DPP-IV (dipeptidyl peptidase-IV) inhibitory peptide screened from yak blood as well as screening method and equipment

Abstract

The invention relates to the technical field of bioactive peptide screening, in particular to a DPP-IV inhibitory peptide screened from yak blood and a screening method and device.The method comprises the steps that yak blood is collected, blood cells are separated, a crude protein solution is obtained through crushing, centrifugation and fluorescent substrate preliminary screening, impurities are removed, marking is conducted, and a protein solution containing a fluorescent mark is prepared; specific polypeptide is fixed on an amino modified glass slide and incubated with a protein solution to prepare a DPP-IV inhibitory peptide solution containing a fluorescence label, signals are collected to extract a first peptide fragment set, sequence information of a second peptide fragment set is generated through sequence comparison and virtual enzymolysis, polypeptide is synthesized, and the inhibition effect is evaluated through incubation, fluorescence intensity detection and incubation in a culture medium. And an efficient and accurate method is provided for screening the DPP-IV inhibitory peptide.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptide screening, and in particular to a DPP-IV inhibitory peptide screened from yak blood, a screening method and a device. Background Art

[0002] DPP-IV inhibitory peptides are a class of peptide substances that can inhibit the activity of dipeptidyl peptidase-IV (DPP-IV). They are usually composed of several to dozens of amino acids, and their amino acid sequences have certain specificity and diversity. DPP-IV inhibitory peptides exist in a variety of organisms, such as animal milk, blood, tissues, and plant seeds and fruits, and can also be prepared by chemical synthesis or genetic engineering technology.

[0003] Most DPP-IV inhibitory peptides exert their inhibitory effects by competing with DPP-IV substrates for binding to the active site. They are structurally similar to the natural substrates of DPP-IV and can enter and bind to the active site. However, due to the particularity of their structure, they cannot be effectively cleaved by DPP-IV, thereby preventing the real substrate from binding to DPP-IV and inhibiting the enzymatic activity of DPP-IV.

[0004] Yak blood is rich in proteins, such as hemoglobin, which can produce a large number of peptides after enzymatic hydrolysis. Many bioactive peptides have DPP-IV inhibitory activity, and the active products of homologous proteins are very likely to have similar biological functions. Bovine hemoglobin hydrolysate has been reported to have good DPP-IV inhibitory activity. As a homologous protein of bovine hemoglobin, yak hemoglobin also has the potential to produce DPP-IV inhibitory peptides.

[0005] The patent document with publication number "CN117486970A" discloses a novel DPP-IV inhibitory peptide derived from yak hemoglobin, a discovery method and an application thereof. The DPP-IV inhibitory peptide of the above invention is MKHL; the molecular formula of the DPP-IV inhibitory peptide is C23H41N7O5S. The novel DPP-IV inhibitory peptide discovered from yak hemoglobin is used as a raw material in the preparation of functional foods for blood sugar regulation, which has the characteristics of no adverse reactions, good screening accuracy and cost saving and efficiency improvement.

[0006] However, existing technologies often rely on a single method for activity verification. It is difficult to comprehensively and accurately evaluate the biological activity of the target polypeptide only through traditional means such as molecular docking, solid-phase synthesis and inhibition rate determination. There may be false positive or false negative results, which cannot fully reflect its effect in the actual physiological environment.

[0007] Therefore, the existing technology has the problem of a single activity verification method. Summary of the invention

[0008] In order to solve the problems existing in the related technologies for screening DPP-IV inhibitory peptides from yak blood, the present invention provides a DPP-IV inhibitory peptide screened from yak blood, a screening method and a device, establishes a multivariate activity verification system, and comprehensively evaluates the DPP-IV inhibitory activity of the target polypeptide from multiple dimensions, so that the result is more reliable.

[0009] The first technical solution of the present invention: a DPP-IV inhibitory peptide screened from yak blood, wherein the DPP-IV inhibitory peptide is DPENF; the molecular structure of the DPP-IV inhibitory peptide is,

[0010]

[0011] The molecular formula of the DPP-IV inhibitory peptide is C 27 H 36 N6O 11 .

[0012] The second technical solution of the present invention: a screening device for screening DPP-IV inhibitory peptides from yak blood, comprising a bottom plate, an oscillating structure is arranged above the bottom plate, a shaking structure is arranged on one side of the oscillating structure, and a soaking structure is arranged on one side of the shaking structure.

[0013] The integration of operations that were originally scattered across different lab benches and required frequent manual sample transfers greatly reduces sample turnover time, lowers the chance of samples being contaminated by the outside world, and improves the continuity and fluency of the entire screening process. It meets multiple key processing needs in one stop and provides hardware support for efficient and accurate screening of DPP-IV inhibitory peptides.

[0014] Preferably, the oscillation structure includes an oscillation frame, which is fixedly connected to the top of the base plate, a driving slide is slidably connected to one side of the inner part of the oscillation frame, a liquid material frame is fixedly connected to the top of the driving slide, a loading plate is provided above the liquid material frame, a driven slide is fixedly connected to the bottom of the loading plate, and the driven slide is slidably connected to the oscillation frame.

[0015] The liquid material frame is filled with glutaraldehyde solution, and the filling plate is filled with a glass slide. The oscillating structure allows the glass slide to be evenly covered and penetrated with glutaraldehyde solution under gentle and regular oscillation, ensuring stable cross-linking of each amino group, avoiding uneven local reactions that affect the peptide fixation effect, improving the quality of slide modification, and further improving the specificity of subsequent peptide screening, reducing experimental errors caused by poor slide treatment.

[0016] Preferably, a driving column is provided on one side of the driving slide, a rotating plate is fixedly connected to the side of the driving column, a transmission plate is rotatably connected to the side of the rotating plate away from the driving column, and one end of the transmission plate away from the rotating plate is rotatably connected to the driven slide.

[0017] Effectively coordinate the movement of the double slides to make the slide oscillate smoothly, prevent the slide from slipping and the solution from splashing due to uneven power, ensure that the chemical reaction is carried out in a stable environment, optimize the preparation process of amino-modified slides, and provide a reliable basis for the subsequent fixation of polypeptides.

[0018] Preferably, a first spring is fixedly connected to one side of the driving slide, and an end of the first spring away from the driving slide is fixedly connected to the oscillation frame, and a second spring is fixedly connected to the side of the driving slide away from the first spring, and an end of the second spring away from the driving slide is fixedly connected to the oscillation frame.

[0019] It provides buffering for the movement of the driving slide, ensures the uniformity of the slide oscillation, improves the quality of the cross-linking reaction of the amino-modified slide, and provides high-quality slides for subsequent experimental links.

[0020] Preferably, a third spring is fixedly connected to one side of the driven slide, and an end of the third spring away from the driven slide is fixedly connected to the oscillation frame, and a fourth spring is fixedly connected to the side of the driven slide away from the third spring, and an end of the fourth spring away from the driven slide is fixedly connected to the oscillation frame.

[0021] It provides buffering for the movement of the driven slide, ensures the uniformity of the slide oscillation, improves the quality of the cross-linking reaction of the amino-modified slide, and provides high-quality slides for subsequent experimental steps.

[0022] Preferably, a filling groove is provided on the upper surface of the filling plate, and a limiting plate is provided at the edge of the filling groove.

[0023] The operator can place the slide quickly and accurately. The limit plate effectively restricts the movement range of the slide during oscillation, preventing the slide from detaching from the loading plate, ensuring the orderly progress of the experiment and reducing experimental interruptions caused by operating errors.

[0024] Preferably, the shaking structure includes a control base, which is fixedly connected to the top of the bottom plate, a servo motor is arranged on the top of the control base, a fixed arc plate is arranged on one side of the servo motor, the bottom end of the fixed arc plate is fixedly connected to the bottom plate, one end of the fixed arc plate is rotatably connected to a rotating column, the output end of the servo motor is fixedly connected to the rotating column, a side of the rotating column away from the fixed arc plate is fixedly connected to a control arc plate, an end of the control arc plate away from the rotating column is rotatably connected to a long arc plate, an end of the long arc plate away from the control arc plate is rotatably connected to a shaking column, the bottom end of the shaking column is rotatably connected to a short arc plate, and an end of the short arc plate away from the shaking column is rotatably connected to the fixed arc plate.

[0025] Driven by servo motors, containers such as centrifuge tubes are shaken in multiple dimensions, and solutions are quickly mixed under the alternating effects of centrifugal force and gravity, such as blood cell lysate and buffer, fluorescent labeling reagent and protein solution, etc., which accelerates the reaction process and improves sample processing efficiency. Gentle shaking avoids cell rupture and protein denaturation, ensuring the quality of experimental samples.

[0026] Preferably, the top end of the shaking column is rotatably connected to a clamping frame, one end of the clamping frame is provided with a circular clamp, and the end of the clamping frame away from the circular clamp is provided with a square clamp.

[0027] It provides firm fixation for conventional round centrifuge tubes and square culture bottles to prevent solution leakage and sample loss caused by loose containers, ensure smooth shaking operations, and meet the shaking requirements of different containers during the screening of DPP-IV inhibitory peptides from yak blood.

[0028] Preferably, the immersion structure includes a water bath tub, which is arranged above the base plate, and a support frame is arranged above the water bath tub, the bottom end of the support frame is fixedly connected to the base plate, a first fixed plate is fixedly connected to the top of the support frame, a first support plate is rotatably connected to the top of the first fixed plate, an end of the first support plate away from the first fixed plate is rotatably connected to the second support plate, a first transmission block is rotatably connected to one side of the second support plate, a first limit frame is fixedly connected to the side of the first transmission block away from the second support plate, a third support plate is rotatably connected to the end of the first transmission block away from the second support plate, a fourth support plate is rotatably connected to the end of the third support plate away from the first transmission block, and a second limit frame is rotatably connected to the end of the fourth support plate away from the third support plate.

[0029] The dialysis bag is suspended in the buffer solution to avoid friction and damage, and impurities cannot penetrate, thus ensuring the purity of the dialysis. The immersion structure can adjust the depth of the dialysis bag as needed to fully soak the dialysis bag, accelerate the diffusion of small molecule impurities, improve the dialysis efficiency, and provide a high-purity protein solution for subsequent experiments.

[0030] Preferably, a fixing hook is fixedly connected to the inner side of the second limiting frame.

[0031] The dialysis bag is firmly fixed to maintain the best dialysis posture, and the buffer solution can freely enter and exit the dialysis bag to ensure that the dialysis process is continuous and efficient.

[0032] Preferably, the second limit frame is rotatably connected to the fifth support plate at one side away from the fourth support plate, the fifth support plate is rotatably connected to the sixth support plate at one end away from the second limit frame, the sixth support plate is rotatably connected to the second transmission block at one end away from the fifth support plate, the second transmission block is fixedly connected to the first limit frame at one side away from the sixth support plate, the second transmission block is rotatably connected to the seventh support plate at one end away from the sixth support plate, the seventh support plate is rotatably connected to the eighth support plate at one end away from the second transmission block, the eighth support plate is rotatably connected to the second fixed plate at one end away from the seventh support plate, and the bottom end of the second fixed plate is fixedly connected to the bottom plate.

[0033] The operator can easily adjust the position of the dialysis bag and accurately reset the dialysis bag after replacing the dialysis buffer to meet the dialysis requirements of different durations and temperatures, ensuring that the dialysis bag is always in the best working condition during the entire dialysis process, providing a guarantee for obtaining high-quality post-dialysis samples.

[0034] Preferably, a servo electric cylinder is rotatably connected to one side of the middle of the first support plate, an end of the servo electric cylinder away from the first support plate is rotatably connected to a third fixed plate, and a bottom end of the third fixed plate is fixedly connected to the bottom plate.

[0035] When the dialysis bag angle needs to be changed after dialysis for different durations or after changing different dialysis buffers, the placement of the dialysis bag can be quickly changed by conveniently controlling the extension and retraction of the servo electric cylinder. This eliminates the need for complex manual adjustments and improves the flexibility and efficiency of experimental operations.

[0036] The third technical solution of the present invention: a method for screening DPP-IV inhibitory peptides from yak blood, comprising the following steps:

[0037] (S01) collecting blood from the vein of an individual yak, injecting the blood into a centrifuge tube containing an anticoagulant, using a shaking mechanism to evenly mix the liquid in the centrifuge tube, and then centrifuging the blood to separate blood cells;

[0038] This step successfully separated the blood cells, providing high-quality raw materials for subsequent protein extraction, maintaining the original state of the blood cells to the greatest extent, ensuring the reliability and stability of the starting samples of the experiment, and effectively avoiding experimental errors caused by sample problems;

[0039] (S02) crushing and centrifuging the blood cells to obtain a crude protein solution, and performing preliminary screening using a fluorescently labeled DPP-IV specific substrate;

[0040] On the one hand, a multi-component lysis buffer and a fine ultrasonic program are used to fully break up blood cells, promote the release of a large amount of protein, and improve the amount and quality of crude protein solution. On the other hand, a microplate reader combined with a fluorescence spectrophotometer detection system is used to preliminarily screen samples based on the inhibition rate as an indicator, and quickly identify crude protein solutions with potential DPP-IV inhibitory activity.

[0041] (S03) placing the dialysis bag in the soaking structure for soaking, removing impurities in the crude protein solution, and labeling it with a fluorescent labeling reagent to prepare a protein solution containing a fluorescent label;

[0042] Through dialysis operation, impurities are efficiently removed, the purity of protein solution is optimized, and pure substrate is provided for subsequent experiments; fluorescent labeling reagents are used to label proteins under precise conditions, giving proteins traceability characteristics;

[0043] (S04) designing a binding polypeptide of the DPP-IV inhibitory peptide, placing the amino-treated glass slide into an oscillating structure for oscillation, and fixing the binding polypeptide on the surface of the amino-treated glass slide;

[0044] The amino-modified glass slide was successfully loaded with DPP-IV binding peptide, forming a stable and highly specific solid-phase reaction platform, which not only ensured the firm binding of the peptide, but also effectively reduced nonspecific adsorption, providing an ideal carrier for subsequent incubation reactions with fluorescently labeled protein solutions.

[0045] (S05) preparing an incubation buffer, adding a fluorescently labeled protein solution to the oscillating structure, and incubating with the amino-treated glass slide to allow the DPP-IV inhibitory peptide to bind to the surface of the amino-treated glass slide and remove unbound proteins;

[0046] The incubation buffer provides optimal conditions for protein-peptide binding, maintains protein activity and reduces nonspecific adsorption; removes unbound proteins to achieve preliminary enrichment of target peptides, laying the foundation for subsequent screening;

[0047] (S06) collecting fluorescence signals, analyzing binding signals of bound polypeptides, and extracting the first peptide segment set on the amino-treated glass slide;

[0048] With the help of high-resolution imaging and microdissection technology of laser confocal microscopy, peptides that strongly bind to the binding polypeptide are locked and obtained as the first peptide set, providing samples for subsequent in-depth exploration of its sequence characteristics, enzyme cleavage sites, etc.

