity of ADAMTS13, characterized in that the method includes at least the step of culturing the recombinant microorganism (C4) or the recombinant cell (C5) in the biological material and isolating the protein for detecting the activity of ADAMTS13.

[0058] Furthermore, the structure of the protein used to detect the activity of ADAMTS13 includes a hairpin structure.

[0059] In a fifth aspect, the invention provides the use of the protein, the biomaterial, the kit, or the method described herein for detecting the activity of ADAMTS13 in the preparation of an enzyme product for detecting the activity of ADAMTS13 protein.

[0060] Furthermore, the samples suitable for use with the product include at least one of blood samples, biological samples, or chemical samples. Optionally, the blood sample can be any blood sample collected from the subject; preferably, the blood sample is selected from at least one of whole blood, plasma, and serum; more preferably, the blood sample is serum.

[0061] The beneficial effects of the present invention include, but are not limited to:

[0062] The protein described in this invention for detecting ADAMTS13 activity can directly measure the ADAMTS13 enzyme activity in blood samples, biological samples, or chemical samples, and is suitable for point-of-care testing (POCT), biological sample screening, and drug development scenarios. Attached Figure Description

[0063] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0064] Figure 1 This is a recombination strategy and three-dimensional structure diagram of the protein used to detect the activity of ADAMTS13 in an embodiment of the present invention, wherein A is a schematic diagram of the open reading frame element of the substrate protein and B is a three-dimensional structure diagram of the protein.

[0065] Figure 2 This is a schematic diagram illustrating the detection of ADAMTS13 enzyme activity at different concentrations in an embodiment of the present invention.

[0066] Figure 3 This is a correlation analysis diagram between the substrate of the present invention and FRETS-VWF73 in an embodiment of the present invention. Detailed Implementation

[0067] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0068] The assay methods for determining ADAMTS13 activity in blood, biological, or chemical samples using the substrates of the present invention are also applicable to assay methods for detecting ADAMTS13 inhibitors in blood, biological, or chemical samples by measuring ADAMTS13 activity using the substrates of the present invention (hereinafter referred to as "the assay methods of the present invention"); and to ADAMTS13 activity assay kits containing the substrates of the present invention (hereinafter referred to as "the kits of the present invention").

[0069] Example 1: Substrate plasmid construction and protein expression

[0070] The nucleotide sequences of all amino acid sequences were codon-optimized using *E. coli* (amino acid sequences as shown in SEQ ID NO. 7, nucleotide sequences as shown in SEQ ID NO. 8), and the resulting gene was synthesized and loaded into the pUC19 backbone to form an amino acid fragment plasmid library. Homologous recombination arm primers were designed (Table 1) to amplify coding sequence fragments 1-6 and the backbone fragment pET51b. Amplification was performed using Q5 high-fidelity polymerase (NEB). After the reaction, the product was digested with DpnI enzyme (NEB), and the amplified fragments were recovered using a DNA recovery kit (NEB). Homologous recombination was then performed on the recovered fragments using NEBuilder HiFi homologous recombination enzyme (NEB) to form a fusion protein (Chain A+B) expression plasmid. The recombination strategy and three-dimensional structure diagram of the protein used to detect ADAMTS13 activity are shown in the figure below. Figure 1 As shown, A is a schematic diagram of the open reading frame element for detecting the substrate protein, and B is a three-dimensional structural diagram of the protein. The recombinant plasmid was transformed into competent Top10 *E. coli* cells (Beyotime Biotechnology Co., Ltd.), and positive clones for ampicillin (Beyotime Biotechnology Co., Ltd.) were screened. The positive clones were confirmed by sequencing of the inserted fragment, and plasmids were extracted from clones containing the correct sequence.

[0071] Table 1: List of primers for fragment amplification

[0072]

[0073] 2. Protein expression and purification

[0074] Chain A and B plasmids were transformed into competent Shuffle T7 E. coli cells, and high-copy-positive clones were selected and amplified to optical density (OD). 600 The protein concentration was approximately 1.5. The mixture was then cooled and induced with IPTG. After induction, the bacterial cells were precipitated, and the supernatant was extracted using bacterial lysis buffer. Chain A+B protein was then purified using a Ni-NTA column for affinity purification, followed by desalting on a dextran gel G25 column. The final protein concentration was adjusted to 2 mg / mL and stored at -80 ℃ for later use.

[0075] Example 2: ADAMTS13 Activity Assay

[0076] 1. Preparation of the reaction system:

[0077] 1) Substrate solution: Take 200 μL of Chain A+B solution (2 mg / mL) and dilute to 10 mL with HEPES buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.2% BSA).

[0078] 2) Standard stock solution (10 µg / mL): 20 μg of recombinant ADAMTS13 protein (R&D system) was dissolved in 2 mL of HEPES buffer, aliquoted into 0.2 mL tubes and stored at -80 °C. It was thawed in an ice bath before use.

[0079] 3) Preparation of standard solutions: Prepare working solutions of 0, 10, 50, 100, 250, 500, and 1000 ng / mL using the stock solution and HEPES buffer.

[0080] 4) Reaction system: Take 200 μL of working solution and add 2 µL of standard solution of each concentration to each well, and repeat the reaction in 3 wells for each well.

