Fluorescent probe for nuclease activity determination and detection reagent and application thereof

Abstract

The invention relates to the technical field of biological enzymes, in particular to a fluorescent probe for nuclease activity determination and a detection reagent and application thereof. One end of the modified DNA oligonucleotide fluorescent probe is provided with a fluorophore (F), the other end of the modified DNA oligonucleotide fluorescent probe is provided with a quenching group (Q), and rapid detection of nuclease can be achieved through design and optimization of a system. When the two groups are close to each other, the fluorescence intensity of the fluorescent molecules is attenuated. When the substrate is cut by nuclease, the head end and the tail end of the substrate are separated, the fluorophore and the quenching group are separated, and the VIC fluorophore emits fluorescence. Because the fluorescence increasing speed is in direct proportion to the nuclease activity, the nuclease activity can be detected in real time or dynamically through fluorescence detection. The method can be used for quantitatively analyzing and detecting the activity content of nuclease in intermediate products, semi-finished products and finished products of various biological products, and is high in sensitivity, good in repeatability and rapid in detection.

Description

Technical Field

[0001] The invention relates to the technical field of biological enzymes, in particular to a fluorescent probe for determining nuclease activity and a detection reagent and application thereof. Background Art

[0002] Nuclease is an enzyme commonly found in the environment and biological materials. It can degrade DNA and RNA and interfere with many experiments. In the fields of bioengineering, vaccine production, pharmaceuticals and food processing, nuclease residues will have a significant impact on product quality. Nucleases can degrade all forms of DNA and RNA and do not have base recognition specificity. Therefore, nucleases are often used to remove nucleic acids from recombinant protein drugs, vaccine samples, cells and gene therapy virus samples to reduce the risk of nucleic acid residue toxicity and improve product safety. In addition, in protein analysis sample preparation, improving recombinant protein purification process, increasing protein yield, cell cryopreservation and other aspects, ideal experimental results can also be achieved by using nucleases to treat nucleic acids.

[0003] In the process of treating biological products with nucleases, trace amounts of nuclease residues may be introduced, which will have a certain impact on the application and safety of subsequent biological products. Therefore, in the safety supervision of biological products, the residual amount of nuclease is one of the important indicators to measure the quality of biological products. Summary of the invention

[0004] The technical solution of the present invention is as follows:

[0005] A fluorescent probe for determining nuclease activity, wherein the sequence of the fluorescent probe is any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7;

[0006] The fluorescent group of the fluorescent probe is any one of VIC, FAM or HEX, and the quenching group of the fluorescent probe is any one of Dabcyl, TAMRA or BHQ1.

[0007] Preferably, the sequence of the fluorescent probe is as shown in SEQ ID NO.6, the fluorescent group of the fluorescent probe is HEX, and the quenching group of the fluorescent probe is BHQ1.

[0008] Preferably, the substrate concentration of the fluorescent probe is any one of 2 μM, 4 μM, 6 μM or 8 μM.

[0009] Preferably, the buffer system of the fluorescent probe reaction includes: 100-400 mM Tris pH 8.0, 500-700 mM KCl, and 20-40 mM MgCl2.

[0010] Preferably, the buffer system for the fluorescent probe reaction includes 100-400 mM Tris pH 8.0, 500-700 mM KCl, 20-40 mM MgCl2 and 0.8% glycerol.

[0011] Preferably, the buffer system of the fluorescent probe reaction includes 100-400 mM Tris pH 8.0, 500-700 mM KCl, 20-40 mM MgCl2, 0.8% glycerol and 200 mg / L ribose.

[0012] Preferably, the buffer system for the fluorescent probe reaction includes 100 mM Tris pH 8.0, 500 mM KCl, 20 mM MgCl2, 0.8% glycerol and 200 mg / L ribose.

[0013] Preferably, the amino acid sequence of the universal nuclease standard used by the fluorescent probe to detect the sample to be tested is shown in SEQ ID NO.9.

[0014] Preferably, the universal nuclease standard used in the fluorescent probe detection of the sample to be tested is diluted to 10 U / mL as follows;

[0015] 273KU / mL is diluted to 1000U / mL, then diluted to 20U / mL, and then diluted to 10U / mL, including:

[0016] Previous concentration sample volume (μL) Dilution volume (μL) Final concentration (U / mL) 10 (starting: 273 KU / mL) 2720 1000 20 980 20 500 500 10

[0017] or

[0018] Previous concentration sample volume (μL) Dilution volume (μL) Final concentration (U / mL) 10 (starting: 273 KU / mL) 2720 1000 50 2450 20 500 500 10

[0019] Any one of .

