CN116239701A - Fusion protein and application thereof - Google Patents

Abstract

The invention relates to the technical field of biomedicine, and specifically discloses a fusion protein and its application. The fusion protein is composed of a polypeptide and 6 histidines, and the polypeptide has at least 9/10, 14/15 or 29/30 sequence identity with the amino acid sequence shown in SEQ ID NO: 1; 6 histidines The acid is located at the C-terminus of the polypeptide. The fusion protein designed in the present invention is derived from the human body's own protein, and its metabolites are amino acids, so it is better than other therapeutic drugs in terms of safety. Moreover, the fusion protein of the present invention can significantly reduce the infarct volume of the brain of tMCAO model mice, and can significantly treat ischemic stroke, and has a longer treatment time window than existing thrombolytic drugs. Compared with the existing therapeutic drugs for ischemic stroke, the human-derived sTREM2-6His fusion protein has a new therapeutic mechanism and target.

Description

A fusion protein and its application

technical field

The present invention relates to the technical field of biomedicine, in particular to a fusion protein and its application, in particular to the application of a fusion protein in the preparation of medicines for treating ischemic stroke.

Background technique

Stroke is a group of diseases that cause brain tissue damage due to sudden rupture of blood vessels in the brain or blood circulation disturbance caused by vascular obstruction, including ischemic stroke and hemorrhagic stroke. Ischemic stroke refers to local brain tissue including Necrosis of nerve cells, glial cells, and blood vessels due to lack of blood supply. Among them, stenosis or occlusion of internal carotid artery and vertebral artery can cause ischemic stroke, most of them are over 40 years old, and more men than women, severe cases can cause death

At present, the preferred treatment for ischemic stroke (commonly known as cerebral infarction) is to achieve reperfusion by intravenous thrombolysis and/or endovascular thrombectomy: intravenous thrombolysis within 4.5 hours after stroke onset can effectively reduce The occurrence of disability in patients; for patients with cerebral ischemia but not infarction in perfusion imaging, the treatment time window can be extended to less than 9 hours; in addition, for patients with extensive large vessel occlusion within 6 hours after stroke onset, and Endovascular thrombectomy within 24 hours of stroke onset was effective in reducing morbidity in patients diagnosed with perfusion imaging. However, both intravenous thrombolysis and endovascular resection must be strictly controlled within the time window of the onset of ischemic stroke. However, in reality, many patients have missed the optimal time window for thrombolysis when they are admitted to the hospital, which delays the timing of effective treatment. , and the effective rate of thrombolytic therapy is extremely low in the late stage of neurological injury in ischemic stroke.

Therefore, the strategy of extending the treatment time window is particularly important, and the neuroprotective strategy has always been a research hotspot in the treatment of ischemic stroke. The purpose of neuroprotective treatment is to delay the death of neurons, so as to buy time to restore cerebral blood flow reperfusion, and rescue reversibly damaged neurons and brain tissue. However, the curative effect of the currently developed neuroprotective drugs in clinical application is lower than that of animal models. This is because these neuroprotective drugs have certain toxicity to the human body, so the dose used in clinical practice is far lower than the effective dose of animal experiments, so that The expected therapeutic effect cannot be achieved. Therefore, the development of neuroprotective drugs targeting new targets has very important practical value.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a fusion protein and its application in the preparation of medicaments for the treatment of cerebral apoplexy. The fusion protein designed in the present invention is derived from the human body's own protein, and its metabolites are amino acids, so it is better than other therapeutic drugs in terms of safety. Moreover, the fusion protein of the present invention can significantly reduce the volume of infarction in the brain of tMCAO model mice, and can significantly treat ischemic stroke, and has a longer treatment time window than existing thrombolytic drugs.

In order to achieve the above object, the technical scheme that the present invention takes is:

The first purpose, the present invention provides a fusion protein, the fusion protein is composed of a polypeptide and 6 histidines, the polypeptide has an amino acid sequence shown in SEQ ID NO:1 at least 9/10, 14/15 or 29/30 sequence identity; 6 histidines are located at the C-terminus of the polypeptide.

The amino acid sequence shown in SEQ ID NO: 1 is HNTTVFQGVAGQSLQVSCPYDSMKHWGRRKAWCRQLGEKGPCQRVVSTHN LWLLSFLRRWNGSTAITDDTLGGLTTITLRNLQPHDAGLYQCQSLHGSEADTL RKVLVEVLADPLDHRDAGDLWFPGESESFEDAHVEH.

The fusion protein designed by the present invention (sTREM2 for short) is derived from the human body's own protein, and its metabolites are amino acids. Therefore, the fusion protein of the present invention is better than other therapeutic drugs in terms of safety, and has a significant effect on ischemic stroke. Treatment, and the treatment time window is longer than that of existing thrombolytic drugs.

