Affinity chromatography ligand for improving hirudin adsorption efficiency

Abstract

The invention discloses an affinity chromatography ligand for improving hirudin adsorption efficiency, and relates to the technical field of biological medicines. Comprising a ligand structure, a microenvironment regulation and control module, a small molecule substance storage and release subunit and a small molecule substance type and content determination unit, the ligand structure is provided with a ligand skeleton and is of a three-dimensional space structure, the ligand skeleton comprises a central connection area and a plurality of branch structures extending outwards from the central connection area, and the tail end of each branch structure is provided with a first functional group specifically bound with hirudin; according to the microenvironment regulation and control module, a microenvironment regulation and control area is arranged in a ligand, and a plurality of microenvironment regulation and control units are arranged in the microenvironment regulation and control area. According to the present invention, the ligand structure comprises the ligand core skeleton having the specific chemical structure, the plurality of branch structures extending outward from the center connection region, and the first functional groups arranged at the tail ends of the branch structures and specifically combined with the hirudin, such that the efficient adsorption of the hirudin is achieved;

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an affinity chromatography ligand for improving the adsorption efficiency of hirudin. Background Art

[0002] As a polypeptide substance with important biological activities, hirudin has broad application prospects in the field of biomedicine. However, the process of extracting and purifying hirudin from natural sources is complex and inefficient, which has become one of the key factors restricting its large-scale application.

[0003] Traditional methods have deficiencies and lack the ability of real-time perception and dynamic regulation of the surrounding environment. These ligands usually have fixed chemical structures and functional groups and are difficult to make corresponding adjustments according to changes in environmental factors.

[0004] In summary, there are many deficiencies in the affinity chromatography ligands in the prior art in terms of the adsorption efficiency of hirudin. Therefore, it is particularly important to develop an affinity chromatography ligand for improving the adsorption efficiency of hirudin. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies existing in the prior art, and to propose an affinity chromatography ligand for improving the adsorption efficiency of hirudin.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] An affinity chromatography ligand for improving the adsorption efficiency of hirudin. This system realizes precise control and regulation of the microenvironment around the ligand by introducing a microenvironment regulation module and an intelligent regulation mechanism, thereby significantly improving the adsorption efficiency of hirudin and the separation and purification effect.

[0008] To solve the above technical problems, the present invention provides the following technical solution: An affinity chromatography ligand for improving the adsorption efficiency of hirudin. This system includes a ligand structure, a microenvironment regulation module, a small molecule storage and release subunit, and a small molecule type and content determination unit;

[0009] The ligand structure: a ligand core skeleton with a specific chemical structure, which is formed by connecting a variety of organic functional groups through chemical bonds to form a three-dimensional spatial structure. The ligand skeleton includes a central connection region and multiple branch structures extending outward from this region. A first functional group that specifically binds to hirudin is provided at the end of each branch structure;

[0010] The microenvironment regulation module: A microenvironment regulation region is set in the ligand. This region is distributed at specific positions on the ligand backbone and has a certain spatial association with the central connection region and the branched structure. The microenvironment regulation region contains multiple microenvironment regulation units, and each microenvironment regulation unit consists of the following parts:

[0011] It has the ability to perceive surrounding environmental factors in real time. Its perception principle is based on an electrochemical perception algorithm, and the specific formula is as follows:

[0012]

[0013] Among them, E 感知 represents the perceived energy value output by the environmental perception subunit. k1 and k2 are weight coefficients. In these experiments, the leech peptide adsorption efficiency was measured at different pH values P i , where i ranges from 1 to n representing different pH measurement points and different ionic strengths I j , and j ranges from 1 to m representing the leech peptide adsorption efficiency at different ionic strength measurement points. It is compared with the preset optimal pH value P 0i and the optimal ionic strength I 0j . According to the influence degree of the change in adsorption efficiency on the overall perception result, the values of k1 and k2 are determined. If it is found that the change in pH value has a greater impact on the adsorption efficiency, the value of k1 is relatively large; if the change in ionic strength has a more significant impact, the value of k2 is relatively large;