[0049] (S07) comparing the sequence of the first peptide set with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence to analyze similarity regions and potential enzyme cleavage sites;

[0050] Through sequence comparison and technical verification, the similarities and differences between the first peptide set and the DPP-IV inhibitory peptide sequence and yak hemoglobin sequence were analyzed, and the similarity regions and potential enzyme cleavage sites were identified;

[0051] (S08) based on the comparison results of the first peptide set, the yak hemoglobin sequence is virtually enzymatically digested, and the enzymatic fragments with the potential to inhibit DPP-IV activity are analyzed to generate a second peptide set;

[0052] With the help of virtual enzymatic hydrolysis technology, the sequence information of the second peptide set is generated to provide rich targets for subsequent experimental verification;

[0053] (S09) using a solid phase chemistry method to prepare a second peptide set, obtain a target peptide, prepare a positive test sample and a negative test sample, put equal volumes of the positive and negative test samples into centrifuge tubes, add a fluorescently labeled binding peptide solution, use a shaking mechanism to mix the liquid in the centrifuge tube evenly, incubate, separate the bound and unbound substances, and collect the fractions;

[0054] The specific binding ability of the synthetic peptides was verified through the prepared positive and negative test samples and the standardized incubation, separation and collection operations. The bound and unbound substances were effectively separated based on the molecular size differences using gel filtration columns, providing reliable samples for subsequent fluorescence intensity detection.

[0055] (S10) detecting the fluorescence intensity of each fraction to verify that the target polypeptide has specific binding ability;

[0056] With the help of high-sensitivity detection and curve analysis of the fluorescence spectrophotometer, the distribution and binding state of the target peptide in each fraction can be clearly seen, and the specific binding ability of the target peptide can be verified with objective data;

[0057] (S11) adding the target polypeptide to a culture bottle containing culture medium, using a shaking structure to evenly mix the liquid in the culture bottle, and then incubating, setting up a blank control group and a positive control group, adding DPP-IV enzyme and a fluorescently labeled DPP-IV specific substrate, detecting the change in fluorescence intensity of the supernatant in the culture medium and calculating the inhibition rate, and evaluating the inhibitory effect of the target polypeptide on intracellular DPP-IV activity.

[0058] By simulating the in vivo cell microenvironment, Caco-2 cells are ensured to be in a good growth state, providing suitable conditions for the target peptide to act. The control setting enhances the comparability and reliability of the experiment; the fluorescence intensity is scanned by an ELISA instrument, the changes in DPP-IV enzyme activity are tracked in real time, and the inhibition rate is calculated, which can quantify the degree of inhibition of DPP-IV activity by the target peptide in the cell, providing data support for the evaluation of its clinical application potential.

[0059] Preferably, in the step (S01), blood is collected from the vein of an individual yak using a disposable sterile syringe and injected into a centrifuge tube containing an anticoagulant.

[0060] Disposable sterile syringes effectively prevent external microorganisms from contaminating blood samples and ensure sample purity; anticoagulant centrifuge tubes are quickly injected to prevent blood coagulation, maintain the original state of blood cells, and reduce experimental errors caused by improper sample collection.

[0061] Preferably, the anticoagulant is heparin sodium with a final concentration of 15 U / mL.

[0062] At this concentration, heparin sodium can inhibit blood coagulation, stabilize the morphology and function of blood cells, ensure the integrity of blood cells during subsequent centrifugation, avoid problems such as blood cell aggregation and rupture caused by excessive or insufficient anticoagulation, and ensure experimental consistency and accuracy.

[0063] Preferably, in the step (S01), the blood is centrifuged by placing the centrifuge tube containing the blood in a high-speed refrigerated centrifuge, centrifuging at a temperature of 4°C and a speed of 3500 rpm for 15 minutes, separating the plasma and blood cells, discarding the plasma, and then washing the blood cells twice with physiological saline. After each wash, the blood is placed in a high-speed refrigerated centrifuge again, centrifuged at a temperature of 4°C and a speed of 3500 rpm for 15 minutes, and the blood cell solution is collected after centrifugation.

[0064] Low-temperature high-speed centrifugation can separate plasma and blood cells, gently remove plasma, and reduce residual proteins; low-temperature saline washing can further remove impurities, maintain the osmotic pressure balance of blood cells, prevent cells from bursting or shrinking, and ensure the activity of blood cells.

[0065] Preferably, the physiological saline solution is NaCl with a concentration of 0.9%, and the temperature of the physiological saline solution is 4°C.

[0066] The NaCl concentration of 0.9% is close to the physiological salt concentration of the human body, which prevents cells from being damaged due to differences in osmotic pressure; the low temperature of 4°C meets the requirements for blood cell preservation, prevents cells from being denatured by heat, and ensures that blood cells maintain good activity and integrity during the washing process, providing high-quality cell raw materials for subsequent experiments.

[0067] Preferably, in the step (S02), the crude protein solution is obtained after the blood cells are crushed and centrifuged, by pouring the blood cell solution into an ultrasonic crusher, adding a lysis buffer, and crushing the blood cells by ultrasonication in an ice bath, and then pouring the crushed blood cell solution into a centrifuge tube, placing the centrifuge tube into a high-speed refrigerated centrifuge, and centrifuging at a temperature of 4°C and a speed of 12000 rpm for 20 minutes, and collecting the supernatant after centrifugation as the crude protein solution.

[0068] The ultrasonic disruptor uses high-frequency vibration in an ice bath environment to break up blood cells, and cooperates with lysis buffer to dissolve cell membranes and accelerate protein release; subsequent low-temperature and high-speed centrifugation can precipitate impurities such as cell fragments to obtain the supernatant, which is the crude protein solution, providing rich and pure protein raw materials for subsequent screening of DPP-IV inhibitory peptides.

[0069] Preferably, the lysis buffer is Tris-HCl buffer with a final concentration of 20 to 50 mmol / L and a pH of 7.4 to 8.0.

[0070] Tris-HCl buffer in this concentration and pH range can effectively maintain the acid-base balance of the solution, close to the physiological environment within the cell, provide stable conditions for cell membrane lysis and protein release, prevent protein denaturation due to acid-base changes, ensure the activity of the extracted protein, and facilitate subsequent experimental screening.

[0071] Preferably, the lysis buffer contains NaCl at a final concentration of 150 mmol / L.

[0072] NaCl at a concentration close to physiological concentration provides a suitable ionic environment for proteins, stabilizes the forces between and within protein molecules, maintains the natural three-dimensional structure of proteins, ensures their biological activity, makes the extracted crude protein solution more valuable for research, and improves the success rate of subsequent screening.

[0073] Preferably, the lysis buffer contains Triton X-100 at a final concentration of 0.5% to 1%.

[0074] As a surfactant, Triton X-100 can effectively reduce the surface tension of cell membranes, assist the ultrasonic disruptor in destroying the integrity of cell membranes, make intracellular proteins easier to dissolve, and have little effect on protein function within this concentration range, thereby improving protein extraction efficiency and providing sufficient protein source for subsequent experiments.

[0075] Preferably, the lysis buffer contains EDTA at a final concentration of 1 to 5 mmol / L.

[0076] EDTA strongly binds to metal ions, blocking enzymatic reactions involving metal ions and avoiding protein hydrolysis, ensuring that the extracted crude protein solution has sufficient protein content and intact structure, which is conducive to the subsequent screening of fully functional DPP-IV inhibitory peptides.

[0077] Preferably, the lysis buffer contains AEBSF at a final concentration of 0.1 to 1 mmol / L.

[0078] AEBSF specifically inhibits serine proteases to reduce protein degradation, ensuring that the extracted proteins can be used for subsequent experimental screening and improving experimental reliability.

[0079] Preferably, the lysis buffer contains PMSF at a final concentration of 1 mmol / L.

[0080] PMSF effectively inhibits serine proteases and takes effect quickly in the early stage of cell disruption, preventing proteins from being hydrolyzed at the moment of release, ensuring the integrity of proteins in crude protein solutions, and providing high-quality protein raw materials for subsequent experiments.

[0081] Preferably, the lysis buffer contains Leupeptin at a final concentration of 1 to 10 μg / mL.

[0082] Leupeptin can inhibit a variety of proteases, including serine and cysteine ​​proteases, etc., continuously protecting the protein from degradation during the entire protein extraction process, improving the quality of the crude protein solution, and increasing the probability of screening active DPP-IV inhibitory peptides.

[0083] Preferably, the lysis buffer contains E-64 at a final concentration of 1 to 10 μg / mL.

[0084] E-64 precisely inhibits cysteine ​​proteases, building a tight protective network to ensure that the activity of various proteases is effectively inhibited during protein extraction, thereby protecting protein integrity and facilitating subsequent experiments.

[0085] Preferably, the lysis buffer contains Pepstatin A at a final concentration of 1 to 10 μg / mL.

[0086] Pepstatin A effectively inhibits aspartic proteases and comprehensively inhibits various intracellular proteases, ensuring that the protein is not degraded throughout the entire process from cell release to the preparation of crude protein solution, providing high-quality protein for subsequent screening.

[0087] Preferably, the lysis buffer contains 1,10-Phenanthroline at a final concentration of 1 to 5 mmol / L.

[0088] 1,10-Phenanthroline binds to metal ions, reducing the interference of metal ions on protein structure and function. In the complex cell lysis environment, it stabilizes the protein, ensuring that the extracted protein can be used for subsequent screening experiments and improving the success rate.

[0089] Preferably, the ultrasonic disruptor performs ultrasonication for 3 seconds each time with an interval of 5 seconds, for a total of 10 times.

[0090] Ultrasonic energy is used to effectively break up blood cells, and intermittent operation is used to provide sufficient heat dissipation time to prevent local overheating from causing protein denaturation. This improves cell disruption efficiency while ensuring protein activity, providing high-quality crude protein solutions for subsequent experiments.

[0091] Preferably, in the step (S02), a fluorescently labeled DPP-IV specific substrate is used for preliminary screening, and the crude protein solution, DPP-IV enzyme and fluorescently labeled DPP-IV specific substrate are placed in a microplate reader and mixed, incubated at 37° C. for 30 minutes, and the fluorescence intensity is detected using a fluorescence spectrophotometer. A blank control group and an experimental group are set up, and the fluorescence intensity of the experimental group relative to the blank group is compared, and the inhibition rate is calculated. Samples are screened according to the inhibition rate, and the crude protein solution corresponding to the sample is collected.

[0092] Through temperature-controlled incubation with an ELISA instrument and high-sensitivity detection with a fluorescence spectrophotometer, combined with control experimental design, the degree of inhibition of crude protein on DPP-IV enzyme activity can be quantitatively analyzed. Screening with inhibition rate as an indicator can quickly identify samples with high potential and improve the early efficiency of the screening process.

[0093] Preferably, the blank control group contains DPP-IV enzyme and fluorescently labeled DPP-IV specific substrate, and the experimental group contains crude protein solution, DPP-IV enzyme and fluorescently labeled DPP-IV specific substrate.

[0094] By setting up a control group, the only variable between the experimental group and the blank group was the crude protein solution, and other interfering factors were eliminated. The inhibitory effect of the crude protein on the DPP-IV enzyme activity was reflected by comparing the difference in fluorescence intensity, ensuring the reliability and scientificity of the screening results.

[0095] Preferably, the specific fluorescent substrate is Gly-Pro-AMC.

[0096] Gly-Pro-AMC can specifically react with the DPP-IV enzyme. The change in fluorescence intensity produced during the reaction is closely related to the enzyme activity. The fluorescence signal is clear and stable, easy to detect and quantify, providing a powerful tool for accurately judging the inhibitory activity of crude protein and reducing the probability of false positive or false negative results.

[0097] Preferably, the screening criterion for the sample is an inhibition rate higher than 40%.

[0098] Using this as the boundary for screening can quickly discard most of the samples with low or no activity, focus on a few high-potential samples, improve the success rate of subsequent experiments, and save time and costs.

[0099] Preferably, in the step (S03), impurities in the crude protein solution are removed by soaking the dialysis bag in distilled water for 10 to 15 minutes. After the soaking is completed, the crude protein solution is transferred to the dialysis bag, and the dialysis bag is placed in a dialysis buffer solution. The protein solution is stirred at a speed of 50 to 100 rpm in a constant temperature water bath environment at 25°C until the solutions inside and outside the dialysis bag are fully exchanged and small molecule impurities diffuse into the dialysis buffer solution outside the dialysis bag.

[0100] Using the dialysis principle, based on the characteristics of small molecule impurities that can freely enter and exit the dialysis bag driven by concentration difference, impurities can be removed gently and effectively. This process does not change the main components and activity of the protein solution, providing pure protein substrates for subsequent fine experiments such as fluorescent labeling and protein-peptide binding, ensuring the accuracy and reliability of experimental results.

[0101] Preferably, in the step (S03), the fluorescent labeling reagent is used for labeling by adding the fluorescent labeling reagent and reaction buffer to the protein solution, stirring the protein solution at a speed of 50-100 rpm in a constant temperature water bath at 25°C to carry out the labeling reaction.

[0102] In a mild reaction environment, the fluorescent labeling reagent stably binds to the protein, giving the protein a fluorescent label. This facilitates real-time monitoring of the protein in subsequent experiments, and reflects the degree of protein participation in the reaction through changes in the intensity of the fluorescent signal, providing an intuitive basis for in-depth exploration of the interaction between the protein and other components, and improving the accuracy and convenience of experimental operations.

[0103] Preferably, in the step (S03), the protein solution containing the fluorescent marker is prepared by transferring the protein solution to a dialysis bag, dialyzing the protein solution using a dialysis buffer in a constant temperature water bath at 25°C, removing the dialysis bag after the dialysis is completed, preparing the protein solution containing the fluorescent marker, and transferring the protein solution containing the fluorescent marker to a centrifuge tube.

[0104] The second dialysis process continuously optimizes the purity of the protein solution, completely removes unbound fluorescent markers and potential impurities, ensures that the protein solution containing fluorescent markers has a single component and stable properties, and provides high-quality reaction substrates for subsequent incubation reactions with polypeptide slides, ensuring the smooth progress of the experiment and improving the credibility of the experimental results.

[0105] Preferably, the fluorescent labeling agent is Cy3 fluorescent dye.

[0106] Cy3 fluorescent dye has the advantages of high fluorescence intensity, good photostability, and little interference with protein structure and activity. After labeling the protein, it can continue to emit stable fluorescence over a long experimental period, facilitating accurate observation of protein dynamics during the experiment, providing reliable fluorescence signals, and improving experimental accuracy.

[0107] Preferably, the reaction buffer is a Tris-HCl buffer with a pH value of 7.0 to 9.0.

[0108] The Tris-HCl buffer in this pH range is compatible with the isoelectric point of the protein, which is beneficial to maintaining the native conformation and activity of the protein. At the same time, it provides a suitable chemical environment for the fluorescent labeling reagent and promotes the stable binding of the two.

[0109] The 2-hour reaction provides enough time for the fluorescent labeling reagent to fully contact and bind to the protein molecules, ensuring the comprehensiveness and effectiveness of the labeling.

