CN115177722A - Recombinant cholinesterase preparation and application thereof - Google Patents

Recombinant cholinesterase preparation and application thereof Download PDF

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CN115177722A
CN115177722A CN202210760804.6A CN202210760804A CN115177722A CN 115177722 A CN115177722 A CN 115177722A CN 202210760804 A CN202210760804 A CN 202210760804A CN 115177722 A CN115177722 A CN 115177722A
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recombinant
cholinesterase
leu
fusion protein
glu
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黄跃进
杨盼
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Shanghai Jenomed Biotech Co ltd
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Shanghai Jenomed Biotech Co ltd
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Abstract

The invention provides a recombinant cholinesterase product, which comprises any one of recombinant cholinesterase, a first mixed enzyme, a second mixed enzyme, recombinant cholinesterase-albumin fusion protein, recombinant cholinesterase-Fc fusion protein and recombinant transdermal short peptide-cholinesterase fusion protein. The recombinant cholinesterase product solves the potential risk problems of accuracy, stability and long-term safety of human expression of exogenous genes in a human BChE gene weight-reducing method. The invention also provides a use method of the recombinant cholinesterase product, and application of the recombinant cholinesterase product in neutralizing and degrading nerve agents, neutralizing and degrading organophosphorus agents, hydrolyzing hunger hormone and resisting wrinkles.

Description

Recombinant cholinesterase preparation and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant cholinesterase product and application thereof.
Background
Cholinesterases fall into two broad classes, those which preferentially hydrolyze acetylcholine esters such as acetylcholine, whose enzymatic activity is sensitive to the chemical inhibitor BW284C51, known as acetylcholinesterase (AChE), or acetylcholine acetylhydrolase. Acetylcholinesterase, also known as true, specific, pure, erythroid, or type I cholinesterase, is a membrane-bound glycoprotein and exists in the form of different molecules in erythrocytes, nerve endings, lung, spleen, and cerebral gray matter. Acetylcholinesterase is used in vivo primarily to hydrolyze acetylcholine. Those enzymes which preferentially hydrolyze other esters such as butyrylcholine, whose enzymatic activity is sensitive to the chemical inhibitor tetraisopropyl pyrophosphoroamide (ISO-OMPA), are known as butyrylcholinesterase (BChE, EC 3.1.1.8).
Butyrylcholinesterase is also known as pseudobutyrylcholinesterase or non-specific butyrylcholinesterase. Butyrylcholinesterase is further classified according to its charge, hydrophobicity, interaction with cell membranes or extracellular structures, and subunit composition. The enzyme is also called plasma type, serotype, benzoyl type, vacuity pseudotype or II type cholinesterase, and has more than 11 isoenzyme variants. Butyrylcholinesterase preferentially uses butyrylcholine and benzoylcholine as their in vitro reaction substrates. The enzyme is present in mammalian plasma, liver, pancreas, intestinal mucosa, central nervous system white matter, smooth muscle and heart. The specific function of butyrylcholinesterase is not clear and it has no known specific natural substrate, although it also hydrolyzes acetylcholine.
Human plasma butyrylcholinesterase is a globular tetrameric serine esterase with a human blood half-life of 12 days. It can neutralize and degrade nerve agents and other organophosphorous toxic agents in the blood, preventing these agents from binding to acetylcholinesterase. The use of exogenous butyrylcholinesterase to prevent inactivation of acetylcholinesterase is a potential strategic consideration in the prevention and treatment of poisoning by nerve and organophosphorous agents. But the enzyme concentration in human plasma is very low (2 mg/l), and isolation and purification directly from human plasma is of low commercial value, as well as the risk of transmission of infectious pathogens including viruses such as hepatitis c and aids.
Ghrelin (Ghrelin) is an endogenous Ghrelin hormone, secreted mainly by the human gastric mucosa, and is an endogenous ligand for growth hormone secretagogue receptors. Has effects in promoting growth hormone release, stimulating appetite, regulating energy metabolism, and reducing aggression. Ghrelin levels increase before meals, decrease after meals, and induce satiety when present at higher levels. In some bariatric procedures, the patient's ghrelin levels are reduced, thus causing satiety prior to a normal meal. How to reduce ghrelin levels in humans is one of the ways that obese patients lose weight. In the prior art, the human body has potential risk problems of excessive reaction possibly generated by a virus vector for mediating a BChE gene weight-reducing method and accuracy, stability and long-term safety of exogenous gene in-vivo expression.
Therefore, there is a need to provide a novel recombinant cholinesterase preparation and the application thereof, which avoid the above problems in the prior art.
Disclosure of Invention
The invention aims to provide a recombinant cholinesterase product and application thereof, and solves the potential risk problems that a human body possibly generates excessive reaction to a virus vector of a method for mediating BChE gene weight loss and exogenous gene expression has accuracy, stability and long-term safety.
In order to achieve the above objects, the recombinant cholinesterase preparation of the present invention comprises any one of recombinant cholinesterase, a first mixed enzyme, a second mixed enzyme, a recombinant cholinesterase-albumin fusion protein, a recombinant cholinesterase-Fc fusion protein and a recombinant transdermal short peptide-cholinesterase fusion protein, wherein the first mixed enzyme comprises a transdermal short peptide and a recombinant cholinesterase, and the second mixed enzyme comprises a nano-chip and a recombinant cholinesterase.
The recombinant cholinesterase product has the beneficial effects that: the recombinant cholinesterase product comprises any one of recombinant cholinesterase, a first mixed enzyme, a second mixed enzyme, recombinant cholinesterase-albumin fusion protein, recombinant cholinesterase-Fc fusion protein and recombinant transdermal short peptide-cholinesterase fusion protein, wherein the first mixed enzyme comprises transdermal short peptide and recombinant cholinesterase, and the second mixed enzyme comprises nano-chip and recombinant cholinesterase. The recombinant cholinesterase product solves the potential risk problems that a human body possibly generates excessive reaction to a virus vector of a method for mediating BChE gene weight loss and the accuracy, stability and long-term safety of exogenous gene expression exist.
Preferably, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein are derived from any one of an animal expression system, a plant expression system, a bacterial expression system and a yeast expression system.
Preferably, the animal expression system is selected from any one of an insect expression system and a mammalian expression system.
Preferably, the mammalian expression system comprises any one or more of mammary gland bioreactor systems (milk) from mouse, rat, rabbit, goat, sheep, pig, camel, yak, buffalo and cow.
Preferably, the recombinant cholinesterase preparation further comprises an oral preparation promoter and a transdermal absorption promoter. The beneficial effects are that: the recombinant cholinesterase product can enter a human body more easily, so that a better weight-losing effect is achieved.
Preferably, the oral preparation promoter includes any one or more of protease inhibitor, acidic pH modifier, chelate, surfactant, bile salt, aromatic alcohol, piperazine derivative, ionic liquid, enterotoxin peptide derivative, anionic polymer, cationic polymer, vulcanized polymer, polymer particle, inorganic particle, micelle, liposome, drug crystal, nanofiber and exosome.
Further preferably, the protease inhibitor comprises any one or more of N-acetylcysteine, camostat mesylate, soybean trypsin inhibitor and aprotinin.
Further preferably, the acidic pH modifier includes any one or more of citric acid, fumaric acid, itaconic acid, and tartaric acid.
Further preferably, the chelate compound includes any one or more of ethylenediaminetetraacetic acid (EDTA), diethyltriaminepentaacetic acid (DTPA), and ethyleneglycol bis (2-aminoethyl ether) tetraacetic acid (EGTA).
Further preferably, the surfactant includes one or more of Sodium Dodecyl Sulfate (SDS), sodium 8- (2-hydroxybenzamido) caprylate (SNAC), pyridinium Propane Sulfonate (PPS), and palmitoyl carnitine.
Further preferably, the bile salt includes one or more of sodium deoxycholate, sodium taurocholate, sodium glycodeoxycholate and sodium tauryl dihydrofluoride.
Further preferably, the aromatic alcohol includes one or more of propyl gallate, butyl hydroxy toluene and butyl hydroxy anisole.
Further preferably, the piperazine derivative includes any one or two or more of 1-phenylpiperazine, 1-methyl-4-phenylpiperazine and 1- (4-methylphenyl) piperazine.
Further preferably, the ionic liquid comprises one or more of choline, a puerarin salt, nicotinic acid and trigonelline.
Further preferably, the enterotoxin peptide derivative includes any one or both of a cell-locked toxin and a clostridium perfringens enterotoxin peptide.
Further preferably, the anionic polymer comprises any one or more of carbopol, polyacrylic resin (Eudragit L100) and hypromellose acetate succinate (HPMCAS).
