Application of mosquito salivary gland-based Serpin protein in preparation of antithrombotic drugs
By extracting Serpin protein from the salivary glands of Aedes albopictus and preparing anti-thrombotic drugs, the problem of insufficient anticoagulant research was solved, effective anticoagulant effects and drug development were achieved, and the research and development of anti-thrombotic drugs and the prevention and treatment of mosquito-borne diseases were promoted.
Patent Information
- Application Number
- CN202510756788.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-09
- Publication Date
- 2025-09-23
AI Technical Summary
In the prior art, there is insufficient research on the anticoagulant function of Serpin protein in the salivary glands of Aedes albopictus, which limits the development and application of antithrombotic drugs.
Serpin protein is extracted from the salivary glands of Aedes albopictus, its anticoagulant effect is verified through in vitro and in vivo experiments, and antithrombotic drugs are prepared, including the steps of collecting salivary glands, extracting RNA, reverse transcription, PCR amplification, cloning expression, and protein purification.
Serpin protein significantly prolongs the APTT, PT and TT of the blood, reduces the Fib content, interferes with the blood coagulation process through multiple mechanisms, provides the possibility of new anti-thrombotic drugs, improves drug research and development efficiency and reduces costs.
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Figure CN120678890A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of protein engineering, and in particular to application of mosquito salivary gland-based Serpin protein in the preparation of anti-thrombotic drugs. Background Art
[0002] Aedes albopictus, also known as the Asian tiger mosquito, is a widespread mosquito species in hot and humid environments due to its strong adaptability and rapid reproduction. It is a vector of various infectious diseases, including dengue fever. During the blood-feeding process, these mosquitoes secrete substances from their salivary glands that prevent blood clotting, a property that has piqued the interest of researchers. Previous studies have shown that salivary gland homogenates from A. albopictus can significantly inhibit platelet aggregation and prolong clotting times, including APTT, PT, and TT, indicating the presence of substances that prevent blood clotting. However, relatively little research has been conducted on anticoagulant proteins in the salivary glands of A. albopictus, particularly those related to Serpin proteins, which have been understudied.
[0003] Current research on salivary gland proteins in Aedes albopictus primarily focuses on immune-related proteins, including those involved in mosquito autoimmunity and those that induce inflammatory responses in humans. For example, the D7 family of proteins and the 34k2 salivary protein can elicit immune responses in humans, while C-type lectins participate in mosquito innate immunity and environmental adaptation. Despite extensive research on immune-related proteins, significant gaps remain in the study of anticoagulant proteins in Aedes albopictus. In particular, the role of serpin proteins in anticoagulation remains underappreciated and underutilized.
[0004] The problem with existing technologies is that, while it is known that anticoagulants are present in the salivary glands of Aedes albopictus, the specific composition and mechanism of action of these substances are insufficiently understood. Serpin proteins, in particular, are known to have anticoagulant functions in other insects and organisms, but research into this function in Aedes albopictus is relatively underdeveloped. Furthermore, existing research has largely focused on immune proteins, while research on serpin proteins directly involved in the anticoagulant process is relatively lacking. This limits our ability to fully understand the anticoagulant proteins in the salivary glands of Aedes albopictus and hinders the development of new antithrombotic drugs based on these proteins.
[0005] Therefore, the present invention provides the use of Serpin protein based on mosquito salivary glands in the preparation of anti-thrombotic drugs, and deeply explores the anti-coagulant function of Serpin protein in the salivary glands of Aedes albopictus, which is of great significance for preventing and treating mosquito-borne diseases and developing new anti-thrombotic drugs. Summary of the Invention
[0006] The purpose of the present invention is to solve the above problems and provide the use of Serpin proteins based on mosquito salivary glands in the preparation of antithrombotic drugs, so as to solve the problems in the prior art such as insufficient research on anticoagulant proteins in the salivary glands of Aedes albopictus, limitations on the development of antithrombotic drugs, limitations on the application of natural anticoagulants, and verification of the safety and effectiveness of antithrombotic drugs, thereby promoting the research and development of antithrombotic drugs and the prevention and treatment of mosquito-borne diseases.
