WO2022116378A1

WO2022116378A1 - Method for cleaving dna using soybean extract bowman-birk inhibitor

Info

Publication number: WO2022116378A1
Application number: PCT/CN2021/073010
Authority: WO
Inventors: 杨兆琪; 龚森怡; 尚孟迪; 金坚
Original assignee: 江南大学
Priority date: 2020-12-02
Filing date: 2021-01-21
Publication date: 2022-06-09
Also published as: CN112553274A; CN112553274B

Abstract

Disclosed is a method for cleaving DNA using a soybean extract Bowman-Birk inhibitor, which belongs to the technical field of medicines. Provided is a new use of a soybean Bowman-Birk trypsin inhibitor BBI-A. The BBI-A is used to cleave DNA, and the cleavage efficiency can reach 100% due to the optimization of conditions, such that DNA can be effectively cleaved. A new possibility is provided for preparing a DNA structural probe and potentially preparing an anti-tumor drug.

Description

Method for cutting DNA by Bowman-Birk inhibitor of soybean extract

technical field

The invention relates to a method for cutting DNA by Bowman-Birk inhibitor of soybean extract, and belongs to the technical field of medicine.

Background technique

Nucleic acid is an important biological macromolecule and an indispensable component of all known life forms. It plays a very important role in biological growth, development and reproduction. Nucleic acid cleaving agents play a vital role in the field of nucleic acid chemistry research. In the field of biochemistry and molecular biology, the research on nucleic acid cleaving agents has received extensive attention. Through the research on nucleic acid cleaving agents, some important information in organisms can be obtained. , provides the mechanism of action of nucleases in vivo, and has great research value in medicine and biology.

Bowman–Birk inhibitor (BBI) is a typical natural protease inhibitor isolated from soybean. So far, five BBI subtypes (A, B, C-II, D-II and E-I), according to existing research, BBI-A protease inhibitor in plant is very similar to α-1 antitrypsin in human, both of them have higher anti-cancer effect because of this feature Activity, plays a vital role in the protection of their respective bodies. Because BBI-A protease inhibitors have such activities that are closely related to life, more and more people are paying attention to the study of BBI-A.

According to the existing experimental results, the research on BBI-A at home and abroad mainly focuses on the extraction, separation, structure, biological activity and application prospects of BBI-A itself, especially in the fields of anti-inflammatory and anti-virus. Less involved in other areas.

Therefore, further excavating the potential functions of BBI-A will be able to help make better use of BBI-A.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the applicant of the present invention provides a BBI-A to cut DNA, the purpose of the present invention is to provide a kind of application of BBI-A in the cutting of plasmid DNA, and realize a new DNA cutting method. The BBI-A of the present invention has a cleavage effect on DNA and the optimal conditions for the cleavage of DNA by BBI-A are determined.

The invention provides a DNA cutting method, which is characterized in that the DNA is cut by using soybean Bowman-Birk trypsin inhibitor.

In one embodiment of the present invention, the amino acid sequence of the soybean Bowman-Birk trypsin inhibitor (BBI-A) is shown in SEQ ID NO.1.

In one embodiment of the present invention, 1-15 mg/mL of BBI-A is added to the reaction system.

In one embodiment of the present invention, the reaction system contains metal ions.

In an embodiment of the present invention, the concentration of the metal ions is 0.1-10 mmol/L.

In one embodiment of the present invention, the metal ions include Mg ²⁺ , Cu ²⁺ , Zn ²⁺ and/or Ca ²⁺ .

In one embodiment of the present invention, the reaction time is not less than 0.5h.

In one embodiment of the present invention, the reaction temperature is 35-40°C.

The present invention provides a DNA cleavage kit, which is characterized in that the kit comprises buffer, soybean Bowman-Birk trypsin inhibitor and metal ions.

In one embodiment of the present invention, the buffer is BR buffer.

In an embodiment of the present invention, the concentration of the BR buffer is 120 mmol/L and the pH is 7.0.

