Application of fluorescence signal decline caused by recombinase combined probe in enzyme activity detection and detection method

Abstract

The invention discloses an application of fluorescence signal decline caused by recombinase combined with a probe in enzyme activity detection and a detection method, and belongs to the technical field of enzyme activity detection.Based on the principle that the fluorescence signal decline caused by combination of the recombinase and the probe, the relationship between the fluorescence signal decline when the recombinase is combined with the probe is established; compared with other existing detection methods, the detection method disclosed by the invention has the advantages of simplicity in operation, high detection efficiency, accuracy in quantification, high reliability, low cost and the like, and can be industrially applied on a large scale.

Description

Technical Field

[0001] The invention relates to the field of enzyme activity detection, and in particular to an application of a recombinase-binding probe to induce fluorescence signal reduction in enzyme activity detection and a detection method. Background Art

[0002] Recombinase plays an important role in gene recombination, DNA (deoxyribonucleic acid) repair and replication. When performing its function, it needs to interact with single-stranded binding proteins and other proteins. Common types of recombinases include RecA, UvsX, RAD51, etc. (industry standard terms, no official Chinese name). Such enzymes play an important role in isothermal amplification systems. Isothermal amplification systems are often carried out at lower temperatures. The DNA in the isothermal amplification system exists in a stable double-stranded state. When the primer probe anneals with the target homologous region, it cannot be achieved by random collision like the PCR (polymerase chain reaction) amplification system. It can only rely on biological functional enzymes to bind the primer probe and achieve recombination in the template homologous region.

[0003] The molecular functions of recombinases include ATP (adenosine triphosphate) binding, ATP-dependent DNA damage sensor activity, single-stranded DNA binding activity, double-stranded DNA binding activity, etc. Taking T4 bacteriophage UvsX as an example, the enzyme can first bind to a single-stranded DNA and bind to a double-stranded homologous region, at which time it exerts double-stranded DNA binding activity. In addition, after UvsX binds to single-stranded DNA, it can exert the function of ATPase to degrade ATP. Recombinases have diverse functions and play an important role in isothermal molecular diagnosis. Most of the enzyme activity detection methods currently published can only be used for qualitative analysis, such as Chinese patent CN107893103A, and some can be used for quantitative analysis, such as Chinese patent CN108118085A. However, the detection method is complicated to operate and has high cost (involving biotin labeling and magnetic beads, etc.). The final quantitative result also depends on detection methods such as electrophoresis, which takes a long time and is not easy to promote. The quantitative analysis accuracy of electrophoresis results is not enough, and the repeatability is affected by many factors. Therefore, it is considered to develop a recombinase detection method that is simple to operate and quantitatively accurate. Summary of the invention

[0004] The purpose of the present invention is to provide an application and detection method of a recombinant enzyme binding probe that causes a decrease in fluorescence signal in enzyme activity detection in response to the above-mentioned problems. The detection method obtained by this application is simple to operate, low in cost, and short in detection time. Quantitative detection can be completed in only 10 minutes. The results of multiple repeated experiments are highly consistent, which is easy to promote and overcomes the shortcomings of existing detection methods.

[0005] The technical solution adopted by the present invention is as follows: an application in quantitative detection of recombinase activity by utilizing the property that the fluorescence signal of the probe decreases after the recombinase binds to the probe.

[0006] Furthermore, the recombinase is a recombinase that has not been modified with a quencher.

[0007] Furthermore, the recombinase is one or more combinations of RecA, UvsX, and RAD51, preferably UvsX enzyme.

[0008] Furthermore, the probe is one.

[0009] Furthermore, the probe carries only one luminescent group.

[0010] Furthermore, the activity of the recombinase is quantitatively calculated by using the decrease in the fluorescence signal of the probe after the recombinase binds to the probe.

[0011] Furthermore, in the quantitative detection process, different definitions of enzyme activity of the recombinase are assigned according to different situations of probe excess or recombinase excess, and the quantitative detection results of the enzyme activity under the definition are calculated.

[0012] Further, when the probe is in excess, the enzyme activity of the recombinase is defined as: the amount of recombinase required to saturate bind to 1 pmol of the probe with only one luminescent group at the 5' end and reduce its fluorescence signal is 1 U; when the recombinase is in excess, the enzyme activity of the recombinase is defined as: saturated binding to a probe with only one luminescent group at the 5' end causes a decrease in fluorescence signal by X%, which is considered to be a specific activity of 10X U / mg, where 10 is a custom constant.