[0007] In order to achieve the above-mentioned purpose, the technical solution of the present invention is as follows: the use of mosquito salivary gland-based Serpin protein in the preparation of anti-thrombotic drugs, including the anti-thrombotic drug containing Serpin protein extracted from the salivary glands of Aedes albopictus, and the Serpin protein can effectively prolong the APTT, PT and TT of the blood, reduce the Fib content in the blood, and interact with coagulation factor X.
[0008] Furthermore, the Serpin protein has been verified to have an anticoagulant effect through in vitro and in vivo experiments.
[0009] Furthermore, the preparation method of the antithrombotic drug comprises the following steps:
[0010] S1: Collection of salivary glands of female Aedes albopictus;
[0011] S2: Extract salivary gland RNA and reverse transcribe to synthesize cDNA;
[0012] S3: Amplify the Serpin gene using PCR technology;
[0013] S4: Clone the amplified Serpin gene into the expression vector;
[0014] S5: Transform host cells and induce expression of Serpin protein;
[0015] S6: Purify the expressed Serpin protein and verify its activity.
[0016] Furthermore, the antithrombotic drug can be used to treat diseases caused by blood coagulation, including but not limited to deep vein thrombosis, pulmonary embolism and myocardial infarction.
[0017] Another object of the present invention is to provide a method for using the above-mentioned antithrombotic drug, which comprises administering the antithrombotic drug to a patient in an effective dose to prolong blood coagulation time and reduce the risk of thrombosis.
[0018] Furthermore, the administration of the antithrombotic drug includes but is not limited to intravenous injection, subcutaneous injection or oral administration.
[0019] Another object of the present invention is to provide a method for detecting the interaction between Serpin protein in the salivary glands of Aedes albopictus and coagulation factor X, wherein the method comprises using a pull-down technique to verify the direct binding of Serpin protein to coagulation factor X.
[0020] Another object of the present invention is to provide a use of an antithrombotic drug based on the Serpin protein of the salivary gland of Aedes albopictus, which includes preventing and treating thrombosis-related diseases.
[0021] Compared with the existing technology, the present invention has the following beneficial effects: The present invention innovatively utilizes Serpin protein from the salivary glands of Aedes albopictus as the core component of an antithrombotic drug. First, this approach breaks through the limitations of traditional antithrombotic drug development. By seeking new anticoagulant components from nature, this serpin protein extracted from organisms not only possesses natural anticoagulant properties but can also effectively prolong blood APTT, PT, and TT, and reduce blood Fib levels. This natural bioactive component provides new directions and possibilities for the development of antithrombotic drugs. Second, this approach ensures the anticoagulant effect of serpin protein through dual validation through in vitro and in vivo experiments. This rigorous scientific validation process provides a solid foundation for the safety and efficacy of the drug and also provides reliable data support for clinical application. In addition, the preparation method provided by this approach has clear steps and high efficiency. From collecting salivary glands of Aedes albopictus to the final purification of the expressed serpin protein and activity verification, the entire process is scientific, systematic, and repeatable. This not only improves the efficiency of drug development but also reduces costs, making large-scale production possible. Finally, this protocol also provides a method for administering these antithrombotic drugs. This flexibility provides more options for clinical treatment and greater convenience for patients. By administering antithrombotic drugs to patients at effective doses, blood clotting time can be significantly prolonged and the risk of thrombosis can be reduced. This is of great significance for the prevention and treatment of diseases such as deep vein thrombosis, pulmonary embolism, and myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Schematic diagram of the four coagulation results in an embodiment of the present invention;
[0023] Figure A shows the PT (prothrombin time) values of the experimental group and the control group; Figure B shows the APTT (activated partial thromboplastin time) values of the experimental group and the control group; Figure C shows the Fib (fibrinogen) content of the experimental group and the control group; Figure D shows the TT (clotting time) values of the experimental group and the control group;
[0024] Figure 2 Schematic diagram of the construction of a mouse thrombosis model and the therapeutic effect of Serpin protein in an embodiment of the present invention;
[0025] Figure A shows the length of the tail thrombus in the experimental and control groups; Figure B shows the mass of the tail thrombus in the experimental and control groups.
