Method for collecting active proteins of water leeches, and detection method, analysis method and application thereof

Abstract

The application discloses a method for collecting active proteins of water leeches, a detection method, an analysis method and application thereof, and relates to the technical field of biological medicines. The application provides the water leech seedlings treated by hunger and the living host treated by emptying, so that the water leech seedlings adsorb and suck the host, and then the active proteins are injected into the body of the host; the sample of the host after being sucked is collected, and then is made into a freeze-dried powder after being frozen and dried, so that the active proteins of the water leeches are obtained; the proteins are extracted from the freeze-dried powder, and then are subjected to enzymolysis treatment to generate peptide segments; the liquid chromatography-mass spectrometry technology is used to detect the peptide segments; and the mass spectrometry data are subjected to database searching analysis based on the protein sequence database of the water leeches and the host, so that the active proteins from the water leeches are identified and the relative content is determined. The application utilizes the characteristic that the active proteins are injected into the body of the host in the sucking process of the water leeches, and realizes efficient enrichment of the target proteins. In combination with the proteomics technology, high-throughput and high-sensitivity protein identification is realized.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for collecting active proteins from leeches, as well as its detection method, analytical method, and applications. Background Technology

[0002] Leeches are a type of annelid with significant medicinal value, and the active proteins they secrete possess pharmacological effects such as anticoagulation, thrombolysis, and anti-inflammation. The traditional method for extracting active proteins from leeches involves directly obtaining salivary secretions from live leeches, inducing saliva secretion through physical or electrical stimulation, and then collecting the saliva. Chinese patent CN 121202953A discloses a method for extracting anticoagulant proteins from leeches, which involves preparing an induction solution and using physical stimulation to induce saliva secretion. However, this method is relatively cumbersome and susceptible to human interference. Furthermore, commonly used methods for identifying active proteins in leeches include whole tissue extraction, salivary gland omics analysis, and homologous sequence alignment, but these methods suffer from problems such as unclear targets, numerous interferences, and low efficiency. Summary of the Invention

[0003] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a method for collecting active proteins in leeches, as well as a detection method, analytical method and application thereof, specifically a method and application for collecting and detecting active proteins in leeches during the blood-sucking process.

[0004] Existing methods for extracting and identifying active proteins from leeches suffer from poor targeting, numerous interferences, low efficiency, and difficulty in obtaining protein information directly related to blood-feeding behavior. This invention, based on the biological characteristic of leeches injecting active proteins into the host during feeding, proposes a novel method for enriching active proteins by leeches feeding on specific small animals or their tissues (such as earthworms, tadpoles, and frog skin), and then combining this with high-throughput detection using proteomics technology.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for collecting active proteins in leeches, comprising the following steps: Provide starved leech seedlings and emptied live hosts; The leech seedlings are mixed with the live host, so that the leech seedlings adsorb and feed on the live host, thereby injecting active proteins into the live host. Remove the leech larvae from the live host and collect the live host after it has been ingested; The live host that has been ingested is freeze-dried to produce freeze-dried powder, which yields leech active protein.

[0006] In one specific embodiment of the present invention, the leech seedlings are selected from one or more of Japanese medicinal leeches, Tianjin medicinal leeches, and apiary leeches, and have a weight of 0.05-0.2g; The starvation treatment method includes subjecting the leech seedlings to a static starvation treatment for at least 2 weeks.

[0007] In one specific embodiment of the present invention, the live host is a small animal with a tiny or slender body, selected from one or more of earthworms, eel larvae, tadpoles, mole crickets, and amphipods; The method for emptying the intestinal contents includes: resting the live host for at least one week to empty the intestinal contents and washing with sterile water.

[0008] In one specific embodiment of the present invention, the ratio of the number of leech seedlings to the number of the live host is not less than 20:1.

[0009] In one specific embodiment of the present invention, the mixing method includes: injecting water to a depth of 5-10 cm into an insect cage, placing the leech larvae and the live host into the insect cage, allowing the leech larvae to freely attach to the live host; and ending the feeding process when more than half of the leech larvae have consumed their fill and automatically detach from the live host.

[0010] In one specific embodiment of the present invention, the freeze-drying method includes: pre-freezing the ingested live host at -40°C under normal pressure for 3 hours; freeze-drying under vacuum from -35°C to 25°C for 2 hours at a rate of 5°C increase; and finally freeze-drying under vacuum at 30°C for 5 hours.

[0011] Secondly, the present invention provides a leech active protein obtained by the method described above.

