CN112675293A - Application of HrpNECb protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof - Google Patents

Application of HrpNECb protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof Download PDF

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CN112675293A
CN112675293A CN202011633933.6A CN202011633933A CN112675293A CN 112675293 A CN112675293 A CN 112675293A CN 202011633933 A CN202011633933 A CN 202011633933A CN 112675293 A CN112675293 A CN 112675293A
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protein
hrpnecb
metabolism
receptors
amino acid
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吴伯骥
吴保珍
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Kunming Rsd Technology Co ltd
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Kunming Rsd Technology Co ltd
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Abstract

The invention discloses an application of a HrpNECb protein in pharmacy for recognizing and activating a plurality of types of receptors and/or membrane proteins and signal paths thereof and causing cascade biological effects, and relates to the field of biomedicine, wherein the amino acid sequence of the HrpNECb protein is shown as SEQ ID NO. 1. The HrpNECb protein is used as a ligand protein molecule rich in a plurality of epitopes (linear and conformational) special structures, can recognize, activate and combine membrane receptors, membrane proteins, information channels and metabolic channels of various animals in a cross-boundary manner, and is a special multi-epitope ligand protein with brand new functions, brand new action mechanisms and brand new application prospects.

Description

Application of HrpNECb protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof
Technical Field
The invention relates to the field of biomedicine, in particular to application of HrpNECb protein in pharmacy for recognizing and activating multiple receptors and/or membrane proteins and signal paths thereof and causing cascade biological effects.
Background
Molecular biology is the science of studying life phenomena at the molecular level, elucidating the nature of various life phenomena by studying the structure, function and metabolism of biological macromolecules, and its content covers the entire course of life. DNA, RNA and proteins are three important biological macromolecules that are the molecular basis for life phenomena. The genome determines what life is, the proteome determines what life can do, and the metabolome determines what life actually happens. Modern life science, biotechnology and medical biotechnology, especially proteomics and metabonomics, have been developed rapidly, the ideas of understanding, diagnosing, preventing and controlling, treating and recovering diseases are updated, new understanding and new ways of novel efficient and safe medicines are created, the development of modern medicine enters a brand-new stage, and a wide application prospect is opened up.
The receptor theory of modern life science is one of the basic theories of pharmacodynamics, and is an important basis for explaining the controllable physiological process and pathological process of life, the pharmacological action mechanism of drugs and the structural effect relationship of drug molecules from the molecular level. The ligand is a signal substance which has no other direct functions except for recognizing, binding and activating the receptor, cannot participate in metabolism to produce useful products, does not directly induce any cellular activity, and has no characteristics of enzyme.
The signal path (cell communication) is a communication mechanism for transmitting and receiving information in cells or cells of a multicellular organism with high accuracy and high efficiency, and a rapid cell physiological and biochemical reaction is caused by amplification or gene activity is started, and then a series of cell physiological and biochemical activities are generated to coordinate the activities of various tissues, so that the unified whole life can comprehensively react to changeable internal and external environments, and the coordinated joint mechanism for growth, development, defense and metabolism is built by systems, tissues, organs, cells, subcells, molecules and sub-molecules of a living organism.
The receptor is a functional protein for mediating cell signal transduction, can recognize certain trace substances in the surrounding environment (intracellular and extracellular environments), is recognized and combined with the trace substances, is activated, and triggers subsequent physiological and biochemical reactions through a signal amplification system. Receptors are biological macromolecules composed of cell membranes and intracellular proteins, nucleic acids, lipids, polysaccharides, and the like. Receptors are a broad concept in cell biology, meaning any biological macromolecule capable of binding to hormones, neurotransmitters, drugs or signaling molecules both inside and outside the cell and causing a change in cell function, in which case the signaling molecule is called a ligand. There are hundreds of different signaling molecules in multicellular organisms that transmit information between and within cells, including proteins, amino acid derivatives, nucleotides, cholesterol, fatty acid derivatives, and soluble gas molecules. Receptors present on the plasma membrane of cells are called membrane receptors, the chemical nature of which is for the most part sugar mosaics; receptors located in the cytosol and nucleus, called intracellular receptors, are all DNA binding proteins.
