Recombinant antiplatelet thrombolysin protein and nucleotide sequences therefor, preparation method therefor and use thereof

Abstract

Provided are a recombinant antiplatelet thrombolysin protein and nucleotide sequences therefor, a preparation method therefor and a use thereof. The provided recombinant antiplatelet thrombolysin protein is a high-purity, high-quality and high-activity protein obtained by optimization of the amino acid sequences of a natural antiplatelet thrombolysin protein, protein expression and purification. Using GPIb as a therapeutic target, compared with the natural antiplatelet thrombolysin protein, the recombinant antiplatelet thrombolysin protein has significant activity, and provides a new direction and a new perspective for the development of antiplatelet drugs.

Description

A recombinant antiplatelet thrombolytic protein, its nucleotide sequence, preparation method and application Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a recombinant antiplatelet thrombolytic protein, its nucleotide sequence, preparation method, and application. Background Technology

[0002] With the evolution of social structures driven by globalization, urbanization, and an aging population, cardiovascular diseases pose a serious threat to people's health. The root cause of cardiovascular disease is the formation and development of thrombi. A thrombus is a semi-clotted substance that forms on the surface of the heart and blood vessel lining in a living body. Thrombus formation can occur in arterial or venous circulation, disrupting blood flow and causing infarction in different organs and systems. In-depth research into the molecular mechanisms of coagulation has advanced the understanding of targeted therapies for thrombosis and facilitated further clinical decision-making. Among these advancements, the interaction between platelet glycoprotein Ib (GPIb) and von Willebrand factor (vWF) is a crucial initial and necessary step in thrombus formation.

[0003] Platelet glycoprotein Ib, also known as GPIb, is an adhesion receptor expressed on the platelet membrane, composed of four transmembrane subunits: GPIbα, GPIbβ, GPIX, and GPV. Von von Willebrand factor (vWF) is a key ligand that anchors platelets to the exposed subendothelial layer at the site of injury. The extracellular domain of GPIbα contains the binding site for vWF. Once activated by high shear stress (related to arterial circulation), vWF undergoes a conformational change and binds to GPIbα, initiating platelet adhesion during hemostasis and thrombus formation. This adhesion is a crucial step in hemostasis and thrombus formation.

[0004] The development of antithrombotic drugs has gone through three stages: anticoagulants, thrombolytics, and platelet aggregation inhibitors. Anticoagulants, due to their inability to directly dissolve thrombi and the non-specificity of their anticoagulant effect, are prone to causing bleeding side effects. While thrombolytics offer rapid and direct thrombolysis, their short plasma half-life often leads to bleeding with prolonged use, and the re-infarction rate after thrombolysis is relatively high. Since platelet aggregation is the final pathway for all thrombus formation, inhibiting platelet aggregation can most effectively inhibit thrombus formation and prevent re-infarction, with minimal impact on the coagulation system. Therefore, the development of antithrombotic drugs that inhibit platelet aggregation has a promising future.

[0005] Based on the mechanism of action of platelet membrane receptor GPIb and its ligand vWF, blocking the binding of GPIb to vWF prevents platelets from adhering to the vascular endothelial cell wall, thereby inhibiting platelet aggregation. Moreover, this effect is unrelated to fibrinogen and has no direct impact on the blood coagulation system, meaning that bleeding side effects are likely minimal. It is noteworthy that in cases of vascular endothelial damage, the interaction between platelet GPIb and plasma or endothelial cell vWF under high shear stress conditions reduces platelet flow velocity, thus initiating thrombus formation. Therefore, developing targeted drugs against GPIb may offer greater advantages in treating arterial thrombosis and preventing re-infarction.

[0006] Natural antiplatelet thrombolytic agents are polypeptide drugs extracted from the venom of the sharp-nosed viper (Viper fasciatus) in southern Anhui. They have antiplatelet adhesion and aggregation effects, but they still have the following drawbacks:

[0007] (1) The source of raw materials is limited and the composition is complex, with a low proportion of the main components, making it difficult to establish effective raw material control measures;

[0008] (2) The raw materials are expensive and the purification process is complex, resulting in a low recovery rate, which affects the scale and cost of the process;

[0009] (3) There are batch-to-batch differences in raw materials, which can easily lead to batch-to-batch differences in products;

[0010] (4) The post-translational modifications of natural proteins are complex, while existing quality control measures are limited, which increases the risk of clinical drug use. Summary of the Invention

[0011] The purpose of this application is to provide a recombinant antiplatelet thrombolytic protein, its nucleotide sequence, preparation method, and application, in order to solve the aforementioned problems of natural antiplatelet thrombolytic proteins.

[0012] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0013] In a first aspect, this application provides a recombinant antiplatelet thrombolytic protein, which is obtained by mutating the first 3 amino acids at the N-terminus of the B chain of a natural antiplatelet thrombolytic protein; wherein...

[0014] The amino acid sequence of the A chain of the natural antiplatelet thrombolytic protein is shown in SEQ ID NO.1, and the amino acid sequence of the B chain is shown in SEQ ID NO.2.

[0015] In one alternative embodiment, the mutation includes the removal of the first three amino acids GFC at the N-terminus, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.3;

[0016] And / or, the mutation includes replacing the 3rd amino acid at the N-terminus with a serine, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.4;

[0017] And / or, the mutation includes replacing the first 3 amino acids at the N-terminus with GAD, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.5;

[0018] And / or, the mutation includes replacing the first 3 amino acids at the N-terminus with D instead of GFC, and the amino acid sequence of the B chain of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO. 6;

[0019] And / or, the mutation includes replacing the N-terminal 3rd amino acid with alanine, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.7.

[0020] Secondly, this application provides a nucleotide sequence composition for encoding the above-mentioned recombinant antiplatelet thrombolytic protein, comprising a first nucleotide sequence for encoding an A-chain amino acid sequence and a second nucleotide sequence for encoding a B-chain amino acid sequence.

[0021] Thirdly, this application provides an expression cassette composition, including a first expression cassette and a second expression cassette, wherein the first expression cassette is used to express the first nucleotide sequence and the second expression cassette is used to express the second nucleotide sequence.

[0022] Fourthly, this application provides a recombinant plasmid composition, comprising a first recombinant plasmid and a second recombinant plasmid, wherein the first recombinant plasmid contains the aforementioned first nucleotide sequence or first expression cassette, and the second recombinant plasmid contains the aforementioned second nucleotide sequence or second expression cassette.

[0023] Fifthly, this application provides a recombinant plasmid, including a third recombinant plasmid, wherein the vector of the third recombinant plasmid includes at least two expression frames, containing the first nucleotide sequence and the second nucleotide sequence described above, or containing the first expression frame and the second expression frame described above.

[0024] Sixthly, this application provides a recombinant cell, which is obtained by co-transfecting mammalian cells with the above-mentioned recombinant plasmid composition, or by transfecting mammalian cells with the above-mentioned recombinant plasmid.

[0025] Seventhly, this application provides a method for preparing the above-mentioned recombinant antiplatelet thrombolytic protein, comprising the following steps:

[0026] The recombinant cells were cultured, and the supernatant was collected and purified.

[0027] Eighthly, this application provides a medicament, the active ingredient of which includes the above-mentioned recombinant antiplatelet thrombolytic protein.