[0012] In one specific embodiment of the present invention, the leech active protein contains elastase inhibitor HMEI01, elastase inhibitor HMEI04, elastase inhibitor HMEI22, destabilase 2, serine protease inhibitor guamerin and serine protease inhibitor lefaxin, as well as at least 300 other proteins whose functions are yet to be determined.

[0013] Thirdly, the present invention provides the use of leech active proteins in the preparation of anticoagulant and / or thrombolytic and / or anti-inflammatory drugs.

[0014] Fourthly, the present invention provides a method for detecting the aforementioned leech active protein, comprising the following steps: Proteins are extracted from the active protein of the leech, and the proteins are then enzymatically hydrolyzed to generate peptides; The peptide was detected using liquid chromatography-mass spectrometry to obtain mass spectrometry data; Mass spectrometry data were searched and analyzed using protein sequence databases of leeches and their hosts to identify active proteins from leeches and determine their relative abundance.

[0015] In one specific embodiment of the present invention, the protein extraction method adopts the phenol method, and the molecular weight distribution of the extract is analyzed by using SDS-PAGE gel with a mass concentration of 4%-12%.

[0016] In one specific embodiment of the present invention, the enzymatic hydrolysis method includes: adding chloroacetic acid, trypsin and LysC enzyme to the protein, and hydrolyzing with shaking at 37°C and 1500 rpm for 1-2 hours; the hydrolyzed peptides are desalted using SOLA™ SPE 96-well plates; Before mass spectrometry detection, the enzymatically digested peptides were mixed with the iRT internal standard peptides at a volume ratio of 20:1.

[0017] In one specific embodiment of the present invention, the mass ratio of protein, chloroacetic acid, trypsin and LysC enzyme is 9~11:6.1:0.1:0.1.

[0018] In one specific embodiment of the present invention, the method of library search analysis includes: using a host and leech protein sequence database as the search target, using the iRT sequence as the internal reference sequence, and using DIA-NN software to perform library search analysis on the mass spectrometry sequencing spectral data to identify each peptide and its relative expression level.

[0019] Fifthly, the present invention provides a method for analyzing the active proteins of leeches, comprising the following steps: Based on the search results data, the LFQ of each leech protein in each sample is obtained, and the average LFQ value among multiple samples is calculated as the relative expression level information of each leech protein. Functional annotation of leech proteins was performed using blastp software and international protein databases, and the annotation results were uploaded to the PANTHER database for functional clustering. WolfPsort software was used to analyze the subcellular localization of each leech protein to further infer its functional activity characteristics; Based on the above functional and expression data, leech protein sequences were screened to obtain representative active proteins.

[0020] In one specific embodiment of the present invention, based on the results of the functional annotation and functional clustering, the leech active proteins are divided into 12 categories, of which catalytic active proteins account for 37.2% and binding active proteins account for 36.2%.

[0021] In one specific embodiment of the present invention, the leech active protein contains the following representative active proteins: Hnip04.1552, Hnip03.1277, Hnip03.342, Htia03.853, Hnip03.1808, Hnip05.1166, Hnip03.360, Hnip02.231, Hnip09.787, and Htia05.1479.

[0022] This invention has at least one of the following beneficial effects: This invention utilizes the characteristic of leeches injecting active proteins into the host during feeding to achieve highly efficient enrichment of target proteins. The enriched proteins are entirely released into the host by the leeches during natural feeding, eliminating other human interference. Furthermore, this invention combines proteomics technology to detect and analyze the enriched proteins, achieving high-throughput and high-sensitivity protein identification. It is suitable for research on the pharmacological mechanisms of leeches and the development of novel drugs, possessing significant scientific and commercial value. The treated hosts (such as earthworms, eel larvae, and mole crickets) contain leech active proteins, enhancing their medicinal or health-promoting value. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the process for collecting, detecting, and analyzing leech proteins.

[0024] Figure 2 This is a sequence diagram of the leech protein from Example 2.

[0025] Figure 3 This is a functional annotation diagram of leech protein in Example 3. Detailed Implementation

[0026] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0027] like Figure 1 As shown, this invention provides a method for collecting, detecting, and analyzing leech proteins, including the following steps: I. Leech Protein Collection Leech preparation: Select young leeches of Japanese medicinal leech, Tianjin medicinal leech, or Filipino leech, weighing 0.05–0.2g, and keep them in a state of starvation for more than 2 weeks. It is preferable to use leech seedlings that have hatched from egg cocoons, and keep them in a state of starvation for 1–2 weeks after hatching.

[0028] Host preparation: Select earthworms, tadpoles, bullfrogs, mole crickets, or amphipods as hosts, and allow them to rest for one week to empty their intestinal contents. During this period, wash with sterile water at least three times to reduce microbial contamination.

