Human serum albumin adhesion peptide and its use

Abstract

The application discloses a kind of recombinant human serum albumin-atipuase nano medicine (rHSA-HSAbp-rt-PA) and its preparation method and application.The nano medicine includes atipuase (rt-PA), human serum albumin adhesion peptide (HSAbp) and recombinant human serum albumin (rHSA), wherein the sequence of the human serum albumin adhesion peptide is VGPLGPHYYYCAADLWRL.Recombinant human serum albumin-atipuase nano medicine is connected by human serum albumin adhesion peptide, avoids the step of chemical crosslinking, and the preparation method is simple.The half-life of the recombinant human serum albumin-atipuase nano medicine disclosed in the application is up to 29 min, the treatment effect is better, and the application prospect in the treatment of thrombus diseases is wide.

Description

[0001] This invention application is a divisional application of Chinese patent application filed on June 4, 2025, entitled "A method for preparing and applying recombinant human serum albumin-alteplase nanomedicine" with application number "202510734281.1". Technical Field

[0002] This invention relates to the field of nanomedicine technology, specifically to a method for preparing and applying recombinant human serum albumin-alteplase. Background Technology

[0003] Thrombotic diseases (such as stroke, acute myocardial infarction, pulmonary embolism, and deep vein thrombosis) are among the leading causes of disability and death worldwide. Thrombolysis is a crucial treatment for thrombotic diseases, and even with the widespread use of interventional procedures today, thrombolytic therapy remains irreplaceable, especially in stroke treatment. In recent years, the emergence of new thrombolytic drugs has provided clinicians with a wider range of options, but they may not have an in-depth understanding of each drug.

[0004] Currently, common thrombolytic drugs have undergone multiple updates and iterations. The first generation of thrombolytic drugs is represented by alteplase. Alteplase (rt-PA) is a recombinant tissue plasminogen activator (t-PA) prepared by genetic engineering technology. It is a single-chain serine protease composed of 527 amino acids, but its half-life is extremely short, only 4-5 minutes, requiring continuous intravenous infusion. To solve the problem of its short half-life, many studies have been carried out, mainly including (1) tissue plasminogen activator mutants: represented by reteplase and tenecteplase; (2) tissue plasminogen activator fusion proteins: CN113416724A discloses a tPA. 146Y -HSA-FBP fusion protein, which contains a partial sequence of HSA, and its biocompatibility is increased by the partial sequence of HSA; (3) rtPA modified by nano-targeted delivery system: rtPA is extended by coating it with polymer or liposomes, and rtPA-containing nanoparticles are modified with antibodies, peptides, biomembranes or magnetic effects to give rtPA thrombus targeting and increase local drug concentration. More significant therapeutic effect than free rtPA was observed in animals.

[0005] Human serum albumin (HSA), a commonly used drug delivery material, possesses excellent drug-carrying properties due to its unique molecular structure and has been widely applied in the preparation of nanomedicines. For example, albumin-bound paclitaxel drugs have propelled the paclitaxel market to new heights. HSA has abundant amino acid residues and a specific spatial structure, enabling it to bind tightly to active ingredients through various non-covalent interactions, such as hydrogen bonds, hydrophobic interactions, and electrostatic interactions. This ability to prolong the half-life of active ingredients and its targeted nature provides a new approach to addressing the half-life of tissue plasminogen activators. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention discloses a method for preparing and applying recombinant human serum albumin-alteplase nanomedicine. The specific details of this invention are as follows:

[0007] In a first aspect, the present invention provides a recombinant human serum albumin-alteplase nanomedicine (rHSA-HSAbp-rt-PA). The nanomedicine comprises alteplase (rt-PA), human serum albumin adhesion peptide (HSAbp), and recombinant human serum albumin (rHSA), wherein the sequence of the human serum albumin adhesion peptide is VGPLGPHYYYCAADLWRL (SEQ ID NO. 1).

[0008] Furthermore, the molar ratio of alteplase (rt-PA) to recombinant human serum albumin nanomedicine is 0.5:1 to 3:1, preferably, the molar ratio of alteplase (rt-PA) to recombinant human serum albumin is 1:1.

[0009] Furthermore, the alteplase and the human serum albumin adhesion peptide are linked by chemical bonds or non-chemical bonds.

