A substrate modified peptide for detecting ADAMTS13 enzyme activity, and a detection method and application thereof

Abstract

The application relates to the field of biomedical technology, in particular to a substrate modified peptide for detecting ADAMTS13 enzyme activity and a detection method and application. The application provides a substrate modified peptide for detecting ADAMTS13 enzyme activity, the amino acid sequence of the substrate modified peptide is shown as SEQ ID NO. 1, and the modification method is that a 5-FAM group is marked at the 4th amino acid and a Dabcyl group is marked at the 15th amino acid. The substrate modified peptide and the detection method provided by the application are not only high in accuracy, but also significantly improve the detection sensitivity.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and particularly relates to a substrate modified peptide for detecting ADAMTS13 enzyme activity, a detection method and application. BACKGROUND

[0002] Thrombotic thrombocytopenic purpura (TTP) is a severe diffuse thrombotic microangiopathy, which is mainly characterized by thrombocytopenia and microangiopathic hemolytic anemia, and can be accompanied by central nervous system abnormalities, varying degrees of kidney damage and fever. The disease has an acute onset and a dangerous condition, with a mortality rate of more than 90%, and a poor prognosis. At present, plasma exchange therapy can improve the survival rate to a certain extent.

[0003] A number of studies have confirmed that the mechanism of TTP is mainly due to the decrease or lack of plasma ADAMTS13 enzyme content. ADAMTS13 is a plasma protease that can cleave VWF (Von Willebrand Factor) into smaller fragments, thereby eliminating abnormal large VWF polymers. The UL-VWF is derived from vascular endothelial cells, and is synthesized in cells by multiple VWF monomers, first connected tail to tail by the C-terminal disulfide bond to form a dimer, and then connected head to head by the disulfide bond at the N-terminal of the multiple dimers to form a super large molecular weight VWF polymer. The larger the molecular weight of the VWF polymer, the stronger its adhesion activity, the stronger its ability to entrap platelets, and the more thrombosis. When the activity of ADAMTS13 enzyme is reduced or absent, the formation of super large molecular weight VWF increases, platelet consumption increases, and thrombosis increases, thereby causing TTP. Therefore, rapid detection of ADAMTS13 enzyme activity plays a key role in the prevention and treatment of TTP.

[0004] So far, there have been a variety of methods and reagents for detecting ADAMTS13 enzyme activity. There are mainly two types commonly used in clinical practice: FRET-VWF fluorescent substrate assay and residual collagen binding assay. The residual collagen binding assay (VWF-CBA) is to dilute the serum and add it to the enzyme-labeled plate coated with VWF-containing type III collagen after blocking; the FRET-VWF fluorescent substrate assay is to use chemical synthesis or genetic engineering method to obtain a 73-amino acid VWF substrate from D1596 (D, aspartic acid, Asp) to R1688 (R, arginine, Arg) and to label it with fluorescence, and to use energy transfer between different sites to detect ADAMTS13 enzyme activity. A number of clinical studies have shown that the FRET-VWF fluorescent substrate assay is more rapid, simple, accurate and more suitable for clinical detection.

[0005] There are many kinds of fluorescent substrates for detecting ADAMTS13 enzyme activity, but all of them are based on VWF73 fragment. VWF73 fragment is the smallest substrate fragment that can be cut by ADAMTS13 enzyme, which is located in the A2 region of VWF protein and includes 73 amino acids from D1596 to R1688.

[0006] The commonly accepted fluorescent substrate is the immunofluorescence resonance energy transfer method fluorescent substrate established by Kokame. The substrate is chemically synthesized, and a donor fluorescent group Nma is introduced at the Q1599 (Q, glutamine, Gln) site of the VWF protein, and a receptor fluorescent group Dnp is introduced at the N1610 (N, asparagine, Asn) site. When a certain wavelength of laser irradiates the fluorescent substrate, the fluorescent energy emitted by the donor fluorescent group Nma at the Q1599 site is absorbed by the receptor fluorescent group Dnp at the adjacent N1610 site. When the fluorescent substrate is hydrolyzed by ADAMTS13 enzyme, the fluorescent energy emitted by the donor fluorescent group Nma at the Q1599 site cannot be absorbed by the receptor fluorescent group Dnp at the adjacent N1610 site, so that the fluorescent energy emitted by the donor fluorescent group Nma at the Q1599 site can be measured on the detection instrument. However, the Nma fluorescent signal is not strong, which often leads to low detection sensitivity. SUMMARY

[0007] In view of this, the purpose of the present application is to provide a substrate modified peptide for detecting ADAMTS13 enzyme activity with higher sensitivity and accuracy, and a detection method and application thereof.