The present invention provides the application of the BBI-A, or the method, or the kit in cutting DNA.

In one embodiment of the present invention, the DNA includes circular DNA, strand DNA.

The beneficial technical effects of the present invention are:

1. The present invention uses BBI-A and plasmid pUC19 DNA as the main materials, mixes them for reaction, and detects the reaction result of BBI-A on DNA by using DNA agarose gel electrophoresis technology to explore the interaction between BBI-A and DNA. At the same time, the concentration of the reactants, the reaction time, etc. were changed, and the reaction results were determined by gel electrophoresis to determine the optimal conditions for the interaction between BBI-A and DNA, so that the cleavage rate could reach 100%.

2. The BBI-A of the present invention has nuclease activity, can cut DNA to a certain extent, and has certain guiding significance for exploring the role of BBI-A and DNA and designing new DNA structure probes, etc. The field of drug development is possible as new antitumor drugs.

Description of drawings

Figure 1 is a schematic diagram of the best auxiliary metal ion results for DNA cleavage by BBI-A; (A) Lane 1: DNA control group; Lane 2: Mg ²⁺ , 0.1mM; Lane 3: Zn ²⁺ , 0.1mM; Lane 4: Cu ²⁺ , 0.1 mM; Lane 5: Ca ²⁺ , 0.1 mM; Lane 6: added protein without ions; (B) is the cleavage results under different ion conditions.

Figure 2 is a schematic diagram of the optimal concentration of auxiliary metal ions for DNA cleavage by BBI-A; lane 1 in (A): DNA control group; the final concentrations of Cu ²⁺ in lanes 2-6 are: 0.1 mM, 0.5 mM, 1 mM, respectively , 5mM, 10mM; (B) is the cleavage result under the condition of each Cu ²⁺ concentration.

Figure 3 is a schematic diagram of the results of the optimal protein concentration of BBI-A cutting DNA; (A) lanes 1-5 are 1mg/L, 3mg/L, 5mg/L, 10mg/L, 15mg/L BBI- A; (B) are the cleavage results under different BBI-A concentrations.

Figure 4 is a schematic diagram of the results of the optimal reaction time for BBI-A to cut DNA; (A) lane 1 is the DNA control group; the reaction times in lanes 2-6 are: 0.5h, 1h, 1.5h, 2h, 2.5h; (B) are the cleavage results under different reaction times.

Figure 5 is the BBI-A cleavage diagram of the mixed system; lanes 1-2 are the DNA control group, the BBI-A cleavage group, and the 4; n-Butanol was added; lane 5: BBI-A cleavage group, KI was added; lane 6: BBI-A cleavage group, NaN ₃ was added.

Figure 6 is the connection diagram of DNA after cutting; lanes 1-3 are the DNA control group, the BBI-A cutting group, and the connecting group, respectively.

Detailed ways

BBI-A used in the following examples is soybean Bowman-Birk trypsin inhibitor, and its amino acid sequence is shown in SEQ ID NO.1.

Example 1: Effect of ionic species on DNA cleavage by BBI-A

Experiments with different ion species and concentrations can find the best ion species with the best cleavage effect on DNA

(1) Prepare the sample: add it to a 1.5 mL EP tube, and then add 10.5 μL of BR buffer (120 mmol/L, pH 7.0), 5 μL of protein solution (20 mg/ml), and 2 μL of 1 mmol/L metal For the ionic solution, we choose four ions of Mg ²⁺ , Zn ²⁺ , Cu ²⁺ and Ca ²⁺ for experiments. Mix thoroughly with a vortex mixer. After centrifugation, add 2.5 μL of 100 ng/μL pUC19 DNA plasmid. The reaction system is 20 μL in total. After labeling, mix well and centrifuge in a centrifuge. Seal and store at 37°C. Constant temperature metal bath reaction 2h.