[0029] Leech feeding: Fill a 30×30×30cm 100-mesh insect cage with 5–10cm of water. Place leeches and hosts in a ratio of at least 20:1, allowing them to attach freely. After 5–10 minutes, lift the insect cage and continue observing until more than half of the leeches have satiated and detached. Remove any leeches that have not detached using tweezers.

[0030] Sample freeze-drying: Collect the ingested host sample and place it in a low-temperature freeze-dryer. Freeze-drying procedure: Pre-freeze at -40℃ for 3 hours under normal pressure; freeze-dry under vacuum from -35℃ to 25℃ in increments of 5℃ for 2 hours; finally freeze-dry under vacuum at 30℃ for 5 hours. Grind the freeze-dried sample into powder and store at -20℃.

[0031] The innovation of this invention lies in the fact that after leeches feed on earthworms, they inject trace amounts of salivary proteins into the earthworm's body. These proteins are released into the host's body entirely by the leeches during natural feeding, eliminating other human interference. However, the amount of active proteins obtained by this method is extremely small, and cannot be accurately measured using conventional methods such as the thrombin method. Therefore, this invention employs a proteomics approach for detection and analysis.

[0032] II. Leech protein detection Protein extraction: Weigh 0.1–0.3 g of lyophilized powder and extract total protein using the phenol method. Separate the proteins using 4%–12% SDS-PAGE gel electrophoresis and perform preliminary analysis of the protein molecular weight distribution.

[0033] Enzymatic hydrolysis: Chloroacetic acid, trypsin, and LysC enzyme were added to leech antithrombotic protein, and the mixture was hydrolyzed at 37°C and 1500 rpm for 1–2 hours. The hydrolyzed peptides were desalted using SOLA™ SPE 96-well plates.

[0034] Mass spectrometry detection: The enzymatically digested peptides were separated by liquid chromatography and then analyzed by mass spectrometry. Before injection, the iRT internal standard (purchased from ThermoFisher Scientific) was mixed with the sample at a volume ratio of 1:20.

[0035] Library search analysis: Using protein sequence databases of the host and leeches as the search targets, and iRT sequences as internal reference sequences, the DIA-NN software (https: / / github.com / vdemichev / DiaNN) was used to perform library search analysis on the mass spectrometry sequencing data, removing proteins from the host and retaining only protein sequences from leeches.

[0036] III. Leech Protein Analysis Expression level analysis: The Label-Free Quantification (LFQ) of each leech protein in each sample was obtained from the database search analysis results file, and the mean LFQ value among multiple samples was calculated as the relative expression level information of each leech protein.

[0037] Functional analysis: Leech proteins were functionally annotated using blastp software and the UniProt international protein database, and the annotation results were uploaded to the PANTHER database (https: / / pantherdb.org / ) for functional clustering.

[0038] Subcellular localization analysis: The subcellular localization of each leech protein was analyzed using WolfPsort software (https: / / github.com / fmaguire / WoLFPSort.git) to further infer its functional activity characteristics.

[0039] Sequence screening: Combining the above functional and expression data, leech protein sequences are screened to obtain the most representative active proteins.

[0040] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following specific embodiments.

[0041] Example 1: Collection of proteins from Japanese medicinal leeches This embodiment provides a method for collecting active proteins from leeches, specifically a method for collecting active proteins from Japanese medicinal leeches, including the following steps: (1) Leech preparation: Select 400 Japanese medicinal leech seedlings (weighing about 0.1g) and keep them in a state of hunger for 3 weeks.

[0042] (2) Host preparation: Select 20 healthy Eisenia fetida worms, keep them quiet for 7 days to allow them to empty their intestinal contents, and wash them with sterile water every day to reduce microbial contamination.

[0043] (3) Leech feeding: Place 5cm of water in a 30×30×30cm 100-mesh insect cage, and put 20 Eisenia fetidae and 400 Hirudo nipponiae larvae into the cage. Observe the feeding process. After about 8-10 minutes, lift the cage and continue to observe until more than 60% of the leeches are satiated and fall off. Remove any remaining individuals with tweezers.

[0044] (4) Sample freeze-drying: Collected Eisenia fetidae after being fed on and placed in a low-temperature freeze-dryer. Freeze-drying procedure: Pre-freeze at -40℃ for 3 hours under normal pressure; freeze-dry under vacuum from -35℃ to 25℃ for 2 hours at a rate of 5℃ increase; finally freeze-dry under vacuum at 30℃ for 5 hours. Grind the freeze-dried sample into powder and store at -20℃ to obtain leech active protein.