[0020] The invention comprises the use of any of the aforementioned fluorescent probes for determining nuclease activity, which is used for detecting nuclease residues during the process of treating biological products with nucleases.

[0021] Compared with the prior art, the beneficial effects achieved by the present invention are:

[0022] The present invention professionally designs a modified DNA oligonucleotide fluorescent probe, which has a fluorescent group (F) at one end and a quenching group (Q) at the other end. At the same time, through the design and optimization of the system, rapid detection of nucleases can be achieved. When the two groups are close to each other, the fluorescence intensity of the fluorescent molecule itself will decay. When the substrate is cut by the nuclease, the head and tail ends of the substrate are separated, the fluorescent group and the quenching group are separated, and the VIC fluorescent group will emit fluorescence. Because the rate of increase in fluorescence is proportional to the activity of the nuclease, the activity of the nuclease can be detected in real time or dynamically through fluorescence detection. It can be used for quantitative analysis and detection of the nuclease activity content in intermediates, semi-finished products and finished products of various biological products, with high sensitivity, good repeatability and rapid detection.

[0023] 1. The preferred fluorescent probe can be used as a substrate for the activity determination of the universal nuclease, showing good linearity within a certain concentration range, with R2 ≥ 0.98; 2. The preferred fluorescent probe and its detection reagent optimization can improve the high-sensitivity detection of the universal nuclease, with a detection limit of up to 0.1 U / mL; 3. The preferred fluorescent probe and its detection system can achieve rapid and effective detection of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-1;

[0026] Figure 2 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-2;

[0027] Figure 3 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-3;

[0028] Figure 4 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-4;

[0029] Figure 5 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-5;

[0030] Figure 6 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-6;

[0031] Figure 7 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-7;

[0032] Figure 8 This is a diagram showing the effect of nuclease cleavage on the fluorescent substrate FS-8;

[0033] Fig. 9 It is the standard curve of nuclease to fluorescent substrate FS-1;

[0034] Fig.10 It is the standard curve of nuclease to fluorescent substrate FS-2;

[0035] Fig.11 It is the standard curve of nuclease to fluorescent substrate FS-3;

[0036] Fig.12 It is the standard curve of nuclease to fluorescent substrate FS-4;

[0037] Fig.13 It is the standard curve of nuclease to fluorescent substrate FS-5;

[0038] Fig.14 It is the standard curve of nuclease to fluorescent substrate FS-6;

[0039] Fig.15 It is the standard curve of nuclease to fluorescent substrate FS-7;

[0040] Fig.16 is the enzyme activity progress curve obtained at 2 μM substrate concentration;

[0041] Fig.17 is the enzyme activity progress curve obtained at 4 μM substrate concentration;

[0042] Fig.18 is the enzyme activity progress curve obtained at 6 μM substrate concentration;

[0043] Fig.19 is the enzyme activity progress curve obtained at 8 μM substrate concentration;

[0044] Fig. 20 is the standard curve corresponding to 2 μM substrate concentration;

[0045] Fig.21 is the standard curve corresponding to 4 μM substrate concentration;

[0046] Fig. 22 is the standard curve corresponding to 6 μM substrate concentration;

[0047] Fig.23 is the standard curve corresponding to 8 μM substrate concentration;

[0048] Fig.24 The three-dimensional structure of the universal nuclease;

[0049] Fig.25 Comparison of different dilution methods of standard enzyme;

[0050] Fig.26Linear range verification 2 test standard curve Figure 1 ;

[0051] Fig. 27 Linear range verification 2 test standard curve Figure 2 ;

[0052] Fig.28 Schematic diagram of the substrate standard curve of Thermo kit;

[0053] Fig.29 Schematic diagram of the Yaxin substrate standard curve. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] The nuclease standard in the following examples refers to: (Yaxin, RSN20).

[0056] 1. Experimental process

[0057] 1.1 Reaction system preparation

[0058] 1.1.1 Preparation of reaction premix: Prepare the reaction premix according to Table 1. Dispense 20 μL / well onto a black ELISA plate.