The ligand is a signal substance which has no other direct functions except for recognizing, binding and activating the receptor, cannot participate in metabolism to produce useful products, does not directly induce any cellular activity, and has no characteristics of enzyme.
The combination of ligand and receptor is the process of intermolecular recognition and activation, which depends on the actions of ion coordination bond, hydrogen bond, pi-pi stacking action, electrostatic action, hydrophobic action, van der waals force, etc. with the complementation and the interaction degree of the two molecular spatial structures, the distance between the interacting groups is shortened and the acting force is greatly increased, so the interactivity and the complementarity of the ligand and the receptor molecular spatial structures are the main factors of specific combination, i.e. the epitope concept adopted by the invention. The same ligand may correspond to two or more different receptors, and binding of the same ligand to different types of receptors results in different cellular responses. After the ligand is combined with the receptor, related series of physiological activities are initiated, no matter whether the ligand is endogenous or exogenous, after the ligand is combined with the receptor, the ligand and the receptor form a ligand-receptor combination surface or a compound, so that information is transmitted, and through conduction and transduction, rapid cell physiological and biochemical reactions are initiated through amplification, or gene activities are initiated, a series of cascade reactions occur later to coordinate the activities of various tissues, organs and cells, so that the unified whole life makes comprehensive reactions to changeable internal and external environments.
In 2008, Leader et al first proposed ideas classified according to protein pharmacological actions and classified protein drugs into four major classes: protein medicine for treating diseases with the enzyme activity and regulating activity of protein; ② protein drugs with special targeting activity; ③ recombinant protein vaccines; and fourthly, the recombinant protein medicine for diagnosis. Of these, the first and second classes are mainly used in basic protein therapy, and the third and fourth classes emphasize the use of proteins in vaccines and diagnostic applications. After a century of exploration and zigzag development, protein drugs have matured one step by one step and have a great significance in pharmaceutical industry and clinical application. They have important effects on almost all disease fields such as tumors, infections, autoimmune diseases, metabolic genetic diseases, various senile diseases and degenerative diseases, and are becoming important therapeutic, prophylactic and diagnostic drugs in the 21 st century. The wide application of biotechnology with recombinant DNA technology as the core is expected to give protein drugs a wider development space in the next 30 years: the recombinant protein drug will gradually replace the non-recombinant protein; the restructuring and in-vitro and in-vivo modification become conventional; products expressed with mammalian cell systems will predominate; the non-injectable route of administration of protein drugs is receiving increasing attention; biomimic drugs and biosimilar drugs will be most likely. (Zhuxun, functional classification and development trend of protein drugs, volume 5, No.1 of 2.2010, Chinese medicinal biotechnology, Chin Med Biotechnol, February 2010, Vol.5, No. 1).
It has been shown that recognition binding of ligand to the receptor is determined by key amino acid residues of linear or conformational ligand binding epitopes, e.g., phenylalanine (Phe 82), isoleucine (Ile 83) and valine (Val 85) of the FIGV linear ligand binding epitope of the polypeptide 82-85 of boFc γ 2R are key amino acid residues for recognition of binding to the bovine IgG2 receptor, and further, for example, threonine (Thr 142), asparagine (Asn 143), leucine (Leu 144), glycine (Gly 148) and isoleucine (Ile 149) of the TNLSHNGI linear ligand binding epitope of the polypeptide 142-149 of boFc γ RI are key amino acid residues for recognition of binding to the bovine IgG1 receptor; for another example, alanine (Ala 98), glutamic acid (Gln 99), valine (Val 101), valine (Val 102) and asparagine (Asn 103) of the AQRVVN linear ligand binding epitope at positions 98-103 of boFc γ rliii are key amino acid residues for recognition of binding to the bovine IgG1 receptor.