[0014] The small molecule substance storage and release subunit: It is connected to the environmental perception subunit and decides whether to release or absorb specific small molecule substances according to the perceived energy value output by the environmental perception subunit. Its decision on release or absorption is based on the following algorithm formula:

[0015]

[0016] Among them, S 决策 represents the decision variable of the small molecule substance storage and release subunit. When S 决策 > 1, it decides to release small molecule substances. When S 决策 < 1, it decides to absorb small molecule substances. E 阈值 is determined by statistical analysis of the perceived energy value when the adsorption efficiency significantly decreases, that is, the E 感知 values when the leech peptide adsorption efficiency significantly decreases under different environmental fluctuations are measured in multiple experiments, and their average value is taken as E 阈值 , and C 初始 is the concentration of the small molecule substances initially stored in the small molecule substance storage and release subunit;

[0017] The small molecule species and content determination unit: The types and contents of small molecules stored in the small molecule storage and release subunit are determined based on the analysis of the optimal environmental conditions for hirudin adsorption. By conducting a detailed study on the molecular structure, charge distribution of hirudin, and its adsorption behavior under different environmental conditions, it is determined that under different pH values and ionic strengths, alkaline buffers need to be stored and released to regulate the environment. When the ionic strength is too high, specific ions need to be stored and released to reduce the ionic strength.

[0018] Furthermore, the central connection region of the ligand core skeleton is composed of a specific ring structure, which is connected to multiple branched structures by chemical bonds. The ring structure is prepared by an organic synthesis process, and the specific steps are as follows:

[0019] Select specific organic raw materials A and B, where:

[0020] Raw material A: 3,5-dihydroxybenzoic acid is selected as raw material A, which has multiple reactive sites, namely two hydroxyl groups and one carboxyl group. These reactive sites can participate in subsequent chemical reactions, providing necessary connection points and reaction bases for the formation of the ring structure;

[0021] Raw material B: 1,3-propanediamine is selected as raw material B. The amino groups at both ends have strong reactivity and can undergo a condensation reaction with the carboxyl group in raw material A, thereby gradually constructing the ring structure;

[0022] The selection of reaction solvent C is based on the optimization of reaction activity and selectivity. When solvent C is dimethyl sulfoxide (DMSO), it can promote the reaction between raw materials A and B, improving the reaction yield and the purity of the product;

[0023] Then, the reaction is carried out at a specific reaction temperature and reaction time. The specific situation is as follows:

[0024] Reaction temperature: The reaction is carried out in the temperature range of 80°C - 100°C. Within this temperature range, the molecular motion of raw materials A and B intensifies, enhancing their reactivity. However, it will not cause too many side reactions or raw material decomposition problems due to excessive temperature. After multiple experimental comparisons, when the temperature is set at 90°C, the reaction selectivity and yield can reach a relatively ideal balance state;

[0025] Reaction time: The reaction time is set to 10 - 16 hours. In the initial stage of the reaction, as time goes by, the condensation reaction between raw materials A and B gradually proceeds, and the cyclic structure is gradually formed. When the reaction time reaches about 12 hours, through the monitoring of the reaction process, it is found that both the production amount and purity of the product reach relatively stable and high levels. Continuing to extend the time to 16 hours, although the product amount may increase slightly, considering the production efficiency factor, it is comprehensively determined that 10 - 16 hours is a more appropriate reaction time range.

[0026] Furthermore, the first functional group that specifically binds to hirudin is a modified amino acid residue derivative. The preparation process of this derivative is as follows: Select a specific natural amino acid as the basic raw material, chemically modify it, introduce a specific functional group on the side-chain amino group of lysine, and achieve this modification process through the chemical reaction formula: Lys-NH2 + CH2O + HCOOH → Lys-NH-CH2-COOH, where Lys represents lysine. Then, further derivatize the modified amino acid, connect another specific functional group to the carboxyl group, and complete the derivatization through another chemical reaction formula. After such modification and derivatization, the obtained amino acid residue derivative is used as the first functional group, which has unique chemical properties. It can both specifically bind to a specific region on the hirudin molecule and maintain a relatively stable binding ability under different environmental conditions, providing an important guarantee for improving the adsorption efficiency of hirudin.