Further preferably, the cationic polymer includes any one or two or more of osmotin, N-trimethyl chitosan (TMC) and microcrystalline cellulose (MCC).
Further preferably, the vulcanized polymer includes either or both of chitosan-thioglycolic acid and a stabilized thiolated polymer (PAA-cys-2 MNA).
Further preferably, the polymer particles include any one or more of polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), phenylethylamine (PEA), and Polycaprolactone (PCL).
Further preferably, the inorganic particles include any one or two or more of alumina, gold, and silica.
Further preferably, the micelle comprises any one or more of polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer (Pluronics), tripropylene glycol (TPG) and Oral Anticoagulant (OAC).
It is further preferred that the liposomes comprise synthetic Lipid Nanoparticles (LNPs), lipid polyplexes (LPPs) and certain natural liposomes comprise any of soy phospholipids, egg yolk phospholipids, and milk fat globule phospholipids of animal milks and siRNA lipid nanoparticles.
Further preferably, the drug crystal includes either or both of a crystalline lipase and a Lipid Nanocrystal (LNC).
Further preferably, the nanofibers comprise either or both of fish sarcoplasmic protein fibers and hyaluronic acid nanofibers.
Further preferably, the exosome refers to a vesicular body with a double-layer membrane structure, which is shed from various cell membranes or secreted by cells, i.e. a "carrier" for intercellular communication, and is responsible for the transfer of substances between different cells.
Preferably, the transdermal absorption enhancer comprises any one or more than two of a surfactant, a dimethyl sulfoxide derivative, a pyrrolidone derivative, a azone compound, an alcohol compound and an aliphatic compound.
Preferably, the recombinant cholinesterase comprises any one of recombinant acetylcholinesterase, recombinant butyrylcholinesterase, isozyme of recombinant cholinesterase and a variant of recombinant cholinesterase.
Preferably, the sequence of the recombinant butyrylcholinesterase is shown as SEQ ID No.1, and comprises a goat beta-casein signal peptide sequence and a human butyrylcholinesterase amino acid sequence.
Further preferably, the goat β -casein signal peptide sequence is capable of promoting secretion of the recombinant butyrylcholinesterase into animal milk or culture cell supernatant.
Preferably, the transdermal short peptide is positioned at the N terminal or the C terminal of the recombinant transdermal short peptide-cholinesterase fusion protein.
Preferably, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein further comprise a flexible linker peptide.
Preferably, the sequence of the recombinant cholinesterase-albumin fusion protein is shown in SEQ ID No. 2.
Preferably, the sequence of the recombinant cholinesterase-Fc fusion protein is shown in SEQ ID No. 3.
Preferably, the sequence of the recombinant transdermal short peptide-cholinesterase fusion protein is shown as SEQ ID No. 4.
Preferably, the sequence of the flexible connecting peptide is shown in SEQ ID No.5 or SEQ ID No. 6.
Preferably, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein further comprise a tag sequence convenient for protein purification, wherein the tag sequence is any one or more than two of His, flag, MBP, GST, HA and Myc.
Preferably, the purity of the recombinant cholinesterase, the transdermal short peptide, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein is 90-99%.
Preferably, the transdermal short peptide is prepared by artificial synthesis.
Preferably, the carrier of the recombinant cholinesterase preparation is any one of an adhesive patch, a polymer scaffold and a hydrogel.
Still another object of the present invention is to provide a method for using a recombinant cholinesterase preparation, which is administered orally, transdermally, intradermally, subcutaneously, lymphadenely, intramuscularly, intraperitoneally, or intravenously into a human body.
The use method of the recombinant cholinesterase product has the beneficial effects that: the recombinant cholinesterase product enters a human body in any one mode of oral administration, transdermal absorption, intradermal injection, subcutaneous injection, lymph node injection, intramuscular injection, intraperitoneal injection and intravenous injection, and solves the potential risk problems that the human body possibly generates excessive reaction to a virus vector of a method for mediating BChE gene weight loss and exogenous gene expression has accuracy, stability and long-term safety. The application method of the recombinant cholinesterase product solves the problems of side effects caused by the local injection of the Botox and skin bruising caused by an improper injection method.
Preferably, the transdermal absorption comprises a physical-chemical method, and the physical-chemical method comprises any one or more of an ion introduction technology, an electrotransfection technology, an electroporation technology, an ultrasonic introduction technology, a microneedle technology, a needle-free technology and a chemical transfection technology.
The invention also aims to provide application of the recombinant cholinesterase preparation in neutralizing and degrading nerve agents, organophosphorus agents, hunger hormone and anti-wrinkle.
The application of the recombinant cholinesterase product has the beneficial effects that: the application of the recombinant cholinesterase product in neutralizing and degrading nerve agents and organophosphorus agents can reduce or eliminate the toxic action of nerve agents and organophosphorus agents. The application of the recombinant cholinesterase product in hydrolyzing the ghrelin is to apply the recombinant cholinesterase product to a human body, so that the level of the ghrelin in the human body is reduced, the weight of the human body is reduced, and the aggressive behavior is reduced. The recombinant cholinesterase product solves the potential risk problems of the excessive reaction possibly generated by a human body to a virus vector of a mediated BChE gene weight-losing method and the accuracy, stability and long-term safety existing in the expression of an exogenous gene in vivo in the aspects of reducing the weight of the human body and reducing the aggressive behavior. The recombinant cholinesterase product enables human skin to be smooth and eliminates fine wrinkles through transdermal absorption or intradermal injection.
Preferably, the recombinant cholinesterase preparation is intended for repeated use.
Preferably, the repeated use period includes daily use, every other day use or about 1 to 26 weeks apart to enhance the application effect.
Drawings
FIG. 1 is a photograph of an electrophoretogram showing the recombinant butyrylcholinesterase of the present invention after purification by SDS-PAGE and Coomassie blue staining;
FIG. 2 is a photograph showing an electrophoretogram of recombinant butyrylcholinesterase of the present invention by non-denaturing gel electrophoresis and active staining;
FIG. 3 is a photograph of an electrophoretogram of recombinant butyrylcholinesterase-albumin fusion protein and recombinant butyrylcholinesterase-Fc fusion protein according to an embodiment of the present invention, which is displayed by denaturing gel electrophoresis;
FIG. 4 is a graph of simulated dynamic mechanical data of the enzymatic activity of human plasma butyrylcholinesterase enzyme of the prior art;
FIG. 5 is a graph of simulated dynamic mechanical data of enzyme activity of recombinant butyrylcholinesterase enzyme according to an embodiment of the present invention;
FIG. 6 is a graph showing the results of stability test of recombinant butyrylcholinesterase preparations according to the embodiment of the present invention;
FIG. 7 is a graph showing the results of the rat grip test in example 4 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The use of "including" and "comprising" and similar words, as used herein, is intended to mean that the element or item preceding the word comprises the element or item listed after the word and its equivalents, without excluding other elements or items.
In order to produce a large amount of medicinal recombinant cholinesterase, a pharmaceutical platform of an animal mammary gland bioreactor is established, a plurality of recombinant butyrylcholinesterase (rBChE) positive rabbit lines are generated, a plurality of rabbit milk rBChE high-expression lines are screened from the rabbit positive rabbit lines, and a part of protein is purified. The enzyme kinetic parameters of the produced rBChE are the same as those of human plasma BChE, the antitoxic effect of the nerve agent is equivalent, and particularly, the enzyme is found to have excellent antitoxic effect on a novel toxin agent NovieJoke for the first time in the world. rBChE also hydrolyzes ghrelin, a hunger hormone secreted by mammals, and causes weight loss and reduced aggressive behavior in mammals. In the prior art, the problem of low commercial value exists in the direct separation and purification of BChE from human plasma for detoxification of nerve agents and organophosphorus agents. Some people hydrolyze ghrelin by using a BChE gene therapy method in which a virus vector carries a BChE gene and is injected into a body, but the human body possibly generates an over-reaction to the virus vector mediating the BChE gene weight-reducing method, and the accuracy, stability and long-term safety of the expression of an exogenous gene in the body have potential risk problems. Human plasma butyrylcholinesterase can also hydrolyze cocaine drugs, but the expression level of recombinant butyrylcholinesterase produced by CHO cell system expression is not high (0.005-0.035 g/L), and CMC production can encounter technical bottlenecks. Therefore, the expression and production of the recombinant butyrylcholinesterase by the CHO cells have the problem of low cocaine drug hydrolysis potency. The action mechanism of cholinesterase is similar to that of botulinum toxin, and the cholinesterase can hydrolyze or inactivate acetylcholine at neuromuscular junction to cause muscle relaxation.