[0028] Ninthly, this application provides the use of the above-mentioned recombinant antiplatelet thrombolytic protein in the preparation of a drug, wherein the drug has at least one of the following functions:

[0029] Antagonizes the binding of GPIb to its receptor;

[0030] Antiplatelet adhesion and / or aggregation;

[0031] Antithrombotic;

[0032] Treatment and / or prevention of cardiovascular disease;

[0033] Treatment and / or prevention of stroke, preferably ischemic stroke;

[0034] Reduce the extent of cerebral infarction in stroke;

[0035] Lowering neurobehavioral scores in stroke patients;

[0036] Reduce the number of microthrombi in stroke patients;

[0037] Reduces the number of CD41-positive areas, CD41 cumulative optical density, and fibrin-positive vessels in stroke patients;

[0038] Reduce the expression levels of ICAM-1 and P-selectin in stroke patients.

[0039] In one alternative embodiment, the drug is a chemical drug or a biological agent, which can be prepared by artificial synthesis, microbial synthesis or cell culture.

[0040] In one alternative embodiment, the drug comprises recombinant antiplatelet thrombolytic protein and pharmaceutically acceptable excipients. Specifically, the selection of excipients can be made by those skilled in the art based on actual needs and dosage forms, and the present invention does not impose specific limitations in this regard.

[0041] In one alternative embodiment, the drug comprises an oral formulation, an inhaled formulation, or an injectable formulation.

[0042] In one alternative embodiment, the oral formulation includes at least one of tablets, capsules, pills, granules, drop pills, microcapsules, or micropellets.

[0043] In one alternative embodiment, the recombinant antiplatelet thrombolytic protein in the oral formulation or injection has a mass-to-volume concentration of 0.5 μg / mL to 3 mg / mL.

[0044] In one alternative embodiment, when the oral or injectable formulation is administered, the dosage of recombinant antiplatelet thrombolytic protein is 0.1–1000 μg / kg.

[0045] Based on the above technical solution, this application has at least the following beneficial effects:

[0046] 1. The recombinant antiplatelet thrombolytic protein provided in this application is a high-purity, high-quality, and high-activity protein obtained by optimizing the amino acid sequence of natural antiplatelet thrombolytic protein, expressing and purifying it. With GPIb as the therapeutic target, it has significant activity compared to natural antiplatelet thrombolytic protein, providing a new direction and a new perspective for the development of antithrombotic drugs.

[0047] Specifically, this application constructs a sequence and transfects a stable mammalian cell line, employs modern fed-batch culture, and obtains the target protein component through several purification steps. The main advantages are as follows:

[0048] (1) Material control: All materials used are conventional biopharmaceutical materials, and there are complete quality control measures;

[0049] (2) Mammalian cells are used for secretory expression. The supernatant has a relatively simple composition and high expression level. Conventional chromatography packing materials can be used to achieve a high yield. The upstream and downstream processes are easy to scale up.

[0050] (3) Under the given process conditions, the recombinant protein process is easy to control and the post-translational modification mode is relatively simple, with small batch-to-batch differences and controllability.

[0051] 2. The recombinant antiplatelet thrombolytic protein provided in this application is a GPIb inhibitor, which has a high affinity for human CD42b / GPIbα but does not bind to vWF. Studies have shown that this recombinant antiplatelet thrombolytic protein is a potential effective drug for stroke patients, which may be beneficial in the treatment of ischemic stroke, and has been demonstrated in animal models to have a protective effect against local cerebral ischemia / reperfusion injury.

[0052] Specifically, while intravenous or intra-arterial rt-PA thrombolysis has shown promise in improving neurological outcomes, many patients remain unsuitable for this treatment. Furthermore, thrombolytic therapy generally carries a bleeding tendency (heparin 5%-12%, tirofiban approximately 3%, rt-PA approximately 3%). In addition to the bleeding risk, rt-PA can cause angioedema leading to airway obstruction and does not improve prognosis. There are currently unmet clinical needs regarding intraoperative medication for stroke treatment, necessitating the development of new drugs for stroke therapy.

[0053] This application discloses the use of the aforementioned recombinant antiplatelet thrombolytic protein in the preparation of drugs related to stroke treatment. This recombinant antiplatelet thrombolytic protein reduces the infarct size in model rats, significantly lowers neurological behavioral scores, significantly reduces the number of microthrombi in the infarcted brain, significantly reduces CD41-positive areas, CD41 cumulative optical density, and the number of fibrin-positive vessels in the infarcted brain, and significantly reduces the expression levels of ICAM-1 and P-selectin. Simultaneously, it has a low risk of bleeding, which is beneficial for improving the prognosis of stroke patients. Attached Figure Description

[0054] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0055] Figure 1 is a schematic diagram of the p2MG2T empty carrier in Embodiment 2 of this application;

[0056] Figure 2 is a schematic diagram of the recombinant plasmid in Example 2 of this application;

[0057] Figure 3 is a graph of the non-reducing SDS-PAGE detection results in Example 3 of this application;

[0058] Figure 4 is a growth curve of different mutants in fed-batch culture in a cell pool in Example 4 of this application;

[0059] Figure 5 is a schematic diagram of one-step purification in Example 4 of this application;

[0060] Figure 6 is a schematic diagram of the two-step purification process in Example 4 of this application;

[0061] Figure 7 is a graph of the non-reducing SDS-PAGE detection results of the purified protein in Example 5 of this application;

[0062] Figure 8 is a graph showing the relative activity detection results of the purified protein in Example 5 of this application;

[0063] Figure 9 is the live cell density growth curve during the upstream cell culture process in Example 7 of this application;

[0064] Figure 10 is a cell viability curve during the upstream cell culture process in Example 7 of this application;

[0065] Figure 11 is the titration curve of recombinant protein yield during the upstream cell culture process in Example 7 of this application;

[0066] Figure 12 is a graph showing the results of one-step downstream purification chromatography in Example 7 of this application;

[0067] Figure 13 is a graph showing the results of the two-step downstream purification chromatography in Example 7 of this application.

[0068] Figure 14 is a graph showing the results of cerebral infarction range and cerebral infarction inhibition rate in Embodiment 8 of this application;

[0069] Figure 15 is a graph of the animal neurobehavioral scoring results in Example 8 of this application;

[0070] Figure 16 is a graph showing the number of microthrombi on the infarct side in Embodiment 8 of this application;

[0071] Figure 17 is an immunohistochemical result of CD41 on the infarct side in Example 8 of this application;

[0072] Figure 18 is a graph showing the Fibrin-positive vessel count results on the infarct side in Example 8 of this application;

[0073] Figure 19 is a graph showing the P-selectin dosage results on the infarcted side in Example 8 of this application.

[0074] Figure 20 is a diagram showing the expression results of ICAM-1 on the infarcted side in Example 8 of this application. Detailed Implementation

[0075] To further illustrate the technical means and results adopted by this application to achieve the intended inventive purpose, the following preferred embodiments are used to describe in detail the specific implementation methods, technical solutions, and features according to this application. Specific features, structures, or characteristics in the various embodiments described below can be combined in any suitable form.

[0076] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0077] Antiplatelet thrombolytic proteins extracted from natural raw materials are difficult to control in terms of raw material quality, resulting in low yields and small batches. Based on the study of natural protein structure, this application mutates some sites while maintaining the original main sequence, transfects the synthesized sequence into mammalian cells, and obtains recombinant protein components through downstream purification steps such as three-step purification.

[0078] This invention aims to solve the following problems:

[0079] (1) Obtain optimized recombinant proteins with structures similar to natural proteins;

[0080] (2) A stable cell line was obtained, and protein molecules with anti-platelet aggregation activity could be obtained from the culture supernatant;

[0081] (3) Establish extraction processes for upstream cell culture, cell fluid treatment, capture, and purification of recombinant antiplatelet thrombolytic agents.

