Enzyme activity detection method of T7 RNA polymerase

Abstract

The invention discloses an enzyme activity detection method of T7RNA polymerase. The invention also provides an enzyme activity detection kit of the T7RNA polymerase, and an application of the kit in enzyme activity detection of the T7RNA polymerase. The method for detecting the enzyme activity of the T7RNA polymerase has the advantages of being low in detection cost, short in detection time, easy to operate and the like, in addition, a dsDNA template of the double T7 promoters is adopted in the method, and the efficiency and the lower detection limit of enzyme activity detection can be remarkably improved. The enzyme activity detection method of the T7RNA polymerase is not only suitable for determination of pure enzyme liquid of the T7RNA polymerase, but also suitable for determination of crude enzyme liquid of recombinant bacteria, meets detection requirements in industrial large-scale in-vitro RNA transcription production, and has wide application prospects in the fields of biology, medicines, pesticides, detection and the like.

Description

Technical Field

[0001] This invention belongs to the field of enzyme technology, specifically, it relates to a method for detecting the enzyme activity of T7 RNA polymerase. Background Technology

[0002] T7 RNA polymerase is a highly specific enzyme derived from T7 bacteriophage, primarily used in in vitro transcription reactions to synthesize RNA molecules. Large-scale RNA synthesis can be achieved using T7 RNA polymerase, making it widely used in medicine, agriculture, and other fields. For example, in medicine, this enzyme is widely used in the development of novel RNA drugs (mRNA vaccines, RNA aptamers, and antisense RNA, etc.). Furthermore, in green agriculture, large-scale in vitro transcription technology using this enzyme is crucial for the widespread application of nucleic acid pesticides.

[0003] Accurately calibrating the activity of T7 RNA polymerase in large-scale in vitro transcription processes is crucial for achieving process stability and optimization. While commercially available products of this enzyme exist, different manufacturers use varying methods for activity calibration. Traditional commercial T7 RNA polymerase activity is defined by calibrating the incorporation of substrate (AMP or GMP) per unit time, using isotope labeling. This method suffers from high raw material costs and radioactive contamination. Recently, patent application CN114574547A disclosed a kit and method for detecting T7 RNA polymerase activity. This method quantifies enzyme activity by detecting the amount of pyrophosphate generated during the transcription reaction. However, this detection process requires the expensive PPi sensor and fluorescent quantitative reagent, and necessitates fluorescent detection equipment. In addition, patent application CN116855576A uses an inorganic pyrophosphatase PPase and purine nucleoside phosphorylase PNPase dual-enzyme coupling reaction system to determine the content of pyrophosphate, and then calibrates the activity of T7 RNA polymerase. The coupling substrate used, 7-methyl-6-thioguanosine MESG, is a modified nucleotide and also has a high price.

[0004] In summary, current methods for detecting T7 RNA polymerase activity are costly due to the need for specialized instruments, special substrates, or fluorescent markers. Therefore, there is an urgent need to develop a simple, efficient, and low-cost method for detecting T7 RNA polymerase activity to meet the detection requirements of large-scale industrial in vitro RNA transcription production. Summary of the Invention

[0005] To address the shortcomings of existing methods for detecting T7 RNA polymerase activity, this invention provides a simple, efficient, and low-cost method for detecting T7 RNA polymerase activity.

[0006] In one aspect, the present invention provides a method for detecting the enzyme activity of T7 RNA polymerase, the method comprising the following steps:

[0007] S1: dsDNA template required for T7 RNA polymerase transcription reaction by PCR amplification;

[0008] S2: Prepare a T7 RNA polymerase transcription reaction solution system containing the DNA template in S1 and T7 RNA polymerase standards of different concentrations, carry out the transcription reaction, and obtain the reaction solutions of each standard and the blank control reaction solution;

[0009] S3: Take each reaction solution from S2, and determine the absorbance difference ΔA before and after the reaction using the xyleneamine blue method to plot the enzyme activity standard curve of T7 RNA polymerase; the abscissa of the enzyme activity standard curve of T7 RNA polymerase is the absorbance difference ΔA, and the ordinate is the enzyme activity of T7 RNA polymerase, or the ordinate of the enzyme activity standard curve of T7 RNA polymerase is the absorbance difference ΔA, and the abscissa is the enzyme activity of T7 RNA polymerase; the detection wavelength is 590-620 nm.