In view of the problems in the prior art, an embodiment of the present invention provides a recombinant cholinesterase preparation, comprising any one of a recombinant cholinesterase, a first mixed enzyme, a second mixed enzyme, a recombinant cholinesterase-albumin fusion protein, a recombinant cholinesterase-Fc fusion protein and a recombinant transdermal short peptide-cholinesterase fusion protein, wherein the first mixed enzyme comprises a transdermal short peptide and a recombinant cholinesterase, and the second mixed enzyme comprises a nano-chip and a recombinant cholinesterase.
Specifically, the recombinant cholinesterase product comprises any one of recombinant cholinesterase, a first mixed enzyme, a second mixed enzyme, recombinant cholinesterase-albumin fusion protein, recombinant cholinesterase-Fc fusion protein and recombinant transdermal short peptide-cholinesterase fusion protein, wherein the first mixed enzyme comprises transdermal short peptide and recombinant cholinesterase, and the second mixed enzyme comprises nano-chip and recombinant cholinesterase. The recombinant cholinesterase product solves the potential risk problems that a human body possibly generates excessive reaction to a virus vector of a mediated BChE gene weight-losing method and the accuracy, stability and long-term safety exist in exogenous gene expression. Meanwhile, the recombinant cholinesterase product can solve the problems of low commercial value of BChE directly separated and purified from human plasma and low titer of recombinant butyrylcholinesterase produced by CHO cell expression.
In some embodiments of the invention, the recombinant cholinesterase-albumin fusion protein can increase the half-life of the recombinant cholinesterase preparation in a human body, and enhance the effectiveness of the recombinant cholinesterase preparation.
In some embodiments of the present invention, the recombinant cholinesterase-Fc fusion protein not only exerts the biological activity of the fusion protein, but also has some antibody properties, such as inducing antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent cell-mediated phagocytosis.
In some embodiments of the invention, the transdermal short peptide is TD-1 or other transdermal short peptide.
In some embodiments of the invention, TD-1 consists of 11 amino acids and is the first transdermal enhancing peptide discovered by phage display technology.
In some embodiments of the present invention, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein are derived from any one of an animal expression system, a plant expression system, a bacterial expression system and a yeast expression system.
In some embodiments of the invention, the animal expression system is selected from any one of an insect expression system and a mammalian expression system.
In some embodiments of the invention, the insect expression system comprises an insect cell expression system.
In still other embodiments of the present invention, the mammalian expression system comprises a mammalian cell expression system.
In other embodiments of the invention, the mammalian expression system comprises any one or more of a mammary bioreactor system (milk) from a mouse, rat, rabbit, goat, sheep, pig, camel, yak, buffalo, and cow.
Specifically, the animal mammary gland bioreactor system is characterized in that a regulatory sequence of a mammary gland specific expression milk protein gene is utilized to construct an expression vector, the expression vector is introduced into a female non-human mammal gene of reproductive age, and a recombinant protein is expressed in mammary gland tissues or mammary ducts of the female non-human mammal of reproductive age in a positioning manner; or transfecting said expression vector into mammary tissue or a mammary duct of said female non-human mammal of child-bearing age; specific exogenous gene is directed to express in animal mammary gland specifically and efficiently so as to obtain exogenous active protein from secreted milk.
In some possible embodiments of the invention, the plant expression system, the bacterial expression system, the yeast expression system, the insect cell expression system and the mammalian cell expression system are genetically engineered.
In yet other possible embodiments of the invention, the mammalian mammary expression system and the plant expression system are not genetically engineered.
In some embodiments of the invention, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein, and the recombinant transdermal short peptide-cholinesterase fusion protein are derived from animal milk.
In some more specific embodiments of the invention, the animal milk comprises any one or more of milk from mice, rats, rabbits, goats, sheep, pigs, camels, yaks, buffalos and cows.
In some more specific embodiments of the invention, the insect cell expression system is an SF9 cell line.
In some more specific embodiments of the invention, the mammalian cell expression system is a Chinese hamster ovary cell line (CHO), including but not limited to CH0-K1, CH0-DG44, and CH0-S.
In some more specific embodiments of the invention, the mammalian cell expression system is a baby hamster kidney cell line (BHK).
In some more specific embodiments of the invention, the mammalian cell expression system is a cow mammary epithelial cell MAC-T cell line, a cow mammary epithelial cell BME-UV1 cell line, and other cow, goat, and rabbit primary mammary epithelial cells and mammary epithelial cell lines.
In other more particular embodiments of the invention, the human cell expression system is the HEK293 cell line.
In some embodiments of the present invention, the recombinant cholinesterase comprises any one of a recombinant acetylcholinesterase, a recombinant butyrylcholinesterase, an isozyme of a recombinant cholinesterase, and a variant of a recombinant cholinesterase.
In some embodiments of the invention, the recombinant cholinesterase comprises a full-length recombinant acetylcholinesterase, a full-length recombinant butyrylcholinesterase, and an isozyme of the full-length recombinant cholinesterase or a variant of the full-length recombinant cholinesterase.
In still other embodiments of the present invention, the recombinant cholinesterase comprises a fragmented recombinant acetylcholinesterase, a fragmented recombinant butyrylcholinesterase, and an isozyme of a fragmented recombinant cholinesterase or a variant of a fragmented recombinant cholinesterase.
In some embodiments of the invention, the recombinant cholinesterase preparation further comprises a pharmaceutically acceptable excipient.
In some embodiments of the invention, the recombinant cholinesterase preparation further comprises an oral formulation promoter and a transdermal absorption promoter. The recombinant cholinesterase product can enter a human body more easily, so that a better weight reducing effect is achieved.
In some embodiments of the present invention, the oral formulation promoter includes any one or more of a protease inhibitor, an acidic pH modifier, a chelate, a surfactant, a bile salt, an aromatic alcohol, a piperazine derivative, an ionic liquid, an enterotoxin peptide derivative, an anionic polymer, a cationic polymer, a vulcanized polymer, a polymer particle, an inorganic particle, a micelle, a liposome, a drug crystal, a nanofiber, and an exosome.
In still other embodiments of the present invention, the protease inhibitor comprises any one or more of N-acetylcysteine, camostat mesylate, soybean trypsin inhibitor, and aprotinin.
In still other embodiments of the present invention, the acidic pH modifier comprises any one or more of citric acid, fumaric acid, itaconic acid, and tartaric acid.
In still other embodiments of the present invention, the chelate compound comprises any one or more of ethylenediaminetetraacetic acid (EDTA), diethyltriaminepentaacetic acid (DTPA), and ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA).
In still other embodiments of the present invention, the surfactant includes any one or more of Sodium Dodecyl Sulfate (SDS), sodium 8- (2-hydroxybenzamido) caprylate (SNAC), pyridinium Propanesulfonate (PPS), and palmitoyl carnitine.
In still other embodiments of the present invention, the bile salt includes any one or more of sodium deoxycholate, sodium taurocholate, sodium glycodeoxycholate, and sodium tauryl dihydrofluoride.
In some embodiments of the present invention, the aromatic alcohol includes one or more of propyl gallate, butyl hydroxy toluene and butyl hydroxy anisole.
In still other embodiments of the present invention, the piperazine derivative includes any one or more of 1-phenylpiperazine, 1-methyl-4-phenylpiperazine and 1- (4-methylphenyl) piperazine.
In still other embodiments of the present invention, the ionic liquid comprises one or more of choline, a puerarin salt, nicotinic acid, and trigonelline.
In still other embodiments of the invention, the enterotoxin peptide derivatives include either or both of a cell-locked toxin and a clostridium perfringens enterotoxin peptide.
In still other embodiments of the present invention, the anionic polymer comprises any one or more of carbopol, polyacrylic resin (Eudragit L100), and hypromellose acetate succinate (HPMCAS).
In still other embodiments of the present invention, the cationic polymer comprises any one or more of osmotin, N-trimethyl chitosan (TMC), and microcrystalline cellulose (MCC).
In still other embodiments of the present invention, the vulcanized polymer comprises either or both of chitosan-thioglycolic acid and a stabilized thiolated polymer (PAA-cys-2 MNA).
In still other embodiments of the present invention, the polymer particles include any one or more of poly (lactic-co-glycolic acid) (PLGA), poly (lactic acid) (PLA), phenylethylamine (PEA), and Polycaprolactone (PCL).
In still other embodiments of the present invention, the inorganic particles include any one or two or more of alumina, gold, and silica.
In still other embodiments of the present invention, the micelle comprises any one or more of polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer (Pluronics), tripropylene glycol (TPG), and Oral Anticoagulant (OAC).
In still other embodiments of the present invention, the liposomes comprise synthetic Lipid Nanoparticles (LNPs), lipid polyplexes (LPPs), and certain natural liposomes comprise any of soybean phospholipids, egg yolk phospholipids, and milk fat globule phospholipids of animal milk and siRNA lipid nanoparticles.