[0027] Furthermore, the distribution of the microenvironment regulation region on the ligand core backbone follows the spatial layout principle, which is determined based on the spatial orientation of the hirudin molecule approaching the ligand and the structural stability of the ligand itself. The microenvironment regulation region is distributed at a position on the ligand core backbone close to the end of the branched structure and relatively far from the central connection region. When the hirudin molecule binds to the ligand, the microenvironment regulation region can more directly affect the local environment where the hirudin molecule is located, and at the same time, it will not affect the overall structural stability of the ligand due to being too close to the central connection region. Through computer simulation experiments, the hirudin adsorption process under different layout schemes is simulated, and the adsorption efficiency changes under various environmental fluctuations are observed. Finally, this specific spatial layout principle is determined, providing favorable spatial conditions for the microenvironment regulation region to effectively play its role.

[0028] Furthermore, in addition to being able to sense the pH value and ionic strength, the external environment sensing subunit also has the ability to sense temperature changes. Its sensing of temperature changes is based on a thermal sensing algorithm, and the formula is as follows:

[0029] E 感知温度 = k3×(T - T0) 2

[0030] Among them, E 感知温度 represents the temperature perception energy value output by the environmental perception subunit. k3 is a weight coefficient. In these experiments, the hirudin adsorption efficiency at different temperatures T was measured and compared with the preset optimal temperature T0. The value of k3 was determined according to the degree of influence of the change in adsorption efficiency on the overall perception result. When the temperature change has a greater impact on the adsorption efficiency, the value of k3 is relatively large. And the small molecule substance storage and release subunit will decide whether to release or absorb specific small molecule substances according to the comprehensive result of the temperature perception energy value, the pH value, and the ion strength perception energy value. When the temperature rises and the pH value is on the low side at the same time, the small molecule substance storage and release subunit will select to release appropriate small molecule substances according to the calculation result of the comprehensive perception energy value to achieve a comprehensive regulation of the microenvironment around the ligand and better adapt to the adsorption conditions of hirudin.

[0031] Furthermore, the small molecule substances stored in the small molecule substance storage and release subunit also include small molecule compounds with biological activities. The selection of the bioactive small molecule compounds is based on in-depth research on the biological activity and adsorption mechanism of hirudin. By analyzing the interaction between hirudin and other biomolecules in vivo, it is found that certain bioactive small molecule compounds can affect the adsorption behavior of hirudin. Therefore, these bioactive small molecule compounds are included in the storage range of the small molecule substance storage and release subunit, and their optimal storage content and release conditions are determined through experiments according to different environmental conditions and hirudin adsorption requirements. Under certain specific environmental conditions, when the hirudin adsorption efficiency is low, releasing a certain amount of specific nucleotide derivatives may improve the adsorption efficiency because these derivatives may have specific interactions with hirudin molecules or other molecules in their surrounding environment, thereby promoting the binding of hirudin to the ligand.

[0032] Furthermore, the connection method between the ligand and the chromatography medium adopts a method combining chemical bonding and physical adsorption. The chromatography medium is pretreated, and specific functional groups are introduced on its surface through chemical reactions. One end of the ligand reacts with the epoxy group on the surface of the chromatography medium through chemical bonding to form a firm chemical bond connection. There is also a physical adsorption effect between other parts of the ligand and the surface of the chromatography medium. This way of combining chemical bonding and physical adsorption, on the one hand, ensures the firm attachment of the ligand on the chromatography medium and prevents it from falling off due to water flow factors during use. On the other hand, it also gives the ligand a certain degree of freedom when interacting with hirudin, enabling the ligand to better play its functions of microenvironment regulation and adsorption. Through a large number of experimental comparisons, the ligand using this connection method is significantly superior to the ligand connected by simply using chemical bonding or physical adsorption in terms of hirudin adsorption efficiency.