[0082] The present application will be further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0083] Abbreviation Reference Table:

[0084]

[0085] Example 1: Sequence optimization design strategy for recombinant antiplatelet thrombolytic protein

[0086] Based on the primary structure study of natural antiplatelet thrombolytic protein and information from the GeneBank database, the β-chain amino acid sequence of the unoptimized antiplatelet thrombolytic protein was determined as follows:

[0087] GFCCPLRWSSYEGHCYLVVKEKKTWEDAEKFCTEQRKGGHLVSVHSREEADFLVHLAYPILDLSLIWMGLSNMWNDCKREWSDGTKLDFKSWAKTSDCLIGKTDGDNQWLNMDCSKKHYFVCKFKL.

[0088] In the above β-chain amino acid sequence, the third amino acid at the N-terminus (cysteine) does not participate in the formation of a disulfide bond and is a free thiol group. The presence of a free thiol group may pose a significant risk to product stability. Mass spectrometry and purity studies show that the presence of this free thiol group may lead to the following problems:

[0089] (1) The incorrect cleavage of the signal peptide leads to the loss of the first 3 amino acids of the β chain (GFC);

[0090] (2) It produces certain modifications, such as glutathione modification;

[0091] (3) Two heterodimer molecules further form a tetramer through free thiol groups.

[0092] Protein structure prediction revealed that the N-terminus of the β-chain shown above was not located on the binding surface between the target protein and the drug targets (vWF and GPIb). Further analysis showed that the deletion of the three amino acids at the N-terminus had little impact on protein activity. Therefore, removing this third amino acid (cysteine) was considered to improve the quality of the target protein. To screen for the optimal mutation method, five β-chain mutants as shown in Table 1 were designed in this embodiment. Plasmids were constructed using these five mutants, and CHO-K1 cells were transfected to construct five different mutant cell pools. After fed-batch culture, small-scale samples were prepared for yield calculation and quality analysis, and suitable mutants were selected for subsequent studies.

[0093] Table 1. Mutant design of recombinant antiplatelet thrombolytic β chain

[0094]

[0095] The amino acid sequences involved in the embodiments of this application are as follows:

[0096] The amino acid sequence of the α chain (also known as the A chain) of the natural antiplatelet thrombolytic protein (SEQ ID NO.1):

[0097] DVDCLPGWSAYDQSCYRVFKLLKTWDDAEKFCTERPKGGHLVSIESAGERDFVAQLVSENKQTDNVWLGLKIQSKGQQCSTEWTDGSSVSYENFSEYQSKKCFVLEKNTGFRTWLNLNCGSEYTFVCKSPP.

[0098] The amino acid sequence of the β chain (also known as the B chain) of the natural antiplatelet thrombolytic protein (SEQ ID NO.2):

[0099] GFCCPLRWSSYEGHCYLVVKEKKTWEDAEKFCTEQRKGGHLVSVHSREEADFLVHLAYPILDLSLIWMGLSNMWNDCKREWSDGTKLDFKSWAKTSDCLIGKTDGDNQWLNMDCSKKHYFVCKFKL.

[0100] The amino acid sequence of the recombinant antiplatelet thrombolytic protein β chain (also known as B chain) mutant delGFC (SEQ ID NO.3):

[0101] CPLRWSSYEGHCYLVVKEKKTWEDAEKFCTEQRKGGHLVSVHSREEADFLVHLAYPILDLSLIWMGLSNMWNDCKREWSDGTKLDFKSWAKTSDCLIGKTDGDNQWLNMDCSKKHYFVCKFKL.

[0102] The amino acid sequence of the recombinant antiplatelet thrombolytic protein β chain (also known as B chain) mutant C3S (SEQ ID NO.4):

[0103] GFSCPLRWSSYEGHCYLVVKEKKTWEDAEKFCTEQRKGGHLVSVHSREEADFLVHLAYPILDLSLIWMGLSNMWNDCKREWSDGTKLDFKSWAKTSDCLIGKTDGDNQWLNMDCSKKHYFVCKFKL.

[0104] The amino acid sequence (SEQ ID NO.5) of the recombinant antiplatelet thrombolytic protein β chain (also known as B chain) mutant GFC2GAD:

[0105] GADCPLRWSSYEGHCYLVVKEKKTWEDAEKFCTEQRKGGHLVSVHSREEADFLVHLAYPILDLSLIWMGLSNMWNDCKREWSDGTKLDFKSWAKTSDCLIGKTDGDNQWLNMDCSKKHYFVCKFKL.

[0106] The amino acid sequence of the recombinant antiplatelet thrombolytic protein β chain (also known as B chain) mutant GFC2D (SEQ ID NO.6):

[0107] DCPLRWSSYEGHCYLVVKEKKTWEDAEKFCTEQRKGGHLVSVHSREEADFLVHLAYPILDLSLIWMGLSNMWNDCKREWSDGTKLDFKSWAKTSDCLIGKTDGDNQWLNMDCSKKHYFVCKFKL.

[0108] The amino acid sequence of the recombinant antiplatelet thrombolytic protein β chain (also known as B chain) mutant C3A (SEQ ID NO.7):

[0109] GFACPLRWSSYEGHCYLVVKEKKTWEDAEKFCTEQRKGGHLVSVHSREEADFLVHLAYPILDLSLIWMGLSNMWNDCKREWSDGTKLDFKSWAKTSDCLIGKTDGDNQWLNMDCSKKHYFVCKFKL.

[0110] Nucleotide sequence of the α chain (also known as the A chain) of a natural antiplatelet thrombolytic protein containing the TNFR signal peptide (SEQ ID NO. 8):

[0111] Atggcccccgtggccgtgtgggccgccctggccgtgggcctggagctgtgggccgccgcccacgccgacgtggactgcctgcccggctggtccgcctacgaccagtcctgctacagggtgttcaagctgctgaagacctgggacgacgccgagaagttctgcaccgagaggcccaagggcggccacctggtgtccatcgagtccgccggcgagagggacttcgtggcccagctggtgtccgagaacaagcagaccgacaacgtgtggctgggcctgaagatccagtccaagggccagcagtgctccaccgagtggaccgacggctcctccgtgtcctacgagaacttctccgagtaccagtccaagaagtgcttcgtgctggagaagaacaccggcttcaggacctggctgaacctgaactgcggctccgagtacaccttcgtgtgcaagtcccccccctga.

[0112] Nucleotide sequence of β chain (also known as B chain) of natural anti-platelet thrombolysin containing TNFR signal peptide (SEQ ID NO. 9):

[0113] AtggcccccgtggccgtgtgggccgccctggccgtgggcctggagctgtgggccgccgcccacgccggcttcTGCtgccccctgaggtggtcctcctacgagggccactgctacctggtggtgaaggagaagaagacctgggaggacgccgagaagttctgcaccgagcagaggaagggcggccacctggtgtccgtgcactccagggaggaggccgacttcctggtgcacctggcctaccccatcctggacctgtccctgatctggatgggcctgtccaacatgtggaacgactgcaagagggagtggtccgacggcaccaagctggacttcaagtcctgggccaagacctccgactgcctgatcggcaagaccgacggcgacaaccagtggctgaacatggactgctccaagaagcactacttcgtgtgcaagttcaagctgtga。

[0114] Nucleotide sequence of mutant delGFC containing TNFR signal peptide (SEQ ID NO. 10):