In still other embodiments of the present invention, the drug crystal comprises any one of a crystalline lipase and a Lipid Nanocrystal (LNC).
In still other embodiments of the present invention, the nanofibers comprise either or both of fish sarcoplasmic protein fibers and hyaluronic acid nanofibers.
In still other embodiments of the present invention, the exosome refers to a vesicular-like body with a double-layer membrane structure, which is shed from various cell membranes or secreted by cells, i.e., a "carrier" for intercellular communication, and is responsible for the transfer of substances between different cells.
In some embodiments of the present invention, the transdermal absorption enhancer includes any one or more of a surfactant, a dimethyl sulfoxide derivative, a pyrrolidone derivative, a azone compound, an alcohol compound, and an aliphatic compound.
In some embodiments of the present invention, the transdermal absorption enhancer may be further selected from any one or both of dimethyl sulfoxide and pyrrolidone.
In some embodiments of the invention, the sequence of the recombinant butyrylcholinesterase is shown as SEQ ID No.1, and comprises a goat beta-casein signal peptide sequence and a human butyrylcholinesterase amino acid sequence.
In other more specific embodiments of the invention, the goat β -casein signal peptide sequence is capable of promoting secretion of the recombinant butyrylcholinesterase into animal milk or culture cell supernatant.
In some embodiments of the invention, the transdermal short peptide is located at the N-terminus or C-terminus of the recombinant transdermal short peptide-cholinesterase fusion protein.
In some embodiments of the invention, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein, and the recombinant transdermal short peptide-cholinesterase fusion protein further comprise a flexible linker peptide.
In some embodiments of the invention, the recombinant cholinesterase-albumin fusion protein has a sequence shown in SEQ ID No. 2.
In some embodiments of the invention, the recombinant cholinesterase-Fc fusion protein has a sequence as shown in SEQ ID No. 3.
In some embodiments of the invention, the sequence of the recombinant transdermal short peptide-cholinesterase fusion protein is shown as SEQ ID No. 4.
In some embodiments of the invention, the sequence of the flexible connecting peptide is shown in SEQ ID No.5 or SEQ ID No. 6.
In some embodiments of the present invention, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein further comprise a tag sequence for facilitating protein purification, wherein the tag sequence is any one or more than two of His, flag, MBP, GST, HA and Myc.
In some embodiments of the present invention, the purity of the recombinant cholinesterase, the transdermal short peptide, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein is 90-99%.
In some embodiments of the invention, the transdermal short peptide is prepared by artificial synthesis.
In some embodiments of the present invention, the carrier of the recombinant cholinesterase preparation is any one of an adhesive patch, a polymer scaffold and a hydrogel.
In the embodiment of the invention, the application method of the recombinant cholinesterase product is provided, and the recombinant cholinesterase product is introduced into a human body by any one of oral administration, transdermal absorption, intradermal injection, subcutaneous injection, lymph node injection, intramuscular injection, intraperitoneal injection and intravenous injection.
Specifically, the recombinant cholinesterase product enters a human body through any one of oral administration, transdermal absorption, intradermal injection, subcutaneous injection, lymph node injection, intramuscular injection, intraperitoneal injection and intravenous injection, and the recombinant cholinesterase product solves the potential risk problems that the human body possibly generates excessive reaction to a virus vector of a method for mediating BChE gene weight loss and the exogenous gene expression has accuracy, stability and long-term safety respectively. Meanwhile, the recombinant cholinesterase product solves the problems of low commercial value of BChE which is directly separated and purified from human plasma and low titer of recombinant butyrylcholinesterase produced by CHO cell expression. The application method of the recombinant cholinesterase product solves the problems of side effects caused by the local injection of the Botox and skin bruising caused by an improper injection method.
More specifically, the subcutaneous injection comprises injection at multiple sites on human skin, with a spacing of about 0.5-10 cm, and an injection amount of the recombinant cholinesterase preparation of 0.1-1000 units, to smooth human skin and eliminate fine wrinkles.
In some embodiments of the invention, the transdermal absorption comprises a physical-chemical method, the physical-chemical method comprises any one or more of an iontophoresis technique, an electrotransfection technique, an electroporation technique, an ultrasonic wave introduction technique, a microneedle technique, a needle-free technique, and a chemical transfection technique.
In some embodiments of the invention, the microneedle technology comprises nanocrystalline nano-wafer microneedle technology.
In some embodiments of the invention, there is provided a use of a recombinant cholinesterase preparation for neutralizing and degrading a nerve agent, neutralizing and degrading an organophosphorous agent, hydrolyzing ghrelin, hydrolyzing a cocaine substance, and preparing an anti-wrinkle medicament.
In some embodiments of the invention, the anti-wrinkle effect is to reduce or eliminate wrinkles on human skin.
Specifically, the application of the recombinant cholinesterase preparation in neutralizing and degrading nerve agents and organophosphorus agents can reduce or eliminate the toxic action of nerve agents and organophosphorus agents. The application of the recombinant cholinesterase product in hydrolysis of the ghrelin is that the recombinant cholinesterase product is applied to a human body, so that the level of the ghrelin in the human body is reduced, the weight of the human body is reduced, and the aggressive behavior is reduced. In order to reduce or even eliminate side effects caused by the topical injection of the Botox and skin bruising caused by improper injection methods, the recombinant cholinesterase preparation is absorbed through the skin or injected into the skin in a transdermal mode, so that the skin of a human body is smooth and fine wrinkles are eliminated. The recombinant cholinesterase product solves the potential risk problems that a human body possibly generates excessive reaction to a virus vector of a mediated BChE gene weight-losing method and the accuracy, stability and long-term safety exist in exogenous gene expression. Meanwhile, the recombinant cholinesterase product solves the problems of low commercial value of BChE which is directly separated and purified from human plasma and low titer of recombinant butyrylcholinesterase produced by CHO cell expression. The application method of the recombinant cholinesterase product solves the problems of side effects caused by the local injection of the Botox and skin bruising caused by an improper injection method.
In some embodiments of the invention, the recombinant cholinesterase preparation is intended for repeated use.
In some embodiments of the invention, the repeated use period comprises daily use, every other day use, or about 1 to 26 weeks apart, to enhance the application effect.
Example 1
Introducing the recombinant butyrylcholinesterase (rBChE, shown in SEQ ID No. 1) protein coding nucleic acid plasmid into female reproductive rabbit genes, positioning and expressing the rBChE in mammary tissues of the female reproductive rabbits, and collecting rabbit milk. The Ellman assay method is used for detecting the expression concentration of the rabbit milk rBChE, the high-efficiency expression of the rabbit milk rBChE (0.24-12 g/L, see table 1 in detail) is realized in a plurality of positive rabbit lines, the serum concentration of the rabbit milk rBChE is more than 6000 times higher than that of the human plasma BChE, a world record (table 2) is created, the technical bottleneck of mass production is broken through, and an unexpected optimal technical effect is obtained. The primary rabbit milk pure product containing rBChE is purified by affinity chromatography and ion exchange chromatography. The washed rabbit milk primary was loaded on a procainamide affinity column equilibrated in advance with 10mM phosphate buffer (pH 7.2), 1mM EDTA and 140mM NaCl. The column was washed with 10 bed volumes of the same equilibration buffer and the protein was eluted with 10mM phosphate buffer (pH 7.2), 1mM EDTA and 500mM NaCl. The eluate was loaded onto an HQ50 ion exchange chromatography column, which was equilibrated with the same buffer. The eluate (containing rBChE) was collected, followed by washing the column with 10mM phosphate buffer (pH 7.2), 1mM EDTA and 1M NaCl to remove any trapped impurities. Purified rBChE was sterile filtered and stored at-20 ℃. Purified proteins were tested for BChE activity and total protein concentration was determined.
TABLE 1 expression concentration of rabbit milk rBChE of different rabbit lines
Rabbit line Rabbit milk rBChE expression concentration
D152 12.01g/L
D161 0.63g/L
D173 0.24g/L
D185 4.11g/L
TABLE 2 expression production of rBChE in different systems
Figure BDA0003724002700000171
Meanwhile, respectively introducing the recombinant butyrylcholinesterase-albumin fusion protein (shown as SEQ ID No. 2) protein coding nucleic acid plasmid and the recombinant butyrylcholinesterase-Fc fusion protein (shown as SEQ ID No. 3) protein coding nucleic acid plasmid into a female breeding mouse gene, respectively positioning and expressing the recombinant butyrylcholinesterase-albumin fusion protein and the recombinant butyrylcholinesterase-Fc fusion protein in mammary tissue of the female breeding mouse, and collecting mouse milk. The expression of the mouse milk recombinant butyrylcholinesterase-albumin fusion protein and the expression of the recombinant butyrylcholinesterase-Fc fusion protein are detected by an Ellman assay detection method by taking the recombinant butyrylcholinesterase expressed in rabbit milk as a control, and the expression concentration of the mouse milk is approximately equivalent to that of the rabbit milk recombinant butyrylcholinesterase respectively (see figure 3 for details).
