A polypeptide derived from East Asian scorpion and its application

Abstract

This invention discloses a polypeptide derived from the East Asian scorpion and its applications, belonging to the field of functional protein technology. The amino acid sequence of the East Asian scorpion-derived polypeptide is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5. This invention also provides the application of the East Asian scorpion-derived polypeptide with the amino acid sequence shown in SEQ ID NO.1 in the preparation of procoagulant drugs and the application of the East Asian scorpion-derived polypeptide with the amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5 in the preparation of anticoagulant drugs. The East Asian scorpion-derived polypeptide provided by this invention has good application value in the development of antithrombotic or procoagulant drugs.

Description

Technical Field

[0001] This invention relates to the field of functional protein technology, and in particular to a polypeptide derived from the East Asian scorpion and its applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The coagulation system activates prothrombin through both intrinsic and extrinsic pathways, promoting the conversion of fibrinogen into fibrin. The anticoagulation system, on the other hand, relies on antithrombin-III, the protein C pathway, and the fibrinolytic system for dynamic regulation. Maintaining the dynamic balance between blood coagulation and anticoagulation is crucial for normal vascular function: when this balance is disrupted, excessive coagulation can lead to thrombosis and cardiovascular events, while insufficient coagulation can result in massive bleeding, such as deep intraoperative hemorrhage; both pose serious threats to health. Therefore, developing safe and effective anticoagulants and procoagulants is of great significance for maintaining coagulation balance and ensuring the success of clinical treatment.

[0004] Existing anticoagulants have significant limitations: conventional chemical drugs such as warfarin require strict restriction of vitamin K intake or rely on hepatic drug-metabolizing enzymes, making them prone to drug interactions; while peptide drugs are highly specific and easily metabolized, some have side effects such as thrombocytopenia and bleeding, and naturally derived anticoagulant peptides are difficult to mass-produce due to their low content and high purification costs. Regarding procoagulant drugs, there is an urgent clinical need. Commonly used snake venom thrombins (such as tebuconazole) have the advantages of rapid onset and significant efficacy, but they rely on specific snake species, and long-term use may face resource limitations or immunogenicity risks. Therefore, the development of new procoagulant drugs is urgently needed.

[0005] Scorpions, as a traditional medicinal material, possess the effects of calming wind and relieving spasms, clearing the meridians and relieving pain. They are widely used in the treatment of infantile convulsions, hemiplegia, and other diseases. The main medicinal species is the East Asian scorpion (also known as the Ma's scorpion). Currently, active components with coagulation-regulating functions have been found in the venom of the East Asian scorpion. However, existing technologies mostly focus on crude extracts or mixed components of the East Asian scorpion, and there are very few reports of single polypeptides with clear sequences and significant activities, which is difficult to meet the clinical demand for diversified development of anticoagulant and procoagulant drugs. Summary of the Invention

[0006] In view of this, the present invention provides a polypeptide derived from the East Asian scorpion and its application. The present invention identifies and identifies five polypeptides, MmTX70, MmTX118, MmTX67, MmTX106 and MmTX126, from the East Asian scorpion gene transcriptome. The recombinant polypeptide obtained by prokaryotic expression of its sequence showed procoagulant or anticoagulant activity in in vitro experiments.

[0007] In a first aspect, the present invention provides a polypeptide derived from the East Asian scorpion, the amino acid sequence of which is shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5.

[0008] Specifically, the amino acid sequence of MmTX70 (SEQ ID NO. 1) is as follows:

[0009] QLVTGCVFWQCRNSCLSKGYRGGGMCQGIIEKKCYCIR.

[0010] The amino acid sequence of MmTX118 (SEQ ID NO. 2) is as follows:

[0011] KKNGYPADDDGCKIACFFRKNGCPFACQKLNGSTGSCDIVKKACKCEGLPDNAKLWDQTKQCNKK.

[0012] The amino acid sequence of MmTX67 (SEQ ID NO. 3) is as follows:

[0013] KTSCNHLKRCAKYGFYRNCTECCKQHKHSGGYCTMYKCLCKI.

[0014] The amino acid sequence of MmTX106 (SEQ ID NO. 4) is as follows:

[0015] GDGYIRNVDDGCKLSCFLQNEMCNRECKIRGAYYGYCWSWGISCWCEGLPDNKLWKQETNTCRGKK.

