Jellyfish punching toxin protein as well as separation and purification method and application thereof
Through the combination of low-temperature high-speed centrifugation, ultracentrifugation and gel filtration column Superdex 75 16/600 GL and anion exchange column Mono Q, the sand jellyfish piercing toxin protein was successfully isolated and purified, solving the failure to effectively separate the existing technology. The problem of purifying lethal piercing toxin proteins has laid the foundation for in-depth study of its structure, function and mechanism of action and the development of sand jellyfish sting treatment drugs.
Patent Information
- Application Number
- CN202411759379.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-03
- Publication Date
- 2025-02-07
AI Technical Summary
The prior art has failed to effectively isolate and purify lethal piercing toxin proteins from sand jellyfish venom, resulting in the inability to conduct in-depth research on its structure, function and mechanism of action, and the inability to develop therapeutic drugs for sand jellyfish stings.
The sand jellyfish perforated toxin protein was isolated and purified by combining the combination of low-temperature high-speed centrifugation, ultracentrifugation and gel filtration column Superdex 75 16/600 GL and anion exchange column Mono Q.
The successful separation of high-efficiency and high-resolution perforated toxin protein from sand jellyfish venom, with a molecular weight of about 45kDa, laying the foundation for in-depth study of its structure, function and mechanism of action, and the development of therapeutic drugs for sand jellyfish stings.
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Figure CN119390802A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biochemistry, and in particular relates to a sand jellyfish pore-piercing toxin protein and a separation and purification method and application thereof. Background Art
[0002] Sand jellyfish ( Nemopilema nomurai ) is a cnidarian belonging to the order Rhizostomata and the family Rhizostomidae. It is a poisonous marine animal. Sand jellyfish contain a large number of tentacles. Nematocysts exist on the surface of the tentacles of sand jellyfish. They can secrete toxins. After piercing the skin, the venom can be injected into humans or other animals to achieve predation and defense functions. When human skin comes into contact with the tentacles of sand jellyfish, stinging symptoms will appear, such as: burning pain and itching, blisters, rashes, tissue necrosis, and severe allergic reactions may occur when the sting is severe, as well as systemic reactions including fever, difficulty breathing, muscle weakness, etc. There is a very small probability of death due to cardiac arrest, acute pulmonary edema, etc. after the sting.
[0003] The toxin of sand jellyfish is mainly a complex mixture of various protein toxins, peptides, etc. Current studies have shown that the toxin of sand jellyfish has various toxic effects, including lethal toxicity, blood toxicity, skin toxicity, kidney toxicity, etc. There is currently no emergency medicine for sand jellyfish stings. The main reason is that there is no purification method for separating lethal toxin protein from sand jellyfish venom, and it is impossible to obtain pure lethal toxin of sand jellyfish, and study its properties, functions and antidotes. This application has established a method for separating and purifying perforating toxin protein from sand jellyfish venom for the first time, laying the foundation for in-depth research on the structure, function, mechanism of action of sand jellyfish perforating toxin protein and the development of its antidote. Summary of the invention
[0004] The object of the present invention is to provide a method for preparing a jellyfish from a sand jellyfish ( N.nomurai ) venom, and a method for separating lethal punching toxin from the venom of sand jellyfish ( N.nomurai ) The perforating toxin protein isolated from the venom of the sand jellyfish lays the foundation for in-depth research on the lethal toxin of the sand jellyfish and the development of drugs to treat sand jellyfish stings.
[0005] To achieve the above object, the present invention first provides a technical solution: a method for separating and purifying the pore-piercing toxin protein of a sand jellyfish, comprising the following steps: (1) subjecting the toxin of the sand jellyfish to low-temperature high-speed centrifugation and collecting the supernatant for later use; (2) subjecting the supernatant obtained in step (1) to ultracentrifugation, and collecting the precipitate for later use; (3) The precipitate obtained in step (2) is redissolved and centrifuged again at high speed, and the supernatant is collected for later use; (4) pumping the supernatant obtained in step (3) into a Superdex 75 16 / 600 GL gel filtration chromatography column for separation at low temperature, washing with a buffer solution, and collecting an eluate with an elution volume range of 70 ml to 75 ml to obtain the crude protein of the sand jellyfish pore toxin; (5) The supernatant obtained in step (4) is concentrated and pumped into a Mono Q anion exchange chromatography column for separation at low temperature, and then rinsed with a buffer solution, and the eluate with an elution volume range of 18 ml to 20 ml is collected to obtain the sand jellyfish pore toxin protein.
[0006] Preferably, the elution solution used in step (4) is 15-25 mM PBS, pH 7.5-8.5, 0.15 M NaCl; the elution volume is 120-180 mL, the flow rate is 0.6-1 mL / min, and the temperature is 2-6 °C.