FIG. 1 is a photograph of an electrophoretogram showing the recombinant butyrylcholinesterase of the present invention after purification by SDS-PAGE and Coomassie blue staining; FIG. 2 is a photograph showing an electrophoretogram of recombinant butyrylcholinesterase of the present invention by non-denaturing gel electrophoresis and active staining; FIG. 3 is a photograph of an electrophoretogram of recombinant butyrylcholinesterase-albumin fusion protein and recombinant butyrylcholinesterase-Fc fusion protein according to an embodiment of the present invention, which is displayed by denaturing gel electrophoresis; FIG. 4 is a graph of simulated dynamic mechanical data of the enzymatic activity of human plasma butyrylcholinesterase enzyme of the prior art; FIG. 5 is a graph of enzyme activity simulation dynamic mechanical data of recombinant butyrylcholinesterase of the present invention.
After the recombinant butyrylcholinesterase is purified and subjected to denaturing SDS-PAGE electrophoresis and Coomassie brilliant blue staining, the molecular weight of the monomeric recombinant butyrylcholinesterase is about 90kd, the purity is higher than 95%, and the activity is higher than 700U/ml, as shown in figure 1.
The recombinant butyrylcholinesterase is subjected to non-denaturing gel electrophoresis and an activity staining method, and rabbit milk samples containing different concentrations of recombinant butyrylcholinesterase are mixed with 0.1mM 50-mer proline (Polyproline), and assembled into tetramers after being treated overnight at 37 ℃. Sample 1 was human plasma butyrylcholinesterase (200 mU, untreated); sample 2 is a positive rabbit milk sample (recombinant butyrylcholinesterase 1000 u/ml); sample 3 is a positive rabbit milk sample (recombinant butyrylcholinesterase 300 u/ml); sample 4 is a positive rabbit milk sample (recombinant butyrylcholinesterase 18 u/ml); sample 5 is a positive rabbit milk sample (recombinant butyrylcholinesterase 1000u/ml, no proline treatment); sample 6 was a sample of freshly purified rabbit milk (1000 u/ml, not treated with proline), see FIG. 2.
Referring to fig. 3, a photograph of an electrophoretogram of recombinant butyrylcholinesterase-albumin fusion protein and recombinant butyrylcholinesterase-Fc fusion protein according to an embodiment of the present invention, which is displayed by denaturing gel electrophoresis, is shown. Sample 1 is the protein size standard; samples 2-4 were all positive rabbit milk dilution samples containing recombinant butyrylcholinesterase protein (white arrow); sample 5 is a diluted sample of murine milk containing recombinant butyrylcholinesterase-Fc fusion protein (grey arrow); sample 6 is a diluted sample of rat milk containing recombinant butyrylcholinesterase-albumin fusion protein (black arrow); samples 7-8 were negative mouse milk dilution sample controls.
Referring to FIGS. 4 and 5, the Michaelis constants of human plasma butyrylcholinesterase and recombinant butyrylcholinesterase were determined, wherein the Km value is 120 μm/L and the Km value is 121 μm/L, which indicates that the enzyme kinetics characteristics of human plasma butyrylcholinesterase and recombinant butyrylcholinesterase are the same.
Example 2
1. Anti-virus experiment of rabbit milk rBChE mice against nerve agent soman
Mice were grouped and injected with 2.5mg/ml,5mg/ml,10mg/ml rBChE solutions, respectively, half an hour before 1-fold and 2-fold shuttle lethal doses, respectively. And (4) analyzing results: 5mg/ml rBChE was resistant to 1-fold soman lethal dose.
6 mice were first intraperitoneally injected with a 1-fold soman lethal dose, followed immediately by intravenous injection of 10mg/ml rBChE solution, and the first aid effect was observed. 6 mice survived 3. It indicates that the first aid is effective. After 1-fold of the soman lethal dose, rBChE solution was injected in one minute, and the injection time required may vary from mouse to mouse due to individual differences.
2. Anti-virus experiment of rabbit milk rBChE and human plasma BChE mice against nerve agent Novieke
NovieJoke is a binary organophosphorus series nerve agent developed by the Soviet Union, and has the four characteristics of no detection, no protection, safety of users, no meeting of the rules of the chemical industry and the like. At ordinary times, the pesticide is disguised to be separately stored and mixed before use to become the high-efficiency nerve agent, and the toxicity of the nerve agent is 5 to 8 times higher than that of sarin, VX, soman and the like. Mice were injected subcutaneously with norvqiaoke poison, intravenously injected with rabbit milk rBChE and human plasma BChE, observed for behavioral changes and intoxication symptoms in animals, and for treatment effects.
The experimental results determine the anti-virus potency of rabbit milk HuBChE: after the rabbit milk HuBChE of 100mg/kg is injected intravenously, the 10 times lethal dose of Novovian Joke poisoning can be cured (Table 3). HuBChE, human serum at 20mg/kg, was administered intravenously to treat a 10-fold lethal dose of NovieJoke intoxication (Table 4).
TABLE 3 anti-toxin potency of Rabbit milk rBChE
Novier Qiaoge Rabbit milk rBChE Mouse Survival Survival rate
10LD 100mg/kg 4 4 100％
TABLE 4 antitoxic potency of human plasma BChE
Novajqiaoke (Nuoweiwe Qiaoke) Human plasma BChE Mouse Survival Survival rate
2LD 20mg/kg 8 8 100％
As can be seen from tables 3 and 4: (1) Rabbit milk rBChE and human plasma BChE can both effectively cure mice poisoned by Nuowei Qiaoke. (2) the rabbit milk rBChE and human plasma BChE have very high anti-virus potency. (3) The anti-virus titer of rabbit milk rBChE and human plasma BChE is basically equivalent.
Example 3
Purified rabbit milk rBChE samples were mixed with polyethylene glycol products mPEG-pLE50 (P50) and mPEG113-pLE100 (P100), respectively, and stirred at 4 ℃ for 30 minutes. The original purified rBChE sample and the above mixture sample were divided into 250ul, sealed and placed in a 37 ℃ incubator, and a part of the sample was taken on day 1, day 4, day 8, day 39, day 89 and day 179, respectively, and assayed for BChE activity by Ellman assay.
FIG. 6 is a diagram showing the results of the stability experiment of the recombinant butyrylcholinesterase preparation of the embodiment of the invention.
And (4) analyzing results: the enzyme activity stability of the original purified rBChE sample was good at 37 ℃ in an incubator over a half year period, see FIG. 6.
Example 4
FIG. 7 is a graph showing the results of the rat grip test in example 4 of the present invention.
The mechanism of action of cholinesterases is theoretically similar to that of botulinum toxin (Botox), which all hydrolyze or inactivate acetylcholine at the neuromuscular junction, causing muscle relaxation. The rat in vivo grip test was used mainly to compare the paralytic effects of Botox and recombinant cholinesterase preparations on muscle relaxation. Saline or test article (rBChE, rAChE, botox) was injected intramuscularly into both forelimb muscles of SD rats (100 ul/time), 0, 8, 24, 48, 72 hours after injection, and the grip was measured using a digital grip apparatus. Forelimb grasping force was measured by holding the nape and tail of the neck, placing the forelimb on the tension rod, and gently pulling the rat back until the tension rod was released. Each rat was tested 5 times in succession. The average of five trials was recorded as the grip of the rat. The data were summarized and analyzed by analytical software, see fig. 7.
As can be seen from FIG. 7, the rat grip of rBChE at different doses was relatively reduced 8 hours after rat injection, and substantially returned to normal 24 hours later. While the rats showed a relative decrease in grip 48 hours after the Botox injection and essentially returned to normal after 72 hours. The results of the experiments show that rBChE injection provides a more early onset of muscle relaxation than Botox injection, with substantially the same time to recovery of grip.
Example 5
24 obese mice models (strain background: C57BL/6J mice) with the age of 10-12 weeks, which are constructed based on high-fat diet feeding, are purchased from the Ji extract medicine health, and after the mice arrive, the time for recovery is about 2-4 days. The obese mice were divided into 3 groups of 8 mice each, and high fat diet was maintained during intraperitoneal injection (per 100g of common feed, 20g of lard, 20g of milk powder, 1 egg, 10 drops of cod liver oil, 250g of soybean sprouts were added, and the feed was prepared and then baked.