[0013] Furthermore, the present invention also includes a method for quantitatively detecting recombinase activity, comprising the following steps: A. Design a probe with only one luminescent group so that the recombinase to be detected can bind to it and cause its fluorescence signal to decrease; B. Using different gradient recombinase concentrations as the horizontal axis and the fluorescence decrease after the recombinase at each concentration binds to the quantitative probe as the vertical axis, a linear regression curve between the recombinase concentration and the fluorescence decrease was established; C. Configure the probe reaction system to establish a standard curve 1 between the probe and the fluorescence height, add recombinase to the probe system, establish a standard curve 2 between the probe and the fluorescence height after the recombinase saturates and binds to the probe, and establish a standard curve between different concentrations of probe and the fluorescence decrease using the fluorescence height difference between the standard curve 1 and the standard curve 2 as the ordinate and different probe molar amounts as the abscissa; D. According to different situations of probe excess or recombinase excess, different definitions of enzyme activity are assigned to the recombinase, and the quantitative detection results of enzyme activity under this definition are calculated.

[0014] Furthermore, when the probe is in excess, the enzyme activity of the recombinase is defined as: the amount of recombinase required to saturately bind to 1 pmol of the probe with only one luminescent group at the 5' end and reduce its fluorescence signal is 1 U; when calculating the enzyme activity, the probe amount is substituted into the standard curve of different probe concentrations and fluorescence reduction, and the calculated fluorescence reduction is substituted into the linear regression curve to calculate the quantitative data of the enzyme activity.

[0015] Furthermore, when the recombinant enzyme is in excess, the enzyme activity of the recombinant enzyme is defined as: saturated binding to a probe with only one luminescent group at the 5' end, resulting in a decrease in fluorescence signal by X%, is considered to be a specific activity of 10X U / mg, where 10 is a custom constant; when calculating the enzyme activity, the slope K of the standard curve of different probe concentrations and the decrease in fluorescence is calculated, and the slope K1 of the standard curve, the ratio of K / K1 is the X value, the protein concentration is converted to mass, and the enzyme activity corresponding to 1μL of protein stock solution is X×1μL / 10 6 .

[0016] Furthermore, the concentration of the recombinant enzyme is 0 ng / μL-500 ng / μL.

[0017] Further, in step B, the amount of the quantitative probe was 10 pmol.

[0018] Furthermore, the probe reaction system includes: a probe, a reaction buffer and H2O, and the reaction buffer includes Tris-HCl (Tris hydrochloride, a buffer salt), potassium acetate, magnesium acetate, and DTT (dithiothreitol).

[0019] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. The present invention is based on the principle that the binding of recombinase to the probe will cause the fluorescence signal of the probe to decrease, and establishes the relationship between the binding of the recombinase to the probe and the decrease in the fluorescence signal, thereby quantitatively detecting the activity of the recombinase. The recombinase is not modified with a quencher, and the quantification is accurate.

[0020] 2. Compared with other existing detection methods, the detection method of the present invention has the advantages of simple operation, accurate quantitative analysis, and can be widely used. At the same time, it is more accurate than the concentration unit. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the annealing process between the probe structure 1 of the present invention and the substrate; Figure 2 for Figure 1 Scatter plot of the relationship between the fluorescence decrease of the probe in the system and the concentration of UvsX enzyme; Figure 3 Schematic diagram of the annealing process between the probe structure 2 of the present invention and the substrate; Figure 4 for Figure 3 Scatter plot of the relationship between the fluorescence decrease of the probe in the system and the concentration of UvsX enzyme; Figure 5 for Figure 4 Scatter plot of the relationship between the fluorescence decrease and the UvsX enzyme concentration when the system lacks ATPγ-S reaction; Figure 6 It is a scatter plot showing the relationship between the fluorescence decrease amount and the UvsX enzyme concentration when the probe structure of the present invention reacts in the system; Figure 7a It is a scatter plot of the relationship between the fluorescence decrease and the UvsX enzyme concentration when the probe of the present invention and the buffer system form a reaction system; Figure 7b It is a scatter plot of the relationship between the fluorescence decrease and the UvsX enzyme concentration when the probe of the present invention, the buffer system and the UvsX enzyme form a reaction system; Figure 7c A scatter plot of the relationship between the fluorescence decrease and the UvsX enzyme concentration when the reaction system is composed of the probe of the present invention, the buffer system, and the heat-inactivated UvsX enzyme; Figure 8 It is a schematic diagram of the principle of fluorescence reduction when the probe structure of the present invention binds to the UvsX enzyme; Fig. 9 This is a result diagram showing the effect of RecA enzyme on the fluorescent signal of the probe of the present invention; Fig.10 This is a result diagram showing the effect of RAD51 protease on the fluorescence signal of the probe of the present invention; Fig.11 This is a trend diagram showing the correlation between the fluorescence decrease and the enzyme concentration when the probe of the present invention binds to the UvsX enzyme; Fig.12a It is a standard curve diagram of fluorescence signals corresponding to different probe concentrations of the present invention; Figure 12b It is a standard curve diagram of the fluorescence signal corresponding to different probe concentrations when the UvsX enzyme of the present invention is saturated with the probe; Fig.12c It is a standard curve diagram of different probe concentrations and the decrease in fluorescence signal when the UvsX enzyme of the present invention saturates and binds to the probe; Fig.13a This is a graph showing the effect of UvsX on the fluorescence signal of the probe of the present invention; Fig.13b This is a graph showing the effect of gp32 on the fluorescence signal of the probe of the present invention; Fig.13c This is a diagram showing the effect of BSA on the fluorescence signal of the probe of the present invention; Fig.14a This is a test result diagram of the effect of ATP on the UvsX-probe complex; Fig.14bThis is a test result diagram of the effect of ATP on the gp32-probe complex; Fig.14c This is a test result diagram of the effect of ATP on the BSA-probe complex; Fig.15 The three batches of UvsX were amplified in an isothermal system according to their concentration and enzyme activity. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0023] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0024] The following terms or definitions are provided merely to aid understanding of the present invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0025] Unless otherwise defined hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the present invention are intended to be the same as those generally understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the present invention.

