Organophosphorus hydrolase central targeting delivery strategy based on receptor mediation and fusion protein and application of organophosphorus hydrolase central targeting delivery strategy

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses an organophosphorus hydrolase central targeting delivery strategy based on receptor mediation and fusion protein and application of the organophosphorus hydrolase central targeting delivery strategy. The fusion protein ANG-OPHDS5 comprises a targeting peptide Angiopep-2, a connecting fragment and organophosphorus hydrolase, and the amino acid sequence of the fusion protein ANG-OPHDS5 is as shown in SEQ ID NO: 1. The fusion protein can realize efficient central targeting delivery through transendocytosis mediated by an LRP1 receptor highly expressed on a blood brain barrier. Experiments show that the fusion protein can effectively degrade organophosphorus toxicants, retain high enzyme activity, remarkably relieve brain tissue damage in an animal model, and show good brain targeting and detoxification effects. The invention further relates to nucleic acid for coding the fusion protein, an expression vector, a recombinant host cell microorganism, a preparation method of the fusion protein and application of the fusion protein in preparation of drugs for relieving or treating organophosphorus poisoning.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to a receptor-mediated targeted delivery, a fusion protein, an organophosphorus hydrolase and a central target delivery strategy and application thereof. BACKGROUND

[0002] Organophosphorus poisoning is an important public health problem and the main type of sudden poisoning event. Organophosphorus toxic molecules can invade the human body through various routes such as skin, eyes, inhalation, etc., and it is very difficult to prevent. The toxicity is strong and the effect is rapid, and it has a wide toxic effect on the liver, lungs, kidneys, brain and heart. Especially for the central nervous system, it can cause irreversible damage. Timely removal or degradation of toxic molecules entering the body, especially the central system, is the first important link in anti-poisoning rescue. Organophosphorus compounds have obvious lipophilicity and can penetrate the blood-brain barrier and other biological membranes. Through the study of in vivo metabolic transformation and toxicological mechanism, it is found that the detoxification system existing in the body itself can be detoxified through binding or degradation before the toxic molecules act on the toxic targets from entering the blood circulation. However, the effect is very limited, and high-dose organophosphorus toxic molecules cannot be effectively removed in time, causing poisoning symptoms and even death. At present, the drugs used in clinical treatment of organophosphorus poisoning mainly include anticholinergic drugs and cholinesterase reactivators. Among them, although anticholinergic drugs such as atropine can quickly relieve symptoms, due to the lack of objective indicators for drug use, the side effects are large, and there is still no ideal dosage regimen. Cholinesterase reactivators such as oxime drugs cannot be efficiently delivered to the central nervous system, and cannot activate the central nervous system acetylcholinesterase in time, so that the central nervous system damage caused by poisoning cannot be repaired, causing delayed multiple neurological diseases. This suggests that in the face of the threat of organophosphorus poisoning, the development of efficient and less toxic organophosphorus poisoning rescue drugs is still a world problem that needs to be solved urgently.

[0003] In recent years, domestic and foreign have carried out in-depth exploration on biological scavengers of organophosphorus compounds. Organophosphorus hydrolase (OPH), as a new generation of biological scavenger, can rapidly catalyze the degradation of organophosphorus compounds by breaking ester bonds. It not only has a broad spectrum of substrates, but also has no loss during the catalytic process, and has great potential to develop into a high-efficiency organophosphorus scavenger and poisoning rescue drug. However, although the natural OPH discovered so far has high catalytic activity for organophosphorus compounds, it is difficult to effectively cross the blood-brain barrier as a biological macromolecule. If it is to be further studied as an organophosphorus poisoning rescue drug, the first bottleneck problem to be solved is how to efficiently cross the blood-brain barrier and achieve central target delivery. The current mainstream strategy for crossing the blood-brain barrier is to use high-expression specific receptors to transport drugs into the brain through "receptor-mediated transcytosis". SUMMARY

[0004] The present application aims to overcome the defects of large side effects and low delivery efficiency of existing drugs for treating organophosphorus poisoning, and provide a low-toxicity, target-delivery organophosphorus fusion protein. Another object of the present application is to provide a preparation method of the above-mentioned protein and its application in detecting or scavenging organophosphorus compounds and treating organophosphorus poisoning.

[0005] To achieve the above-mentioned objects, the present application adopts the following technical solutions:

[0006] The present application provides a fusion protein in the first aspect, comprising a targeting peptide, a connecting segment and an organophosphorus hydrolase.

[0007] Further, the amino acid sequence of the fusion protein comprises the targeting peptide, the connecting segment and the organophosphorus hydrolase in a certain direction.

[0008] Further, the direction is from N-terminal to C-terminal or from C-terminal to N-terminal.

[0009] In the specific embodiment of the present application, the direction is from N-terminal to C-terminal.

[0010] Further, the organophosphorus hydrolase comprises a wild-type organophosphorus hydrolase or an organophosphorus hydrolase mutant.

[0011] In the present application, the mutation can refer to one or more conservative amino acid substitutions or one or more non-conservative amino acid substitutions, deletions or insertions. The sequence after mutation is different from the wild-type sequence, wherein the mutations do not abolish the biological activity of the wild-type sequence. The conservative substitution usually includes substitution of one amino acid by another amino acid with similar characteristics, such as substitution within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Other conservative amino acid substitutions are known in the art. Non-conservative substitutions, such as replacement of basic amino acids by hydrophobic amino acids, are also well known in the art.

[0012] Further, the wild-type organophosphorus hydrolase comprises OPH, PTE, OPPA, MHP, etc.

[0013] Further, the organophosphorus hydrolase mutant comprises OPH-DS5, OpdA-E132Q, OPAA-H254R, MPH-L271F, etc.

[0014] Further, the amino acid sequence of the organophosphorus hydrolase is any one of A1) to A5):

[0015] A1) the amino acid sequence is 29-367 of SEQ ID NO: 1;

[0016] A2) the amino acid sequence of a protein having organophosphorus hydrolase activity obtained by adding, deleting, substituting, or inserting one or more amino acid residues in the amino acid sequence of A1);

[0017] A3) the amino acid sequence of a protein having organophosphorus hydrolase activity and having 80% or more identity to the amino acid sequence of any one of A1) to A2);

[0018] A4) the amino acid sequence of a fusion protein obtained by fusing a protein tag to the carboxy terminus and / or amino terminus of the protein of any one of A1) to A3).