[0010] Furthermore, the alteplase and human serum albumin adhesion peptide are linked by cross-linking and / or fusion protein methods.

[0011] Furthermore, the cross-linking method includes at least one of chemical cross-linking, physical cross-linking, enzymatic cross-linking, and photocross-linking.

[0012] Furthermore, the cross-linking agent in the chemical cross-linking method includes at least one of glutaraldehyde, genipin, and carbodiimide (EDC / NHS); the physical cross-linking method includes at least one of ionic cross-linking and thermally induced cross-linking; and the enzyme in the enzymatic cross-linking method includes at least one of transglutaminase and horseradish peroxidase (HRP).

[0013] Furthermore, the alteplase and the human serum albumin adhesion peptide are linked by a fusion protein, preferably the human serum albumin adhesion peptide is linked to the N-terminus or C-terminus of the alteplase.

[0014] Furthermore, the human serum albumin adhesion peptide is linked to recombinant human serum albumin via chemical bonds or non-chemical bonds.

[0015] In a specific embodiment of the present invention, the human serum albumin adhesion peptide is linked to recombinant human serum albumin via a non-chemical bond.

[0016] In a second aspect, the present invention provides a composition comprising at least the recombinant human serum albumin-alteplase nanomedicine and pharmaceutically acceptable additives.

[0017] Furthermore, the dosage form of the composition includes at least one of nanosuspension, nanoemulsion, and lyophilized powder.

[0018] Furthermore, alteplase in this invention can be replaced with other protein drugs. Preferably, the protein drugs include at least one of cytokine drugs, enzyme drugs, antibody drugs, hormone drugs, and vaccine protein drugs.

[0019] More preferably, the cytokine drugs include at least one of interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony-stimulating factor (CSF), and erythropoietin (EPO); the enzyme drugs include at least one of adenosine deaminase, phenylalanine lyase, pancreatic enzyme, and terepramase; and the hormone drugs include at least one of insulin, growth hormone, and gonadotropins.

[0020] A third aspect of the present invention provides the use of the recombinant human serum albumin-alteplase nanomedicine or the composition thereof in the preparation of medicaments for the prevention and / or treatment of thrombotic diseases.

[0021] Furthermore, the thrombotic diseases include at least one of ischemic stroke, myocardial infarction, peripheral artery disease, thromboangiitis obliterans, deep vein thrombosis, pulmonary embolism, portal vein thrombosis, cerebral venous sinus thrombosis, disseminated intravascular coagulation, thrombotic microangiopathy, and antiphospholipid antibody syndrome.

[0022] In a fourth aspect, the present invention provides a method for preparing the recombinant human serum albumin-alteplase nanomedicine, the method comprising the following steps:

[0023] S1: Human serum albumin adhesion peptide and alteplase are linked together by cross-linking or recombinant genetic engineering technology;

[0024] S2: Mix the HSAbp-rt-PA prepared in step S1 with the recombinant human serum albumin solution to obtain the recombinant human serum albumin-alteplase nanomedicine.

[0025] Furthermore, the cross-linking method in step S1 includes at least one of chemical cross-linking, physical cross-linking, enzymatic cross-linking, and photocross-linking.

[0026] Furthermore, the cross-linking agent in the chemical cross-linking method includes at least one of glutaraldehyde, genipin, and carbodiimide (EDC / NHS); the physical cross-linking method includes at least one of ionic cross-linking and thermally induced cross-linking; and the enzyme in the enzymatic cross-linking method includes at least one of transglutaminase and horseradish peroxidase (HRP).

[0027] Furthermore, the specific operation of linking human serum albumin adhesion peptide and alteplase together using recombinant genetic engineering technology in step S1 is as follows:

[0028] S1-1: Link human serum albumin adhesion peptide to the N-terminus or C-terminus of alteplase to obtain the amino acid sequence of the fusion protein;

[0029] S1-2: Optimize the nucleotides of the fusion protein according to the codon preference of the recombinant cells;

[0030] S1-3: The nucleotides of the optimized fusion protein are introduced into recombinant cells to express the corresponding fusion protein.

[0031] Furthermore, the method also includes a step of purifying the recombinant human serum albumin-alteplase nanomedicine.