(2) Preparation of 1% agarose gel: Weigh 1 g of solid agarose and add it to a 250 mL conical flask containing 100 mL of 1×TAE solution. Repeat heating with a microwave oven and then shake to dissolve the solid completely. until the solution is clear and transparent. When the agarose solution is placed at room temperature and gradually cooled to below 60°C, add 7 μL of EB solution (ethidium bromide), shake the conical flask to make the solution evenly mixed, and then slowly pour the agarose solution into the prepared level. In the plastic box, insert the comb. Allow the gel to dry naturally and remove the baffle and comb.

(3) Sample loading: Take out the fully reacted sample from the thermostat, pipet 4 μL of 6×Loading Buffer into the centrifuge tube, and use a vortex mixer to fully shake and mix evenly before centrifuging. After the solution is evenly mixed, draw the sample and add it to the spotting well.

pUC19 DNA: 250ng, final concentration of protein solution: 5mg/mL, 37°C, 2h. In Figure 1, lane 1: DNA control (plasmid pUC19 DNA before treatment); lanes 2-5: Mg ²⁺ , Zn ²⁺ , Cu ²⁺ , Ca ²⁺ with a final concentration of 0.1 mmol/L respectively; lanes 6: Does not contain metal ions.

(4) Electrophoresis: Tighten the lid of the electrophoresis apparatus in the correct direction, turn on the switch, turn on the power, adjust the voltage to 100V, the current to 260mA, and run the gel for 40min, so that the cleaved pUC19 plasmid DNA and its fragments migrate in the agarose gel .

(5) Photographing and data analysis: The agarose gel was photographed by using a gel imager and the original file was saved, and the original file was analyzed by software. Under the condition of ion concentration of 1 mmol/L, the effect of the four ions on the effect of assisted BBI-A on DNA cleavage is not much different, and they can basically reach 43%. Among them, the effect of Cu ²⁺ assisted BBI-A on the cleavage of pUC19 DNA plasmid is better than that of other ions. With three ions, a cutting rate of 52.8% was achieved. Therefore, Cu ²⁺ , which has a better effect among the four metal ions, was selected as the experimental auxiliary ion for the experiment.

Example 2: Effect of ion concentration on DNA cleavage by BBI-A

The effect of ion concentration on DNA cleavage by BBI-A was examined using gel electrophoresis. Cu ²⁺ was chosen as the auxiliary ion for the reaction, and experiments were carried out with different ion concentrations (0.1 mmol/L, 0.5 mmol/L, 1 mmol/L, 5 mmol/L, 10 mmol/L).

(1) Prepare the sample: add it to a 1.5mL EP tube, and sequentially add 10.5μL of BR buffer (120mmol/L, pH7.0), 5μL of target compound solution (20mg/ml), and 2μL of sulfuric acid of different concentrations Copper solution (1mmol/L, 5mmol/L, 10mmol/L, 50mmol/L, 100mmol/L). Mix thoroughly with a vortex mixer, add 2.5 μL of 100 ng/μL pUC19 DNA plasmid after centrifugation, label the number, mix well, then centrifuge with a centrifuge, seal and place in a constant temperature metal bath at 37°C for 2 hours of reaction .

(2) Preparation of 1% agarose gel: Weigh 1 g of solid agarose and add it to a 250 mL conical flask containing 100 mL of 1×TAE solution. Repeat heating with a microwave oven and then shake to dissolve the solid completely. until the solution is clear and transparent. When the solution was gradually cooled to below 60°C at room temperature, 7 μL of EB solution (ethidium bromide) was added, the conical flask was shaken to make the solution evenly mixed, and then the agarose solution was slowly poured into the prepared horizontal plastic frame. , insert the comb. Allow the gel to dry naturally and remove the baffle and comb.

(3) Sample loading: Take out the fully reacted sample from the thermostat, pipet 4 μL of 6×Loading Buffer into the centrifuge tube, and use a vortex mixer to fully shake and mix evenly before centrifuging. After the solution is evenly mixed, pipette 20 μL of the sample vertically and carefully into the spotting well slot.

pUC19 DNA: 250ng, final concentration of protein solution: 5mg/mL, 37°C, reaction for 2h. In Figure 2, lane 1: DNA control (plasmid pUC19 DNA before treatment); lane 2-6: Cu ²⁺ concentrations were 0.1 mmol/L, 0.5 mmol/L, 1 mmol/L, 5 mmol/L, and 10 mmol/L, respectively.