[0008] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0009] The present application provides a substrate modified peptide for detecting ADAMTS13 enzyme activity, and the amino acid sequence of the substrate modified peptide is shown as SEQ ID NO. 1. The modification method is that the 4th amino acid is labeled with a 5-FAM group and the 15th amino acid is labeled with a Dabcyl group.

[0010] As a preferred, the amino acid sequence of the substrate modified peptide is obtained by mutating the 1596-1632 and 1644-1668 amino acids of the VWF protein.

[0011] As a preferred, the mutation sites are the 1599th and 1610th amino acids of the VWF protein; the glutamine at the 1599th position is mutated to lysine, and the asparagine at the 1610th position is mutated to lysine.

[0012] The present application also provides the application of the substrate modified peptide for detecting ADAMTS13 enzyme activity in detecting ADAMTS13 enzyme activity.

[0013] The application also provides a method for detecting ADAMTS13 enzyme activity, comprising the following steps:

[0014] (1) mixing the sample to be detected with a substrate modification peptide to obtain a mixture;

[0015] (2) detecting the fluorescence intensity of the mixture at 490 nm and 520 nm, and calculating the ADAMTS13 enzyme activity according to the fluorescence intensity value;

[0016] The substrate modification peptide is the substrate modification peptide.

[0017] Preferably, the initial concentration of the substrate modification peptide is 1-3 μmol / l.

[0018] Preferably, the volume ratio of the sample to be detected to the substrate modification peptide is 1:8-12.

[0019] The application has the following beneficial effects:

[0020] The application provides a substrate modification peptide for detecting ADAMTS13 enzyme activity, wherein the amino acid sequence of the substrate modification peptide is shown as SEQ ID NO. 1, and the modification method is that the 4th amino acid is labeled with a 5-FAM group and the 15th amino acid is labeled with a Dabcyl group. When a certain wavelength laser irradiates the modification peptide, the fluorescence group emits energy, and the energy is just absorbed by the quenching group. When the modification peptide is enzymolyzed by ADAMTS13 enzyme, the fluorescence group and the quenching group are separated from each other, and the emitted energy can be detected. Fluorescein is widely used, including fluorescence microscopy, flow cytometry, etc. The fluorescence group used in the application is the commonly used 5-FAM, which has strong fluorescence signal and lower cost. The quenching group Dabcyl is used in combination with 5-FAM, which has good quenching effect and can greatly improve the sensitivity of the experiment. The substrate modification peptide of the application has higher sensitivity and better accuracy compared with the fluorescence substrate of the immunofluorescence resonance energy transfer method established by Kokame.

[0021] The detection principle of the application is energy resonance transfer of two adjacent fluorescence groups. When a certain wavelength laser irradiates the sample to be detected and the substrate mixture, if the ADAMTS13 enzyme activity in the sample is very low and cannot cut the modification peptide, the two modification groups are close to each other, the fluorescence emitted by the 5-FAM of the 4th amino acid is just absorbed by the 15th Dabcyl modification group, and no energy is emitted; when the ADAMTS13 enzyme activity in the sample is very high, the modification peptide is cut by ADAMTS13, the two modification groups are separated from each other, the energy is emitted, and the ADAMTS13 enzyme activity is detected. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1Dynamic curve graph of the reaction of the substrate modification peptide of Example 1 with different concentrations of standard sample;

[0023] Figure 2 Standard curve of the substrate modification peptide of Example 1 (left), and curve of detecting 2.5% standard sample concentration (right);

[0024] Figure 3 Dynamic curve graph of the reaction of the fluorescent substrate of Comparative Example 1 with different concentrations of standard sample;

[0025] Figure 4 Standard curve of the fluorescent substrate of Comparative Example 1 (left), and curve of detecting 10% standard sample concentration (right). DETAILED DESCRIPTION

[0026] The present application provides a substrate modification peptide for detecting ADAMTS13 enzyme activity, and the amino acid sequence of the substrate modification peptide is shown as SEQ ID NO. 1, and the modification method is that the 4th amino acid is labeled with a 5-FAM group and the 15th amino acid is labeled with a Dabcyl group.

[0027] SEQ ID NO. 1 is: DREKAPNLVYMVTGKPASDEIKRLPGDIQVVPIGVGPWPNAPILIQDFETLPREAPDLVLQR.