[0115] ATGGCCCCCGTGGCCGTGTGGGCCGCCCTGGCCGTGGGCCTGGAGCTGTGGGCCGCCGCCCACGCCTGCCCCCTGAGGTGGTCCTCCTACGAGGGCCACTGCTACCTGGTGGTGAAGGAGAAGAAGACCTGGGAGGACGCCGAGAAGTTCTGCACCGAGCAGAGGAAGGGCGGCCACCTGGTGTCCGTGCACTCCAGGGAGGAGGCCGACTTCCTGGTGCACCTGGCCTACCCCATCCTGGACCTGTCCCTGATCTGGATGGGCCTGTCCAACATGTGGAACGACTGCAAGAGGGAGTGGTCCGACGGCACCAAGCTGGACTTCAAGTCCTGGGCCAAGACCTCCGACTGCCTGATCGGCAAGACCGACGGCGACAACCAGTGGCTGAACATGGACTGCTCCAAGAAGCACTACTTCGTGTGCAAGTTCAAGCTGTGA。

[0116] Nucleotide sequence of mutant C3S containing TNFR signal peptide (SEQ ID NO.11):

[0117] Atggcccccgtggccgtgtgggccgccctggccgtgggcctggagctgtgggccgccgcccacgccggcttctcctgccccctgaggtggtcctcctacgagggccactgctacctggtggtgaaggagaagaagacctgggaggacgccgagaagttctgcaccgagcagaggaagggcggccacctggtgtccgtgcactccagggaggaggccgacttcctggtgcacctggcctaccccatcctggacctgtccctgatctggatgggcctgtccaacatgtggaacgactgcaagagggagtggtccgacggcaccaagctggacttcaagtcctgggccaagacctccgactgcctgatcggcaagaccgacggcgacaaccagtggctgaacatggactgctccaagaagcactacttcgtgtgcaagttcaagctgtga。

[0118] Nucleotide sequence of mutant GFC2GAD containing TNFR signal peptide (SEQ ID NO.12):

[0119] ATGGCCCCCGTGGCCGTGTGGGCCGCCCTGGCCGTGGGCCTGGAGCTGTGGGCCGCCGCCCACGCCGGCGCCGACTGCCCCCTGAGGTGGTCCTCCTACGAGGGCCACTGCTACCTGGTGGTGAAGGAGAAGAAGACCTGGGAGGACGCCGAGAAGTTCTGCACCGAGCAGAGGAAGGGCGGCCACCTGGTGTCCGTGCACTCCAGGGAGGAGGCCGACTTCCTGGTGCACCTGGCCTACCCCATCCTGGACCTGTCCCTGATCTGGATGGGCCTGTCCAACATGTGGAACGACTGCAAGAGGGAGTGGTCCGACGGCACCAAGCTGGACTTCAAGTCCTGGGCCAAGACCTCCGACTGCCTGATCGGCAAGACCGACGGCGACAACCAGTGGCTGAACATGGACTGCTCCAAGAAGCACTACTTCGTGTGCAAGTTCAAGCTGTGA。

[0120] Nucleotide sequence of mutant GFC2D containing TNFR signal peptide (SEQ ID NO. 13):

[0121] ATGGCCCCCGTGGCCGTGTGGGCCGCCCTGGCCGTGGGCCTGGAGCTGTGGGCCGCCGCCCACGCCGACTGCCCCCTGAGGTGGTCCTCCTACGAGGGCCACTGCTACCTGGTGGTGAAGGAGAAGAAGACCTGGGAGGACGCCGAGAAGTTCTGCACCGAGCAGAGGAAGGGCGGCCACCTGGTGTCCGTGCACTCCAGGGAGGAGGCCGACTTCCTGGTGCACCTGGCCTACCCCATCCTGGACCTGTCCCTGATCTGGATGGGCCTGTCCAACATGTGGAACGACTGCAAGAGGGAGTGGTCCGACGGCACCAAGCTGGACTTCAAGTCCTGGGCCAAGACCTCCGACTGCCTGATCGGCAAGACCGACGGCGACAACCAGTGGCTGAACATGGACTGCTCCAAGAAGCACTACTTCGTGTGCAAGTTCAAGCTGTGA。

[0122] Nucleotide sequence of mutant C3A containing the Albumin signal peptide (SEQ ID NO.14):

[0123] ATGAAGTGGGTGACCTTCATCTCCCTGCTGTTCCTGTTCTCCTCCGCCTACTCCGGCTTCGCCTGCCCCCTGAGGTGGTCCTCCTACGAGGGCCACTGCTACCTGGTGGTGAAGGAGAAGAAGACCTGGGAGGACGCCGAGAAGTTCTGCACCGAGCAGAGGAAGGGCGGCCACCTGGTGTCCGTGCACTCCAGGGAGGAGGCCGACTTCCTGGTGCA CCTGGCCTACCCCATCCTGGACCTGTCCCTGATCTGGATGGGCCTGTCCAACATGTGGAACGACTGCAAGAGGGAGTGGTCCGACGGCACCAAGCTGGACTTCAAGTCCTGGGCCAAGACCTCCGACTGCCTGATCGGCAAGACCGACGGCGACAACCAGTGGCTGAACATGGACTGCTCCAAGAAGCACTACTTCGTGTGCAAGTTCAAGCTGTGA.

[0124] It should be noted that the nucleotide sequences of SEQ ID NO. 8–14 consist of the start codon ATG, the nucleotide sequence of the corresponding signal peptide, the nucleotide sequence of the mutant, and the stop codon TGA.

[0125] Example 2 Construction of Recombinant Antiplatelet Thrombolytic Protein Gene Vector (Recombinant Plasmid)

[0126] Based on the sequence optimization design approach in Example 1, the nucleotide sequence of the α chain was kept unchanged and combined with five β chain mutants to construct recombinant plasmids. The nucleotide sequences of the five β chain mutants were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The p2MG2T vector containing a dual expression cassette was used in plasmid construction, and its map is shown in Figure 1.

[0127] During the construction of recombinant plasmids, the specific construction methods referred to the conventional methods of molecular biology gene cloning. Finally, sequencing confirmed that the five plasmids were successfully constructed. Among them, the α chain and β chain were inserted at the same position in the five recombinant plasmids. Taking the recombinant plasmid of C3S mutant as an example, the map of the recombinant plasmids is shown in Figure 2.

[0128] Example 3 Expression and screening of recombinant antiplatelet thrombolytic protein corresponding to different mutants

[0129] The recombinant plasmids constructed in Example 2 were used to transfect *E. coli*, and high-quality recombinant plasmids were then extracted for cell transfection. Five recombinant plasmids were transfected into CHO-K1 cells, and MSX was used for selection until cell viability recovered to over 90%. After removing the selection pressure, the cells were cultured until viability approached 100%, and then used for fed-batch culture. The expression in the supernatant was analyzed using a 4–20% gradient non-reducing SDS-PAGE. The specific procedure is as follows:

[0130] 1. Fed culture

[0131] Culture medium: 50 mL EXCELL Advanced CHO-K1 Fed-batch medium.

[0132] Feeding: 3% (V / V) BalanCD CHO-K1 Feed4, 0.3% (V / V) Cell Boost7b, starting from day 3, feed every 2 days, glucose controlled at 8g / L, culture for 9 days.

[0133] 2. Protein purification

[0134] (1) Sample pretreatment: The cell pool supernatant was adjusted to pH 4.6 with 5M acetic acid, centrifuged at 4℃ and 4000g for 5 minutes and then filtered through a 0.22μm filter.