[0016] The amino acid sequence of MmTX126 (SEQ ID NO. 5) is as follows:

[0017] SMFDDGYPVKNGCRISCIPDEHDDLCEQFCKKNKAETGGCDFDADACKCWGELGGMEIWEPKSSECKSWNDNLITKILEN.

[0018] Secondly, the present invention provides a coding gene that encodes the aforementioned polypeptide derived from the East Asian scorpion, the nucleotide sequence of which is shown in SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9 or SEQ ID NO. 10.

[0019] Specifically, the nucleotide sequence encoding MmTX70 (SEQ ID NO. 6) from the 5' end to the 3' end is as follows:

[0020] CAGCTTGTGACCGGTTGTGTTTTCTGGCAGTGTCGTAATAGCTGTCTGAGCAAAGGCTATCGCGGTGGTGGTATGTGCCAGGGTATTATTGAAAAGAAATGCTACTGCATCCGC.

[0021] The nucleotide sequence encoding MmTX118 (SEQ ID NO. 7) from the 5' end to the 3' end is as follows:

[0022] AAAAAGAATGGTTATCCAGCAGATGATGATGGTTGTAAGATAGCTTGCTTTTTCCGTAAGAATGGTTGCCCTTTTGCATGCCAAAAACTGAATGGCAGTACTGGTTCTTGTGATATAGTGAAAAAAGCATGCAAATGTGAAGGACTTCCAGATAATGCAAAACTTTGGGATCAAACCAAACAATGCAACAAAAAATAA.

[0023] The nucleotide sequence encoding MmTX67 (SEQ ID NO. 8) from the 5' end to the 3' end is as follows:

[0024] AAAACTAGCTGCAATCATTTAAAGCGATGTGCAAAGTATGGATTTTATCGAAATTGTACCGAATGCTGTAAACAACATAAACATTCAGGAGGTTATTGTACAATGTATAAATGCCTATGTAAAATATAA.

[0025] The nucleotide sequence encoding MmTX106 (SEQ ID NO. 9) from the 5' end to the 3' end is as follows:

[0026] GGTGACGGTTATATTCGTAATGTTGATGATGGCTGCAAGCTGAGCTGTTTTCTGCAGAATGAAATGTGCAATCGTGAATGTAAGATCCGTGGTGCCTATTATGGCTATTGCTGGAGCTGGGGTATTAGTTGCTGGTGCGAAGGCCTGCCGGATAATAAGCTGTGGAAACAGGAAACCAATACCTGTCGCGGTAAAAAATAA.

[0027] The nucleotide sequence encoding MmTX126 (SEQ ID NO. 10) from the 5' end to the 3' end is as follows:

[0028] TCAATGTTCGATGATGGATATCCAGTTAAAAACGGCTGCCGAATTTCATGCATACCAGATGAGCATGATGATCTTTGCGAACAATTCTGCAAGAAGAATAAAAGCAGAAACAGGTGGATGTGACTTTGATGCATGCATGCAAATGTTGGGGAGAATTAGGTGGAATGGAAATATGGGAACCTAAATCATCAGAATGCAAGAGTTGGAATGACAATCTAATAACAAAGATTTTGGAAAATTAA.

[0029] The present invention does not impose any special restrictions on the source of the encoding gene; it can be prepared using conventional methods in the art or synthesized by a biotechnology company.

[0030] Thirdly, the present invention provides a recombinant plasmid having the above-mentioned coding gene inserted into it.

[0031] In this invention, the original plasmid of the recombinant plasmid includes pET-32a.

[0032] In this invention, the recombinant plasmid is preferably constructed using the following method: the coding gene is ligated into the vector pET-32a; the method of ligating the coding gene into the vector pET-32a is preferably homologous recombination to construct the plasmid.

[0033] Fourthly, the present invention provides a recombinant bacterium comprising the above-mentioned recombinant plasmid.

[0034] In this invention, the original strain of the recombinant bacteria is preferably Escherichia coli, more preferably Escherichia coli BL21(DE3) or Escherichia coli Shuffle T7 strain.

[0035] Fifthly, the present invention provides a procoagulant pharmaceutical composition comprising a polypeptide derived from the East Asian scorpion with the amino acid sequence SEQ ID NO. 1.

[0036] In a sixth aspect, the present invention provides the use of the above-described procoagulant drug composition in the preparation of procoagulant drugs.

[0037] In a seventh aspect, the present invention provides an anticoagulant pharmaceutical composition comprising a polypeptide derived from the East Asian scorpion with an amino acid sequence as shown in SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5.