[0019] The term "protein tag" refers to a polypeptide or protein fused and expressed with a target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking, and / or purification of the target protein. The protein tag includes His tag, Flag tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0020] In a specific embodiment of the present application, the organophosphorus hydrolase is OPH-DS5.

[0021] Further, the targeting peptide can specifically bind to a receptor.

[0022] Further, the receptor is a central nervous system receptor, a cardiovascular system receptor, a tumor tissue receptor, an inflammatory tissue receptor, a liver tissue receptor, a lung tissue receptor, a kidney tissue receptor.

[0023] Further, the central nervous system receptor includes an acetylcholine receptor, a low-density lipoprotein receptor, a glucose transport receptor, a dopamine receptor cell receptor.

[0024] Further, the cardiovascular system receptor includes NRP-1, Integrin α5β1; the tumor tissue receptor includes EGFR, VEGFR; the inflammatory tissue receptor includes ICAM-1, VCAM-1, TLR; the liver tissue receptor includes ASGPR; the lung tissue receptor includes ACE2, SP-B; the kidney tissue receptor includes Megalin, Cubilin.

[0025] Further, the central nervous system receptor is a low-density lipoprotein receptor.

[0026] Further, the targeting peptide includes Angiopep-2, RVG29, T7, THR, GLUT1-BP.

[0027] Further, the amino acid sequence of the targeting peptide is any one of B1) to B3):

[0028] B1) the amino acid sequence is SEQ ID NO: 3;

[0029] B2) an amino acid sequence with targeting delivery function obtained by adding, deleting, substituting, or inserting one or more amino acid residues in the amino acid sequence shown in B1);

[0030] B3) an amino acid sequence corresponding to a polypeptide having 80% or more identity to any one of the amino acid sequences shown in B1) and B2) and having targeting delivery function.

[0031] In a specific embodiment of the present application, the targeting peptide is Angiopep-2.

[0032] Further, the linker segment is located between the targeting peptide and the organophosphorus hydrolase;

[0033] Further, the linker segment comprises a flexible linker segment, a rigid linker segment, a cleavable linker segment, and a water-soluble short peptide.

[0034] Further, the linker segment is a flexible linker segment.

[0035] Still further, the amino acid sequence of the linker segment is any one of C1) to C4):

[0036] C1) the amino acid sequence is SEQ ID NO: 4;

[0037] C2) the amino acid sequence is GGGGS, GGGGSGGGGS, or GGGGSGGGGSGGGGS;

[0038] C3) an amino acid sequence for connecting the targeting peptide and the organophosphorus hydrolase obtained by adding, deleting, substituting, or inserting one or more amino acid residues in any one of the amino acid sequences shown in C1) to C2);

[0039] C4) an amino acid sequence for connecting the targeting peptide and the organophosphorus hydrolase having 80% or more identity to any one of the amino acid sequences shown in C1) to C3).

[0040] In a specific embodiment of the present application, the amino acid sequence of the linker segment is SGGRGGA.

[0041] The second aspect of the present application provides any one of the following biomaterials:

[0042] (1) a nucleic acid molecule or a vector thereof, the nucleic acid molecule or the vector thereof encoding the protein according to the first aspect of the present application;

[0043] (2) A recombinant host cell comprising the nucleic acid molecule of (1) and / or a vector thereof.

[0044] Further, the nucleic acid molecule includes a DNA molecule or an RNA molecule; the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. In the present application, the nucleic acid molecule can also refer to a codon-optimized nucleic acid molecule, and can also refer to a nucleic acid molecule obtained by substitution and / or deletion and / or addition of one or several nucleotides, as long as it is a nucleic acid molecule capable of encoding the fusion protein of the first aspect of the present application, which falls within the scope of the present application.

[0045] Further, the nucleic acid molecule is any one of the following D1) to D4):

[0046] D1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2;

[0047] D2) a DNA molecule having the nucleotide sequence of 7 to 1104 of SEQ ID NO: 2;

[0048] D3) a DNA molecule having the nucleotide sequence of 85 to 1104 of SEQ ID NO: 2;

[0049] D4) a DNA molecule having 80% or more identity to any one of the DNA molecules of D1) to D3) and encoding the same protein.

[0050] D5) a DNA molecule obtained by adding a regulatory sequence necessary for protein coding to any one of the DNA molecules of D1) to D4); the regulatory sequence includes a promoter, and / or a restriction site located after the promoter, and / or a termination signal of transcription and translation.

[0051] Further, the type of the vector is not limited, for example, plasmid, phagemid, phage derivative, animal virus, and cosmid, which can be changed depending on the host cell to be introduced. Virus vector technology is well known in the art, and introduction can be made by standard techniques such as infection, transfection, transduction, or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, liposome transfection, cell microinjection, and viral vector. In some embodiments, viruses that can be used as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus. In addition, in order to evaluate the expression of a protein of interest (e.g., a monoclonal antibody), the vector introduced into the cell can also include either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of expression cells from a cell population sought to be transfected or infected by the viral vector.

[0052] Further, the vector can be an expression vector, a cloning vector, or an integration vector. A typical cloning vector contains a transcription and translation terminator, a start sequence, and a promoter that can be used to regulate the expression of a desired nucleic acid sequence. An integration vector contains a component that integrates the target sequence into the genome of a cell. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vector usually contains sequences for plasmid maintenance and for cloning and expression of foreign nucleotide sequences. The sequences usually include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of nucleic acids encoding a monoclonal antibody to be expressed, and an optional marker element.

[0053] Further, the vector includes pET28a, pET32a, or pET22b.

[0054] Still further, the vector is pET28a.

[0055] Further, the recombinant host cell includes eukaryotic cells and prokaryotic cells.

[0056] Further, the eukaryotic cell includes mammalian cells, insect cells, plant cells, yeast cells.

[0057] Further, the prokaryotic cell includes bacteria, actinomycetes, or cyanobacteria.

[0058] Further, the bacteria include gram-negative bacteria or gram-positive bacteria;

[0059] Further, the gram-negative bacteria include Escherichia, Salmonella, Erwinia, Pseudomonas, or Caulobacter.

[0060] Further, the gram-positive bacteria include Lactococcus, Streptococcus, Enterococcus.