(4) Electrophoresis: Close the lid of the electrophoresis apparatus in the correct direction, turn on the switch, turn on the power, adjust the voltage to 100V, the current to 260mA, and run the gel for 40min to allow the cut DNA and its fragments to migrate in the agarose gel.

(5) Photograph and data analysis: by using a gel imager to photograph the agarose gel and save the original file, use software to analyze the original file, as can be seen from Figure 2, Cu ²⁺ of different concentrations (from 0.1mmol /L～10mmol/L) had little effect on the results of assisted BBI-A cleavage of DNA.

Example 3: Effects of BBI-A concentration changes on DNA cleavage under ion-free conditions

Since the cleavage efficiency of DNA was not greatly improved by ions, the reaction system was further optimized under ion-free conditions.

(1) Prepare the sample: add it to a 1.5 mL EP tube, add 20 mg/ml protein solution to 1 μL, 3 μL, 5 μL, 10 μL, and 15 μL in turn, and then add BR buffer (120 mmol/L, pH 7.0) to make up 17.5 μL. Mix thoroughly with a vortex mixer, add 2.5 μL of 100 ng/μL pUC19 DNA plasmid after centrifugation, make a serial number mark, mix well, then centrifuge with a centrifuge, seal it and place it in a constant temperature metal bath at 37°C for 2 hours .

pUC19 250ng, 37℃, 2h. In Figure 3, lane 1: DNA control; lane 2-6: BBI-A solution final concentrations are 1mmol/L, 3mmol/L, 5mmol/L, 10mmol/L, 15mmol/L, respectively.

(5) Photographing and data analysis: The agarose gel is photographed by using a gel imager and the original file is saved, and the original file is analyzed by software, as shown in Figure 3, under the condition of no ions, with the increase of the concentration , the cutting effect is obviously getting better and better, when the protein concentration reaches 15mmol/L, the cutting rate can reach 100%.

Example 4: Effect of reaction time on DNA cleavage by BBI-A

The effect of reaction time on DNA cleavage by BBI-A was investigated.

(1) Prepare the sample: add it to a 1.5mL EP tube, and then add 10.5μL of BR buffer (120mmol/L, pH7.0), 5μL of protein (20mg/mL) solution, and 2μL of 1mmol/L sulfuric acid copper solution. Mix thoroughly with a vortex mixer. After centrifugation, add 2.5 μL of 100 ng/μL pUC19 DNA plasmid with a final concentration of 5 mg/mL of protein. After labeling, mix well and then centrifuge in a centrifuge. Seal it. React in a constant temperature metal bath at 37°C for 0.5-2.5h respectively.

In Figure 4, lane 1: DNA control (plasmid pUC19 DNA before treatment); lane 2-6: 0.5h, 1h, 1.5h, 2h, 2.5h.

(5) Photographing and data analysis: The agarose gel is photographed by using a gel imager and the original file is saved, and the original file is analyzed by software. As can be seen from Figure 4 above, with the prolongation of the reaction time, the cleavage effect of BBI-A on DNA became more and more obvious. And from the figure, we can see that the DNA of the control group is almost not broken, and the sample added with BBI-A has a cleavage rate of 52.4% even if it is only reacted for 2 hours. Good cutting action.

Example 5: DNA cleavage mechanism verification

(1) Cutting experiment

A free radical scavenger was added to the reaction system to observe the changes of the cleavage results to judge the mechanism of DNA cleavage by BBI-A. It contains BR buffer (120 mmol/L, pH 7.0), the concentration of DNA is 100 ng/μL, the concentration of protein solution is 20 mg/ml, and the concentration of DMSO, n-butanol, KI and NaN ₃ solution is 1 M. After adding the reagents in sequence according to the following table, shake and centrifuge them and place them in a constant temperature water bath for 2 hours at a constant temperature of 37°C.