[0010] S4: Take the T7 RNA polymerase sample to be tested, prepare the T7 RNA polymerase transcription reaction solution system, carry out the transcription reaction, and obtain the test sample reaction solution and the blank control reaction solution;

[0011] S5: Take each reaction solution from S4 and determine the absorbance difference ΔA of the reaction solution before and after the reaction using the xyleneamine blue method. Substitute ΔA into the standard curve obtained in S3 to calculate the enzyme activity of T7 RNA polymerase in the T7 RNA polymerase sample to be tested. The detection wavelength is 590-620 nm.

[0012] In one or more embodiments, the detection wavelength is 605 nm.

[0013] In one or more embodiments, in steps S3 and S5, prior to the xyleneamine blue determination, the reaction solution is diluted by an appropriate factor so that the magnesium ion concentration in the diluted reaction solution is in the range of 0.5 to 8.0 mM.

[0014] In one or more embodiments, the absorbance difference ΔA is calculated by the formula ΔA = A1 - A0, where A1 is the absorbance of each standard reaction solution or the test sample reaction solution at the detection wavelength, and A0 is the absorbance of the blank control reaction solution at the detection wavelength.

[0015] In one or more embodiments, in step S1, the dsDNA template has a T7 promoter sequence in both 5'-3' directions of the two single strands, and the length of the dsDNA template is 200-1500 bp.

[0016] In one or more embodiments, in step S1, the sequence of a single strand in the dsDNA template is shown in SEQ ID NO: 1.

[0017] In another aspect, the present invention provides a T7 RNA polymerase activity assay kit, the kit comprising: (1) T7 RNA polymerase standard; (2) T7 RNA polymerase buffer; (3) dsDNA template; (4) four NTPs; (5) xyleneamine blue stock solution; and (6) alkaline reagent stock solution;

[0018] The T7 RNA polymerase buffer is an aqueous solution containing magnesium ion salt, 50 mM dithiothreitol, 25 mM spermidine and 400 mM Tris-HCl, with a pH of 7.5 to 8.5; wherein the magnesium ion salt is selected from magnesium acetate, magnesium chloride or magnesium sulfate, and the concentration of magnesium ions in the aqueous solution is 40 mM to 80 mM.

[0019] The xyleneamine blue stock solution is an ethanol solution containing 3% (w / v) xyleneamine blue II;

[0020] The alkaline reagent storage solution is an aqueous solution containing 1% (w / v) Tween-20, 20 mM borax and 20 mM sodium hydroxide, with a pH of 9.5 to 10.5.

[0021] In one or more embodiments, the dsDNA template has a T7 promoter sequence in the 5'-3' direction on both single strands, and the length of the dsDNA template is 200-1500 bp.

[0022] In one or more embodiments, in step S1, the sequence of a single strand in the dsDNA template is shown in SEQ ID NO: 1.

[0023] In another aspect, the present invention provides the application of a kit as described in any embodiment herein in the detection of T7 RNA polymerase activity.

[0024] Preferably, the kit is used according to the method described in any embodiment of this document.

[0025] The principle of the T7 RNA polymerase activity detection method provided by this invention is to indirectly detect enzyme activity by detecting the amount of magnesium ions consumed per unit time in the solution. The reagents and equipment required for this detection are relatively common. Compared with isotope detection and fluorescence detection methods, this method has the advantages of low detection cost, short detection time, and simple operation. Furthermore, the T7 RNA polymerase activity detection method provided by this invention uses a dsDNA template with a dual T7 promoter, which can significantly improve the efficiency and detection limit of enzyme activity detection. Moreover, using dsDNA with a bidirectional T7 promoter as a template, the two RNA single strands produced by T7 RNA polymerase catalysis can complement each other during enzyme activity detection, thereby reducing the impact of product degradation on enzyme activity determination. The T7 RNA polymerase activity detection method provided by this invention is applicable not only to the determination of pure T7 RNA polymerase enzyme solution but also to the determination of crude enzyme solution from recombinant bacteria, meeting the detection needs of large-scale industrial in vitro RNA transcription production, and has broad application prospects in the fields of biology, medicine, pesticides, and detection. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the principle of magnesium-involved in vitro transcription reaction.