[0110] Regular replacement of the dialysis buffer can maintain a large concentration difference between the inside and outside of the dialysis bag, accelerate the diffusion of small molecule impurities, and improve dialysis efficiency. The 4-6 hours of dialysis time is sufficient to remove most impurities, so that the final fluorescent-labeled protein solution can reach extremely high purity, providing pure and reliable protein samples for subsequent experiments.

[0111] Preferably, in the step (S04), the binding polypeptide of the DPP-IV inhibitory peptide is designed by using SWISS-MODEL, inputting the DPENF sequence, constructing a three-dimensional model, downloading polypeptide data from the Antimicrobial Peptides Database, using Python script in combination with the Pandas library to write a screening program, setting screening conditions, running the screening program, extracting polypeptide sequences that meet the conditions as ligand models, using AutoDock Vina, importing the ligand model and the three-dimensional model of the DPENF sequence, docking, outputting the docking score ranking, screening out the top 10 to 20 polypeptide sequences, analyzing the interaction between the screened polypeptide sequences and the DPENF sequence, screening out polypeptide sequences that have positively charged amino acids forming stable electrostatic pairs near the acidic residues of DPENF and have hydrophobic stacking effects with the aromatic ring of phenylalanine, preparing the screened polypeptide sequences by solid phase chemical synthesis, detecting the binding ability of the synthetic polypeptides to DPENF by surface plasmon resonance technology, calculating the binding constant and dissociation constant, and using the polypeptide sequences with a dissociation constant less than 100 nM as binding polypeptides.

[0112] With the help of SWISS-MODEL to build models and database screening, combined with Python scripts for automated screening, and then docking and scoring with AutoDockVina, peptide sequences with high binding potential to the DPENF sequence can be quickly and accurately located, reducing the workload and time of screening, avoiding human errors, improving screening efficiency and accuracy, and ensuring the acquisition of high-quality binding peptide candidate groups. By analyzing the interaction between the peptide and the DPENF sequence, peptides that can form stable electrostatic pairs near acidic residues and have hydrophobic stacking effects with the aromatic ring of phenylalanine are screened out, so that the screened peptides can form stable and specific binding with DPENF, reduce nonspecific binding interference, enhance the ability to identify and bind DPP-IV inhibitory peptides in complex sample environments, and improve detection accuracy. The peptides were prepared by solid-phase chemical synthesis, and the binding constant and dissociation constant were determined by surface plasmon resonance technology. Peptides with a dissociation constant of less than 100nM were selected as binding peptides, providing quantitative data support for the binding stability of the peptide and DPENF, ensuring stable function in subsequent experiments and improving the screening success rate.

[0113] Preferably, the screening condition is that the polypeptide length is 8 to 10 amino acids, and contains one positively charged amino acid, one aromatic amino acid and one amino acid that can form a hydrogen bond.

[0114] The peptide length is limited to 8-10 amino acids, so that the selected peptides have a moderate molecular size. They will not lack sufficient structural diversity and binding sites due to being too short, making it difficult to effectively interact specifically with the DPP-IV inhibitory peptide; nor will they be too complex due to being too long, increasing the difficulty, cost and complexity of subsequent experimental operations. This helps to facilitate subsequent preparation, characterization and application research while maintaining a certain activity, thereby improving overall R&D efficiency.

[0115] On the other hand, the requirement that the peptide contains a positively charged amino acid, an aromatic amino acid, and an amino acid that can form a hydrogen bond is based on the profound application of the principle of intermolecular interaction. Positively charged amino acids can form stable ionic bonds with acidic residues that may exist on the DPP-IV inhibitory peptide through electrostatic attraction, thereby enhancing the tightness of the binding; aromatic amino acids can generate specific affinity with the corresponding aromatic structure region on the target molecule by virtue of their unique π-π stacking or hydrophobic effect, further stabilizing the binding; and amino acids that can form hydrogen bonds use hydrogen bonds, a strong intermolecular force, to interconnect with suitable donor or acceptor groups on the surface of the DPP-IV inhibitory peptide molecule, enriching the binding mode and strengthening the binding stability and specificity between the peptide and the DPP-IV inhibitory peptide from multiple dimensions.

[0116] Preferably, in the step (S04), the binding polypeptide is fixed on the surface of the amino glass slide by first washing the amino glass slide with anhydrous ethanol, drying it under a nitrogen gas flow, and then immersing the amino glass slide in a glutaraldehyde solution with a concentration of 0.5% to 2%, and shaking it for 30 to 120 minutes to allow the glutaraldehyde to cross-link with the amino groups on the surface of the amino glass slide, thereby introducing active aldehyde groups on the surface of the amino glass slide. After the reaction is completed, the amino glass slide is taken out, washed with PBS buffer, and the binding polypeptide is dissolved in PBS buffer to a concentration of 0.1 to 1 mg. / mL of binding polypeptide solution, drip the binding polypeptide solution onto the surface of the amino-treated glass slide to make the binding polypeptide solution evenly cover the amino-treated glass slide, place the amino-treated glass slide in a sealed container containing water, react for 2 to 4 hours, so that the binding polypeptide is covalently bound to the aldehyde group on the surface of the amino-treated glass slide through the amino group to be fixed, and after fixation, wash the amino-treated glass slide with PBS buffer to remove unbound polypeptide and impurities, then place it in a BSA solution with a concentration of 1 to 5%, incubate for 1 to 2 hours, then wash the amino-treated glass slide with PBS buffer, and blow dry it under a nitrogen gas flow.

[0117] The impurities on the surface of the glass slide are removed by washing with anhydrous ethanol, and nitrogen blowing is used to ensure a dry environment; glutaraldehyde cross-linking provides active sites for peptide fixation and enhances binding stability; the peptide solution is combined with the glass slide to react under appropriate conditions to ensure that the bound peptide is evenly and firmly attached and reduce the risk of falling off; subsequent PBS washing is used to remove unbound substances, BSA incubation is used to block nonspecific sites, and nitrogen blowing is used to keep the glass slide clean, jointly creating a solid phase carrier with strong specificity and high stability.

[0118] Preferably, in the step (S05), the incubation buffer is prepared by selecting a Tris solution with a pH value of 7.4, adding BSA to make the final concentration of BSA reach 0.05%, and then filtering and sterilizing with a 0.22 μm filter membrane to prepare the incubation buffer.

[0119] The pH 7.4 Tris solution is close to the physiological environment, which is conducive to maintaining the natural conformation and activity of the protein; 0.05% BSA, as a blocking agent, can occupy potential nonspecific adsorption sites on the surface of the slide, reduce nonspecific binding of the protein solution, and sterilize with a 0.22μm filter membrane to provide a sterile and stable environment for the incubation reaction, thereby improving the reaction specificity and efficiency.

[0120] Preferably, in the step (S05), the DPP-IV inhibitory peptide is bound to the surface of the amino-treated glass slide by covering the surface of the amino-treated glass slide with a fluorescently labeled protein solution, incubating at a temperature of 37° C. and a humidity of 50% for 1 hour, and then rinsing the amino-treated glass slide with an incubation buffer to remove unbound proteins, leaving only the specifically bound DPP-IV inhibitory peptide on the surface of the amino-treated glass slide.

[0121] 37℃ is close to human body temperature, and 50% humidity simulates the mild and humid in vivo microenvironment, which is conducive to the full interaction between proteins and peptides and increases the probability of binding; the incubation buffer rinse utilizes its component characteristics to effectively remove unbound proteins, so that most of the proteins retained on the slide are specifically bound fluorescently labeled DPP-IV inhibitory peptides, improving the quality of subsequent analysis samples.

[0122] Preferably, the fluorescence signal is collected in the step (S06) by placing the incubated glass slide on the stage of a laser confocal microscope, scanning the surface of the glass slide point by point, collecting the fluorescence signal intensity of each pixel point, and generating a fluorescence image.

[0123] With its high resolution and deep focusing capabilities, the laser confocal microscope can distinguish fluorescent signals at different positions on the slide and scan point by point without missing any details. The generated fluorescent image fully presents the distribution and intensity information of protein and polypeptide binding, providing an intuitive basis for identifying strong binding areas.

[0124] Preferably, in the step (S06), the binding signal of the bound polypeptide is analyzed by analyzing the fluorescence signal to identify the strong binding signal region, and the peptides captured in the strong binding signal region are extracted from the amino-treated glass slide using micro-cutting technology to form a first peptide set.

[0125] Through fluorescence signal analysis, strong binding areas are delineated, and micro-cutting technology is used for fine manipulation at the microscopic level to accurately separate target peptides, ensuring the high purity of the first peptide set extracted.

[0126] Preferably, in the step (S07), the sequence of the first peptide set is compared with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence, and the sequence of the first peptide set, the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence are input into BlastP for comparison analysis, the expected threshold is set to 0.001, the BLOSUM62 matrix is ​​selected, the penalty for opening the gap is set to 11, and the penalty for extending the gap is set to 1, and the comparison results are output in a tabular form, including the query sequence identifier, the target sequence identifier, the comparison score, the expected threshold, the similarity percentage, and the start and end positions of the comparison area, and the results with a similarity percentage higher than 40% are screened, and the start and end positions of the comparison area are checked. The potential cleavage sites were screened in the similarity region of the first peptide segment set and within the range of 5 to 10 amino acids upstream and downstream thereof according to the information of cleavage sites in the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence. The potential cleavage sites were analyzed using PeptideCutter, and short peptide fragments containing potential cleavage sites were synthesized. The cleavage products were detected by enzyme cleavage reaction, mass spectrometry and electrophoresis technology to verify the authenticity of the potential cleavage sites. The site-directed mutagenesis technology was used to change the amino acids in the similarity region of the first peptide segment set, and the effects of the polypeptides before and after mutation on the DPP-IV enzyme activity and the changes in the interaction with yak hemoglobin were observed to verify the accuracy of the similarity region.

[0127] Verify the authenticity of restriction sites and the accuracy of similarity regions from different angles to improve the targeted nature of research and development.

[0128] Preferably, in the step (S08), the yak hemoglobin sequence is subjected to virtual enzymatic hydrolysis by using PeptideCutter to simulate enzymatic hydrolysis, and fragments with potential DPP-IV inhibitory activity are screened out from the generated enzymatic fragments, which are the second peptide segment set.

[0129] With built-in enzyme cutting rules and algorithms, PeptideCutter analyzes the yak hemoglobin sequence, screens out potentially active enzymatic fragments, and organizes them into a second peptide set, providing rich targets for subsequent experimental verification.

[0130] Preferably, in the step (S09), the positive test sample and the negative test sample are prepared by adding the target polypeptide powder into PBS buffer to prepare a positive test sample with a concentration of 50 μg / mL, and the negative test sample is PBS buffer.

[0131] By comparing changes in fluorescence and other signals, the binding ability of the target peptide can be intuitively presented.

[0132] Preferably, in the step (S09), equal volumes of positive and negative test samples are placed in centrifuge tubes respectively, and 50 μL of the positive test sample and 50 μL of the fluorescently labeled DPP-IV inhibitory peptide solution are aspirated using a micropipette, and the aspirated samples are transferred to a silanized 0.5 mL sterile microcentrifuge tube for mixing, and 50 μL of the negative test sample and 50 μL of the fluorescently labeled DPP-IV inhibitory peptide solution are aspirated again, and the aspirated samples are transferred to another sterile microcentrifuge tube for mixing.

[0133] The micropipette accurately controls the sample volume to ensure the accurate mixing ratio of each sample.

[0134] Preferably, the step (S09) is to separate the bound and unbound substances after incubation and collect the fractions, wherein the positive sample and the negative sample incubated at 37° C. for 30 minutes are transferred to two 100 μL micro-injectors, a Sephadex G-25 gel filtration chromatography column is selected, and is vertically fixed on an Agilent 1260 Infinity II chromatograph, 3 mL of PBS buffer is selected, and the Sephadex G-25 gel filtration chromatography column is rinsed at a flow rate of 0.8 mL / min, and the mixed reaction liquid of the positive sample and the negative sample is respectively injected into the injection ports of the two Sephadex G-25 gel filtration chromatography columns using two 100 μL micro-injectors, and the Agilent 1260 Infinity II chromatograph is started to allow the mixed reaction liquid of the positive sample and the negative sample to enter the Sephadex G-25 gel filtration chromatography column for separation, and a Model 10000 micro-injector is connected at the outlet of the Sephadex G-25 gel filtration chromatography column. 2110 fraction collector, one fraction is collected every 1.2 mL, and each component in the mixed reaction solution of the positive sample and the negative sample is eluted from the Sephadex G-25 gel filtration column in turn and collected in the fraction collection tube.

[0135] The Sephadex G-25 gel filtration column is based on the molecular sieving effect, which distinguishes molecules of different sizes, elutes and collects the bound and unbound substances in sequence, and improves the purity of each fraction, providing a more single and reliable sample for subsequent fluorescence intensity detection.

[0136] Preferably, the 3 mL PBS buffer comprises 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4, and is prepared by adding 800 mL deionized water to a 1 L volumetric flask, stirring magnetically until completely dissolved, adjusting the pH value to 7.4 with a Mettler Toledo precision pH meter, and finally adjusting the volume to 1 L with deionized water, and filtering and sterilizing with a 0.22 μm filter membrane.

[0137] PBS buffer has precise composition and appropriate pH. It is sterile and pure after sterile filtration, providing a stable buffer system for the operation of gel filtration chromatography columns, ensuring that the separation process is not disturbed by impurities, pH fluctuations, etc., ensuring the separation effect of conjugates and unconjugated substances, and improving experimental reliability.

[0138] Preferably, the fluorescence intensity of each fraction is detected in the step (S10), and each collected fraction is sequentially transferred to a quartz cuvette with an optical path of 1 cm, and placed in a Shimadzu RF-5301PC fluorescence spectrophotometer sample cell for fluorescence intensity detection, and the fluorescence intensity value corresponding to each fraction is recorded, and a curve graph of the fluorescence intensity changing with the fraction is generated, and the fluorescence intensity curve graph is analyzed.

[0139] Shimadzu RF-5301PC fluorescence spectrophotometer can capture the changes in fluorescence intensity of each fraction with its high sensitivity. The generated curve graph comprehensively reflects the distribution and binding state of the target peptide. The binding ability is quantified through data analysis, providing strong support for subsequent in-depth research on its inhibitory effect on DPP-IV activity.

[0140] Preferably, in the step (S11), the target polypeptide is added to the culture bottle containing the culture medium, and the Caco-2 cell suspension is transferred to a 15 mL centrifuge tube containing 9 mL of complete culture medium, centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, and the precipitated Caco-2 cells are resuspended with 2 mL of complete culture medium, and the Caco-2 cell suspension is transferred to the culture bottle, and the culture bottle is placed in a 37°C, 5% CO2 incubator for culture, and the growth state of the Caco-2 cells is observed under an inverted microscope every day. When the confluence of the Caco-2 cells reaches 80% to 90%, the old culture medium in the culture bottle is discarded, and the surface of the Caco-2 cells is rinsed with 2 mL of PBS buffer to remove the residual culture medium and dead cells. Add 1 mL of 0.25% trypsin-0.02% EDTA digestion solution, place the culture flask in a 37°C incubator for digestion for 2 to 3 minutes, until the Caco-2 cells become round and begin to fall off, add 2 mL of DMEM high-glucose medium containing 10% FBS to terminate digestion, transfer the Caco-2 cell suspension to a 15 mL centrifuge tube with a pipette, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add complete medium to resuspend the cells, and then inoculate the Caco-2 cells into a 96-well culture plate. After the Caco-2 cells grow in the 96-well culture plate for 4 to 6 hours, use an analytical balance to weigh the target polypeptide powder, dissolve it with DMSO, and dilute it with complete medium to make the final concentration of the target polypeptide 10 μM.