Harpin is a protein encoded by genes in the gram-negative bacterial "hypersensitive response and pathogenicity (hrp)" gene cluster that is similar in nature and function, rich in glycine, free of cystine, sensitive to protease, thermostable, and capable of causing hypersensitive responses in non-host plants. Allergic reactions (HR) are manifested by rapid, local atrophy and necrosis of infected tissues of non-host plants, thereby limiting the spread of pathogenic bacteria and inducing systemic resistance, which is a common manifestation and effective way for plants to resist pathogen infection. After research for over thirty years, these encoded proteins have been acknowledged by biologists, plant pathologists and application researchers in the field, Harpin hypersensitive proteins belonging to resistance-inducing proteins for inducing plant systemic resistance have become the contents reported by gene hrpNEccs and its expression product hrpNEccs protein which can safely induce plants to generate disease resistance, insect repellency, stress resistance, promote plant growth and development and improve yield in the field of plant protection, for example, patent publication No. CN1687420, entitled gene hrpNEccs for encoding plant multifunctional activity and broad-spectrum resistance cell signal factors, and its expression product hrpNEccs protein.
The HrpNECb protein (Genebank ID: ABD22989.1) is an expression product of hrpNECb gene (gene registration number: bankit698770AY939927), which is composed of 370 amino acid residues, a non-enzymatic protein having a primary, secondary and tertiary structure without quaternary structure, free of cystine and cysteine, rich in glycine, isoelectric point pI 5.43, molecular weight Mw 36636.21Da, GenBank ID: ABD 22989.1. The conserved domain of the HrpNECb protein consists of 201 amino acids and is positioned at the C-terminal of the protein, 170-370; the alpha-helical structures 44-64, 110-; beta-sheet structures 10-15, 255-256; do-structures 1-11, 13-43, 67-95, 99-139, 157-174, 197-216, 340-341, 364-370.
The structural domain is a region with a specific structure and an independent function in biological macromolecules, in particular to an independent stable structural region formed by combining different secondary structures and super-secondary structures in protein, the structural domain is also a functional unit of the protein, and in multi-structural-domain protein, different structural domains are often associated with different functions; the secondary and supersecondary structures of proteins are maintained mainly by hydrogen bonds, and include alpha helices, beta sheets, beta turns, random coils, do-structures, etc., alpha helices being repetitive structures with phi and psi near-57 deg. and-47 deg. for each alpha-carbon in the helix, respectively. Each coil of helix occupies 3.6 amino acid residues, the residues rise by 0.54nm along the direction of the helical axis, each residue rotates by 100 degrees around the axis and rises by 0.15nm along the axis, hydrogen bonds are formed between adjacent coils, and the orientation of the hydrogen bonds is almost parallel to the helical axis; beta sheet: the beta-folded sheets are laterally gathered by two or more extended polypeptide chains (or a plurality of peptide segments of one polypeptide chain), and form a zigzag sheet structure through regular hydrogen bonds between N-H and C ═ O on main chains of adjacent peptide chains; the do-structure is a structural region of inherently disordered proteins (IDPs for short), has a wide allosteric effect, serves as a flexible connection region, stores various conformations and motion states, and is widely involved in and regulates transcription, translation, cell division, protein aggregation and cell signal transduction with high repeatability, chargeability, easiness in combination, spatial superiority and high coordination, and particularly participates in a self-assembly regulation process.
The HrpNECb protein is a multidomain protein, forms a special structure of a plurality of linear and conformational epitopes, and different domains are often associated with different functions, thereby determining the application of the protein in the pharmacy for recognizing and activating various types of receptors, membrane proteins, signal paths and metabolic paths and causing multifunctional cascade biological effects. However, there is no report on this.
Disclosure of Invention
The invention aims to: in view of the problems, the invention provides the application of the HrpNECb protein in the pharmacy for recognizing and activating various types of receptors, membrane proteins and signal paths thereof of animals and causing cascade biological effects.
The technical scheme adopted by the invention is as follows:
the application of the HrpNECb protein in the pharmacy for recognizing and activating various types of receptors and/or membrane proteins of animals and signal paths thereof and causing cascade biological effects, wherein the amino acid sequence of the HrpNECb protein is shown as SEQ ID NO. 1.