Among them, the purpose of adding 6 histidines to the C-terminus of the polypeptide in the present invention is to efficiently purify the fusion protein, and the obtained fusion protein can reduce the volume of infarction in tMCAO model mice. The 6 histidines added to the fusion protein have no toxic effect. After the same experimental test, the striatum was injected with 6His polypeptide (6 histidine) or the fusion protein. It is suggested that the 6His polypeptide and its fusion protein with sTREM2 (the polypeptide shown in SEQ ID NO: 1) are safe.

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

The inventors of the present invention have found through extensive research and experiments that the newly designed fusion protein of the present invention (referred to as the human-derived sTREM2-6His fusion protein) and the existing human-derived TREM2 protein are two completely different proteins, and the former is secreted Soluble protein, 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 by ADAM10 or ADAM17, and this The sTREM2 in the fusion protein designed by the invention technology selects the protein sequence of His19-His157 in TREM2. Since the cleavage site of ADAM10 or ADAM17 is at the His157-Ser158 peptide bond of TREM2, this design greatly improves The structure of endogenous sTREM2 naturally produced by the body is restored, and the protein sequence that has a therapeutic effect on ischemic stroke is determined to be the original protein sequence of endogenous sTREM2, which is not only safe, but also can be used for future development. It provides a basis for the therapeutic strategy of releasing endogenous sTREM2 in patients with ischemic stroke to reduce brain damage after ischemia.

The second object is that the present invention provides the application of the above-mentioned fusion protein in the preparation of a medicament for the treatment of cerebral apoplexy.

sTREM2 and TREM2 used in the present invention are two different substances. TREM2 is an innate immune receptor specifically expressed on microglial cells in the brain parenchyma, and is a receptor protein with a single transmembrane structure; sTREM2 is an extracellular soluble protein released after the extracellular domain of TREM2 is hydrolyzed by integrin and metalloproteinase ADAM10 or ADAM17.

Previous studies have shown that sTREM2 can be produced through proteolysis in local tissues and then enter the cerebrospinal fluid after passing through the damaged blood-brain barrier. Therefore, in some neurodegenerative diseases, sTREM2 in the cerebrospinal fluid can be observed to increase, so it is proposed that sTREM2 It can be used as an inflammatory biomarker of AD, and studies have found that sTREM2 can improve the pathological phenotype of AD by regulating the function of microglial cells in AD models. However, the role of sTREM2 in ischemic stroke is still unclear, and the fusion protein designed by the present invention has clarified the mechanism of action. The experiment of the present invention can directly show that the human sTREM2-6His fusion protein is directly located in On neurons in ischemic brain tissue, the neuroprotective effect of sTREM2 on ischemic stroke is an immune regulatory mechanism independent of TREM2.

As a preferred embodiment of the application of the present invention, the stroke is ischemic stroke.

As a preferred embodiment of the application of the present invention, the fusion protein is injected into the striatum brain region of the brain through stereotaxic injection.

The striatum is an important brain region that regulates motor control and cognitive functions. The present invention directly acts on the human-derived sTREM2-6His fusion protein on the striatum brain tissue on the ischemic side to slow down the damage caused by ischemic stroke. Symptoms such as limb hemiplegia, sensory, language and cognitive impairment have a great potential role.

As a preferred embodiment of the application of the present invention, the administration time of the fusion protein is 35 minutes after middle artery embolization.

As a preferred embodiment of the application of the present invention, the dosage of the fusion protein is 5-6 μg.

As a preferred embodiment of the application of the present invention, the injection speed of the fusion protein is 200-300 nL/min.

After verification by animal experiments, the applicant simulates human ischemic stroke using a mouse model of transient middle cerebral artery occlusion (tMCAO). When the middle cerebral artery of C57BL/6 mice was embolized for 35 minutes (the growth rate of mice aged 1-6 months was 45 times that of humans, which was equivalent to 26.25 hours after human ischemic stroke), the brain striae Stereotaxic injection of human-derived sTREM2-6His fusion protein solution into the brain region of the shape body, and found that it can significantly reduce the infarct volume of the brain of tMCAO model mice. It can be seen that the human sTREM2-6His fusion protein has a significant therapeutic effect on ischemic stroke, and has a longer treatment time window than the existing thrombolytic drugs.