[0026] Figure 3 2. It is a schematic diagram of the results of a whole blood coagulation test in an embodiment of the present invention;
[0027] Figure A shows the blood clot area obtained by the whole blood coagulation test of the blood of mice in the experimental group and the control group; Figure B shows the blood clot mass obtained by the whole blood coagulation test of the blood of mice in the experimental group and the control group;
[0028] Figure 4 Schematic diagram of the coagulation factor expression analysis results in an embodiment of the present invention;
[0029] Figure 5 Schematic diagram of the in vitro interaction verification between Serpin protein and coagulation factor X in an embodiment of the present invention. DETAILED DESCRIPTION
[0030] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.
[0031] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.
[0032] Experimental materials: EZ-10 Total RNA Mini-Preps Kit (Sangon Biotech, Shanghai, China), AMV First Strand cDNA Synthesis Kit (Sangon Biotech, Shanghai, China), Rapid Agarose Gel DNA Recovery Kit II (spin column type) (BioTeke, Beijing, China), pET-30 (ɑ+) (Solarbio, Beijing, China) plasmid, DH5α competent cells (TakaraBio Inc., Dalian, China), Plasmid Miniprep Kit (BEIWO, Hangzhou, China), IPTG (biosharp, Beijing, China) inducible expression protein, four coagulation test kits (purchased from Tianyue Biotechnology Co., Ltd., Baoding, China), carrageenan (purchased from MACKLIN, Shanghai, China).
[0033] Example 1: Acquisition and expression of Aedes albopictus salivary gland Serpin protein
[0034] 1) Collect and process Aedes albopictus
[0035] Female Aedes albopictus mosquitoes were collected using the human-baited landing method. This method uses humans as bait to attract mosquitoes to land for collection. The collected mosquitoes are then placed in a -20°C environment to freeze them unconscious for dissection. Once the mosquitoes are stunned, their salivary glands are collected. Because female Aedes albopictus mosquitoes secrete salivary gland secretions during blood feeding, our main research focus is on the Serpin proteins in their salivary glands.
[0036] 2) RNA extraction and cDNA synthesis
[0037] Total RNA was extracted from the salivary glands using the Total RNA Mini-Preps Kit. After RNA extraction, the RNA concentration was measured using a spectrophotometer to ensure that the amount of RNA used in subsequent experiments was accurate and appropriate. The extracted RNA was reverse transcribed into cDNA using the AMV First Strand cDNA Synthesis Kit.
[0038] 3) PCR amplification of Serpin gene
[0039] Specific primers for the Aedes albopictus Serpin gene were designed and PCR was used to amplify the Serpin gene using Aedes albopictus salivary gland cDNA as a template. The amplification system consisted of 2× Taq Plus MasterMix, forward primer, reverse primer, cDNA, and sterile water. Gene amplification was performed on a Biometra TOne PCR instrument using a program that included 30 cycles of pre-denaturation, denaturation, annealing, and extension. Amplified products were verified by agarose gel electrophoresis, and the pure Aedes albopictus salivary gland Serpin gene fragment was recovered using the Quick Agarose Gel DNA Extraction Kit II (spin column format). The forward and reverse primers are listed in Table 1.
