A CXCR-2 mutant and its preparation method and application

Abstract

The present invention discloses a CXCR-2 mutant and a preparation method and application thereof, and the application of the CXCR-2 mutant as a drug for clearing brain damage. The brain damage includes cerebral hematoma. Chemokine receptor 2 (CXCR-2) is expressed in microglial cells in some central nervous system diseases. After CXCR-2 binds to the ligand chemokine 2 (CXCL2), it can promote the transformation of microglial cells to M1 type, block the CXCL2-CXCR-2 signaling pathway, and reverse this transformation, promoting the transformation of M1 to M2 type. The present invention is designed according to the sequence of CXCR-2, and a new protein sequence is obtained. The sequence can be recombinantly expressed as a protein to obtain a recombinant protein with good water solubility. In a rat model of cerebral hemorrhage induced by collagenase, the recombinant protein can promote the absorption of rat cerebral hematoma. It can effectively improve the prognosis of cerebral hemorrhage.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and in particular relates to a CXCR-2 mutant and a preparation method and application thereof. Background Art

[0002] In the acute phase of ICH, surgical evacuation may be more risky than effective due to the relatively small size of the hematoma. Furthermore, the surgical procedure inevitably affects normal brain tissue and nerves, leading to irreversible side effects. Medical intervention for ICH primarily involves the use of antihypertensive and hemostatic drugs, but these are intended to control hematoma expansion rather than promote its resorption. The use of hemostatic drugs also carries the risk of thromboembolic events, and their long-term benefits are uncertain. In recent years, with the advancement of precise stereotactic brain technology, minimally invasive surgery combined with in situ drug delivery has effectively improved the therapeutic efficacy of ICH. Studies have shown that minimally invasive surgery and thrombolytic therapy performed within 72 hours after ICH are associated with improved neurological function 180 days later. Therefore, the development of drugs that can promote hematoma evacuation and allow in situ treatment via the surgical corridor established by minimally invasive surgery could provide a new approach to the treatment of ICH and potentially improve its efficacy.

[0003] Existing research indicates that intracerebral hematoma clearance occurs primarily through three pathways: the microglia / macrophage system is the first line of defense against intracerebral hemorrhage and the primary phagocytic system mediating hematoma clearance; some scavenger receptors (such as CD36, CD47, and SRA) participate in endocytosis, playing a crucial role in maintaining brain homeostasis and phagocytosis, further promoting hematoma clearance; and upstream regulatory systems (such as Nrf2 and PPAR-γ) can also indirectly promote hematoma clearance. Increasing evidence indicates that microglia play a key role in hematoma clearance. When a hematoma forms, microglia are rapidly activated and can polarize into two phenotypes: M1 and M2. The M1 phenotype expresses a range of inflammatory factors, leading to neuronal apoptosis, disruption of neural tracts, and damage to the blood-brain barrier. In contrast, the M2 phenotype primarily expresses anti-inflammatory factors, promoting the phagocytosis of dying cells and blood debris, thereby facilitating hematoma clearance. Therefore, developing drugs that can regulate the polarization of microglia, the first line of defense against cerebral hematoma, to M2 type will help promote the clearance of cerebral hematoma and is also the focus of current neurological research on secondary brain injury after cerebral hemorrhage. Summary of the Invention

[0004] The present invention discloses a CXCR-2 mutant and a preparation method thereof, wherein the mutant can enhance the binding with the ligand chemokine 2 and promote the absorption of cerebral hematoma. The second object of the present invention is to provide an application of the CXCR-2 mutant.

[0005] In order to achieve the above first object, the technical solution of the present invention is: a CXCR-2 mutant, characterized in that: the amino acid sequence is MGEIRVDNFS LEDFFSGDID SYNYSSDPPF TLSDAAPCPS ANLDINRYAT TTTYTQTTQQSQTGQSQTMQ TTQYNRSTCS VTDTYQQQQA TADQYYAQTQ PTWAASKVNG WIFGSFLCKT YSYQQETTYYSSTQQQACTS MDRYLAIVHA TSTLIQKRHL VKYTCTTMWY QSQTQSQPTY TQRTTVKANP STVVCYENIGNNTSKWRTTQ RTQPQTYGYQ QPQQTMQYCY GFTLRTLFKA HMGQKHRAMR TTYATTQTYQ QCWQPYQTTQYTDTLMRTKL IKETCERQNE INKALEATET QGYQHSCQQP TTYAFIGQKF RHGLLKIMAN YGLVSKEFLAKEGRPSFVGSSSANTSTTLHHHHHHHHHH.

