Water-soluble membrane proteins, recombinant vectors, recombinant host bacteria and their modification methods and applications

Abstract

This invention belongs to the field of protein engineering and biomedicine, and particularly relates to a water-soluble membrane protein, a recombinant vector, a recombinant host bacterium, and their modification methods and applications. The method involves the following steps: First, an interface mutant is constructed based on the SQTY code, and its water solubility and ligand binding ability are evaluated. If the requirements are not met, multiple low-impact transmembrane regions are screened, and after mutation modification, the interface mutant is introduced to construct a single-transmembrane combined mutant, whose water solubility and ligand binding ability are evaluated. If the requirements are still not met, the multiple low-impact transmembrane regions are combined in pairs, and the interface mutant is introduced to construct various double-transmembrane combined mutants, whose water solubility and ligand binding ability are evaluated, and the optimal double-transmembrane combined mutant is selected. This method rationally mutates CXCR4 in stages to achieve water solubility, minimizing changes to the protein's structure and other physicochemical properties, thereby maintaining or even enhancing its binding ability to the ligand CXCL12.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering and biomedicine, and particularly relates to a water-soluble membrane protein, a recombinant vector, a recombinant host bacterium, and its modification methods and applications. Background Technology

[0002] With the emergence of clinical needs for the prognosis and treatment of cerebral hemorrhage, targeted intervention on the hematoma microenvironment of cerebral hemorrhage, especially the blocking of abnormal activation of the CXCL12 / CXCR4 signal axis, is not only beneficial to reduce secondary brain injury caused by cerebral hemorrhage, but also helps to achieve synergistic effects of neuroprotection and neurorepair, thereby reducing the medical and economic burden on patients, families and society.

[0003] Targeted intervention systems for cerebral hemorrhage need to be optimized for greater specificity and efficiency. Traditional biomembrane biomimetic systems typically use CXCR4-rich mesenchymal stem cell membranes or neural stem cell membranes to encapsulate drugs, aiming to achieve homing intervention to the cerebral hemorrhage lesion area. However, due to the diverse types of receptor proteins on natural cell membranes, the difficulty in completely preserving and correctly presenting these receptor proteins during membrane preparation, and the instability of their expression levels, these biomimetic systems generally suffer from significant multi-effects but insufficient specificity in practical applications. In contrast, using the directly biosynthesized membrane receptor protein CXCR4 for targeted intervention in cerebral hemorrhage offers advantages such as targeted specificity and controllable action compared to traditional biomembrane biomimetic targeting systems.

[0004] CXCR4 belongs to the G protein-coupled receptor (GPCR) family and has a transmembrane α-helix structure. In its sequence, hydrophobic amino acids such as leucine, isoleucine, valine, and phenylalanine are continuously and densely distributed, forming a large area of ​​hydrophobic region. Due to its strong hydrophobicity, CXCR4 can only maintain its active conformation in the cell membrane environment and is difficult to use directly to construct targeted systems in vitro.

[0005] Although existing research has successfully achieved water-soluble modification of CXCR4 based on QTY Code technology, due to the limitations of CXCR4... QTYAll seven transmembrane regions were modified using QTY Codes. Due to the excessive modification ratio, the affinity for ligands decreased by about 5 times. For example, the design strategies of Chinese patent documents CN106459174A and CN113929766A, as well as US patent document US20230265164A1, are all "overall replacement and indiscriminate modification". They perform batch and indiscriminate replacement of hydrophobic amino acids in the complete α-helical domains (mainly transmembrane α-helices) of natural membrane proteins. That is, hydrophobic amino acids such as L, I, V, and F in the transmembrane domains are uniformly replaced with hydrophilic nonionic amino acids such as Q, T, and Y (N and S can be used to replace L, I, and V). In other words, the protein mutation is completed in one go to obtain a water-soluble variant. The only goal is to achieve water solubility and binding ability with natural ligands. As a result, the excessive modification ratio leads to a decrease in affinity for ligands by about 5 times. This also led to its application in targeted cancer therapy; however, due to the excessively high modification rate (mutation rate of 22.7%), its affinity for the ligand decreased by approximately 5-fold. It is evident that preserving affinity function can ensure CXCR4's ability to block the CXCL12 / CXCR4 axis in vivo.

[0006] Therefore, it is necessary to develop a water-soluble membrane protein, recombinant vector, recombinant host bacteria, and their modification methods and applications based on the crystal structure of the CXCR4 receptor protein, so as to improve the affinity between water-soluble (Soluble CXCR4) SCXCR4 and CXCL12 and provide new ideas for the development of novel hematoma targeted intervention systems. Summary of the Invention

[0007] The purpose of this invention is to provide a high-affinity water-soluble membrane protein, a recombinant vector, a recombinant host bacterium, and its modification method and application, which can minimize the impact on affinity during the water-soluble modification of CXCR4, retain or even enhance the original affinity, further enhance the binding affinity of CXCR4 to SDF-1 / CXCL12, achieve more efficient targeted enrichment in the cerebral hemorrhage lesion area, enhance the blocking efficacy of the CXCL12 / CXCR4 axis, effectively intervene in secondary brain injury, and ultimately achieve synergistic therapeutic effects of neuroprotection and neurorepair.

[0008] To solve the aforementioned technical problems, the present invention specifically adopts the following technical solution: The first aspect of this invention is to provide a method for modifying a high-affinity water-soluble membrane protein, the specific steps of which are: S1: Based on the crystal structure of the CXCR4 receptor protein, the spatial distribution characteristics of amino acids in its transmembrane region and at the transmembrane interface were analyzed. Then, site-directed water-soluble mutagenesis was performed on the CXCR4 transmembrane interface sites using QTY code to construct the SQTY interface mutant CXCR4. SQTY And evaluate the obtained interface mutant CXCR4SQTY If the water solubility and ligand binding ability meet the requirements, a high-affinity water-soluble membrane protein is obtained; if the requirements are not met, proceed to step S2. S2: Based on the structure-activity relationship of the interaction between CXCR4 and CXCL12, multiple transmembrane regions of CXCR4 were analyzed and ranked, and at least three transmembrane regions with the least impact on ligand binding affinity were screened out; then, QTY codes were used for mutation to obtain single transmembrane mutants; then, the single transmembrane mutants were compared with the interface mutant CXCR4. SQTY Site-directed water-soluble mutagenesis was performed to obtain a single-transmembrane co-mutant. The water solubility and ligand binding ability of the obtained single-transmembrane co-mutant were evaluated. If the requirements were met, the high-affinity water-soluble membrane protein was obtained by screening according to the screening conditions. If the requirements were still not met, the process was changed to step S3. S3: The single transmembrane mutants obtained in step S2 are combined in pairs to construct multiple combined mutants; then, the multiple combined mutants are respectively combined with the interface mutant CXCR4 obtained in step S1. SQTY Site-directed water-soluble mutagenesis was performed to construct a variety of transmembrane joint mutants. The water solubility and ligand binding ability of the obtained transmembrane joint mutants were further evaluated. The optimal transmembrane joint mutant was selected from the transmembrane joint mutants that met the requirements, which is the high-affinity water-soluble SCXCR4 mutant. The condition for meeting the requirements in steps S1, S2 and S3 is that the average total hydrophilicity coefficient of the mutant is less than 0.

[0009] Preferably, the screening conditions in steps S1, S2, and S3 are as follows: the change in binding free energy between the mutant and CXCL12 is less than that of the transmembrane mutant CXCR4. QTY or even less than the change in the binding free energy of the CXCR4 receptor protein to CXCL12 or / and the mutation rate of the mutant is less than 15%.

[0010] Preferably, the screening condition in step S1 is that the change in the binding free energy of the mutant to CXCL12 is less than that of the transmembrane mutant CXCR4. QTY It is, or even less than, the change in the binding free energy of the CXCR4 receptor protein to CXCL12.

[0011] Preferably, the screening conditions in steps S2 and S3 are: the change in binding free energy between the mutant and CXCL12 is less than that of the transmembrane variant CXCR4. QTY Even smaller than the change in the binding free energy of the CXCR4 receptor protein to CXCL12 and the mutation rate of the mutant is less than 15%.

[0012] Preferably, in steps S2 and S3, when the average total hydrophilicity coefficient (GRAVY) of the mutant is less than 0, a low mutation rate (not higher than 15%) and a change in the binding free energy (Δ) between the mutant and CXCL12 are further considered. i Screening was conducted using the condition that G) less than -15.2 kcal / mol (the change in the binding free energy of CXCR4 receptor protein to CXCL12), and finally, high-affinity water-soluble SCXCR4 mutants were selected.

[0013] Using the above technical solution, prioritizing a low mutation rate and avoiding modification of functional amino acids, the CXCR4 protein is rationally modified in stages using interfacial amino acids as a starting point for water solubility modification. This ultimately achieves water solubility of CXCR4 while minimizing changes to the protein's structure and other physicochemical properties, thereby maintaining or even enhancing its binding affinity to the ligand CXCL12. This successfully designs a high-affinity, water-soluble SCXCR4 protein. The process mainly consists of three stages. The first stage involves optimizing the interfacial amino acids. Since the water solubility of GPCR proteins is primarily related to the hydrophilicity and hydrophobicity of their interfacial amino acids, QTY site-directed mutagenesis is performed only on the interfacial amino acids. Simultaneously, the water solubility and CXCL12 binding affinity of the mutant are predicted, prioritizing initial water solubility with a low mutation rate to minimize interference with the ligand binding region. If the requirements are met, i.e., the transmembrane mutant CXCR4 is successfully created. SQTY If the average total hydrophilicity coefficient (GRAVY) is less than 0, screening is performed. The screening criteria are: the change in free energy (Δ) between the mutant and CXCL12. i G) less than -12.9 kcal / mol (CXCR4) QTY Change in binding free energy with CXCL12 (Δ) i If the free energy of binding between the natural CXCR4 receptor protein and CXCL12 is less than -15.2 kcal / mol (G), then a high-affinity water-soluble membrane protein is obtained. The second stage: If only the interface amino acid mutation fails to achieve water solubility, an interface mutant is introduced into the low-impact transmembrane region for combined mutation modification. This is not an indiscriminate modification of all transmembrane regions; rather, based on the structure-activity relationship of the CXCR4-CXCL12 interaction, the seven transmembrane regions of CXCR4 are systematically ordered, and the three transmembrane regions with the least impact on ligand binding affinity are screened out. After mutation modification, they are then combined with interface amino acids to gradually construct multiple mutants of "single transmembrane region + interface amino acid" to achieve a balance between improved water solubility and retained affinity. If the criteria are met, i.e., the total average hydrophilicity coefficient of the single transmembrane combined mutant is less than 0, then further screening is conducted based on a low mutation rate (not higher than 15%) and a free energy change of less than -15.2 kcal / mol (the free energy change of binding between the mutant and CXCL12). This results in a high-affinity water-soluble membrane protein. If the criteria are not met, the third stage of modification begins. The third stage: If the design expectations are still not met, the three transmembrane regions with low impact are combined in pairs, and interfacial amino acids are introduced for joint mutation. This gradually constructs multiple mutants of "two transmembrane regions + interfacial amino acids". If the second stage still does not meet the criteria, the scope of transmembrane region modification is gradually expanded. The water solubility and ligand binding ability of the obtained two-transmembrane joint mutants are further evaluated to determine whether the requirements are met. When the average total hydrophilicity coefficient of the obtained two-transmembrane joint mutants is less than 0, they are screened based on a low mutation rate (less than 15%) and a free energy change of less than -15.2 kcal / mol between the two-transmembrane joint mutants and CXCL12. Finally, a high-affinity water-soluble membrane protein is obtained through screening, rather than blindly modifying the entire structure.