[0026] As used in the present invention, the terms "comprising", "including", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps.

[0027] The term “consisting of” is considered as a preferred embodiment of the term “comprising.” If hereinafter a group is defined as comprising at least a certain number of embodiments, this is also to be understood as disclosing a group which preferably consists of these embodiments only.

[0028] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0029] The terms "approximately" and "substantially" in the present invention represent the accuracy interval that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0030] The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably a polymeric molecule, comprising ribonucleic acid, deoxyribonucleic acid or analog units thereof. The nucleic acid may be single-stranded or double-stranded. A single-stranded nucleic acid may be a nucleic acid of one strand of a denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid may be a single-stranded nucleic acid that is not derived from any double-stranded DNA.

[0031] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0032] Example 1 Discovery of the interaction between recombinase and probe During the experimental research, the inventor accidentally discovered that there is an interaction relationship between the recombinase and the probe, that is, it was found that under certain conditions, the recombinase will bind to the probe, thereby causing the probe fluorescence signal to decrease. Therefore, it is conceived to use this discovery to quantitatively detect the activity of the recombinase. In addition, the recombinase of the present invention is a recombinase that has not been chemically modified. Chemical modification has a functional impact on proteins, and complete modification cannot be guaranteed. When the above two situations exist, the accuracy of the final enzyme activity quantification will be affected. In order to avoid such situations, shorten the test cycle, and reduce the complexity of the operation, the recombinase of the present invention is not chemically modified or modified with a quencher.

[0033] To this end, the applicant designed and implemented the following exploratory experiments: 1. Influence of probe structure According to the conventional probe design method, the inventors designed two typical probe structures CZM-P1 and CZM-P2 as shown in Table 1. The two probes can be annealed to the same template. The template sequence is as follows: Figure 1 As shown, the 3'-end modification groups are close to each other, and the fluorescence is quenched after they are close to each other. The recombination principle is as follows Figure 1 shown.

[0034] Table 1 Probe sequence list

[0035] An enzyme activity test system was configured, in which the recombinant enzyme used commercial UvsX, and the test gradient was 0 ng / μL-600 ng / μL. When the gradient was 0 ng / uL, in order to ensure the consistency of the buffer environment in the system, the storage solution was used as a substitute. The components and addition amounts of the enzyme activity test system are shown in Table 2 below, and the reaction buffer preparation information is shown in Table 3.

[0036] Table 2 Components of enzyme activity test system 1

[0037] Table 3 Reaction buffer (10x reaction buffer) preparation details

[0038] The test results show that as the concentration of UvsX enzyme increases, the fluorescence height of the test system becomes lower and lower, and the fluorescence difference becomes higher and higher (such as Figure 2 As shown in the figure, the result was repeatedly verified, and the data was unstable. The reason is that the primer annealing efficiency of CZM-P1 and CZM-P2 is inconsistent, which will result in that for the same template, no matter which primer achieves homologous recombination alone, the fluorescence signal will not decrease effectively. Therefore, the decrease in fluorescence signal is not equivalent to the recombination function of UvsX. The probe structure cannot accurately test the enzyme activity. In other words, the design of multiple probe structures cannot use the above findings to accurately quantify the activity of the recombinase.