[0032] Furthermore, the method also includes the step of freeze-drying and refrigerating the recombinant human serum albumin-alteplase nanomedicine.

[0033] The beneficial effects of the present invention include, but are not limited to:

[0034] The recombinant human serum albumin-alteplase nanomedicine disclosed in this invention is linked by human serum albumin adhesion peptides, avoiding the chemical cross-linking step, and the preparation method is simple. The recombinant human serum albumin-alteplase nanomedicine of this invention has a long half-life of 29 minutes, resulting in better therapeutic effects and broad application prospects in the treatment of thrombotic diseases. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0036] Figure 1 This is a gel electrophoresis image of the nanomedicine (rHSA-HSAbp-rt-PA) in an embodiment of the present invention.

[0037] Figure 2 This is a schematic diagram of the cytotoxicity test results of rHSA-HSAbp-rt-PA and HSAbp-rt-PA in the embodiments of the present invention.

[0038] Figure 3 This is a schematic diagram of the "blood drug activity-time curve" results for rHSA-HSAbp-rt-PA and HSAbp-rt-PA in the embodiments of the present invention. Detailed Implementation

[0039] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0040] Recombinant human serum albumin (rHSA): Produced by Tonghua Anruit Biopharmaceutical Co., Ltd., batch number ART103L-240124-01. This rHSA has the same amino acid sequence as human serum albumin.

[0041] Alteplase: Ertonil, purchased from Boehringer Ingelheim.

[0042] Example 1: Screening of human serum albumin-binding peptides

[0043] Using bioinformatics methods, the spatial structural characteristics of monoclonal antibodies or nanobodies against human serum albumin (HSA) and the surface charge of HSA were analyzed to design a series of peptides that can bind to HSA. After direct synthesis using chemical synthesis, the affinity between the peptide molecules and HSA was determined using surface plasmon resonance (SPR) technology. The specific steps are as follows: An EDC / NHS mixture (1:1 ratio) was injected at a flow rate of 10 μL / min to activate the carboxyl groups on the chip surface, forming reactive ester groups. HSA solution (flow rate 10 μL / min) was injected into the activated chip surface to covalently bind to the activated carboxyl groups. 1M ethanolamine (pH 8.5) was injected at a flow rate of 10 μL / min to block unreacted ester groups and prevent non-specific binding. The chip surface was rinsed with PBS buffer (flow rate 10 μL / min) until the baseline stabilized. Then, 0.1 μM peptide solution (flow rate 30 μL / min) was sequentially injected into the activated chip, and the binding signal was monitored. Human serum albumin adhesion peptide sequences with equilibrium dissociation constants less than 0.01 μM were screened.

[0044] Following the above steps, we screened out the human serum albumin adhesion peptide VGPLGPHYYYCAADLWRL (SEQ ID NO.1) and named it HSAbp.

[0045] Example 2: Preparation of fusion protein HSAbp-rt-PA

[0046] HSAbp was linked to the N-terminus of a recombinant tissue plasminogen activator (sequence shown in SEQ ID NO.2) to form the fusion protein HSAbp-rt-PA (sequence shown in SEQ ID NO.3).

[0047] According to the E. coli preference, the nucleotide sequence encoding the fusion protein HSAbp-rt-PA was codon optimized to obtain the corresponding nucleotide sequence (as shown in SEQ ID NO.4).

[0048] The fusion protein HSAbp-rt-PA was cloned into the expression vector pET-22b (containing the His tag) by a commissioned biotechnology company. The expression vector pET-22b was then used to transform BL21(DE3) Escherichia coli, which were then plated on LB agar plates containing ampicillin and incubated at 37°C for 12 hours.

[0049] Pick a single colony and inoculate it into 5 mL of LB liquid medium (containing Amp), and incubate at 37°C with shaking for 12 hours. Transfer to 500 mL of LB medium at a 1:100 ratio and incubate at 37°C until OD (outcome limit) is reached. 600 =0.6. Add IPTG to a final concentration of 0.5 mM, induce at 25°C for 16 hours, collect the bacterial cells, centrifuge at 4°C, 8000×g for 10 minutes, discard the supernatant, and wash the bacterial pellet with PBS buffer (pH 7.4). Resuspend the bacterial cells in lysis buffer (containing 1 mM PMSF and 10 mM imidazole), and sonicate on ice (300W, 3 seconds on, 5 seconds off, for 20 minutes). Centrifuge at 4°C, 12000×g for 30 minutes, and collect the supernatant.

