A fusion protein and use thereof

Abstract

The application relates to the field of biological medicine, and particularly discloses a fusion protein and application thereof. The fusion protein is composed of a polypeptide and six histidines, the polypeptide has sequence consistency of at least 9 / 10, 14 / 15 or 29 / 30 with the amino acid sequence shown in SEQ ID NO:1, and the six histidines are located at the C-terminal of the polypeptide. The fusion protein designed in the application is derived from a human body protein, the metabolic product is an amino acid, and therefore the safety is better than that of other therapeutic drugs. Moreover, the fusion protein can significantly reduce the infarction volume of the brain of a tMCAO model mouse, has a significant treatment on ischemic stroke, and has a longer treatment time window than that of an existing thrombolytic drug. Compared with the existing ischemic stroke treatment drug, the human sTREM2-6His fusion protein has a brand-new treatment mechanism and target.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a fusion protein and its application, specifically the application of a fusion protein in the preparation of drugs for treating ischemic stroke. Background Technology

[0002] Stroke is a group of diseases caused by sudden rupture or blockage of blood vessels in the brain, resulting in impaired blood circulation and damage to brain tissue. It includes ischemic stroke and hemorrhagic stroke. Ischemic stroke refers to the necrosis of local brain tissue, including nerve cells, glial cells, and blood vessels, due to insufficient blood supply. Stenosis or occlusion of the internal carotid artery and vertebral artery can cause ischemic stroke, which is more common in people over 40 years of age and more prevalent in men than women. Severe cases can lead to death.

[0003] Currently, the preferred treatment for ischemic stroke (commonly known as cerebral infarction) is intravenous thrombolysis and / or endovascular thrombectomy to achieve reperfusion. Intravenous thrombolysis within 4.5 hours of stroke onset can effectively reduce disability. For patients with ischemic brain tissue but no infarction on perfusion imaging, the treatment window can be extended to within 9 hours. In addition, endovascular thrombectomy within 6 hours of stroke onset can effectively reduce disability rates in patients with extensive large vessel occlusion, and within 24 hours of stroke onset in patients diagnosed by perfusion imaging. However, both intravenous thrombolysis and endovascular thrombectomy must be strictly controlled within the time window of ischemic stroke onset. In reality, many patients have missed the optimal thrombolysis time window upon admission, delaying effective treatment. Furthermore, thrombolytic therapy has extremely low efficacy in the later stages of neurological damage in ischemic stroke.

[0004] Therefore, strategies to extend the treatment window are particularly important, and neuroprotective strategies have long been a research hotspot in the treatment of ischemic stroke. The goal of neuroprotective therapy is to delay neuronal death, buying time to restore cerebral blood flow and reperfusion, and salvaging reversibly damaged neurons and brain tissue. However, the efficacy of currently developed neuroprotective drugs in clinical applications is lower than in animal models. This is because these neuroprotective drugs have certain toxicity to humans, so the dosage used in clinical practice is far lower than the effective dosage in animal experiments, thus failing to achieve the expected therapeutic effect. Therefore, developing neuroprotective drugs targeting new specific targets has significant practical value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art by providing a fusion protein and its application in the preparation of drugs for treating stroke. The fusion protein designed in this invention is derived from the human body's own proteins, and its metabolites are amino acids, thus exhibiting superior safety compared to other therapeutic drugs. Furthermore, the fusion protein of this invention can significantly reduce the infarct volume in the brain of tMCAO model mice, demonstrating significant therapeutic effect on ischemic stroke, and exhibiting a longer therapeutic window than existing thrombolytic drugs.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] Firstly, the present invention provides a fusion protein composed of a polypeptide and six histidine residues, wherein the polypeptide has a sequence identity with at least 9 / 10, 14 / 15 or 29 / 30 of the amino acid sequence shown in SEQ ID NO:1; the six histidine residues are located at the C-terminus of the polypeptide.

[0008] The amino acid sequence shown in SEQ ID NO:1 is HNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHN LWLLSFLRRWNGSTAITDDTLGGTLTITLRNLQPHDAGLYQCQSLHGSEADTL RKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEH.

[0009] The fusion protein (sTREM2) designed in this invention is derived from the human body's own protein, and its metabolites are amino acids. Therefore, the fusion protein of this invention is safer than other therapeutic drugs, has a significant therapeutic effect on ischemic stroke, and has a longer therapeutic window than existing thrombolytic drugs.