[0014] This phased modification achieves water solubility of membrane receptor proteins under low mutation rate conditions, effectively avoiding the structural damage and functional loss problems caused by traditional whole-body water solubility modification; it maintains a high affinity for CXCL12, and its binding affinity is about 5 times higher than that of traditional high mutation rate water solubility strategies.

[0015] Preferably, the crystal structure of the CXCR4 receptor protein in step S1 is obtained from a publicly available structure database or through homology modeling and structure prediction methods. During the analysis process, molecular dynamics simulations are used to assist in the spatial conformation analysis of the transmembrane region and its interfacial amino acids, thereby improving the accuracy of spatial distribution feature analysis. Simultaneously with site-directed mutagenesis, the water solubility and CXCL12 binding affinity of the mutant are predicted to achieve initial water solubility at a low mutation rate.

[0016] Preferably, in steps S1, S2, and S3, the water solubility of the mutant is evaluated using the GRAVY index, and the evaluation results are used to determine whether the mutant meets the requirements, thereby deciding whether to proceed to the next stage of design. If the average total hydrophilicity coefficient (GRAVY) is less than 0, the requirements are met, and a mutant meeting the requirements is determined, and screening is performed. Otherwise, in steps S1 and S2, the design continues to the next stage. The GRAVY index is a quantitative value of the overall hydrophobicity / hydrophilicity of the protein calculated from the amino acid sequence, used to objectively evaluate the water solubility of the protein.

[0017] Preferably, in steps S1, S2, and S3, the change in the binding free energy (Δ) between the mutant and CXCL12 is used. i G) Assess the affinity of the mutant, when Δ i G is less than the Δ of natural CXCR4. i G (-15.2 kcal / mol) satisfies the requirement of high affinity.

[0018] Preferably, in steps S1, S2, and S3, if the average total hydrophilicity coefficient (GRAVY) is less than 0, the next stage of design is not performed. Instead, a low mutation rate (less than 15%) and a change in the free energy of the mutant binding to CXCL12 (Δ) are used. i G) Screening was conducted with a condition of less than -15.2 kcal / mol to identify high-affinity water-soluble SCXCR4 mutants.

[0019] Preferably, in step S1, the spatial distribution characteristics of amino acids at the transmembrane interface are analyzed for the seven transmembrane regions TM1-TM7, combined with the CXCR4 "barrel" conformation.

[0020] Preferably, in step S2, based on the structure-activity relationship of CXCR4 and CXCL12 binding, the influence of different transmembrane regions on ligand binding affinity is evaluated, analyzed, and ranked. The three transmembrane regions with the least influence are selected for QTY Code mutation, and the obtained mutants are then combined in pairs to construct various combined mutants. The results show that TM1, TM2, and TM5 are the three transmembrane regions with the least influence on binding affinity.

[0021] Among them, the structure-activity relationship (SAR) based on the binding of CXCR4 and CXCL12 refers to the intrinsic connection and correspondence between the molecular structure of a protein (such as the spatial arrangement of the transmembrane regions of CXCR4 and the key amino acid sites) and its biological activities (such as the binding affinity to the ligand CXCL12 and its signal transduction ability).

[0022] Preferably, the evaluation analysis in step S2 includes the distribution of key binding residues, the degree of participation of transmembrane regions in ligand binding, and their impact on overall conformational stability.

[0023] Preferably, in step S2, the contributions of the seven transmembrane regions of CXCR4 to the interaction between CXCR4 and the ligand CXCL12 are analyzed and ranked according to their contribution, thereby selecting the three transmembrane regions with the least impact on ligand binding affinity. In some specific embodiments, the three transmembrane regions with the least impact on ligand binding affinity are selected as TM1, TM2, and TM5.

[0024] Preferably, in step S3, when constructing a combined mutant using QTY Code, the overall conformation of CXCR4 and the ligand binding interface are kept basically unchanged, while the transmembrane interface and the hydrophobic amino acid in the transmembrane region are combined and mutated in stages. In step S3, when constructing the dual transmembrane joint mutant, the transmembrane region interface amino acid is used as the starting point, and the transmembrane region is gradually superimposed and expanded, thereby achieving a constrained expansion of the modification range.

[0025] Preferably, the staged combined mutation mentioned in step S3 refers to mutation sites selected from hydrophobic amino acids at the transmembrane interface and hydrophobic amino acids of TM1 and TM5. This staged mutation design strategy only modifies the interface and hydrophobic residues, thereby achieving non-destructive function while ensuring water solubility and activity.

[0026] Preferably, in step S3, QTY Code is used to perform site-specific water-soluble joint mutation modification on the above-mentioned paired sites. Starting from the interfacial amino acid, the modification ranges of TM1, TM2, and TM5 are gradually superimposed to construct a variety of joint mutants with "two transmembrane regions + interfacial sites". The water solubility and ligand binding ability of each mutant are evaluated simultaneously. Under the premise of low mutation rate, a high-affinity water-soluble SCXCR4 is finally constructed.

[0027] Preferably, the amino acid mutation rate of the obtained SCXCR4 mutant that meets the requirements is not higher than 15%, and the mutation sites are hydrophobic amino acids at the transmembrane interface and hydrophobic amino acids of TM1 and TM5.

[0028] Preferably, the mutation rate of the obtained SCXCR4 mutant is 12.8%, which is much lower than that of the CXCR4 constructed by the traditional whole-body water-soluble modification strategy. QTY The mutation rate was 22.7% to maximize the preservation of the natural structural features and biological functions of CXCR4 while achieving water solubility.

[0029] Preferably, the binding affinity of the obtained SCXCR4 mutant to CXCL12 is higher than that of CXCR4. QTYIt improves by about 5 times and is superior to the natural CXCR4 protein.

[0030] A second aspect of the present invention is to provide a high-affinity, water-soluble SCXCR4 protein, wherein the SCXCR4 protein is a mutant obtained by the method described in the first aspect of the present invention, and its amino acid sequence is shown in SEQ ID NO:1.

[0031] Preferably, the SCXCR4 protein is a recombinantly expressible functional protein that can exist stably in vitro and maintain its biological activity.

[0032] Preferably, the SCXCR4 protein has a 6×His tag introduced at its C-terminus to facilitate subsequent Ni-NTA affinity purification.

[0033] Preferably, the nucleotide sequence encoding the water-soluble membrane protein SCXCR4 is further optimized and modified to obtain the nucleotide sequence shown in SEQ ID NO:2.

[0034] A third aspect of the present invention is to provide a high-affinity, water-soluble recombinant SCXCR4 protein, which is encoded by a nucleotide sequence obtained by optimizing and modifying the nucleotide sequence encoding the SCXCR4 protein described in the second aspect of the present invention, wherein the optimized and modified nucleotide sequence is shown in SEQ ID NO: 2.

[0035] Preferably, the 5' end of the optimized and sequence-modified gene sequence of the SCXCR4 protein (as shown in SEQ ID NO: 2) is supplemented with the recognition sequence of restriction endonuclease BamHI (GGATCC) and the 3' end is supplemented with the recognition sequence of restriction endonuclease XhoI (CTCGAG) to obtain the target gene sequence of the recombinant SCXCR4 protein, as shown in SEQ ID NO: 3.

[0036] A fourth aspect of the present invention is to provide a recombinant expression vector pET-20b(+)-SCXCR4, wherein the recombinant expression vector contains the nucleotide sequence described in the third aspect of the present invention.

[0037] Preferably, the recombinant vector is a pET-20b(+) prokaryotic expression vector.

[0038] Preferably, the recombinant vector contains a nucleotide sequence encoding the SCXCR4 protein as described in SEQ ID NO: 2, which has been optimized with host codons to improve the expression efficiency of the SCXCR4 protein in the host bacteria.

[0039] A fifth aspect of the present invention is to provide a recombinant host bacterium BL21(DE3)-pET-20b(+)-SCXCR4, wherein the recombinant host bacterium comprises the recombinant expression vector described in the fourth aspect of the present invention, and the host bacterium is *Escherichia coli* BL21(DE3). The recombinant host bacterium is capable of efficiently expressing the SCXCR4 protein, and the resulting protein exists in a soluble form.

[0040] A sixth aspect of the invention relates to the use of a high-affinity, water-soluble SCXCR4 protein in the preparation of a medicament for blocking the CXCL12 / CXCR4 signaling axis.

[0041] Preferably, the drug is applied to the area of ​​cerebral hemorrhage lesion and intervenes in secondary brain injury by effectively blocking the CXCL12 / CXCR4 axis, thereby promoting the recovery of voluntary motor function after cerebral hemorrhage and improving behavioral performance.

[0042] Preferably, the drug is used for the prognostic treatment of cerebral hemorrhage, that is, a drug for targeted intervention of secondary brain injury after cerebral hemorrhage. The SCXCR4 protein in the drug reaches its peak drug content in brain tissue 2 hours after administration to the brain tissue, with a brain tissue uptake rate of 21.38±1.48%.