(1) Injecting normal saline into abdominal cavity: injecting 100ul of normal saline for x15 times half an hour before meals every morning;
(2) High dose rBChE (2.5 mg/ml) was injected intraperitoneally: administering 100ul rBChE x15 times before meal half an hour before meal every morning;
(3) Low dose rBChE (0.6 mg/ml) was injected intraperitoneally: the half hour before meals were injected with 100ul rBChE x15 times every other morning.
Measurement indexes are as follows: during the experiment, tail blood was taken at the end of each week to determine plasma BChE concentrations, acylghrelin and Desacyl-ghrelin concentrations, and the ratio of Desacyl/acyl ghrelin.
Other indexes are as follows: according to the weight and the length of the mouse, calculating the Lee's index:
Figure BDA0003724002700000211
and fat index (%): body fat weight (grams)/body weight (grams) x100.
Statistical treatment: processing was performed using SPSS statistical software. All parameters were used for each group of mice: mean ± standard deviation (X ± SD) and differences between groups were analyzed by variance, significant t-test.
The experimental result shows that compared with the obese mice injected with the normal saline, the obese mice injected with the recombinant butyrylcholinesterase product in the same period in the abdominal cavity have reduced appetite, weight and aggressive behavior.
Although the embodiments of the present invention have been described in detail hereinabove, it is apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it is to be understood that such modifications and variations fall within the scope and spirit of the present invention as set forth in the following claims. Moreover, the invention as described herein is capable of other embodiments and of being practiced or of being carried out in various ways.
Sequence listing
<110> Shanghai 26688
<120> recombinant cholinesterase preparation and application thereof
<130> 2022.06.30
<140> HRCN22L31014A
<141> 2022-06-30
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Met Lys Val Leu Ile Leu Ala Cys Leu Val Ala Leu Ala Leu Ala Glu
1 5 10 15
Asp Asp Ile Ile Ile Ala Thr Lys Asn Gly Lys Val Arg Gly Met Asn
20 25 30
Leu Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro Tyr
35 40 45
Ala Gln Pro Pro Leu Gly Arg Leu Arg Phe Lys Lys Pro Gln Ser Leu
50 55 60
Thr Lys Trp Ser Asp Ile Trp Asn Ala Thr Lys Tyr Ala Asn Ser Cys
65 70 75 80
Cys Gln Asn Ile Asp Gln Ser Phe Pro Gly Phe His Gly Ser Glu Met
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Trp Asn Pro Asn Thr Asp Leu Ser Glu Asp Cys Leu Tyr Leu Asn Val
100 105 110
Trp Ile Pro Ala Pro Lys Pro Lys Asn Ala Thr Val Leu Ile Trp Ile
115 120 125
Tyr Gly Gly Gly Phe Gln Thr Gly Thr Ser Ser Leu His Val Tyr Asp
130 135 140
Gly Lys Phe Leu Ala Arg Val Glu Arg Val Ile Val Val Ser Met Asn
145 150 155 160
Tyr Arg Val Gly Ala Leu Gly Phe Leu Ala Leu Pro Gly Asn Pro Glu
165 170 175
Ala Pro Gly Asn Met Gly Leu Phe Asp Gln Gln Leu Ala Leu Gln Trp
180 185 190
Val Gln Lys Asn Ile Ala Ala Phe Gly Gly Asn Pro Lys Ser Val Thr
195 200 205
Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Leu His Leu Leu
210 215 220
Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Gln Ser Gly
225 230 235 240
Ser Phe Asn Ala Pro Trp Ala Val Thr Ser Leu Tyr Glu Ala Arg Asn
245 250 255
Arg Thr Leu Asn Leu Ala Lys Leu Thr Gly Cys Ser Arg Glu Asn Glu
260 265 270
Thr Glu Ile Ile Lys Cys Leu Arg Asn Lys Asp Pro Gln Glu Ile Leu
275 280 285
Leu Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val Asn
290 295 300
Phe Gly Pro Thr Val Asp Gly Asp Phe Leu Thr Asp Met Pro Asp Ile
305 310 315 320
Leu Leu Glu Leu Gly Gln Phe Lys Lys Thr Gln Ile Leu Val Gly Val
325 330 335
Asn Lys Asp Glu Gly Thr Ala Phe Leu Val Tyr Gly Ala Pro Gly Phe
340 345 350
Ser Lys Asp Asn Asn Ser Ile Ile Thr Arg Lys Glu Phe Gln Glu Gly
355 360 365
Leu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser Ile
370 375 380
Leu Phe His Tyr Thr Asp Trp Val Asp Asp Gln Arg Pro Glu Asn Tyr
385 390 395 400
Arg Glu Ala Leu Gly Asp Val Val Gly Asp Tyr Asn Phe Ile Cys Pro
405 410 415
Ala Leu Glu Phe Thr Lys Lys Phe Ser Glu Trp Gly Asn Asn Ala Phe
420 425 430
Phe Tyr Tyr Phe Glu His Arg Ser Ser Lys Leu Pro Trp Pro Glu Trp
435 440 445
Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Leu Pro
450 455 460
Leu Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Leu Ser Arg
465 470 475 480
Ser Ile Val Lys Arg Trp Ala Asn Phe Ala Lys Tyr Gly Asn Pro Asn
485 490 495
Glu Thr Gln Asn Asn Ser Thr Ser Trp Pro Val Phe Lys Ser Thr Glu
500 505 510
Gln Lys Tyr Leu Thr Leu Asn Thr Glu Ser Thr Arg Ile Met Thr Lys
515 520 525
Leu Arg Ala Gln Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Lys Val
530 535 540
Leu Glu Met Thr Gly Asn Ile Asp Glu Ala Glu Trp Glu Trp Lys Ala
545 550 555 560
Gly Phe His Arg Trp Asn Asn Tyr Met Met Asp Trp Lys Asn Gln Phe
565 570 575
Asn Asp Tyr Thr Ser Lys Lys Glu Ser Cys Val Gly Leu
580 585
<210> 2
<211> 1134
<212> PRT
<213> Artificial Sequence
<400> 2
Met Lys Val Leu Ile Leu Ala Cys Leu Val Ala Leu Ala Leu Ala Glu
1 5 10 15
Asp Asp Ile Ile Ile Ala Thr Lys Asn Gly Lys Val Arg Gly Met Asn
20 25 30
Leu Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro Tyr
35 40 45
Ala Gln Pro Pro Leu Gly Arg Leu Arg Phe Lys Lys Pro Gln Ser Leu
50 55 60
Thr Lys Trp Ser Asp Ile Trp Asn Ala Thr Lys Tyr Ala Asn Ser Cys
65 70 75 80
Cys Gln Asn Ile Asp Gln Ser Phe Pro Gly Phe His Gly Ser Glu Met
85 90 95
Trp Asn Pro Asn Thr Asp Leu Ser Glu Asp Cys Leu Tyr Leu Asn Val
100 105 110
Trp Ile Pro Ala Pro Lys Pro Lys Asn Ala Thr Val Leu Ile Trp Ile
115 120 125
Tyr Gly Gly Gly Phe Gln Thr Gly Thr Ser Ser Leu His Val Tyr Asp
130 135 140
Gly Lys Phe Leu Ala Arg Val Glu Arg Val Ile Val Val Ser Met Asn
145 150 155 160
Tyr Arg Val Gly Ala Leu Gly Phe Leu Ala Leu Pro Gly Asn Pro Glu
165 170 175
Ala Pro Gly Asn Met Gly Leu Phe Asp Gln Gln Leu Ala Leu Gln Trp
180 185 190
Val Gln Lys Asn Ile Ala Ala Phe Gly Gly Asn Pro Lys Ser Val Thr
195 200 205
Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Leu His Leu Leu
210 215 220
Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Gln Ser Gly
225 230 235 240
Ser Phe Asn Ala Pro Trp Ala Val Thr Ser Leu Tyr Glu Ala Arg Asn
245 250 255
Arg Thr Leu Asn Leu Ala Lys Leu Thr Gly Cys Ser Arg Glu Asn Glu
260 265 270
Thr Glu Ile Ile Lys Cys Leu Arg Asn Lys Asp Pro Gln Glu Ile Leu
275 280 285
Leu Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val Asn
290 295 300
Phe Gly Pro Thr Val Asp Gly Asp Phe Leu Thr Asp Met Pro Asp Ile
305 310 315 320
Leu Leu Glu Leu Gly Gln Phe Lys Lys Thr Gln Ile Leu Val Gly Val
325 330 335
Asn Lys Asp Glu Gly Thr Ala Phe Leu Val Tyr Gly Ala Pro Gly Phe
340 345 350
Ser Lys Asp Asn Asn Ser Ile Ile Thr Arg Lys Glu Phe Gln Glu Gly
355 360 365
Leu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser Ile
370 375 380
Leu Phe His Tyr Thr Asp Trp Val Asp Asp Gln Arg Pro Glu Asn Tyr
385 390 395 400
Arg Glu Ala Leu Gly Asp Val Val Gly Asp Tyr Asn Phe Ile Cys Pro
405 410 415
Ala Leu Glu Phe Thr Lys Lys Phe Ser Glu Trp Gly Asn Asn Ala Phe
420 425 430
Phe Tyr Tyr Phe Glu His Arg Ser Ser Lys Leu Pro Trp Pro Glu Trp
435 440 445
Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Leu Pro
450 455 460
Leu Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Leu Ser Arg
465 470 475 480
Ser Ile Val Lys Arg Trp Ala Asn Phe Ala Lys Tyr Gly Asn Pro Asn
485 490 495
Glu Thr Gln Asn Asn Ser Thr Ser Trp Pro Val Phe Lys Ser Thr Glu
500 505 510
Gln Lys Tyr Leu Thr Leu Asn Thr Glu Ser Thr Arg Ile Met Thr Lys
515 520 525
Leu Arg Ala Gln Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Lys Val
530 535 540
Gly Gly Gly Gly Ser Asp Ala His Lys Ser Glu Val Ala His Arg Phe
545 550 555 560
Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe
565 570 575
Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val
580 585 590
Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala
595 600 605
Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys
610 615 620
Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys
625 630 635 640
Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp
645 650 655
Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met
660 665 670
Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu
675 680 685
Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu
690 695 700
Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala
705 710 715 720
Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp
725 730 735
Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu
740 745 750
Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu
755 760 765
Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val
770 775 780
Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu
785 790 795 800
Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn
805 810 815
Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu
820 825 830
Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro
835 840 845
Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val
850 855 860
Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu
865 870 875 880
Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu
885 890 895
Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala
900 905 910
Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro
915 920 925
Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe
930 935 940
Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr
945 950 955 960
Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser
965 970 975
Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala
980 985 990
Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln
995 1000 1005
Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys
1010 1015 1020
Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu
1025 1030 1035 1040
Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe
1045 1050 1055
Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile
1060 1065 1070
Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala
1075 1080 1085
Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val
1090 1095 1100
Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu
1105 1110 1115 1120
Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu
1125 1130
<210> 3
<211> 782
<212> PRT
<213> Artificial Sequence
<400> 3
Met Lys Val Leu Ile Leu Ala Cys Leu Val Ala Leu Ala Leu Ala Glu
1 5 10 15
Asp Asp Ile Ile Ile Ala Thr Lys Asn Gly Lys Val Arg Gly Met Asn
20 25 30
Leu Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro Tyr
35 40 45
Ala Gln Pro Pro Leu Gly Arg Leu Arg Phe Lys Lys Pro Gln Ser Leu
50 55 60
Thr Lys Trp Ser Asp Ile Trp Asn Ala Thr Lys Tyr Ala Asn Ser Cys
65 70 75 80
Cys Gln Asn Ile Asp Gln Ser Phe Pro Gly Phe His Gly Ser Glu Met
85 90 95
Trp Asn Pro Asn Thr Asp Leu Ser Glu Asp Cys Leu Tyr Leu Asn Val
100 105 110
Trp Ile Pro Ala Pro Lys Pro Lys Asn Ala Thr Val Leu Ile Trp Ile
115 120 125
Tyr Gly Gly Gly Phe Gln Thr Gly Thr Ser Ser Leu His Val Tyr Asp
130 135 140
Gly Lys Phe Leu Ala Arg Val Glu Arg Val Ile Val Val Ser Met Asn
145 150 155 160
Tyr Arg Val Gly Ala Leu Gly Phe Leu Ala Leu Pro Gly Asn Pro Glu
165 170 175
Ala Pro Gly Asn Met Gly Leu Phe Asp Gln Gln Leu Ala Leu Gln Trp
180 185 190
Val Gln Lys Asn Ile Ala Ala Phe Gly Gly Asn Pro Lys Ser Val Thr
195 200 205
Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Leu His Leu Leu
210 215 220
Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Gln Ser Gly
225 230 235 240
Ser Phe Asn Ala Pro Trp Ala Val Thr Ser Leu Tyr Glu Ala Arg Asn
245 250 255
Arg Thr Leu Asn Leu Ala Lys Leu Thr Gly Cys Ser Arg Glu Asn Glu
260 265 270
Thr Glu Ile Ile Lys Cys Leu Arg Asn Lys Asp Pro Gln Glu Ile Leu
275 280 285
Leu Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val Asn
290 295 300
Phe Gly Pro Thr Val Asp Gly Asp Phe Leu Thr Asp Met Pro Asp Ile
305 310 315 320
Leu Leu Glu Leu Gly Gln Phe Lys Lys Thr Gln Ile Leu Val Gly Val
325 330 335
Asn Lys Asp Glu Gly Thr Ala Phe Leu Val Tyr Gly Ala Pro Gly Phe
340 345 350
Ser Lys Asp Asn Asn Ser Ile Ile Thr Arg Lys Glu Phe Gln Glu Gly
355 360 365
Leu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser Ile
370 375 380
Leu Phe His Tyr Thr Asp Trp Val Asp Asp Gln Arg Pro Glu Asn Tyr
385 390 395 400
Arg Glu Ala Leu Gly Asp Val Val Gly Asp Tyr Asn Phe Ile Cys Pro
405 410 415
Ala Leu Glu Phe Thr Lys Lys Phe Ser Glu Trp Gly Asn Asn Ala Phe
420 425 430
Phe Tyr Tyr Phe Glu His Arg Ser Ser Lys Leu Pro Trp Pro Glu Trp
435 440 445
Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Leu Pro
450 455 460
Leu Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Leu Ser Arg
465 470 475 480
Ser Ile Val Lys Arg Trp Ala Asn Phe Ala Lys Tyr Gly Asn Pro Asn
485 490 495
Glu Thr Gln Asn Asn Ser Thr Ser Trp Pro Val Phe Lys Ser Thr Glu
500 505 510
Gln Lys Tyr Leu Thr Leu Asn Thr Glu Ser Thr Arg Ile Met Thr Lys
515 520 525
Leu Arg Ala Gln Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Lys Val
530 535 540
Gly Gly Gly Gly Ser Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
545 550 555 560
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
565 570 575
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Glu Pro
580 585 590
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
595 600 605
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
610 615 620
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
625 630 635 640
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
645 650 655
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
660 665 670
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
675 680 685
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
690 695 700
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
705 710 715 720
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
725 730 735
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
740 745 750
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
755 760 765
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
770 775 780
<210> 4
<211> 605
<212> PRT
<213> Artificial Sequence
<400> 4
Met Lys Val Leu Ile Leu Ala Cys Leu Val Ala Leu Ala Leu Ala Ala
1 5 10 15
Cys Ser Ser Ser Pro Ser Lys His Cys Gly Gly Gly Gly Gly Ser Glu
20 25 30
Asp Asp Ile Ile Ile Ala Thr Lys Asn Gly Lys Val Arg Gly Met Asn
35 40 45
Leu Thr Val Phe Gly Gly Thr Val Thr Ala Phe Leu Gly Ile Pro Tyr
50 55 60
Ala Gln Pro Pro Leu Gly Arg Leu Arg Phe Lys Lys Pro Gln Ser Leu
65 70 75 80
Thr Lys Trp Ser Asp Ile Trp Asn Ala Thr Lys Tyr Ala Asn Ser Cys
85 90 95
Cys Gln Asn Ile Asp Gln Ser Phe Pro Gly Phe His Gly Ser Glu Met
100 105 110
Trp Asn Pro Asn Thr Asp Leu Ser Glu Asp Cys Leu Tyr Leu Asn Val
115 120 125
Trp Ile Pro Ala Pro Lys Pro Lys Asn Ala Thr Val Leu Ile Trp Ile
130 135 140
Tyr Gly Gly Gly Phe Gln Thr Gly Thr Ser Ser Leu His Val Tyr Asp
145 150 155 160
Gly Lys Phe Leu Ala Arg Val Glu Arg Val Ile Val Val Ser Met Asn
165 170 175
Tyr Arg Val Gly Ala Leu Gly Phe Leu Ala Leu Pro Gly Asn Pro Glu
180 185 190
Ala Pro Gly Asn Met Gly Leu Phe Asp Gln Gln Leu Ala Leu Gln Trp
195 200 205
Val Gln Lys Asn Ile Ala Ala Phe Gly Gly Asn Pro Lys Ser Val Thr
210 215 220
Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val Ser Leu His Leu Leu
225 230 235 240
Ser Pro Gly Ser His Ser Leu Phe Thr Arg Ala Ile Leu Gln Ser Gly
245 250 255
Ser Phe Asn Ala Pro Trp Ala Val Thr Ser Leu Tyr Glu Ala Arg Asn
260 265 270
Arg Thr Leu Asn Leu Ala Lys Leu Thr Gly Cys Ser Arg Glu Asn Glu
275 280 285
Thr Glu Ile Ile Lys Cys Leu Arg Asn Lys Asp Pro Gln Glu Ile Leu
290 295 300