[0061] Further, the Escherichia include Escherichia coli, Escherichia gergusonii, Escherichia hermanii;

[0062] Further, the Escherichia coli is Escherichia coli BL21.

[0063] The third aspect of the present application provides a conjugate, wherein the conjugate comprises the fusion protein according to any one of the first aspect of the present application.

[0064] Further, the conjugate is obtained by coupling the fusion protein according to the first aspect of the present application with a functional molecule.

[0065] Further, the coupling includes covalent coupling and non-covalent coupling.

[0066] Further, the covalent coupling includes amide bond, ester bond, thioether bond, disulfide bond, coupling bond between amino and carboxyl or thiol, maleimide-thiol linkage, aldehyde-amino Schiff base bond or its reductive amination bond.

[0067] Further, the non-covalent coupling includes electrostatic interaction, hydrophobic interaction, hydrogen bond interaction, van der Waals force, affinity tag-ligand interaction, biotin-avidin binding or metal-histidine coordination interaction.

[0068] Further, the functional molecule includes fluorescent dye, liposome, nanoparticle or cell penetrating peptide.

[0069] Further, the liposome includes phosphatidylserine, cholesterol and PEG-modified lipid.

[0070] Further, the nanoparticle includes PLGA, PEG-PLGA, gold nanoparticle, magnetic iron oxide.

[0071] Further, the cell penetrating peptide includes Tat, Penetratin, Arg8, Transportan.

[0072] Further, the fluorescent dye includes Cy5.5, Cy7, FITC.

[0073] Further, the fluorescent dye is Cy5.5.

[0074] Further, the fluorescent dye is covalently coupled to the fusion protein.

[0075] The fourth aspect of the present application provides a pharmaceutical composition comprising the fusion protein according to the first aspect of the present application, the biomaterial according to the second aspect of the present application, or the conjugate according to the third aspect of the present application. The fusion protein and / or the biomaterial and / or the conjugate is present in the pharmaceutical composition in a therapeutically effective amount.

[0076] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0077] The pharmaceutically acceptable carriers and / or excipients described in the present application include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly water-soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl cellulose, etc.). Using these materials, various dosage forms can be prepared, including, but not limited to, injection, tablet, capsule, oral liquid, granule, ointment, cream, ointment, eye drop, suspension, and other external or topical administration dosage forms. It can be a general preparation, sustained-release preparation, controlled-release preparation, and various microparticle drug delivery systems. In order to prepare a unit administration dosage form into an injection preparation, such as a solution, an emulsion, a lyophilized powder, and a suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid ester, etc. In addition, in order to prepare an isotonic injection solution, an appropriate amount of sodium chloride, glucose, or glycerol can be added to the injection preparation, and in addition, a conventional cosolvent, a buffer, a pH adjuster, etc. can be added. In addition, if necessary, a coloring agent, a preservative, a flavoring agent, a sweetener, or other materials can also be added to the pharmaceutical preparation. In order to prepare a unit administration dosage form into a tablet, various carriers commonly known in the art can be widely used. As examples of the carriers, there are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders such as water, glycerol, polyethylene glycol, ethanol, propyl alcohol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants such as dry starch, alginate, agar powder, alginic acid, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors such as sucrose, glycerol triestearate, cocoa butter, hydrogenated oil, etc.; absorption promoters such as quaternary ammonium salt, sodium dodecyl sulfate, etc.; lubricants such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablet can be further prepared into a coated tablet, such as a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, or a double-layer tablet and a multi-layer tablet. In order to prepare a unit administration dosage form into a pill, various carriers commonly known in the art can be widely used. As examples of the carriers, there are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth gum, gelatin, ethanol, liquid sugar, rice paste or batter, etc.; disintegrants such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intracavity injection, etc. The above administration routes are preferably injection administration. When a patient has skin, mucous membrane or eye contamination with organophosphorus and has local poisoning symptoms, an external use or topical administration dosage form can be used.

[0078] The pharmaceutical composition of the present application can be administered orally, intravenously, intra-arterially, intraperitoneally, intrathecally, transdermally, via the respiratory tract, rectally, topically, etc. In the above methods, the subject can be a mammal. The mammal can be selected from the group consisting of bovine, equine, feline, canine, leporine, porcine, camelid, rodent and primate, including but not limited to, bovine, equine, caprine, ovine, feline, leporine, porcine, camelid, alpaca, rat, mouse, guinea pig, non-human primate (such as simian, monkey, baboon, chimpanzee) and human, preferably bovine, equine, canine, caprine, ovine, porcine, camelid, rat, mouse, monkey and human.

[0079] In some embodiments, the therapeutically effective amount of the fusion protein and / or the biomaterial and / or the multimeric fusion protein of the present application can be variously prescribed according to the formulation method, administration method, age, weight, sex, disease state, diet, administration time, administration route, excretion rate and reaction sensitivity of the patient, etc., and a skilled physician can easily determine the prescription and the prescription for the desired therapeutically effective amount.

[0080] The fifth aspect of the present application provides any one of the following methods:

[0081] (1) A method for preparing the fusion protein of the first aspect of the present application, the preparation method comprising introducing the nucleic acid molecule or a vector thereof in the biomaterial of the second aspect of the present application into a recombinant host cell in the biomaterial of the second aspect of the present application, culturing and expressing the fusion protein of the first aspect of the present application, and extracting and purifying the fusion protein;

[0082] (2) A method for detecting and / or removing organophosphorus compounds using the fusion protein of any one of the first aspect of the present application, characterized in that the fusion protein detects and / or removes organophosphorus compounds in a sample in a reaction system;

[0083] Further, the preparation method of the fusion protein further comprises a purification method of the fusion protein of the first aspect. The purification method comprises using the properties of the fusion protein itself or the tag carried by the fusion protein to select a suitable purification method to enrich the fusion protein to be purified. In some embodiments, affinity purification, gel filtration, ion exchange purification, hydrophobic interaction, size exclusion chromatography, electrophoresis can be used for purification. In a specific embodiment of the present application, the purification method is affinity purification.

[0084] Further, the sample includes air, water, soil, food or biological fluid.

[0085] Further, the reaction system further includes a buffer, a chromogenic agent, an immobilized reagent.