[0043] Preferably, the drug is a drug that promotes the recovery of voluntary motor function and improves behavioral performance after cerebral hemorrhage. After treatment with the drug (SCXCR4 protein), the recovery of voluntary motor function and improvement of behavioral performance can be effectively promoted as early as day 7 after cerebral hemorrhage.

[0044] Preferably, the drug is one that inhibits the abnormal activation of the CXCL12 / CXCR4 axis caused by cerebral hemorrhage, reduces the expression of inflammatory factors, and promotes the transformation of the immune phenotype to an anti-inflammatory / reparative type. The overall intervention effect is superior to that of CXCR4. QTY protein.

[0045] Compared with the prior art, the present invention has the following beneficial technical effects: (1) This study proposes a phased rational design strategy based on SQTY Code, and the modification logic has clear progressiveness and precision. First, the "barrel" crystal structure of the CXCR4 receptor protein is analyzed to identify the hydrophobic amino acids at the transmembrane interface, and SQTY Code is used to mutate them. If water solubility is not achieved, the subsequent steps are carried out. Second, based on the structure-activity relationship of the CXCR4-CXCL12 interaction, the low-impact transmembrane region is identified, and QTY Code mutations are introduced into it to gradually construct a "single transmembrane region + interface amino acid" mutant to achieve a balance between improved water solubility and retention of affinity. If the requirements are still not met, a "two transmembrane regions + interface amino acid" mutant is gradually constructed, that is, the scope of transmembrane region modification is gradually expanded, rather than blindly and comprehensively modified. Water solubility of membrane receptor protein is achieved under low mutation rate conditions, which effectively avoids the problems of structural damage and loss of function caused by traditional overall water solubility modification.

[0046] (2) The SCXCR4 protein constructed in this invention has an amino acid mutation rate of only 12.8% and still maintains a high affinity for CXCL12. Its binding affinity is about 5 times higher than that of the traditional high mutation rate water solubility strategy, thus achieving synergistic optimization of water solubility and biological function.

[0047] (3) The SCXCR4 protein provided by the present invention is a soluble functional receptor protein that can be stably and efficiently expressed in a prokaryotic expression system, which reduces the dependence of membrane protein preparation on complex eukaryotic systems and membrane biomimetic systems, and significantly improves preparation efficiency and reproducibility.

[0048] (4) The SCXCR4 protein provided by the present invention can be used for targeted intervention of secondary brain injury after cerebral hemorrhage. By blocking or regulating the CXCL12 / CXCR4 signal axis, it helps to inhibit the abnormal recruitment of inflammatory cells and improve the lesion microenvironment. It can effectively promote the recovery of spontaneous motor function and the improvement of behavioral performance after cerebral hemorrhage as early as the 7th day after cerebral hemorrhage. Thus, it provides a safe, efficient and targeted technical solution for the prognosis and treatment of cerebral hemorrhage. Attached Figure Description

[0049] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0050] Figure 1The above are the SDS-PAGE results of the recombinant SCXCR4 protein expressed by the vector in this invention. Figure 2 The image shows the results of micro-thermal surge (MST) detection of the SCXCR4 recombinant protein of this invention. Figure 3A This invention explores the contact angle analysis of CXCR4. QTY CXCR4, which differs from the recombinant SCXCR4 protein in its hydrophilicity. QTY Representative images of the contact angle of SCXCR4; Figure 3B CXCR4 in this invention QTY Quantitative statistical analysis of contact angle with SCXCR4; Figure 4A This invention uses an SD rat model of cerebral hemorrhage to investigate CXCR4. QTY Representative fluorescence distribution images of rat brain tissue in each group after drug administration, in the difference in targeting efficacy with recombinant SCXCR4 protein; Figure 4B This invention uses an SD rat model of cerebral hemorrhage to investigate CXCR4. QTY Quantitative analysis of fluorescence intensity in rat brain tissue after administration in groups that showed differences in targeting efficacy with recombinant SCXCR4 protein; Figure 5A This invention provides a quantitative statistical analysis of the fluorescence intensity of major organs and brain tissue in an SD rat model of cerebral hemorrhage 2 hours after drug administration. Figure 5B This invention analyzes the proportion of in vitro fluorescence signal in brain tissue to the total signal of major organs in a rat model of cerebral hemorrhage 2 hours after drug administration. Figure 6A In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY Representative immunofluorescence micrographs of CXCR4 in brain tissue of each experimental group 3 days after treatment with recombinant SCXCR4 protein. The scale bar of the Merge image is 100 μm and the scale bar of the Enlarge image is 25 μm. Figure 6B In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY Quantitative analysis of fluorescence intensity of CXCR4 in brain tissue of each experimental group 3 days after treatment with recombinant SCXCR4 protein; Figure 6C In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTYRepresentative immunofluorescence micrographs of CXCR12 in brain tissue of each experimental group 3 days after treatment with recombinant SCXCR4 protein. The scale bar of the Merge image is 100 μm, and the scale bar of the Enlarge image is 25 μm. Figure 6D In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY Quantitative analysis of fluorescence intensity of CXCR12 in brain tissue of each experimental group 3 days after treatment with recombinant SCXCR4 protein; Figure 7A In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY IL-6 expression levels in the brain tissue surrounding hematoma in rats of each group after 3 days of treatment with recombinant SCXCR4 protein; Figure 7B In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY TNF-α expression levels in the brain tissue surrounding hematoma in rats of each group after 3 days of treatment with recombinant SCXCR4 protein; Figure 7C In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY IL-1β expression levels in brain tissue surrounding hematoma in rats of each group after 3 days of treatment with recombinant SCXCR4 protein; Figure 8A In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY Representative immunofluorescence microscopic images of CD86 in the brain hemorrhage lesion area of ​​rats in each group after 3 days of treatment with recombinant SCXCR4 protein. The scale bar of the Merge image is 100 μm, and the scale bar of the Enlarge image is 25 μm. Figure 8B In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY Quantitative analysis of CD86 fluorescence intensity in brain tissue of rats in each group after 3 days of treatment with recombinant SCXCR4 protein; Figure 9A In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY Representative immunofluorescence microscopic images of CD206 in the brain hemorrhage lesion area of ​​rats in each group after 3 days of treatment with recombinant SCXCR4 protein. The scale bar of the Merge image is 100 μm, and the scale bar of the Enlarge image is 25 μm. Figure 9B In this invention, CXCR4 was administered to a rat model of SD intracerebral hemorrhage. QTY Quantitative analysis of CD206 fluorescence intensity in brain tissue of rats in each group after 3 days of treatment with recombinant SCXCR4 protein; Figure 10 H&E staining evaluation of brain tissue after cerebral hemorrhage modeling and treatment according to the present invention, scale bar is 200μm; Figure 11A This is a schematic diagram of the apparatus for conducting an open field experiment after rat modeling according to the present invention; Figure 11B The present invention provides CXCR4 respectively QTY Representative activity heatmaps of rats on days 1-7 after cerebral hemorrhage modeling, 3 days after treatment with recombinant SCXCR4 protein, with colors transitioning from blue (low-frequency activity) to red (high residence time); Figure 11C The present invention provides CXCR4 respectively QTY Statistical analysis of the mean velocity of rats on days 1-7 after cerebral hemorrhage modeling 3 days after treatment with recombinant SCXCR4 protein; Figure 11D The present invention provides CXCR4 respectively QTY Statistical analysis of the total movement distance of rats on days 1-7 after cerebral hemorrhage modeling, 3 days after treatment with recombinant SCXCR4 protein. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0052] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0053] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0054] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4%, more typically + / -3%, more typically + / -2%, even more typically + / -1%, even more typically + / -0.5%.

[0055] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0056] Definition of the noun: QTY Code (QTY encoding / QTY password) is a protein engineering technology proposed by MIT in 2018. Its core is to replace hydrophobic amino acids with hydrophilic amino acids, transforming hydrophobic membrane proteins into water-soluble proteins while retaining their structure and function.

[0057] SQTY (Surface QTY) precisely limits the QTY Code replacement rules to the interface residues of protein-protein / protein-ligand, replacing hydrophobic amino acids at the transmembrane interface rather than the entire chain or transmembrane region. The core is to only change the interface, not the core, thereby regulating affinity / water solubility / stability.

[0058] Tryptone: peptone; Tris: Tris(hydroxymethyl) aminomethane; EDTA: Ethylenediaminetetraacetic acid. Tris-HCl: Tris-hydroxymethylaminomethane hydrochloride buffer; SDS: Sodium dodecyl sulfate; PMSF: Benzyl sulfonyl fluoride; SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis; Triton X-100 is a nonionic surfactant; Tween-20: Polysorbate 20 / Polyoxyethylene dehydrated sorbitan monolaurate; β-ME: β-mercaptoethanol; IPTG (isopropyl-β-D-thiogalactoside): It is the most commonly used protein expression inducer in molecular biology. Its core function is to precisely initiate the expression of exogenous genes regulated by the lac operon in prokaryotic systems (such as Escherichia coli).

[0059] ddH2O: Abbreviation for double-distilled water, which refers to high-purity laboratory water obtained through two distillations. It can effectively remove non-volatile impurities, some ions and microorganisms, and is often used in experimental scenarios where high water purity is required. Δ i G: The full name is interface solvation free energy gain, which refers to the change in solvation free energy when the interface is formed, that is, the change in binding free energy. It is the core indicator for measuring the binding stability. A negative value usually indicates that the interaction has thermodynamic spontaneity. The more negative the value, the stronger the binding stability of the protein complex and the higher the intermolecular affinity.