The HrpNECb protein is rich in a plurality of linear and conformational epitope structures, and refers to a functional group consisting of amino acid residues capable of being recognized and combined with cell membrane receptors, membrane proteins and the like, wherein the functional group consists of the following amino acid residues which can be recognized, combined and activated with the receptors and are rich in proton-donating amino acid residues or proton-accepting amino acid residues; further, containing one to more hydrophobic non-polar amino acid residues, containing one to more acidic positively charged, basic negatively charged amino acid residues, containing one to more amido polar uncharged amino acid residues, containing one to more polar uncharged amino acid residues; further, amino acid residues that are proton-rich (excluding methionine residues) or proton-accepting (including methionine residues): glutamic acid, asparaginic acid, lysine, histidine, methionine, serine, threonine, tyrosine and arginine, which can be identified and activated with corresponding amino acid residues of the multi-type receptor protein in a hydrogen bond mode to form a binding surface or a compound; further, hydrophobic apolar amino acid residues: valine, leucine, isoleucine, alanine and phenylalanine can form a tight combination surface or compound with various types of receptors by nonpolar hydrophobic and van der waals force; acidic positively charged, basic negatively charged amino acid residues: the aspartyl acid, the glutamic acid, the lysine and the arginine can form a tight combination surface or a compound with various types of receptors through ionic bonds; amide group polar uncharged amino acid residue: the amide groups of asparagine and glutamine can form a bonding surface or a compound with a cysteine recognition region Pam3 CSK4 of a receptor through stronger hydrogen bonds; polar uncharged amino acid residues: serine forms a tight joint surface or compound with a multi-type receptor through a polar hydrogen bond with strong hydrogen bond; further, the full sequence of the HrpNEcb protein has 370 amino acid residues, wherein the critical amino acid residues are 226: 94 hydrophobic nonpolar amino acid residues, 41 polar uncharged amino acid residues, 44 amido amino acid residues, 47 acidic positively charged and basic negatively charged amino acid residues, and the key amino acid accounts for 61 percent of the total sequence; the conserved structural region of the HrpNECb protein has 200 amino acid residues: wherein the key amino acid residues comprise 138, 51 hydrophobic nonpolar amino acid residues, 19 polar uncharged amino acid residues, 28 amido amino acid residues, 40 acidic positively charged and basic negatively charged amino acid residues, and the key amino acid accounts for 69 percent of the conserved domain; the alpha-helical region of the HrpNECb protein has 71 amino acid residues, 52 key amino acid residues: 27 hydrophobic nonpolar amino acid residues, 7 polar uncharged amino acid residues, 8 amido amino acid residues, 10 acidic positively charged and basic negatively charged amino acid residues, and the key amino acid accounts for 73 percent of the alpha-helical structure; furthermore, hypersensitive proteins such as HrpNECc, HrpNECa, HrpNECb, HrpNECh, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNECa, HrpNSam, HrpNBag, HrpNPas and HrpNECnt are screened, cloned and prepared from Erwinia and Pseudomonas, and analysis of the molecular structures according to bioinformatics shows that the hypersensitive proteins have similar structural characteristics, structural evolution trends and structures similar to those of the multi-epitope HrpNECb ligand protein: contains one or more hydrophobic non-polar amino acid residues, contains one or more polar uncharged amino acid residues, contains one or more amide polar uncharged amino acid residues, contains one or more acidic positively charged and basic negatively charged amino acid residues; further, hydrophobic apolar amino acid residues: valine, leucine, isoleucine, alanine, phenylalanine, methionine, polar uncharged amino acid residues: serine, amido polar uncharged amino acid residues: asparagine, glutamine, acidic positively charged, basic negatively charged amino acid residues: aspartyl acid, glutamic acid, lysine, histidine, arginine; furthermore, the above-mentioned key amino acid residues account for 62.3% -73.7% of the total sequence of these protein molecules, 61% -74% of the conserved domain, and 66.2% -79% of the alpha-helical structure; further, the amino acid residues (generally referred to as key amino acid residues) of the HrpNEcb protein, not limited to these amino acid residues, can achieve complementarity, interactivity and specific recognition, activation and combination of the spatial structure and the electrical property of the ligand and receptor molecules through hydrogen bonds, ionic bonds, hydrophobicity, non-polarity, polarity and van der waals force, form a tight binding surface or complex with multiple types of receptors, can cause the change of the conformation, energy, electrical property and information of the receptor molecules, and can amplify and express a series of biological effects through signal conduction and transduction.