[0086] Further, the buffer includes Tris-HCl, HEPES, PBS, Glycine-NaOH.

[0087] Further, the chromogenic agent includes p-nitrophenyl ester reagent (such as p-nitrophenyl phosphate pNPP, p-nitrophenyl acetate pNPA, p-nitrophenyl parathion pNPD), Ellman reagent.

[0088] Further, the immobilized reagent includes a membrane, a gel, a magnetic microsphere or a porous material.

[0089] The sixth aspect of the present application provides any one of the following applications:

[0090] (1) the application of the fusion protein of any one of the first aspect of the present application, the biomaterial of the second aspect of the present application, the conjugate of the third aspect of the present application, the pharmaceutical composition of the fourth aspect of the present application, and the method of the fifth aspect of the present application in the preparation of a drug for treating or alleviating the symptoms caused by organophosphorus compound poisoning;

[0091] (2) the application of the fusion protein of any one of the first aspect of the present application, the biomaterial of the second aspect of the present application, the conjugate of the third aspect of the present application, the pharmaceutical composition of the fourth aspect of the present application, and the method of the fifth aspect of the present application in the preparation of a drug for detecting and / or removing organophosphorus compounds.

[0092] Further, the organophosphorus compound includes a nerve agent and a pesticide.

[0093] Further, the nerve agent includes a G-type agent or a VX nerve agent.

[0094] Further, the organophosphorus compound includes a compound containing a P-O bond, a P-CN bond, a P-F bond, a P-S bond; specifically, the organophosphorus compound containing a P-O bond and connected to a 4-nitrophenyl group (such as parathion, methyl parathion) is hydrolyzed into p-nitrophenol; the compound containing a P-CN bond (such as tabun) can be hydrolyzed to obtain hydrocyanic acid; the organophosphorus compound containing a P-F bond (such as sarin, soman, cyclosarin) can be hydrolyzed to obtain fluoride; the organophosphorus compound containing a P-S bond (such as malathion, VX nerve agent (O-ethyl-S-[2-(diisopropylamino)ethyl] methyl thiophosphate), dimethoate, phorate, ethoprophos, terbufos) can be hydrolyzed to obtain some sulfides.

[0095] Further, the symptoms include acute poisoning, intermediate syndrome, delayed multiple psychosis, chronic neuropsychiatric syndrome. The acute poisoning in the application has a onset time of several minutes to several hours after exposure to toxic environment, and the clinical symptoms are usually M-like symptoms (salivation, lacrimation, polyhidrosis, increased bronchial secretion, diarrhea, vomiting, miosis, bronchospasm, bradycardia), N-like symptoms (fasciculation, muscle weakness, paralysis, dyspnea), central symptoms (headache, disturbance of consciousness, convulsion, coma); the intermediate syndrome has a onset time of 24-96 hours after acute poisoning, and the clinical symptoms are usually weakness of neck muscles, proximal limb muscles, respiratory muscles, and cranial nerve palsy; the delayed multiple psychosis has a onset time of 1-3 weeks after poisoning, and the clinical symptoms are usually symmetrical numbness, pain, weakness of distal lower limbs, muscle atrophy in later stage, disappearance of tendon reflex, and movement disorder; the chronic neuropsychiatric syndrome appears in long-term low-dose exposure, and the clinical symptoms are usually memory loss, insomnia, emotional fluctuation, attention disorder, depression, anxiety, and the like.

[0096] The application has the advantages and beneficial effects that:

[0097] (1) Achieving central target delivery and high-efficiency detoxification dual functions: by fusing a targeting peptide (such as Angiopep-2) and a flexible linker fragment at the N-terminus of an organophosphorus hydrolase mutant (OPH-DS5), a fusion protein capable of specifically binding to central nervous system receptors (such as low-density lipoprotein receptors) is constructed, achieving receptor-mediated trans-blood-brain barrier delivery. The fusion protein maintains high catalytic activity while significantly improving its enrichment efficiency and bioavailability in brain tissue, effectively removing organophosphorus compounds entering the central nervous system.

[0098] (2) Having detection and removal integrated functions, wide application range: the fusion protein can catalyze the hydrolysis of various organophosphorus compounds in a buffer system and produce a color reaction, achieving rapid colorimetric detection and synchronous degradation of organophosphorus pollutants in air, water, soil, food and biological fluids, the reaction is sensitive and easy to operate, and is suitable for environmental monitoring, food safety and emergency protection fields.

[0099] (3) Constructing a multifunctional coupling system to expand application potential: the fusion protein of the application can be further coupled with a fluorescent dye, a liposome, a nanoparticle or a cell-penetrating peptide through a covalent bond or non-covalent interaction to form a composite system with imaging, targeted delivery and stable release capabilities, providing a diversified application basis for the fusion protein in the fields of drug development, in vivo imaging and biosensor construction. BRIEF DESCRIPTION OF DRAWINGS

[0100] Figure 1Vector map of recombinant expression vector pET28a-ANG-OPHDS5

[0101] Figure 2 SDS-PAGE map of protein ANG-OPHDS5 after purification; wherein, 1: Protein ladder, 2: Protein ANG-OPHDS5;

[0102] Figure 3 Standard curve of para-nitrophenol, the hydrolysis product of paraoxon;

[0103] Figure 4 Enzymatic activity determination of protein ANG-OPHDS5 and RVG29-OPHDS5-His with paraoxon as substrate;

[0104] Figure 5 In vivo imaging map of Cy5.5-labeled protein OPH-DS5 and ANG-OPHDS5 in mice;

[0105] Figure 6 CD31-labeled map of blood vessels in brain tissue;

[0106] Figure 7 Co-localization map of protein and blood vessels;

[0107] Figure 8 In vivo imaging map of Cy5.5-labeled protein OPH-DS5 and ANG-OPHDS5 in brain tissue of mice;

[0108] Figure 9 Effect of pre-injection of different proteins on pathological changes in brain tissue caused by 10-fold lethal dose of ethyl paraoxon (HE staining);

[0109] Figure 10 Effect of pre-injection of different proteins on apoptotic damage in brain tissue caused by 10-fold lethal dose of ethyl paraoxon (TUNEL staining). DETAILED DESCRIPTION

[0110] The present application is further described in conjunction with the following embodiments. The following description is merely intended to be illustrative of the present application and is not intended to limit the scope of the present application. Any modification of the described embodiments, which comes within the scope of the present application are intended to be included therein.

