A protein combination for detecting ADAMTS13 activity and application thereof

Abstract

The present application relates to the field of proteins, in particular to a protein combination for detecting ADAMTS13 activity and application thereof.The protein combination for detecting ADAMTS13 activity provided by the present application is composed of two protein subchains Chain A and Chain B, wherein Chain A comprises a circularly arranged GFP as shown in SEQ ID NO.1, a V Part1 of a vWF variant as shown in SEQ ID NO.2 and a negative chain polypeptide as shown in SEQ ID NO.5; Chain B comprises a nanobody as shown in SEQ ID NO.6, a V Part2 of a vWF (D1596-R1668) variant as shown in SEQ ID NO.3 and a positive chain polypeptide as shown in SEQ ID NO.4.The protein combination for detecting ADAMTS13 activity provided by the present application can directly determine the ADAMTS13 enzyme activity in a blood sample, a biological sample or a chemical sample, and is suitable for clinical point-of-care testing, 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 combination for detecting ADAMTS13 activity and its application. 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 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 denaturing agents for unfolding. However, there are no denaturing agents 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. The synthesis of substrates with 3 polypeptides 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, which is difficult to meet the needs of large-scale TTP screening, and is not suitable for the clinical needs of emergency diagnosis and treatment of emergency room patients; (4) Magnetic microparticle chemiluminescence detection: 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 combination based on green fluorescent protein spatial conformation recombination technology for detecting the activity of ADAMTS13, thereby achieving rapid and accurate detection of ADAMTS13 activity.

[0006] In a first aspect, the present invention provides a protein assembly for detecting the activity of ADAMTS13, wherein the protein assembly for detecting the activity of ADAMTS13 comprises two protein daughter chains, Chain A and Chain B, wherein...

[0007] Chain A includes a circularly arranged GFP (cpGFP) as shown in SEQ ID NO.1, V Part 1 of the vWF (D1596-R1668) variant as shown in SEQ ID NO.2, and a negative-chain polypeptide as shown in SEQ ID NO.5;

[0008] Chain B includes a nanobody as shown in SEQ ID NO. 6, V Part 2 of the vWF (D1596-R1668) variant as shown in SEQ ID NO. 3, and a positive-chain polypeptide as shown in SEQ ID NO. 4.

[0009] The sequences of each part are as follows:

[0010] SEQ ID NO: 1:

[0011] NVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVN GHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEY

[0012] SEQ ID NO: 2:

[0013] DREQAPNVVYMVTGNPTSDEIKRLPGDIQLVPIG

[0014] SEQ ID NO: 3:

[0015] VAPNANVQELKRIGWPNAPILIQDFETLPREAPDLVLQR

[0016] SEQ ID NO: 4:

[0017] AQLKKKIAALKKKNAQLKWKIAALKKKLA

[0018] SEQ ID NO: 5:

[0019] SQLEKEIAALEKENAQLEWEIAALEKELA

[0020] SEQ ID NO: 6:

[0021] QVQLVESGGGLVRPGGSLRLSCVDSERTSYPMGWFRRAPGKEREFVASITWSGIDPTYADSVADRFTTSRDVANNTLYLQMNSLKHEDTAVYYCAAKAPVGNSSSPYDFDYWGQGTQVTVS

[0022] 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.

[0023] In a specific embodiment of the present invention, the various parts are directly connected.

[0024] Furthermore, the amino acid sequence of the protein used to detect ADAMTS13 activity includes at least one of the following:

[0025] B1) The amino acid sequence of Chain A includes the amino acid sequence shown in SEQ ID NO.7, and the amino acid sequence of Chain B includes the amino acid sequence shown in SEQ ID NO.8;

[0026] B2) 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 B1).

[0027] SEQ ID NO: 7:

[0028] MNVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLP VPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYDREQAPNVVYMVTGNPTSDEIKRLPGDIQLVPIGAQLKKKIAALKKKNAQLKWKIAALKKKLA

[0029] SEQ ID NO: 8:

[0030] MQVQLVESGGGLVRPGGSLRLSCVDSERTSYPMGWFRRAPGKEREFVASITWSGIDPTYADSVADRFTTSRDVANNTLYLQMNSLKHEDTAVYYCAAKAPVGNSSSPYDFDYWGQGTQVTVSVAPNANVQELKRIGWPNAPILIQDFETLPREAPDLVLQRSQLEKEIAALEKENAQLEWEIAALEKELA

[0031] Furthermore, the sequence of the tag protein is HHHHHH.