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

The invention provides a fusion protein and its application in the preparation of drugs for the treatment of cerebral apoplexy. The fusion protein designed in the invention is derived from the human body's own protein, and its metabolites are amino acids, so it is safer than other therapeutic drugs. excellent. Moreover, the fusion protein of the present invention can significantly reduce the volume of infarction in the brain of tMCAO model mice, and can significantly treat ischemic stroke, and has a longer treatment time window than existing thrombolytic drugs. Compared with the existing therapeutic drugs for ischemic stroke, the human sTREM2-6His fusion protein has a new therapeutic mechanism and target.

Description of drawings

Figure 1 is a graph showing the effect of human-derived sTREM2-6His fusion protein on the model of transient middle cerebral artery embolism;

Figure 2 is the results of immunohistochemical (IHC-Fr) experiments on frozen sections of mouse brains treated in different groups (the lower right is 100 μm).

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are commercially available unless otherwise specified.

Example 1. Effect of human-derived sTREM2-6His fusion protein on transient middle cerebral artery embolism model

Design of human-derived sTREM2-6His fusion protein: the human-derived sTREM2-6His fusion protein of the present invention consists of a polypeptide (sTREM2 for short) as shown in SEQ ID NO: 1 and 6 histidines (6His polypeptide for short), 6 The two histidines are located at the C-terminus of the polypeptide shown in SEQ ID NO:1.

Specific steps:

(1) The experimental subjects were C57BL/6 mice, 8-10 weeks old, 30 mice, SPF grade, weighing 26-28 g, free to eat and drink, and the animals were adapted to the experiment for one week.

(2) Establishment of transient middle cerebral artery occlusion (tMCAO) model: adult male C57BL/6 mice with a body weight of 20 to 30 g were selected for modeling: the mice were anesthetized (intraperitoneal injection of 1% ammonium sodium barbital, 45 mg/kg), and after it completely lost consciousness, it was fixed on the operating table in a supine position; the skin was cut from the middle of the neck, and the common carotid artery (CCA), carotid artery, and carotid artery were separated. The external carotid artery (ECA), internal carotid artery (internal carotid artery, ICA), the two small arterial branches on the ECA were scalded with an electric iron; the distal end of the ECA was knotted with 5-0 surgical thread, and the proximal Use 4-0 surgical thread to make a dead knot at the end, and use 5-0 surgical thread to make a slipknot in the middle; use 4-0 surgical thread to make a slipknot for the CCA; then insert the thread plug (Pingdingshan Yushun Biotechnology Co., Ltd.) from the ECA, and tighten for 5 -0 surgical thread to fix the suture, then cut the 4-0 suture at the proximal end and continue to insert the suture to the bifurcation of ECA and ICA. Cut off the distal end of the external carotid artery, pull the stump to make the thread plug turn around, and then insert the thread plug homeopathically until the resistance stops, and finally tighten the 4-0 surgical thread on the common carotid artery. During the bleeding period, the mice were placed in an incubator. After the ischemia was over, the suture was pulled out to achieve reperfusion, and the skin was sutured. The mice were sacrificed 24 hours after reperfusion, and the brains were removed for TTC (triphenyltetrazolium chloride, triphenyltetrazolium chloride) staining, and the infarct volume ratio of ischemic stroke was calculated.

(3) The experimental groups are as follows:

Note: Physiological saline is the solvent used to dissolve the 6His polypeptide and fusion protein in this experiment; heat-inactivated sTREM2-6His is the sTREM2-6His fusion protein that has been heat-inactivated for 1.5 hours to eliminate the influence of inorganic ions in the fusion protein solution .

(4) Striatal administration method (stereotaxic injection): Stereotaxic location on the striatum coordinate point on the ischemic side after 35 minutes of middle cerebral artery embolization in mice (x-distance from sagittal suture left/right 2.5 mm, y - 0.5 mm from the front porch and z - 3.5 mm deep), and the fusion protein sTREM2-6His (5 μg, 2 μl)/mouse or its control solution was injected using a Hamilton microsyringe at a speed of 200 nL/min.

Experimental results:

As shown in Figure 1, the infarct volume percentage of the ischemic cerebral hemisphere in the tMCAO+Vehicle group was significantly higher than that in the Sham+Vehicle group, and no infarction occurred in the brain tissue of the mice in the sham operation (sham) group, which proved that The establishment of mouse tMCAO model was successful. In the tMCAO group, the Vehicle group was used as the control. The results showed that the sTREM2-6His fusion protein significantly reduced the infarct volume of the cerebral hemisphere of the ischemic side, and the influence of the solvent for dissolving sTREM2-6His was excluded; while the injection of 6His polypeptide or heat quenching Live fusion protein (heat-inactivatd sTREM2-6His) has no significant effect on brain tissue infarction caused by tMCAO, which clarifies that sTREM2-6His fusion protein significantly reduces the infarct volume of cerebral hemisphere on the ischemic side, which is It is caused by the action of the active protein sTREM2 in the fusion protein, and the influence of inorganic salt ions in the fusion protein solution can be excluded. In addition, in the sham operation (sham) group, injection of 6His polypeptide or sTREM2-6His fusion protein had no damaging effect on the brain tissue of mice, which clarified that 6His polypeptide and its fusion protein with sTREM2 had no harmful effect on brain tissue. All have no toxic effects and are safe. Therefore, the above preliminary experimental results suggest that sTREM2 may become a key molecule in improving the pathological state of ischemic stroke.