[0141] The in vivo microenvironment of 37°C and 5% CO2 was simulated to ensure the normal growth, adhesion and metabolism of Caco-2 cells. The target peptide was properly dissolved and diluted with DMSO to ensure that the target peptide was evenly dispersed and the activity was stable, creating conditions for accurately evaluating its inhibitory effect on intracellular DPP-IV activity.

[0142] Preferably, the complete culture medium is made of DMEM high-glucose culture medium and 10% FBS, 1% NEAA, and 1% Pen-Strep in a volume ratio.

[0143] DMEM high-glucose culture medium provides rich carbon sources and other nutrients; 10% FBS supplements growth factors, amino acids, etc. to promote cell proliferation; 1% NEAA makes up for the lack of essential amino acids; 1% Pen-Strep prevents microbial contamination. The three work together to create an environment suitable for cell growth, ensure cell vitality, provide a stable cell source for the experiment, and improve the credibility of the experiment.

[0144] Preferably, a blank control group and a positive control group are set in the step (S11), and a 96-well culture plate is divided into a target polypeptide group, a blank control group and a positive control group, and 6 replicate wells are set for each group. The target polypeptide solution is aspirated with a multichannel pipette and added to each replicate well of the target polypeptide group. The sample volume for each replicate well is 100 μL, 100 μL of complete culture medium is added to the blank control group, and a binding polypeptide solution with a concentration of 10 μM is added to the positive control group.

[0145] Multiple control groups were set up, and each group of duplicate wells ensured data repeatability; the target peptide group detected the effect of the target peptide, the blank control group excluded interference from the culture medium, and the positive control group introduced known binding peptides for comparison. By comparing the changes in indicators such as fluorescence intensity in different groups, the inhibitory activity of the target peptide was clearly presented.

[0146] Preferably, in the step (S11), the fluorescence intensity change of the supernatant in the culture medium is detected and the inhibition rate is calculated, and the 96-well culture plate of the target polypeptide group and the positive control group is placed in a 37°C, 5% CO2 incubator for incubation for 24 hours, and the state of the Caco-2 cells is observed under an inverted microscope every 4 to 6 hours. After the incubation, the 96-well culture plate is taken out from the incubator, and a DPP-IV enzyme solution with a concentration of 0.1U / mL is added to each duplicate well of the target polypeptide group and the positive control group, and the amount added to each duplicate well is 50μL, and Gly-Pro-AMC with a final concentration of 50μM is added, and the amount added to each duplicate well is 50μL. The 96-well culture plate is placed in a microplate reader, and the fluorescence intensity is scanned once every 2 to 3 minutes for a total of 30 to 60 minutes. The fluorescence intensity value of each duplicate well in each scan is recorded, and a curve atlas of fluorescence intensity change over time is generated, and the average fluorescence intensity and inhibition rate are calculated.

[0147] The ELISA scanner scans and captures the changes in fluorescence intensity over time. By calculating the inhibition rate, the inhibitory effect of the target peptide on intracellular DPP-IV activity is quantified, providing core data for preclinical research and promoting its transformation to clinical application.

[0148] Preferably, the calculation formula for the mean fluorescence intensity is:

[0149]

[0150] In the formula, I i is the fluorescence intensity of the ith replicate well, n is the number of replicate wells, I 平 is the mean fluorescence intensity.

[0151] Preferably, the calculation formula of the inhibition rate is:

[0152]

[0153] In the formula, I 空白 is the average fluorescence intensity of the blank control group, I 目标多肽 is the average fluorescence intensity of the target peptide group, I 阳性 is the average fluorescence intensity of the positive control group, and IR is the inhibition rate.

[0154] The present invention has the following beneficial effects:

[0155] (1) With the help of an ELISA combined with a fluorescence spectrophotometer, the crude protein solution was screened using the inhibition rate as an indicator using the specific fluorescent substrate Gly-Pro-AMC. The inhibitory effect of the protein on the DPP-IV enzyme activity was reflected through a control experiment design, and samples with potential DPP-IV inhibitory activity were quickly identified, which reduced the processing of invalid samples, improved the efficiency of early screening, and pointed out the direction for subsequent research.

[0156] (2) Through multi-step fine processing, the bound peptides are fixed on the amino-treated glass slide to form a solid-phase reaction platform with strong specificity and high stability, reducing nonspecific adsorption and creating ideal conditions for subsequent incubation reactions with fluorescently labeled protein solutions, thereby achieving preliminary enrichment of the target peptides.

[0157] (3) Prepare positive and negative test samples, perform incubation in a standardized manner, use gel filtration columns to accurately separate the bound and unbound substances, and collect fractions. Use a fluorescence spectrophotometer to detect the fluorescence intensity of each fraction, and use objective data to verify the specific binding ability of the synthetic peptide, laying a solid foundation for subsequent in-depth research on the inhibitory effect on DPP-IV activity and ensuring the scientificity and reliability of the screening results.

[0158] (4) In a simulated in vivo cellular microenvironment, the target peptide was applied to Caco-2 cells. Through multiple control settings and rigorous sample addition, incubation, fluorescence intensity scanning, and inhibition rate calculation processes, the inhibitory degree of the target peptide on DPP-IV activity in cells was accurately quantified, providing key data support for the evaluation of its clinical application potential and effectively promoting the transformation process from basic research to clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] Figure 1 It is a schematic diagram of the overall structure of the present invention;

[0160] Figure 2 It is a schematic diagram of the oscillation structure of the present invention;

[0161] Figure 3 is a schematic diagram of a loading plate of the present invention;

[0162] Figure 4 It is a schematic diagram of the shaking structure of the present invention;

[0163] Figure 5 is a schematic diagram of a fixed arc plate of the present invention;

[0164] Figure 6 It is a schematic diagram of the soaking structure of the present invention;

[0165] Figure 7 is a schematic diagram of a second limiting frame of the present invention;

[0166] Figure 8 It is a schematic diagram of the molecular structure of the DPP-IV inhibitory peptide of the present invention;

[0167] Fig. 9 is a schematic diagram of the 3D structure of the DPP-IV inhibitory peptide of the present invention;

[0168] Fig.10 It is a schematic diagram of the in vitro chemical analysis results of the DPP-IV inhibitory peptide concentration and the DPP-IV inhibition rate of the present invention;

[0169] Fig.11 is a schematic diagram of the interaction site between the DPP-IV inhibitory peptide of the present invention and DPP-IV;

[0170] Fig.12 It is a schematic diagram of the molecular mechanism by which the DPP-IV inhibitory peptide of the present invention inhibits DPP-IV activity.

[0171] The marks in the accompanying drawings are: 100, bottom plate; 200, oscillation structure; 201, oscillation frame; 203, driving slide; 2031, driving column; 2032, rotating plate; 2033, transmission plate; 202, first spring; 2021, second spring; 204, driven slide; 2022, third spring; 2023, fourth spring; 205, filling plate; 2051, filling slot; 2052, limit plate; 300, shaking structure; 301, control base; 302, servo motor; 303, fixed arc plate; 3041, rotating column; 304, control arc plate; 305, long arc plate; 306, shaking column; 307, short arc plate; 308, clamping frame; 30 81. round fixture; 3082. square fixture; 400. immersion structure; 401. water bath; 402. support frame; 403. first fixed plate; 4031. first support plate; 4032. second support plate; 4051. first transmission block; 405. first limit frame; 4033. third support plate; 4034. fourth support plate; 406. second limit frame; 4061. fixed hook; 407. fifth support plate; 4071. sixth support plate; 4052. second transmission block; 4072. seventh support plate; 4073. eighth support plate; 4074. second fixed plate; 206. liquid material frame; 404. servo electric cylinder; 4041. third fixed plate. DETAILED DESCRIPTION

[0172] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but they are not intended to limit the present invention.

[0173] The DPP-IV inhibitory peptide screened from yak blood has a sequence of DPENF; the molecular structure of the DPP-IV inhibitory peptide is,

[0174]

[0175] The molecular formula of DPP-IV inhibitory peptide is C 27 H 36 N6O 11 .

[0176] Screening equipment for screening DPP-IV inhibitory peptides from yak blood, including Figure 1 The bottom plate 100 shown in FIG. 1 has a bottom plate 100 provided above the bottom plate 100. Figure 2 The oscillating structure 200 shown in FIG. 1 has a side provided with a Figure 4 The shaking structure 300 shown in FIG. 1 has a side provided with a Figure 6 An immersion structure 400 is shown.

[0177] The oscillation structure 200 includes an oscillation frame 201, which is fixedly connected to the top of the bottom plate 100. A driving slide 203 is slidably connected to one side of the inner part of the oscillation frame 201. A liquid material frame 206 is fixedly connected to the top of the driving slide 203. The top of the liquid material frame 206 is provided with Figure 3 As shown in the loading plate 205 , a driven slide 204 is fixedly connected to the lower part of the loading plate 205 , and the driven slide 204 is slidably connected to the oscillation frame 201 .

[0178] A driving column 2031 is provided on one side of the driving slide 203, and a rotating plate 2032 is fixedly connected to the side of the driving column 2031. A side of the rotating plate 2032 away from the driving column 2031 is rotatably connected to a transmission plate 2033, and one end of the transmission plate 2033 away from the rotating plate 2032 is rotatably connected to the driven slide 204.

[0179] A first spring 202 is fixedly connected to one side of the driving slide 203, and an end of the first spring 202 away from the driving slide 203 is fixedly connected to the oscillation frame 201. A second spring 2021 is fixedly connected to the side of the driving slide 203 away from the first spring 202, and an end of the second spring 2021 away from the driving slide 203 is fixedly connected to the oscillation frame 201.

[0180] A third spring 2022 is fixedly connected to one side of the driven slide 204, and one end of the third spring 2022 away from the driven slide 204 is fixedly connected to the oscillation frame 201. A fourth spring 2023 is fixedly connected to the side of the driven slide 204 away from the third spring 2022, and one end of the fourth spring 2023 away from the driven slide 204 is fixedly connected to the oscillation frame 201.

[0181] A filling groove 2051 is formed on the upper surface of the filling plate 205 , and a limiting plate 2052 is disposed at the edge of the filling groove 2051 .

[0182] The shaking structure 300 includes a control base 301, which is fixedly connected to the top of the bottom plate 100. A servo motor 302 is arranged on the top of the control base 301, and a side of the servo motor 302 is arranged as follows: Figure 5 The fixed arc plate 303 shown in the figure has its bottom end fixedly connected to the bottom plate 100, one end of the fixed arc plate 303 is rotatably connected to a rotating column 3041, the output end of the servo motor 302 is fixedly connected to the rotating column 3041, the side of the rotating column 3041 away from the fixed arc plate 303 is fixedly connected to a control arc plate 304, one end of the control arc plate 304 away from the rotating column 3041 is rotatably connected to a long arc plate 305, one end of the long arc plate 305 away from the control arc plate 304 is rotatably connected to a shaking column 306, the bottom end of the shaking column 306 is rotatably connected to a short arc plate 307, and one end of the short arc plate 307 away from the shaking column 306 is rotatably connected to the fixed arc plate 303.

[0183] The top end of the shaking column 306 is rotatably connected to a clamping frame 308 , one end of the clamping frame 308 is provided with a circular clamp 3081 , and the end of the clamping frame 308 away from the circular clamp 3081 is provided with a square clamp 3082 .

[0184] The immersion structure 400 includes a water bath 401, which is arranged above the bottom plate 100. A support frame 402 is arranged above the water bath 401. The bottom end of the support frame 402 is fixedly connected to the bottom plate 100. A first fixed plate 403 is fixedly connected above the support frame 402. A first support plate 4031 is rotatably connected above the first fixed plate 403. An end of the first support plate 4031 away from the first fixed plate 403 is rotatably connected to a second support plate 4032. The second support plate One side of the first transmission block 4051 is rotatably connected to the first support plate 4032, and the first transmission block 4051 is fixedly connected to the first limit frame 405 on the side away from the second support plate 4032. The end of the first transmission block 4051 away from the second support plate 4032 is rotatably connected to the third support plate 4033, and the end of the third support plate 4033 away from the first transmission block 4051 is rotatably connected to the fourth support plate 4034. The end of the fourth support plate 4034 away from the third support plate 4033 is rotatably connected to the fourth support plate 4034. Figure 7 A second limiting frame 406 is shown.

[0185] A fixing hook 4061 is fixedly connected to the inner side of the second limiting frame 406 .

[0186] The side of the second limiting frame 406 away from the fourth supporting plate 4034 is rotatably connected to the fifth supporting plate 407, the end of the fifth supporting plate 407 away from the second limiting frame 406 is rotatably connected to the sixth supporting plate 4071, the end of the sixth supporting plate 4071 away from the fifth supporting plate 407 is rotatably connected to the second transmission block 4052, the side of the second transmission block 4052 away from the sixth supporting plate 4071 is fixedly connected to the first limiting frame 405, the end of the second transmission block 4052 away from the sixth supporting plate 4071 is rotatably connected to the seventh supporting plate 4072, the end of the seventh supporting plate 4072 away from the second transmission block 4052 is rotatably connected to the eighth supporting plate 4073, the end of the eighth supporting plate 4073 away from the seventh supporting plate 4072 is rotatably connected to the second fixed plate 4074, and the bottom end of the second fixed plate 4074 is fixedly connected to the bottom plate 100.

[0187] A servo electric cylinder 404 is rotatably connected to one side of the middle of the first support plate 4031 , and an end of the servo electric cylinder 404 away from the first support plate 4031 is rotatably connected to a third fixing plate 4041 . The bottom end of the third fixing plate 4041 is fixedly connected to the bottom plate 100 .