[0033] Furthermore, there is an interaction relationship among multiple branched structures of the ligand backbone, which is achieved by setting specific interaction groups on the branched structures. These interaction groups include, but are not limited to, hydrogen bond-forming groups and electrostatic interaction groups. When the ligand is not bound to hirudin, an internal structural stability mechanism is formed among these interaction groups, keeping the overall structure of the ligand stable. When the hirudin molecule approaches the ligand, these interaction groups will be adjusted according to the specific situation of the hirudin molecule. Some groups may temporarily interrupt the interaction to provide a more suitable binding space for the hirudin molecule. At the same time, other groups may form new interactions with the corresponding regions on the hirudin molecule, thereby enhancing the binding ability between the ligand and hirudin. Through computer simulation and experimental verification, this interaction relationship among the branched structures optimizes the overall structure and function of the ligand, playing an important role in improving the adsorption efficiency of hirudin.

[0034] Furthermore, during the preparation of the ligand, an integrated process flow is adopted for the synthesis and assembly of each component. This process flow systematically integrates the synthesis of the ligand core backbone, the introduction of the first functional group, the construction of the microenvironment regulation region, and the connection step between the ligand and the chromatography medium. In the same reaction system, the synthesis of the ligand core backbone and the introduction of the first functional group are carried out sequentially in a predetermined order. By reasonably controlling the reaction conditions, the smooth completion of these two steps is ensured. On the basis of the formed ligand core backbone and the first functional group, the microenvironment regulation region is constructed. Similarly, through precise control of the relevant reaction conditions, the high-quality construction of the microenvironment regulation region is achieved. The prepared ligand is connected to the chromatography medium by a combination of chemical bonding and physical adsorption. This integrated process flow not only improves the preparation efficiency of the ligand but also ensures the good synergistic effect among the components of the ligand, providing a strong guarantee for improving the adsorption efficiency of hirudin.

[0035] The beneficial effects of the present invention are as follows:

[0036] 1. The present invention realizes the efficient adsorption of hirudin through the ligand structure, including a ligand core backbone with a specific chemical structure, multiple branched structures extending outward from the central connection region, and a first functional group specifically binding to hirudin provided at the end of each branched structure. In addition, the microenvironment regulation module in the ligand can sense and adjust the microenvironment around the ligand in real time to optimally adapt to the adsorption conditions of hirudin, thereby further improving the adsorption efficiency. This design makes this affinity chromatography ligand have significant advantages in the field of biological separation and purification, and can extract and purify hirudin from complex biological samples more quickly and effectively.

[0037] 2. The present invention adopts an integrated process flow in the preparation process, systematically integrating the synthesis of the ligand core skeleton, the introduction of the first functional group, the construction of the microenvironment regulation region, and the connection step between the ligand and the chromatography medium. This integrated preparation method not only improves the preparation efficiency of the ligand, but also ensures good synergy among the components. In addition, specific interaction groups are set between multiple branched structures of the ligand skeleton to form a stable internal structure, enabling the ligand to have good stability and durability while maintaining high adsorption efficiency. This stability and durability are crucial for the long-term use of the bioseparation and purification process, ensuring that the ligand can still maintain excellent performance after multiple uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a flowchart of the operation of an affinity chromatography ligand for improving the adsorption efficiency of hirudin proposed by the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] The technical solutions of the present invention will be further described in detail below in conjunction with the specific embodiments.

[0040] Example 1:

[0041] This example describes that in a biochemical laboratory, researchers are committed to separating and purifying hirudin from a complex biological sample containing hirudin for subsequent drug research and development experiments. The amount of sample to be processed in the experiment is relatively small, but the purity requirement for hirudin is extremely high.