[0027] Figure 2 This is an agarose gel electrophoresis result of the dsDNA template required for the T7 RNA polymerase transcription reaction.

[0028] Figure 3 This is a scan image of the xyleneamine blue colorimetric reaction of different concentrations of magnesium ions within the detection wavelength range of 500–700 nm.

[0029] Figure 4 It is a standard curve for magnesium ion concentration at a detection wavelength of 605 nm.

[0030] Figure 5 This is a graph showing the absorbance difference of T7 RNA polymerase transcription reaction solutions containing different magnesium ion concentrations at a wavelength of 605 nm.

[0031] Figure 6 This is a graph showing the absorbance difference of T7 RNA polymerase transcription reaction solutions containing different enzyme concentrations at a wavelength of 605 nm. Detailed Implementation

[0032] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0033] In this invention, the "xyleneamine blue method" refers to a clinical diagnostic technique for determining the magnesium ion content in serum. This method is simple and low-cost, and suitable for semi-automatic or fully automated biochemical analyzers. The principle of this method is that, under alkaline conditions, magnesium ions in serum react with xyleneamine blue to form a colored complex. This complex has a maximum absorption peak at a wavelength of 505 nm, and its absorbance is positively correlated with the magnesium concentration in the sample. Therefore, currently commercially available reagent kits and related patent applications (e.g., CN108169224A and CN110672518A) report detection wavelengths between 505 nm and 540 nm.

[0034] In this invention, "xyleneamine blue working solution" refers to a liquid obtained by mixing an appropriate amount of xyleneamine blue storage solution and an alkaline reagent storage solution in equal volumes. In specific use, after mixing the two solutions, the mixture is allowed to stand at room temperature for 10 minutes before reacting with a liquid containing magnesium ions.

[0035] In this invention, the "T7 RNA polymerase" possesses transcriptional activity that catalyzes the production of RNA using DNA as a template. During this reaction, for every 1 nmol NTP (nucleoside triphosphate) consumed per unit time, 1 nmol of pyrophosphate is produced. Each 1 nmol of pyrophosphate reacts with 2 nmol of magnesium ions to form magnesium pyrophosphate precipitate. Therefore, the enzyme activity of T7 RNA polymerase can be indirectly determined by measuring the consumption of magnesium ions per unit time. In in vitro transcription experiments, magnesium ions typically act as a cofactor, helping to stabilize the binding of the enzyme to the DNA template and promoting the polymerization of substrate nucleoside triphosphates (NTPs). Furthermore, magnesium ions help maintain the three-dimensional structure of T7 RNA polymerase, ensuring the effectiveness of its active site and facilitating efficient catalysis. The principle of magnesium-involved in vitro transcription is illustrated in formulas (I), (II), and (...). Figure 1 As shown.

[0036]

[0037] In this invention, the principle of determining the enzyme activity of T7 RNA polymerase using the "xyleneamine blue method" is as follows: T7 RNA polymerase has transcriptional activity that catalyzes the production of RNA using DNA as a template. During this reaction, for every 1 nmol of NTP (nucleoside triphosphate) consumed per unit time, 1 nmol of pyrophosphate is produced, and each 1 nmol of pyrophosphate reacts with 2 nmol of magnesium ions to form magnesium pyrophosphate precipitate. By removing the magnesium pyrophosphate precipitate from the reaction system through high-speed centrifugation, and detecting the difference in magnesium ion concentration before and after the reaction, the amount of NTP consumed and the amount of PPi (pyrophosphate) generated can be indirectly determined, thereby determining the enzyme activity. The magnesium ion concentration before and after the reaction can be determined by the xyleneamine blue method. The principle of this method is that magnesium ions in an alkaline medium can react with xyleneamine blue to form a water-soluble purple-red complex. In the wavelength range of 590–620 nm, the absorbance of the xyleneamine blue-magnesium complex is inversely proportional to the concentration of magnesium ions in the solution. That is, as the transcription reaction proceeds, magnesium ions in the system are gradually consumed, and the absorbance of the xyleneamine blue method will increase. The increase in absorbance is positively correlated with the amount of magnesium ion consumption. Subsequently, the increase in absorbance is also positively correlated with enzyme activity. The correlation between the increase in absorbance and enzyme activity can be quantified by constructing a standard curve.