[0050] The Ni-NTA column was equilibrated with binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). The crude extract was loaded onto the column at a flow rate of 1 mL / min to bind the His-tagged protein. Non-specifically bound proteins were washed sequentially with buffers containing 20 mM and 50 mM imidazole. The nanobody was eluted with buffer containing 250 mM imidazole, and the elution peak was collected. The eluent was transferred to a dialysis bag (molecular weight cutoff 25 kDa), and dialyzed against PBS for 24 hours (4°C, buffer changes 3 times) to remove imidazole, yielding the purified fusion protein HSAbp-rt-PA. Gel electrophoresis showed that the molecular weight of the fusion protein HSAbp-rt-PA was approximately 61 kDa.

[0051] Example 3: Construction of a nanomedicine (rHSA-HSAbp-rt-PA)

[0052] Weigh 100 mg of rHSA and dissolve it in 10 mL of PBS (pH 7.4) to prepare a 10 mg / mL rHSA solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4 °C for later use.

[0053] Weigh 100 mg of the fusion protein HSAbp-rt-PA and dissolve it in 10 mL of PBS (pH 7.4) to prepare a 10 mg / mL HSAbp-rt-PA solution. Filter the solution through a 0.22 μm filter membrane for sterilization and store at 4 °C for later use.

[0054] HSAbp-rt-PA and rHSA were mixed at a molar ratio of 0.5 to 3:1 and reacted for 3-5 minutes. The reaction solution was then placed in a dialysis bag (MWCO 100kDa) for storage to obtain the (rHSA-HSAbp-rt-PA) stock solution. The solution was dialyzed in 1L PBS for 24 hours (with 3 changes of solution). The purified nanoparticle suspension was then detected by gel electrophoresis, frozen (-80℃, 2 hours), and then freeze-dried for 24 hours to obtain a solid powder.

[0055] The results are as follows Figure 1 As shown.

[0056] Depend on Figure 1 It was observed that HSAbp-rt-PA:rHSA bound completely to rHSA when the molar ratio was 1:1. At molar ratios of 2:1 and 3:1, a light-colored band of approximately 190 kDa appeared. Therefore, it was inferred that HSA contains two binding sites for the fusion protein HSAbp-rt-PA, but one of them has weaker binding affinity. The rHSA-HSAbp-rt-PA fusion protein with a 1:1 molar ratio of HSAbp-rt-PA:rHSA was selected for further experiments.

[0057] Example 4: Cytotoxicity assays of rHSA-HSAbp-rt-PA and HSAbp-rt-PA (CCK-8 assay)

[0058] MTT / CCK-8 assay: HUVEC cells were seeded (10 4Cells / wells were adhered to the culture medium and then divided into groups: blank group (cell-free), cell control group (HUVEC cells + complete medium), alteplase group (1, 2.5, 5, 10, 20, 50 μg / mL), HSAbp-rt-PA fusion protein group (equivalent rt-PA concentrations of 1, 2.5, 5, 10, 20, 50 μg / mL), rHSA-HSAbp-rt-PA nanoparticle group (equivalent rt-PA concentrations of 1, 2.5, 5, 10, 20, 50 μg / mL), and rHSA group (drug-free rHSA NPs, excluding carrier toxicity). After 24 h of incubation, 10 μL of CCK-8 working solution (10% CCK-8 + 90% serum-free medium) was added to each well, and the absorbance at 450 nm was measured after 1 h. Experiments were independently repeated ≥3 times to exclude edge effects. The experimental results were statistically analyzed using SPSS, and the results are shown in Table 1. Figure 2 As shown.

[0059] Calculate cell viability:

[0060]

[0061] Table 1

[0062]

[0063] Wherein, * indicates P<0.05 compared with the cell control group, ** indicates P<0.01 compared with the cell control group, # indicates P<0.05 compared with the rt-PA group, and ## indicates P<0.01 compared with the rt-PA group.

[0064] According to Table 1 and Figure 2 It can be seen that the cytotoxicity of the HSAbp-rt-PA group (rHSA-HSAbp-rt-PA group) is much lower than that of the rt-PA group.