[0135] (2) One-step purification

[0136] Chromatography column: Capto MMC BestChrom EzScreen;

[0137] Column volume: 4.2 mL, 10 cm × 0.7 cm;

[0138] Equilibration buffer: 25mM NaPO4, 50mM NaAc, 25mM Tris, 60mM NaCl, pH 4.6;

[0139] Elution buffer: 25mM NaPO4, 50mM NaAc, 25mM Tris, 60mM NaCl, pH 9.0;

[0140] Regeneration solution: 1M NaCl, 0.5M NaOH;

[0141] Elution gradient:

[0142] Table 2 One-step purification elution gradient

[0143]

[0144] (2) Two-step purification

[0145] Chromatography column: POROS XS BestChrom EzScreen;

[0146] Column volume: 4.2 mL, 10 × 0.7 cm;

[0147] Equilibration buffer: 50mM NaAc, pH 5.5;

[0148] Elution buffer: 50mM NaAc, 1M NaCl, pH 5.5;

[0149] Regeneration solution: 1M NaCl, 0.5M NaOH;

[0150] Elution gradient:

[0151] Table 3 Two-step purification elution gradient

[0152]

[0153] Preliminary analysis of the cell culture supernatant using non-reducing SDS-PAGE was performed, and the results are shown in Figure 3. Lane 1 represents the delGFC mutant, lane 2 the C3S mutant, lane 3 the C3A mutant, lane 4 the GFC2GAD mutant, and lane 5 the GFC2D mutant. M represents the marker. Figure 3 shows that the band of the C3A mutant is diffuse, presumably due to uneven cleavage of the Albamin signal peptide; therefore, this mutant was discarded. The remaining four mutants were analyzed using the TNFR signal peptide, and no abnormalities such as band diffusion were observed; these mutants can proceed to the next screening stage.

[0154] Example 4: Expression and purification of recombinant antiplatelet thrombolytic protein corresponding to different screened mutants.

[0155] 1. Performance comparison test of different mutants in fed-batch cell culture

[0156] The growth curves of the DelGFC mutant, C3S mutant, GFC2GAD mutant, and GFC2D mutant in fed-batch cell culture showed no significant differences, and the maximum cell density was approximately 15 × 10^6 cells / mL, as shown in Figure 4.

[0157] 2. Expression and purification of recombinant proteins from different mutants

[0158] After 10 days of fed-batch culture in the cell pool, the supernatant was harvested for protein purification and analysis. The specific steps for protein purification are detailed in Example 3. The process and results of the first-step purification are shown in Figure 5, and the process and results of the second-step purification are shown in Figure 6. In Figures 5 and 6, the red lines represent the collected elution fractions. The purified protein was analyzed using non-reducing SDS-PAGE, and the results are shown in Figure 7. M represents the marker, lane 1 represents the delGFC mutant, lane 2 represents the C3S mutant, lane 3 represents the GFC2GAD mutant, and lane 4 represents the GFC2D mutant. Figure 7 shows that the non-reducing SDS-PAGE indicates high purity of the target protein, but a weak, unknown band appears above the main band of the target protein. The band below may represent overexpressed, mispaired single strands.

[0159] 3. Results of purified protein yield, RP-HPLC purity, and Tm detection.

[0160] RP-HPLC purity determination: A C3 column was used with a UV wavelength of 280 nm. Mobile phase A consisted of 0.05% TFA + 20% acetonitrile-water, and mobile phase B consisted of 0.05% TFA + 90% acetonitrile-water. The sample loading volume was 175 μg. After two-step purification, the RP-HPLC purity and Tm value of the four constructed mutants showed no significant differences. The N-terminal sequence coverage was 100%, with no truncation. The GFC2GAD and GFC2D mutants showed higher yields. Specific results are shown in Table 4.

[0161] Table 4. Results of purified protein yield, RP-HPLC purity, and Tm detection.

[0162]

[0163] Example 5: Detection of biological activity based on flow cytometry

[0164] The isolation of fresh human platelets involves numerous ethical and legal considerations, making the development of practical and alternative assessment and detection methods crucial. Recombinant antiplatelet thrombolytic protein can competitively bind to GPⅠb-Ⅸ-Ⅴ on platelets, thereby inhibiting platelet aggregation and producing anticoagulant and antithrombotic effects. Therefore, measuring the binding strength of recombinant antiplatelet thrombolytic protein to GPⅠb-Ⅸ-Ⅴ on platelets can reflect the antiplatelet aggregation effect of drugs. Based on this scientific principle, cells expressing GPⅠb-Ⅸ-Ⅴ were used in place of platelets in experiments. Flow cytometry was employed to examine the binding affinity of recombinant antiplatelet thrombolytic protein to cells expressing GPⅠb-Ⅸ-Ⅴ, thus determining the antiplatelet aggregation effect of drugs.

[0165] Specific experimental procedure: Frozen DAMI cells were removed from the liquid nitrogen tank, thawed, and passaged until the cells reached the logarithmic growth phase, adjusting the cell density to 3–4E6 / mL. The diluted and mixed DAMI cells were added to 96-well plates, with 50 μL of cell suspension added to each well (resulting in 150,000–200,000 cells / well). Then, 50 μL of serially diluted recombinant antiplatelet thrombolytic protein was added (the highest concentration was 100 μg / mL, followed by 4-fold serial dilutions, for a total of 10 concentration gradients). A blank control and a negative control (using PBS instead of the sample) were also set up. After incubating at room temperature for 1 h, add 200 μL PBS, mix well, centrifuge at 1200 rpm for 6 min, and discard the supernatant. Add the antibody antagonizing recombinant antiplatelet thrombolytic protein (1B9 mouse monoclonal antibody), dilute 1:2000, 100 μL / well, and incubate at 4℃ for 1 h. Add another 200 μL PBS, mix well, centrifuge at 1200 rpm for 6 min, and discard the supernatant. Add 100 μL of secondary antibody and incubate at 4℃ in the dark for 0.5 h. Add another 200 μL PBS to each well, mix well, centrifuge at 1200 rpm for 6 min, and discard the supernatant. Finally, resuspend the cells in 150 μL PBS and perform flow cytometry to detect the cells. Calculate the relative activity.

[0166] The method for calculating relative activity is as follows: Plot the concentrations of the reference standard and the test sample on the x-axis, and the average fluorescence intensity (MFI) or positive rate on the y-axis. Fit a four-parameter curve to obtain the corresponding EC50 values. Using the reference standard as 100%, calculate the activity of the sample relative to the reference standard. The specific formula is:

[0167] The relative binding activity (%) of the sample = (reference EC50 / sample EC50) × 100%.

[0168] The cell pool was fed with feed and cultured using the above method, and the purified samples were tested. The test results are shown in Figure 8, and the relative binding activities of each recombinant protein are shown in Table 5.

[0169] Table 5. Calculation results of relative binding activity of recombinant proteins

[0170]

[0171] In summary, by maintaining the original α sequence and modifying the β sequence through mutation, all four mutants were able to produce recombinant antiplatelet thrombolytic protein during cell culture with feed. The activity of the four mutants was basically the same according to the flow cytometry detection method based on DAMI cells developed by the institute.

[0172] Example 6: Inhibitory effect of recombinant antiplatelet thrombolytic protein on platelet aggregation activity

[0173] 1. Blood sample collection

[0174] Take several vacuum blood collection tubes containing anticoagulant, and immediately invert them to mix thoroughly after blood collection.

[0175] 2. Sample preparation

[0176] (1) Preparation of platelet-rich plasma (PRP): The anticoagulated blood sample is centrifuged at 1000 rpm for 6 minutes using a horizontal centrifuge. After the centrifuge stops naturally, the sample is removed and the upper layer is carefully aspirated into a glass bottle, which is platelet-rich plasma.