[0039] To solve the above-mentioned probe structure problem, the inventors improved the probe structure and designed a probe CZM-P3. At this time, a probe modified with a FAM group (a green fluorescent group) was integrated into the template through amplification. The recombination principle is as follows: Figure 3 , design a pair of amplification primers, the sequences of which are shown in Table 4, where KZ-F1 and KZ-R1 are Figure 3 The double-stranded sequence in the middle is used as a substrate to synthesize a 74bp sequence with a FAM group at the 5' end of one side of the sequence. In this form, when the probe is recombined to the target position, the BHQ1 (a fluorescence quenching group) group of CZM-P3 is close to the FAM group on the template, and the fluorescence is quenched. The successful recombination of each probe will correspond to a decrease in fluorescence.

[0040] Table 4 KZ-F1, KZ-R1, CAM-P3 sequence details

[0041] The enzyme activity test system was configured, and the UvsX gradient still used 0 ng / μL-600 ng / μL, of which the 0 ng / uL control used UvsX storage solution. The components and addition amounts of the enzyme activity test system are shown in Table 5 below.

[0042] Table 5 Components of enzyme activity test system 2

[0043] The test results are as follows Figure 4 ,With the increasing concentration of UvsX enzyme, the decrease of fluorescence height gradually increases, and repeated tests are relatively stable.

[0044] In order to confirm that the decrease in fluorescence signal is indeed caused by the recombination of the probe, a set of comparative experiments 1 was set up, in which ATPγ-S (adenosine 5′-[γ-thio] triphosphate tetralithium salt) in the reaction buffer (10x reaction buffer) in the configuration system was removed; since ATPγ-S is an ATP analog, it is required to provide energy for UvsX to achieve recombination, and the hydrolysis time of this substance is long, which prevents the rapid generation of ADP (adenosine diphosphate) and causes the separation of the protein-nucleic acid complex. Therefore, the performance of UvsX enzyme in the system without adding ATPγ-S was tested. The configuration system is as shown in Table 4 above, and only ATPγ-S in the reaction buffer (10x reaction buffer) was removed. The test results are as follows Figure 5 When ATPγ-S is not provided in the system, the fluorescence change trend is similar to Figure 4 The comparative test showed that under this probe structure, the decrease in the fluorescence signal in the test result was not caused by the recombination of the UvsX enzyme and the template, but by the binding of the UvsX enzyme to the probe.

[0045] To verify the above assumption, a set of comparative experiments 2 was set up. No template was added to the system, and only the probe CZM-P4 with FAM group and UvsX enzyme were retained to test whether the system would have fluorescence signal quenching. The reaction system is shown in Table 6. When the corresponding gradient UvsX enzyme was added to the system, the fluorescence signal decreased regularly, and the degree of decrease (such as Figure 6 shown) and Figure 4 , Figure 5 The results were verified repeatedly and the data were relatively stable.

[0046] Table 6 Components of the enzyme activity test system

[0047] Among them, the CZM-P4 probe sequence is shown in Table 7 below.

[0048] Table 7 CZM-P4 sequence details

[0049] In addition, three groups of experimental examples were set up, in which the first group of experimental reaction systems only included the probe and the buffer system, the second group of experimental reaction systems included the probe, the buffer system and the UvsX enzyme, and the third group of reaction systems included the probe, the buffer system and the equal amount of UvsX enzyme after heat inactivation, in which the heat inactivation treatment method was 80°C for 20 min. The test results are as follows Figure 7a , 7b , 7c, the results showed that the fluorescence decrease in the third group of experiments was basically consistent with that in the first group of experiments, that is, after inactivation, the UvsX enzyme ( Figure 7c ) has a similar effect on the fluorescence of the probe as that without UvsX enzyme ( Figure 7a) are consistent, while the second group of experiments ( Figure 7b ), that is, the fluorescence signal decreased to a certain extent after adding UvsX enzyme, indicating that the heat-inactivated UvsX enzyme cannot act on the single-stranded probe, proving that the decrease in fluorescence signal is caused by the binding of UvsX enzyme to the probe. Analysis of the cause may be caused by the binding of UvsX enzyme to the nucleic acid sequence and FAM group.

[0050] 2. Determination of the probe structure of the present invention According to the detection results of the comparative experiments 2 and 3, the present invention uses the phenomenon that the fluorescence signal decreases when the probe only has a FAM group as the principle of the UvsX enzyme activity detection method. That is, when the probe structure CZM-P4 is used, when the UvsX enzyme is added, its binding to the probe will cause the probe fluorescence signal to decrease. The principle is as follows: Figure 8 shown.

[0051] In order to explore whether the probe structure itself will affect the above phenomenon, the present invention further designed probes CZM-P5 and CZM-P6 according to the probe structure, and their sequences are shown in Table 8.