Leu Asn Glu Ala Phe Val Val Pro Tyr Gly Thr Pro Leu Ser Val Asn
305 310 315 320
Phe Gly Pro Thr Val Asp Gly Asp Phe Leu Thr Asp Met Pro Asp Ile
325 330 335
Leu Leu Glu Leu Gly Gln Phe Lys Lys Thr Gln Ile Leu Val Gly Val
340 345 350
Asn Lys Asp Glu Gly Thr Ala Phe Leu Val Tyr Gly Ala Pro Gly Phe
355 360 365
Ser Lys Asp Asn Asn Ser Ile Ile Thr Arg Lys Glu Phe Gln Glu Gly
370 375 380
Leu Lys Ile Phe Phe Pro Gly Val Ser Glu Phe Gly Lys Glu Ser Ile
385 390 395 400
Leu Phe His Tyr Thr Asp Trp Val Asp Asp Gln Arg Pro Glu Asn Tyr
405 410 415
Arg Glu Ala Leu Gly Asp Val Val Gly Asp Tyr Asn Phe Ile Cys Pro
420 425 430
Ala Leu Glu Phe Thr Lys Lys Phe Ser Glu Trp Gly Asn Asn Ala Phe
435 440 445
Phe Tyr Tyr Phe Glu His Arg Ser Ser Lys Leu Pro Trp Pro Glu Trp
450 455 460
Met Gly Val Met His Gly Tyr Glu Ile Glu Phe Val Phe Gly Leu Pro
465 470 475 480
Leu Glu Arg Arg Asp Asn Tyr Thr Lys Ala Glu Glu Ile Leu Ser Arg
485 490 495
Ser Ile Val Lys Arg Trp Ala Asn Phe Ala Lys Tyr Gly Asn Pro Asn
500 505 510
Glu Thr Gln Asn Asn Ser Thr Ser Trp Pro Val Phe Lys Ser Thr Glu
515 520 525
Gln Lys Tyr Leu Thr Leu Asn Thr Glu Ser Thr Arg Ile Met Thr Lys
530 535 540
Leu Arg Ala Gln Gln Cys Arg Phe Trp Thr Ser Phe Phe Pro Lys Val
545 550 555 560
Leu Glu Met Thr Gly Asn Ile Asp Glu Ala Glu Trp Glu Trp Lys Ala
565 570 575
Gly Phe His Arg Trp Asn Asn Tyr Met Met Asp Trp Lys Asn Gln Phe
580 585 590
Asn Asp Tyr Thr Ser Lys Lys Glu Ser Cys Val Gly Leu
595 600 605
<210> 5
<211> 10
<212> PRT
<213> Artificial Sequence
<400> 5
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
1 5 10
<210> 6
<211> 5
<212> PRT
<213> Artificial Sequence
<400> 6
Gly Gly Gly Gly Ser
1 5

Claims (21)

1. A recombinant cholinesterase preparation, comprising any one of recombinant cholinesterase, a first mixed enzyme, a second mixed enzyme, recombinant cholinesterase-albumin fusion protein, recombinant cholinesterase-Fc fusion protein and recombinant transdermal short peptide-cholinesterase fusion protein, wherein the first mixed enzyme comprises transdermal short peptide and recombinant cholinesterase, and the second mixed enzyme comprises nano chip and recombinant cholinesterase.
2. The recombinant cholinesterase preparation of claim 1, wherein the recombinant cholinesterase, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein, and the recombinant transdermal short peptide-cholinesterase fusion protein are derived from any one of an animal expression system, a plant expression system, a bacterial expression system, and a yeast expression system.
3. The recombinant cholinesterase preparation of claim 2, wherein the animal expression system is selected from any one of an insect expression system and a mammalian expression system.
4. The recombinant cholinesterase preparation according to claim 3, wherein the mammalian expression system comprises any one or more of a mammary bioreactor system (milk) from mouse, rat, rabbit, goat, sheep, pig, camel, yak, water buffalo and cow.
5. The recombinant cholinesterase preparation of claim 1, further comprising an oral formulation enhancer and a transdermal absorption enhancer.
6. The recombinant cholinesterase preparation of claim 5, wherein the oral formulation promoter comprises any one or more of a protease inhibitor, an acidic pH modifier, a chelate, a surfactant, a bile salt, an aromatic alcohol, a piperazine derivative, an ionic liquid, an enterotoxin peptide derivative, a cationic polymer, an anionic polymer, a vulcanized polymer, a polymer particle, a micelle, a liposome, a drug crystal, a nanofiber and an exosome.
7. The recombinant cholinesterase preparation according to claim 5, wherein the transdermal absorption enhancer comprises one or more of surfactants, dimethyl sulfoxide derivatives, pyrrolidone derivatives, azone compounds, alcohol compounds and fat compounds.
8. The recombinant cholinesterase preparation according to claim 1, wherein the recombinant cholinesterase comprises any one of a recombinant acetylcholinesterase, a recombinant butyrylcholinesterase, an isozyme of a recombinant cholinesterase, and a variant of a recombinant cholinesterase.
9. The recombinant cholinesterase preparation according to claim 8, wherein the sequence of the recombinant butyrylcholinesterase is shown in SEQ ID No. 1.
10. The recombinant cholinesterase preparation of claim 1, wherein the short transdermal peptide is located at the N-terminus or C-terminus of the recombinant short transdermal peptide-cholinesterase fusion protein.
11. The recombinant cholinesterase preparation of claim 1, wherein the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein, and the recombinant transdermal short peptide-cholinesterase fusion protein further comprise a flexible linker peptide.
12. The recombinant cholinesterase preparation of claim 11, wherein the recombinant cholinesterase-albumin fusion protein has the sequence shown in SEQ ID No. 2.
13. The recombinant cholinesterase preparation of claim 11, wherein the recombinant cholinesterase-Fc fusion protein has the sequence shown in SEQ ID No. 3.
14. The recombinant cholinesterase preparation of claim 11, wherein the sequence of the recombinant transdermal short peptide-cholinesterase fusion protein is as shown in SEQ ID No. 4.
15. The recombinant cholinesterase preparation of claim 11, wherein the flexible linker peptide has the sequence shown in SEQ ID No.5 or SEQ ID No. 6.
16. The recombinant cholinesterase preparation according to claim 1, wherein the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein and the recombinant transdermal short peptide-cholinesterase fusion protein further comprise a tag sequence for facilitating protein purification, wherein the tag sequence is any one or more than two of His, flag, MBP, GST, HA and Myc.
17. The recombinant cholinesterase preparation of claim 1, wherein the purity of the recombinant cholinesterase, the transdermal short peptide, the recombinant cholinesterase-albumin fusion protein, the recombinant cholinesterase-Fc fusion protein, and the recombinant transdermal short peptide-cholinesterase fusion protein is 90-99%.
18. The recombinant cholinesterase preparation of claim 1, wherein the carrier of the recombinant cholinesterase preparation is any one of a sticker, a polymer scaffold and a hydrogel.
19. A method for using a recombinant cholinesterase preparation, comprising administering the recombinant cholinesterase preparation of any one of claims 1 to 18 to a human body by any one of oral administration, transdermal absorption, intradermal injection, subcutaneous injection, lymph node injection, intramuscular injection, intraperitoneal injection, and intravenous injection.
20. The method of using the recombinant cholinesterase preparation of claim 19, wherein the transdermal absorption comprises a physico-chemical method comprising any one or more of iontophoresis, electrotransfection, electroporation, ultrasound introduction, microneedle, needle-free, chemical transfection.
21. Use of a recombinant cholinesterase preparation according to any one of claims 1 to 18 for neutralizing and degrading nerve agents, neutralizing and degrading organophosphate agents, hydrolysing ghrelin and anti-wrinkle.
CN202210760804.6A 2022-06-30 2022-06-30 Recombinant cholinesterase preparation and application thereof Pending CN115177722A (en)

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TW112120546A TW202402322A (en) 2022-06-30 2023-06-01 Recombinant cholinesterase product and application thereof

Applications Claiming Priority (1)

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CN115177722A true CN115177722A (en) 2022-10-14

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