[0177] (2) Preparation of anemic platelet plasma (PPP): Centrifuge the blood sample that has been aspirated for PRP again at 4000 rpm for 10 minutes. After the centrifuge stops naturally, take out the sample and carefully aspirate the upper layer into a glass bottle. This is anemic platelet plasma. The plasma should be clear and transparent.

[0178] (3) Preparation of inducing agent: Take the 125mg / mL RIS solution that has been dispensed and place it on an ice pack for later use.

[0179] 3. Testing

[0180] (1) Control group test

[0181] Add a small magnetic rod and 350 μL of PRP to the sample cup, and preheat it in the thermostatic well. Add a small magnetic rod and 350 μL of PPP to another cup, label it, and preheat it. Set the platelet aggregator (LBY-NJ4, Shanghai Pulisheng Instruments) to TEST mode. Insert the PPP cup into the test channel and press the corresponding channel key. The instrument will automatically detect the zero point; the value displayed in the window is the relative concentration of platelets in the PPP plasma. After the value stabilizes, press the corresponding channel key. Remove the PPP cup and place the PRP cup inside. The value displayed in the window represents the relative concentration of platelets in the tested plasma (PRP). After the value stabilizes, press the corresponding channel key, and the window will display Ris.

[0182] (2) Sample group detection

[0183] Add a small magnetic rod and 350 μL of PRP to the sample cup and preheat it in the thermostat. Insert the PPP cup into the test channel and press the corresponding channel key. The instrument will automatically detect the zero point, and the value displayed in the window is the relative concentration of platelets in the PPP plasma. After the value stabilizes, press the corresponding channel key. Remove the PPP cup and place the PRP cup in it. The value displayed in the window represents the relative concentration of platelets (PRP) in the tested plasma. After the value stabilizes, use a microsyringe to add the appropriate volume of drug to the bottom of the cup. After the value stabilizes, press the corresponding channel key, and the window will display Ris.

[0184] (3) Use a microsyringe to draw 7.2 μL of 125 mg / mL ristoctycin solution into the bottom of the cup. Immediately press the corresponding channel key to enter the platelet aggregation test state. The window displays the timing time or the maximum platelet aggregation rate. After the test is completed, the instrument will automatically print out the results and record the maximum aggregation rate of the corresponding well.

[0185] 4. Result Calculation

[0186] Platelet aggregation inhibition percentage = (platelet aggregation percentage of control tube - platelet aggregation percentage of sample tube) / platelet aggregation percentage of control tube × 100%, with two consecutive lower concentrations with inhibition rates greater than 95% defined as one activity unit. The results are shown in Table 6.

[0187] Table 6. Calculation results of the inhibitory titer of recombinant protein on platelet aggregation activity.

[0188]

[0189] It is evident that by maintaining the original α sequence and modifying the β sequence through mutation, all four mutants were able to produce recombinant antiplatelet thrombolytic protein during cell culture with feed. The platelet aggregation inhibition activity of the four mutants could be detected by traditional blood-based turbidimetric assay.

[0190] Example 7: Scale-up assay of recombinant antiplatelet thrombolytic protein

[0191] Based on a comparison of the performance and product quality of four mutants in shake-tube culture, a scale-up experiment of recombinant antiplatelet thrombolytic agent was conducted. This example uses the C3S mutant as an example to illustrate the establishment process of the scale-up experimental system. Multiple single-cloning experiments were performed on the C3S transfected cell pool, and a tertiary cell bank was established for small-scale process development and subsequent production.

[0192] The scale-up process of recombinant antiplatelet thrombolytic agent 15L (batch U4001-200917-B151) is as follows:

[0193] 1. Upstream cell culture

[0194] (1) Take one cell from the constructed working cell bank, place it in a 37℃ water bath for 1-3 min until completely thawed, resuspend it in preheated culture medium, centrifuge to collect the cell pellet, and seed the centrifuged cells into a 125mL shake flask at a live cell density of 0.3E6 cells / mL. Then, culture and expand the cells in 500mL and 5L shake flasks in sequence. During the cell expansion process, maintain a CO2 concentration of 5% and a humidity of 75%RH.

[0195] (2) The expanded cells are seeded into the Applikon mechanically stirred bioreactor. The pH and dissolved oxygen are maintained within a constant range. The concentrations of glutamine and glucose are adjusted based on the daily monitoring results. On the third day, an appropriate amount of feed solution and defoamer are added. Fermentation is stopped when fermentation reaches the 12th day or the cell viability is below 80%.

[0196] (3) The fermentation broth is filtered through a deep filter to obtain the supernatant, which is then used for downstream purification.

[0197] (4) Based on the above optimization conditions, the upstream scale-up process and real-time results were tracked and confirmed using indicators such as the live cell density growth curve, cell viability curve, and recombinant protein yield titration, as shown in Figures 9-11. Figure 9 shows the live cell density growth curve during the upstream cell culture process, Figure 10 shows the cell viability curve during the upstream cell culture process, and Figure 11 shows the recombinant protein yield titration curve during the upstream cell culture process.

[0198] 2. Downstream purification

[0199] (1) One-step tomography:

[0200] Chromatography packing material: CaptoMMC, CV=402mL;

[0201] Chromatography equipment: AKTA Pure 150;

[0202] Sample preparation: Take 9605g of the supernatant obtained in step (3) above, add 59g of 2M citric acid solution, adjust the pH to 4.63, and slowly add 3763g of water for injection with a conductivity of 9441μs / cm while stirring. Filter the solution with a 0.22μm filter for sterilization. The specific parameters of the one-step chromatography are shown in Table 7, and the results after the one-step chromatography are shown in Figure 12.

[0203] Table 7. One-step chromatography method for downstream purification

[0204]

[0205] (2) Two-step chromatography:

[0206] Chromatography packing material: CaptoQ, CV=185mL;

[0207] Chromatography equipment: AKTA Pure 150;

[0208] Sample preparation: Take 801.06 g of the collected solution from the first-step chromatography, slowly add 801.03 g of water for injection while stirring, add 7.77 g of 1M citric acid, adjust the pH to 6.49, and the conductivity to 4937 μs / cm. The specific parameters for the second-step chromatography are shown in Table 8, and the results after the second-step chromatography are shown in Figure 13.

[0209] Table 8. Two-step chromatography method for downstream purification

[0210]

[0211] (3) Three-step tomography:

[0212] Chromatography packing material: SP-HP, CV=193mL;

[0213] Chromatography equipment: AKTA Pure 150;

[0214] Sample preparation: Take 1666g of the collected solution from the two-step chromatography above, add 19.15g of 1M citric acid, and adjust the pH to 5.01. The specific parameters for the three-step chromatography are shown in Table 9.

[0215] Table 9. Three-step chromatography method for downstream purification

[0216]

[0217] Using the aforementioned upstream and downstream purification process for recombinant antiplatelet thrombolytic protein, the proportion of the main peak in the SEC-HPLC of the recombinant protein showed an increasing trend during the purification process. After two-step purification, the purity reached 98.71%, which meets the general requirement of >95% for biopharmaceuticals. Charge isomer detection also showed an increasing trend in the main peak. The recovery rate of the three-step purification was over 80%, which can meet the needs of preclinical and commercial production. See Table 10 for details.

[0218] Table 10 Detection results of 15L pilot-scale protein sample from batch U4001-200917-B151

[0219]

[0220] The activity of recombinant antiplatelet thrombolytic agent in 3L small-scale and 15L scale-up samples was detected using cellular and blood methods. The results are shown in Table 11. It can be seen that the relative activity is in the range of 70%-130%, and its inhibition rate on platelet aggregation in blood is non-inferior to that of the natural protein (Source: Zhaoke Pharmaceutical (Hefei) Co., Ltd.).