[0052] Table 8 CZM-P4 to CZM-P6 sequence details

[0053] According to the experimental method of the above probe CZM-P4, a verification test was conducted on the probes CZM-P5 and CZM-P6. When the corresponding gradient UvsX enzyme was added to the system, the fluorescence signal decreased regularly, and the degree of decrease was similar to that of Figure 6 Basically consistent. The results were verified repeatedly, and the data were relatively stable. This shows that the UvsX enzyme can cause fluorescence quenching when it binds to probes with different sequences and fluorescent modification groups, and the structure of the probe itself does not have a significant effect on the above phenomenon, that is, the sequence structure of the probe itself does not affect this phenomenon (the binding of the recombinase to the probe will cause the fluorescence signal to decrease). Therefore, the probe designed by the present invention does not make special requirements for the probe sequence.

[0054] Furthermore, in the above-mentioned validation experiments, the fluorescence detection data of probes CZM-P4, CZM-P5 and CZM-P6 showed that the degree of fluorescence decrease of different probe systems was different, resulting in different linear relationship correlations. In order to facilitate experimental exploration and verification, the probe CZM-P4 with the best linear relationship correlation was subsequently used as the experimental probe.

[0055] Furthermore, in order to explore whether the type of recombinase would affect this phenomenon, the inventors used the representative RecA protein (NEB, M0249S) and RAD51 protein (Huamei Biotechnology CSB-EP019263HU) of the recombinase family as test objects, replacing the UvsX enzyme in the enzyme activity test system 3 in Table 6. The test results showed that when the corresponding gradients of RecA protein and RAD51 protein were added to the system, the RecA protein would cause the fluorescence signal to decrease regularly (such as Fig. 9 ), although RAD51 in the recombinase family did not cause a regular decrease in fluorescence signal (as shown Fig.10 However, the enzyme's quality inspection report shows that the activity of the protein has not been tested, so this phenomenon may be caused by inactivity or low activity. We speculate that all enzymes in the recombinase family can cause a decrease in the fluorescence signal. This phenomenon is common among recombinases and can be used to perform quantitative analysis on various recombinases.

[0056] Example 2 Drawing a linear regression curve of UvsX enzyme and fluorescence reduction 1. UvsX enzyme concentration screening Since the UvsX enzyme-binding probe in the recombinant enzyme showed a good linear relationship in the decrease of the fluorescence signal, the UvsX enzyme was used as the test object in this example to explore the effect of the concentration of the UvsX enzyme.

[0057] When the probe amount is quantitative (8pmol), the UvsX enzyme is screened. When the probe concentration is fixed, as the UvsX enzyme concentration increases, the change in the fluorescence decrease curve first rises and then enters a plateau phase (all probes are saturated and the fluorescence signal no longer decreases further). Therefore, as the UvsX enzyme concentration increases, there will be a linear rise phase. The slope of this rise phase is positively correlated with the enzyme activity of the isoconcentration enzyme. In order to select the appropriate linear phase, the UvsX enzyme concentration gradient screening is required. The screening gradient is shown in Table 9 below, where the highest concentration is 1520ng / μL. Taking gradient 2 as an example, the scatter plot of UvsX enzyme concentration and fluorescence signal decrease is shown in Fig.11 As shown, the linear rising stage of the curve corresponds to the concentration gradient of 0ng / μL-380ng / μL, which is consistent with expectations. The trend was tested repeatedly, and the results were basically consistent. The near-linear rising stage was located at 0ng / μL-380ng / μL. For the convenience of calculation, the integer gradient was re-taken, and 0ng / μL, 100ng / μL, 200ng / μL, 300ng / μL, 400ng / μL, and 500 ng / μL were retested. The results of multiple retests showed that the range of 0ng / μL-400ng / μL was a near-linear rising period, and the slopes of the four repetitions were relatively close, indicating that the fluorescence decrease in the concentration gradient showed a relatively stable increase.

[0058] Table 9 Linear interval statistics

[0059] Since the 7th and 8th linear regression coefficients R 2 The lower value indicated that the method was unstable among replicates, so the amount of probe was adjusted to determine the optimal amount of probe for the linear regression curve reaction system.

[0060] 2. Determination of probe concentration When the concentration gradient of UvsX enzyme was determined to be 0ng / μL-400ng / μL, the amount of probe was screened. In the enzyme activity measurement system, the probe needs to be in excess, so the probe concentration was adjusted on the basis of 8pmol. The test results are shown in Table 10 below. When the amount of probe was further increased to 10pmol, the slope increased further (the increase in slope indicates an increase in △Rn). When the amount of probe was increased to 12pmol again, the effect was consistent with 10pmol. Although the slope K1 values ​​of the repeated results were different, the trend was stable, indicating that when the probe was added to 10pmol, it was already in a saturated state, and the final probe amount was selected to be 10pmol. Therefore, the linear regression curve of UvsX enzyme and fluorescence decrease is y=5.0505x+19.933, where R 2 It is 0.9793, which meets the standard.