[0042] 3,5-dihydroxybenzoic acid is selected as raw material A, 1,3-propanediamine is selected as raw material B, and dimethyl sulfoxide (DMSO) is used as reaction solvent C. The reaction is carried out at a temperature of 90 °C for 12 hours to successfully synthesize the ligand core skeleton with a cyclic structure. The selection of this temperature and time is based on previous experiments, which can ensure that the raw materials react fully, the cyclic structure is complete, and the product purity is high, providing a stable basis for the introduction of subsequent functional groups.

[0043] Based on lysine as the basic raw material, specific functional groups are introduced onto the side-chain amino group through chemical reactions, and another functional group is connected to the carboxyl group to obtain an amino acid residue derivative that specifically binds to hirudin as the first functional group. This process strictly controls the reaction conditions to ensure accurate functional group modification and stable chemical properties, enabling it to effectively bind to hirudin in different environments.

[0044] The microenvironment regulation regions are distributed at positions near the ends of the ligand core backbone and relatively far from the central connection region. This layout is determined through computer simulation experiments, which can ensure that when the hirudin molecule approaches the ligand, the microenvironment regulation regions can effectively affect its local environment without affecting the overall structural stability of the ligand. During the construction process, advanced nanotechnology is used to precisely control the distribution and composition of the microenvironment regulation units, enabling them to have a sensitive environmental perception ability.

[0045] The chromatography medium is pretreated to introduce specific functional groups on its surface. One end of the ligand is reacted with the epoxy groups on the surface of the chromatography medium through chemical bonding to form a strong chemical bond connection, and the other parts of the ligand maintain a certain degree of physical adsorption with the surface of the chromatography medium. This connection method has been verified through a large number of comparative experiments, which can not only ensure the firm attachment of the ligand but also give it the freedom required when interacting with hirudin, effectively improving the adsorption efficiency.

[0046] The complex biological sample containing hirudin is pretreated to remove impurity particles, adjust the pH value to be close to the preset optimal pH value (such as pH 7.5), and control the ionic strength within an appropriate range (such as 0.1 - 0.2 mol / L). Then, the sample is slowly loaded onto the chromatography column filled with the affinity chromatography ligand to ensure sufficient contact between the sample and the ligand.

[0047] The environmental perception subunit in the microenvironment regulation module is used to monitor the changes in pH value, ionic strength, and temperature in real time. The environmental perception subunit operates based on the electrochemical perception algorithm (for pH value and ionic strength) and the thermal perception algorithm (for temperature), and calculates the perceived energy value through a preset formula.

[0048] Among them, E 感知 represents the perceived energy value output by the environmental perception subunit, and k1 and k2 are weight coefficients. In these experiments, the hirudin adsorption efficiency at different pH values P i , where i ranges from 1 to n representing different pH measurement points and different ionic strengths I j , where j ranges from 1 to m representing different ionic strength measurement points, is compared with the preset optimal pH value P 0i and the optimal ionic strength I 0j to determine the values of k1 and k2 according to the influence degree of the adsorption efficiency change on the overall perception result. If it is found that the change in pH value has a greater impact on the adsorption efficiency, the value of k1 is relatively large; if the change in ionic strength has a more significant impact, the value of k2 is relatively large. For example, when the environmental pH value deviates from the optimal pH value by 0.2 units, the perceived energy value changes significantly, triggering the response mechanism of the small molecule storage and release subunit.

[0049] The small molecule storage and release subunit determines whether to release or absorb specific small molecules by comparing the sensed energy value with a pre-set energy threshold.

[0050] Among them, S 决策 represents the decision variable of the small molecule storage and release subunit. When S 决策 > 1, it decides to release small molecules. When S 决策 < 1, it decides to absorb small molecules. E 阈值 is determined by statistical analysis of the sensed energy value when the adsorption efficiency significantly decreases, that is, the E 感知 value when the hirudin adsorption efficiency significantly decreases under different environmental fluctuations is measured in multiple experiments, and its average value is taken as E 阈值 , and C 初始 is the concentration of the small molecules initially stored in the small molecule storage and release subunit. For example, when the pH value drops to 7.3 (lower than the optimal pH value), the sensed energy value exceeds the threshold, and the unit releases an appropriate amount of alkaline buffer (such as HEPES) to adjust the environmental pH value and restore it to the range favorable for hirudin adsorption. During the whole process, the microenvironment around the ligand is continuously optimized to improve the hirudin adsorption efficiency.