[0038] Eighthly, the present invention provides the use of the above-described anticoagulant drug composition in the preparation of anticoagulant drugs.

[0039] In a ninth aspect, the present invention provides the application of a polypeptide derived from the East Asian scorpion, with an amino acid sequence as shown in SEQ ID NO. 1, in the preparation of a procoagulant drug.

[0040] In a tenth aspect, the present invention provides the use of polypeptides derived from the East Asian scorpion, with amino acid sequences such as SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5, in the preparation of anticoagulant drugs.

[0041] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0042] This invention identifies and successfully constructs prokaryotic expression systems for five polypeptides (MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126) from the venom of the East Asian scorpion for the first time. This overcomes the industrialization bottlenecks of low content and difficult purification associated with traditional natural extraction methods, facilitating large-scale production. The recombinant polypeptides obtained through heterologous expression in this invention exhibit procoagulant or anticoagulant activity in experiments: MmTX70 shortens fibrin formation time via an endogenous pathway, exhibiting a procoagulant effect; while MmTX118, MmTX67, MmTX106, and MmTX126 prolong fibrin formation time and clot formation time, demonstrating good anticoagulant activity, especially MmTX67 and MmTX106. The East Asian scorpion-derived polypeptides provided by this invention have significant application value in the development of antithrombotic or procoagulant drugs. Attached Figure Description

[0043] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0044] Figure 1 These are Tricine-SDS-PAGE results of five East Asian scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 in Example 1 of this invention. Among them, A is the Tricine-SDS-PAGE result of MmTX70, MmTX118, MmTX67, and MmTX126, and B is the Tricine-SDS-PAGE result of MmTX106.

[0045] Figure 2 The results of the determination of the inhibitory effect of five East Asian scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106 and MmTX126 on activated partial thromboplastin time (APTT) in Example 2 of the present invention are as follows;

[0046] Figure 3 The results show the inhibitory effects of five East Asian scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 on prothrombin time (PT) in Example 2 of this invention.

[0047] Figure 4 The results of the determination of the inhibitory effect of five East Asian scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106 and MmTX126 on thrombin time (TT) in Example 2 of the present invention are as follows;

[0048] Figure 5 The results (25 μM) show the inhibitory effect of five East Asian scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 on recalcification time in Example 3 of this invention.

[0049] Figure 6 The results (50 μM) show the inhibitory effect of five East Asian scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 on recalcification time in Example 3 of this invention.

[0050] Figure 7 The results show the inhibitory effects of five East Asian scorpion polypeptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 on thrombus formation in Example 4 of this invention. Detailed Implementation

[0051] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0052] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0053] In the following examples, Tricine-SDS-PAGE is a high-resolution electrophoresis technique for separating small molecule proteins or peptides. Its name is a combination of the abbreviations of three core components (Tricine, SDS, and PAGE), and the meanings and functions of each part are as follows: Tricine represents N-tris(hydroxymethyl)methylglycine, which is an amphoteric buffer; SDS represents sodium dodecyl sulfate, which is an anionic surfactant; and PAGE represents polyacrylamide gel electrophoresis.

[0054] Example 1

[0055] This embodiment provides five East Asian scorpion polypeptides, MmTX70, MmTX118, MmTX67, MmTX106 and MmTX126, which were obtained by molecular cloning technology and prokaryotic expression and purification in Escherichia coli.

[0056] 1. Constructing recombinant plasmids

[0057] Using East Asian scorpion cDNA as a template, the nucleotide sequences of MmTX118, MmTX67, and MmTX126 (as shown in SEQ ID NO. 7, 8, and 10, respectively) were obtained by PCR and ligated to the pET32a vector via homologous recombination. The gene sequences of MmTX70 and MmTX106 were codon-optimized, and the optimized gene sequences (as shown in SEQ ID NO. 6 and 9) were synthesized using whole-genome synthesis technology and ligated to the pET32a vector to construct plasmids. A recombinant enterokinase site (GACGACGACGACAAG, SEQ ID NO. 11) was introduced at the 5' end of each gene. The recombinant plasmids pET32a-MmTX70, pET32a-MmTX118, pET32a-MmTX67, pET32a-MmTX106, and pET32a-MmTX126 were constructed.