[0060] Example: The method for modifying this high-affinity water-soluble membrane protein includes the following steps: S1: Based on the crystal structure of the CXCR4 receptor protein, the spatial distribution characteristics of amino acids in its transmembrane region and at the transmembrane interface were analyzed. Then, site-directed water-soluble mutagenesis was performed on the CXCR4 transmembrane interface sites using QTY code to construct the SQTY interface mutant CXCR4. SQTY And evaluate the obtained interface mutant CXCR4 SQTY If the water solubility and ligand binding ability do not meet the requirements, proceed to step S2; The crystal structure of the CXCR4 receptor protein in step S1 is obtained from a public structure database or through homology modeling and structure prediction methods. During the analysis, molecular dynamics simulations are used to assist in the analysis of the spatial conformation of amino acids in the transmembrane region and its interface, so as to improve the accuracy of the spatial distribution characteristics analysis. In step S1, while performing site-directed mutagenesis, the water solubility and binding affinity of the mutant to CXCL12 are predicted to achieve preliminary water solubility at a low mutation rate. The screening criteria in step S1 are: the change in binding free energy between the mutant and CXCL12 is less than that of the transmembrane mutant CXCR4. QTY This is, or even less than, the change in the binding free energy of the CXCR4 receptor protein to CXCL12; In step S1, the spatial distribution characteristics of amino acids at the transmembrane interface are analyzed for the seven transmembrane regions TM1-TM7, combined with the CXCR4 "barrel" conformation.

[0061] S2: Based on the structure-activity relationship of the interaction between CXCR4 and CXCL12, multiple transmembrane regions of CXCR4 were analyzed and ranked, and at least three transmembrane regions with the least impact on ligand binding affinity were screened out; then, QTY codes were used for mutation to obtain single transmembrane mutants; then, the single transmembrane mutants were compared with the interface mutant CXCR4. SQTY Perform site-directed water-soluble mutagenesis to obtain a single-transmembrane combined mutant, and evaluate the water solubility and ligand binding ability of the obtained single-transmembrane combined mutant. If the requirements are still not met, proceed to step S3. In step S2, based on the structure-activity relationship of CXCR4 and CXCL12 binding, the influence of different transmembrane regions on ligand binding affinity is evaluated and ranked. The three transmembrane regions with the least influence are selected for QTY Code mutation, and the resulting mutants are then combined in pairs to construct various combined mutants. The results show that TM1, TM2, and TM5 are the three transmembrane regions with the least influence on binding affinity.

[0062] In some specific embodiments, the analysis in step S2 includes the distribution of key binding residues, the degree of involvement of transmembrane regions in ligand binding, and their impact on overall conformational stability.

[0063] In some specific embodiments, step S2 involves analyzing the contribution of the seven transmembrane regions of CXCR4 to the interaction between CXCR4 and the ligand CXCL12, ranking them according to their contribution, and then selecting the three transmembrane regions with the least impact on ligand binding affinity. In some specific embodiments, the three transmembrane regions with the least impact on ligand binding affinity are identified as TM1, TM2, and TM5.

[0064] S3: The single transmembrane mutants obtained in step S2 are combined in pairs to construct multiple combined mutants; then, the multiple combined mutants are respectively combined with the interface mutant CXCR4 obtained in step S1. SQTY Site-directed water-soluble mutagenesis was performed to construct a variety of transmembrane joint mutants. The water solubility and ligand binding ability of the obtained transmembrane joint mutants were then evaluated to screen out the optimal transmembrane joint mutant, which is the high-affinity water-soluble SCXCR4 mutant. The criteria for satisfying the requirements in steps S1, S2 and S3 are: the average total hydrophilicity coefficient of the mutant is less than 0. The screening criteria in steps S2 and S3 are as follows: the change in binding free energy between the mutant and CXCL12 is less than that of the transmembrane variant CXCR4. QTY Even smaller than the change in the binding free energy of the CXCR4 receptor protein to CXCL12 and the mutation rate of the mutant is less than 15%.

[0065] In step S3, when constructing a combined mutant using QTY Code, the overall conformation of CXCR4 and the ligand binding interface are kept basically unchanged, while the transmembrane interface and the hydrophobic amino acid in the transmembrane region are combined and mutated in stages. In step S3, when constructing the dual transmembrane joint mutant, the transmembrane region interface amino acid is used as the starting point, and the transmembrane region is gradually superimposed and expanded, thereby achieving a constrained expansion of the modification range. The staged combined mutation mentioned in step S3 refers to mutation sites selected from hydrophobic amino acids at the transmembrane interface and hydrophobic amino acids of TM1 and TM5. This staged mutation design strategy only modifies the interface and hydrophobic residues, thereby achieving non-destructive function while ensuring water solubility and activity.

[0066] In some specific embodiments, in step S3, QTY Code is used to perform site-specific water-soluble joint mutation modification on the above-mentioned paired sites. Starting from the interface amino acid, the modification range of TM1, TM2, and TM5 is gradually superimposed to construct a variety of joint mutants with "two transmembrane regions + interface sites" in sequence. The water solubility and ligand binding ability of each mutant are evaluated simultaneously. Under the premise of low mutation rate, a high-affinity water-soluble SCXCR4 is finally constructed.

[0067] In some specific embodiments, the amino acid mutation rate of the obtained SCXCR4 mutant that meets the requirements is not higher than 15%, and the mutation sites are hydrophobic amino acids at the transmembrane interface and hydrophobic amino acids of TM1 and TM5.

[0068] In some specific embodiments, the mutation rate of the obtained SCXCR4 mutant was 12.8%, which is much lower than that of the CXCR4 constructed by the traditional whole-body water-soluble modification strategy. QTY The mutation rate was 22.7% to maximize the preservation of the natural structural features and biological functions of CXCR4 while achieving water solubility.

[0069] In some specific embodiments, the binding affinity of the obtained SCXCR4 mutant to CXCL12 is compared to that of CXCR4. QTY It improves by about 5 times and is superior to the natural CXCR4 protein.

[0070] In steps S1, S2, and S3, the water solubility of the mutant is assessed using the GRAVY index. Based on the assessment results, it is determined whether the mutant meets the requirements, thus deciding whether to proceed to the next stage of design. The GRAVY index is a quantitative value of the overall hydrophobicity / hydrophilicity of the protein calculated from the amino acid sequence, used to objectively evaluate the strength of the protein's water solubility.

[0071] In some specific embodiments, the water solubility of the mutant is evaluated by the GRAVY index in steps S1, S2 and S3, and the mutant is determined to meet the requirements based on the evaluation results, thereby deciding whether to proceed to the next stage of design; if the total average hydrophilicity coefficient (GRAVY) is less than 0, it is considered to meet the requirements, and it is determined that a mutant that meets the requirements has been obtained; otherwise, the next stage of design is proceeded in steps S1 and S2.

[0072] Preferably, in steps S1, S2, and S3, if the average total hydrophilicity coefficient (GRAVY) is less than 0, the next stage of design is not performed. Instead, the design is based on the change in the binding free energy (Δ) between the mutant and CXCL12. i G) Using less than -15.2 kcal / mol as the screening condition, high-affinity water-soluble SCXCR4 mutants were screened out.

[0073] In some specific embodiments, when the average total hydrophilicity coefficient (GRAVY) is less than 0 in steps S2 and S3, the next stage of design is not performed. Instead, a low mutation rate (less than 15%) and a change in the free energy of the mutant binding to CXCL12 (Δ) are used. i G) Using less than -15.2 kcal / mol as the screening condition, high-affinity water-soluble SCXCR4 mutants were screened out.

[0074] The design and expression of high-affinity, water-soluble SCXCR4 protein, the detection of affinity and hydrophilicity of recombinant SCXCR4 protein, and its applications are discussed in detail below.

[0075] The materials used are as follows: Strains: Escherichia coli BL21(DE3) competent cells were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0076] Carrier: pET The 20(+) plasmid is an E. coli expression vector purchased from Sangon Biotech (Shanghai) Co., Ltd. The promoter of this plasmid is T7, the vector size is 3586 bp, and the vector is resistant to ampicillin.

[0077] 10×T4 DNA ligase buffer: Weigh 500 mmol / L Tris-HCl, 100 mmol / L MgCl2, 100 mmol / L DTT, and 10 mmol / L ATP. Dissolve thoroughly in deionized water (ddH2O). Adjust the pH to 7.8 with NaOH (at 25℃). Make up to the corresponding volume, then filter through a 0.22 μm filter membrane for sterilization. Aliquot and store at -20℃ in the dark for later use. The LB solid and liquid media used in the following examples were all prepared in the laboratory, and their formulas are as follows: LB liquid medium: Weigh 5.0 g / L yeast extract, 10.0 g / L NaCl and 10.0 g / L Tryptone, add deionized water (ddH2O) to completely dissolve and make up to volume, and sterilize at 121℃ and 0.1 MPa for 15-30 min; Ampicillin stock solution (1000X): Weigh 5.0 g of ampicillin sodium, dissolve in deionized water (ddH2O) and bring the volume to 50 mL to prepare a 100 mg / mL (1000X) stock solution. After sterilization by filtration through a 0.22 μm membrane, aliquot and store at -20°C protected from light for later use. LB solid medium: Weigh 5.0 g / L yeast extract, 10.0 g / L NaCl, 10.0 g / L Tryptone, and 15.0 g / L agar. Dissolve and bring to volume with deionized water (ddH2O). Autoclave at 121℃ and 0.1 MPa for 15-30 min. After cooling to 50-60℃, add ampicillin stock solution (1000X) to a final concentration of 1X. Mix well, pour into plates, and allow to solidify at room temperature for later use. IPTG stock solution (1M, 1000X): Weigh 238.3 mg / mL of IPTG, add deionized water (ddH2O) to completely dissolve and bring to volume, filter sterilize using a 0.22 μm sterile filter, and store in aliquots at -20℃. Lysis buffer: Weigh 6.1 mg / mL Tris and 17.5 g / L NaCl, and measure 10 mL / L Tween-20. Add deionized water (ddH2O) to completely dissolve and make up to volume. Adjust the pH to 8.0 and store at 4℃ for later use. Buffer 1: Weigh 6.1 g / L Tris, 2.9 g / L NaCl, 1.1 g / L CaCl2 and 120 g / L urea, and measure 1 mL / L of Tritium X-100. Add deionized water (ddH2O) to completely dissolve and bring to volume. Adjust the pH to 7.4 and store at room temperature. Buffer 2: Weigh 6.1 g / L Tris, 58.4 g / L NaCl, 1.1 g / L CaCl2 and 120 g / L urea, and measure 1 mL / L of Tritium X-100. Add deionized water (ddH2O) to completely dissolve and bring to volume. Adjust the pH to 7.4 and store at room temperature. Binding solution: Weigh 6.1 g / L Tris, 8.8 g / L NaCl and 480.5 g / L urea, add 1.4 mL / L β-ME using a pipette, add deionized water (ddH2O) to completely dissolve and make up to volume, adjust the pH to 8.0, and store at room temperature; Refolding buffer: Prepare fresh before use. Weigh 6.1 g / L Tris, 0.9 g / L reduced glutathione, 0.6 g / L oxidized glutathione, 1.5 g / L EDTA and 87.1 g L-arginine, add deionized water (ddH2O) to completely dissolve and bring to volume. Adjust the pH to 9.0 and store at 4°C for later use. Dialysis buffer: Weigh 6.1 g / L Tris, 1.5 g / L EDTA and 87.1 g L-arginine, add deionized water (ddH2O) to completely dissolve and make up to volume, adjust the pH to 9.0, and store at 4℃ for later use; Tissue lysis buffer: Weigh 50 mM Tris-HCl, 0.9% NaCl, 0.1% SDS and 1 mM PMSF (add fresh before use), add deionized water (ddH2O) to dissolve completely, and adjust the pH to 7.5.