[0028] In the present application, the amino acid sequence of the substrate modification peptide is obtained by mutating the 1596th-1632nd and 1644th-1668th amino acids of VWF protein.

[0029] In the present application, the mutation sites are the 1599th and 1610th amino acids of VWF protein; the glutamine at the 1599th position is mutated into lysine, and the asparagine at the 1610th position is mutated into lysine.

[0030] The present application also provides the use of the substrate modification peptide for detecting ADAMTS13 enzyme activity in detecting ADAMTS13 enzyme activity.

[0031] The present application also provides a method for detecting ADAMTS13 enzyme activity, comprising the following steps:

[0032] (1) mixing a sample to be detected with a substrate modification peptide to obtain a mixture;

[0033] (2) detecting the fluorescence intensity of the mixture at 490 nm and 520 nm, and calculating the ADAMTS13 enzyme activity according to the fluorescence intensity value;

[0034] The substrate modification peptide is the substrate modification peptide.

[0035] The sample to be tested is blood plasma to be tested; the blood plasma to be tested is blood plasma after dilution or without dilution; the diluent is 5mmol / l Bis-Tris, 25mmol / l CaCl2, 0.005% Tween-20 with pH 6.0. The dilution ratio is 0-400 times.

[0036] In the present application, the initial concentration of the substrate modification peptide is 1-3 μmol / l, preferably 2 μmol / l.

[0037] In the present application, the volume ratio of the sample to be tested to the substrate modification peptide is 1:8-12, preferably 1:10.

[0038] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0039] Example 1 A substrate modification peptide for detecting ADAMTS13 enzyme activity

[0040] The substrate modification peptide is chemically synthesized by Shanghai Hong Peptide Biotechnology Co., Ltd., which comprises VWF protein positions 1596-1632 and 1644-1668, a total of 62 amino acids; wherein the 1599th amino acid is converted from glutamine (Gln, Q) to lysine (Lys, K) and modified with a 5-FAM fluorescent group; the 1610th amino acid is converted from asparagine (Asn, N) to lysine (Lys, K) and modified with a quenching group Dabcyl. The relative molecular mass of the substrate modification peptide is 7484.44, and the purity is 95.99%. The amino acid sequence of the substrate modification peptide is shown in SEQ ID NO. 1 (i.e. the modification method for SEQ ID NO. 1 is that the 4th amino acid is labeled with a 5-FAM group and the 15th amino acid is labeled with a Dabcyl group).

[0041] Comparative Example 1 Fluorescent substrate of immunofluorescence resonance energy transfer method established by Kokame

[0042] The fluorescent substrate is chemically synthesized, which comprises VWF protein positions D1596-R1688, a total of 73 amino acids as shown in SEQ ID NO. 2; a donor fluorescent group Nma is introduced at the Q1599 position of the VWF protein, and an acceptor fluorescent group Dnp is introduced at the N1610 position. (i.e. the modification method for SEQ ID NO. 2 is that the 4th amino acid is labeled with an Nma group and the 15th amino acid is labeled with a Dnp group).

[0043] The amino acid sequence of SEQ ID NO. 2 is: DREXAPNLVYMVTGXPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQR.

[0044] wherein X is a 2,3-diaminopropionic acid residue modified by -2-(N-).

[0045] Sensitivity of the substrate-modified peptide for detecting ADAMTS 13 enzyme activity in Example 2

[0046] Twenty normal human sodium citrate-anticoagulated platelet-poor plasmas were collected and mixed as normal pooled plasma. The normal pooled plasma and diluent were mixed to prepare the activity standard, and the preparation of the activity standard is shown in Table 1. The diluent was 5 mmol / 1 Bis-Tris, 25 mmol / 1 CaCl2, 0.005% Tween-20, pH 6.0.

[0047] Table 1 Activity standard

[0048] Standard Mixed plasma (μl) Reaction buffer (μl) 100％ 20 180 80％ 16 184 60％ 12 188 40％ 8 192 20％ 4 196 10％ 2 198 5％ 1 199 2.5％ 0.5 199.5 0 0 200

[0049] One hundred microliters of each gradient standard was added to 100 microliters of 2 micromol / 1 substrate-modified peptide of Example 1. The absorbance value of the activity standard was measured at an excitation wavelength of 490 nm and an emission wavelength of 520 nm, and the absorbance value was read every 5 min, for 12 times, for a total reaction time of 1 h. The dynamic curve of the reaction of the substrate-modified peptide with different concentrations of the standard is shown in Figure 1 The standard curve (left) (y = 4E-0.5x 2 + 0.0014x - 0.1246, R 2 = 0.9992) of the substrate-modified peptide and the curve for detecting 2.5% standard concentration (right) are shown in Figure 2