[0059] Table 1

[0060] Components Single reaction volume (μL) 58×(μL) Substrate containing fluorescent probe (4 μM) 10 580 10×Buffer 10 580 total 20 /

[0061] 1.1.2 Add enzyme: Preheat the microplate reader, use a dispenser to quickly add the standard and sample to the corresponding wells on the microplate at 80 μL / well, and test immediately. Note: Due to the extremely fast reaction speed, the sample should be added quickly when the microplate reader reaches 37°C, and the test should be performed immediately to avoid abnormal detection due to excessively high fluorescence values.

[0062] Note: The entire operation was performed on ice.

[0063] 1.2 Program Setup and Data Analysis

[0064] 1.2.1 The settings of the microplate reader are shown in Table 2 below: Once the settings are completed, the template can be saved and called later.

[0065] Table 2

[0066]

[0067]

[0068] 1.2.2 Data Analysis

[0069] 1) Select the linear reaction stage of the standard solution and the test solution, use time as the horizontal axis and RFU as the vertical axis, and calculate the linear regression equations of the standard solution and the test solution respectively. The slope is the RFU change rate of the standard solution and the test solution. The correlation coefficient of the linear regression equation of each 1mL standard solution containing 0.2U should not be less than 0.95. If the instrument can directly calculate Vmax and R2, the data given by the instrument can be directly used for analysis.

[0070] 2) Calculate the linear regression equation with the standard concentration as the horizontal axis and the corresponding RFU change rate (or Vmax calculated by the instrument) as the vertical axis. The correlation coefficient should be no less than 0.98. Calculate the residual amount of nuclease in the test sample based on the fluorescence intensity change rate of the test sample solution (or Vmax calculated by the instrument) and the dilution factor.

[0071] 2. Experimental Results

[0072] 1. Screening of substrates

[0073] The sequence of FS fluorescent substrate is shown in Table 3 below

[0074] Table 3

[0075]

[0076] Eight fluorescent substrates were designed, in which the fluorescent group was located at the 3' end and the quenching group was located at the 5' end. The selected fluorescent groups were VIC, FAM, and HEX, and the selected quenching groups were Dabcyl, TAMRA, and BHQ1. Nucleases with concentrations of 10U / mL, 5U / mL, and 2.5U / mL were selected to test different substrates.

[0077] The buffer for diluting the nuclease here is Buffer 3 in the optimization of the buffer system.

[0078] like Figures 1 to 8 As shown in the figure, the results show that the nuclease standard has good enzymatic cleavage effect on the fluorescent substrates FS-1, FS-2, FS-3, FS-4, FS-5, FS-6, and FS-7, but has no obvious enzymatic cleavage effect on the substrate FS-8. Figures 9 to 15 As shown: Curves drawn by FS-1, FS-2, FS-3, FS-4, FS-6, and FS-7 substrates, R 2 All of them are greater than 0.98, indicating that the universal nuclease has good selectivity for these substrates, that is, the enzyme has high specificity and efficiency in recognizing and acting on these substrates.2 Although it is less than 0.98, the reason may be that the early reaction was relatively fast and the Vmax value of the reaction could not be accurately captured.

[0079] 2. Optimization of Buffer System

[0080] The buffer formula is shown in Table 4 below.

[0081] Table 4

[0082] Buffe name formula Buffer-1 <![CDATA[100 mM Tris pH 8.0, 500 mM KCl, 20 mM MgCl2, 0.8% glycerol, 200 mg / L ribose]]> Buffer-2 <![CDATA[100 mM Tris pH 8.0, 500 mM KCl, 20 mM MgCl2, 0.8% glycerol]]> Buffer-3 <![CDATA[100mM Tris pH8.0,500mM KCl,40mM MgCl2]]> Buffer-4 <![CDATA[400 mM Tris pH8.0, 700 mM KCl, 25 mM MgCl2, 0.8% glycerol, 200 mg / L ribose]]>

[0083] Screen the reaction Buffer 1, Buffer 2, Buffer 3 and Buffer 4. Dilute the universal nuclease standard to 10U / mL, 5U / mL, 2.5U / mL, 1.25U / mL, 0.625U / mL, 0.3125U / mL, 0.15625U / mL, and add them to the system Buffer 1, Buffer 2, Buffer 3 and Buffer 4 for reaction, draw the standard curve, and calculate the recovery rate. The experimental data are shown in Tables 5 and 6.