[0007] Preferably, the eluent A used in step (5) is 15-25 mM PBS, pH 7.5-8.5; the eluent B is 15-25 mM PBS, pH 7.5-8.5, 1M NaCl; the elution volume is 30-40 ml, the flow rate is 1.0-1.5 mL / min, and the temperature is 2-6 °C.
[0008] The invention also provides the sand jellyfish pore-perforating toxin protein obtained by the method, and the molecular weight of the protein is about 45 kDa.
[0009] The present invention further provides the use of the sand jellyfish pore-perforating toxin protein obtained by the method in developing drugs for treating sand jellyfish stings.
[0010] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention establishes a new method for preparing crude toxin of sand jellyfish, which can effectively separate impurity proteins and is conducive to subsequent separation and purification; 2. The present invention can separate the toxin protein that punches holes in the cell membrane from the venom of the sand sea jellyfish by only two-step purification using a gel filtration chromatography column Superdex 75 16 / 600 GL and an anion exchange chromatography column Mono Q, and has the advantages of high efficiency and high resolution. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 The SDS-PAGE comparison results of the components separated in Example 2 of the present invention and the components obtained by the traditional method; Figure 2 For the sand jellyfish ( N. nomurai) Chromatographic results of gel filtration chromatography column Superdex 75 16 / 600 GL for separation of piercing toxin from venom; Figure 3 For the sand jellyfish ( N.nomurai ) Chromatographic results of Mono Q anion exchange column for separation of piercing toxins from venom; Figure 4 For the sand jellyfish ( N.nomurai ) Schematic diagram of SDS-PAGE results of separating piercing toxin from venom; Figure 5 This is a transmission electron microscope (TEM) observation of the holes formed on the cell membrane surface by the separated and purified pore-perforating toxin protein in Example 2 of the present invention. DETAILED DESCRIPTION
[0012] In order to facilitate the understanding of the present invention, the present invention is described in more detail below in conjunction with the accompanying drawings and specific embodiments. Preferred embodiments of the present invention are provided in the accompanying drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.
[0013] Example 1 The method for separating and purifying the pore-piercing toxin protein of the sand jellyfish provided in this embodiment comprises the following steps: (1) Centrifuge the venom of the sand sea jellyfish at 10,000 g for 20 minutes at 2°C and collect the supernatant for later use; (2) Ultracentrifuge the supernatant at 200,000 g for 20 min at 2°C, discard the supernatant, and collect the precipitate for later use; (3) Resuspend the precipitate and centrifuge again at 10,000 g for 20 min at 2 °C. Collect the supernatant for later use. (4) Pump 1 mL of the supernatant of the sand jellyfish into a Superdex 75 16 / 600 GL gel filtration column for separation at 2 °C at a flow rate of 0.6 mL / min. Use 15 mM PBS, pH 7.5, 0.15 M NaCl to rinse 120 mL. Collect the eluate in the elution range of 70 ml-75 ml and concentrate the eluate. (5) Pump 1 mL of the sand jellyfish concentrate into a Mono Q anion exchange chromatography column for separation at 2°C with a flow rate of 1.0 mL / min. Use eluent A: 15 mM PBS, pH 7.5; eluent B: 15 mM PBS, pH 7.5, 1 M NaCl for rinsing for 30 mL. Collect the eluate in the elution range of 18 ml to 20 ml to obtain the sand jellyfish pore-forming toxin protein.
[0014] Example 2 The method for separating and purifying the pore-piercing toxin protein of the sand jellyfish provided in this embodiment comprises the following steps: (1) Centrifuge the venom of the sand sea jellyfish at 12,000 g for 30 minutes at 4°C and collect the supernatant for later use; (2) Ultracentrifuge the supernatant at 200,000 g for 20 min at 4 °C, discard the supernatant, and collect the precipitate for later use; (3) Resuspend the precipitate and centrifuge again at 12,000 g for 20 min at 4 °C. Collect the supernatant for later use. (4) Pump 1 mL of the supernatant of the sand jellyfish into a Superdex 75 16 / 600 GL gel filtration column for separation at 4°C at a flow rate of 0.8 mL / min. Use 20 mM PBS, pH 8.0, 0.15 M NaCl to rinse 120 mL. Collect the eluate in the elution range of 70 ml to 75 ml and concentrate the eluate. (5) Pump 1 mL of the sand jellyfish concentrate into a Mono Q anion exchange chromatography column for separation at 4°C with a flow rate of 1.2 mL / min. Use eluent A: 20 mM PBS, pH 8.0; eluent B: 20 mM PBS, pH 8.0, 1 M NaCl to rinse 35 mL, and collect the eluate in the elution range of 18 ml to 20 ml to obtain the sand jellyfish pore-forming toxin protein.