[0006] The method for producing CXCR-2 mutant protein is characterized by comprising the following steps:

[0007] (1) Preparation of recombinant plasmid;

[0008] (2) Vector transformation: Remove BL21(DE3) competent cells from -80°C and thaw on ice; add plasmid DNA to BL21(DE3) strain, mix gently, and incubate on ice; heat at 42°C for 2-30 min, then place on ice; add room temperature LB culture medium, shake and incubate at 200 rpm, 37°C; spread the bacterial solution on LB agar plates containing ampicillin and incubate at 37°C overnight;

[0009] (3) Protein expression: Select a single colony from the agar plate and inoculate it into LB medium containing ampicillin, shake and culture at 200 rpm, 37°C overnight; inoculate the bacterial solution into LB medium for expansion culture, shake and culture at 200 rpm, 37°C until the OD600 of the bacterial solution reaches 0.6-0.8; add IPTG to a final concentration of 0.5 mM and shake and culture at 37°C for induction;

[0010] (4) Lysis and washing:

[0011] Collect the bacterial solution after IPTG induction, centrifuge at 8000 rpm, 4°C, and collect the precipitate; add B-PER solution, ultrasonically disrupt in an ice bath, mix and repeat the lysis once; centrifuge at 11500 rpm, 4°C, and discard the supernatant; add Buffer 1 to wash the precipitate, ultrasonicate, centrifuge at 11500 rpm, 4°C, 25 min, and discard the supernatant; add Buffer 2 and ultrasonicate; centrifuge at 11500 rpm, 4°C, and discard the supernatant; add ml Buffer 1 and ultrasonicate; centrifuge at 11500 rpm, 4°C, 25 min, and discard the supernatant;

[0012] (5) Protein purification:

[0013] Add DeB to dissolve the precipitate, and suspend it overnight using magnetic stirring or a chromatography cabinet; centrifuge at 11500 rpm and 4°C, and collect the supernatant; purify by Ni column chromatography, collect the purified sample, and dialyze using a dialysis bag or ultrafiltration tube to remove DeB to obtain the purified protein.

[0014] Use of a CXCR-2 mutant as a drug for clearing brain damage.

[0015] The brain injury includes a cerebral hematoma.

[0016] Beneficial effects:

[0017] Chemokine receptor-2 (CXCR-2) is expressed in microglial cells in some central nervous system diseases. Binding of CXCR-2 to its ligand, chemokine 2 (CXCL2), promotes the transformation of microglial cells to the M1 phenotype. Blocking the CXCL2-CXCR-2 signaling pathway can reverse this transformation and promote the transition from M1 to M2. This invention, based on the CXCR-2 sequence, has developed a novel protein sequence that can be recombinantly expressed to produce a highly water-soluble recombinant protein. In a rat model of collagenase-induced intracerebral hemorrhage, this recombinant protein has been shown to promote the absorption of brain hematomas, effectively improving the prognosis of intracerebral hemorrhage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the result of plasmid sequencing.

[0019] Figure 2 This is the SDS-PAGE analysis of the recombinant protein.

[0020] Figure 3 This is a Western Blot analysis of the purified recombinant protein.

[0021] Figure 4 This is the sagittal view of the brain of rats in the blank control group.

[0022] Figure 5 Sagittal images of the brain of rats in the CXCR-2 intervention group.

[0023] Figure 6 This is the sagittal view of the brain of rats in the recombinant protein intervention group.

[0024] Figure 7 Sagittal view of normal rat brain. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] Example 1

[0027] CXCR-2 mutant, the amino acid sequence is MGEIRVDNFS LEDFFSGDID SYNYSSDPPF TLSDAAPCPSANLDINRYAT TTTYTQTTQQ SQTGQSQTMQTTQYNRSTCS VTDTYQQQQA TADQYYAQTQ PTWAASKVNGWIFFGSFLCKTYSYQQETTYY SSTQQQACTS MDRYLAIVHA TSTLIQKRHL VKYTCTTMWYQSQTQSQPTYTQRTTVKANP STVVCYENIG NNTSKWRTTQ RTQPQTYGYQQPQQTMQYCY GFTLRTLFKA HMGQKHRAMRTTYATTQTYQ QCWQPYQTTQYTDTLMRTKL IKETCERQNE INKALEATET QGYQHSCQQPTTYAFIGQKFRHGLLKIMAN YGLVSKEFLA KEGRPSFVGS SSANTSTTL HHHHHHHHHH

[0028] Prepare as follows:

[0029] 1. The recombinant plasmid was synthesized by entrusting GenScript to use pET-22b as the vector. Figure 1 The results of plasmid sequencing.