[0084] Table 5

[0085]

[0086] Table 6

[0087]

[0088]

[0089] The results showed that the correlation coefficients of the linear regression equations drawn by the four buffers were no less than 0.98. The recovery rate of Buffer 1 was better than that of Buffer 2 and Buffer 4, and Buffer 2 and Buffer 4 were better than that of Buffer 3.

[0090] 3. Substrate concentration optimization

[0091] FS-6 was selected for substrate concentration optimization, and the substrate concentrations were 2μM, 4μM, 6μM, and 8μM, respectively.

[0092] The nuclease used here is a nuclease standard, and the buffer used to dilute the nuclease here is Buffer 1 in the optimization of the buffer system.

[0093] like Figure 16 to Figure 23 The results showed that the enzyme activity curves obtained with substrate concentrations of 2μM, 4μM, 6μM and 8μM were all good, with R 2 All are greater than 0.98, so substrate concentrations of 2 μM, 4 μM, 6 μM and 8 μM are preferred.

[0094] 4. Dilution of Standards

[0095] The amino acid sequence of the universal nuclease is shown in SEQ ID NO.9:

[0096] MDTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQKSDLNQGA WARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN

[0097] The three-dimensional structure diagram is as follows Fig.24 shown.

[0098] 4.1 Dilution of standard enzyme: Dilute the stock solution of universal nuclease standard to 10U / mL according to the following three methods.

[0099] (1) Dilution method 1: 273 KU / mL was diluted to 10 KU / mL, then diluted to 1 KU / mL, then diluted to 100 U / mL, then diluted to 20 U / mL, and then diluted to 10 U / mL, as shown in Table 7.

[0100] Table 7

[0101] Previous concentration sample volume (μL) Dilution volume (μL) Final concentration (U / mL) 10 (starting: 273 KU / mL) 260 10000 10 90 1000 50 450 100 140 560 20 350 350 10

[0102] (2) Dilution method 2: 273 KU / mL was diluted to 1000 U / mL, then diluted to 20 U / mL, and then diluted to 10 U / mL, as shown in Table 8.

[0103] Table 8

[0104] Previous concentration sample volume (μL) Dilution volume (μL) Final concentration (U / mL) 10 (starting: 273 KU / mL) 2720 1000 20 980 20 500 500 10

[0105] (3) Dilution method three: 273 KU / mL was diluted to 1000 U / mL, then diluted to 20 U / mL, and then diluted to 10 U / mL, as shown in Table 9.

[0106] Table 9

[0107] Previous concentration sample volume (μL) Dilution volume (μL) Final concentration (U / mL) 10 (starting: 273 KU / mL) 2720 1000 50 2450 20 500 500 10

[0108] 4.2 Experimental Results

[0109] As shown in Table 10, Table 11 and Fig.25 shown.

[0110] Table 10

[0111]

[0112] Table 11

[0113]

[0114] 4.3 Experimental Conclusion

[0115] The overall fluorescence value of dilution scheme 1 is low and the RSD is large; there is little difference between dilution schemes 2 and 3. Therefore, dilution schemes 2 and 3 are preferred.

[0116] 5. Methodological Validation

[0117] The relevant validation process refers to the four guidelines of the Chinese Pharmacopoeia (2020 edition): 9101 Guidelines for Validation of Analytical Methods.

[0118] 5.1 Linear range

[0119] 5.1.1 Verification Purpose

[0120] Confirm that the test value is proportional to the concentration of the standard.

[0121] 5.1.2 Verification Method

[0122] Use 0.1×Buffer1 as the diluent to dilute the universal nuclease standard to 10U / mL, 5U / mL, 2.5U / mL, 1U / mL, 0.5U / mL, 0.25U / mL, and 0.1U / mL. Perform two tests on the above 7 samples, and make a standard curve and test the results.

[0123] 5.1.3 Acceptability criteria

[0124] At least 5 consecutive standard solution concentration points were used to generate a standard curve, R 2 The value should be ≥ 0.98.

[0125] 5.1.4 Verification results

[0126] The standard curve of the second test is as follows Fig.26 , Fig. 27 shown.