[0081] 5) Reaction and measurement: The reaction was carried out in a completely black 96-well plate and incubated at 37 °C using the heating module of the microplate reader for 60 min. Starting from 30 min, the fluorescence signal intensity was read every 2 minutes.

[0082] 2. The results are as follows Figure 2 The results showed that after 30 minutes of incubation, significant differences in fluorescence signals were observed among the different concentration groups. For example, the fluorescence signal in the 1000 ng / mL group (1147.9 ± 103.3 AU) was significantly higher than that in the 20 ng / mL group (250.3 ± 28.7 AU). This dose-dependent signal difference persisted until the last detection time point of 60 minutes, indicating that the substrate was efficiently cleaved by ADAMTS13, and the fluorescence signal increased with the release of the cleavage product. This cleavage was dose-dependent and the dynamic range covered a wide range from healthy individuals to TTP patients, demonstrating broad diagnostic applicability. It should be noted that in shorter reaction times, the difference was less significant in the low concentration groups due to interference from the background fluorescence signal (190.7 ± 44.17 AU). Therefore, for extremely rapid detection (reaction time less than 25 minutes), optimization of the background signal of the reaction system is necessary.

[0083] For the fluorescence values ​​at 30, 40, 50, and 60 min, linear regression analysis of enzyme content versus fluorescence intensity was performed. The results, as shown in Table 2, all showed good linearity (R²>0.98). It should be noted that there is a possibility of a hook effect in the reaction system at the highest concentration (1000 ng / mL). The reaction system needs to be optimized before implementation. However, this risk can be avoided by controlling the reaction time.

[0084] Table 2. Data from linear regression analysis of enzyme content and fluorescence intensity

[0085]

[0086] Example 3: Serum assay for ADAMTS13 enzyme activity

[0087] This example tested 25 clinical plasma samples containing sodium citrate (3.2%), and compared the substrate of this invention with the FRETS-VWF73 (AnaSpec) substrate. Except for the FRETS-VWF73 substrate concentration of 0.25 mg / mL, the excitation wavelength of 340 nm, and the emission wavelength of 490 nm, all other experimental conditions were consistent with those in Example 2, with an incubation time of 45 min. The results, based on 1 IU / mL as 100% activity, are shown in Table 3. Statistical analysis was performed on the data of the substrate of this invention and FRETS-VWF73 (…). Figure 3 The linear regression equation is y = 1.0213x + 1.7568, the Pearson correlation coefficient is r = 0.9970, and the coefficient of determination is R² = 0.9940, indicating that the data obtained by the substrate of this invention and the FRETS-VWF73 detection method are highly correlated and the two methods have good consistency. Therefore, the ADAMTS13 enzyme activity detection based on the substrate of this invention can be used as an alternative to the detection method based on the FRETS-VWF73 substrate.

[0088] Table 3. Detection of ADAMTS13 activity in plasma from 25 human cases

[0089]

[0090] This invention discloses a novel substrate basic element, combination method, preparation method, and detection method for detecting ADAMTS13 activity based on a homogeneous fluorescence method. Those skilled in the art can refer to the above description and appropriately modify the process parameters to implement and apply the technology of this invention. This patent cannot exhaustively describe all embodiments. However, any modifications, supplements, or similar alternatives derived therefrom are still within the protection scope of this invention.

Claims

1. A protein for detecting the activity of ADAMTS13, characterized in that, The amino acid sequence of the protein used to detect ADAMTS13 activity is selected from at least one of the following: A1) The amino acid sequence is as shown in SEQ ID NO.7; A2) An amino acid sequence of a fusion protein with the same function obtained by attaching a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

2. A biomaterial, characterized in that, The biomaterial is selected from at least one of the following: B1) A nucleic acid molecule containing the protein encoding the protein of claim 1 for detecting ADAMTS13 activity; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Recombinant cells containing the nucleic acid molecule described in B1), or recombinant cells containing the expression cassette described in B2), or recombinant cells containing the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule described in B1) is shown in SEQ ID NO.

8.

4. The biomaterial according to claim 2, characterized in that, The recombinant microorganisms described in B4 include at least one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

5. The biomaterial according to claim 2, characterized in that, The recombinant cells described in B5 include at least one type of hamster ovary cells and silkworm ovary cells.

6. A reagent kit, characterized in that, The kit comprises at least the protein for detecting ADAMTS13 activity as described in claim 1 or the biological material as described in any one of claims 2-5.

7. A method for preparing the protein of claim 1 for detecting ADAMTS13 activity, characterized in that, The method includes at least the step of culturing the recombinant microorganism (B4) or the recombinant cell (B5) in any of the biological materials of claims 2-5, and isolating the protein used to detect the activity of ADAMTS13.

8. The use of the protein for detecting ADAMTS13 activity according to claim 1, or the biomaterial according to any one of claims 2-5, or the kit according to claim 6, or the method according to claim 7, in the preparation of an enzyme product for detecting the activity of ADAMTS13 protein.

9. The application according to claim 8, characterized in that, The sample suitable for the product is selected from at least one of biological or chemical samples.

10. The application according to claim 8, characterized in that, The sample suitable for use with the product is a blood sample.

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