6-phosphofructokinase, preparation method thereof and application of 6-phosphofructokinase in screening and killing active substances of ampullaria gigas

Abstract

The invention provides 6-phosphofructokinase, a preparation method thereof and application of the 6-phosphofructokinase in screening and killing active substances of ampullaria gigas, and belongs to the technical field of biology, and the sequence of the 6-phosphofructokinase is shown as SEQ ID No.1. A compound capable of killing the ampullaria gigas can be rapidly screened out aiming at a PFK target spot of the ampullaria gigas, and the 6-phosphofructokinase can be applied to screening and killing active substances of the ampullaria gigas. Compared with traditional bred ampullaria gigas screening, the screening method is high in speed and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a 6-phosphofructokinase, its preparation method, and its application in screening active substances for killing golden apple snails. Background Technology

[0002] Golden apple snail ( Pomacea canaliculata Snails are invasive aquatic snails that cause serious damage to agriculture and the ecological environment. Traditional snail eradication methods suffer from environmental pollution and toxicity to non-target organisms. Therefore, developing an efficient, environmentally friendly, and highly specific snail eradication method is of great significance.

[0003] Existing methods for screening molluscicidal activity involve live in vivo screening, which suffers from drawbacks such as high dosage, long screening cycles, and unclear target sites for active substances. Furthermore, the snails may exhibit apparent death, leading to inaccurate results. Pyrospirin compounds primarily act on the liver of *Pomacea canaliculata*, inhibiting 6-phosphofructokinase in the liver.

[0004] Phosphofructokinase 6-kinase (PFK) is an important antioxidant enzyme that plays a crucial role in the survival and reproduction of the golden apple snail. Heterologous expression technology can be used to express the PFK gene from the golden apple snail in host cells, thereby enabling the screening of substances with molluscicidal activity. Summary of the Invention

[0005] This invention discloses a 6-phosphofructokinase, its preparation method, and its application in screening active substances for killing golden apple snails.

[0006] To achieve the above objectives, the first aspect of the present invention provides a 6-phosphofructokinase, the sequence of which is shown in SEQ ID No. 1.

[0007] A second aspect of the present invention provides a recombinant plasmid comprising the 6-phosphofructokinase described in the first aspect.

[0008] A third aspect of the present invention provides a recombinant strain comprising the 6-phosphofructokinase described in the first aspect.

[0009] A fourth aspect of the present invention provides a nucleotide encoding 6-phosphofructokinase as described in the first aspect, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0010] The fifth aspect of this invention provides a method for preparing 6-phosphofructokinase, comprising the following steps: (1) The gene encoding the 6-phosphofructokinase was cloned, and its nucleotide sequence is shown in SEQ ID No. 2. The gene was introduced into Escherichia coli to construct an expression system and obtain the recombinant expression strain. (2) The expression conditions are as follows: Escherichia coli OD cultured at 37℃ 600 The value was adjusted to 0.5-0.6, and induction was performed at 16℃ and 30℃ for 15-18 h; IPTG concentration was 0.2 mM. (3) Take 1 mL of culture from each induction sample, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL TBS. Collect the remaining culture, centrifuge at 4000 r / min for 10 min, discard the culture supernatant, and resuspend the bacterial pellet in TBS; (4) After adding PMSF to the resuspension to a final concentration of 1 mM and performing ultrasonic disruption, the supernatant and precipitate were respectively added to TBS for resuspension. (5) Perform 12% SDS-PAGE analysis; (6) The bacterial cells identified as inclusion bodies were resuspended in 40 ml of TBS at pH 8.0; ultrasonically disrupted for 2 min at 15% power until completely broken down without particles; (7) Add 500 μl of 20% Triton (final concentration 0.25%) to the above liquid; invert the centrifuge tube, mix thoroughly, and let stand for 10 min; (8) Centrifugation: 4℃, 10000rpm, 5min, then discard the supernatant; (9) The precipitate was resuspended in 4 ml TBS at pH 8.0 and sonicated for 2 min at 15% power until it was completely broken down into particles. (10) Add 16 ml of denaturing mother liquor TBS, 10 M urea, and pH 8.0 to the above liquid; (11) Place the centrifuge tubes in a rotary mixer and rotate for 2-3 hours to denature them; (12) Centrifugation: 4℃, 10000rpm, 5min, the supernatant is denatured protein.