[0045] Example 2: Detection of Hirudin from Japanese Medical Leeches This embodiment provides a method for detecting active proteins in leeches, specifically for detecting active proteins from Japanese medicinal leeches collected in Example 1, including the following steps: (1) Protein extraction: Take 0.2g of powder, extract protein by phenol method, separate by 6% SDS-PAGE gel electrophoresis, and preliminarily analyze the molecular weight distribution of protein.

[0046] (2) Enzymatic hydrolysis: 6.1 mg chloroacetic acid, 0.1 mg trypsin and 0.1 mg LysC enzyme were added to 10 mg leech antithrombotic protein and enzymatically hydrolyzed for 1.5 hours at 37°C and 1500 rpm. The enzymatically hydrolyzed peptides were desalted using SOLA™ SPE 96-well plates.

[0047] (3) Proteome sequencing: Peptide separation and detection were performed using LC-MS / MS with iRT internal standard calibration. The specific method was as follows: enzymatically digested peptides were separated by liquid chromatography and then sent to a mass spectrometer for detection. A nano-liquid chromatography (C18 column, 300 nL / min, 90-minute gradient) coupled with an Orbitrap Astral mass spectrometer was used. MS1 resolution was 120K; MS2 used DDA mode with a cycle time of 1 second and dynamic exclusion for 20 seconds. Before injection, the iRT internal standard (purchased from ThermoFisher Scientific) was mixed with the sample at a volume ratio of 1:20.

[0048] (4) Proteome database search: Using host and leech protein sequence databases as search targets, and iRT sequences as internal reference sequences, the mass spectrometry sequencing spectral data was analyzed using DIA-NN software (https: / / github.com / vdemichev / DiaNN). Host-derived proteins were removed, retaining only leech-derived protein sequences, and each peptide and its relative expression level were identified. In this embodiment, the protein databases of Eisenia fetida and Hirudo medicinalis were searched, identifying 320 active proteins derived from leeches, including various anticoagulant and anti-inflammatory peptides.

[0049] like Figure 2 As shown, this invention detected some active proteins from *Hirudo medicinalis*, of which six are known antithrombotic proteins: HMEI01, HMEI04, and HMEI22 are elastase inhibitors with anti-inflammatory effects; destabilase2 is an destabilizing enzyme with thrombolytic activity; guamerin is a multifunctional serine protease inhibitor with both anticoagulant and tissue-protective functions; and lefaxin is also a serine protease inhibitor with anticoagulant activity. The remaining sequences have not been specifically studied and require further functional annotation.

[0050] Example 3: Analysis of proteins in Japanese medicinal leeches This embodiment provides an analytical method for leech active proteins, specifically for functional annotation of the Japanese medicinal leech active proteins detected in Example 2, including the following steps: (1) Expression level analysis: Quantitative analysis showed that the relative expression level of these proteins was 7.6 × 10⁻⁶. 6 The expression levels of different proteins varied significantly, even though each protein had an LFQ value of 6.7 × 10⁻⁶ units. For example, among the three detected HMEI family members (HMEI01, HMEI04, and HMEI22), HMEI04 had an LFQ value of 6.7 × 10⁻⁶ units. 6 The LFQ of HMEI01 and HMEI22 are only 1.0 × 10⁻⁶ respectively. 6 and 1.0×10 6 .

[0051] (2) Functional analysis: Using the detected Japanese medicinal leech protein as the search sequence, the sequence was compared with all sequences in the UniProt international protein database using blastp software to obtain the UniProt ID of the target sequence with the smallest E value. The UniProt ID of the target sequence (annotation result) was uploaded to the PANTHER database (https: / / pantherdb.org / ) for GO functional clustering. The results of functional annotation and clustering are as follows: Figure 3 As shown, the proteins of the Japanese medicinal leech can be divided into 12 categories, among which catalytic activity (accounting for 37.2%) and binding (accounting for 36.2%) are absolutely dominant.

[0052] (3) Subcellular localization: The subcellular localization information of each Japanese medicinal leech protein was analyzed using the WolfPsort software. The study found that the Japanese medicinal leech protein contained 41 exotropic active proteins.

[0053] (4) Sequence screening: Based on expression levels, functional annotations, and subcellular localization data, leech protein sequences were screened to obtain the most representative exotropic active proteins. Table 1 lists the top 10 representative exotropic active proteins from Japanese medicinal leeches. Functional searches revealed that they are all directly or indirectly related to antithrombotic functions. Therefore, the leech proteins collected in this invention can serve as candidate proteins for new drug development.

[0054] Table 1. Representative exoproteins of 10 types from Japanese medicinal leeches, along with their functions and expression levels. In summary, this invention enriches active proteins by leeches feeding on specific hosts and achieves efficient identification by combining proteomics technology, providing reliable technical support for the research and development of leech proteins.