[0111] Example 1 Construction of recombinant expression vector and expression strain of organic phosphorus hydrolase fusion protein ANG-OPHDS5

[0112] 1.1 Construction of recombinant vector pET28a-ANG-OPHDS5

[0113] Based on the amino acid sequence of OPH-DS5 disclosed in the patent CN118516331A, the sequence of Angiopep-2 (SEQ ID NO: 3 TFFYGGSRGKRNNFKTEEY) and the connecting fragment (SEQ ID NO: 4 SGGRGGA) are connected at the N-terminal of OPH-DS5 to obtain the organic phosphorus hydrolase mutant fusion protein ANG-OPHDS5 with the amino acid sequence of SEQ ID NO: 1, wherein the amino acids at positions 1-2 have no functional role and only serve as the N-terminal of the protein for initiating the transcription and translation process of the protein, the amino acids at positions 3-28 are the targeting peptide and the connecting sequence, and the amino acids at positions 29-367 are the organic phosphorus hydrolase mutant OPH-DS5.

[0114] SEQ ID NO: 1 consists of 367 amino acids, and the amino acid sequence is specifically as follows:

[0115] MATFFYGGSRGKRNNFKTEEYSGGRGGAMAMITNSGDRINTVRGPITISEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRRARAAGVRTIVDVSTFDIGRDVSLLAEVSRAADVHIVAATGLWFDPPLSMRLRSVEELTQFFLREIQYGIEDTGIRAGIIKVATTGKATPFQELVLRAAARASLATGVPVTTHTAASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTALAARGYLIGLDHIPHSAIGLEDNASASALLGIRSWQTRALLIKALIDQGYMKQILVSNDWLFGFSSYVTNIMDVMDSVNPDGMAFIPLRVIPFLREKGVPQETLAGITVTNPARFLSPTLRAS

[0116] The coding sequence of the fusion protein ANG-OPHDS5 is subjected to E. coli codon optimization and whole gene synthesis, and the nucleotide sequence of the coding gene is SEQ ID NO: 2; the DNA sequence obtained by synthesis is used to replace the small fragment between the NcoI and EcoRI enzyme cutting sites of the cloning vector pET28a (+) (the whole gene synthesis and plasmid construction are completed by Universal Biological (Anhui) Co., Ltd.), and the other part of the sequence remains unchanged to obtain a recombinant vector, which is named pET28a-ANG-OPHDS5. The map of the recombinant vector pET28a-ANG-OPHDS5 is as follows:Figure 1 as shown.

[0117] SEQ ID NO: 2:

[0118]

[0119] 1.2 Construction of recombinant expression strain

[0120] The recombinant vector pET28a-ANG-OPHDS5 was mixed with E. coli DH5a competent cells, incubated in ice water bath for 30 min, then heat shocked at 42 °C for 90 s, and finally incubated in ice water bath for 2 min; the incubated E. coli was inoculated into 500 μL LB liquid medium (including yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L), and cultured at 37 °C with 220 r / min shaking for 45 min. An appropriate amount of bacterial liquid was spread on LB plates (containing 2% agar) containing kanamycin sulfate (Kan, 100 μg / mL), and cultured at 37 °C for 18 h. Single colonies were picked and positive clones were selected for further sequencing verification. The plasmid in the correct strain was extracted, and E. coli BL21(DE3) competent cells were transformed using the same method as above. Single colonies were picked and the plasmid was extracted for sequencing verification. The recombinant expression strain, named BL21-ANG-OPHDS5, was obtained.

[0121] The single colony of the expression strain was picked and the plasmid was extracted. The universal primers (T7 / T7 ter) were used for sequencing, and the coding gene ang-ophds5 was 1104 bp in size, with 100% consistency with the corresponding synthesized gene sequence, proving that the recombinant expression strain BL21-ANG-OPHDS5 was successfully constructed.

[0122] Example 2 Preparation of organic phosphorus hydrolase fusion protein ANG-OPHDS5

[0123] 2.1 Preparation of E. coli culture medium and bacterial liquid recovery

[0124] 2.1.1 Preparation of culture medium

[0125] Prepare Luria-Bertani (LB) culture solution (for E. coli growth): weigh 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, dissolve in deionized water and make up to 1 L, and sterilize at 121 °C under high pressure for 25 min.

[0126] Prepare Terrific Broth (TB) culture solution (for E. coli growth and target protein expression): weigh 12 g of tryptone, 24 g of yeast extract, and 4 mL of glycerol, dissolve in deionized water and make up to 900 mL, and sterilize at 121 °C under high pressure for 25 min.

[0127] Prepare the phosphate buffer: weigh 2.31 g of potassium phosphate dibasic, 12.54 g of potassium phosphate monobasic, dissolve in deionized water and dilute to 100 mL, and sterilize under high pressure at 121 ℃ for 25 min. Before use, add the phosphate buffer to the culture medium at a volume ratio of 10:1 (culture medium: phosphate buffered saline).

[0128] 2.1.2 BL21-ANG-OPHDS5 bacterial liquid recovery

[0129] Take 50 μL of BL21-ANG-OPHDS5 bacterial liquid and inoculate it into 100 mL of LB (containing Kan 50 μg / mL) culture medium, and incubate it at 37 ℃ on a constant temperature shaker at a speed of 180 rpm for 16 h. According to the proportion of 100 mL of recovered bacterial liquid inoculated into 1 L of TB culture medium, inoculate the recovered bacterial liquid after 16 h of culture into TB culture medium, and incubate it at 37 ℃ on a constant temperature shaker at a speed of 180 rpm for 5 h. Take out the bacterial liquid that has been cultured at 37 ℃ for 5 h, add 250 mL of inducer 1 M IPTG (concentration in culture medium is 0.25 mM), and put it into a shaker at 25 ℃, 180 rpm for 24 h. Centrifuge the bacterial liquid that has been cultured for 24 h, the centrifugation conditions are 4 ℃, 9000 rpm, 3 min, collect the bacterial body and store it at -80 ℃.