[0015] Example 3 The method for separating and purifying the pore-piercing toxin protein of the sand jellyfish provided in this embodiment comprises the following steps: (1) Centrifuge the venom of the sand sea jellyfish at 14,000 g for 40 minutes at 6°C and collect the supernatant for later use; (2) Ultracentrifuge the supernatant at 200,000 g at 6°C for 20 min, discard the supernatant, and collect the precipitate for later use; (3) Resuspend the precipitate and centrifuge again at 10,000 g for 20 min at 6 °C. Collect the supernatant for later use. (4) Pump 1 mL of the supernatant of the sand jellyfish into a Superdex 75 16 / 600 GL gel filtration column for separation at 6°C at a flow rate of 1.0 mL / min. Use 25 mM PBS, pH 8.5, 0.15 M NaCl to wash 120 mL, collect the eluate in the elution range of 70 ml-75 ml, and concentrate the eluate; (5) Pump 1 mL of the sand jellyfish concentrate into a Mono Q anion exchange chromatography column for separation at 6°C with a flow rate of 1.5 mL / min. Use eluent A: 25 mM PBS, pH 8.5; eluent B: 25 mM PBS, pH 8.5, 1 M NaCl to rinse 40 mL. Collect the eluate in the elution range of 18 ml to 20 ml to obtain the sand jellyfish pore-forming toxin protein.
[0016] Example 4 Determination of the Sand Jellyfish Hole-Piercing Toxin Protein Obtained in Example 2 (1) The pore-forming toxin protein of the sand jellyfish obtained by Superdex 75 16 / 600 GL gel filtration column was subjected to chromatography. The chromatographic results are shown in Figure 2 shown.
[0017] (2) The pore-forming toxin protein of the sand jellyfish obtained by using the Mono Q anion exchange column was subjected to chromatography. The chromatographic results are shown in Figure 3 shown.
[0018] The toxin protein was analyzed by SDS-PAGE electrophoresis. Figure 4 As shown, the molecular weight of the toxin protein is approximately 45 kDa.
[0019] Example 5 Analysis of the pore-perforating effect of the sand jellyfish pore-perforating toxin protein isolated by the method of the present invention like Figure 5 As shown, the pore-perforating toxin protein separated and purified in Example 2 of the present invention can form holes with a pore size of about 13 nm on the cell membrane surface. The toxin protein lays a foundation for in-depth research on the structure, function, mechanism of action of the sand jellyfish pore-perforating toxin protein and the development of its antidote.
Claims
1. A method for separating and purifying the toxin protein of sand jellyfish, characterized in that: The following steps are involved: (1) subjecting the toxin of the sand jellyfish to low-temperature high-speed centrifugation and collecting the supernatant for later use; (2) subjecting the supernatant obtained in step (1) to ultracentrifugation, and collecting the precipitate for later use; (3) The precipitate obtained in step (2) is redissolved and centrifuged again at high speed, and the supernatant is collected for later use; (4) pumping the supernatant obtained in step (3) into a Superdex 75 16 / 600 GL gel filtration chromatography column for separation at low temperature, washing with a buffer solution, and collecting an eluate with an elution volume range of 70 ml to 75 ml to obtain the crude protein of the sand jellyfish pore toxin; (5) The supernatant obtained in step (4) is concentrated and pumped into a Mono Q anion exchange chromatography column for separation at low temperature, and then rinsed with a buffer solution, and the eluate with an elution volume range of 18 ml to 20 ml is collected to obtain the sand jellyfish pore toxin protein.
2. The method for separating and purifying the sand jellyfish pore-piercing toxin protein according to claim 1, characterized in that: The elution solution used in step (4) is 15-25 mM PBS, pH 7.5-8.5, 0.15M NaCl; the elution volume is 120-180 mL, the flow rate is 0.6-1 mL / min, and the temperature is 2-6°C.
3. The method for separating and purifying the sand jellyfish pore-piercing toxin protein according to claim 1, characterized in that: The eluent A used in step (5) is 15-25 mM PBS, pH 7.5-8.5; the eluent B is 15-25 mM PBS, pH 7.5-8.5, 1M NaCl; the elution volume is 30-40 ml, the flow rate is 1.0-1.5 mL / min, and the temperature is 2-6 °C.
4. A sand jellyfish perforating toxin protein obtained by the method according to any one of claims 1 to 3, characterized in that: Its molecular weight is approximately 45 kDa.
5. The use of the sand jellyfish pore-piercing toxin protein according to claim 4, characterized in that: The sand jellyfish pore-perforating toxin protein is used for developing drugs for treating sand jellyfish stings.