[0032] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein of Chain A after adding the tag is shown in SEQ ID NO.9:

[0033] MNVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPW PTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYDREQAPNVVYMVTGNPTSDEIKRLPGDIQLVPIGAQLKKKIAALKKKNAQLKWKIAALKKKLAHHHHHH

[0034] The amino acid sequence of the fusion protein after Chain B is tagged is shown in SEQ ID NO: 10:

[0035] MQVQLVESGGGLVRPGGSLRLSCVDSERTSYPMGWFRRAPGKEREFVASITWSGIDPTYADSVADRFTTSRDVANNTLYLQMNSLKHEDTAVYYCAAKAPVGNSSSPYDFDYWGQGTQVTVSVAPNANVQELKRIGWPNAPILIQDFETLPREAPDLVLQRSQLEKEIAALEKENAQLEWEIAALEKELAHHHHHH

[0036] Furthermore, the protein used to detect the activity of ADAMTS13 can form a structure of two parallel polypeptides consisting of a positive-chain polypeptide and a negative-chain polypeptide.

[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 a combination of proteins 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 as follows:

[0044] The nucleotide sequence of Chain A includes the nucleotide sequence shown in SEQ ID NO.11, and the nucleotide sequence of Chain B includes the nucleotide sequence shown in SEQ ID NO.12.

[0045] SEQ ID NO.11:

[0046] ATGAATGTATATATTAAAGCCGATAAGCAAAAAAATGGGATCAAAGCGAACTTTCACATTCGACACAACATCGAAGATGGTGGCGTTCAGCTCGCGTATCACTACCAGCAAAACACGCCCATCGGTGATGGCCCTGTTCTCCTTCCTGATAACCATTATTTAAGCGTTCAATCGAAACTCAGTAAAGATCCTAACGAAAAACGGGACCATATGGTGCTGCTGGAATTTGTGACCGCTGCCGGCATCACGTTGGGCATGGATGAACTGTACAAGGGAGGCACGGGTGGCAGCATGGTTTCTAAAGGAGAAGAGCTGTTTACGGGCGTCGTGCCTATTCTGGTGGAACTCGATGGTGATGTGAATGGACATAAATTTTCGGTTAGCGGCGAGGGCGAAGGCGACGCGACATATGGAAAACTGACCCTCAAGTTCATTTGCACTACCGGTAAACTGCCCGTTCCTTGGCCTACCTTGGTAACCACGTTGACCTATGGCGTCCAGTGCTTTTCACGTTATCCGGATCATATGAAACAGCATGATTTTTTTAAGTCAGCAATGCCGGAAGGCTACATTCAAGAACGTACTATCTTCTTCAAAGATGATGGCAATTACAAAACCCGTGCCGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGTATTGAACTCAAAGGCATTGATTTTAAAGAAGACGGAAACATCCTCGGTCACAAATTGGAATACGATCGCGAACAAGCACCCAACGTTGTGTATATGGTCACCGGTAATCCTACATCAGACGAAATCAAACGGCTGCCTGGCGATATTCAGCTGGTGCCAATTGGTGCCCAGCTGAAAAAGAAAATTGCGGCACTGAAGAAGAAGAATGCACAGTTAAAATGGAAAATTGCGGCACTGAAAAAGAAATTAGCGCACCACCACCACCACCAC

[0047] SEQ ID NO.12:

[0048] ATGCAGGTTCAGCTGGTAGAATCTGGCGGAGGGCTGGTGCGCCCGGGCGGCTCACTCCGTCTGAGCTGCGTGGATAGTGAACGCACGTCATACCCGATGGGGTGGTTCCGTCGTGCCCCGGGCAAAGAACGTGAATTTGTGGCGTCT ATCACTTGGTCGGGGATTGATCCGACCTACGCCGATTCCGTGGCGGATCGCTTTACGACCTCACGCGATGTGGCTAATAATACTCTCTATTTACAGATGAACAGTTTAAAACATGAAGATACCGCGGTATATTACTGCGCCGCCAAA GCCCCGGTAGGAAACTCATCTAGCCCTTATGACTTTGATTACTGGGGCCAAGGTACTCAAGTCACGGTTTCAGTCGCACCGAATGCCAACGTCCAGGAGTTAAAACGTATTGGCTGGCCAAATGCCCCAATTCTGATACAAGACTTC GAAAACTCTTCCTCGCGAAGCTCCCGACCTGGTGCTTCAACGCTCACAACTAGAGAAAGAGATTGCGGCGTTGGAAAAAGAAAACGCCCAACTGGAATGGGAAATTGCCGCCTTGGAAAAAGAATTAGCGCACCACCACCACCACCAC

[0049] 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.

[0050] 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.

[0051] In a third aspect, the present invention provides a kit comprising at least the protein combination for detecting the activity of ADAMTS13 or the biological material described herein.

[0052] Furthermore, the kit also includes a solvent for diluting the protein combination used to detect the activity of ADAMTS13, optionally the solvent being at least one of HEPES buffer, MES buffer, PBS buffer, Bis-Tris buffer, and Tris-HCl buffer, optionally with a pH range of at least one of 5.0-9.0.

[0053] 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.

[0054] 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.

[0055] Furthermore, the kit also includes a coagulation inhibitor, preferably, the coagulation inhibitor comprising at least one of heparin and sodium citrate.

[0056] 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.

[0057] 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 cpGFP tube opening, and cpGFP resumes luminescence.

[0058] 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.

[0059] 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.

[0060] In a fourth aspect, the present invention provides a method for preparing the protein combination for detecting the activity of ADAMTS13, the method comprising at least the steps of culturing the recombinant microorganism (C4) or the recombinant cells (C5) in the biological material and isolating the protein combination for detecting the activity of ADAMTS13.

[0061] Furthermore, the structure of the protein assembly used to detect the activity of ADAMTS13 includes a double-stranded structure.

[0062] In a fifth aspect, the present invention provides the use of the protein combination for detecting the activity of ADAMTS13, or the biological material, or the kit, or the method thereof, in the preparation of an enzyme product for detecting the activity of ADAMTS13 protein.

[0063] Further, the sample suitable for the product includes 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.

[0064] The beneficial effects of the present invention include, but are not limited to:

[0065] The protein combination for detecting ADAMTS13 activity described in this invention 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

[0066] 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:

[0067] Figure 1 This is a fusion strategy and three-dimensional structure diagram of the protein combination 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 elements of Chain A and Chain B, and B is a three-dimensional structure diagram of Chain A and Chain B.

[0068] Figure 2 This is a schematic diagram showing the results of the matching concentration determination of protein Chain A and protein Chain B in an embodiment of the present invention.

[0069] Figure 3 This is a schematic diagram illustrating the detection of ADAMTS13 enzyme activity at different concentrations in an embodiment of the present invention.

[0070] Figure 4 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

[0071] 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.

[0072] 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").

[0073] Example 1: Substrate plasmid construction and protein combinatorial expression

[0074] 1. Plasmid recombination

[0075] Nucleotide sequences of all amino acid sequences were optimized using *E. coli* codons, and genes were synthesized and loaded into the pUC19 backbone to form corresponding amino acid fragment plasmid libraries. Primers with overlapping start and end points were designed (Table 1) to amplify coding sequence fragments 1-6 and backbone fragments pET28-1 and pET28-2. Amplification was performed using a high-fidelity polymerase. After the reaction, the products were digested with DpnI enzyme, and the amplified fragments were recovered using a DNA recovery kit. Sequence fragments 1, 2, and 4 were combined with the pET28-1 backbone, and sequence fragments 6, 3, and 5 were combined with the pET28-2 backbone. Homologous recombination was performed on the recovered fragments using a homologous recombination enzyme to form Chain A and Chain B expression plasmids. The fusion strategy and three-dimensional structure diagram of the protein combination used to detect ADAMTS13 activity are shown below. Figure 1 As shown, A is a schematic diagram of the open reading frame elements of Chain A and Chain B, and B is a three-dimensional structural diagram of Chain A and Chain B. The recombinant plasmid was transformed into competent Top10 *E. coli* cells, and kanamycin-positive clones were screened. Positive clones were amplified and confirmed by insertion fragment sequencing. Clones containing the correct sequence underwent plasmid extraction. The amino acid sequence of Chain A includes the amino acid sequence shown in SEQ ID NO. 9, and the amino acid sequence of Chain B includes the amino acid sequence shown in SEQ ID NO. 10. The nucleotide sequence of Chain A includes the nucleotide sequence shown in SEQ ID NO. 11, and the nucleotide sequence of Chain B includes the nucleotide sequence shown in SEQ ID NO. 12.