[0078] 5×Loading Buffer: Weigh 1.25 mL of 1.0 mol / L Tris-HCl (pH 6.8), 0.5 g of SDS, 25 mg of bromophenol blue, 2.5 mL of glycerol, and 250 μ L of β-ME. Dissolve them thoroughly in deionized water (ddH2O) and bring the volume to 5 mL. Aliquot the solution and store at -20 °C protected from light for later use.

[0079] The rats used in the following examples were SD rats, purchased from Chongqing Enswell Biotechnology Co., Ltd.

[0080] Unless otherwise specified in this application, all experimental reagents are conventional reagents in the art, which can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; all experimental methods not specifically specified are conventional methods in the art, and relevant experimental manuals, such as the Molecular Cloning Experimental Manual or the instructions of relevant reagent manufacturers, can be consulted.

[0081] Example 1: Design and expression of high-affinity water-soluble SCXCR4 protein 1. Protein design The inventors constructed a high-affinity, water-soluble SCXCR4 protein using a phased, rationally designed mutation strategy, and introduced a 6×His tag at the C-terminus of the protein to facilitate subsequent Ni-NTA affinity purification. The amino acid sequence of SCXCR4 is shown in SEQ ID NO: 1, and it is 358 amino acids in length.

[0082] The amino acid sequence of the high-affinity, water-soluble SCXCR4 protein is shown in SEQ ID NO: 1: MEGISIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKTYQPTTYSTTYQTGTTGNGQTTQTMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWATDAVANWYFGNFLCKAVHVTYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYTGVWTPALQLTTPDFTFANVSE ADDRYICDRFYPNDLWTTTYQYQHTMTGQTQPGTTTQSCYCIIISKLSHSKGHQKRKALKTTTILIQAFFACWLPYYTGISTDSFILLEIIKQGCEFENTVHKWISITEAQAFYHCCLNPTLYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSSHHHHHH.

[0083] 2. Construction of expression carrier The amino acid sequence of the high-affinity, water-soluble SCXCR4 protein was translated into its corresponding nucleotide sequence. Based on the amino acid sequence of the SCXCR4 protein, the resulting nucleotide sequence was optimized using E. coli codons, and bases were added to optimize the gene sequence structure to improve expression efficiency in the host. The final high-affinity, water-soluble SCXCR4 protein coding gene sequence, shown in SEQ ID NO: 2, is 1087 bases in length. A restriction endonuclease BamHI recognition sequence (GGATCC) was added to the 5' end of the SCXCR4 coding gene sequence, and a restriction endonuclease XhoI recognition sequence (CTCGAG) was added to the 3' end, resulting in the target gene sequence of the recombinant SCXCR4 protein, shown in SEQ ID NO: 3. The target gene sequence was then synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0084] The nucleotide sequence (1087 bp) of the high-affinity, water-soluble SCXCR4 protein is shown in SEQ ID NO: 2.

[0085] The target gene sequence (1099 bp) of the SCXCR4 recombinant protein is shown in SEQ ID NO: 3:

[0086] The pET-20b(+) plasmid was used as the expression vector. The pET-20b(+) plasmid and the synthesized target gene were double-digested using restriction endonucleases BamHI and XhoI, respectively. The digestion system (20 μL) consisted of: 16 μL ddH2O, 2 μL 10×T4 DNA ligase buffer, 1 μL of the empty pET-20b(+) plasmid to be digested, 0.5 μL of restriction endonuclease BamHI, and 0.5 μL of XhoI. Digestion conditions: 37℃ for 3 h, followed by inactivation at 80℃ for 20 min.

[0087] The digested pET-20b(+) plasmid and target gene were recovered separately. The target gene was ligated into the pET-20b(+) vector using T4 DNA ligase. Ligation system (20 μL): 2 μL 10×T4 DNA ligase buffer, 0.2 μL T4 DNA ligase, 3 μL pET-20b(+), 1 μL target gene, and ddH2O to a final volume of 20 μL. Ligation conditions: overnight at 16°C.

[0088] The ligation product was transformed into an *E. coli* DH5α clone strain using a heat shock method. The transformation method is as follows: Remove DH5α competent cells from the -80℃ freezer and place them on ice for about 5 minutes to allow the glycerol to fully thaw. Then, add an appropriate amount of ligation product to the competent cells, gently pipette 3-4 times to mix, and incubate on ice for 30 minutes. After drying the outer wall of the centrifuge tube, heat-shock the sample at 42℃ for 90 seconds, then immediately transfer it back to an ice bath to cool for 2 minutes. Under aseptic conditions, add 800 μL of LB liquid medium to the transformation system and incubate at 37℃ and 150 rpm for 45 minutes. After incubation, collect the cells by centrifugation at 8000 rpm for 5 minutes, discard some of the supernatant, resuspend the cells in about 100 μL of LB liquid medium, and evenly spread them on LB solid medium containing 100 μg / mL ampicillin. Invert the culture dishes and incubate at 37℃ for 10-16 hours.

[0089] Single colonies were picked and inoculated into LB liquid medium containing 100 μg / mL ampicillin. After shaking culture at 37°C for 10-16 h, the culture was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis to identify positive clones.

[0090] The vector of the positive clone with the correct sequence was extracted using a plasmid extraction kit (Omega, D6943-01) according to the kit instructions, resulting in the recombinant vector pET-20b(+)-SCXCR4.

[0091] 3. Construction of recombinant host bacteria The concentration of the recombinant vector pET-20b(+)-SCXCR4 was adjusted to 100 ng / μL for later use. E. coli BL21(DE3) competent cells stored at -80℃ were thawed on ice. 50 μL of competent cells were added to a pre-chilled centrifuge tube, along with 3 μL of the vector solution. The mixture was gently mixed and incubated on ice for 10 min. The centrifuge tube was then heat-shocked in a 42℃ water bath for 60-90 s and immediately returned to ice for 3 min. 600 μL of antibiotic-free LB liquid medium was added to the transformation system, and the cells were incubated at 37℃ and 200 rpm for 1 h. After incubation, 50-100 μL of the bacterial culture was evenly spread onto LB solid medium containing ampicillin (1×) and incubated at 37℃ for 12-16 h. Single colonies were picked and inoculated into LB liquid medium containing ampicillin (1×) and incubated overnight at 37℃ and 120 rpm for 12-16 h. When the bacterial solution OD 600 When the concentration reaches 0.6-0.8, the bacterial culture is mixed with 50% glycerol at a volume ratio of 1:1 to prepare glycerol bacteria, and stored at -80℃ for later use.

[0092] 4. Expression, purification, and refolding of recombinant SCXCR4 protein Expression of SCXCR4 recombinant protein: 30 μL of recombinant host bacteria preserved in glycerol was inoculated into 30 mL of LB liquid medium containing ampicillin (1×) and cultured at 37℃ and 120 rpm with shaking for 12–16 h. OD of the bacterial culture was then measured. 600 Once the bacterial growth reaches 0.6-0.8, it is then inoculated onto 3L of LB liquid medium containing ampicillin (1×) and cultured at 37℃ and 200rpm for 4 hours. When the OD of the bacterial culture reaches 0.6-0.8... 600 When the concentration reaches 0.6-0.8 again, add IPTG stock solution (1M, 1000×) to make the final concentration 1mM, and induce expression at 37℃ and 200rpm for 4h. After induction, centrifuge at 4℃ and 8000×g for 5min, discard the supernatant, and collect the bacterial cells.

[0093] Extraction and purification of SCXCR4 recombinant protein: Collected bacterial cells were resuspended in an appropriate amount of lysis buffer and homogenized using a high-pressure homogenizer at 700-800 Pa. The lysed solution was centrifuged at 4℃ and 8000×g for 5 min, the supernatant was discarded, and the precipitate inclusion bodies were collected. The inclusion bodies were washed sequentially with Buffer 1, Buffer 1, Buffer 2, and Buffer 1 again, with each wash performed by centrifugation at 4℃ and 11500 rpm for 25 min at 4℃. The supernatant was discarded, and the precipitate was retained. The final precipitate was added to binding buffer, thoroughly resuspended, and incubated overnight at 4℃ for 12-16 h. Subsequently, purification was performed using Ni-NTA affinity chromatography: After pre-equilibration of Ni-NTA beads, they were shaken and bound to the sample at 4℃ for 2 h. The samples were then subjected to abnormal washing with imidazole-containing binding buffers at concentrations of 0 mM, 10 mM, and 20 mM to remove non-specific proteins. Finally, the target protein was eluted with binding buffer containing 250 mM imidazole, and the eluent was collected.

[0094] Refolding of SCXCR4 recombinant protein: The concentration of eluted protein was determined using the BCA method, and the protein concentration was adjusted to 1-3 mg / mL using binding buffer. The purified protein was placed in a dialysis bag with a 10 kDa molecular weight cutoff and dialyzed in refolding buffer at 4°C for 12-16 h. The dialysis buffer was then replaced, and dialyzing continued for 24 h, with the dialysis buffer replaced twice during this period. Afterward, the protein was transferred to L-arginine buffer prepared with PBS (pH 7.5) and dialyzed for another 24 h at 4°C. After dialysis, the protein was centrifuged at 11500 rpm for 25 min at 4°C. The supernatant was collected, and the precipitate was discarded to obtain correctly folded SCXCR4 recombinant protein.