Table 1. Reaction systems discussed in cleavage mechanism

As shown in Figure 5, in the presence of hydroxyl radical quenchers such as DMSO and tert-butanol, the cleavage of DNA by BBI-A was not inhibited, indicating that there were no hydroxyl radicals in the BBI-A mixture, which did not belong to the mechanism of radical cleavage. In the presence of the singlet oxygen quencher sodium azide and the superoxide quencher potassium iodide, the cleavage of DNA by BBI-A was significantly inhibited. Therefore, we can preliminarily determine that the cleavage mechanism of BBI-A cutting DNA may be hydrolytic cleavage or oxidative cleavage.

(2) Connection experiment

The control group contained 250 ng DNA; the cutting group contained 2.5 μL DNA (100 ng/μL), 10 μL BBI-A solution (20 mg/mL), and 7.5 μL BR buffer (120 mmol/L, pH 7.0); the ligation group was cutting After the group was purified, the sample volume was concentrated to 15 μL and added to a centrifuge tube, then 2 μL of ligase buffer, 0.5 μL of T4 DNA ligase, and 2.5 μL of deionized water were added to form a total volume of 20 μL of sample for re-ligation experiments. .

Table 2 ligation reaction system

As shown in Figure 6, No. 1 is the DNA control, experimental group 1 is a group that did not add DNA T4 ligase for normal reaction, and experimental group 2 is a group that added DNA T4 ligase to react for 2 hours after purification, as shown in the experimental results It can be seen that lane 2 is the experimental result of purification and re-ligation after cleavage. From the situation of the bands in lane 2, it can be seen that there is no band with a slower mobility than the cleavage band, which proves that no ligation product DNA is regenerated, which indicates that the added T4 DNA ligase No effect, the cut DNA is not ligated again. It is known that hydrolytic cleavage only destroys phosphodiester bonds, and does not cause complete damage to DNA, and the cleaved DNA can be reconnected by T4 DNA ligase. It can be seen that the DNA cleavage by BBI-A is caused by singlet oxygen and superoxide, which belongs to oxidative cleavage.

Example 6: Preparation of DNA cleavage kit

The kit contains BR buffer (120mmol/L, pH 7.0), BBI-A, and metal ions, including Mg ²⁺ , Cu ²⁺ , Zn ²⁺ or Ca ²⁺ ; when used, BBI-A The final concentration of metal ions is added to 1-15 mg/mL, and the metal ion concentration is preferably 0.1-10 mM.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims

A method for cleaving DNA, characterized in that the DNA is cleaved with soybean Bowman-Birk trypsin inhibitor.
The method according to claim 1, wherein the amino acid sequence of the soybean Bowman-Birk trypsin inhibitor is shown in SEQ ID NO.1.
The method according to claim 2, wherein the concentration of soybean Bowman-Birk trypsin inhibitor in the reaction system is 1-15 mg/mL.
The method according to claim 3, wherein the reaction system contains metal ions.
The method according to claim 4, wherein the concentration of the metal ions is 0.1-10 mmol/L.
The method of claim 5, wherein the metal ions include Mg 2+ , Cu 2+ , Zn 2+ and/or Ca 2+ .
The method according to claim 6, wherein the reaction time is not less than 0.5h.
A DNA cleavage kit, characterized in that the kit comprises buffer, soybean Bowman-Birk trypsin inhibitor and metal ions.
The kit according to claim 8, wherein the metal ions comprise Mg 2+ , Cu 2+ , Zn 2+ and/or Ca 2+ .
The kit according to claim 9, wherein the buffer is a BR buffer.
Soybean Bowman-Birk trypsin inhibitor, or the method of any one of claims 1-7, or the application of any one of the kits of claims 8-10 in cutting DNA.
The application according to claim 11, wherein the DNA comprises circular DNA and strand DNA.