[0038] In this invention, the "T7 RNA polymerase activity assay kit" includes T7 RNA polymerase standard, T7 RNA polymerase buffer, dsDNA template, NTP mixture, xyleneamine blue stock solution, and alkaline reagent stock solution. Each component can be obtained commercially or prepared from commercially available materials. Each solution component is prepared from an RNase-free liquid. The T7 RNA polymerase standard is a quantitatively quantified T7 RNA polymerase used to catalyze transcription reactions. Reaction solutions of transcription reactions involving different T7 RNA polymerase standards are subjected to a xyleneamine blue colorimetric reaction. The relationship between the absorbance of the colorimetric reaction and the enzyme activity in the reaction solution can be used to plot a standard curve of T7 RNA polymerase activity. The dsDNA template refers to the double-stranded DNA template required for the transcription reaction catalyzed by T7 RNA polymerase, containing a T7 promoter sequence in the 5'-3' direction on one of the single strands or on both single strands. The dsDNA template can be linear dsDNA or circular dsDNA, with a length of 200–1500 bp. NTP refers to the four nucleoside triphosphates required for transcription.

[0039] Example 1: Preparation of dsDNA template required for T7 RNA polymerase transcription reaction

[0040] The transcription reaction of T7 RNA polymerase requires four nucleotide triphosphates (NTPs) and a dsDNA template containing a T7 promoter. This invention utilizes a dsDNA template with T7 promoter sequences in both 5'-3' directions of its two single strands, i.e., a dsDNA template with a bidirectional T7 promoter sequence, which significantly improves the efficiency and detection limit of enzyme activity assay. Furthermore, using a dsDNA with a bidirectional T7 promoter as a template allows for complementary pairing of the two RNA single strands produced by T7 RNA polymerase during enzyme activity detection, thereby reducing the impact of product degradation on enzyme activity assays.

[0041] This embodiment uses the commonly used eGFP sequence as an example. Using primers eGFP-F and eGFP-R (sequences shown in Table 1), and a vector (pET28a-eGFP, purchased from Wuhan Miaoling Biotechnology Co., Ltd.) carrying the eGFP gene sequence as a template, dsDNA with a bidirectional T7 promoter was obtained by PCR amplification, which served as a template for the subsequent T7 RNA polymerase transcription reaction.

[0042] Table 1: Primers for PCR amplification of dsDNA template fragments

[0043] Primer name Primer sequence (5'-3') eGFP-F taatacgactcactataggggATGGTGAGCAAGGGCGAG (SEQ ID NO: 2) eGFP-R taatacgactcactatagggGGTAGTGGTCGGCGAGC (SEQ ID NO: 3)

[0044] The PCR amplification system is shown in Table 2. The amplification conditions were 98℃ for 10 s; 58℃ for 5 s; 72℃ for 3 s; for a total of 30 cycles. The final amplified dsDNA template was analyzed by agarose gel electrophoresis, and the results are as follows: Figure 2 As shown in the image; where "Marker" represents the marker nucleic acid and "eGFP" represents the eGFP dsDNA template fragment obtained by PCR. The result shows a single, clear band, indicating high purity of the dsDNA template, with a band size of approximately 590 bp, consistent with expectations. After purification using a commercial purification kit, the concentration of the dsDNA template was determined to be 200 ng / μL by NanoDrop, and it was stored at -20℃.