[0051] After the adsorption reaches equilibrium, a specific eluent (such as a buffer containing a certain concentration of NaCl) is used for elution to elute the hirudin adsorbed on the ligand and collect the eluent. After detection, the purity of the purified hirudin reaches over 95%, meeting the high-purity requirements for drug R & D experiments.

[0052] Example 2:

[0053] This example describes that in a highly specialized pharmaceutical laboratory, a team of multidisciplinary experts is dedicated to the research on the structure and function of hirudin. Their goal is to deeply understand the biological activity mechanism of hirudin, especially its interaction with specific ligands, in order to develop new drugs or therapies.

[0054] First, based on computer simulation technology, the researchers designed a theoretical affinity chromatography ligand structure that can efficiently bind to hirudin according to the patent description and the three-dimensional structure prediction of hirudin. Subsequently, in the laboratory, chemical synthesis experts precisely synthesized the ligand using advanced organic synthesis methods and verified its structural correctness and purity by means of nuclear magnetic resonance (NMR) and mass spectrometry (MS).

[0055] The synthesized ligand is firmly attached to the chromatography medium (such as agarose beads) through chemical bonding technology to ensure uniform ligand distribution and high stability. Hirudin is extracted and purified from natural sources or recombinant expression systems to prepare a sample solution with an appropriate concentration. Meanwhile, a series of control samples are prepared for evaluating non-specific adsorption.

[0056] The sample solution containing hirudin is slowly passed through a chromatography column packed with ligand-modified chromatography medium, and the elution curve is recorded to observe the adsorption and desorption behaviors of hirudin. Using the environmental sensing subunit,

[0057] where, E 感知 represents the sensing energy value output by the environmental sensing subunit, and k1 and k2 are weighting coefficients. In these experiments, the hirudin adsorption efficiencies at different pH values P i , where i ranges from 1 to n representing different pH measurement points and different ionic strengths I j , where j ranges from 1 to m representing different ionic strength measurement points, are measured. The hirudin adsorption efficiencies are compared with the preset optimal pH value P 0i and the optimal ionic strength I 0j . According to the influence degree of the change in adsorption efficiency on the overall sensing result, the values of k1 and k2 are determined. If it is found that the change in pH value has a greater impact on the adsorption efficiency, the value of k1 is relatively large; if the change in ionic strength has a more significant impact, the value of k2 is relatively large. Continuously monitor the changes in pH value, ionic strength, temperature, and possibly oxygen concentration during the experimental process to ensure the consistency of experimental conditions. Through the small molecule storage and release subunit, where, S 决策 represents the decision variable of the small molecule storage and release subunit. When S 决策 > 1, it is decided to release small molecules; when S 决策 < 1, it is decided to absorb small molecules. E 阈值 is a preset energy threshold. Monitor and record the release or absorption of small molecules (such as cofactors, inhibitors) that may be involved during the experimental process to evaluate their impact on the hirudin-ligand interaction.

[0058] Collect and analyze the chromatography experimental data, including adsorption efficiency, desorption conditions, evaluate the binding strength and selectivity between hirudin and the ligand. By comparing and analyzing the experimental results under different environmental conditions, clarify the specific impacts of pH value, ionic strength, and temperature factors on the hirudin adsorption efficiency, establish a mathematical model for quantitative description, and combine computer simulation with experimental data to explore the molecular mechanism of the hirudin-ligand interaction, including key contact points and types of interaction forces, providing a theoretical basis for designing more efficient affinity chromatography strategies or drug development.