[0011] (13) The crude enzyme solution of denatured 6-phosphofructokinase obtained in step (12) was purified multiple times using Ni-NTA Binding-Buffer (TBS, 0 mM imidazole, 8M urea, pH 8.0) to obtain purified 6-phosphofructokinase protein enzyme.

[0012] The sixth aspect of this invention provides the application of the 6-phosphofructokinase described in the first aspect in screening for active substances that kill golden apple snails.

[0013] The seventh aspect of this invention provides a method for screening active substances that kill golden apple snails using 6-phosphofructokinase, characterized by comprising the following steps: (1) In a 96-well plate, add an appropriate amount of recombinant 6-phosphofructokinase solution to each well, and then add solutions of different concentrations of the compounds to be screened, setting a single concentration gradient of 10 μg / mL to evaluate the dose dependence of the compounds; at the same time, a blank control group is set up. (2) The PFK enzyme activity reaction principle is to catalyze the fructose-6-phosphate and ATP to produce fructose-1,6-bisphosphate and ADP. Pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to NAD+ in sequence. The NADH decrease rate is measured at 340 nm to reflect the PFK activity.

[0014] (3) Enzyme activity definition: One enzyme activity unit is defined as the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP into 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute by 1 mg of tissue protein.

[0015] (4) Use an ELISA reader to measure the absorbance of each well at a wavelength of 340 nm and record the data.

[0016] Through the above technical solution, the present invention can achieve at least the following beneficial effects: This invention can rapidly screen compounds that can kill golden apple snails by targeting their PFK sites, which is faster and less costly than traditional screening methods for farmed golden apple snails. Attached Figure Description

[0017] Figure 1 These are SDS-PAGE images of fermentation supernatant and whole-cell protein in this invention. In the images, M: protein molecular weight standard; 1: supernatant induced by 16°C; 2: total protein induced by 16°C; 3: supernatant induced by 30°C; 4: total protein induced by 30°C. Figure 2 This is an SDS-PAGE analysis diagram of protein purification in this invention. In the diagram, M: protein molecular weight standard; 1: loaded protein (supernatant); 2: flow-through; 3: 25mM imidazole elution buffer; 4: 50mM imidazole elution buffer; 5: 100mM imidazole elution buffer; 6-7: 500mM imidazole elution buffer. Figure 3 This is a protein dialysis SDS-PAGE analysis diagram from the present invention. In the diagram, M: protein molecular weight standard; 1: loaded protein (supernatant); Figure 4 This is a bar chart showing the statistical effect of the compounds in this invention on the inhibitory effect of recombinant PFK enzyme. Detailed Implementation

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. Example

[0019] 1. Extraction of PFK expression gene from Pomacea canaliculata: Three primer pairs (P1, P2, and P3) were designed (Table 1) to retrieve the 6-phosphofructokinase gene from *Pomacea canaliculata* as the starting sequence (GenBank: PX625995, sequence source: NCBI). Codon optimization was performed to account for the codon bias of *E. coli*, and the amino acid sequence was named PFK. Based on the structural characteristics of the pET28a expression vector, a comprehensive design scheme was developed, adding restriction enzyme sites NcoⅠ and XhoⅠ to both ends of the PFK gene sequence. The optimized gene sequence was then inserted into the expression vector, and the recombinant plasmid was named pET28a-PFK, synthesized by Beijing Qingke Biotechnology Co., Ltd. The optimized protein sequence is shown in SEQ ID No. 1. The optimized nucleotide sequence is shown in SEQ ID No. 2.