[0061] Table 10 Statistical table of the effect of different probe amounts on enzyme activity test results

[0062] Example 3: Plotting a standard curve of probe and fluorescence reduction 1. Establish a standard curve between the probe and the fluorescence height 1 To establish a standard curve 1 between the probe and the fluorescence height, the reaction system was configured as shown in Table 11 below. The probe amount was set to 0 pmol, 1 pmol, 2 pmol, 3 pmol, 4 pmol, and 5 pmol to obtain a standard curve 1 between the probe amount and the fluorescence height (as shown in Table 11). Fig.12a As shown in the figure), the obtained standard curve 1 is y=840.89x+1.0595, where R 2 It is 0.9927, which meets the standard.

[0063] Table 11 Probe and fluorescence reaction system

[0064] 2. Establish a standard curve of the fluorescence height after the probe and UvsX enzyme saturate bind to the probe A standard curve 2 of the fluorescence height after the probe and the UvsX enzyme saturatedly bound to the probe was established. The reaction system was configured as shown in Table 11 below. The probe amount was set to 0 pmol, 1 pmol, 2 pmol, 3 pmol, 4 pmol, and 5 pmol to obtain a standard curve 2 of the fluorescence height after the probe amount and the UvsX enzyme saturated bound to the probe (as shown in Table 11). Figure 12b As shown), the obtained standard curve 2 is y=514.65x+26.167, where R 2 It is 0.9854, which meets the standard.

[0065] 3. Draw a standard curve of the probe and the fluorescence decrease The fluorescence height difference between standard curve 1 and standard curve 2 was used as the ordinate, and the molar amount of different probes was used as the abscissa to establish the standard curve of different concentrations of probes and the fluorescence decrease (e.g. Fig.12c ), the standard curve is y=330.75x+9.5, where R 2 It is 0.9609, which meets the standard.

[0066] Example 4 Calculation of enzyme activity (taking UvsX enzyme as an example) 1. When the probe is excessive When the probe is in excess, the enzyme activity of UvsX is defined as: 1U of UvsX enzyme is required to saturate the binding with 1pmol of the probe having only one FAM modification group at the 5' end and reduce its fluorescence signal.

[0067] The enzyme activity calculation process is: When the amount of probe is 1pmol, substitute the amount of probe into y=330.75x+9.5, and the fluorescence decrease is 330. (The intercept of the standard curve with the y-axis is the experimental system error, that is, the deviation between the theoretical amount of probe and the actual amount of probe. Therefore, when calculating the fluorescence signal decrease corresponding to 1pmol of probe, the intercept is not included in the calculation, the same below.) Substituting the fluorescence decrease of 330 into the linear regression curve y=5.0505x+19.933, the amount of UvsX enzyme required to achieve a fluorescence decrease of 330 can be obtained, which is 65.34 ng / μL.

[0068] The concentration unit of the UvsX enzyme amount was converted into a mass unit, i.e., 65.34 ng / μL×20 μL / 1000000=0.00131 mg.

[0069] Therefore, the enzyme activity corresponding to 1 μL of protein stock solution is 5 mg / mL×1 μL / 1000 / 0.00131 mg / U=3.83U.

[0070] Therefore, the specific activity is 3.83 U / 5 mg / mL × 1 μL / 1000 = 765.23 U / mg.

[0071] 2. When UvsX enzyme is excessive When UvsX enzyme is in excess, the enzyme activity of UvsX enzyme is defined as: saturated binding to a probe with only one luminescent group at the 5' end resulting in a decrease in fluorescence signal by X%, which is considered to be 10X U / mg, where 10 is a custom constant.

[0072] The enzyme activity calculation process is: Combined with Example 3 and Fig.12a , 12b , 12c, it can be obtained that after the UvsX enzyme saturates with the probe, the probe fluorescence signal decreases by 39.33% (taking the quotient of the slope values ​​in the equation y=330.75x+9.5 and y=840.89x+1.0595), and the specific activity of the UvsX enzyme is obtained to be 10×39.33=393.3U / mg.

[0073] The enzyme activity corresponding to 1uL of protein stock solution is 5mg / mL×1μL / 1000×393.3U / mg=1.97U.

[0074] It can be seen that different definitions will result in different enzyme activity data calculated. In actual application, the method can be selected according to the actual situation.