[0127] 5.1.5 Verification conclusion

[0128] In the concentration range of 0.1U / mL to 5U / mL nuclease standard, a standard curve was generated with 6 consecutive concentration points. 2 Value ≥ 0.98.

[0129] 5.2 Accuracy

[0130] 5.2.1 Verification Purpose

[0131] Accuracy refers to the degree of closeness between the result determined by the established method and the true value or reference value, generally expressed as recovery rate (%). Accuracy should be tested within the specified linear range.

[0132] 5.2.2 Verification Method

[0133] Select dilution samples for spike recovery verification. According to the pharmacopoeia standards, perform spike tests at high, medium and low concentrations. Test the nuclease standards at high (2.5U / mL), medium (1U / mL) and low (0.5U / mL) concentrations respectively, repeat 3 times for each test, and test 2 parallels each time. Use the test results of at least 9 samples for evaluation.

[0134] 5.2.3 Acceptable standards

[0135] The recovery rate of the test sample should be between 50% and 150%.

[0136] 5.2.4 Verification results

[0137] The verification results are shown in Table 12.

[0138] Table 12

[0139]

[0140] 5.2.5 Verification conclusion

[0141] The recovery rate of the test sample was between 50% and 150%.

[0142] 5.3 Repeatability

[0143] 5.3.1 Verification Purpose

[0144] Precision refers to the closeness of the results obtained by multiple sampling and measurement of the same uniform sample under specified measurement conditions. Precision is generally expressed as deviation, standard deviation or relative standard deviation. The precision of the results obtained by the same analyst under the same conditions is called repeatability.

[0145] 5.3.2 Verification Method

[0146] Three concentrations of nuclease standards were tested, high (2.5 U / mL), medium (1 U / mL) and low (0.5 U / mL), and each test was repeated 3 times, with 2 parallels each time. The results of at least 9 samples were used for evaluation.

[0147] 5.3.3 Acceptability criteria

[0148] The detection RSD of high, medium and low concentrations should all be <30%.

[0149] 5.3.4 Verification results

[0150] The verification results are shown in Table 13 below.

[0151] Table 13

[0152]

[0153] 5.3.5 Verification conclusion

[0154] The detection RSDs of high, medium and low concentrations were all <30%.

[0155] 5.4 Intermediate precision

[0156] 5.4.1 Verification Purpose

[0157] Precision refers to the closeness between the results obtained from multiple samplings of the same uniform sample under specified measurement conditions. Precision is generally expressed as deviation, standard deviation or relative standard deviation. The precision between the results of measurements under different conditions in the same laboratory, such as different time, different analysts, different equipment, etc., is called intermediate precision.

[0158] 5.4.2 Verification Method

[0159] Two experimenters independently tested the test products at different times.

[0160] Three concentrations of nuclease standards were tested, high (2.5 U / mL), medium (1 U / mL) and low (0.5 U / mL), and each test was repeated 3 times, with 2 parallels each time. The values ​​independently tested by 2 experimenters were calculated for mean and RSD values ​​at the same time, and the RSD values ​​of the three concentrations of high (2.5 U / mL), medium (1 U / mL) and low (0.5 U / mL) were all less than 30%.

[0161] 5.4.3 Acceptability criteria

[0162] The values ​​independently tested by two experimenters were used to calculate the mean and RSD values, and the RSD was < 30%.

[0163] 5.4.4 Verification results

[0164] The verification results are shown in Table 14 below.

[0165] Table 14

[0166]

[0167] 5.4.5 Verification conclusion

[0168] The values ​​independently tested by two experimenters were used to calculate the mean and RSD values, and the RSD was < 30%.

[0169] 5.5 Limit of quantitation

[0170] 5.5.1 Verification Purpose

[0171] The limit of quantitation refers to the lowest amount of the substance in the sample that can be quantitatively determined, and the results should meet the accuracy and precision requirements. When analyzing trace or trace amounts of drugs, and quantitatively determining drug impurities and degradation products, the limit of quantitation of the method should be determined.

[0172] 5.5.2 Verification Method

[0173] The test samples at concentrations of 0.5 U / mL, 0.25 U / mL, 0.2 U / mL, and 0.1 U / mL were tested, and the results of 8 samples were used for evaluation at each concentration.