[0065] Example 5: Activity Experiment of rHSA-HSAbp-rt-PA and HSAbp-rt-PA

[0066] The activity of rHSA-HSAbp-rt-PA and HSAbp-rt-PA solutions was detected using a human t-PA ELISA kit (purchased from Shanghai Huabang Biotechnology Co., Ltd.). The equivalent rt-PA concentration was 1 μg / mL, with rt-PA as a reference. The results are shown in Table 2.

[0067] Table 2

[0068] Group rt-PA group HSAbp-rt-PA group rHSA-HSAbp-rt-PA group Activity (IU / mL) 756±24 714±17 708±11

[0069] The results showed that the activities of rHSA-HSAbp-rt-PA and HSAbp-rt-PA were not significantly different from those of rt-PA.

[0070] Example 6: Detection of in vitro thrombolysis using rHSA-HSAbp-rt-PA and HSAbp-rt-PA

[0071] Fresh blood from healthy individuals was placed in an anticoagulant tube. Thrombin (1-5 U / mL) and CaCl2 (10-20 mM) were added to the anticoagulant whole blood. After mixing, the mixture was injected into a silicone tube or a 96-well plate and incubated at 37°C for 1-2 hours to form a thrombus. The thrombus was gently removed, rinsed three times with PBS, and the surface moisture was blotted dry with filter paper. The thrombus was weighed (recorded as W0). The thrombus was then placed in a PBS solution containing thrombolytic drugs, with an equal volume of PBS used for the control group. The mixture was incubated at 37°C with constant shaking (50-100 rpm) for 2 hours. The reaction was terminated, and any remaining thrombus was removed, rinsed with PBS, blotted dry, and weighed (recorded as W1). The thrombolysis rate was calculated. The experimental groups consisted of rHSA-HSAbp-rt-PA and HSAbp-rt-PA, while the positive control was alteplase (rt-PA). The concentration of rt-PA, the active ingredient in rHSA-HSAbp-rt-PA, HSAbp-rt-PA, and alteplase, was the same (10 μg / mL). The negative control was an equal volume of PBS solution. Formula: The results are shown in Table 3.

[0072] Table 3

[0073]

[0074] ** indicates that the p-value is ≤0.01 compared to the PBS group.

[0075] As shown in Table 3, statistical analysis revealed no significant differences in the in vitro thrombolytic effects of rHSA-HSAbp-rt-PA, HSAbp-rt-PA, and alteplase.

[0076] Example 7: Half-life detection of rHSA-HSAbp-rt-PA and HSAbp-rt-PA

[0077] Twenty adult SD rats, half male and half female (n=10 per group), were randomly divided into two groups. Each group received an intravenous bolus injection of rHSA-HSAbp-rt-PA or HSAbp-rt-PA (the clinically equivalent dose of rt-PA was 0.9 mg / kg). Blood samples were collected via the tail vein at 0 (baseline), 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, and 120 minutes. Serum was collected, and the biological activity of rt-PA retained in the serum was detected using a human t-PA ELISA kit (purchased from Shanghai Huabang Biotechnology Co., Ltd.). Blood drug activity-time curves were plotted, and data fitting was performed using software to analyze pharmacokinetic parameters. The results are shown in Table 4. Figure 3 As shown.

[0078] Table 4

[0079] PK parameters HSAbp-rt-PA rHSA-HSAbp-rt-PA <![CDATA[T 1 / 2β (min)]]> 18.36 29.14 <![CDATA[T peak (min)]]> 10.49 16.26

[0080] From Table 4 and Figure 3 It can be seen that the half-life of rHSA-HSAbp-rt-PA and HSAbp-rt-PA is significantly longer than that of rt-PA (half-life 4-5 min), and the half-life of rHSA-HSAbp-rt-PA is longer than that of HSAbp-rt-PA. It is speculated that HSAbp-rt-PA alone is partially cleared after entering the blood and before it binds to HSA in the plasma.