[0059] Based on the current findings, further optimize the ligand structure to improve the selectivity and efficiency of affinity chromatography, explore the interaction network between hirudin and other biomolecules, provide a more comprehensive perspective for understanding its complex biological activities, conduct in vivo experiments to verify the in vitro research results, and lay a solid foundation for the clinical application of hirudin.

[0060] As mentioned above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, shall be covered by the protection scope of the present invention.

Claims

1. An affinity chromatography ligand for improving the adsorption efficiency of hirudin, characterized in that, It includes a ligand structure, a microenvironment regulation module, a small molecule storage and release subunit, and a small molecule type and content determination unit; The ligand structure: It has a ligand backbone with a three-dimensional spatial structure. The ligand backbone includes a central connection region and multiple branch structures extending outward from the central connection region. A first functional group that specifically binds to hirudin is provided at the end of each branch structure; The microenvironment regulation module: A microenvironment regulation region is provided in the ligand. The microenvironment regulation region contains multiple microenvironment regulation units; The small molecule storage and release subunit: It is connected to the environment perception subunit and decides whether to release or absorb specific small molecules according to the perceived energy value output by the environment perception subunit; The small molecule type and content determination unit: The types and contents of small molecules stored in the small molecule storage and release subunit are determined according to the analysis of the optimal environmental conditions for hirudin adsorption.

2. The affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 1, characterized in that The ligand backbone of the ligand structure is formed by connecting various organic functional groups through chemical bonds; The microenvironment regulation region of the microenvironment regulation module is distributed at the set positions of the ligand backbone, has a spatial association with the central connection region and the branch structures. The microenvironment regulation unit has the ability to perceive surrounding environmental factors in real time, and its perception principle is based on an electrochemical perception algorithm. The specific formula is as follows: Among them, E 感知 represents the perceived energy value output by the environmental perception subunit, and k1 and k2 are weighting coefficients, based on the measured different pH values P i , where i ranges from 1 to n representing different pH measurement points and different ionic strengths I j , where j ranges from 1 to m representing the hirudin adsorption efficiency under different ionic strength measurement points, compared with the preset optimal pH value P 0i and the optimal ionic strength I 0j for comparison. The values of k1 and k2 are determined according to the influence degree of the change in adsorption efficiency on the overall perception result. If it is found that the change in pH value has a greater impact on the adsorption efficiency, the value of k1 is relatively large; if the change in ionic strength has a more significant impact, the value of k2 is relatively large; The small molecule storage and release subunit makes the decision to release or absorb based on the following algorithm formula: Among them, S 决策 represents the decision variable of the small molecule storage and release subunit. When S 决策 > 1, it decides to release small molecule substances. When S 决策 < 1, it decides to absorb small molecule substances. E 阈值 is determined by statistical analysis of the perceived energy value when the adsorption efficiency significantly decreases, specifically: measuring the E 感知 value when the hirudin adsorption efficiency significantly decreases under different environmental fluctuations through multiple experiments, and taking the average value as E 阈值 , C 初始 is the concentration of the small molecule substances initially stored in the small molecule storage and release subunit; The small molecule type and content determination unit: When the pH value is on the low side, it stores and releases alkaline buffers to adjust the environment. When the ionic strength is too high, it stores and releases corresponding ions to reduce the ionic strength.

3. An affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, characterized in that, The central connection region of the ligand core backbone is composed of a specific ring structure. This ring structure is connected to multiple branch structures through chemical bonds. The ring structure is prepared by an organic synthesis process. The specific steps are as follows: Select specific organic raw materials A and B, where: Raw material A: 3,5-dihydroxybenzoic acid is selected as raw material A; Raw material B: 1,3-propanediamine is selected as raw material B, which is used to undergo a condensation reaction with the carboxyl group in raw material A to construct the ring structure; Reaction solvent C; Then, the reaction is carried out at a specific reaction temperature and reaction time. The specific situation is as follows: Reaction temperature: The reaction is carried out in the temperature range of 80°C - 100°C; Reaction time: The reaction time is set to 10 - 16 hours.