[0045] Table 2: PCR amplification solution system for dsDNA template fragments

[0046] Components Dosage (μL) 2x PrimerSTAR Mixture 25 Forward primer eGFP-F (25mM) 1.0 Reverse primer eGFP-R (25mM) 1.0 pET28a-eGFP plasmid (20 ng / μL) 0.5 <![CDATA[ddH2O]]> Add to a total volume of 50 μL

[0047] The sequence of one single strand of the dsDNA template to be amplified is as follows:

[0048] (SEQ ID NO: 1)

[0049] Example 2: Optimization of detection wavelength in the xyleneamine blue colorimetric reaction

[0050] In the xyleneamine blue method for colorimetric reaction, considering the significant difference between the absorption peaks corresponding to different concentrations of magnesium ions in the liquid, this invention performs full-wavelength scanning on the colorimetric reaction solutions corresponding to different concentrations of magnesium ions.

[0051] Full-wavelength scanning procedure: Equal volumes of xyleneamine blue stock solution and alkaline reagent stock solution were mixed and incubated at 37°C for 5 min. Magnesium acetate solutions of concentrations from 0 to 10.0 mM were prepared. 2.5 μL of magnesium acetate solutions of different concentrations were added to 250 μL of xyleneamine blue working solution in an ELISA plate and mixed well. After incubation at 37°C for 2 min, the plate was shaken for 5 s on a TECAN microplate reader. The absorbance of the colorimetric reaction solutions corresponding to different concentrations of magnesium ion solutions was scanned in the wavelength range of 500–700 nm.

[0052] Scan results as follows Figure 3As shown, in the wavelength range of 590–620 nm, the difference in light absorption peaks between the colorimetric reaction solutions corresponding to different concentrations of magnesium ions is relatively large, reaching its peak at a wavelength of 605 nm. Under a single detection wavelength, different light absorption values ​​in the colorimetric reaction correspond to different magnesium ion concentrations in the solution; from Figure 3 It can be seen that the difference between the absorption peaks at 605 nm is significantly greater than the difference between the absorption peaks at a single wavelength within the detection wavelength range of 505 nm to 540 nm in traditional methods. Therefore, selecting a detection wavelength of 605 nm can further improve the sensitivity of T7 RNA polymerase activity detection.

[0053] Example 3: Construction of a standard curve for magnesium ion concentration

[0054] Based on the optimization results of selecting 605nm as the detection wavelength in Example 2, a magnesium ion standard curve was plotted with magnesium ion concentration on the x-axis and absorbance (OD) at 605nm on the y-axis. The results are as follows: Figure 4 As shown in the figure, within the concentration range of 0.5–8.0 mM, there is a good linear relationship between magnesium ion concentration and absorbance, with a coefficient of determination R0. 2 The xyleneamine blue colorimetric reaction detection method with a detection wavelength of 605 nm and a value of 0.998 can accurately determine the concentration of magnesium ions in the solution.

[0055] Example 4: Optimization of magnesium ion concentration in T7 RNA polymerase transcription reaction

[0056] In in vitro transcription experiments, it is crucial to control the magnesium ion concentration within a reasonable range. Both excessively low and excessively high magnesium ion concentrations can inhibit the activity of T7 RNA polymerase, thereby affecting mRNA synthesis efficiency. Therefore, proper control of the magnesium ion concentration is essential for efficient and specific transcription reactions.

[0057] Transcription reaction: T7 RNA polymerase transcription reaction solution systems containing different concentrations of magnesium ions (0, 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 120 mM) were prepared; a solution without T7 RNA polymerase standard was used as a blank control; the specific formulations are shown in Table 3. After thorough mixing of each solution, the reaction was carried out at 37℃ for 1 h, and then terminated by treatment at 85℃ for 10 min; all reaction solutions were then centrifuged at 12000 rpm for 10 min, the supernatant was collected and diluted 10-fold with RNase-free H2O for the determination of magnesium ion concentration.