[0174] 5.5.3 Acceptability criteria

[0175] The recovery rate limit met 50%-150%, and RSD<30%.

[0176] 5.5.4 Verification results

[0177] The verification results are shown in Table 15.

[0178] Table 15

[0179]

[0180]

[0181] 5.5.5 Verification conclusion

[0182] The recovery rate limit of the detection result of 0.2U / mL nuclease concentration meets 50%-150%, RSD<30%, so the quantitative limit of this kit is 0.2U / mL.

[0183] 6. Actual sample testing application

[0184] 6.1 Materials and methods

[0185] Samples: Two types of sample stock solutions of nucleases used in the production process were selected, with three batches of each.

[0186] Method: Follow the experimental procedure in part 1 and calculate the residual nuclease in the sample using the standard curve.

[0187] 6.2 Results

[0188] The results are shown in Table 16. From the results, it can be seen that the standard curve linearity of the Yaxin universal nuclease (Yaxin, RSN20) matched with the Thermo Fisher kit substrate (the substrate contains a conventionally designed fluorescent probe) (Thermo Fisher, AM1970) and the fluorescent substrate FS-6 of the present application is good, R 2 >0.98. In addition, residual nuclease activity can be detected in all six batches of stock solutions, and the difference in the residual nuclease in the samples tested with the two substrates is not significant. This indicates that Yaxin universal nuclease (Yaxin, RSN20) can be used as a standard for the fluorescent substrate method, and the fluorescent substrate FS-6 of this application can be comparable to the Thermo Fisher kit substrate (Thermo Fisher, AM1970).

[0189] Thermo kit substrate standard curve is as follows Fig.28 As shown, the substrate standard curve of this application is as follows Fig.29 shown.

[0190] Table 16

[0191]

[0192] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0193] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for determining nuclease activity, characterized in that: The sequence of the fluorescent probe is any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7; The fluorescent group of the fluorescent probe is any one of VIC, FAM or HEX, and the quenching group of the fluorescent probe is any one of Dabcyl, TAMRA or BHQ1.

2. A fluorescent probe for determining nuclease activity according to claim 1, characterized in that: The sequence of the fluorescent probe is shown in SEQ ID NO.6, the fluorescent group of the fluorescent probe is HEX, and the quenching group of the fluorescent probe is BHQ1.

3. A fluorescent probe for determining nuclease activity according to claim 1, characterized in that: The substrate concentration of the fluorescent probe is any one of 2 μM, 4 μM, 6 μM or 8 μM.

4. A fluorescent probe for determining nuclease activity according to claim 1, characterized in that: The buffer system of the fluorescent probe reaction includes: 100-400mM Tris pH8.0, 500-700mM KCl, and 20-40mM MgCl2.

5. A fluorescent probe for determining nuclease activity according to claim 4, characterized in that: The buffer system of the fluorescent probe reaction includes 100-400 mM Tris pH 8.0, 500-700 mM KCl, 20-40 mM MgCl2 and 0.8% glycerol.

6. A fluorescent probe for determining nuclease activity according to claim 5, characterized in that: The buffer system of the fluorescent probe reaction includes 100-400mM Tris pH8.0, 500-700mM KCl, 20-40mM MgCl2, 0.8% glycerol and 200mg / L ribose.

7. A fluorescent probe for determining nuclease activity according to claim 6, characterized in that: The buffer system of the fluorescent probe reaction includes 100mM Tris pH8.0, 500mM KCl, 20mM MgCl2, 0.8% glycerol and 200mg / L ribose.

8. A fluorescent probe for determining nuclease activity according to claim 1, characterized in that: The amino acid sequence of the universal nuclease standard used by the fluorescent probe to detect the sample to be tested is shown in SEQ ID NO.

9.

9. A fluorescent probe for determining nuclease activity according to claim 1, characterized in that: The universal nuclease standard used in the fluorescent probe detection of the sample to be tested is diluted to 10U / mL as follows; 273KU / mL is diluted to 1000U / mL, then diluted to 20U / mL, and then diluted to 10U / mL, including: or Any one of .

10. Use of a fluorescent probe for determining nuclease activity as claimed in any one of claims 1 to 9, characterized in that: Used to detect residual nucleases during the process of treating biological products with nucleases.

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