4. An affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, characterized in that, The first functional group that specifically binds to hirudin is a modified amino acid residue derivative. The preparation process of this derivative is as follows: Select a specific natural amino acid as the basic raw material, chemically modify it, introduce a functional group on the side-chain amino group of lysine, and carry out the modification through the chemical reaction formula: Lys-NH2 + CH2O + HCOOH → Lys-NH-CH2-COOH, where Lys represents lysine. Then, carry out derivatization treatment on the modified amino acid, connect another functional group to the carboxyl group, and complete the derivatization through another chemical reaction formula. After the modification and derivatization treatment, the obtained amino acid residue derivative is used as the first functional group.

5. The affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, characterized in that, The distribution of the microenvironment regulation region on the ligand core skeleton follows the spatial layout principle, and the determination method of the spatial layout principle is as follows: The microenvironment regulation region is distributed at a position on the ligand core skeleton close to the end of the branched structure and relatively far from the central connection region. When the hirudin molecule binds to the ligand, the microenvironment regulation region can more directly affect the local environment where the hirudin molecule is located, and at the same time, it will not affect the overall structural stability of the ligand due to being too close to the central connection region. Through computer simulation experiments, the hirudin adsorption process under different layout schemes is simulated, and the change of adsorption efficiency under various environmental fluctuations is observed, and finally the spatial layout principle is determined.

6. The affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, characterized in that, The perception of temperature change by the external environment perception subunit is based on a thermal perception algorithm, and the formula is as follows: E 感知温度 = k3×(T - T0) 2 Among them, E 感知温度 represents the temperature perception energy value output by the environmental perception subunit, and k3 is the weight coefficient; By measuring the hirudin adsorption efficiency at different temperatures T and comparing it with the preset optimal temperature T0, the value of k3 is determined according to the influence degree of the adsorption efficiency change on the overall perception result. When the temperature change has a greater impact on the adsorption efficiency, the value of k3 is relatively large, and the small molecule substance storage and release subunit determines whether to release or absorb specific small molecule substances according to the comprehensive result of the temperature perception energy value and the pH value and ionic strength perception energy value.

7. An affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, characterized in that, The small molecule substances stored in the small molecule substance storage and release subunit also include small molecule compounds with biological activity, and the determination method of the small molecule compounds is as follows: By analyzing the interaction between hirudin and other biomolecules in the organism, it is found that the bioactive small molecule compound affects the adsorption behavior of hirudin. The bioactive small molecule compound with this function is included in the storage range of the small molecule substance storage and release subunit, and its optimal storage content and release conditions are determined through experiments according to different environmental conditions and hirudin adsorption requirements.

8. An affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, characterized in that, The connection method between the ligand and the chromatography medium adopts a method combining chemical bonding and physical adsorption. The chromatography medium is pretreated, functional groups are introduced on the surface through chemical reactions, and one end of the ligand is reacted with the epoxy group on the surface of the chromatography medium by chemical bonding to form a firm chemical bond connection.

9. An affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, characterized in that, There is an interaction relationship between multiple branched structures of the ligand skeleton, and the interaction groups include hydrogen bond formation groups and electrostatic interaction groups.

10. The affinity chromatography ligand for improving the adsorption efficiency of hirudin according to claim 2, wherein, During the preparation of the ligand, an integrated process flow is adopted for the synthesis and assembly of the components. This process flow systematically integrates the synthesis of the ligand core skeleton, the introduction of the first functional group, the construction of the microenvironment regulation region, and the connection step between the ligand and the chromatography medium. In the same reaction system, the synthesis of the ligand core skeleton and the introduction of the first functional group are carried out in sequence according to the predetermined order. By controlling the reaction conditions, the microenvironment regulation region is constructed on the basis of the formed ligand core skeleton and the first functional group. By controlling the reaction conditions, the prepared ligand is connected to the chromatography medium by a method combining chemical bonding and physical adsorption.

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