[0095] 5. SDS-PAGE detection of proteins Prepare an SDS-PAGE protein electrophoresis gel with a separating gel concentration of 10% and a stacking gel concentration of 5%, as follows: Separating gel 10% (10 mL): 3.3 mL of 30% acrylamide solution, 4 mL of ddH2O, 2.5 mL of 1.5 M Tris (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate solution, and 0.004 mL of TEMED.

[0096] 5% (5 mL) stacking gel: 0.83 mL 30% acrylamide solution, 3.4 mL ddH2O, 0.63 mL 1.5 M Tris (pH 6.8), 0.05 mL 10% SDS, 0.05 mL 10% ammonium persulfate solution, 0.005 mL TEMED.

[0097] Sample preparation: Take 20 μL of purified protein solution, add 5× Loading Buffer, vortex to mix, and place in a 100℃ metal bath for 10 min to denature the protein. After denaturation, centrifuge and load 10 μL of the sample. Electrophoresis parameters: constant voltage 70V, 120V after entering the separating gel.

[0098] SDS-PAGE results are as follows Figure 1 As shown, from Figure 1 The results show that the SCXCR4 protein was successfully expressed, and the molecular weight of the recombinant SCXCR4 protein is approximately 40 kDa, which is consistent with the expected protein molecular weight.

[0099] Example 2: Determination of the affinity and hydrophilicity of SCXCR4 recombinant protein 1. SCXCR4 recombinant protein affinity assay Micro-thermophoretic kinetics (MST) assay: CXCL12 protein was labeled with NHS-fluorescent dye at a molar ratio of 1:5-1:10. After desalting or dialysis, the concentration and labeling rate were measured. Unlabeled SCXCR4 protein was diluted to 10-15 concentration gradients from 10 nM to 10 μM, and labeled protein was diluted to 1-10 nM. The two were mixed 1:1 and incubated. After setting the instrument temperature to 25℃ and other parameters, the sample was injected into the capillary to measure the thermophoretic kinetic signal. The data was processed using the instrument software, the normalized fluorescence signal change was calculated, and the Kd value was obtained by nonlinear least squares fitting. The experiment was repeated three times, and the average value and standard deviation were used to verify the results. The mutation rate of the recombinant SCXCR4 protein was controlled at 12.8%, and the total average hydrophilicity coefficient (GRAVY value) was further reduced to -0.27, thus achieving a significant leap in hydrophilicity. Simultaneously, the affinity of SCXCR4 for binding to the ligand CXCL12 was detected, and the results are as follows: Figure 2 As shown, the dissociation constant (Kd) of SCXCR4 and CXCL12 is 3.4 ± 1.0 nM. This is significantly higher than the reported values ​​for natural CXCR4 (~5 nM) and CXCR4... QTY (17.3±4.2 nM), SCXCR4, after achieving receptor water solubility, still exhibited a significantly enhanced CXCL12 binding affinity, compared to CXCR4. QTY The binding affinity is increased by about 5 times, and its binding ability is even slightly better than that of natural CXCR4 (Table 1 and Table 2).

[0100] Table 1. Recombinant SCXCR4 protein, native CXCR4, and CXCR4 QTY Affinity parameters when combined with CXCL12 Table 2 Recombinant SCXCR4 protein, native CXCR4 and CXCR4 QTY Changes in free energy of combination with CXCL12 2. Hydrophilicity detection of SCXCR4 recombinant protein Contact angle detection: The lyophilized recombinant protein powder was ground, evenly spread on the surface of a glass slide, and flattened and fixed. After leveling the stage, a drop of deionized water was dropped onto the sample surface using the static drop method. An image was acquired immediately after the droplet contacted the sample surface. The contact angle was calculated by fitting the droplet profile using ImageJ, and the average of the contact angles on both sides was taken as the result of a single measurement. Each sample was measured at least three times at different locations. The results are as follows: Figure 3A and Figure 3B As shown, Figure 3A It can be seen that, compared with SCXCR4, CXCR4 QTY The water droplets spread out larger and have a smaller contact angle, from Figure 3B Quantitative statistical analysis of the contact angle also shows that SCXCR4 has a larger contact angle.

[0101] Example 3: SCXCR4 protein was used as a CXCL12 / CXCR4 inhibitor for targeted intervention therapy of cerebral hemorrhage.

[0102] 1. Establishment of a model of cerebral hemorrhage A rat model of intracerebral hemorrhage (ICH) was established using type IV collagenase induction. The specific steps were as follows: Healthy male SD rats (300-320g) were selected, anesthetized, and fixed on a stereotaxic instrument. They were placed on a heated pad to maintain body temperature (approximately 37℃). Hair on the head was shaved and routinely disinfected. A 1cm incision was made in the midline of the scalp to expose the skull. Using the anterior fontanelle as a reference point, a hole was drilled 0.2mm posterior to the anterior fontanelle and 3.5mm lateral to the right side using a skull drill. Guided by the stereotaxic instrument, the tip of a micro-injection needle was aligned with the center of the hole. After gently touching the dura mater as the zero point, the needle was slowly inserted vertically to a depth of 5.5mm (DV), with the tip positioned in the right basal ganglia region. 0.6U of type VII collagenase solution was slowly and evenly injected. After injection, the needle was left in place for 5-10 minutes to reduce backflow and leakage, then slowly withdrawn. The skull hole was sealed with bone wax, and the scalp incision was sutured. The animals were fed normally after recovery.

[0103] 2. Small animal live imaging The recombinant protein was labeled with Cy5.5 fluorescent dye, and in vivo near-infrared fluorescence imaging was performed on small animals. The specific steps are as follows: Following the reagent instructions, Cy5.5 dye (excitation wavelength 680 nm, emission wavelength 710 nm) was dissolved in DMSO to prepare a 10 mM stock solution. After thorough dissolution, it was aliquoted and stored at -40°C, avoiding repeated freeze-thaw cycles during use. The working solution was prepared according to the instructions, mixing Cy5.5 and protein at a molar ratio of dye:protein = 10:1. The mixture was reacted at room temperature in the dark for 1 hour, gently inverting and mixing every 15 minutes during the reaction to avoid vigorous shaking / vortexing and reduce the risk of protein inactivation and aggregation. After labeling, the mixture was dialyzed at 4°C in the dark to remove unlabeled dye molecules and then sterilized using a 0.22 μm filter. A type VII collagenase-induced cerebral hemorrhage model was established in SD rats. Cy5.5-labeled protein was injected via tail vein 24 hours after modeling, with the dosage calculated based on rat body weight, at 0.375 mg / kg of Cy5.5-labeled protein injected via tail vein. The drug uses a single Cy5.5-labeled protein as the active ingredient. The drug solution concentration is 0.075 mg / mL, which is prepared according to the routine injection volume of 5 mL / kg in mouse tail veins. That is, the drug solution containing 0.375 mg is injected at a concentration of 0.075 mg / mL.

[0104] The experiment was divided into three groups: sham surgery + CXCR4. QTY Treatment group (sham surgery modeling and CXCR4 injection) QTY Protein therapy, namely Sham+CXCR4 QTY Group), cerebral hemorrhage + CXCR4 QTY Protein therapy group (brain hemorrhage modeling and CXCR4 injection) QTY Protein-based treatment, namely ICH+CXCR4 QTY The group consists of two groups: the cerebral hemorrhage + SCXCR4 protein treatment group (cerebral hemorrhage modeling and SCXCR4 protein injection for treatment, i.e., the ICH + SCXCR4 group).

[0105] At 1, 2, 4, and 6 hours post-injection, images were taken using the PerkinElmer IVIS LuminaIII small animal in vivo imaging system to detect the mean radiant efficiency (Avg.) signal intensity in the brains of SD rats. Representative fluorescence distribution images of the brain tissue of each group after drug administration are shown below. Figure 4A As shown; quantitative analysis of fluorescence intensity in brain tissue is as follows. Figure 4B As shown. Figure 4A The results showed that at a dosage of 0.375 mg / kg, CXCR4 QTY Both SCXCR4 and other proteins can achieve homing to the lesion area of ​​cerebral hemorrhage. Figure 4BFurther quantitative analysis showed that the ICH+SCXCR4 group reached the peak signal intensity in the brain 2 hours after drug administration, with an average radiant efficiency (Avg.) of (6.996±1.083)×10⁻⁶. 7 [p / s / cm² / sr] / [μW / cm²], significantly higher than Sham+CXCR4 QTY Group ((5.640±0.521)×10) 7 [p / s / cm² / sr] / [μW / cm²]) ( p <0.001), and significantly higher than ICH+CXCR4. QTY Group ((6.102±1.058)×10) 7 [p / s / cm² / sr] / [μW / cm²]) ( p <0.01). And ICH+CXCR4 QTY The peak signal in the brain of the group occurred at 4 hours.

[0106] In addition, animals were sacrificed 2 hours after drug administration, and the heart, liver, spleen, lungs, kidneys, and brain were rapidly removed for in vitro fluorescence imaging to detect the fluorescence distribution and enrichment in each organ. Quantitative statistical analysis of fluorescence intensity in major organs and brain tissue is as follows: Figure 5A As shown. The results indicate that there were no significant differences in signal distribution among the groups in major non-target organs such as the heart, liver, spleen, lungs, and kidneys. p >0.05), indicating CXCR4 QTY Neither SCXCR4 nor SCXCR4 proteins showed significant non-specific organ enrichment; however, 2 hours after administration, the mean radiant efficiency signal intensity (Avg. Radiant Efficiency) in brain tissue of the ICH+SCXCR4 group was (1.070±0.418)×10⁻⁶. 7 [p / s / cm² / sr] / [μW / cm²]), significantly higher than Sham+CXCR4. QTY Group ((1.078±0.47)×10) 7 [p / s / cm² / sr] / [μW / cm²]) ( p <0.001), and significantly higher than ICH+CXCR4. QTY Group ((0.979±0.352)×10) 7 [p / s / cm² / sr] / [μW / cm²]) ( p <0.01, consistent with the trend in in vivo imaging results. Further analysis revealed the proportion of brain tissue signal to total signal in major organs 2 hours after drug administration (…). Figure 5B The study found that the proportion in the ICH+SCXCR4 group was 21.38±1.48%, which was significantly higher than that in the Sham+CXCR4 group.QTY Group (13.05±0.43%) p <0.001), and significantly higher than ICH+CXCR4. QTY Group (14.25±1.93%) p <0.01).