[0020] Table 1. Primer information for P1, P2, and P3

[0021] SEQ ID No. 1: MAEVASPARRYSRKDSVLATPGKEGQLVDFGAWAGNSIAVFTSGGDSQGMNAAVRAVVRMGFYLGCKVYLVKEGYQGMVDGTENIVEATWSSVSGILQLGGTVIGSARCKEFRERQGRLKAAENLLKFNITNLVVIGGDGSLTGADLFRQEWSSLLQELVQNGTVTQEQAIQCQHLNIVGMVGSIDNDFCGTDMTIGTDSALHRIIECVDAISTTASSHQRAFVLEVMGRHCGYLALVAALASEADWVFIPEWPPEGDWRTTLCNKLATERNLGQRLNIVLVAEGAMDREGNAISADDVKNVLVERLHYDTRVTVLGHVQRGGSPSAFDRVLGSRMGAEAVLALMDAGPDTPACVVSLEGNQTVRVPLMECVARTKAVQAAMDERNFEEAVRLRGKSFQNNLNTYRLLSKLRPPSSLCTQQTHRNLAVMNVGAPACGMNAAVRSFVRLGLTQGFRILAIEESFDGLVAGRVKPFTWQQVQGWAACGGSLLGTKKQLAHEVGLGNIAEKLREFKIDGLLIVGGFEAFHSAYELGQNRAQYPAFCIPICVIPCTISNNVPGTDFSLGADTALNEISDICDRIKQSATGTKRRVFVVETMGGFCGYLATLSALAAGADAAYIYEEKTTISDLRDDVYHLKDKILNAGVQRGLVLRNENANPNYTTSFIHQLFAEEGKGVFSVRMNVLGHMQQGGVPSPFDRNYGTKMSAKCVDWFKEHINKNTTQSGRVEARRPDSVVLLGMQKRFLYFSPIEELAINVDFNKRLPLEQWWLKLRPLLRILAKHKVGAYQAEAELADLDEMTTSDNYHHHHHH*; SEQ ID No. 2:

[0022] 2. Expression and purification of recombinant PFK protein 2.1 Transformation of the recombinant vector into Escherichia coli BL21-DE3 Add 1 μL of plasmid to 100 μL of competent bacteria and place on ice for 20 min. Heat shock at 42°C for 45-60 sec, then immediately place on ice for 5 min, and add 600 μL of ILB culture medium. Shake at 37°C and 220 rpm for 1 h, centrifuge, and spread the entire mixture onto LB agar plates containing 50 μg / mL Kan at a final concentration of 0.1 V / V% (v / v), and incubate inverted at 37°C overnight.

[0023] 2.2 Identification of IPTG-induced expression of recombinant bacterial fusion protein (1) Pick a single clone from the transformed plate and inoculate it into 4 mL of LB liquid medium (test tube filling), and add 0.1 V / V% of antibiotic solution containing 50 μg / mL Kan. Shake overnight at 37°C and 220 r / min.

[0024] (2) The next day, inoculate 100 mL of LB liquid medium at a 1:100 ratio, with the antibiotic ratio as described above; shake at 37°C and 220 rpm until the bacterial cell OD reaches zero. 600 The effective value was 0.5-0.8; two parallel inoculations were performed.

[0025] (3) a. Add IPTG to one portion to a final concentration of 0.2 mM, and shake overnight at 16°C and 220 r / min to induce protein expression. b. Add IPTG to the other portion to a final concentration of 0.2 mM, and shake overnight at 30°C and 220 r / min to induce protein expression.

[0026] (4) Stop shaking the bacteria. Take 1 mL of culture from each induction sample, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL TBS. Collect the remaining culture, centrifuge at 4000 r / min for 10 min, discard the culture supernatant, and resuspend the bacterial pellet in TBS. Add PMSF to the resuspended solution to a final concentration of 1 mM, sonicate to disrupt the culture, and then take the supernatant and pellet solution separately and resuspend them in TBS.

[0027] (5) Perform 12% SDS-PAGE analysis; the banding is shown in the attached image after Coomassie brilliant blue staining. Figure 1 As shown.

[0028] 2.3 Inclusion body degeneration (1) The bacterial cells identified as inclusion bodies were resuspended in 40 mL of TBS at pH 8.0; ultrasonically disrupted for 2 min at 15% power until no particles were present.

[0029] (2) Add 500 μL of 20% Triton (final concentration of 0.25%) to the above liquid; invert the centrifuge tube, mix thoroughly, and let stand for 10 min.