[0010] In this invention, adding six histidine residues to the C-terminus of the peptide is for the purpose of efficiently purifying the fusion protein. The resulting fusion protein can reduce the infarct volume in tMCAO model mice. The six histidine residues added to the fusion protein have no toxic effects. In the same experimental tests, no infarct foci were found in the brains of sham-operated mice whose striatum was injected with the 6His peptide (6 histidine residues) or the fusion protein. This suggests that the 6His peptide and the fusion protein formed by it and sTREM2 (as shown in SEQ ID NO:1) are safe.

[0011] As a preferred embodiment of the fusion protein of the present invention, the fusion protein is composed of a polypeptide as shown in SEQ ID NO:1 and 6 histidines, wherein the 6 histidines are located at the C-terminus of the polypeptide as shown in SEQ ID NO:1.

[0012] Through extensive research and experimentation, the inventors of this invention discovered that the newly designed fusion protein (referred to as the human sTREM2-6His fusion protein) and the existing human TREM2 protein are two completely different proteins. The former is a secreted, soluble protein, while the latter is a receptor protein with a single transmembrane domain. In nature, endogenous sTREM2 is a soluble protein released into the extracellular space after the extracellular domain of TREM2 is hydrolyzed and cleaved by ADAM10 or ADAM17. However, the sTREM2 in the fusion protein designed by this invention is selected from TREM2. The His19-His157 protein sequence in TREM2 is designed to closely resemble the structure of naturally occurring endogenous sTREM2 because the cleavage site of ADAM10 or ADAM17 is located at the His157-Ser158 peptide bond in TREM2. This design confirms that the protein sequence with therapeutic effects on ischemic stroke is the original endogenous sTREM2 protein sequence. This approach is safe and can provide a basis for future development of therapeutic strategies that reduce post-ischemic brain damage by increasing the release of endogenous sTREM2 in ischemic stroke patients.

[0013] Secondly, the present invention provides the application of the above-mentioned fusion protein in the preparation of drugs for treating stroke.

[0014] The sTREM2 and TREM2 used in this invention are two different substances. TREM2 is an innate immune receptor specifically expressed on microglia in the brain parenchyma, and is a receptor protein with a single transmembrane structure; while sTREM2 is an extracellular soluble protein released after the extracellular domain of TREM2 is hydrolyzed and cleaved by integrins and metalloproteinases ADAM10 or ADAM17.

[0015] Previous studies have shown that sTREM2 can be produced through proteolysis in local tissues and then cross the damaged blood-brain barrier to enter the cerebrospinal fluid. Therefore, elevated sTREM2 levels in the cerebrospinal fluid can be observed in some neurodegenerative diseases, suggesting that sTREM2 could serve as an inflammatory biomarker for Alzheimer's disease (AD). Furthermore, some studies have found that sTREM2 improves the pathological phenotype of AD by regulating microglial function in AD models. However, the role of sTREM2 in ischemic stroke remains unclear. The fusion protein designed in this invention clarifies this mechanism of action. Experiments in this invention directly demonstrate that the human sTREM2-6His fusion protein is directly localized to neurons in ischemic brain tissue, and the neuroprotective effect of sTREM2 on ischemic stroke is an immunomodulatory mechanism independent of TREM2.

[0016] In a preferred embodiment of the application described in this invention, the stroke is an ischemic stroke.

[0017] As a preferred embodiment of the application described in this invention, the fusion protein is injected stereotactically into the striatum region of the brain.

[0018] The striatum is an important brain region that regulates motor control and cognitive function. This invention directly applies the human-derived sTREM2-6His fusion protein to the striatum on the ischemic side, which has great potential to alleviate symptoms such as hemiplegia, sensory, language and cognitive impairment caused by ischemic stroke.

[0019] In a preferred embodiment of the application described in this invention, the fusion protein is administered 35 minutes after the middle cerebral artery embolization.

[0020] In a preferred embodiment of the application described in this invention, the dosage of the fusion protein is 5–6 μg.

[0021] In a preferred embodiment of the application described in this invention, the injection rate of the fusion protein is 200–300 nL / min.