[0055] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for collecting active proteins from leeches, characterized in that, Includes the following steps: Provide starved leech seedlings and emptied live hosts; The leech seedlings are mixed with the live host, allowing the leech seedlings to adsorb and consume the live host, thereby injecting active proteins into the live host. Remove the leech larvae from the live host and collect the live host after it has been ingested; The live host that has been ingested is freeze-dried to produce freeze-dried powder, which yields leech active protein.

2. The method according to claim 1, characterized in that, The leech seedlings are selected from one or more of Japanese medicinal leeches, Tianjin medicinal leeches, and Filipino leeches, and weigh 0.05-0.2g; The starvation treatment method includes: subjecting the leech seedlings to a static starvation treatment for at least 2 weeks; The live host is a small or slender animal, selected from one or more of earthworms, eel larvae, tadpoles, mole crickets, and amphipods; The method for emptying the intestinal contents includes: resting the live host for at least one week to empty the intestinal contents and washing with sterile water.

3. The method according to claim 1, characterized in that, The ratio of the number of leech seedlings to the number of live hosts is not less than 20:1; The mixing method includes: injecting water to a depth of 5-10 cm into an insect cage, placing the leech larvae and the live host into the insect cage, allowing the leech larvae to freely attach to the live host; and ending the feeding process when more than half of the leech larvae have consumed their fill and automatically detach from the live host. The freeze-drying method includes: pre-freezing the ingested live host at -40°C under normal pressure for 3 hours; freeze-drying under vacuum from -35°C to 25°C for 2 hours at a rate of 5°C increase; and finally freeze-drying under vacuum at 30°C for 5 hours.

4. A leech-active protein, characterized in that, The leech active protein obtained by the method according to any one of claims 1 to 3 contains elastase inhibitor HMEI01, elastase inhibitor HMEI04, elastase inhibitor HMEI22, destabilase 2, serine protease inhibitor guamerin and serine protease inhibitor lefaxin, as well as at least 300 other proteins whose functions are yet to be determined.

5. The use of the leech active protein of claim 4 in the preparation of anticoagulant and / or thrombolytic and / or anti-inflammatory drugs.

6. A method for detecting leech active proteins according to claim 4, characterized in that, Includes the following steps: Proteins are extracted from the active protein of the leech, and the proteins are then enzymatically hydrolyzed to generate peptides; The peptide was detected using liquid chromatography-mass spectrometry to obtain mass spectrometry data; Mass spectrometry data were searched and analyzed using protein sequence databases of leeches and their hosts to identify active proteins from leeches and determine their relative abundance.

7. The detection method according to claim 6, characterized in that, The protein was extracted using the phenol method, and the molecular weight distribution of the extract was analyzed using SDS-PAGE gel with a mass concentration of 4%-12%. The enzymatic hydrolysis method includes: adding chloroacetic acid, trypsin, and LysC enzyme to the protein, and hydrolyzing with shaking at 37°C and 1500 rpm for 1-2 hours; the hydrolyzed peptides are then desalted using SOLA™ SPE 96-well plates. Before mass spectrometry detection, the enzymatically digested peptides were mixed with the iRT internal standard peptides at a volume ratio of 20:

1. The method for searching and analyzing the database includes: using protein sequence databases of the host and leeches as the search targets, using iRT sequences as internal reference sequences, and using DIA-NN software to perform database search and analysis on the mass spectrometry sequencing spectral data to identify each peptide and its relative expression level.

8. A method for analyzing the active protein of leeches according to claim 4, characterized in that, Includes the following steps: According to the search results data described in claim 6, the LFQ of each leech protein in each sample is obtained, and the average LFQ value among multiple samples is calculated as the relative expression level information of each leech protein. Functional annotation of leech proteins was performed using blastp software and international protein databases, and the annotation results were uploaded to the PANTHER database for functional clustering. WolfPsort software was used to analyze the subcellular localization of each leech protein to further infer its functional activity characteristics; Based on the above functional and expression data, leech protein sequences were screened to obtain representative active proteins.

9. The analytical method according to claim 8, characterized in that, Based on the functional annotation and functional clustering results, the leech active proteins were divided into 12 categories, of which catalytic active proteins accounted for 37.2% and binding active proteins accounted for 36.2%.

10. The analytical method according to claim 8, characterized in that, The leech active proteins contain the following representative active proteins: Hnip04.1552, Hnip03.1277, Hnip03.342, Htia03.853, Hnip03.1808, Hnip05.1166, Hnip03.360, Hnip02.231, Hnip09.787, and Htia05.1479.

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