[0030] (3) Centrifugation: 4℃, 10000rpm, 5min; discard the supernatant.

[0031] (4) The precipitate was resuspended in 4 ml of TBS at pH 8.0 and sonicated for 2 min at 15% power until it was completely broken down into particles.

[0032] (5) Add 16 mL of denatured mother liquor TBS, 10 M urea, and pH 8.0 to the above liquid.

[0033] (6) Insert the centrifuge tubes into a rotary mixer and rotate for 2-3 hours to denature them.

[0034] (7) Centrifugation: 4℃, 10000rpm, 5min; the supernatant is denatured protein.

[0035] 2.4 Ni column affinity purification (1) Purification was performed using a gravity column. The denatured solution was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA Binding-Buffer (TBS, 0 mM imidazole, 8M urea, pH 8.0).

[0036] (2) Rinse with 3-5 column volumes of Binding-Buffer to remove unbound proteins.

[0037] (3) Elute the target protein with Ni-NTA Elution-Buffer (TBS, 25 mM imidazole, 8M urea, pH 8.0) and collect the eluent.

[0038] (4) Elute the target protein with Ni-NTA Elution-Buffer (TBS, 50 mM imidazole, 8M urea, pH 8.0) and collect the eluent.

[0039] (5) Elute the target protein with Ni-NTA Elution-Buffer (TBS, 100 mM imidazole, 8M urea, pH 8.0) and collect the eluent.

[0040] (6) Elute the target protein with Ni-NTA Elution-Buffer (TBS, 500 mM imidazole, 8M urea, pH 8.0) and collect the eluent.

[0041] (7) Perform SDS-PAGE analysis, and the results are attached. Figure 2 As shown.

[0042] (8) After the protein solution collected above is identified and confirmed as the target band, it is added to the dialysis bag and dialyzed overnight using TBS.

[0043] (9) Perform SDS-PAGE analysis, and the results are attached. Figure 3 As shown.

[0044] 2.4 Recombinant protease activity analysis (1) In 96-well plates, the recombinant protease activity was detected using a phosphofructokinase (PFK) / fructose-6-phosphokinase kit.

[0045] (2) PFK reaction principle: It catalyzes the reaction of fructose-6-phosphate and ATP to produce fructose-1,6-bisphosphate and ADP. Pyruvate kinase and lactate dehydrogenase further catalyze the oxidation of NADH to NAD in sequence. + The rate of NADH decrease can be measured at 340 nm to reflect the activity of PFK.

[0046] (3) Enzyme activity definition: One enzyme activity unit is defined as the conversion of 1 nmol fructose-6-phosphate and 1 nmol ATP into 1 nmol fructose-1,6-bisphosphate and 1 nmol ADP per minute by 1 mg of tissue protein.

[0047] 2.5 Establishment and optimization of inhibitor screening system (1) The compounds are numbered with letters and Arabic numerals according to the order of their synthesis.

[0048] (2) First, we investigated whether all compounds inhibited recombinant 6-phosphofructokinase at a single concentration of 10 μg / mL.

[0049] (3) The preparation method of the stock solution of the compound used for screening research is as follows: weigh 5 mg of the test compound, add 1 mL of dimethyl sulfoxide to dissolve it, prepare a stock solution of 5000 μg / mL, and then dilute it 100 times to prepare a stock solution of 50 μg / mL.

[0050] (4) Inhibitor screening experiments were performed in 96-well plates using a phosphofructokinase (PFK) / fructose-6-phosphokinase kit.

[0051] (5) Each well reaction system contains: 1 μL of the test compound solution and 19 μL of phosphofructokinase solution. First, mix the test compound with the phosphofructokinase solution and incubate at room temperature (25℃) for 30 min. Then add reagents 1, 2, 3 and 4 from the kit, mix evenly, and let stand at room temperature (25℃) in the dark for 30 min. Measure the absorbance value at 450 nm.

[0052] (6) A blank control (inhibitor-free system) was set up. The inhibition rate of each compound on the activity of 6-phosphofructokinase was calculated according to Formula 1: Formula 1:

[0053] (7) Compounds with an inhibition rate >50% were set as positive in the initial screening and dose-effect verification was performed.