[0081] Example 8: Animal model experiments using rHSA-HSAbp-rt-PA and HSAbp-rt-PA

[0082] Middle Cerebral Artery Occlusion (MCAO) Model: Adult mice were fasted for 12 hours prior to the experiment, but water was allowed. Mice were anesthetized by intraperitoneal injection of 5% chloral hydrate (0.1 mL / 10 g body weight). After anesthesia took effect, the mice were fixed supine on the operating table. A midline incision was made in the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated. The ECA was ligated proximal to the heart, and sutures were threaded under the CCA and ICA for later use. A small incision was made approximately 3-4 mm from the bifurcation of the ECA, and a pre-treated suture plug (approximately 0.26-0.28 mm in diameter) was inserted into the ICA through the ECA incision to a depth of approximately 18-20 mm, blocking blood flow to the middle cerebral artery and inducing cerebral ischemia. The ischemia time was controlled to 60 minutes, after which the suture plug was slowly removed, allowing reperfusion of blood flow.

[0083] Drug administration: The experimental group was administered rHSA-HSAbp-rt-PA and HSAbp-rt-PA, while the positive control group was administered alteplase (rt-PA). The concentrations of rt-PA, the active ingredient in rHSA-HSAbp-rt-PA, HSAbp-rt-PA, and alteplase were the same. The negative control group was administered an equal volume of PBS solution. During cerebral ischemia-reperfusion, each group was injected via the tail vein at a relative dose of 0.9 mg / kg body weight. Twenty-four hours after reperfusion, the rats were anesthetized with 5% chloral hydrate, decapitated, and the brain was removed. Brain slices were placed in 2% TTC staining solution and stained at 37°C for 8 minutes in the dark. Images were taken, and the infarct volume was calculated.

[0084] Percentage of infarct volume = (Volume of contralateral hemisphere - Volume of non-infarcted area in ipsilateral hemisphere) / Volume of contralateral infarcted hemisphere × 100%.

[0085] The results are shown in Table 5.

[0086] Table 5

[0087] Group PBS group rt-PA group HSAbp-rt-PA group rHSA-HSAbp-rt-PA group Cerebral infarction area (%) 31.85 21.47* 17.39**# 15.08**#

[0088] Where * indicates P<0.05 compared to the PBS group, ** indicates P<0.01 compared to the PBS group, # indicates P<0.05 compared to the rt-PA group, and ## indicates P<0.01 compared to the rt-PA group.

[0089] As shown in Table 5, the rHSA-HSAbp-rt-PA and HSAbp-rt-PA groups were significantly more effective than the rt-PA group in treating cerebral infarction.

[0090] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A human serum albumin adhesion peptide, characterized in that, The amino acid sequence of the human serum albumin adhesion peptide is VGPLGPHYYYCAADLWRL (SEQ ID NO. 1).

2. The use of the human serum albumin adhesion peptide as described in claim 1 in the preparation of alteplase (rt-PA) drugs with extended half-life.

3. The application according to claim 2, characterized in that, The alteplase (rt-PA) and human serum albumin adhesion peptide are linked by a fusion protein, with the human serum albumin adhesion peptide attached to the N-terminus of the alteplase (rt-PA).

4. The application according to any one of claims 2-3, characterized in that, The drug also includes pharmaceutically acceptable excipients or additives.

5. A fusion protein, characterized in that, The fusion protein is composed of the human serum albumin adhesion peptide as described in claim 1 and alteplase (rt-PA), and the amino acid sequence of the fusion protein is shown in SEQ ID NO.

3.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule is the nucleic acid molecule encoding the fusion protein of claim 5.

7. The nucleic acid molecule according to claim 6, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.

4.

8. A method for preparing the fusion protein as described in claim 5, characterized in that, Includes the following steps: (1) The nucleotide sequence of human serum albumin adhesion peptide was linked to the 5' end of the alteplase (rt-PA) nucleotide sequence and constructed onto the target body to obtain a recombinant vector; (2) The recombinant vector was transferred into the target strain, and the recombinant engineered strain was obtained by screening. (3) The recombinant engineered bacteria are fermented and cultured, the bacterial cells are collected and lysed, and the fusion protein described in claim 5 is purified.

9. The use of the fusion protein as described in claim 5 in the preparation of alteplase (rt-PA) nanomedicines with extended half-life.

10. The application according to claim 9, characterized in that, The dosage form of the nanomedicine includes at least one of nanosuspension, nanoemulsion, and lyophilized powder.

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