[0058] Determination of magnesium ion concentration changes using the xyleneamine blue method: Take 2.5 μL of the diluted reaction supernatant and add it to 250 μL of xyleneamine blue working solution. Incubate at 37℃ for 2 min, and then measure the OD value (A1) of each reaction solution at 605 nm. Using the OD value (A0) of the blank control reaction supernatant, calculate the absorbance difference ΔA (ΔA = A1 - A0) of each reaction solution at 605 nm. Three replicates were set up for each concentration determination.

[0059] The results of the magnesium ion concentration change measurement are as follows: Figure 5 As shown, and in combination Figure 4 The magnesium ion standard curve shows that ΔA reflects the enzyme activity of T7 RNA polymerase under corresponding magnesium ion concentrations. A larger ΔA indicates stronger catalytic activity of T7 RNA polymerase under that magnesium ion concentration. Both excessively high and low magnesium ion concentrations affect enzyme activity. When the magnesium ion concentration ranges from 40 to 80 mM, the effect of magnesium ion concentration on enzyme activity is relatively small, with a magnesium ion concentration of approximately 60 mM being relatively optimal.

[0060] Table 3: Optimization of transcription reaction solution system by magnesium ion concentration

[0061] Components volume Final concentration / mass / enzyme activity <![CDATA[10×T7 buffer (without Mg 2+ ions)]]> 5μL 1× dsDNA template containing the T7 promoter sequence 5μL Approximately 6μg NTP mixture (50mM each) 5μL 5mM each T7 RNA polymerase standard (50 U / μL) 2μL 100U 1M magnesium acetate solution 0, 0.5 μL to 6 μL 0, 10~120mM <![CDATA[RNase-free H2O]]> Make up to 50 μL /

[0062] Example 5: Determination of the standard curve of T7 RNA polymerase activity

[0063] Transcription reaction: T7 RNA polymerase transcription reaction solutions containing different enzyme activities (0, 10 U, 20 U, 30 U, 40 U, ​​50 U, 60 U, 70 U) were prepared; a solution without T7 RNA polymerase standard was used as a blank control; see Table 4 for specific formulations. After thorough mixing of all solutions, the reaction was carried out at 37℃ for 1 h, and then terminated by treatment at 85℃ for 10 min; all reaction solutions were then centrifuged at 12000 rpm for 10 min, the supernatant was collected and diluted 10-fold with RNase-free H2O for the determination of magnesium ion concentration.

[0064] Determination of magnesium ion concentration changes using the xyleneamine blue method: 2.5 μL of diluted reaction supernatant was added to 250 μL of xyleneamine blue working solution. After incubation at 37 °C for 2 min, the OD value (A1) of each reaction solution was measured at 605 nm. The OD value of the blank control reaction supernatant was used as A0. The absorbance difference ΔA (ΔA = A1 - A0) at 605 nm was calculated for each reaction solution. Three replicates were performed for each concentration determination.

[0065] Table 4: Standard Curve Transcription Reaction Solution System

[0066] Components volume Final concentration / mass / enzyme activity <![CDATA[10×T7 buffer (containing 0.6 M Mg 2+ ions)]]> 5μL 1× dsDNA template containing the T7 promoter sequence 5μL Approximately 6ug NTP mixture (50mM each) 2.5μL 5mM each T7 RNA polymerase standard (10 U / μL) 0, 1~7μL 0，10～70U <![CDATA[RNase-free H2O]]> Make up to 50 μL /

[0067] Plot a graph with the T7 RNA polymerase standard enzyme activity (U) on the x-axis and the absorbance difference ΔA (ΔA = A1 - A0) at 605 nm on the y-axis, as shown below. Figure 6 The standard curve is shown.

[0068] Depend on Figure 6 It is evident that within the enzyme activity range of 10–70 U, the enzyme activity of T7 RNA polymerase exhibits a good linear relationship with the absorbance difference, with a coefficient of determination R0. 2 It is 0.9928.