[0040] Table 1 Forward primer and reverse primer of amplification system
[0041]
[0042] Aedes albopictus salivary gland Serpin gene fragment:
[0043]
[0044] 4) Gene cloning and expression vector construction
[0045] Primers for the Aedes albopictus Serpin gene containing EcoRI and HandIII restriction sites were designed, as shown in Table 2 below. The purified Serpin gene product was used as a template to amplify the target gene. The pET-30(ɑ+) plasmid was digested with enzymes, and the linearized plasmid was ligated with the Serpin gene containing the restriction sites. The ligation product was then introduced into DH5α competent cells and cultured in LB solid medium containing kanamycin. After a single colony grew, it was picked for colony PCR verification.
[0046] Table 2 Primers for Serpin genes of Aedes albopictus
[0047]
[0048] 5) Protein expression and verification
[0049] Positive colonies were picked and placed in LB liquid medium containing kanamycin at 37°C, and IPTG was added to induce protein expression. After protein induction, the bacteria were broken, and SDS-PAGE electrophoresis and Western blot were performed to verify the protein expression. The Serpin protein of the salivary gland of Aedes albopictus was expressed in large quantities, and the protein was dried to a powder state using a freeze dryer for storage.
[0050] Example 2: Evaluation of the effect of Aedes albopictus salivary gland Serpin protein on blood coagulation
[0051] Step 1: Collection of blood samples
[0052] 1. Blood collection: Venous blood samples were collected from the elbow vein of healthy adults.
[0053] 2. Anticoagulant treatment: Sodium citrate is used as an anticoagulant and mixed with blood in a ratio of 1:9 to prevent blood clotting.
[0054] Step 2: Preparation of blood samples
[0055] 1. Allocate blood sample: Divide the anticoagulated blood sample into two parts.
[0056] 2. Preparation of control group and experimental group:
[0057] Control group: an aliquot of blood was mixed with phosphate buffered saline (PBS) at a ratio of 1:1.
[0058] Experimental group: Another portion of blood was mixed with the Serpin protein solution from the salivary glands of Aedes albopictus in a 1:1 ratio.
[0059] Step 3: Blood sample processing
[0060] The mixed blood samples of the control group and experimental group were incubated in a 37°C water bath for 5 minutes to simulate in vivo conditions.
[0061] Step 4: Coagulation analysis
[0062] Coagulation function analysis: The two groups of blood samples were analyzed using a coagulation function test kit, including prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib), and clotting time (TT).
[0063] Step 5: Data Analysis
[0064] Compare the four coagulation parameters between the control and experimental groups to assess the effects of serpins on blood coagulation. Observe whether the experimental group has significantly prolonged PT, APTT, and TT compared to the control group, suggesting that serpins may have an anticoagulant effect. Observe whether the experimental group has decreased Fib levels compared to the control group, further suggesting that serpins may affect the blood coagulation process.
[0065] Experimental conclusion: The experimental results are as follows Figure 1 As shown, Serpin protein from the salivary glands of Aedes albopictus has a significant effect on the blood coagulation process. Compared with blood treated with PBS alone, blood samples treated with Serpin protein showed a significant prolongation of APTT, PT and TT, indicating that Serpin protein delays blood coagulation by inhibiting multiple steps in the coagulation cascade. At the same time, the reduction in fibrinogen (Fib) content further suggests that Serpin protein may reduce the raw materials for clot formation, thereby affecting the final stage of blood coagulation. These results collectively support the use of Serpin protein from the salivary glands of Aedes albopictus as a potential anti-thrombotic therapeutic drug, which interferes with the blood coagulation process through multiple mechanisms and provides a strong experimental basis for the research and development of anti-thrombotic drugs.
[0066] Example 3: Construction of mouse thrombosis model and Serpin protein treatment
[0067] Mice were injected with 100mg / kg of carrageenan, a substance known to induce thrombosis. After injection, the mice were kept at 8°C for 12 hours, which helps the carrageenan form thrombi in the mice's tails. After thrombi were formed, the mice were randomly divided into two groups: an experimental group and a control group. The experimental group mice received an intraperitoneal injection of a serpin protein solution, while the control group mice received an equal amount of PBS (phosphate buffered saline) intraperitoneally, with injections every 12 hours for six consecutive times.