[0188] The method for screening DPP-IV inhibitory peptides from yak blood comprises the following steps:

[0189] (S01) collecting blood from the vein of an individual yak, injecting the blood into a centrifuge tube containing an anticoagulant, using a shaking structure 300 to evenly mix the liquid in the centrifuge tube, and then centrifuging the blood to separate blood cells;

[0190] (S02) crushing and centrifuging the blood cells to obtain a crude protein solution, and performing preliminary screening using a fluorescently labeled DPP-IV specific substrate;

[0191] (S03) placing the dialysis bag into the soaking structure 400 for soaking, removing impurities in the crude protein solution, and labeling it with a fluorescent labeling reagent to prepare a protein solution containing a fluorescent label;

[0192] (S04) designing a binding polypeptide of the DPP-IV inhibitory peptide, placing the amino-treated glass slide into the oscillation structure 200 for oscillation, and fixing the binding polypeptide on the surface of the amino-treated glass slide;

[0193] (S05) preparing an incubation buffer, adding a fluorescently labeled protein solution to the oscillating structure 200, and incubating with the amino-treated glass slide to allow the DPP-IV inhibitory peptide to bind to the surface of the amino-treated glass slide and remove unbound proteins;

[0194] (S06) collecting fluorescence signals, analyzing binding signals of bound polypeptides, and extracting the first peptide segment set on the amino-treated glass slide;

[0195] (S07) comparing the sequence of the first peptide set with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence to analyze similarity regions and potential enzyme cleavage sites;

[0196] (S08) based on the comparison results of the first peptide set, the yak hemoglobin sequence is virtually enzymatically digested, and the enzymatic fragments with the potential to inhibit DPP-IV activity are analyzed to generate a second peptide set;

[0197] (S09) using solid phase chemistry to prepare a second peptide set, obtain a target peptide, prepare a positive test sample and a negative test sample, put equal volumes of the positive and negative test samples into centrifuge tubes, add a fluorescently labeled binding peptide solution, use a shaking mechanism 300 to mix the liquid in the centrifuge tube evenly, separate the bound and unbound substances after incubation, and collect the fractions;

[0198] (S10) detecting the fluorescence intensity of each fraction to verify that the target polypeptide has specific binding ability;

[0199] (S11) adding the target polypeptide to a culture bottle containing culture medium, using a shaking mechanism 300 to evenly mix the liquid in the culture bottle, and then incubating, setting up a blank control group and a positive control group, adding DPP-IV enzyme and a fluorescently labeled DPP-IV specific substrate, detecting the change in fluorescence intensity of the supernatant in the culture medium and calculating the inhibition rate, and evaluating the inhibitory effect of the target polypeptide on intracellular DPP-IV activity.

[0200] In step (S01), blood is collected from the vein of the individual yak using a disposable sterile syringe.

[0201] The anticoagulant was heparin sodium with a final concentration of 15 U / mL.

[0202] In step (S01), the blood is centrifuged by placing the centrifuge tube containing the blood in a high-speed refrigerated centrifuge, centrifuging at a temperature of 4°C and a speed of 3500 rpm for 15 minutes, separating the plasma and blood cells, discarding the plasma, and then washing the blood cells twice with physiological saline. After each washing, the blood is placed in a high-speed refrigerated centrifuge again, centrifuged at a temperature of 4°C and a speed of 3500 rpm for 15 minutes, and the blood cell solution is collected after centrifugation.

[0203] The physiological saline solution is NaCl with a concentration of 0.9% and a temperature of 4°C.

[0204] In step (S02), the blood cells are crushed and centrifuged to obtain a crude protein solution, which is obtained by pouring the blood cell solution into an ultrasonic crusher, adding a lysis buffer, and crushing the blood cells by ultrasonication in an ice bath, and then pouring the crushed blood cell solution into a centrifuge tube, placing the centrifuge tube into a high-speed refrigerated centrifuge, and centrifuging at a temperature of 4° C. and a speed of 12000 rpm for 20 minutes, and collecting the supernatant after centrifugation as the crude protein solution.

[0205] The lysis buffer is Tris-HCl buffer with a final concentration of 20-50 mmol / L and a pH of 7.4-8.0.

[0206] The lysis buffer contained NaCl at a final concentration of 150 mmol / L.

[0207] The lysis buffer contains Triton X-100 at a final concentration of 0.5% to 1%.

[0208] The lysis buffer contains EDTA at a final concentration of 1-5 mmol / L.

[0209] The lysis buffer contains AEBSF at a final concentration of 0.1 to 1 mmol / L.

[0210] The lysis buffer contained PMSF at a final concentration of 1 mmol / L.

[0211] The lysis buffer contains Leupeptin at a final concentration of 1 to 10 μg / mL.

[0212] The lysis buffer contains E-64 at a final concentration of 1 to 10 μg / mL.

[0213] The lysis buffer contains Pepstatin A at a final concentration of 1 to 10 μg / mL.

[0214] The lysis buffer contains 1,10-Phenanthroline at a final concentration of 1 to 5 mmol / L.

[0215] The ultrasonic disruptor was used for 3 seconds each time with 5 seconds interval, for a total of 10 times.

[0216] In step (S02), a fluorescently labeled DPP-IV specific substrate is used for preliminary screening, which is to put the crude protein solution, DPP-IV enzyme and the fluorescently labeled DPP-IV specific substrate into a microplate reader and mix them, incubate them at 37° C. for 30 minutes, detect the fluorescence intensity using a fluorescence spectrophotometer, set a blank control group and an experimental group, compare the fluorescence intensity of the experimental group relative to the blank group, calculate the inhibition rate, screen the samples according to the inhibition rate, and collect the crude protein solution corresponding to the samples.

[0217] The blank control group contained DPP-IV enzyme and fluorescently labeled DPP-IV specific substrate, and the experimental group contained crude protein solution, DPP-IV enzyme and fluorescently labeled DPP-IV specific substrate.

[0218] The fluorescently labeled DPP-IV specific substrate is Gly-Pro-AMC.

[0219] The screening criteria for samples was an inhibition rate higher than 40%.

[0220] In step (S03), impurities in the crude protein solution are removed by soaking the dialysis bag in distilled water for 10 to 15 minutes, then transferring the crude protein solution to the dialysis bag, placing the dialysis bag in a dialysis buffer, and stirring the protein solution at a temperature of 25° C. and a rotation speed of 50 to 100 rpm until the solutions inside and outside the dialysis bag are fully exchanged and small molecule impurities diffuse into the dialysis buffer outside the dialysis bag.

[0221] In step (S03), the fluorescent labeling reagent is used for labeling, and the fluorescent labeling reagent and reaction buffer are added to the protein solution, and the protein solution is stirred at a temperature of 25° C. and a rotation speed of 50-100 rpm to perform a labeling reaction for 2 hours.

[0222] In step (S03), a protein solution containing a fluorescent marker is prepared by transferring the protein solution to a dialysis bag and dialyzing it with a dialysis buffer at a temperature of 25°C. The dialysis buffer is replaced every 1 to 2 hours, and the dialysis time is 4 to 6 hours. After the dialysis is completed, the dialysis bag is taken out, and the solution in the dialysis bag is the protein solution containing a fluorescent marker.

[0223] The fluorescent labeling agent is Cy3 fluorescent dye.

[0224] The reaction buffer is a Tris-HCl buffer with a pH of 7.0 to 9.0.

[0225] In step (S04), the binding polypeptide of the DPP-IV inhibitory peptide is designed by using SWISS-MODEL, inputting the DPENF sequence, constructing a three-dimensional model, downloading polypeptide data from the Antimicrobial Peptides Database, using Python scripts in combination with the Pandas library to write a screening program, setting screening conditions, running the screening program, extracting polypeptide sequences that meet the conditions as ligand models, using AutoDock Vina, importing the ligand model and the three-dimensional model of the DPENF sequence, docking, outputting the docking score ranking, screening out the top 10 to 20 polypeptide sequences, analyzing the interaction between the screened polypeptide sequences and the DPENF sequence, screening out polypeptide sequences that have positively charged amino acids forming stable electrostatic pairs near the acidic residues of DPENF and have hydrophobic stacking effects with the aromatic ring of phenylalanine, using solid phase chemical synthesis to prepare the screened polypeptide sequences, using surface plasmon resonance technology to detect the binding ability of the synthetic polypeptide to DPENF, calculating the binding constant and dissociation constant, and using the polypeptide sequence with a dissociation constant less than 100 nM as the binding polypeptide.

[0226] The screening conditions are that the polypeptide length is 8 to 10 amino acids and contains one positively charged amino acid, one aromatic amino acid and one amino acid that can form hydrogen bonds.

[0227] In step (S04), the binding polypeptide is fixed on the surface of the amino glass slide by first washing the amino glass slide with anhydrous ethanol, drying it under a nitrogen gas flow, and then immersing the amino glass slide in a glutaraldehyde solution with a concentration of 0.5% to 2%, shaking it for 30 to 120 minutes to allow the glutaraldehyde to cross-link with the amino groups on the surface of the amino glass slide, thereby introducing active aldehyde groups on the surface of the amino glass slide. After the reaction is completed, the amino glass slide is taken out, washed with a PBS buffer, and the binding polypeptide is dissolved in the PBS buffer to a concentration of 0.1 to 1 mg / mL. The amino-treated glass slide is placed in a sealed container containing water and reacted for 2 to 4 hours to allow the binding polypeptide to covalently bind to the aldehyde group on the surface of the amino-treated glass slide through the amino group for fixation. After fixation, the amino-treated glass slide is washed with PBS buffer to remove unbound polypeptides and impurities, and then placed in a BSA solution with a concentration of 1 to 5% and incubated for 1 to 2 hours. The amino-treated glass slide is then washed with PBS buffer and blown dry under a nitrogen gas flow.

[0228] In step (S05), the incubation buffer solution is prepared by selecting a Tris solution with a pH value of 7.4, adding BSA to make the final concentration of BSA reach 0.05%, and then filtering and sterilizing with a 0.22 μm filter membrane to prepare the incubation buffer solution.

[0229] In step (S05), the DPP-IV inhibitory peptide is bound to the surface of the amino-treated glass slide by covering the surface of the amino-treated glass slide with a fluorescently labeled protein solution, incubating at 37° C. and 50% humidity for 1 hour, and then rinsing the amino-treated glass slide with an incubation buffer to remove unbound proteins, leaving only the specifically bound DPP-IV inhibitory peptide on the surface of the amino-treated glass slide.

[0230] In step (S06), the fluorescence signal is collected by placing the amino-treated glass slide on the stage of the laser confocal microscope, scanning the surface of the amino-treated glass slide point by point, collecting the fluorescence signal intensity of each pixel point, and generating a fluorescence image.

[0231] In step (S06), the binding signal of the bound polypeptide is analyzed by identifying the strong binding signal area based on the fluorescence signal analysis, and the peptides captured in the strong binding signal area are extracted from the amino-treated glass slide using micro-cutting technology to form a first peptide set.

[0232] In step (S07), the sequence of the first peptide set is compared with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence, and the sequence of the first peptide set, the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence are input into BlastP for comparison analysis, the expected threshold is set to 0.001, the BLOSUM62 matrix is ​​selected, the penalty for opening the gap is set to 11, and the penalty for extending the gap is set to 1. The comparison results are output in a tabular form, including the query sequence identifier, the target sequence identifier, the comparison score, the expected threshold, the similarity percentage, and the start and end positions of the comparison region. The results with a similarity percentage higher than 40% are screened, and the start and end positions and amino acids of the comparison regions are checked. Acid sequence, according to the information of restriction site in DPP-IV inhibitory peptide sequence and yak hemoglobin sequence, potential restriction sites were screened in the similarity region of the first peptide set and within 5 to 10 amino acids upstream and downstream, and PeptideCutter was used to analyze the potential restriction sites, and short peptide fragments containing potential restriction sites were synthesized. The restriction products were detected by mass spectrometry and electrophoresis technology to verify the authenticity of the potential restriction sites, and the site-directed mutagenesis technology was used to change the amino acids in the similarity region of the first peptide set, and the effects of the polypeptides before and after mutation on the DPP-IV enzyme activity and the changes in the interaction with yak hemoglobin were observed to verify the accuracy of the similarity region.

[0233] In step (S08), the yak hemoglobin sequence is subjected to virtual enzymatic hydrolysis by using PeptideCutter to simulate enzymatic hydrolysis, and fragments with potential DPP-IV inhibitory activity are screened out from the generated enzymatic hydrolysis fragments, which are the second peptide segment set.

[0234] In step (S09), the positive test sample and the negative test sample are prepared by adding the target polypeptide powder into PBS buffer to prepare a positive test sample with a concentration of 50 μg / mL, and the negative test sample is PBS buffer.

[0235] In step (S09), equal volumes of positive and negative test samples are placed in centrifuge tubes, respectively. A micropipette is used to aspirate 50 μL of the positive test sample and 50 μL of the fluorescently labeled DPP-IV inhibitory peptide solution, and the mixture is transferred to a silanized 0.5 mL sterile microcentrifuge tube for mixing. 50 μL of the negative test sample and 50 μL of the fluorescently labeled DPP-IV inhibitory peptide solution are aspirated again, and the mixture is transferred to another sterile microcentrifuge tube for mixing.

[0236] In step (S09), the bound and unbound substances are separated after incubation and the fractions are collected, and the positive sample and the negative sample incubated at 37° C. for 30 minutes are transferred to two 100 μL micro-injectors, a Sephadex G-25 gel filtration chromatography column is selected, and is vertically fixed on an Agilent 1260 Infinity II chromatograph, 3 mL of PBS buffer is selected, and the Sephadex G-25 gel filtration chromatography column is rinsed at a flow rate of 0.8 mL / min, and the mixed reaction liquid of the positive sample and the negative sample is respectively injected into the injection ports of the two Sephadex G-25 gel filtration chromatography columns using two 100 μL micro-injectors, and the Agilent 1260 Infinity II chromatograph is started to allow the mixed reaction liquid of the positive sample and the negative sample to enter the Sephadex G-25 gel filtration chromatography column for separation, and the Sephadex G-25 gel filtration chromatography column is used for separation. The outlet of the G-25 gel filtration column was connected to a Model 2110 fraction collector, and one fraction was collected every 1.2 mL. The components in the mixed reaction solution of the positive sample and the negative sample were eluted from the Sephadex G-25 gel filtration column in turn and collected in the fraction collection tube.

[0237] 3 mL of PBS buffer includes 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4. Use a 1 L volumetric flask, add 800 mL of deionized water, stir magnetically until completely dissolved, adjust the pH value to 7.4 with a Mettler Toledo precision pH meter, and finally make up to 1 L with deionized water and filter through a 0.22 μm filter membrane for sterilization.

[0238] In step (S10), the fluorescence intensity of each fraction is detected by sequentially transferring the collected fractions into a quartz cuvette with an optical path of 1 cm, placing the cuvette into a Shimadzu RF-5301PC fluorescence spectrophotometer sample cell for fluorescence intensity detection, recording the fluorescence intensity value corresponding to each fraction, generating a curve graph of fluorescence intensity versus fraction change, and analyzing the fluorescence intensity curve graph.

[0239] In step (S11), the target polypeptide is added to a culture bottle containing culture medium, and the Caco-2 cell suspension is transferred to a 15 mL centrifuge tube containing 9 mL of complete culture medium, centrifuged at 1000 rpm for 5 minutes, the supernatant is discarded, and the precipitated Caco-2 cells are resuspended with 2 mL of complete culture medium, and the Caco-2 cell suspension is transferred to a culture bottle, and the culture bottle is placed in a 37°C, 5% CO2 incubator for culture, and the growth state of the Caco-2 cells is observed under an inverted microscope every day. When the confluence of the Caco-2 cells reaches 80% to 90%, the old culture medium in the culture bottle is discarded, and the surface of the Caco-2 cells is rinsed with 2 mL of PBS buffer to remove the residual culture medium and dead cells, and 1 mL of PBS buffer is added. L of 0.25% trypsin-0.02% EDTA digestion solution, place the culture flask in a 37°C incubator for digestion for 2 to 3 minutes, until the Caco-2 cells become round and begin to fall off, add 2mL of DMEM high-glucose medium containing 10% FBS to terminate the digestion, transfer the Caco-2 cell suspension to a 15mL centrifuge tube with a pipette, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add complete medium to resuspend the cells, and then inoculate the Caco-2 cells into a 96-well culture plate. After the Caco-2 cells grow in the 96-well culture plate for 4 to 6 hours, use an analytical balance to weigh the target polypeptide powder, dissolve it with DMSO, and dilute it with complete medium to make the final concentration of the target polypeptide 10μM.