[0107] The above results indicate that, compared with CXCR4 QTY Compared to other proteins, the SCXCR4 protein exhibits higher in vivo targeting efficiency, enabling faster and more precise accumulation in the cerebral hemorrhage lesion area. Furthermore, current literature reports that similar targeted drugs for cerebral hemorrhage typically require doses of 2-5 mg / kg, with peak homing time to the lesion ranging from 2-12 hours. In contrast, the SCXCR4 protein requires only a dose of 0.375 mg / kg to achieve effective homing and specific accumulation in the cerebral hemorrhage lesion area within 2 hours post-administration. Therefore, the SCXCR4 protein offers the advantages of low dosage and high targeting efficiency. Simultaneously, using the directly biosynthesized membrane receptor protein CXCR4 for targeted intervention in cerebral hemorrhage offers advantages such as targeted specificity and controllable action compared to traditional biomimetic targeting systems.

[0108] 3. Immunofluorescence staining analysis of brain tissue Brain tissue was extracted from SD rats treated with recombinant protein for 3 days, paraffin sections were prepared, and the expression of CXCL12, CXCR4, CD86, and CD206 in the brain tissue was analyzed by immunofluorescence.

[0109] The experiment was divided into four groups: sham surgery + PBS treatment group (sham surgery modeling followed by PBS injection for treatment, i.e., Sham + PBS group), cerebral hemorrhage + PBS treatment group (cerebral hemorrhage modeling followed by PBS injection for treatment, i.e., ICH + PBS group), and cerebral hemorrhage + CXCR4 group. QTY Protein therapy group (brain hemorrhage modeling and CXCR4 injection) QTY Protein-based treatment, namely ICH+CXCR4 QTY The group consists of two groups: the cerebral hemorrhage + SCXCR4 protein treatment group (cerebral hemorrhage modeling and SCXCR4 protein injection for treatment, i.e., the ICH + SCXCR4 group).

[0110] The specific steps are as follows: Place paraffin-embedded sections in an oven at 65°C for 30-60 minutes. Immediately after dewaxing, dewax the sections in xylene three times, 10 minutes each time, at room temperature. After dewaxing, rehydrate the sections twice in anhydrous ethanol for 5 minutes each time; then hydrate them in 95%, 85%, 75%, and 50% ethanol for 5 minutes each time; finally, wash them 1-2 times in deionized water (ddH2O) for 5 minutes each time. Place the sections in Tris-EDTA antigen retrieval solution (1X) and microwave at 90°C for 20 minutes. Cool to room temperature and wash three times with PBST for 5 minutes each time. Permeabilize with 0.5% Triton X-100 at room temperature for 15 minutes, then wash three times with PBST for 5 minutes each time. Block with 0.1% BSA solution at room temperature for 1-2 hours. After diluting the primary antibody to the concentration recommended in the instructions, the antibody was added to the slide surface to completely cover the tissue. Incubation was carried out overnight at 4°C for 12-16 hours, followed by three 10-minute washes with PBST. Alexa Fluor 555-labeled secondary antibody, matching the primary antibody source, was diluted 1:500, added to the slide surface to completely cover the tissue, and incubated at room temperature in the dark for 1 hour. Washing was carried out three times with PBST for 10 minutes each. The entire process was kept in the dark. Nuclear staining was completed with a DAPI-containing anti-fluorescence quenching mounting medium, and the slide was mounted. After standing at room temperature for approximately 5 minutes, imaging and subsequent analysis were performed using a fluorescence microscope. Representative immunofluorescence micrographs of CXCR4 are shown below. Figure 6A As shown, its fluorescence signal is mainly located in the cell membrane, accompanied by diffuse granular fluorescence in the cytoplasm. Quantitative analysis of CXCR4 fluorescence intensity is as follows: Figure 6B As shown; representative immunofluorescence micrographs of CXCL12 are as follows. Figure 6C As shown, its fluorescence is mainly distributed in the extracellular space and perinuclear cytoplasm, consistent with the characteristics of extracellular secreted soluble proteins; quantitative analysis of CXCL12 fluorescence intensity is as follows. Figure 6D As shown. From Figures 6A-6D The results showed that, compared with the Sham+PBS group, the expression levels of CXCL12 and CXCR4 in the brain tissue of the ICH+PBS group were significantly increased (p<0.001), demonstrating that intracerebral hemorrhage can significantly activate the CXCL12 / CXCR4 signaling axis. In contrast, the ICH+CXCR4 group showed significantly higher expression levels. QTY Both the ICH+SCXCR4 group and the CXCR4 group significantly reduced the expression levels of CXCL12 and CXCR4, indicating that both water-soluble mutant proteins have a certain signal transduction blocking effect. Among them, the blocking effect of SCXCR4 protein was significantly better than that of CXCR4. QTY(p<0.01) The expression levels of CXCL12 and CXCR4 in the brain tissue after treatment were close to those in the Sham+PBS group, showing a stronger ability to block abnormal activation of the CXCL12 / CXCR4 axis induced by cerebral hemorrhage.

[0111] 4. ELISA detection of inflammatory factor content in brain tissue surrounding cerebral hemorrhage lesions Total protein was extracted from brain tissue surrounding cerebral hemorrhage lesions, and the levels of IL-6, IL-1β, and TNF-α inflammatory factors in the brain tissue were determined by ELISA.

[0112] The experiment was divided into four groups: sham surgery + PBS treatment group (sham surgery modeling followed by PBS injection for treatment, i.e., Sham + PBS group), cerebral hemorrhage + PBS treatment group (cerebral hemorrhage modeling followed by PBS injection for treatment, i.e., ICH + PBS group), and cerebral hemorrhage + CXCR4 group. QTY Protein therapy group (brain hemorrhage modeling and CXCR4 injection) QTY Protein-based treatment, namely ICH+CXCR4 QTY The group consists of two groups: the cerebral hemorrhage + SCXCR4 protein treatment group (cerebral hemorrhage modeling and SCXCR4 protein injection for treatment, i.e., the ICH + SCXCR4 group).

[0113] The specific steps are as follows: SD rats were sacrificed 3 days after recombinant protein treatment, with all procedures performed on ice. Approximately 1-3g of brain tissue surrounding the cerebral hemorrhage lesion was rapidly isolated, gently rinsed 1-2 times in pre-chilled PBS to remove residual blood, blotted dry, and immediately flash-frozen in liquid nitrogen, then stored at -80℃ for later use. The -80℃ tissue samples were then slowly thawed on ice, and pre-chilled lysis buffer was added at a ratio of tissue:tissue lysis buffer = 1:9 (w / v). The samples were then sonicated at 4℃. The homogenate was centrifuged at 5000×g for 10 min at 4℃, and the supernatant was collected, aliquoted, and stored at -80℃ for analysis. Total protein concentration was determined using the BCA method, and all samples were adjusted to a uniform concentration of 1-3 mg / mL. Following the instructions of the ELISA kit (Shanghai Gaining Biotechnology), IL-6, TNF-α, and IL-1β inflammatory factors were detected. The ELISA quantitative results for IL-6 are shown below. Figure 7A As shown; the ELISA quantitative results of TNF-α are as follows. Figure 7B As shown; the ELISA quantitative results of IL-1β are as follows. Figure 7C As shown. The results showed that, compared with the ICH+PBS group, the ICH+CXCR4 group... QTY Although the levels of IL-6 and TNF-α in the tissue surrounding the hematoma in the treatment group showed a decreasing trend, the differences did not reach statistical significance. p >0.05). Although CXCR4QTY Immunofluorescence analysis showed a certain inhibitory trend on IL-6 and TNF-α, but its overall anti-inflammatory effect in the peri-hematoma tissue remained limited. In contrast, the levels of IL-6, IL-1β, and TNF-α in the peri-hematoma tissue of the ICH+SCXCR4 treatment group were significantly lower than those of the ICH+PBS group. p <0.05).

[0114] Representative immunofluorescence images of CD86 are as follows: Figure 8A As shown, the fluorescence signal was mainly located on the surface of the microglia membrane; simultaneously, significant positive fluorescence signals were observed in the nucleus and perinuclear cytoplasm, suggesting microglia activation, intracellular endocytosis of CD86 receptors on the membrane surface, and nuclear transport. Quantitative analysis results of the fluorescence signal are as follows: Figure 8B As shown, Figure 8A and Figure 8B The results showed that the CD86 fluorescence intensity was significantly increased in the ICH+PBS group compared with the Sham+PBS group. p <0.001), indicating a significantly enhanced pro-inflammatory immunophenotype in the acute phase of cerebral hemorrhage. (CXCR4) QTY After protein intervention treatment, CD86 expression was significantly downregulated ( p <0.001), while SCXCR4 protein intervention further inhibited CD86 expression, and its level was significantly lower than that of the ICH+PBS group ( p <0.001) and ICH+CXCR4 QTY Group( p The value <0.001 indicates that the SCXCR4 protein has a superior regulatory capacity in inhibiting pro-inflammatory immune phenotype polarization.

[0115] Representative immunofluorescence images of CD206 are shown below. Figure 9A As shown, the fluorescence signal was mainly located on the surface of the microglia membrane; simultaneously, significant positive fluorescence signals were observed in the nucleus and perinuclear cytoplasm, suggesting microglia activation, intracellular endocytosis of CD206 receptors on the membrane surface, and nuclear transport. Quantitative analysis results of the fluorescence signal are as follows: Figure 9B As shown, Figure 9A and Figure 9B The results showed that, compared with the Sham+PBS group, the expression of CD206 in the brain tissue of the ICH+PBS group was significantly increased. p <0.001), indicating that anti-inflammatory / repair-related immune responses are activated after cerebral hemorrhage. Based on this, CXCR4... QTY Protein therapy can further enhance CD206 expression ( p <0.001), while the CD206 level reached its highest level after SCXCR4 protein treatment, significantly higher than that in the ICH+PBS group ( p<0.001) and ICH+CXCR4 QTY Group( p <0.001), indicating that it is more conducive to promoting the enhancement of anti-inflammatory / repair immune phenotype.