[0054] 2.6 Validation of the dose-response effect of positive initial screening (1) The test method of immersion killing snails was adopted.

[0055] (2) Experimental group: Weigh 10 mg of the test compound into a 5 mL capped round-bottom centrifuge tube, add 900 μL of dimethyl sulfoxide (DMSO) and 300 μL of Tween 20 into the centrifuge tube, and obtain the experimental stock solution after it is fully dissolved.

[0056] (3) Add each of the above stock solutions into a water bucket, dilute with water to 1L, and stir thoroughly.

[0057] (4) Blank control group: (a) 1L of water; (b) 1L of water with 300μL of Tween 20 added; (c) 1L of water with 900μL of DMSO added; (d) 1L of water with 300μL of Tween 20 and 900μL of DMSO added.

[0058] (5) Randomly select 8 adult snails that are active and similar in size, put them into the center of the bottom of each group of buckets, and seal the bucket with a lid (or plastic mesh) to prevent them from escaping.

[0059] (6) Observe the climbing behavior of golden apple snails in the bucket at 12-hour intervals. After soaking for 72 hours in each group of experiments, record the number of dead snails and remove the dead snails in time to avoid contaminating the experimental solution. For golden apple snails whose vitality status cannot be accurately determined, they can be placed in dechlorinated tap water. If the snail cannot extend its antennae within 1 hour, floats in the dechlorinated tap water, and does not respond to human stimulation with a needle-like object, or cannot open its mouth and move within 30 minutes, it is determined to be a dead snail. The 72-hour mortality rate of golden apple snails is calculated according to Formula 2, and the results are shown in Table 2 below.

[0060] Formula 2:

[0061] Table 2. Structures and 72-hour mortality rates of five compounds: C9, C10, C11, C12, and C14.

[0062] The preparation methods for the above six compounds are as follows: 1.

[0063] 1.88 g (10 mmol) of 3-amino-4-bromophenol was dissolved in 10 mL of dichloromethane (DCM). 1 mL of triethylamine (Et3N) was added and stirred until completely dissolved. 2.07 g (11 mmol) of 3,4-dichlorophenyl isocyanate was dissolved in 10 mL of DCM and added dropwise to the above reaction solution. The mixture was stirred at room temperature for 6 h, and the reaction progress was monitored by thin-layer chromatography (TLC) until the starting material was completely consumed. After the reaction was complete, the solid product was collected by vacuum filtration and washed repeatedly with water and DCM to remove impurities. Finally, it was dried in a vacuum drying oven. The solid product was characterized as follows: 1 mL of triethylamine (Et3N) was added to dichloromethane (DCM) and stirred until completely dissolved. 12 mmol of 3,4-dichlorophenyl isocyanate was dissolved in 10 mL of DCM and added dropwise to the above reaction solution. The mixture was stirred at room temperature for 6 h, and the reaction progress was monitored by thin-layer chromatography (TLC) until the starting material was completely consumed. After the reaction was completed, the solid product was collected by vacuum filtration and washed repeatedly with water and DCM to remove impurities. Finally, it was dried in a vacuum drying oven to obtain the solid product characterized as follows: 1-(5-bromo-2-hydroxyphenyl)-3-(3,4-dichlorophenyl)urea (NL-6c), gray solid, yield 70.5%.

[0064] 2.

[0065] 2-Amino-5-chloro-1,3,4-thiadiazole (1.0 mmol, 135.6 mg) and 3-chlorophenyl isocyanate (1.1 mmol, 168.3 mg) were added to a 100 mL reaction flask, followed by the addition of 20 mL of anhydrous acetonitrile. The reaction was carried out at 80°C for 5–6 h. After the reaction was complete, the precipitate was collected by vacuum filtration, washed with a small amount of anhydrous acetonitrile, and yielded 205.3 mg of a dry white solid, representing a yield of 71.0%.

[0066] 3.