[0240] The complete culture medium was made of DMEM high glucose medium and 10% FBS, 1% NEAA, and 1% Pen-Strep in a volume ratio.

[0241] In step (S11), a blank control group and a positive control group are set up, and a 96-well culture plate is divided into a target polypeptide group, a blank control group and a positive control group, and 6 replicate wells are set up in each group. The target polypeptide solution is aspirated with a multichannel pipette and added to each replicate well of the target polypeptide group. The amount of each replicate well is 100 μL, 100 μL of complete culture medium is added to the blank control group, and a binding polypeptide solution with a concentration of 10 μM is added to the positive control group.

[0242] In step (S11), the fluorescence intensity change of the supernatant in the culture medium is detected and the inhibition rate is calculated. The 96-well culture plate of the target polypeptide group and the positive control group is placed in a 37° C., 5% CO2 incubator for incubation for 24 hours, and the state of the Caco-2 cells is observed under an inverted microscope every 4 to 6 hours. After the incubation, the 96-well culture plate is taken out from the incubator, and a DPP-IV enzyme solution with a concentration of 0.1 U / mL is added to each duplicate well of the target polypeptide group and the positive control group, and the amount of each duplicate well is 50 μL. Gly-Pro-AMC with a final concentration of 50 μM is added, and the amount of each duplicate well is 50 μL. The 96-well culture plate is placed in a microplate reader, and the fluorescence intensity is scanned once every 2 to 3 minutes for a total of 30 to 60 minutes. The fluorescence intensity value of each duplicate well in each scan is recorded, and a curve atlas of fluorescence intensity change over time is generated, and the average fluorescence intensity and inhibition rate are calculated.

[0243] The calculation formula for the mean fluorescence intensity is:

[0244]

[0245] In the formula, I i is the fluorescence intensity of the ith replicate well, n is the number of replicate wells, I 平 is the mean fluorescence intensity.

[0246] The calculation formula of inhibition rate is:

[0247]

[0248] In the formula, I 空白 is the average fluorescence intensity of the blank control group, I 目标多肽 is the average fluorescence intensity of the target peptide group, I 阳性 is the average fluorescence intensity of the positive control group, and IR is the inhibition rate.

[0249] The method for screening DPP-IV inhibitory peptides from yak blood comprises the following steps:

[0250] (S01) collecting blood from the vein of an individual yak, injecting the blood into a centrifuge tube containing an anticoagulant, using a shaking structure (300) to evenly mix the liquid in the centrifuge tube, and then centrifuging the blood to separate blood cells;

[0251] (S02) crushing and centrifuging the blood cells to obtain a crude protein solution, and performing preliminary screening using a fluorescently labeled DPP-IV specific substrate;

[0252] (S03) placing the dialysis bag into the soaking structure (400) for soaking, removing impurities in the crude protein solution, and labeling it with a fluorescent labeling reagent to prepare a protein solution containing a fluorescent label;

[0253] (S04) designing a binding polypeptide of the DPP-IV inhibitory peptide, placing the amino-treated glass slide in an oscillating structure (200) for oscillation, and fixing the binding polypeptide on the surface of the amino-treated glass slide;

[0254] (S05) preparing an incubation buffer, adding a fluorescently labeled protein solution to the oscillating structure (200), and incubating with the amino-treated glass slide to allow the DPP-IV inhibitory peptide to bind to the surface of the amino-treated glass slide and remove unbound proteins;

[0255] (S06) collecting fluorescence signals, analyzing binding signals of bound polypeptides, and extracting the first peptide segment set on the amino-treated glass slide;

[0256] (S07) comparing the sequence of the first peptide set with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence to analyze similarity regions and potential enzyme cleavage sites;

[0257] (S08) based on the comparison results of the first peptide set, the yak hemoglobin sequence is virtually enzymatically digested, and the enzymatic fragments with the potential to inhibit DPP-IV activity are analyzed to generate a second peptide set;

[0258] (S09) using a solid phase chemical method to prepare a second peptide set, obtain a target peptide, prepare a positive test sample and a negative test sample, put equal volumes of the positive and negative test samples into centrifuge tubes, add a fluorescently labeled binding peptide solution, use a shaking structure (300) to evenly mix the liquid in the centrifuge tube, incubate, separate the bound and unbound substances, and collect the fractions;

[0259] (S10) detecting the fluorescence intensity of each fraction to verify that the target polypeptide has specific binding ability;

[0260] (S11) adding the target polypeptide to a culture bottle containing culture medium, using a shaking structure (300) to evenly mix the liquid in the culture bottle, and then incubating, setting up a blank control group and a positive control group, adding DPP-IV enzyme and a fluorescently labeled DPP-IV specific substrate, detecting the change in fluorescence intensity of the supernatant in the culture medium and calculating the inhibition rate, evaluating the inhibitory effect of the target polypeptide on intracellular DPP-IV activity, and obtaining the DPP-IV inhibitory peptide DPENF.

[0261] In the step (S01), the blood is centrifuged by placing the centrifuge tube containing the blood into a high-speed refrigerated centrifuge, separating the plasma and blood cells, discarding the plasma, and then washing the blood cells with physiological saline. After each washing, the blood is placed in a high-speed refrigerated centrifuge again, and the blood cell solution is collected after centrifugation;

[0262] In the step (S02), the crude protein solution is obtained after the blood cells are crushed and centrifuged, which comprises pouring the blood cell solution into an ultrasonic crusher, adding a lysis buffer, crushing the blood cells by ultrasonication in an ice bath, pouring the crushed blood cell solution into a centrifuge tube, placing the centrifuge tube in a high-speed refrigerated centrifuge, and collecting the supernatant as the crude protein solution after centrifugation;

[0263] In the step (S02), a fluorescently labeled DPP-IV specific substrate is used for preliminary screening, and the crude protein solution, DPP-IV enzyme and the fluorescently labeled DPP-IV specific substrate are placed in a microplate reader and mixed. After incubation, the fluorescence intensity is detected using a fluorescence spectrophotometer, a blank control group and an experimental group are set, the fluorescence intensity of the experimental group relative to the blank group is compared, the inhibition rate is calculated, the samples are screened according to the inhibition rate, and the crude protein solution corresponding to the sample is collected.

[0264] In the step (S03), impurities in the crude protein solution are removed by soaking the dialysis bag in distilled water for 10 to 15 minutes, then transferring the crude protein solution to the dialysis bag, placing the dialysis bag in a dialysis buffer, and stirring the protein solution until the solutions inside and outside the dialysis bag are fully exchanged and small molecule impurities diffuse into the dialysis buffer outside the dialysis bag;

[0265] The step (S03) of using a fluorescent labeling reagent for labeling is to add a fluorescent labeling reagent and a reaction buffer to the protein solution, stir the protein solution, and perform a labeling reaction;

[0266] In the step (S03), the protein solution containing the fluorescent marker is prepared by transferring the protein solution to a dialysis bag and dialyzing it with a dialysis buffer. The dialysis buffer is replaced every 1 to 2 hours, and the dialysis time is 4 to 6 hours. After the dialysis is completed, the dialysis bag is taken out, and the solution in the dialysis bag is the protein solution containing the fluorescent marker.

[0267] In the step (S04), the binding polypeptide of the DPP-IV inhibitory peptide is designed by using SWISS-MODEL, inputting the DPENF sequence, constructing a three-dimensional model, downloading polypeptide data from the Antimicrobial Peptides Database, using Python scripts in combination with the Pandas library to write a screening program, setting screening conditions, running the screening program, extracting polypeptide sequences that meet the conditions as ligand models, using AutoDock Vina, importing the ligand model and the three-dimensional model of the DPENF sequence, docking, outputting the docking score ranking, screening out the top 10 to 20 polypeptide sequences, analyzing the interaction between the screened polypeptide sequences and the DPENF sequence, screening out polypeptide sequences that have positively charged amino acids forming stable electrostatic pairs near the acidic residues of DPENF and have hydrophobic stacking effects with the aromatic ring of phenylalanine, preparing the screened polypeptide sequences by solid phase chemical synthesis, detecting the binding ability of the synthetic polypeptides to DPENF by surface plasmon resonance technology, calculating the binding constant and dissociation constant, and using the polypeptide sequences with a dissociation constant less than 100 nM as binding polypeptides;

[0268] The screening condition is that the polypeptide length is between 8 and 10 amino acids, and contains one positively charged amino acid, one aromatic amino acid and one amino acid that can form a hydrogen bond.

[0269] In the step (S04), the binding polypeptide is fixed on the surface of the amino glass slide by first washing the amino glass slide with anhydrous ethanol, drying it under a nitrogen gas flow, and then immersing the amino glass slide in a glutaraldehyde solution with a concentration of 0.5% to 2%, shaking it for 30 to 120 minutes to cross-link the glutaraldehyde with the amino groups on the surface of the amino glass slide, and introducing active aldehyde groups on the surface of the amino glass slide. After the reaction is completed, the amino glass slide is taken out, washed with a PBS buffer, and the binding polypeptide is dissolved in the PBS buffer to prepare a concentration of 0.1 to 1 mg / m L of binding polypeptide solution, dripping the binding polypeptide solution onto the surface of the amino glass slide, so that the binding polypeptide solution evenly covers the amino glass slide, placing the amino glass slide in a sealed container containing water, reacting for 2 to 4 hours, so that the binding polypeptide is covalently bound to the aldehyde group on the surface of the amino glass slide through the amino group, and fixed, after the fixation is completed, the amino glass slide is washed with PBS buffer to remove unbound polypeptide and impurities, and then placed in a BSA solution with a concentration of 1 to 5%, incubated for 1 to 2 hours, and then washed with PBS buffer, and placed under a nitrogen gas flow to dry.

[0270] In the step (S05), the DPP-IV inhibitory peptide is bound to the surface of the amino-treated glass slide by incubating the surface of the amino-treated glass slide with a fluorescently labeled protein solution, and then washing the amino-treated glass slide with an incubation buffer to remove unbound proteins, leaving only the specifically bound DPP-IV inhibitory peptide on the surface of the amino-treated glass slide;

[0271] In the step (S06), the fluorescent signal is collected by placing the amino glass slide on the stage of the laser confocal microscope, scanning the surface of the amino glass slide point by point, collecting the fluorescent signal intensity of each pixel point, and generating a fluorescent image;

[0272] In the step (S06), the binding signal of the bound polypeptide is analyzed by identifying the strong binding signal area based on the fluorescence signal analysis, and the peptides captured in the strong binding signal area are extracted from the amino-treated glass slide using micro-cutting technology to form a first peptide set.

[0273] In the step (S07), the sequence of the first peptide segment set is compared with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence, and the sequence of the first peptide segment set, the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence are input into BlastP for comparison analysis, the expected threshold is set to 0.001, the BLOSUM62 matrix is ​​selected, the penalty for opening the gap is set to 11, and the penalty for extending the gap is set to 1, and the comparison results are output in a table form, including the query sequence identifier, the target sequence identifier, the comparison score, the expected threshold, the similarity percentage, and the start and end positions of the comparison area. The results with a similarity percentage higher than 40% are screened, and the start and end positions of the comparison area and the amino acid sequence are checked. According to the information of restriction sites in the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence, potential restriction sites were screened in the similarity region of the first peptide set and within 5 to 10 amino acids upstream and downstream, and PeptideCutter was used to analyze the potential restriction sites, and short peptide fragments containing potential restriction sites were synthesized. The restriction products were detected by enzyme cleavage reaction, mass spectrometry and electrophoresis technology to verify the authenticity of the potential restriction sites, and the site-directed mutagenesis technology was used to change the amino acids in the similarity region of the first peptide set, and the effects of the polypeptides before and after mutation on the DPP-IV enzyme activity and the changes in the interaction with yak hemoglobin were observed to verify the accuracy of the similarity region.

[0274] In the step (S08), the yak hemoglobin sequence is subjected to virtual enzymatic hydrolysis, which uses PeptideCutter to simulate enzymatic hydrolysis, and screens out fragments with potential DPP-IV inhibitory activity from the generated enzymatic hydrolysis fragments, which are the second peptide segment set;

[0275] In the step (S09), the positive test sample and the negative test sample are prepared by adding the target polypeptide powder into a PBS buffer to prepare the positive test sample, and the negative test sample is a PBS buffer;

[0276] In the step (S09), equal volumes of positive and negative test samples are placed in centrifuge tubes respectively, and the positive test sample and the fluorescently labeled DPP-IV inhibitory peptide solution are aspirated using a micropipette, transferred to a silanized sterile microcentrifuge tube for mixing, and the negative test sample and the fluorescently labeled DPP-IV inhibitory peptide solution are aspirated again, transferred to another sterile microcentrifuge tube for mixing;

[0277] The step (S09) is to separate the bound and unbound substances after incubation and collect fractions, wherein the incubated positive sample and negative sample are transferred to two injectors, a Sephadex G-25 gel filtration chromatography column is selected, and is vertically fixed on an Agilent 1260 Infinity II chromatograph, a PBS buffer is selected to rinse the Sephadex G-25 gel filtration chromatography column, and the mixed reaction liquid of the positive sample and the negative sample is respectively injected into the injection ports of the two Sephadex G-25 gel filtration chromatography columns using two injectors, and the Agilent 1260 Infinity II chromatograph is started to allow the mixed reaction liquid of the positive sample and the negative sample to enter the Sephadex G-25 gel filtration chromatography column for separation, and a Model 2110 fraction collector is connected to the outlet of the Sephadex G-25 gel filtration chromatography column, and a fraction is collected at equal volume intervals, and each component in the mixed reaction liquid of the positive sample and the negative sample is eluted from the Sephadex G-25 gel filtration chromatography column in turn and collected in a fraction collection tube.