[0050] Sensitivity of the fluorescent substrate for immunofluorescence resonance energy transfer method established by Kokame in Comparative Example 2

[0051] According to the technical solution of Reference Example 2, the substrate-modified peptide of Example 1 therein was replaced by the fluorescent substrate of Comparative Example 1, and the absorbance value of the activity standard was measured at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The other steps were the same as those of Example 2. The dynamic curve of the reaction of the fluorescent substrate with different concentrations of the standard is shown in Figure 3 The standard curve (left) (y = 0.0155x 2 + 0.2898x + 1.1031, R 2 = 0.9937) of the fluorescent substrate and the curve for detecting 10% standard concentration (right) are shown in​Figure 4 as shown.

[0052] as shown. Figure 1 , Figure 3 As shown, the two substrates have different differences in the cleavage activity of ADAMTS13 enzyme, the energy released by the substrate modification peptide of Example 1 is more than 10 times that of the fluorescent substrate of Comparative Example 1. The detection sensitivity of the fluorescent substrate of Comparative Example 1 is 10%, and the sensitivity of the substrate modification peptide of Example 1 can be less than 5%.

[0053] As shown. Figure 2 , Figure 4 As shown, the fluorescence amplification curve of the substrate modification peptide of Example 1 is more stable in low concentration activity detection, the result is more reliable, and the sensitivity is higher.

[0054] Accuracy of the substrate modification peptide for detecting ADAMTS13 enzyme activity in Example 3

[0055] Take 8 different plasma samples, 1-3 are clinically diagnosed thrombotic thrombocytopenic purpura patients, and 4-8 are normal human control plasma. Each takes 20 μl of plasma, adds 180 μl of diluent, and obtains samples 1-8 to be tested.

[0056] Take 100 μl of each of samples 1-8 to be tested and mix with 100 μl of 2 μmol / l substrate modification peptide of Example 1 to obtain mixtures 1-8. Under the conditions of excitation wavelength 490 nm and emission wavelength 520 nm, the fluorescence intensity of reaction systems 1-8 is detected, the absorbance value is read every 5 min, and the total reaction time is 1 h. The curve is obtained, and the slope is measured.

[0057] The reaction slope is brought into the corresponding standard curve (see Figure 2 left, standard curve of substrate modification peptide) to calculate the ADAMTS13 enzyme activity of the plasma to be tested. The calculation results are shown in Table 2.

[0058] Accuracy of the fluorescent substrate of Comparative Example 3 Kokame established by immunofluorescence resonance energy transfer method

[0059] According to the technical scheme of Example 3, the fluorescent substrate of Comparative Example 1 is replaced by the substrate modification peptide of Example 1, and the fluorescence intensity of the reaction system is detected under the conditions of excitation wavelength 340 nm and emission wavelength 450 nm. The other steps are the same as in Example 3.

[0060] The reaction slope is brought into the corresponding standard curve (see Figure 4 left, standard curve of fluorescent substrate) to calculate the ADAMTS13 enzyme activity of the plasma to be tested. The calculation results are shown in Table 2.

[0061] Table 2 Detection of ADAMTS13 enzyme activity of the plasma to be tested

[0062]

[0063] It can be seen from Table 2 that the fluorescent substrate of Comparative Example 1 cannot be accurately quantified under the condition of less than 10% activity, and the sensitivity is low. The substrate modified peptide of Example 1 and the enzyme detection method are completely consistent with the clinical performance, and can provide a powerful tool for the diagnosis of clinical TTP.

[0064] As can be seen from the above examples and comparative examples, the application provides a substrate modified peptide for detecting ADAMTS13 enzyme activity and a detection method and application, the amino acid sequence of the substrate modified peptide is shown as SEQ ID NO. 1, and the modification method is that the 4th amino acid is labeled with a 5-FAM group and the 15th amino acid is labeled with a Dabcyl group. The substrate modified peptide and the detection method of the application have high accuracy and high sensitivity.

[0065] The above only describes the preferred embodiments of the present application, and it should be pointed out that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A substrate-modified peptide for detecting ADAMTS 13 enzyme activity, characterized in that, The amino acid sequence of the substrate modification peptide is shown as SEQ ID NO. 1, and the modification method is that the 4th amino acid is labeled with a 5-FAM group and the 15th amino acid is labeled with a Dabcyl group.

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