[0075] Example 5 Specificity Verification In order to verify whether the phenomenon of fluorescence signal decrease caused by recombinase binding probe is unique to recombinase, single-chain binding protein gp32 (can bind single chain) and BSA (calf serum albumin, non-binding protein) were compared with it under the same conditions. The test system is shown in Table 12. Each protein was prepared according to 0ng / μL-250ng / μL. The test results are shown in Fig.13a , Fig.13b , Fig.13c The results showed that both UvsX enzyme and gp32 caused a decrease in fluorescence signal, but BSA protein did not show this phenomenon, indicating that this phenomenon is not unique to recombinant enzymes. It is possible that any non-sequence-specific binding to single-stranded nucleic acids can cause a decrease in fluorescence signal. Therefore, the enzyme activity detection method established based on recombinant enzymes in the present invention may be extended to all proteins that interact with single-stranded nucleic acids.

[0076] Table 12 Specificity test system table

[0077] However, when this method is used to test proteins of unknown origin, it can only distinguish whether there is enzyme activity, but it is difficult to distinguish whether it is the activity of the recombinant enzyme. Therefore, it is evaluated whether the presence of ATP will affect the binding of the protein to the probe. In theory, when ADP is present, the UvsX-ssDNA complex decomposes, and ATP, as a high-energy substance, is very easy to decompose. The effect of ATP on the activity of the recombinant enzyme is well known in the art.

[0078] The sample addition system is shown in Table 13. The presence of ATP will cause the fluorescence signal to decrease. The linear relationship between the fluorescence signal decrease y and the ATP concentration x is tested to be y=218.88x-133.08. The effect of ATP on the protein probe is as follows. Fig.14a , 14b As shown in Figures 14c, with the addition of ATP, the fluorescence curve shows a trend of first rising and then falling. The rise is due to the separation of UvsX and the probe caused by the presence of ATP, thereby increasing the fluorescence, while the decline stage is mainly due to the fact that even if the separation of the protein and the probe will restore the fluorescence signal of the probe, the amount of ATP gradually increases, causing the background fluorescence value of the probe to decrease, so it subsequently shows a downward trend. In addition, only the UvsX system will show this trend, indicating that ATP will only affect the UvsX-probe complex, but not the gp32-probe complex, and the recovery height of the fluorescence entering the plateau phase is basically consistent with the decrease in the fluorescence of the probe caused by 250ng / μL protein. Therefore, when using the method of the present invention to quantify the recombinant enzyme, the method can be used to qualitatively test the protein.

[0079] Table 13 Test system for the effect of ATP on protein-probe complex

[0080] Example 6 Reliability verification of the quantitative method of the present invention In order to verify the feasibility of the quantitative method of the recombinant enzyme activity of the present invention, the correlation between the enzyme activity test results of the present invention and the amplification performance was verified in an isothermal amplification system (taking UvsX enzyme as an example).

[0081] The present invention prepares isothermal systems with three batches of UvsX enzymes according to enzyme activity and concentration, and tests the amplification effect. UvsX enzyme is added to the system at 2U according to enzyme activity, and the system is uniformly prepared at 329ng / μL according to concentration. The specific amount of the system is shown in Table 14, the preparation system is shown in Table 15, the template amount is 60 copies / reaction, and the test results are as follows Fig.15 .

[0082] Table 14 Correspondence between concentration and enzyme activity in three batches of UvsX enzyme system

[0083] Table 15 Isothermal amplification system

[0084] In the above table, Exonuclease III is exonuclease III, Tris-Ac is tris-potassium acetate buffer, KOAc is potassium acetate, MgOAc is magnesium acetate, ppk2 is type 2 polyphosphate kinase, pap is polyphosphate adenylate transferase, dNTPs is deoxynucleoside triphosphate, and ASFV is African swine fever virus.

[0085] Combining Table 14 and Figure 12, it can be found that batches 1 and 2 have the same performance according to the enzyme activity preparation system, and batch 2 has slightly worse amplification according to the concentration preparation system. The reason is that compared with the optimal amount (2U), the amount added in batch 2 is slightly higher than the enzyme activity (2.36U), so the amplification effect is slightly reduced. Batch 3 has a larger difference in effect compared with batch 1 in terms of concentration or enzyme activity, because compared with the optimal amount (2U), the amount added in batch 3 is converted into enzyme activity (1.14U) which is lower, so the amplification effect is reduced. Since only the amount of UvsX enzyme is changed in the amplification system, other substances and amounts remain unchanged. Therefore, the difference in amplification performance is only caused by the amount of UvsX enzyme added. It can be seen that the definition of the enzyme activity of UvsX enzyme is correlated with the performance in the system. The quantitative detection method of the present invention can be used to quantitatively detect UvsX enzyme, and the detection results are accurate.