[0068] The two groups of mice were placed in an environment of 24°C for 72 hours to simulate the recovery process under normal physiological conditions. The mice were photographed and their appearance changes were recorded. Blood was drawn from the hearts of the mice for subsequent hematological analysis. The tails of the mice were photographed and the length of the black tails was measured. The black tails of the thrombus were cut off and weighed, which is an intuitive indicator for evaluating thrombosis and treatment effects. The livers of the mice were removed to analyze the expression of thrombosis-related biomarkers.
[0069] The experimental results are as follows Figure 2 As shown, the black tail length of the experimental group of mice treated with Serpin protein was significantly shortened and the thrombus mass was significantly reduced compared with the control group, which indicates that Serpin protein has a significant anti-thrombotic effect.
[0070] By constructing a mouse thrombosis model and comparing the therapeutic effects of the Serpin protein treatment group with those of the control group, the present invention visually observes the therapeutic effect of Serpin protein on thrombosis. The experimental results support the potential of Serpin protein as a potential anti-thrombotic drug, with the potential to reduce thrombosis and improve thrombosis-related symptoms.
[0071] Example 4: Evaluation of the therapeutic effect of Serpin protein on blood coagulation in mice
[0072] Measurement of thrombus length and weight: Directly measure the thrombus formed in the mouse tail, record the thrombus length, and accurately weigh the thrombus from the thrombus portion to quantify the extent of thrombus formation.
[0073] Whole blood coagulation test: Fibrinogen (Fg) solution provided by Yeasen Biotechnology was added to a 96-well plate, and Fib reagent provided by Tianyue was added to the fibrinogen solution. The plates were shaken and incubated overnight at 4°C to form a fibrin clot. The mouse blood was anticoagulated with sodium citrate, and the anticoagulated mouse blood was added to the fibrin clot. The plates were shaken and incubated at 37°C for 90 minutes. The clot was rinsed three times with saline to remove unbound blood components. The mass of the fibrin clot bound to the mouse blood was then weighed, and the area of the clot was measured.
[0074] Coagulation factor expression analysis: Mouse livers were excised and liver RNA was extracted. The extracted RNA was reverse transcribed into cDNA. Specific primers for mouse coagulation factors were designed and used for real-time fluorescence quantitative PCR analysis using 2x Realtime PCR Super Mix (SYBRgreen, with anti-Taq) provided by Mei5bio to assess the expression of coagulation factors in mice. The primers are listed in Table 3 below.
[0075] Table 3 Primer list
[0076]
[0077] Experimental results analysis
[0078] The results of the whole blood agglutination test are as follows Figure 3 As shown, the area and mass of blood clots in mice treated with Serpin protein were significantly reduced compared with those in mice treated with PBS, indicating that Serpin protein can significantly inhibit blood coagulation.
[0079] The results of coagulation factor expression are shown in the attached Figure 4 As shown in the figure, compared with the PBS treatment group, the expression levels of multiple coagulation factors (I, II, III, V, VII, IX, XI, XII and XIII) in the liver of mice treated with Serpin protein were significantly reduced, and the expression levels of coagulation factors VIII and X were also reduced to a certain extent, which further confirmed the inhibitory effect of Serpin protein on the coagulation process.
[0080] Example 5: Verification of the in vitro interaction between Aedes albopictus salivary gland Serpin protein and mouse coagulation factor X
[0081] Step 1: Gene cloning and plasmid construction
[0082] 1. Gene cloning: The coagulation factor X (FⅩ) gene from mouse liver was cloned into the pET-30 (ɑ+) plasmid. The Serpin gene from the salivary gland of Aedes albopictus was cloned into the pET-42 (ɑ+) plasmid.
[0083] 2. Plasmid construction: Using molecular cloning technology, the target gene is correctly inserted into the corresponding expression plasmid to construct a recombinant plasmid for protein expression.