[0058] 2. Prokaryotic expression

[0059] The recombinant plasmids pET-32a-MmTX118, pET-32a-MmTX67, and pET32a-MmTX126 were transformed into *E. coli* BL21(DE3) for expression; the recombinant plasmids pET-32a-MmTX70 and pET-32a-MmTX106 were transformed into *E. coli* ShuffleT7 strain for expression. Single colonies were picked and placed in shake tubes, 5 mL of LB medium and 5 μL of 100 mg / mL ampicillin (AMP) were added, and the mixture was incubated overnight at 37°C and 200 rpm to obtain a seed culture. 1 mL of the seed culture was transferred to 100 mL of LB liquid medium, 50 μL of 100 mg / mL AMP was added, and the mixture was incubated at 37°C and 200 rpm for about 3 h. When OD=0.6, 10 mL of the bacterial culture was transferred to 1000 mL of LB liquid medium, and 1000 μL of AMP was added. AMP was cultured at 37℃ for 4 hours. When the bacterial culture became turbid and reached the logarithmic phase, 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added, and protein expression was induced at 18℃ and 120 rpm for about 18 hours. The culture was then centrifuged at 4℃ and 3900 rpm, the supernatant was discarded, and the five recombinant bacteria were frozen at -80℃.

[0060] 3. Recombinant protein purification

[0061] The bacterial cells were resuspended in lysis buffer containing 10 mM imidazole, sonicated on ice, and the supernatant was collected after centrifugation. The nickel column was equilibrated with wash buffer containing 20 mM imidazole, followed by gradient elution with elution buffers containing different concentrations of imidazole to obtain the target proteins MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126. Protein solubility and expression levels were verified by SDS-PAGE. Recombinant enterokinase (1 U: 1 mg) was used to remove the fusion protein tag TrxA, and protein size was verified by Tricine-SDS-PAGE. The Tricine-SDS-PAGE results are shown below. Figure 1 As shown, from Figure 1 From A, the molecular weight of MmTX70 is consistent with the expected 4.3 kDa, the molecular weight of MmTX118 is consistent with the expected 7.1 kDa, the molecular weight of MmTX67 is consistent with the expected 5.4 kDa, and the molecular weight of MmTX126 is consistent with the expected 9.0 kDa. Figure 1 The molecular weight of MmTX106 obtained from B is consistent with the expected 7.7 kDa.

[0062] Example 2

[0063] This embodiment provides the effects of peptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 in Example 1 on activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT).

[0064] The activated partial thromboplastin time (APTT) test primarily reflects the intrinsic coagulation pathway, and the length of APTT depends mainly on the activity of coagulation factors in this pathway. The prothrombin time (PT) test is mainly used to assess the extrinsic coagulation pathway; if coagulation factors are deficient or their activity is reduced, PT will be prolonged. The principle of the thrombin time (TT) test is based on adding a "standardized" thrombin solution to the tested plasma and observing the time required for plasma coagulation. This reflects the time it takes for fibrinogen in the plasma to convert to fibrin, and helps determine whether there are abnormalities in fibrinogen levels or the presence of anticoagulants.

[0065] In this embodiment, coagulation function tests (APTT, PT, TT) were performed using the kits as instructed by Beijing Zhongchi Weiye Technology Development Co., Ltd., and measured using an XL32OOc fully automated coagulation analyzer. The blank control was physiological saline, and enoxaparin sodium was used as a positive control. The final concentration of all peptides was 25 μM. Results are as follows: Figure 2 , Figure 3 and Figure 4 As shown in the figure, "*", "**", "***", "****", and "ns" are used to represent the range of P-values ​​at different significance levels. "*" indicates a P-value ≤ 0.05; "**" indicates a P-value ≤ 0.01; "***" indicates a P-value ≤ 0.001; and "****" indicates a P-value ≤ 0.0001. The more "*" symbols, the more significant the result. "ns" indicates a P-value > 0.05, meaning the result is not significant.

[0066] Depend on Figure 2 and Figure 3 It was found that APTT was significantly reduced under the action of MmTX70, and significantly increased under the action of MmTX67 and MmTX106, while PT test results were not significant. This indicates that MmTX70, MmTX67, and MmTX106 act on the intrinsic coagulation pathway rather than the extrinsic coagulation pathway, and that MmTX70 has a procoagulant effect, while MmTX67 and MmTX106 have anticoagulant effects. Figure 4 The results showed that MmTX67 and MmTX106 significantly prolonged thrombin time, indicating that MmTX67 and MmTX106 inhibit fibrin formation and have significant anticoagulant activity.

[0067] Example 3

[0068] This embodiment provides the effect of peptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Example 1 on recalcification time.