[0022] Animal experiments have validated that the applicant used a transient middle cerebral occlusion (tMCAO) mouse model to simulate human ischemic stroke. In C57BL / 6 mice, 35 minutes after middle cerebral artery occlusion (the growth rate of 1-6 month old mice is 45 times that of humans, equivalent to 26.25 hours after a human ischemic stroke), a human-derived sTREM2-6His fusion protein solution was stereotactically injected into the striatum region of the brain. This significantly reduced the infarct volume in the tMCAO model mice. Therefore, the human-derived sTREM2-6His fusion protein has a significant therapeutic effect on ischemic stroke, and its therapeutic window is longer than that of existing thrombolytic drugs.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention provides a fusion protein and its application in the preparation of drugs for treating stroke. The fusion protein designed in this invention is derived from the human body, and its metabolites are amino acids, thus exhibiting superior safety compared to other therapeutic drugs. Furthermore, the fusion protein of this invention can significantly reduce the infarct volume in the brain of tMCAO model mice, demonstrating significant therapeutic effects on ischemic stroke, and exhibiting a longer therapeutic window than existing thrombolytic drugs. Compared to existing ischemic stroke treatments, the human-derived sTREM2-6His fusion protein possesses a novel therapeutic mechanism and target. Attached Figure Description

[0025] Figure 1Figure showing the effect of human sTREM2-6His fusion protein on a transient middle cerebral artery embolism model;

[0026] Figure 2 Immunohistochemical (IHC-Fr) results of frozen sections of mouse brains treated in different groups (the subscript on the right is 100 μm). Detailed Implementation

[0027] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0029] Example 1: Effects of human sTREM2-6His fusion protein on a transient middle cerebral artery embolism model

[0030] Design of human-derived sTREM2-6His fusion protein: The human-derived sTREM2-6His fusion protein of the present invention is composed of a polypeptide (abbreviated as sTREM2) as shown in SEQ ID NO:1 and 6 histidines (abbreviated as 6His polypeptide), the 6 histidines being located at the C-terminus of the polypeptide as shown in SEQ ID NO:1.

[0031] Specific steps:

[0032] (1) The experimental subjects were C57BL / 6 mice, 8-10 weeks old, 30 mice, SPF grade, weighing 26-28g, with free access to food and water. The experiment was conducted after the animals had adapted for one week.

[0033] (2) Establishment of a transient middle cerebral occlusion (tMCAO) model: Adult male C57BL / 6 mice weighing 20-30g were selected for modeling. The mice were anesthetized (intraperitoneal injection of 1% sodium pentobarbital, 45mg / kg), and after they were completely unconscious, they were fixed on the operating table in a supine position. The skin was cut open from the middle of the neck, and the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ECA) were separated. The two small arterial branches on the external carotid artery (ICA) and the extracorporeal carotid artery (ECA) were severed using a cauterizing iron. A knot was tied at the distal end of the ECA with 5-0 surgical suture, a knot at the proximal end with 4-0 surgical suture, and a slip knot with 5-0 surgical suture in the middle. A slip knot was tied at the CCA with 4-0 surgical suture. A suture plug (from Pingdingshan Yushun Biotechnology Co., Ltd.) was then inserted into the ECA, and the 5-0 surgical suture was tightened to secure the plug. The 4-0 surgical suture at the proximal end was then cut, and the plug was continued to be inserted until the bifurcation of the ECA and ICA. The distal end of the external carotid artery was severed, and the plug was pulled back by the severed end. The plug was then reinserted until resistance was felt, and finally the 4-0 surgical suture on the common carotid artery was tightened. The mouse was subjected to ischemia for 1 hour, during which time it was placed in an incubator. After the ischemia was relieved, the plug was removed to achieve reperfusion, and the skin was sutured. Mice were sacrificed 24 hours after reperfusion, and their brains were collected and stained with TTC (triphenyltetrazolium chloride) to calculate the infarct volume ratio of ischemic stroke.

[0034] (3) The experimental groups are as follows:

[0035]

[0036]

[0037] Note: Physiological saline was used in this experiment to dissolve the 6His peptide and fusion protein; heat-inactivated sTREM2-6His was the sTREM2-6His fusion protein that had been heat-inactivated for 1.5 hours to eliminate the influence of inorganic ions in the fusion protein solution.

[0038] (4) Striatal administration method (stereotactic injection): After 35 minutes of cerebral artery embolization in mice, the striatum coordinates on the ischemic side were stereotactically located (x - distance from the left / right side of the sagittal suture 2.5 mm, y - distance from the anterior fontanelle 0.5 mm, and z - depth 3.5 mm). The fusion protein sTREM2-6His (5 μg, 2 μl) / mouse or its control solution was injected using a Hamilton microsyringe at a rate of 200 nL / min.