[0084] Step 2: Protein expression and lysis
[0085] 1. Induce expression: The constructed plasmid is transformed into Escherichia coli (E. coli), and the expression of GST-tagged fusion protein (GST protein and GST-Serpin protein) and His-tagged fusion protein (his-FⅩ protein) is induced.
[0086] 2. Bacterial lysis: After induction of expression, the bacteria are collected and lysed to release the fusion protein and obtain a protein mixture containing the target protein.
[0087] Step 3: Pull Down Experiment
[0088] 1. Protein purification: GST and GST-Serpin proteins were purified using GST-tagged protein purification agarose magnetic beads (purchased from Beyotime, Shanghai, China).
[0089] 2.4°C Incubation: Incubate the purified GST protein and GST-Serpin protein with the magnetic beads at 4°C overnight to ensure sufficient binding of the protein to the magnetic beads.
[0090] 3. Washing and adding his-FX protein: After incubation, discard the protein solution, wash the magnetic beads, and add his-FX protein for incubation to allow possible interaction to occur.
[0091] 4. Elution and Western blot verification: After incubation, elution is performed to collect the material bound to the magnetic beads, and Western blot technology is used to verify the interaction and detect whether the his-FⅩ protein binds to the GST-Serpin protein.
[0092] Pull Down and Western blot results are shown in the attached Figure 5 As shown, the results showed that the his-FⅩ protein existed in the band corresponding to the GST-Serpin protein, while the his-FⅩ protein signal was absent in the band corresponding to the GST protein, indicating that the Serpin protein can interact with coagulation factor X in vitro.
[0093] This example, validated by pull-down assays and Western blot analysis, confirms a direct interaction between Serpin and Factor X. This is crucial for understanding Serpin's anticoagulant mechanism and evaluating its potential as an antithrombotic drug. These results support Serpin's ability to interact with Factor X, providing further molecular evidence for its potential as a candidate antithrombotic drug.
[0094] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.
Claims
1. Application of mosquito salivary gland-based Serpin protein in the preparation of antithrombotic drugs, characterized by: The antithrombotic drug contains Serpin protein extracted from the salivary glands of Aedes albopictus. The Serpin protein can effectively prolong the APTT, PT and TT of the blood, reduce the Fib content in the blood, and interact with coagulation factor X.
2. The use according to claim 1, characterized in that: The Serpin protein has been verified to have an anticoagulant effect through in vitro and in vivo experiments.
3. The antithrombotic drug according to claim 1, characterized in that: The preparation method of the antithrombotic drug comprises the following steps: S1: Collection of salivary glands of female Aedes albopictus; S2: Extract salivary gland RNA and reverse transcribe to synthesize cDNA; S3: Amplify the Serpin gene using PCR technology; S4: Clone the amplified Serpin gene into the expression vector; S5: Transform host cells and induce expression of Serpin protein; S6: Purify the expressed Serpin protein and verify its activity.
4. The antithrombotic drug according to claim 1, characterized in that: The antithrombotic drug can be used to treat diseases caused by blood coagulation, including but not limited to deep vein thrombosis, pulmonary embolism and myocardial infarction.
5. A method of using the antithrombotic drug according to any one of claims 1 to 4, characterized in that: The method comprises administering the antithrombotic drug to a patient at an effective dose to prolong blood clotting time and reduce the risk of thrombosis.
6. The method according to claim 5, wherein: The administration of the antithrombotic drug includes but is not limited to intravenous injection, subcutaneous injection or oral administration.
7. A method for detecting the interaction between Serpin protein from the salivary glands of Aedes albopictus and coagulation factor X, characterized by: The method includes using the Pull Down technology to verify the direct binding of Serpin protein to coagulation factor X.
8. A use of an antithrombotic drug based on Serpin protein from the salivary glands of Aedes albopictus, characterized by: The uses include preventing and treating thrombosis-related diseases.