[0221] Table 11 Results of Activity Assay - Turbidimetric Method

[0222]

[0223] Example 8: Therapeutic effect of recombinant antiplatelet thrombolytic protein on a rat model of cerebral ischemia-reperfusion.

[0224] 1. Laboratory animals and reagents

[0225] 127 male SD rats were selected to establish a rat model of cerebral ischemia-reperfusion using the suture occlusion method. ZK052 is a recombinant antiplatelet thrombolytic protein, specifically the mutant C3S ultimately screened in the above examples, batch number: 20210102-1, source: Zhaoke Pharmaceutical (Hefei) Co., Ltd. ZK001 is a natural antiplatelet thrombolytic protein, batch number: 20210104, source: Zhaoke Pharmaceutical (Hefei) Co., Ltd. The experiments and results of the remaining mutants screened in the above examples are similar to those in this example and will not be repeated here.

[0226] 2. Experimental Methods

[0227] The experiment was divided into 7 groups: sham surgery group, model control group, low-dose ZK052 group, medium-dose ZK052 group, high-dose ZK052 group, ZK001 group, and rhTNK-tPA group. The number of animals in the sham surgery group was 16, the number of animals in the model control group, low-dose ZK052 group, medium-dose ZK052 group, and ZK001 group was 18, the number of animals in the high-dose ZK052 group was 20, and the number of animals in the rhTNK-tPA group was 19. In the model control group and each drug administration group, the middle cerebral artery was occluded with a suture plug for 90 minutes, and then the suture plug was pulled out for reperfusion. In the sham surgery group, only the carotid artery was dissected without inserting a suture plug. Animals in the rhTNK-tPA group, ZK001 group, ZK052 low-dose group, ZK052 medium-dose group, and ZK052 high-dose group were administered the drug once immediately after reperfusion (within 5 minutes), at doses of 10 mg / kg, 6 μg / kg, 3 μg / kg, 6 μg / kg, and 12 μg / kg, respectively, with a drug administration volume of 5 mL / kg for each group. The condition of the experimental animals was observed after drug administration, and the following indicators were observed:

[0228] (1) General condition observation;

[0229] (2) Weight;

[0230] (3) Behavioral scores: Bederson scores were taken once each at 1 hour of ischemia and 1 hour of reperfusion on the day of modeling (1 hour after drug administration), and NSS scores were taken once each at 1 day before surgery and 24 hours after surgery.

[0231] (4) Infarction range and infarction inhibition rate: Brains were taken 24 hours after reperfusion for TTC staining to determine the infarction range and calculate the infarction inhibition rate in rats;

[0232] (5) Histopathological changes: microthrombi;

[0233] (6) Immunohistochemical and immunofluorescence detection: P-selectin, ICAM-1, fibrin and CD41.

[0234] 3. Experimental Results

[0235] (1) Extent of cerebral infarction

[0236] The results are shown in Figure 14. All data in the figure are expressed as mean ± standard error (Mean ± SEM). Figure A shows the infarct area, Figure B shows the infarct area inhibition rate, and Figure C shows typical TTC staining results for each group. In the figures, * indicates the P-value compared to the sham-operated group, and & indicates the P-value compared to the model control group.

[0237] As shown in Figure 14, after intervention with ZK052 at doses of 3 μg / kg, 6 μg / kg, and 12 μg / kg, the cerebral infarction range in the animals was 18.282±0.927%, 16.601±1.166%, and 14.327±1.570%, respectively, which were significantly lower than those in the model control group (23.330±0.543%) (P=0.0117, 0.0009, and 0.0001). The cerebral infarction inhibition rates at each dose were 21.635±3.972%, 28.844±4.997%, and 38.589±6.729%, respectively. With increasing dose, the cerebral infarction range tended to decrease, while the cerebral infarction inhibition rate showed a significant increasing trend. In the medium-dose group (6 μg / kg), the cerebral infarction range of animals showed a decreasing trend compared with the same dose of ZK001 (16.601±1.166% Vs. 18.768±0.54%, P=0.1499), and a slight decrease compared with the positive control drug rhTNK-tPA (10 mg / kg) (16.601±1.166% Vs. 17.005±1.449, P=0.3766). At a dose of 12 μg / kg, ZK052 showed a slightly better inhibitory effect on the cerebral infarction range than the positive control drug rhTNK-tPA at 10 mg / kg (14.327±1.570 Vs. 17.005±1.449, P=0.1902).

[0238] The results above suggest that under the conditions of this experiment, ZK052 can effectively reduce the infarct size of rats with ischemic stroke. It exhibits a good dose-response relationship at a dose range of 3-12 μg / kg. At the same dose, ZK052 has a better inhibitory effect on cerebral infarction in rats with ischemia-reperfusion than the control drug ZK001. At doses of 6 μg / kg and 12 μg / kg, ZK052 has a better inhibitory effect on cerebral infarction in rats with ischemia-reperfusion than rhTNK-tPA.

[0239] (2) Neurobehavioral score

[0240] The results are shown in Figure 15. All data in the figure are expressed as mean ± standard error (Mean ± SEM). Figure A shows the Bederson score of the animals 1 hour after ischemia, Figure B shows the Bederson score of the animals 1 hour after ischemia-reperfusion (1 hour after drug administration), and Figure C shows the NSS score of the animals 24 hours after modeling. In the figures, * indicates the P-value compared with the sham-operated group, and & indicates the P-value compared with the model control group.

[0241] As shown in Figure 15, all animals had a Bederson score of 2 or 3 after 1 hour of ischemia. One hour after ischemia-reperfusion (1 hour after drug administration), the Bederson scores of animals in the medium-dose (6 μg / kg) and high-dose (12 μg / kg) ZK052 groups showed a decreasing trend, and were significantly lower than the model control group (2.53±0.12 and 2.50±0.12 vs. 2.94±0.06, P=0.0318 and 0.0217). Twenty-four hours post-surgery, the NSS of the ZK001 group showed no decreasing trend compared to the model control group. Animals treated with 10 mg / kg rhTNK-tPA and 3–12 μg / kg ZK052 showed a certain decreasing trend in NSS, but the differences were not statistically significant.

[0242] The above results suggest that, under the experimental conditions, the test sample ZK052 has the effect of improving neurological function damage in rats with ischemic stroke.

[0243] (3) Histopathological examination

[0244] The results are shown in Figure 16. All data in the figure are expressed as mean ± standard error (Mean ± SEM). In the figure, * indicates the P-value compared with the sham surgery group, and & indicates the P-value compared with the model control group.

[0245] As shown in Figure 16, 24 hours after surgery, significant microthrombi appeared in the brain of the infarcted side of the model control group, with a microthrombus number of 184.00±46.69. The number of microthrombi in the ZK001 and rhTNK-tPA groups was reduced to varying degrees compared with the model control group. Among them, the number of microthrombi in the rhTNK-tPA group was significantly reduced compared with the model control group (54.67±17.94 vs. 184.00±46.69, P=0.0469). The number of microthrombi in the low-dose, medium-dose, and high-dose ZK052 groups showed a dose-related decrease compared with the model control group. Among them, the number of microthrombi in the high-dose ZK052 group was significantly reduced compared with the model control group (41.33±24.01 vs. 184.00±46.69, P=0.0114), and showed a decreasing trend compared with the number of microthrombi in the brain of animals after intervention with 10 mg / kg rhTNK-tPA.