[0069] The recalcification assay involves treating blood or plasma samples with an anticoagulant to remove free calcium ions from the blood, preventing the clotting process from starting normally. When calcium ions are reintroduced, the clotting factors are reactivated, and the clotting process restarts, thus exploring the influence of various factors on the clotting process.

[0070] The specific steps are as follows: Dilute MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 to 125 μM or 250 μM; add 20 μL of sample to a 96-well plate, making two replicates; add 50 μL of thrombocytopenic plasma (PPP), incubate the 96-well plate in a shaker (37℃, 200 rpm) for 10 min, add 30 μL of calcium ion solution at 37℃, and immediately measure the absorbance at 405 nm using a microplate reader. The final sample concentrations are determined to be 25 μM and 50 μM, respectively. For the blank control group, 20 μL of physiological saline was used instead of 20 μL of peptide sample; for the negative control group, 30 μL of physiological saline was used instead of 30 μL of calcium ion solution. Results are as follows: Figure 5 and Figure 6 As shown.

[0071] Depend on Figure 5 and Figure 6 It can be seen that the negative control group maintained the lowest absorbance because no calcium ion solution was added, thus the coagulation reaction was not initiated, resulting in the lowest absorbance. The blank control group, after the addition of calcium ions, initiated the coagulation reaction, and the absorbance gradually increased with coagulation. MmTX70 at 25 μM shortened the fibrin formation time, exhibiting a procoagulant effect, while its effect was not significant at 50 μM. MmTX67 and MmTX106 significantly inhibited fibrin formation at both 25 μM and 50 μM concentrations, demonstrating significant anticoagulant effects. The anticoagulant effect of MmTX67 and MmTX106 at 25 μM was better than that at 50 μM. MmTX118 and MmTX126 slightly prolonged the recalcification time at a concentration of 50 μM. These results indicate that MmTX118, MmTX67, MmTX106, and MmTX126 all prolong the recalcification time. Among them, MmTX67 and MmTX106 showed more significant anticoagulant effects.

[0072] Example 4

[0073] This embodiment provides the effects of peptides MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 from Example 1 on thrombus formation.

[0074] Thromboelastography (TEG) can dynamically monitor the entire blood coagulation process. By detecting and analyzing the strength and stability of blood clots formed by the interaction between fibrin and platelets, TEG can effectively simulate the impact of test samples on the actual coagulation process in the body.

[0075] The specific steps are as follows:

[0076] 32 μL of diluted solutions of MmTX70, MmTX118, MmTX67, MmTX106, and MmTX126 (final concentration 25 μM) were mixed with 310 μL of platelet-deficient plasma (PPP) and incubated at 37°C for 5 min to obtain the mixture. The thromboelastography instrument was turned on, the interface was adjusted, and the beaker was placed. 20 μL of calcium ion solution was added to the beaker, followed by 340 μL of the mixture. The thromboelastography instrument was operated according to the instructions. To eliminate the influence of solvent, PBS (phosphate-buffered saline) and DMSO (dimethyl sulfoxide) groups were set up as controls. The results are as follows. Figure 7 As shown.

[0077] Depend on Figure 7 It can be seen that MmTX70 shortens the blood clot formation time and has procoagulant activity; MmTX118 slightly prolongs the blood clot formation time and has antithrombotic activity; MmTX67, MmTX106, and MmTX126 significantly prolong the blood clot formation time and have significant antithrombotic activity.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide derived from the East Asian scorpion, characterized in that, The amino acid sequence of the polypeptide derived from the East Asian scorpion is shown in SEQ ID NO. 3 or SEQ ID NO.

4.

2. A gene encoding a gene, characterized in that, It encodes the East Asian scorpion-derived polypeptide of claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 8 or SEQ ID NO.

9.

3. A recombinant plasmid, characterized in that, Its insertion contains the coding gene as described in claim 2.

4. A recombinant bacterium, characterized in that, It contains the recombinant plasmid as described in claim 3.

5. An anticoagulant drug composition, characterized in that, It includes polypeptides derived from the East Asian scorpion, with amino acid sequences as shown in SEQ ID NO. 3 or SEQ ID NO.

4.

6. The use of the anticoagulant composition as described in claim 5 in the preparation of anticoagulant drugs.

7. Application of peptides derived from the East Asian scorpion with amino acid sequences as shown in SEQ ID NO. 3 or SEQ ID NO. 4 in the preparation of anticoagulant drugs.

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