[0067] 2-Amino-5-chloro-1,3,4-thiadiazole (1.0 mmol, 135.6 mg) and 3-bromophenyl isocyanate (1.1 mmol, 198.0 mg) were added to a 100 mL reaction flask, followed by the addition of 20 mL of anhydrous acetonitrile. The reaction was carried out at 80°C for 5–6 h. After the reaction was complete, the precipitate was collected by vacuum filtration, washed with a small amount of anhydrous acetonitrile, and yielded 200.8 mg of a dry white solid, representing a yield of 60.2%.

[0068] 4.

[0069] At room temperature, 640 mg (5 mmol) of compound 3-amino-5-chloropyridine was dissolved in 15 mL of DCM, and then 5 mmol of compound p-chlorophenyl isocyanate was added. The mixture was stirred for 5–7 h. After the reaction was complete (monitored by TLC), the mixture was filtered, the solid was washed with water and DCM, and dried to obtain the target compound as a white solid with a yield of 82.6%.

[0070] 5.

[0071] 2-Amino-5-chloro-1,3,4-thiadiazole (1.0 mmol, 135.6 mg) and 4-trifluoromethylphenyl isocyanate (1.1 mmol, 205.8 mg) were added to a 100 mL reaction flask, followed by the addition of 20 mL of anhydrous acetonitrile. The reaction was carried out at 80°C for 5–6 h. After the reaction was complete, the precipitate was collected by vacuum filtration, washed with a small amount of anhydrous acetonitrile, and yielded 189.1 mg of a dry white solid, representing a yield of 58.6%.

[0072] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A 6-phosphofructokinase, characterized in that, The sequence of the 6-phosphofructokinase is shown in SEQ ID No.

1.

2. A recombinant plasmid, characterized in that, The recombinant plasmid contains the 6-phosphofructokinase described in claim 1.

3. A recombinant bacterial strain, characterized in that, The recombinant strain contains the 6-phosphofructokinase described in claim 1.

4. A nucleotide encoding the 6-phosphofructokinase as described in claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID No.

2.

5. The method for preparing 6-phosphofructokinase as described in claim 1, characterized in that, Includes the following steps: (1) The gene encoding the 6-phosphofructokinase was cloned, and its nucleotide sequence is shown in SEQ ID No.

2. The gene was introduced into Escherichia coli to construct an expression system and obtain the recombinant expression strain. (2) The expression conditions are as follows: Escherichia coli OD cultured at 37℃ 600 The value was adjusted to 0.5-0.6, and induction was performed at 16℃ and 30℃ for 15-18 h; IPTG concentration was 0.2 mM. (3) After induction, take 1 mL of culture from each induction sample, centrifuge at 10000 r / min at room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet in 100 μL TBS; (4) After adding PMSF to the resuspension to a final concentration of 1 mM and performing ultrasonic disruption, the supernatant and precipitate were respectively added to TBS for resuspension. (5) Perform 12% SDS-PAGE analysis and Coomassie brilliant blue staining for banding; (6) After resuspending the bacterial cells identified as inclusion bodies in TBS and sonicating them, Triton was added for washing and centrifugation. The precipitate was then resuspended in TBS and sonicated. Subsequently, denaturing buffer containing urea was added for rotational denaturation. The supernatant was then centrifuged to obtain the denatured protein. (7) The denatured protein solution obtained in step (6) was loaded onto a Ni-NTA affinity chromatography column pre-equilibrated with Ni-NTA binding-Buffer using a His-tagged protein purification column to purify the protein to obtain 6-phosphofructokinase protein purification enzyme.

6. The application of the 6-phosphofructokinase as described in claim 1 in screening for active substances that kill golden apple snails.

7. The method for screening active substances that kill golden apple snails using 6-phosphofructokinase as described in claim 1, characterized in that, Includes the following steps: (1) In a 96-well plate, add an appropriate amount of recombinant 6-phosphofructokinase solution to each well, and then add solutions of different concentrations of the compounds to be screened, setting a single concentration gradient of 10 μg / mL to evaluate the dose dependence of the compounds; at the same time, a blank control group is set up. (2) PFK was used to catalyze the reaction of fructose-6-phosphate and ATP to produce fructose-1,6-bisphosphate and ADP for substrate reaction; (3) Use an ELISA reader to measure the absorbance of each well at a wavelength of 340 nm and record the data.

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