[0116] 5. Wood-Eosin (H&E) staining The experiment was divided into four groups: sham surgery + PBS treatment group (sham surgery modeling followed by PBS injection for treatment, i.e., Sham + PBS group), cerebral hemorrhage + PBS treatment group (cerebral hemorrhage modeling followed by PBS injection for treatment, i.e., ICH + PBS group), and cerebral hemorrhage + CXCR4 group. QTY Protein therapy group (brain hemorrhage modeling and CXCR4 injection) QTY Protein-based treatment, namely ICH+CXCR4 QTY The group consists of two groups: the cerebral hemorrhage + SCXCR4 protein treatment group (cerebral hemorrhage modeling and SCXCR4 protein injection for treatment, i.e., the ICH + SCXCR4 group).

[0117] The specific steps are as follows: Paraffin-embedded sections of brain tissue were placed in an oven at 65°C for 30-60 minutes. The sections were then rapidly dewaxed in xylene, repeated three times at room temperature for 10 minutes each time. After dewaxing, the sections were sequentially transferred to anhydrous ethanol for rehydration twice, 5 minutes each time. Subsequently, they were placed in 95%, 85%, 75%, and 50% ethanol for 5 minutes each for hydration. Finally, they were transferred to deionized water (ddH2O) and washed 1-2 times, 5 minutes each time. Hematoxylin staining was performed for 5-10 minutes, followed by rinsing with deionized water (ddH2O) for 15 minutes. Differentiation was performed using acidic differentiation solution for 2-10 seconds until the blue color lightened, then rinsed with deionized water (ddH2O) and allowed to regain its blue color for approximately 10 minutes. Eosin staining was performed for 1-3 minutes, followed by rinsing with deionized water (ddH2O) to remove residual stain. The samples were then subjected to a gradient dehydration process, sequentially placed in 50%, 75%, 85%, 95%, and anhydrous ethanol for 3 minutes each. The sections were treated twice in xylene at room temperature (10 min for the first time, 5 min for the second time) to make the tissue transparent; a suitable amount of neutral resin was added for mounting, covered with a coverslip, and observed and images were acquired under a microscope after curing. Figure 10 As shown, the second row is an enlarged view of the area selected in the first row; the brain tissue structure of the Sham+PBS group was basically intact, with only a very small amount of mechanical damage visible at the needle insertion trajectory; in contrast, the ICH+PBS group showed a large number of red blood cells aggregated in the core area of ​​the lesion, with severe loosening and structural disorder in the peripheral tissue, exhibiting typical acute-phase pathological features; ICH+CXCR4 QTY In the ICH+SCXCR4 treatment group, hematoma lesions remained, but the structures of the adjacent brain tissue were relatively intact. This indicates that CXCR4... QTYSCXCR4 protein may intervene in the secondary injury process after cerebral hemorrhage by blocking the CXCL12 / CXCR4 axis and regulating inflammatory response and immune microenvironment.

[0118] 6. Open Field Test (OFT) The experiment was divided into four groups: sham surgery + PBS treatment group (sham surgery modeling followed by PBS injection for treatment, i.e., Sham + PBS group), cerebral hemorrhage + PBS treatment group (cerebral hemorrhage modeling followed by PBS injection for treatment, i.e., ICH + PBS group), and cerebral hemorrhage + CXCR4 group. QTY Protein therapy group (brain hemorrhage modeling and CXCR4 injection) QTY Protein-based treatment, namely ICH+CXCR4 QTY The group consists of two groups: the cerebral hemorrhage + SCXCR4 protein treatment group (cerebral hemorrhage modeling and SCXCR4 protein injection for treatment, i.e., the ICH + SCXCR4 group).

[0119] The specific steps are as follows: The open field test chamber measures 1m × 1m × 0.5m. After removing the rat from its cage, it is gently placed in the center of the open field with its back to the experimenter. The experimenter quickly leaves, allowing the animal to explore freely for 5-10 minutes while the experiment is recorded. After each animal's test, it is returned to its cage. The bottom, side walls, and all surfaces in the chamber are thoroughly cleaned with 75% ethanol or laboratory disinfectant. The chamber is allowed to dry completely before the next animal is tested to minimize interference from excrement and odor residue. The structure of the open field test apparatus is as follows: Figure 11A As shown.

[0120] Open field thermograms used color gradients to reflect the spatial activity distribution of rats in an open field, with red areas representing locations with longer dwell times (high dwell time) and blue areas representing areas with lower activity frequencies (low-frequency activity). Representative activity thermograms of rats in each group at different time points after cerebral hemorrhage are shown below. Figure 11B As shown, the Sham+PBS group exhibited a larger range of motion at all time points, with dispersed movement trajectories, and was able to enter the central area of ​​the open field, demonstrating normal voluntary movement ability, spatial exploration behavior, and low anxiety-like manifestations. In contrast, the ICH+PBS group showed significantly limited range of motion from day 1 to 7 after intracerebral hemorrhage, with high-intensity signals mainly concentrated in the corners and periphery of the open field, and a significant reduction in exploration of the central area, indicating that intracerebral hemorrhage led to persistent voluntary motor impairment and enhanced anxiety-like behavior. After drug intervention, the ICH+CXCR4... QTYFrom day 3 onwards, the range of motion in the group was larger than that in the ICH+PBS group, and the corner-based loitering phenomenon was partially alleviated. However, the movement trajectory was still mainly distributed along the edge of the open field, with limited exploration of the central region. Notably, the ICH+SCXCR4 group showed a wider range of movement distribution from day 1 after intracerebral hemorrhage, and the range of motion continued to expand over time, showing a clear trend of exploration towards the central region by day 7. This indicates that SCXCR4 protein can not only effectively promote the recovery of voluntary motor ability after intracerebral hemorrhage, but also help alleviate anxiety-like behaviors associated with intracerebral hemorrhage. Further analysis of the average speed and total movement distance of each group yielded the following results: Figure 11C and Figure 11D As shown, on day 7 after cerebral hemorrhage, the mean movement speed and total movement distance of rats in the ICH+SCXCR4 group were 7.24±1.48 cm / s and 21.71±3.74 m, respectively, both significantly higher than those in the ICH+PBS group. p <0.05), and the overall recovery trend is better than ICH+CXCR4. QTY Group( p <0.05).

[0121] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0122] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A method for modifying a high-affinity water-soluble membrane protein, characterized in that, The specific steps are as follows: S1: Based on the crystal structure of the CXCR4 receptor protein, the spatial distribution characteristics of its transmembrane region amino acids and the amino acids at the transmembrane region interface were analyzed. Then, site-directed water-soluble mutagenesis was performed on the CXCR4 transmembrane region interface site using QTY Code to construct the SQTY Code interface mutant CXCR4. SQTY And evaluate the obtained interface mutant CXCR4 SQTY If the water solubility and ligand binding ability meet the requirements, a high-affinity water-soluble membrane protein is obtained; if the requirements are not met, proceed to step S2. S2: Based on the structure-activity relationship of the interaction between CXCR4 and CXCL12, multiple transmembrane regions of CXCR4 were analyzed and ranked, and at least three transmembrane regions with the least impact on ligand binding affinity were screened out; then, QTY code was used to mutate and obtain single transmembrane mutants. Then, the single transmembrane mutant and the interface mutant CXCR4 were compared. SQTY Site-directed water-soluble mutagenesis was performed to obtain a single-transmembrane co-mutant, and the water solubility and ligand binding ability of the obtained single-transmembrane co-mutant were evaluated. If the requirements were met, high-affinity water-soluble membrane proteins were screened to obtain them. If the requirements were still not met, the process was moved to step S3. S3: The single transmembrane mutants obtained in step S2 are combined in pairs to construct multiple combined mutants; then, the multiple combined mutants are respectively combined with the interface mutant CXCR4 obtained in step S1. SQTY Site-directed water-soluble mutagenesis was performed to construct a variety of transmembrane joint mutants. The water solubility and ligand binding ability of the obtained transmembrane joint mutants were further evaluated. The optimal transmembrane joint mutant was selected from the transmembrane joint mutants that met the requirements, which is the high-affinity water-soluble SCXCR4 mutant. The condition for meeting the requirements in steps S1, S2 and S3 is that the average total hydrophilicity coefficient of the mutant is less than 0.

2. The method according to claim 1, characterized in that, In step S3, when constructing a combined mutant using QTY Code, the overall conformation of CXCR4 and the ligand binding interface are kept unchanged, while the transmembrane interface and the hydrophobic amino acid in the transmembrane region are combined and mutated in stages. In step S3, when constructing the dual transmembrane joint mutant, the transmembrane region interface amino acid is used as the starting point, and the transmembrane region is gradually superimposed and expanded, thereby achieving a constrained expansion of the modification range.

3. The method according to claim 1, characterized in that, The screening criteria in steps S1, S2, and S3 are as follows: the change in binding free energy between the mutant and CXCL12 is less than that of the transmembrane mutant CXCR4. QTY Or / and the mutation rate of the mutant is less than 15%.

4. A high-affinity water-soluble membrane protein SCXCR4, characterized in that, The high-affinity water-soluble membrane protein SCXCR4 is a mutant obtained by the method described in any one of claims 1-3, and its amino acid sequence is shown in SEQ ID NO:

1.

5. The high-affinity water-soluble membrane protein SCXCR4 according to claim 4, characterized in that, The water-soluble membrane protein SCXCR4 has a 6×His tag introduced at its C-terminus.

6. The high-affinity water-soluble membrane protein SCXCR4 according to claim 4, characterized in that, The nucleotide sequence encoding the water-soluble membrane protein SCXCR4 was further optimized and modified, and the resulting nucleotide sequence is shown in SEQ ID NO:

2.

7. A recombinant expression vector pET-20b(+)-SCXCR4, characterized in that, The recombinant expression vector contains the nucleotide sequence of claim 6.

8. A recombinant host bacterium BL21(DE3)-pET-20b(+)-SCXCR4, characterized in that, The recombinant host bacterium comprises the recombinant expression vector of claim 7, wherein the host bacterium is Escherichia coli BL21(DE3).

9. The use of the high-affinity water-soluble membrane protein SCXCR4 as described in any one of claims 4-6 in the preparation of a medicament for blocking the CXCL12 / CXCR4 signaling axis.

10. The application according to claim 9, characterized in that, The drug is applied to areas of cerebral hemorrhage lesions and intervenes in secondary brain injury by blocking the CXCL12 / CXCR4 axis, thereby promoting the recovery of voluntary motor function after cerebral hemorrhage and improving behavioral performance.

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