[0278] In the step (S10), the fluorescence intensity of each fraction is detected by sequentially transferring the collected fractions into a quartz cuvette with an optical path of 1 cm, placing the collected fractions into a sample cell of a Shimadzu RF-5301PC fluorescence spectrophotometer for fluorescence intensity detection, recording the fluorescence intensity value corresponding to each fraction, generating a curve spectrum of fluorescence intensity versus fraction change, and analyzing the fluorescence intensity curve spectrum;

[0279] In the step (S11), the target polypeptide is added to a culture bottle containing a culture medium, the Caco-2 cell suspension is transferred to a centrifuge tube containing a complete culture medium, the supernatant is discarded by centrifugation, the precipitated Caco-2 cells are resuspended with the complete culture medium, the Caco-2 cell suspension is transferred to a culture bottle, the culture bottle is placed in an incubator for culture, the growth state of the Caco-2 cells is observed under an inverted microscope every day, and when the confluence of the Caco-2 cells reaches 80% to 90%, the old culture medium in the culture bottle is discarded, and the surface of the Caco-2 cells is rinsed with PBS buffer to remove the residual Culture medium and dead cells, add trypsin-EDTA digestion solution, place the culture flask in the incubator for digestion for 2 to 3 minutes, until the Caco-2 cells become round and begin to fall off, add DMEM high-glucose medium containing FBS to terminate digestion, transfer the Caco-2 cell suspension to a centrifuge tube with a pipette, centrifuge and discard the supernatant, add complete culture medium to resuspend the cells, and then inoculate the Caco-2 cells to the culture plate. After the Caco-2 cells grow on the culture plate for 4 to 6 hours, use an analytical balance to weigh the target polypeptide powder, dissolve it with DMSO, and dilute it with complete culture medium;

[0280] In the step (S11), a blank control group and a positive control group are set up, and the culture plate is divided into a target polypeptide group, a blank control group and a positive control group, and each group is provided with multiple replicate wells. The target polypeptide solution is aspirated with a multichannel pipette and added to each replicate well of the target polypeptide group. Samples are added to each replicate well, complete culture medium is added to the blank control group, and binding polypeptide solution is added to the positive control group.

[0281] Example:

[0282] In this example, the experimental materials are 5 healthy adult yaks, and 8 mL of blood is collected from each yaks, a total of 40 mL, which is extracted using a disposable sterile syringe and injected into a centrifuge tube containing 15 U / mL heparin sodium anticoagulant.

[0283] In this example, after blood treatment, centrifugation, washing, crushing, and re-centrifugation, 1.2 mL of crude protein solution was finally obtained from 40 mL of blood, and the protein concentration was 2.5 mg / mL.

[0284] In this embodiment, a microplate reader and a fluorescence spectrophotometer were used, and the samples were incubated at 37°C for 30 minutes. A blank control group and an experimental group were set up to detect 10 crude protein samples (0.2 mL each). Among them, the inhibition rates of 4 samples were higher than 40%, namely 43%, 47%, 50%, and 52%, respectively, and the inhibition rates of the remaining samples were between 20% and 35%. The crude protein solutions corresponding to the 4 samples with high inhibition rates were collected.

[0285] In this embodiment, the purity of the protein solution after dialysis is increased by about 30%. After fluorescence labeling, the labeling efficiency reaches 85% through fluorescence intensity detection.

[0286] In this example, 150 peptide sequences were screened from the database, and the top 15 were selected for subsequent analysis after docking scoring. Finally, 5 binding peptides with dissociation constants less than 100 nM were determined, and their binding constants were between 20-60 nM.

[0287] In this example, the target polypeptide was added to the culture medium containing Caco-2 cells, and blank and positive control groups were set up, with 6 replicates in each group. After incubation for 24 hours, DPP-IV enzyme and Gly-Pro-AMC were added and scanned with an ELISA reader. The inhibition rate of the positive control group reached 60%. In the target polypeptide group, 3 replicates had an inhibition rate higher than 50%, which were 52%, 55%, and 58%, respectively. The remaining replicates were between 40% and 50%, and the inhibition rate of the blank control group was less than 10%.

[0288] In this embodiment, the DPP-IV inhibitory peptide sequence is DPENF (Asp-Pro-Glu-Asn-Phe), and the molecular formula is C 27 H 36 N6O 11 , the SMILES specification format is:

[0289] N[C@@]([H])(CC(=O)O)C(=O)N1[C@@]([H])(CCC1)C(=O)N[C@@]([H])(CCC( =O)O)C(=O)N[C@@]([H])(CC(=O)N)C(=O)N[C@@H](CC1=CC=C(C=C1))C(=O)O.

[0290] The molecular structure of the DPP-IV inhibitory peptide in this example is as follows Figure 8 As shown, the 3D structure of the DPP-IV inhibitory peptide in this example is as shown Fig. 9 shown.

[0291] The results of in vitro chemical analysis of the DPP-IV inhibitory peptide concentration and DPP-IV inhibition rate in this example are as follows: Fig.10 As shown, the results show that the DPP-IV inhibition rate of DPENF at a low concentration of 0.2 mg / mL is already >20%. After fitting the results, it was found that the dose-effect relationship of DPENF in inhibiting the biological activity of human DPP-IV is y=100 / (1.657x -0.4815 +1). The R of the regression equation 2 =0.9686, which is higher than 0.95, indicating a high fitting accuracy, that is, the inhibitory effect of DPENF on the biological activity of human DPP-IV conforms to the S-shaped dose-effect curve. At the same time, the half maximum inhibitory concentration IC calculated according to this equation is 50=4.60mmol / mL. Existing studies have also found that a variety of peptide sequences similar to DPENF also have certain DPP-IV inhibitory activity, such as the peptide KA (IC 50 =6.27mmol / L), and the peptide EK (IC 50 =3.22mmol / L). In summary, the candidate peptide DPENF was identified as a novel DPP-IV inhibitory peptide discovered from yak blood and had high DPP-IV inhibitory activity.

[0292] The interaction sites between the DPP-IV inhibitory peptide and DPP-IV in this example are as follows Fig.11 As shown, the molecular mechanism by which the DPP-IV inhibitory peptide of this embodiment inhibits DPP-IV activity is as follows Fig.12 As shown, Fig.11 and Fig.12 The non-bonded interaction mode of DPENF with human DPP-IV is shown in 3D and 2D images.

[0293] This example uses the following tools to analyze the physicochemical properties and bioaccessibility indicators of polypeptides:

[0294] Table 1: Indicators, parameters and calculation analysis tools

[0295]

[0296]

[0297] The results showed that the hydrophobicity value of the peptide was 14.45 kcal / mol. Judging from the hydrophobicity range of common peptides, this value is at an upper-middle level, indicating that this peptide has a certain hydrophobic tendency.

[0298] At pH = 7.0, the net charge is -2 and the isoelectric point is 2.92. From the perspective of charge, the negative charge is relatively large, and under physiological conditions (pH is about 7.4), the polypeptide will be negatively charged. This makes it have electrostatic attraction when interacting with positively charged molecules or groups.

[0299] The ToxinPred tool detected no potential toxicity, which clearly ruled out the possibility that the peptide had toxicity risks under common toxicity prediction models, providing important safety guarantees for its use in vivo.

[0300] The water solubility is 5.75 mol / mL. Compared with the common water solubility standards of polypeptides, this polypeptide has a certain water solubility. In practical applications, such as in the preparation of pharmaceutical preparations, it is necessary to consider the effect of its solubility on dosage form selection and administration method.

[0301] The probability of absorption in the human intestine is as high as 75%, which is a relatively high number. This indicates that when the polypeptide passes through the human intestine, a large proportion can be absorbed by intestinal epithelial cells and enter the body's circulatory system, providing strong theoretical support for its oral administration route, which means that it has great potential and feasibility in the development of oral drug preparations.

[0302] It has bitter, sour and umami taste, and the bad taste needs to be masked by adding appropriate sweeteners and spices to increase consumer acceptance.

[0303] In this embodiment, the screening criteria of inhibition rate were tested:

[0304] 1. Activity tracking comparison experiment of samples with different inhibition rates

[0305] In this example, 50 crude protein solution samples from different yaks were selected, which were initially processed and under the same conditions. Each sample was mixed with DPP-IV enzyme and Gly-Pro-AMC, incubated at 37°C for 30 minutes, and then the fluorescence intensity was measured by a fluorescence spectrophotometer. A blank control group was set to calculate the inhibition rate.

[0306] Experimental data showed that the 30 samples with an inhibition rate lower than 40% performed poorly in subsequent peptide binding experiments. For example, after sample A with an inhibition rate of 25% was incubated with a DPP-IV binding peptide containing a DPENF sequence fragment, laser confocal microscopy showed that the average fluorescence intensity of the protein-peptide binding region was only 120 units, while the average background fluorescence intensity was 100 units, and the signal contrast was as low as 1.2, making it almost impossible to distinguish effective binding signals, meaning that the potential inhibitory peptide was difficult to play a role.

[0307] In contrast, the 20 samples with inhibition rates higher than 40% were optimistic. For example, sample B with an inhibition rate of 50% had an average fluorescence intensity of 500 units in the strong binding area on the slide after incubation, an average background fluorescence intensity of 150 units, and a signal contrast ratio of 3.3. The first peptide set extracted by microdissection was sequenced and three characteristic sequences potentially related to DPP-IV inhibitory activity were found, paving the way for subsequent research.

[0308] 2. Long-term cell experiment observation

[0309] In this example, the crude protein solutions corresponding to samples with different inhibition rates were treated and then added to Caco-2 cell culture medium. Blank and positive control groups were set up and incubated in a 37° C., 5% CO2 incubator for 72 hours. The fluorescence intensity of the culture medium supernatant was measured every 12 hours to calculate the inhibition rate.

[0310] In the 30 sample groups with inhibition rates below 40%, the intracellular DPP-IV enzyme activity remained high throughout the whole process. For example, after 72 hours of incubation, the intracellular DPP-IV enzyme activity in the group containing sample C (inhibition rate 30%) was only 5% lower than that in the blank control group, which was almost negligible.

[0311] The 20 sample groups with inhibition rates higher than 40% showed significant effects. Taking sample D (inhibition rate 45%) as an example, after 72 hours of incubation, the intracellular DPP-IV enzyme activity was reduced by 30% compared with the blank control group. Although it was not as good as the positive control group (reduction of 50%), it has proved its potential.

[0312] 3. Large-scale sample screening data analysis

[0313] In this example, 200 yak blood samples were screened, and the inhibition rate and subsequent experimental index data were recorded. Statistics show that for 80 samples with an inhibition rate higher than 40%, the probability of subsequently identifying potential DPP-IV inhibitory activity polypeptide sequences is 35%, and for 120 samples with an inhibition rate lower than 40%, the probability is only 12%. Moreover, 72% of the final target polypeptides of the high inhibition rate samples showed significant activity in the specific binding and intracellular activity inhibition evaluation, meeting the requirements of the early stage of research and development.

[0314] In summary, multiple examples and detailed data show that setting a screening standard with an inhibition rate higher than 40% can accurately screen out low-activity samples, focus on high-potential samples, improve the success rate of subsequent experiments, and lay a solid foundation for the efficient screening of DPP-IV inhibitory peptides.

[0315] In this embodiment, the oscillation frame 201 of the oscillation structure 200 is fixed above the base plate 100 as an overall supporting structure, and the driving slide 203 and the driven slide 204 on one side thereof work in coordination. A driving column 2031 is provided on one side of the driving slide 203, and the side of the driving column 2031 is fixedly connected to a rotating plate 2032. The rotating plate 2032 is rotatably connected to a transmission plate 2033 on the side away from the driving column 2031, and the other end of the transmission plate 2033 is rotatably connected to the driven slide 204, thereby realizing power transmission and linking the two.

[0316] The liquid material frame 206 is fixed above the driving slide 203 and is used to contain glutaraldehyde solution. The filling plate 205 is located above the liquid material frame 206. The driven slide 204 below it is slidably connected to the oscillation frame 201. A filling groove 2051 is provided on the upper surface of the filling plate 205, and a limiting plate 2052 is provided on the edge to facilitate the operator to accurately place the slide. The limiting plate 2052 can constrain the movable range of the slide to prevent it from detaching during oscillation.

[0317] The first spring 202 and the second spring 2021 are connected to both sides of the driving slide 203, and the third spring 2022 and the fourth spring 2023 are connected to both sides of the driven slide 204, which jointly provide a buffer to ensure that the glutaraldehyde solution evenly covers and penetrates the amino groups on the surface of the slide under gentle and uniform oscillation, thereby achieving stable cross-linking modification, avoiding uneven local reactions that affect the fixation effect of the polypeptide, reducing experimental errors caused by poor slide treatment, and improving subsequent screening specificity.

[0318] The shaking structure 300 is fixed on the top of the base plate 100 based on the control base 301, and the servo motor 302 is installed on it. The bottom end of the fixed arc plate 303 is fixed to the base plate 100, and one end is rotatably connected to the rotating column 3041. The output end of the servo motor 302 drives the rotating column 3041 to rotate.

[0319] The rotating column 3041 drives the control arc plate 304 to move, and the control arc plate 304 is linked with the shaking column 306 through the long arc plate 305. The bottom end of the shaking column 306 is rotatably connected with the fixed arc plate 303 through the short arc plate 307, forming a multi-dimensional shaking mechanism.

[0320] The clamping frame 308 at the top of the shaking column 306 is provided with a circular clamp 3081 and a square clamp 3082, which can firmly fix containers such as centrifuge tubes and culture bottles. Driven by the servo motor 302, the container is shaken in multiple dimensions, and the solution is quickly mixed under the alternating action of centrifugal force and gravity, such as blood cell lysate and buffer solution, fluorescent labeling reagent and protein solution, etc., to accelerate the reaction process, and gently shake to avoid cell rupture and protein denaturation, thereby ensuring the quality of experimental samples.

[0321] The water bath 401 of the immersion structure 400 is placed above the base plate 100, the bottom end of the support frame 402 is fixed to the base plate 100, the first fixed plate 403 is rotatably connected to the first support plate 4031 above, the second support plate 4032 is linked to the first transmission block 4051, the first transmission block 4051 drives the first limit frame 405 and the third support plate 4033, the fourth support plate 4034 is linked to the second limit frame 406, and the inner side of the second limit frame 406 is fixedly connected to the fixed hook 4061 for hanging the dialysis bag so that it is suspended in the buffer solution, so that the operator does not directly contact the dialysis bag during the experiment, thereby ensuring the purity of the dialysis.

[0322] One side of the middle part of the first support plate 4031 is rotatably connected to the servo electric cylinder 404, and the other end of the servo electric cylinder 404 is fixedly connected to the bottom end of the third fixed plate 4041. By controlling the extension and retraction of the servo electric cylinder 404, the operator can easily adjust the position of the dialysis bag and accurately reset the dialysis bag after replacing the dialysis buffer to meet the dialysis requirements of different durations and temperatures, ensure that the dialysis bag is fully infiltrated, accelerate the diffusion of small molecule impurities, improve the dialysis efficiency, and provide a high-purity protein solution for subsequent experiments.

[0323] The present invention integrates the originally scattered operation processes through the coordinated operation of the oscillation structure 200, the shaking structure 300, and the immersion structure 400, reduces the risk of sample contamination, improves the consistency, accuracy and efficiency of the screening process, and provides an optimized hardware platform support for screening DPP-IV inhibitory peptides from yak blood, thereby ensuring the smooth progress of the entire screening experiment and the reliability of the results from the hardware level.