[0076] Table 1: List of primers for fragment amplification

[0077]

[0078] 2. Protein expression and purification

[0079] Chain A and Chain 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 concentration was approximately 1.5. The cells were cooled and induced with IPTG. After induction, the bacterial cells were precipitated, and the supernatant was extracted using bacterial lysis buffer. Chain A and Chain B proteins were purified by affinity purification using a Ni-NTA column, desalted using a dextran gel G25 column, and the final protein concentration was adjusted to 1 mg / mL. The cells were then stored at -80 °C for later use.

[0080] 3. Fluorescence detection

[0081] 50 μL of Chain A protein solution (1 mg / mL) was mixed with 0, 10, 50, 100, 150, 200, and 250 μL of Chain B protein solution (1 mg / mL), respectively. The volume was adjusted to 5 mL with HEPES buffer, resulting in a Chain A protein concentration of 10 μg / mL and Chain B protein concentrations of 0, 5, 10, 20, 30, 40, and 50 μg / mL. After incubation at 37 °C for 10 min, the fluorescence signal intensity was measured using a microplate reader (Tecan Infinite Pro2000) with an excitation wavelength of 488 nm and an emission wavelength of 510 nm (n=8).

[0082] The results are as follows Figure 2 As shown, when the Chain B concentration reaches 30 μg / mL, it can block more than 98% of the fluorescence signal of the fused cfGFP.

[0083] Example 2: ADAMTS13 Activity Assay

[0084] 1. Preparation of the reaction system:

[0085] 1) Substrate solution: Take 50 μL Chain A solution (1 mg / mL), 150 μL Chain B solution (1 mg / mL), and 4.8 mL HEPES buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.2% BSA) and mix well.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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 an ELISA reader for 30 min. The fluorescence signal intensity was read every 2 minutes.

[0090] 2. The results are as follows Figure 3As shown, the fluorescence intensity gradually increased over time during the reaction. Taking the 1000 ng / mL concentration group as an example, the increase was slow within 10 minutes (94.9 ± 7.4 AU), accelerating from 30-60 minutes (576.8 ± 47.2 AU to 4725.2 ± 393.2 AU), reflecting the dynamic process of the enzymatic reaction. The fluorescence intensity increased significantly with increasing ADAMTS13 dose, reaching 4725.2 ± 393.2 AU in the 1000 ng / mL group at 60 minutes, much higher than the 25.3 ± 2.1 AU of the 10 ng / mL group. This indicates 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 higher concentration reaction systems, there is a possibility of a hook effect, which needs to be confirmed and ruled out before implementation. Linear regression analysis of enzyme content versus fluorescence intensity was performed on the fluorescence values ​​at different concentrations at time points of 10, 20, 30, 40, 50, and 60 min. The results are shown in Table 2. From 10 to 60 min, all dose ranges showed good linearity (R²>0.92). However, as the reaction time increased, the intercept turned negative, possibly due to the nonlinear growth of the signal in the high-dose range. Therefore, in later applications, attention should be paid to controlling the reaction time, or segmented calculations should be performed for the low and high dose ranges. These steps can be further refined in the development of specific reagent kits and the adaptation of detection equipment, but they do not affect the overall result that the R² for the entire range (0-1 IU / mL) within 60 min is higher than 0.95, proving that the substrate has a strong response to the concentration of ADAMTS13 and is suitable for quantitative detection.

[0091] Different concentrations (0, 10, 50, 100, 250, 500, 1000 ng / mL) of ADAMTS13 were added to the substrate solution and incubated at 37 °C for 60 min. Fluorescence intensity was measured every 2 min.