[0030] 2. Plasmid transformation: Remove BL21(DE3) competent cells from -80°C and thaw on ice; add 100 ng of plasmid DNA to the BL21(DE3) strain, mix gently, and incubate on ice for 30 minutes; heat at 42°C for 90 seconds, then place on ice for 3 minutes; add 100 μl of room temperature LB culture medium and incubate at 37°C with shaking at 200 rpm for 60 minutes; spread the bacterial liquid on LB agar plates containing ampicillin (100 μg / ml) and incubate at 37°C overnight.

[0031] 3. Protein Expression: Select a single colony from an agar plate and inoculate it into 20 ml of LB medium containing ampicillin (final concentration 50 μg / L) and shake culture overnight at 200 rpm, 37°C. Expand the culture by inoculating the bacterial suspension into 1 L of LB medium and shaking culture at 200 rpm, 37°C until the OD600 reaches 0.6-0.8. Add IPTG to a final concentration of 0.5 mM and induce the culture at 37°C for 4 h.

[0032] 4. Lysis and washing: Collect the bacterial liquid after IPTG induction, centrifuge at 8000 rpm, 4°C for 5 min, collect the precipitate; add B-PER solution, ultrasonically disrupt (ice bath) for 25 min, 2s on, 8s off, 30% power, mix and repeat the lysis once; centrifuge at 11500 rpm, 4°C, 25 min, discard the supernatant; add 17 ml of Buffer 1 to wash the precipitate, ultrasonically for 15 min (ice bath), 2s on, 8s off, 30% power; centrifuge at 11500 rpm, 4°C, 25 min, discard the supernatant; add 17 ml of Buffer 2 and ultrasonically for 15 min (ice bath); centrifuge at 11500 rpm, 4°C, 25 min, discard the supernatant; add 17 ml of Buffer 1 and ultrasonically for 15 min (ice bath); centrifuge at 11500 rpm, 4°C, 25 min, discard the supernatant.

[0033] 5. Protein purification: Add 10 ml of DeB to the precipitate after centrifugation to dissolve the precipitate, stir magnetically for 1.5 hours (room temperature) or suspend in a chromatography cabinet overnight (4°C); centrifuge at 11500 rpm, 4°C, 25 minutes, collect the supernatant; purify by Ni column chromatography, collect the purified sample, and dialyze using a dialysis bag or ultrafiltration tube to remove DeB to obtain the purified protein.

[0034] Figure 2 The SDS-PAGE of the recombinant protein, where M1 is the protein molecular weight standard, M2 is the WB marker, PC1 is fetal bovine serum albumin (1 μg), PC2 is fetal bovine serum albumin

[0035] (2 μg), NC is cell lysate without induction, 1 is cell lysate induced for 16 h and 15°C, 2 is cell lysate induced for 4 h and 37°C, NC1 is supernatant of cell lysate without induction, 3 is supernatant of cell lysate induced for 16 h and 15°C, 4 is supernatant of cell lysate induced for 4 h and 37°C, NC2 is precipitate of cell lysate without induction, 5 is precipitate of cell lysate induced for 16 h and 15°C, and 6 is precipitate of cell lysate induced for 4 h and 37°C. Figure 3 This is a Western Blot analysis of the purified recombinant protein.

[0036] Example 2

[0037] Animal experiments

[0038] In the collagenase-induced cerebral hemorrhage rat model, compared with the blank control group and the CXCR-2 intervention group, this recombinant protein can promote the clearance of rat cerebral hematoma.

[0039] Experimental groups (6 rats per group as parallel experiments): ① blank control group, ② CXCR-2 intervention group, ③ this recombinant protein intervention group

[0040] Experimental methods and results:

[0041] (1) Establishment of rat cerebral hemorrhage model

[0042] Rats were weighed and anesthetized with 7% chloral hydrate (5 ml / kg) intraperitoneally. Spontaneous breathing was ensured during the operation. The rats were fixed in a prone position on a cranial stereotaxic apparatus. The scalp was incised, and the anterior fontanel was used as the origin. The needle was placed 0.2 mm forward and 2 mm laterally to the right, with a depth of 3.7 mm as the injection point. The skull was drilled using a skull drill. After the needle was inserted, 0.2 U type IV collagenase was slowly injected into the right striatum at 2 μL / min. After the injection, the needle was retained for 10 min and then slowly withdrawn at 1 mm / min. An injection tube was fixed at the injection site for drug administration.