[0130] 2.2 Extraction and purification of protein ANG-OPHDS5

[0131] Take out the bacterial body stored at -80 ℃, add 100 mL of buffer containing 20 mM HEPES and 0.5 M sodium chloride (pH=8.0) to each 10 g of bacterial body, and completely resuspend the bacterial body at room temperature. Put the bacterial body suspension into an ultrasonic disrupter and perform disruption in an ice bath, use a 12 mm probe, the disruption conditions are: ultrasonic power 75%, disrupt for 5 s, stop for 5 s, disrupt for 40 min, replace the ice bath after 20 min of disruption, centrifuge the bacterial body at 4 ℃, 11000 rpm for 30 min after disruption is completed, collect the supernatant, filter the supernatant with filter paper, and obtain the bacterial liquid supernatant and store it in ice.

[0132] Take 0.4 g of protamine sulfate, dissolve it with HEPES buffer, and dilute it to 20 mL for standby. Take the supernatant of the broken bacteria, and add the prepared protamine sulfate solution drop by drop into the supernatant while stirring and mixing. Then use a horizontal shaker for ice bath oscillation for 30 min, centrifuge at 13751xg and 4°C for 30 min, and take the supernatant. Add ammonium sulfate to the supernatant while stirring for 30 min, so that the supernatant reaches a 50% ammonium sulfate saturation concentration. Ice bath oscillation for 30 min, centrifuge at 11000 rpm and 4°C for 30 min, collect the precipitate, and redissolve it in HEPES buffer. Use a 10 kDa ultrafiltration tube to concentrate it. Use the hydrophobic column HiTrap Phenyl HP to purify the protein. The mobile phase A is pH 6.0 20 mM sodium acetate buffer, the mobile phase B is pH 6.0 20 mM sodium acetate buffer (containing 1 M NaCl), the flow rate is 0.5 mL / min, the maximum column pressure is 0.3 MPa, the flow rate is 2 mL / min, equilibration with mobile phase A for 1.5 column volumes, then gradient elution with the addition of mobile phase B, and the elution peak of the target protein is collected. Use a 10 kDa ultrafiltration centrifuge tube to concentrate the reaction solution to 1 mL to obtain a concentrated sample, which is ready for the next step of purification. Transfer the concentrated sample to a 1.5 mL centrifuge tube, balance, centrifuge at 10000 rpm and 4°C for 10 min, use the cation column Hiscreen Capto S, sample 500 μL each time, and a total of 2 injections. The alarm column pressure is 0.2 MPa, the flow rate of the mobile phase is 1.6 mL / min, the mobile phase A is pH 6.0 20 mM acetic acid sodium solution, the mobile phase B is pH 6.0 20 mM acetic acid sodium solution (containing 1 M sodium chloride), the volume fraction of mobile phase A is set from 100% to 0%, the volume fraction of mobile phase B is set from 0% to 100%, elution for 20 column volumes, collect one tube every 5 mL, collect the corresponding components, and obtain the cation column collected components. Concentrate the cation column collected components by ultrafiltration, at a condition of 5000 r / min, 4°C, to 1 mL. Transfer the concentrated sample to a 1.5 mL centrifuge tube, balance, centrifuge at 10000 rpm and 4°C for 10 min, use the gel filtration chromatography column Superdex 200 column (1 cm x 30 cm), sample 500 μL each time, the mobile phase is pH 6.0 20 mM acetic acid sodium buffer, the flow rate is 0.5 mL / min, the maximum column pressure is 1.9 MPa, equilibration for 1.5 column volumes, sample elution for a total of 1.5 column volumes, collect the elution peak of the target protein, add 5 μL of sample buffer, boil in a water bath for 10 min, then take it out and cool to room temperature, take 20 μL for SDS-PAGE, and the electrophoresis condition is 120 V for 85 min. The experimental results are as follows Figure 3As shown, there are fewer bands in the same lane in SDS-PAGE, indicating that the target protein ANG-OPHDS5 with high purity is obtained after three column chromatography purifications.

[0133] Example 3 Enzymatic activity determination of the organophosphorus hydrolase fusion protein ANG-OPHDS5 using parathion as substrate

[0134] 3.1 Determination of sample concentration by BCA method

[0135] The protein concentration was determined according to the BCA kit instructions (BCA kit Pierce™ BCA Protein Assay Kit, purchased from Thermo Fisher Scientific, item number: 23227).

[0136] 3.2 Preparation of p-nitrophenol standard curve

[0137] The organophosphorus hydrolase can catalyze the hydrolysis of its substrate ethyl parathion to generate p-nitrophenol, and the molar ratio of ethyl parathion to p-nitrophenol is 1:1. Accordingly, the enzyme activity of the sample can be evaluated by determining the amount of p-nitrophenol generated under the same conditions. Different volumes of 6 mM p-nitrophenol solution (20.86 mg of p-nitrophenol was dissolved in 5 mL of deionized water, and the volume was made up to 25 mL in a volumetric flask), supplemented with 1 mL of 50 mM HEPES buffer at pH 8.5, 1 mL of 10% (w / v) trichloroacetic acid and 10% (w / v) sodium carbonate solution were added in turn, and the final concentration of p-nitrophenol was in the range of 0 to 192 μmol / L (8 μmol / L increment) with 13 concentration points. The absorbance at 405 nm was determined, and the experiment was set up with 3 replicate wells, and the average value was taken. The absorbance value was linearly fitted with the p-nitrophenol concentration to draw the standard curve. Figure 4

[0138] 3.3 Determination of enzyme activity

[0139] The concentration of the protein sample was determined by BCA method, and it was diluted to 500 ng / mL using 50 mM HEPES at pH=8.5.