[0092] The fluorescence values ​​at each concentration at time points of 10, 20, 30, 40, 50, and 60 min were statistically analyzed by linear regression analysis of enzyme content and fluorescence intensity, including linear regression parameters (slope, intercept, R²). The results are shown in Table 2.

[0093] Table 2. Data from linear regression analysis of enzyme content and fluorescence intensity

[0094]

[0095] Example 3: Serum assay for ADAMTS13 enzyme activity

[0096] 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 being 1.75 mg / mL, the excitation wavelength being changed to 340 nm, and the emission wavelength being changed to 490 nm, all other experimental conditions were consistent with those in Example 2, with an incubation time of 30 min. The results, based on 1 IU / mL as 100% activity, are shown in Table 3. Statistical analysis of the data from the substrate of this invention and FRETS-VWF73 showed a linear regression (P<0.001), indicating a high correlation between the data obtained from the detection methods using the substrate of this invention and FRETS-VWF73. The two methods exhibit good consistency; therefore, the ADAMTS13 enzyme activity assay based on the substrate of this invention can serve as an alternative to the detection method based on the FRETS-VWF73 substrate.

[0097] Table 3. Detection of ADAMTS13 activity in plasma from 25 human cases

[0098]

[0099] Twenty-five human plasma samples were tested using both the substrate of this invention and the FRETS-VWF73 substrate. Pearson correlation analysis was performed on the results, and the results are as follows: Figure 4 As shown, the ADAMTS13 activity measured by the two methods exhibits a highly linear positive correlation, with the correlation equation being: y = 0.9952x + 1.1474. The Pearson correlation coefficient is r = 0.9825, and the coefficient of determination is R² = 0.9655 r, indicating that the results from the two methods are highly consistent. Therefore, the new substrate developed in this invention has excellent correlation and consistency with the currently recognized gold standard substrate FRETS-VWF73, and can be used as a reliable substrate for detecting ADAMTS13 activity in clinical or research settings.

[0100] 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 assembly for detecting the activity of ADAMTS13, characterized in that, The protein assembly used to detect ADAMTS13 activity consists of two protein sub-chains, Chain A and Chain B, wherein: The amino acid sequence of the protein combination used to detect ADAMTS13 activity is selected from at least one of the following: A1) The amino acid sequence of Chain A is as shown in SEQ ID NO.7, and the amino acid sequence of Chain B is as shown in SEQ ID NO.8; A2) An amino acid sequence of a fusion protein with the same function obtained by linking a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

2. The protein assembly according to claim 1, characterized in that, The amino acid sequence of the fusion protein of Chain A after adding the tag, as described in A2), is shown in SEQ ID NO.9, and the amino acid sequence of the fusion protein of Chain B after adding the tag, as shown in SEQ ID NO:

10.

3. A biomaterial, characterized in that, The biomaterial is selected from at least one of the following: B1) A nucleic acid molecule encoding the protein combination for detecting the activity of ADAMTS13 as described in any one of claims 1-2; 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 B1), or recombinant cells containing the expression cassette described in B2), or recombinant cells containing the recombinant vector described in B3).

4. The biomaterial according to claim 3, characterized in that, The nucleotide sequences of the nucleic acid molecules described in B1) are as follows: the nucleotide sequence of Chain A is as shown in SEQ ID NO.11, and the nucleotide sequence of Chain B is as shown in SEQ ID NO.

12.

5. The biomaterial according to claim 3, characterized in that, The recombinant microorganisms described in B4) or the recombinant cells described in B5) include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

6. A reagent kit, characterized in that, The kit comprises at least the protein combination for detecting ADAMTS13 activity as described in any one of claims 1-2 or the biological material as described in any one of claims 3-5.

7. A method for preparing the protein combination for detecting ADAMTS13 activity as described in any one of claims 1-2, characterized in that, The method includes at least the step of culturing the recombinant microorganism (B4) or the recombinant cell (B5) from any of the biological materials of claims 3-5, and isolating the protein combination used to detect the activity of ADAMTS13.

8. The use of the protein combination for detecting the activity of ADAMTS13 according to any one of claims 1-2, or the biomaterial according to any one of claims 3-5, 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 the ADAMTS13 protein combination.

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

10. The application according to claim 8, characterized in that, Samples suitable for use with the product include blood samples.

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