[0246] The results above suggest that under the conditions of this experiment, ZK052 has a significant inhibitory effect on microthrombus formation in the acute phase of cerebral infarction in rats. It has a good dose-response relationship at a dose of 3-12 μg / kg, and at a dose of 12 μg / kg, its inhibitory effect on microthrombi is slightly better than that of the positive control drug rhTNK-tPA.

[0247] (4) Immunohistochemistry and immunofluorescence

[0248] The results are shown in Figures 17-20. In Figure 7, Figure A shows the percentage of CD41 positive areas, and Figure B shows the cumulative optical density of CD42. All data in the figures are expressed as mean ± standard error (Mean ± SEM). In the figures, * indicates the P-value compared with the sham surgery group, and & indicates the P-value compared with the model control group.

[0249] Fibrin deposition and the formation of microthrombi from secondary fibrin-platelet aggregates are important causes of persistent microcirculatory disturbances after vascular recanalization. This study used CD41 immunohistochemistry and fibrin immunofluorescence to detect fibrin and platelet deposition in the cerebral infarction site. As shown in Figures 17 and 18, 24 hours after modeling, the CD41-positive area in the model control group was significantly larger than that in the sham-operated group (3.19±1.26 vs. 0.27±0.23, P=0.0052), the cumulative optical density of CD41 (IDO) was significantly increased (17703.65±5392.69 vs. 1580.55±1274.74, P=0.0040), and the number of fibrin-positive vessels was significantly increased. After ZK052 dose groups, ZK001, or rhTNK-tPA intervention, the CD41-positive area, CD41 cumulative optical density, and the number of fibrin-positive vessels decreased to varying degrees. Among them, the CD41 positive area, CD41 cumulative optical density and fibrin positive blood vessel number of animals in each dose group of ZK052 decreased with increasing dose, and when the dose was above 6 μg / kg, the CD41 positive area and CD41 cumulative optical density showed a decreasing trend compared with ZK001 and rhTNK-tPA.

[0250] The results above suggest that under the conditions of this experiment, ZK052 has a good inhibitory effect on platelet deposition and fibrin deposition, and has a good dose-response relationship in the dose range of 3~12μg / kg. Moreover, at a dose greater than 6μg / kg, its inhibitory effect on platelet deposition is slightly better than that of ZK001 and rhTNK-tPA.

[0251] In ischemic stroke, ischemia and hypoxia lead to severe local inflammatory responses. Locally expressed P-selectin and ICAM-1 can induce leukocyte aggregation in ischemic brain tissue, activating local immune responses and thus exacerbating ischemic brain injury. This study used immunofluorescence to explore the expression of ICAM-1 and P-selectin. As shown in Figures 19 and 20, the expression levels of ICAM-1 and P-selectin in rats were significantly increased 24 hours after ischemia-reperfusion. After intervention with ZK052, the expression levels of both ICAM-1 and P-selectin in the animals decreased, showing a good dose-response relationship. At doses of 6 μg / kg and 12 μg / kg, the expression levels of P-selectin and ICAM-1 were reduced compared to ZK001 and rhTNK-tPA.

[0252] The results above suggest that, under the conditions of this experiment, ZK052 may have a certain inhibitory effect on the local inflammatory response in ischemic stroke.

[0253] (5) Weight

[0254] Animals in the sham-operated group did not show a significant decrease in body weight 24 hours after surgery. Animals in the model control group and each drug-treated group showed a significant decrease in body weight compared to the sham-operated group 24 hours after surgery (P < 0.0001). The decrease in body weight in each drug-treated group was not significantly different from that in the model control group.

[0255] (6) General condition observation

[0256] No obvious abnormalities were observed in the sham-operated group animals on the day of surgery and one day after surgery. After MCAO modeling, animals in the model control group and each drug administration group all showed symptoms such as gait instability, drooling, and circling, with no differences between groups. 24 hours after surgery, surviving animals mostly showed stroke-related abnormalities such as gait instability, circling, piloerection, drooling, and soiling around the nose, eyes, and mouth, with no significant differences in the types and severity of abnormalities among groups.

[0257] The above description is merely a preferred embodiment of this application; however, the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and its improved concept, should be covered within the scope of protection of this application.

Claims

1. A recombinant antiplatelet thrombolytic protein, characterized in that, The recombinant antiplatelet thrombolytic protein was obtained by mutating the first three amino acids at the N-terminus of the B chain of natural antiplatelet thrombolytic protein; wherein... The amino acid sequence of the A chain of the natural antiplatelet thrombolytic protein is shown in SEQ ID NO.1, and the amino acid sequence of the B chain is shown in SEQ ID NO.

2.

2. The recombinant antiplatelet thrombolytic protein according to claim 1, characterized in that, The mutation includes the removal of the first three amino acids GFC at the N-terminus, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.3; And / or, the mutation includes replacing the 3rd amino acid at the N-terminus with a serine, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.4; And / or, the mutation includes replacing the first 3 amino acids at the N-terminus with GAD, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.5; And / or, the mutation includes replacing the first 3 amino acids at the N-terminus with D instead of GFC, and the amino acid sequence of the B chain of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO. 6; And / or, the mutation includes replacing the N-terminal 3rd amino acid with alanine, and the B-chain amino acid sequence of the recombinant antiplatelet thrombolytic protein is shown in SEQ ID NO.

7.

3. A nucleotide sequence composition for encoding the recombinant antiplatelet thrombolytic protein as described in claim 1 or 2, characterized in that, It includes a first nucleotide sequence for encoding the amino acid sequence of chain A, and a second nucleotide sequence for encoding the amino acid sequence of chain B.

4. An expression frame composition, characterized in that, It includes a first expression frame and a second expression frame, wherein the first expression frame is used to express the first nucleotide sequence as described in claim 3, and the second expression frame is used to express the second nucleotide sequence as described in claim 3.

5. A recombinant plasmid composition, characterized in that, It includes a first recombinant plasmid and a second recombinant plasmid, wherein the first recombinant plasmid contains the first nucleotide sequence as described in claim 3 or the first expression frame as described in claim 4, and the second recombinant plasmid contains the second nucleotide sequence as described in claim 3 or the second expression frame as described in claim 4.

6. A recombinant plasmid, characterized in that, The third recombinant plasmid includes a vector comprising at least two expression frames, containing the first nucleotide sequence and the second nucleotide sequence as described in claim 3, or containing the first expression frame and the second expression frame as described in claim 4.

7. A recombinant cell, characterized in that, The recombinant cells are obtained by co-transfection of mammalian cells with the recombinant plasmid composition of claim 5, or by transfection of mammalian cells with the recombinant plasmid of claim 6.

8. The method for preparing the recombinant antiplatelet thrombolytic protein according to claim 1 or 2, characterized in that, Includes the following steps: The recombinant cells described in claim 7 were cultured, and the supernatant was collected and purified.

9. A drug, characterized in that, The active ingredient of the drug includes the recombinant antiplatelet thrombolytic protein as described in claim 1 or 2.

10. The use of the recombinant antiplatelet thrombolytic protein according to claim 1 or 2 in the preparation of a medicament, wherein the medicament functions as at least one of the following: Antagonizes the binding of GPIb to its receptor; Antiplatelet adhesion and / or aggregation; Antithrombotic; Treatment and / or prevention of cardiovascular disease; Treatment and / or prevention of stroke, preferably ischemic stroke; Reduce the extent of cerebral infarction in stroke; Lowering neurobehavioral scores in stroke patients; Reduce the number of microthrombi in stroke patients; Reduces the number of CD41-positive areas, CD41 cumulative optical density, and fibrin-positive vessels in stroke patients; Reduce the expression levels of ICAM-1 and P-selectin in stroke patients.

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