[0324] The present invention has been described in detail above by general description and specific embodiments, but it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A method for screening DPP-IV inhibitory peptides from yak blood, characterized in that: Includes the following step, (S01) collecting blood from the vein of an individual yak, injecting the blood into a centrifuge tube containing an anticoagulant, using a shaking structure (300) to evenly mix the liquid in the centrifuge tube, and then centrifuging the blood to separate blood cells; (S02) crushing and centrifuging the blood cells to obtain a crude protein solution, and performing preliminary screening using a fluorescently labeled DPP-IV specific substrate; (S03) placing the dialysis bag into the soaking structure (400) for soaking, removing impurities in the crude protein solution, and labeling it with a fluorescent labeling reagent to prepare a protein solution containing a fluorescent label; (S04) designing a binding polypeptide of the DPP-IV inhibitory peptide, placing the amino-treated glass slide in an oscillating structure (200) for oscillation, and fixing the binding polypeptide on the surface of the amino-treated glass slide; (S05) preparing an incubation buffer, adding a fluorescently labeled protein solution to the oscillating structure (200), and incubating with the amino-treated glass slide to allow the DPP-IV inhibitory peptide to bind to the surface of the amino-treated glass slide and remove unbound proteins; (S06) collecting fluorescence signals, analyzing binding signals of bound polypeptides, and extracting the first peptide segment set on the amino-treated glass slide; (S07) comparing the sequence of the first peptide set with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence to analyze similarity regions and potential enzyme cleavage sites; (S08) based on the comparison results of the first peptide set, the yak hemoglobin sequence is virtually enzymatically digested, and the enzymatic fragments with the potential to inhibit DPP-IV activity are analyzed to generate a second peptide set; (S09) using a solid phase chemical method to prepare a second peptide set, obtain a target peptide, prepare a positive test sample and a negative test sample, put equal volumes of the positive and negative test samples into centrifuge tubes, add a fluorescently labeled binding peptide solution, use a shaking structure (300) to evenly mix the liquid in the centrifuge tube, incubate, separate the bound and unbound substances, and collect the fractions; (S10) detecting the fluorescence intensity of each fraction to verify that the target polypeptide has specific binding ability; (S11) adding the target polypeptide to a culture bottle containing culture medium, using a shaking structure (300) to evenly mix the liquid in the culture bottle, and then incubating, setting up a blank control group and a positive control group, adding DPP-IV enzyme and a fluorescently labeled DPP-IV specific substrate, detecting the change in fluorescence intensity of the supernatant in the culture medium and calculating the inhibition rate, evaluating the inhibitory effect of the target polypeptide on intracellular DPP-IV activity, and obtaining the DPP-IV inhibitory peptide DPENF.

2. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 1, characterized in that: In the step (S01), the blood is centrifuged by placing the centrifuge tube containing the blood into a high-speed refrigerated centrifuge, separating the plasma and blood cells, discarding the plasma, and then washing the blood cells with physiological saline. After each washing, the blood is placed in a high-speed refrigerated centrifuge again, and the blood cell solution is collected after centrifugation; In the step (S02), the crude protein solution is obtained after the blood cells are crushed and centrifuged, which comprises pouring the blood cell solution into an ultrasonic crusher, adding a lysis buffer, crushing the blood cells by ultrasonication in an ice bath, pouring the crushed blood cell solution into a centrifuge tube, placing the centrifuge tube in a high-speed refrigerated centrifuge, and collecting the supernatant as the crude protein solution after centrifugation; In the step (S02), a fluorescently labeled DPP-IV specific substrate is used for preliminary screening, and the crude protein solution, DPP-IV enzyme and the fluorescently labeled DPP-IV specific substrate are placed in a microplate reader and mixed. After incubation, the fluorescence intensity is detected using a fluorescence spectrophotometer, a blank control group and an experimental group are set, the fluorescence intensity of the experimental group relative to the blank group is compared, the inhibition rate is calculated, the samples are screened according to the inhibition rate, and the crude protein solution corresponding to the sample is collected.

3. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 1, characterized in that: In the step (S03), impurities in the crude protein solution are removed by soaking the dialysis bag in distilled water for 10 to 15 minutes, then transferring the crude protein solution to the dialysis bag, placing the dialysis bag in a dialysis buffer, and stirring the protein solution until the solutions inside and outside the dialysis bag are fully exchanged and small molecule impurities diffuse into the dialysis buffer outside the dialysis bag; The step (S03) of using a fluorescent labeling reagent for labeling is to add a fluorescent labeling reagent and a reaction buffer to the protein solution, stir the protein solution, and perform a labeling reaction; In the step (S03), the protein solution containing the fluorescent marker is prepared by transferring the protein solution to a dialysis bag and dialyzing it with a dialysis buffer. The dialysis buffer is replaced every 1 to 2 hours, and the dialysis time is 4 to 6 hours. After the dialysis is completed, the dialysis bag is taken out, and the solution in the dialysis bag is the protein solution containing the fluorescent marker.

4. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 1, characterized in that: In the step (S04), the binding polypeptide of the DPP-IV inhibitory peptide is designed by using SWISS-MODEL, inputting the DPENF sequence, constructing a three-dimensional model, downloading polypeptide data from the Antimicrobial Peptides Database, using Python scripts in combination with the Pandas library to write a screening program, setting screening conditions, running the screening program, extracting polypeptide sequences that meet the conditions as ligand models, using AutoDock Vina, importing the ligand model and the three-dimensional model of the DPENF sequence, docking, outputting the docking score ranking, screening out the top 10 to 20 polypeptide sequences, analyzing the interaction between the screened polypeptide sequences and the DPENF sequence, screening out polypeptide sequences that have positively charged amino acids forming stable electrostatic pairs near the acidic residues of DPENF and have hydrophobic stacking effects with the aromatic ring of phenylalanine, preparing the screened polypeptide sequences by solid phase chemical synthesis, detecting the binding ability of the synthetic polypeptides to DPENF by surface plasmon resonance technology, calculating the binding constant and dissociation constant, and using the polypeptide sequences with a dissociation constant less than 100 nM as binding polypeptides; The screening condition is that the polypeptide length is between 8 and 10 amino acids, and contains one positively charged amino acid, one aromatic amino acid and one amino acid that can form a hydrogen bond.

5. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 4, characterized in that: In the step (S04), the binding polypeptide is fixed on the surface of the amino glass slide by first washing the amino glass slide with anhydrous ethanol, drying it under a nitrogen gas flow, and then immersing the amino glass slide in a glutaraldehyde solution with a concentration of 0.5% to 2%, shaking it for 30 to 120 minutes to cross-link the glutaraldehyde with the amino groups on the surface of the amino glass slide, and introducing active aldehyde groups on the surface of the amino glass slide. After the reaction is completed, the amino glass slide is taken out, washed with a PBS buffer, and the binding polypeptide is dissolved in the PBS buffer to prepare a concentration of 0.1 to 1 mg / m L of binding polypeptide solution, dripping the binding polypeptide solution onto the surface of the amino glass slide, so that the binding polypeptide solution evenly covers the amino glass slide, placing the amino glass slide in a sealed container containing water, reacting for 2 to 4 hours, so that the binding polypeptide is covalently bound to the aldehyde group on the surface of the amino glass slide through the amino group, and fixed, after the fixation is completed, the amino glass slide is washed with PBS buffer to remove unbound polypeptide and impurities, and then placed in a BSA solution with a concentration of 1 to 5%, incubated for 1 to 2 hours, and then washed with PBS buffer, and placed under a nitrogen gas flow to dry.

6. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 1, characterized in that: In the step (S05), the DPP-IV inhibitory peptide is bound to the surface of the amino-treated glass slide by incubating the surface of the amino-treated glass slide with a fluorescently labeled protein solution, and then washing the amino-treated glass slide with an incubation buffer to remove unbound proteins, leaving only the specifically bound DPP-IV inhibitory peptide on the surface of the amino-treated glass slide; In the step (S06), the fluorescent signal is collected by placing the amino glass slide on the stage of the laser confocal microscope, scanning the surface of the amino glass slide point by point, collecting the fluorescent signal intensity of each pixel point, and generating a fluorescent image; In the step (S06), the binding signal of the bound polypeptide is analyzed by identifying the strong binding signal area based on the fluorescence signal analysis, and the peptides captured in the strong binding signal area are extracted from the amino-treated glass slide using micro-cutting technology to form a first peptide set.

7. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 1, characterized in that: In the step (S07), the sequence of the first peptide segment set is compared with the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence, and the sequence of the first peptide segment set, the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence are input into BlastP for comparison analysis, the expected threshold is set to 0.001, the BLOSUM62 matrix is ​​selected, the penalty for opening the gap is set to 11, and the penalty for extending the gap is set to 1, and the comparison results are output in a table form, including the query sequence identifier, the target sequence identifier, the comparison score, the expected threshold, the similarity percentage, and the start and end positions of the comparison area. The results with a similarity percentage higher than 40% are screened, and the start and end positions of the comparison area and the amino acid sequence are checked. According to the information of restriction sites in the DPP-IV inhibitory peptide sequence and the yak hemoglobin sequence, potential restriction sites were screened in the similarity region of the first peptide set and within 5 to 10 amino acids upstream and downstream, and PeptideCutter was used to analyze the potential restriction sites, and short peptide fragments containing potential restriction sites were synthesized. The restriction products were detected by enzyme cleavage reaction, mass spectrometry and electrophoresis technology to verify the authenticity of the potential restriction sites, and the site-directed mutagenesis technology was used to change the amino acids in the similarity region of the first peptide set, and the effects of the polypeptides before and after mutation on the DPP-IV enzyme activity and the changes in the interaction with yak hemoglobin were observed to verify the accuracy of the similarity region.

8. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 1, characterized in that: In the step (S08), the yak hemoglobin sequence is subjected to virtual enzymatic hydrolysis, which uses PeptideCutter to simulate enzymatic hydrolysis, and screens out fragments with potential DPP-IV inhibitory activity from the generated enzymatic hydrolysis fragments, which are the second peptide segment set; In the step (S09), the positive test sample and the negative test sample are prepared by adding the target polypeptide powder into a PBS buffer to prepare the positive test sample, and the negative test sample is a PBS buffer; In the step (S09), equal volumes of positive and negative test samples are placed in centrifuge tubes respectively, and the positive test sample and the fluorescently labeled DPP-IV inhibitory peptide solution are aspirated using a micropipette, transferred to a silanized sterile microcentrifuge tube for mixing, and the negative test sample and the fluorescently labeled DPP-IV inhibitory peptide solution are aspirated again, transferred to another sterile microcentrifuge tube for mixing; The step (S09) is to separate the bound and unbound substances after incubation and collect fractions, wherein the incubated positive sample and negative sample are transferred to two injectors, a Sephadex G-25 gel filtration chromatography column is selected, and is vertically fixed on an Agilent 1260 Infinity II chromatograph, a PBS buffer is selected to rinse the Sephadex G-25 gel filtration chromatography column, and the mixed reaction liquid of the positive sample and the negative sample is respectively injected into the injection ports of the two Sephadex G-25 gel filtration chromatography columns using two injectors, and the Agilent 1260 Infinity II chromatograph is started to allow the mixed reaction liquid of the positive sample and the negative sample to enter the Sephadex G-25 gel filtration chromatography column for separation, and a Model 2110 fraction collector is connected to the outlet of the Sephadex G-25 gel filtration chromatography column, and a fraction is collected at equal volume intervals, and each component in the mixed reaction liquid of the positive sample and the negative sample is eluted from the Sephadex G-25 gel filtration chromatography column in turn and collected in a fraction collection tube.

9. The method for screening DPP-IV inhibitory peptides from yak blood according to claim 1, characterized in that: In the step (S10), the fluorescence intensity of each fraction is detected by sequentially transferring the collected fractions into a quartz cuvette with an optical path of 1 cm, placing the collected fractions into a sample cell of a Shimadzu RF-5301PC fluorescence spectrophotometer for fluorescence intensity detection, recording the fluorescence intensity value corresponding to each fraction, generating a curve spectrum of fluorescence intensity versus fraction change, and analyzing the fluorescence intensity curve spectrum; In the step (S11), the target polypeptide is added to a culture bottle containing a culture medium, the Caco-2 cell suspension is transferred to a centrifuge tube containing a complete culture medium, the supernatant is discarded by centrifugation, the precipitated Caco-2 cells are resuspended with the complete culture medium, the Caco-2 cell suspension is transferred to a culture bottle, the culture bottle is placed in an incubator for culture, the growth state of the Caco-2 cells is observed under an inverted microscope every day, and when the confluence of the Caco-2 cells reaches 80% to 90%, the old culture medium in the culture bottle is discarded, and the surface of the Caco-2 cells is rinsed with PBS buffer to remove the residual Culture medium and dead cells, add trypsin-EDTA digestion solution, place the culture flask in the incubator for digestion for 2 to 3 minutes, until the Caco-2 cells become round and begin to fall off, add DMEM high-glucose medium containing FBS to terminate digestion, transfer the Caco-2 cell suspension to a centrifuge tube with a pipette, centrifuge and discard the supernatant, add complete culture medium to resuspend the cells, and then inoculate the Caco-2 cells to the culture plate. After the Caco-2 cells grow on the culture plate for 4 to 6 hours, use an analytical balance to weigh the target polypeptide powder, dissolve it with DMSO, and dilute it with complete culture medium; In the step (S11), a blank control group and a positive control group are set up, and the culture plate is divided into a target polypeptide group, a blank control group and a positive control group, and each group is provided with multiple replicate wells. The target polypeptide solution is aspirated with a multichannel pipette and added to each replicate well of the target polypeptide group. Samples are added to each replicate well, complete culture medium is added to the blank control group, and binding polypeptide solution is added to the positive control group.

10. A screening device for screening DPP-IV inhibitory peptides from yak blood, characterized by: It comprises a bottom plate (100), an oscillating structure (200) is arranged above the bottom plate (100), a shaking structure (300) is arranged on one side of the oscillating structure (200), and a soaking structure (400) is arranged on one side of the shaking structure (300); The oscillation structure (200) comprises an oscillation frame (201), the oscillation frame (201) is fixedly connected to the top of the bottom plate (100), a driving slide (203) is slidably connected to one side of the interior of the oscillation frame (201), a liquid material frame (206) is fixedly connected to the top of the driving slide (203), a loading plate (205) is arranged above the liquid material frame (206), a driven slide (204) is fixedly connected to the bottom of the loading plate (205), and the driven slide (204) is slidably connected to the oscillation frame (201); The shaking structure (300) comprises a control base (301), wherein the control base (301) is fixedly connected to the top of the base plate (100), a servo motor (302) is arranged on the top of the control base (301), a fixed arc plate (303) is arranged on one side of the servo motor (302), the bottom end of the fixed arc plate (303) is fixedly connected to the base plate (100), one end of the fixed arc plate (303) is rotatably connected to a rotating column (3041), and the output end of the servo motor (302) is fixedly connected to the rotating column (3041). The rotating column (3041) is fixedly connected to a control arc plate (304) at one side away from the fixed arc plate (303); the end of the control arc plate (304) away from the rotating column (3041) is rotatably connected to a long arc plate (305); the end of the long arc plate (305) away from the control arc plate (304) is rotatably connected to a shaking column (306); the bottom end of the shaking column (306) is rotatably connected to a short arc plate (307); the end of the short arc plate (307) away from the shaking column (306) is rotatably connected to the fixed arc plate (303).

Images