[0140] ​Take four 5 mL centrifuge tubes, designating tube 1 as the blank control and tubes 2, 3, and 4 as the experimental groups. Add 5 μL of 10 mg / mL ethyl paraoxonate solution and 900 μL of 50 mM pH 8.5 HEPES buffer to each tube. Add 1 mL of 10% trichloroacetic acid to tube 1, followed by 100 μL of the protein solution to be tested (10% trichloroacetic acid inactivates the enzyme and terminates the reaction), and incubate at 37°C. Then, add 100 μL of the protein solution to tubes 2, 3, and 4 sequentially every 30 seconds. After 10 min, remove tube 1, and then remove tubes 2, 3, and 4 sequentially every 30 seconds, immediately adding 10% trichloroacetic acid. Add 1 mL of 10% sodium carbonate solution to each of the four centrifuge tubes for color development. After mixing, take 200 μL of each sample and measure the absorbance at 405 nm using a microplate reader. Measure each sample in triplicate. Experimental results are as follows Figure 5 As shown, enzyme activity (U / mg) = amount of p-nitrophenol (μmol) × (1 mg / (0.1 mL * sample concentration)) / 10 min. A control group was set up for each sample during the assay. The results showed that the activity of OHDS5 in catalyzing the hydrolysis of oxyphosphine was 280.2 U / mg, and the activity of ANG-OPHDS5 was 227.5 U / mg. The fusion protein ANG-OPHDS5 showed a slightly lower activity in catalyzing the hydrolysis of oxyphosphine compared to OHDS5, but it could still efficiently catalyze the hydrolysis of ethyl p-oxyphosphine.

[0141] Example 4: Fluorescent labeling and in vivo imaging of the organophosphorus hydrolase fusion protein ANG-OPHDS5

[0142] 4.1 Cy5.5 fluorescent labeling of the target protein ANG-OPHDS5

[0143] The Cy5.5 fluorescently labeled protein kit (Xi'an Ruixi Biotechnology, catalog number: R-SJH-003) allows for simple and rapid covalent coupling of proteins with Cy5.5 fluorescent dyes via primary amino groups (such as lysine) on the protein surface. The specific method is as follows:

[0144] (1) Take the protein to be labeled, dissolve it in 1 mL of pure water, add Coupling Reagent, mix well and set aside.

[0145] (2) Take Cy5.5-mix solid, dissolve it in Cy5.5-mix solution, and add it to the solution from step (1), and mix thoroughly. If Cy5.5-mix is ​​not fully dissolved, it can be dissolved by ultrasonication in a water bath.

[0146] (3) React at room temperature in the dark for 30 min (stirring or not stirring is acceptable). Extending the reaction time will not affect the coupling efficiency.

[0147] (4) Transfer the reaction solution to an ultrafiltration tube and ultrafilter at 10,000 g for 5 min to remove unreacted Cy5.5-mix. To improve purity, add pure water to resuspend the protein and repeat the ultrafiltration twice.

[0148] (5) The antibody / protein retained by the ultrafiltration tube is resuspended in pure water or PBS to obtain the Cy5.5 fluorescently labeled antibody / protein solution.

[0149] 4.2 In vivo imaging of the organophosphorus hydrolase fusion protein ANG-OPHDS5 Six male C57BL / 6N mice (20 ± 2 g) were randomly divided into three groups (n=2). Each group received an equal volume (300 μL) of physiological saline, OPH-DS5, or ANG-OPHDS5 via tail vein injection. The mice were placed in a small animal in vivo imaging system at 0.5 h and 1 h, respectively, with oxygen and anesthesia connected, and the stage heating switch turned on. The software was then opened, fluorescence mode selected, excitation and emission light from Cy5.5 dye chosen, and the gain adjusted before image acquisition. Figure 5 Simultaneously, based on the overall animal imaging results, the mice underwent cardiac perfusion at 50 min and 100 min. Brain tissue from each group of mice was collected, fixed in paraformaldehyde, dehydrated, sectioned, and the blood vessels were stained with CD31 to observe the positional relationship between autofluorescent proteins and blood vessels. Figure 6 and Figure 7 Nine male C57BL / 6N mice (20 ± 2 g) were randomly divided into three groups (n=3). Each group was injected intravenously with an equal volume (200 μL) of physiological saline, OPHDS5, and ANG-OPHDS5, respectively. Heart perfusion was performed on the mice 1 h later. Brain tissue from each group was harvested and placed in a small animal in vivo imaging system. The software was then opened, fluorescence mode was selected, excitation and emission light of Cy5.5 dye were chosen, and the gain was adjusted before images were acquired. Figure 8 In vivo imaging results showed that, compared with OPH-DS5, ANG-OPHDS5 exhibited certain brain targeting and efficacy. Immunofluorescence detection results of isolated brain tissue showed that ANG-OPHDS5 could co-localize well with brain capillary endothelial cells.

[0150] Example 5 Pharmacodynamic study of organophosphorus hydrolase fusion protein ANG-OPHDS5 Male SD rats (200 ± 20 g) were randomly divided into two groups (n = 1), then, with 10 times the absolute lethal dose of ethyl parathion (6.58 mg / kg) subcutaneously infected each group of mice, 2 min after infection, tail vein injection of two rescue drugs ANG-OPHDS5 and OPH-DS5 at a dose of 120 μg / kg, observe and record the poisoning symptoms of mice and give score (salivation, tremor, convulsions and respiratory distress were recorded 1-4 points); among them, the mice injected with OPH-DS5 rescue drugs appeared whole body tremor at 6 min, lasting 10 seconds, and then returned to normal; the mice in ANG-OPHDS5 group had no any symptoms after infection and drug administration. Two groups of rats were heart perfusion after 24 h, fixed with polyformaldehyde, then the brain tissue was taken out for histological section HE and TUNEL staining Figure 9 and Figure 10 ) The results showed that compared with OPH-DS5 group, the brain tissue apoptosis injury of ANG-OPHDS5 group mice was significantly reduced, which may be due to the brain targeting delivery of ANG-OPHDS5 accelerated the clearance of organophosphorus toxic molecules in brain tissue, and reduced the damage of organophosphorus to brain tissue.

[0151] The above description of the embodiments is only for the purpose of understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. A fusion protein, characterized in that, The fusion protein comprises a targeting peptide, a connecting segment, and an organophosphorus hydrolase; Preferably, the amino acid sequence of the fusion protein comprises, from N-terminus to C-terminus, a targeting peptide, a connecting segment, and an organophosphorus hydrolase.

2. The fusion protein of claim 1, wherein, The organophosphorus hydrolase comprises a wild-type organophosphorus hydrolase or an organophosphorus hydrolase mutant; Preferably, the amino acid sequence of the organophosphorus hydrolase is any one of A1) to A5): A1) the amino acid sequence of SEQ ID NO: 1 from 29th to 367th; A2) the amino acid sequence of a protein having organophosphorus hydrolase activity obtained by adding, deleting, substituting, or inserting one or more amino acid residues in the amino acid sequence of A1); A3) the amino acid sequence of a protein having 80% or more identity with any one of the amino acid sequences of A1) to A2) and having organophosphorus hydrolase activity; A4) the amino acid sequence of a fusion protein obtained by fusing a protein tag to the carboxy terminus and / or amino terminus of any one of the proteins of A1) to A3).