[0043] (2) Drug treatment

[0044] Blank control group: 4 hours after the rats were injected with collagenase, 20 μl of PBS solution was slowly injected through the injection channel.

[0045] CXCR-2 intervention group: 4 hours after the injection of collagenase, 20 μl of CXCR-2 suspension (concentration of about 0.70 mg / ml PBS solution) was slowly injected through the injection channel.

[0046] The recombinant protein intervention group: 4 hours after the rats were injected with collagenase, 20 μl of the recombinant protein solution in PBS (concentration of about 0.70 mg / ml PBS solution) was slowly injected through the injection channel.

[0047] Normal control group: rats were not treated with collagenase, and 20 μl of PBS solution was slowly injected through the injection channel.

[0048] (3) Observation of cerebral hematoma

[0049] 24 hours after the completion of the drug administration, the rats were weighed and anesthetized. The chest and abdominal cavities were opened in the supine position to fully expose the heart and liver. The injection needle was inserted into the left ventricle and the right atrial appendage was cut open. Normal saline was rapidly perfused until the liver turned white. After clear liquid flowed out of the right atrial appendage, 4% paraformaldehyde fixative was switched. The perfusion was first rapid and then slow until the rat's liver became hard and the limbs became rigid. The brain was decapitated and fixed in 4% paraformaldehyde. The brain was cut open in the sagittal plane above the injection site to observe the hematoma. The severity of brain hematoma in rats in the recombinant protein intervention group was significantly lower than that in the blank control group and the CXCR-2 intervention group. Figure 4 This is the sagittal view of the brain of rats in the blank control group. Figure 5 This is the sagittal image of the brain of rats in the CXCR-2 intervention group. Figure 6 This is the sagittal view of the brain of rats in the recombinant protein intervention group. Figure 7 Sagittal view of normal rat brain.

[0050] Example 3

[0051] In the animal neurological function evaluation test, rats treated with recombinant protein scored higher, indicating that recombinant protein can effectively improve the prognosis of cerebral hemorrhage.

[0052] Experimental groups (6 rats per group as parallel experiments): ① blank control group, ② CXCR-2 intervention group, ③ this recombinant protein intervention group

[0053] Experimental methods and results:

[0054] (1) The method for establishing rat cerebral hemorrhage model is the same as above

[0055] (2) The administration method of each test group is the same as above

[0056] (3) Animal neurological function evaluation

[0057] Neurological function of rats in each group was evaluated 24 hours, 2, 4, and 7 days after administration. Evaluation items:

[0058] ① Spontaneous rotation: 0-3 levels, 0 is no rotation, 3 is continuous rotation;

[0059] ② Hindlimb contraction: 0-3, with 0 being immediate contraction and 3 being no retraction after lateral displacement of the hindlimb;

[0060] ③ Bilateral forepaw grasping: 0-3, with 0 being normal grasping and 3 being completely unable to grasp;

[0061] ④Beam walking ability: 0-3 levels, where 0 means being able to quickly pass the scale and 3 means being unable to stand on the scale for more than 10 seconds.

[0062] The results showed that the neurological function evaluation results of rats with cerebral hemorrhage treated with the recombinant protein were significantly better than those of the blank control group and the CXCR-2 intervention group during the recovery period.

[0063] Table 1 shows the results of neurological function evaluation

[0064]

Claims

1. A CXCR-2 mutant, characterized in that: The amino acid sequence is MGEIRVDNFS LEDFFSGDIDSYNYSSDPPF TLSDAAPCPS ANLDINRYAT TTTYTQTTQQ SQTGQSQTMQ TTQYNRSTCS VTDTYQQQQATADQYYAQTQ PTWAASKVNG WIFGSFLCKT YSYQQETTYY SSTQQQACTS MDRYLAIVHA TSTLIQKRHLVKYTCTTMWY QSQTQSQPTY TQRTTVKANP STVVCYENIG NNTSKWRTTQ RTQPQTYGYQ QPQQTMQYCYGFTLRTLFKA HMGQKHRAMR TTYATTQTYQ QCWQPYQTTQ YTDTLMRTKL IKETCERQNE INKALEATETQGYQHSCQQP TTYAFIGQKF RHGLLKIMAN YGLVSKEFLA KEGRPSFVGS SSANTSTTL HHHHHHHHHH.

2. Use of the CXCR-2 mutant according to claim 1 in the preparation of a drug for clearing brain damage, wherein the brain damage is cerebral hematoma.

Images