[0069] Example 6: Determination of enzyme activity in crude enzyme solution of recombinant T7 RNA polymerase expressed in Escherichia coli

[0070] The encoding gene for T7 RNA polymerase was constructed into an *E. coli* expression vector, transformed into *E. coli*, and expressed using IPTG to obtain recombinant bacterial cells accumulating T7 RNA polymerase. 5 g of recombinant bacteria were reconstituted in 40 mL of 20 mM phosphate buffer (pH 7.0), placed on ice, and sonicated to disrupt the cells. The cells were then centrifuged at 12000 rpm for 10 min, and the supernatant was collected as the crude T7 RNA polymerase solution. The crude enzyme solution was then subjected to ultrafiltration three times using the same phosphate buffer to obtain the ultrafiltered crude enzyme solution.

[0071] Transcription reaction: Take 2 μL of ultrafiltration crude enzyme solution and prepare a solution system as shown in Table 5. The final magnesium ion concentration is 60 mM. After the solution is thoroughly mixed, react at 37℃ for 1 h. Then, treat at 85℃ for 10 min to terminate the reaction. Centrifuge the reaction solution after treatment at 85℃ at 12000 rpm for 10 min, collect the supernatant and dilute it 10 times for the determination of magnesium ion concentration.

[0072] Determination of magnesium ion concentration change by xyleneamine blue method: The magnesium ion concentration change was tested using the xyleneamine blue method described in Example 5. Three replicates were performed for each concentration, and the average value was taken as the measured value. The absorbance difference ΔA (ΔA = A1 - A0) at 605 nm was found to be 0.248. Substituting this value into the T7 RNA polymerase activity standard curve obtained in Example 5, the enzyme activity of the crude enzyme solution was calculated to be 36.10 U (2 μL), or 18.05 U / μL.

[0073] Table 5: Transcription reaction solution system for ultrafiltration crude enzyme solution

[0074] Components volume Final concentration / mass / enzyme amount <![CDATA[10×T7 buffer (containing 0.6M Mg 2+ ions)]]> 5μL 1× dsDNA template containing the T7 promoter sequence 5μL Approximately 6μg NTP mixture (50mM each) 5μL 5mM each Ultrafiltration crude enzyme solution 2μL To be tested <![CDATA[RNase-free H2O]]> Make up to 50 μL /

[0075] Example 7: Determination of enzyme activity in purified enzyme solution of recombinant T7 RNA polymerase expressed in Escherichia coli

[0076] 50g of the recombinant bacteria from Example 6 was ultrasonically disrupted, and the enzyme was purified using affinity chromatography and ion exchange chromatography to obtain a purified enzyme solution. SDS-PAGE analysis showed that the protein purity in the purified enzyme solution was greater than 95%, with a concentration of 0.5 μg / μL. The purified enzyme solution was diluted 5 times to obtain a diluted purified enzyme solution.

[0077] Transcription reaction: Take 2 μL of purified enzyme solution dilution to prepare the T7 RNA polymerase reaction system as shown in Table 6. The final magnesium ion concentration is 60 mM. After the solution is thoroughly mixed, react at 37℃ for 1 h. Then, treat at 85℃ for 10 min to terminate the reaction. Centrifuge the reaction solution after treatment at 85℃ at 12000 rpm for 10 min, collect the supernatant and dilute it 10 times for the determination of magnesium ion concentration.

[0078] Determination of magnesium ion concentration change by xyleneamine blue method: The magnesium ion concentration change was tested using the xyleneamine blue method described in Example 5. Three replicates were performed, and the average value was taken as the measured value. The absorbance difference ΔA (ΔA = A1 - A0) at 605 nm was 0.415. Substituting this value into the T7 RNA polymerase activity standard curve obtained in Example 5, and taking into account the dilution factor of the purified enzyme solution, the enzyme activity of the purified solution was calculated to be 290.4 U (2 μL), or 145.2 U / μL.