3. The fusion protein of claim 1, wherein, The targeting peptide can specifically bind to a receptor; Preferably, the receptor is a central nervous system receptor, a cardiovascular system receptor, a tumor tissue receptor, an inflammatory tissue receptor, a liver tissue receptor, a lung tissue receptor, a kidney tissue receptor; Preferably, the central nervous system-specific receptor comprises one or more of an acetylcholine receptor, a low-density lipoprotein receptor, a glucose transport receptor, and a dopamine receptor cell receptor; Preferably, the central nervous system-specific receptor is a low-density lipoprotein receptor; Preferably, the amino acid sequence of the targeting peptide is any one of B1) to B3): B1) the amino acid sequence of SEQ ID NO: 3; B2) the amino acid sequence of a polypeptide having a targeting delivery function obtained by adding, deleting, substituting, or inserting one or more amino acid residues in the amino acid sequence of B1); B3) the amino acid sequence of a polypeptide having 80% or more identity with any one of the amino acid sequences of B1) and B2) and having a targeting delivery function.

4. The fusion protein of claim 1, wherein, The connecting segment is located between the targeting peptide and the organophosphorus hydrolase; Preferably, the amino acid sequence of the connecting segment is any one of C1) to C4): C1) the amino acid sequence of SEQ ID NO: 4; C2) the amino acid sequence of GGGGS, GGGGSGGGGS, or GGGGSGGGGSGGGGS; C3) the amino acid sequence of a segment for connecting the targeting peptide and the organophosphorus hydrolase obtained by adding, deleting, substituting, or inserting one or more amino acid residues in the amino acid sequence of any one of C1) to C2); C4) the amino acid sequence of a segment for connecting the targeting peptide and the organophosphorus hydrolase having 80% or more identity with any one of the amino acid sequences of C1) to C3).

5. A nucleic acid molecule and vectors thereof, characterized in that, The nucleic acid molecule or a vector thereof encodes the fusion protein of claim 1; Preferably, the nucleic acid molecule is any one of D1) to D4): D1) a DNA molecule having the nucleotide sequence of SEQ ID NO: 2; D2) a DNA molecule having a nucleotide sequence of SEQ ID NO: 2 from 7 to 1104; D3) a DNA molecule having a nucleotide sequence of SEQ ID NO: 2 from 85 to 1104; D4) a DNA molecule having 80% or more identity to any one of the DNA molecules described in D1) to D3) and encoding the same protein; D5) a DNA molecule obtained by adding a regulatory sequence necessary for protein coding to any one of the DNA molecules described in D1) to D4); Preferably, the vector comprises pET28a, pET32a or pET22b. Preferably, the vector is pET28a.

6. A recombinant host cell, characterized in that, The recombinant host cell comprises the nucleic acid molecule of claim 5 or a vector thereof; Preferably, the recombinant host cell can efficiently express the fusion protein of claims 1 to 4 in vitro. Preferably, the host cell comprises eukaryotic cells and prokaryotic cells. Preferably, the prokaryotic cell comprises bacteria, actinomycetes or cyanobacteria. Preferably, the bacteria comprise gram-negative bacteria or gram-positive bacteria. Preferably, the gram-negative bacteria comprise Escherichia, Salmonella, Erwinia, Pseudomonas or Caulobacter. Preferably, the Escherichia comprises Escherichia coli, E. gergsonii, E. hermanii. Preferably, the Escherichia coli is Escherichia coli BL21.

7. A conjugate, characterized in that, The conjugate comprises the fusion protein of any one of claims 1 to 4. Preferably, the conjugate is obtained by conjugating the fusion protein of claims 1 to 4 with a functional molecule. Preferably, the conjugation comprises covalent conjugation and non-covalent conjugation. Preferably, the functional molecule comprises a fluorescent dye, a liposome, a nanoparticle or a cell-penetrating peptide. Preferably, the fluorescent dye comprises Cy5.5, Cy7, FITC. Preferably, the fluorescent dye is Cy5.

5. Preferably, the fluorescent dye is covalently conjugated to the fusion protein.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises one or more of the fusion protein of claims 1 to 4 or a conjugate thereof, the nucleic acid molecule of claim 5 or a vector thereof, and the recombinant host cell of claim 7 as an active ingredient. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or adjuvant.

9. A method, comprising any one of the following: (1) A method for preparing the fusion protein according to any one of claims 1 to 4, characterized in that, The preparation method comprises introducing the nucleic acid molecule of claim 5 or a vector thereof into the recombinant host cell of claim 6, culturing and expressing the fusion protein of claim 1, and extracting and purifying the fusion protein; (2) A method for detecting and / or removing organophosphorus compounds using the fusion protein of any one of claims 1 to 4, wherein the fusion protein detects and / or removes organophosphorus compounds in a sample in a reaction system; Preferably, the sample comprises air, water, soil, food or biological fluid. Preferably, the reaction system further comprises a buffer, a chromogenic agent, an immobilized reagent. Preferably, the immobilized reagent comprises a membrane, a gel, a magnetic microsphere or a porous material.

10. Use in any one of the following: (1) the use of the fusion protein according to any one of claims 1-4, the nucleic acid molecule or vector thereof according to claim 5, the conjugate according to claim 7, the pharmaceutical composition according to claim 8, or the method according to claim 9 in the preparation of a medicament for treating or alleviating the symptoms caused by organophosphorus compound poisoning; (2) the use of the fusion protein according to any one of claims 1-4, the nucleic acid molecule or vector thereof according to claim 5, the conjugate according to claim 7, the pharmaceutical composition according to claim 8, or the method according to claim 9 in the preparation of a medicament for detecting and / or eliminating organophosphorus compounds; Preferably, the organophosphorus compounds include nerve agents and pesticides. Preferably, the organophosphorus compounds include compounds containing P-O bonds, P-CN bonds, P-F bonds, P-S bonds. Preferably, the symptoms include acute poisoning, intermediate syndrome, delayed multiple psychosis, and chronic neuropsychiatric syndrome.

Images