[0086] In addition, the amplification performance of batch three shows that when the specific activity test result is too low, even if a sufficient amount of UvsX enzyme is added, the difference cannot be made up. Through the grayscale analysis of the polyacrylamide gel electrophoresis results, the purity of the above three batches of enzymes is more than 95%, indicating that the reason why batch three does not pass the performance is the structure of the UvsX enzyme itself, that is, the protein folding state. The present invention tests five batches of specific activity data (data not shown) and tests the amplification effect in the system. The results show that when the specific activity is <258U / mg, even if the enzyme activity is prepared according to the system, the amplification effect is still not as good as the batch with higher specific activity. Therefore, the enzyme passing standard can be set as ≥258U / mg. Different systems may require inconsistent passing standards, and this standard is for reference only.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An application in quantitative detection of recombinase activity by utilizing the property that the fluorescence signal of the probe decreases after the recombinase binds to the probe, characterized in that: The recombinase is a recombinase not modified by a quencher, the probe is a single piece, and the probe carries only one luminescent group.

2. The use according to claim 1, characterized in that The recombinase is a combination of one or more of RecA, UvsX and RAD51.

3. The use according to claim 1 or 2, characterized in that: The activity of the recombinase is quantitatively calculated by the decrease in the probe fluorescence signal after the recombinase binds to the probe.

4. The use according to claim 3, characterized in that In the quantitative detection process, different definitions of enzyme activity of the recombinase are assigned according to different situations of probe excess or recombinase excess, and the quantitative detection results of the enzyme activity under the definition are calculated.

5. The use according to claim 4, characterized in that When the probe is in excess, the enzyme activity of the recombinase is defined as: 1U of the recombinase required for saturated binding with 1pmol of the probe with only one luminescent group at the 5' end and a decrease in its fluorescence signal; when the recombinase is in excess, the enzyme activity of the recombinase is defined as: saturated binding with a probe with only one luminescent group at the 5' end causes a decrease in fluorescence signal by X%, which is considered to be 10X U / mg, where 10 is a custom constant.

6. A method for quantitatively detecting recombinase activity, characterized in that: The steps include: A. Design a probe with only one luminescent group so that the recombinase to be detected can bind to it and cause its fluorescence signal to decrease; B. Using different gradient recombinase concentrations as the horizontal axis and the fluorescence decrease after the recombinase at each concentration binds to the quantitative probe as the vertical axis, a linear regression curve between the recombinase concentration and the fluorescence decrease was established; C. Configure the probe reaction system to establish a standard curve 1 between the probe and the fluorescence height, add recombinase to the probe system, establish a standard curve 2 between the probe and the fluorescence height after the recombinase saturates and binds to the probe, and establish a standard curve between different concentrations of probe and the fluorescence decrease using the fluorescence height difference between the standard curve 1 and the standard curve 2 as the ordinate and different probe molar amounts as the abscissa; D. According to different situations of probe excess or recombinase excess, different definitions of enzyme activity are assigned to the recombinase, and the quantitative detection results of enzyme activity under this definition are calculated.

7. The method for quantitatively detecting recombinase activity according to claim 6, characterized in that: When the probe is in excess, the enzyme activity of the recombinase is defined as: the amount of recombinase required to saturate the binding with 1 pmol of the probe with only one luminescent group at the 5' end and reduce its fluorescence signal is 1 U; when calculating the enzyme activity, the probe amount is substituted into the standard curve of different probe concentrations and fluorescence reduction, and the calculated fluorescence reduction is substituted into the linear regression curve to calculate the quantitative data of the enzyme activity.

8. The method for quantitatively detecting recombinase activity according to claim 6, characterized in that: When the recombinant enzyme is in excess, the enzyme activity of the recombinant enzyme is defined as: saturated binding to a probe with only one luminescent group at the 5' end, resulting in a decrease in fluorescence signal by X%, which is considered to be 10X U / mg, where 10 is a custom constant; when calculating the enzyme activity, the slope K of the standard curve of different probe concentrations and the decrease in fluorescence is calculated, and the slope K1 of the standard curve, the ratio of K / K1 is the X value, the protein concentration is converted to mass, and the enzyme activity corresponding to 1μL protein stock solution is X×1μL / 10 6 .

9. The method for quantitatively detecting recombinase activity according to claim 6, characterized in that: The concentration of the recombinant enzyme is 0 ng / μL-500 ng / μL.

10. The method for quantitatively detecting recombinase activity according to claim 6, characterized in that: In step B, the amount of the quantitative probe was 10 pmol.

11. The method for quantitatively detecting recombinase activity according to claim 6, characterized in that: The probe reaction system includes: a probe, a reaction buffer and H2O, and the reaction buffer includes Tris-HCl, potassium acetate, magnesium acetate and DTT.

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