[0079] Table 6: Solution system for determining enzyme activity in purified enzyme solution

[0080] Components volume Final concentration / mass / enzyme amount <![CDATA[10×T7 buffer (containing 0.6 M Mg 2+ ions)]]> 5μL 1× dsDNA template containing the T7 promoter sequence 5μL Approximately 6μg NTP mixture (50mM each) 5μL 5mM each Purified enzyme solution dilution 2μL To be tested <![CDATA[RNase-free H2O]]> Make up to 50 μL /

Claims

1. A method for detecting the enzyme activity of T7 RNA polymerase, characterized in that, The method includes the following steps: S1: dsDNA template required for T7 RNA polymerase transcription reaction by PCR amplification; S2: Prepare a T7 RNA polymerase transcription reaction solution system containing the DNA template in S1 and T7 RNA polymerase standards of different concentrations, carry out the transcription reaction, and obtain the reaction solutions of each standard and the blank control reaction solution; S3: Take each reaction solution from S2, and determine the absorbance difference ΔA before and after the reaction using the xyleneamine blue method to plot the enzyme activity standard curve of T7 RNA polymerase; the abscissa of the enzyme activity standard curve of T7 RNA polymerase is the absorbance difference ΔA, and the ordinate is the enzyme activity of T7 RNA polymerase, or the ordinate of the enzyme activity standard curve of T7 RNA polymerase is the absorbance difference ΔA, and the abscissa is the enzyme activity of T7 RNA polymerase; the detection wavelength is 590-620 nm. S4: Take the T7 RNA polymerase sample to be tested, prepare the T7 RNA polymerase transcription reaction solution system, carry out the transcription reaction, and obtain the test sample reaction solution and the blank control reaction solution; S5: Take each reaction solution from S4 and determine the absorbance difference ΔA of the reaction solution before and after the reaction using the xyleneamine blue method. Substitute ΔA into the standard curve obtained in S3 to calculate the enzyme activity of T7 RNA polymerase in the T7 RNA polymerase sample to be tested. The detection wavelength is 590-620 nm.

2. The method as described in claim 1, characterized in that, The detection wavelength is 605nm.

3. The method as described in claim 1, characterized in that, In steps S3 and S5, before the xyleneamine blue determination, the reaction solution is diluted by an appropriate factor so that the magnesium ion concentration in the diluted reaction solution is in the range of 0.5 to 8.0 mM.

4. The method as described in claim 1, characterized in that, The absorbance difference ΔA is calculated by the formula ΔA=A1-A0, where A1 is the absorbance of each standard reaction solution or the test sample reaction solution at the detection wavelength, and A0 is the absorbance of the blank control reaction solution at the detection wavelength.

5. The method according to any one of claims 1-4, characterized in that, In step S1, the dsDNA template has a T7 promoter sequence in both 5'-3' directions of the two single strands, and the length of the dsDNA template is 200-1500 bp.

6. The method as described in claim 5, characterized in that, In step S1, the sequence of a single strand in the dsDNA template is shown in SEQ ID NO:

1.

7. A kit for detecting the activity of T7 RNA polymerase, characterized in that, The kit includes: (1) T7 RNA polymerase standard; (2) T7 RNA polymerase buffer; (3) dsDNA template; (4) four NTPs; (5) xyleneamine blue stock solution; and (6) alkaline reagent stock solution; The T7 RNA polymerase buffer is an aqueous solution containing magnesium ion salt, 50 mM dithiothreitol, 25 mM spermidine and 400 mM Tris-HCl, with a pH of 7.5 to 8.5; wherein the magnesium ion salt is selected from magnesium acetate, magnesium chloride or magnesium sulfate, and the concentration of magnesium ions in the aqueous solution is 40 mM to 80 mM. The xyleneamine blue stock solution is an ethanol solution containing 3% (w / v) xyleneamine blue II; The alkaline reagent storage solution is an aqueous solution containing 1% (w / v) Tween-20, 20 mM borax and 20 mM sodium hydroxide, with a pH of 9.5 to 10.

5.

8. The kit according to claim 7, characterized in that, The dsDNA template has a T7 promoter sequence in both of its single strands along the 5'-3' direction, and the length of the dsDNA template is 200-1500 bp.

9. The reagent kit as described in claim 8, characterized in that, In step S1, the sequence of a single strand in the dsDNA template is shown in SEQ ID NO:

1.

10. The use of a kit as described in any one of claims 7-9 in the detection of T7 RNA polymerase activity.

Images