Embodiment 2, the immunohistochemistry (IHC-Fr) experiment of frozen section of mouse brain

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

1) Sham+6His sham operation group (35 minutes after sham operation, striatum stereotaxic injection of 6His polypeptide);

2) tMCAO+6His group (stereotaxic injection of 6His polypeptide in the striatum at 35 minutes of ischemia);

3) tMCAO+sTREM2-6His group (stereotaxic injection of human sTREM2-6His in the striatum at 35 minutes of ischemia).

Mice in each group were sacrificed 24 hours after reperfusion, and their brains were taken for frozen sections, and immunofluorescent double staining was performed with anti-βⅢ-tubulin antibody and anti-His tag antibody, and then the brain slices were photographed with a confocal microscope. Among them, DAPI (blue) is the nucleus, βⅢ-tubulin (red) is the specific marker of neurons, His (green) shows the human sTREM2-6His, and Merge is the superimposed image of the three channels.

Referring to Figure 2, the results show that βⅢ-tubulin (red) and human sTREM2-6His (green) co-localize in the brain slices of the tMCAO+sTREM2-6His group (yellow co-localization images are produced after merging), and Sham+6His Neither the sham operation group nor the tMCAO+6His group had 6His staining (indicating that the 6His polypeptide injected into the striatum was neither combined with any protein in the brain tissue nor taken up by cells in the brain tissue).

Compared with the Sham+6His sham operation group, the striatal neurons in the brain slices of the tMCAO+6His group showed obvious damage, abnormal shape, and axon reduction, while the striatal neurons in the brain slices of the tMCAO+sTREM2-6His group Compared with the tMCAO+6His group, the morphological changes were normal and the axons increased, showing that the 6His polypeptide itself had no toxic effect on the mouse brain, and sTREM2 had a protective effect on the nerve damage of ischemic stroke. In summary, the development of human-derived sTREM2-6His fusion protein as a new stroke drug with high safety has good prospects and great potential value.

In summary, the fusion protein designed in the present invention is derived from the human body's own protein, and its metabolites are amino acids. Therefore, the fusion protein of the present invention is better than other therapeutic drugs in terms of safety, and has a significant effect on ischemic stroke. The treatment, and the treatment time window is longer than the existing thrombolytic drugs. The present applicant uses a mouse model of transient middle cerebral artery occlusion (tMCAO) to simulate human ischemic stroke. When the middle cerebral artery of C57BL/6 mice was embolized for 35 minutes (the growth rate of mice aged 1-6 months was 45 times that of humans, which was equivalent to 26.25 hours after human ischemic stroke), the brain striae Stereotaxic injection of human-derived sTREM2-6His fusion protein solution into the brain region of the shape body, and found that it can significantly reduce the infarct volume of the brain of tMCAO model mice. It can be seen that the human sTREM2-6His fusion protein has a significant therapeutic effect on ischemic stroke, and has a longer treatment time window than the existing thrombolytic drugs.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that The technical solution of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solution of the present invention.

Claims (8)

1. A fusion protein, characterized in that, the fusion protein is composed of a polypeptide and 6 histidines, and the polypeptide has at least 9/10, 14/15 or 29 of the amino acid sequence shown in SEQ ID NO:1 /30 sequence identity; 6 histidines are located at the C-terminus of the polypeptide. 2. The fusion protein according to claim 1, characterized in that, the fusion protein is composed of a polypeptide as shown in SEQ ID NO:1 and 6 histidines, and the 6 histidines are located as in SEQ ID NO:1 The C-terminus of the indicated polypeptides. 3. The application of the fusion protein according to claim 1 or 2 in the preparation of a medicament for the treatment of stroke. 4. The application according to claim 3, wherein the stroke is ischemic stroke. 5. The application according to claim 3, wherein the fusion protein is injected into the striatum brain region of the brain through stereotaxic injection. 6. The application according to claim 3, wherein the administration time of the fusion protein is 35 minutes after the middle artery embolization. 7. The application according to claim 3, wherein the dosage of the fusion protein is 5-6 μg. 8. The application according to claim 5, wherein the injection speed of the fusion protein is 200-300 nL/min.

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