[0039] Experimental results:

[0040] like Figure 1 As shown, the percentage of infarct volume in the ischemic cerebral hemisphere of the tMCAO+Vehicle group was significantly higher than that in the Sham+Vehicle group, and no infarction was observed in the brain tissue of mice in the sham group, which proves that the establishment of the mouse tMCAO model was successful. The Vehicle group was used as a control in all tMCAO groups. The results showed that the sTREM2-6His fusion protein significantly reduced the infarct volume in the ischemic cerebral hemisphere, excluding the influence of the solvent used to dissolve sTREM2-6His. However, injection of the 6His peptide or heat-inactivated sTREM2-6His had no significant effect on tMCAO-induced brain infarction. This clarifies that the phenomenon of the sTREM2-6His fusion protein significantly reducing the infarct volume in the ischemic cerebral hemisphere is due to the action of the active protein sTREM2 within the fusion protein, and the influence of inorganic salt ions in the fusion protein solution can be excluded. Furthermore, in the sham group, injection of either the 6His peptide or the sTREM2-6His fusion protein had no damaging effect on mouse brain tissue. This confirms that the 6His peptide and its fusion protein with sTREM2 are safe and have no toxic effects on brain tissue. Therefore, the above preliminary experimental results suggest that sTREM2 may be a key molecule in improving the pathological state of ischemic stroke.

[0041] Example 2: Immunohistochemical (IHC-Fr) Experiment of Frozen Sections of Mouse Brain

[0042] Immunohistochemical (IHC-Fr) experiments on frozen sections of mouse brain included the following groups:

[0043] 1) Sham+6His sham surgery group (6His polypeptide was injected into the striatum stereotactically 35 minutes after the sham surgery);

[0044] 2) tMCAO+6His group (6His polypeptide was stereotactically injected into the striatum at 35 minutes of ischemia);

[0045] 3) tMCAO+sTREM2-6His group (human-derived sTREM2-6His was injected into the striatum stereotactic region after 35 minutes of ischemia).

[0046] Twenty-four hours after reperfusion, mice in each group were sacrificed, and their brains were frozen sections. Immunofluorescence double staining was performed using anti-βIII-tubulin antibody and anti-His tag antibody, followed by confocal microscopy imaging of the brain slices. DAPI (blue) represents cell nuclei, βIII-tubulin (red) is a neuronal-specific marker, His (green) shows human sTREM2-6His, and Merge is a three-channel superposition image.

[0047] refer to Figure 2 The results showed that βⅢ-tubulin (red) and human sTREM2-6His (green) colocalized in brain slices of the tMCAO+sTREM2-6His group (a yellow colocalization image was produced after merging), and no 6His staining was found in either the Sham+6His sham-operated group or the tMCAO+6His group (indicating that the 6His peptide injected into the striatum does not bind to any protein in the brain tissue, nor is it taken up by cells in the brain tissue).

[0048] Compared to the Sham+6His sham-operated group, the striatal neurons in the tMCAO+6His group showed significant damage, morphological abnormalities, and a reduction in axons in brain slices. In contrast, the striatal neurons in the tMCAO+sTREM2-6His group showed normal morphology and an increase in axons compared to the tMCAO+6His group. This indicates that the 6His peptide itself has no toxic effect on the mouse brain, while sTREM2 has a protective effect against neurological damage caused by ischemic stroke. In conclusion, the development of human-derived sTREM2-6His fusion protein into a novel, safe, and effective treatment for stroke shows great promise and significant potential value.

[0049] In summary, the fusion protein designed in this invention is derived from the human body's own protein, and its metabolites are amino acids. Therefore, the fusion protein of this invention has superior safety compared to other therapeutic drugs, and it has a significant therapeutic effect on ischemic stroke, with a longer therapeutic window than existing thrombolytic drugs. The applicant used a transient middle cerebral occlusion (tMCAO) mouse model to simulate human ischemic stroke. In C57BL / 6 mice, 35 minutes after middle cerebral artery occlusion (the growth rate of 1-6 month old mice is 45 times that of humans, equivalent to 26.25 hours after ischemic stroke in humans), a human-derived sTREM2-6His fusion protein solution was stereotactically injected into the striatum region of the brain, and it was found that it significantly reduced the infarct volume in the brain of the tMCAO model mice. Therefore, the human-derived sTREM2-6His fusion protein has a significant therapeutic effect on ischemic stroke, with a longer therapeutic window than existing thrombolytic drugs.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of a fusion protein in the preparation of a medicament for treating ischemic stroke, characterized in that, The fusion protein consists of a polypeptide as shown in SEQ ID NO: 1 and 6 histidines at the C-terminal end of the polypeptide as shown in SEQ ID NO:

1.

2. Use according to claim 1, wherein The fusion protein is injected by stereotactic injection into the striatum brain region.

Images