The multifunctional cascade biological effect refers to the obvious expression difference of functional gene groups related to three levels of cell components, molecular functions and biological processes of different organs and tissues, and comprises cell components (including cells, cell knots, cell parts, extracellular matrixes, extracellular matrix components, extracellular regions, extracellular region parts, macromolecular complexes, membranes, membrane parts, membrane closed cavities, organelles, parts of organelles, supramolecular fibers, synapses, synapse parts, antioxidant activity and the like), molecular functions (including binding, catalytic activity, activity of chemoattractants, activity of chemorepellents, activity of electron carriers, activity of metal chaperones, molecular function supervision mechanisms, activity molecular sensors, activity of nucleic acid binding transcription factors, activity of signal sensors, activity of structural molecules, binding of transcription factor activity proteins, protein binding, trafficking activity, etc.), biological processes (including behavior, bioadhesion, bioregulation, cell aggregation, cell death, cellular component organization or biogenesis, cellular processes, detoxification, processes of development, growth, processes of the immune system, localization, motility, metabolic processes, multiple biological processes, processes of multiple cellular organisms, negative regulation of biological processes, positive regulation of biological processes, presynaptic processes involving synaptic transmission, regulation of biological processes, reproduction, reproductive processes, stimulatory responses, rhythmic processes, signaling, single biological processes, etc.) are significantly altered.
Preferably, the receptor proteins include LRRC 1515-leucine repeat membrane protein receptor, HLA-a major histocompatibility complex, class I, class a receptor, LGALS3BP galactose 3 binding protein (receptor), LAMP2 lysosomal associated membrane protein 2 receptor, GNB 2G guanine nucleotide binding protein subunit Beta 2 receptor.
Preferably, the membrane proteins include DSG4 desmoglein, ANXA4 annexin a4, CAPRIN1 cyclin, 1UTRN dystrophin protein, pinin desmoplanin, VAMP-associated protein A, VCL focal adhesion, Ezrin epithelial-type cadherin, PKP3 platelet avidin 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD 2N acetylated alpha linked acidic dipeptidase 2.
Preferably, the signaling pathway comprises one or more of the hsa04152: AMPK signaling pathway, hsa03460: fanconi anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: paque signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, and hsa04390: river horse signaling pathway.
Preferably, the signaling pathway further comprises a metabolic signaling pathway comprising an antiviral, antibacterial, anti-foreign, anti-inflammatory associated metabolic pathway: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa01130 biosynthesis of antibiotics, hsa05131 shigellosis, hsa04612 antigen processing and presentation, hsa05130 pathogenic E.coli infection, hsa05100 bacterial invasion of epithelial cells, hsa05132 Salmonella infection, hsa05169 Barr virus infection, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05166 HTLV-I infection, hsa 05105164 influenza A, hsa05134 legionnaire's disease, hsa05160 hepatitis C, hsa05162 measles, hsa05133 whooping cough, hsa05322 systemic lupus erythematosus, 670 hsa 0404670 leukocyte epithelial migration, hsa 46 Ammi disease, hsa 42 trypanosomiasis, and hsa05200 leukocyte epithelial migration in cancer; including important neurological metabolic pathways: hsa05012 for Parkinson's disease, hsa05016 for Huntington's chorea, hsa05010 for Alzheimer's disease; including nucleic acid, protein, amino acid, sugar, fat metabolism related pathways: hsa03420: nucleotide excision repair, hsa00970: aminoacyl biosynthesis, hsa03430: mismatch repair, hsa01210: 2-oxocarboxylic acid metabolism, hsa03440: homologous recombination, hsa04360: axonal guidance, hsa00051: fructose and mannose metabolism, hsa00565: ether lipid metabolism, hsa00510: N-polysaccharide biosynthesis and hsa04110: cell cycle, hsa03030: DNA replication, hsa03013: RNA transport, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome, hsa04141: endoplasmin processing, hsa04810: regulation of the actin skeleton, hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa04932: nonalcoholic fatty liver (NAFLD), hsa00020: citric acid cycle, hsa00564: glycerophospholipid, hsa 0008: cholesterol biosynthesis, hsa 05015: biogenesis monitored, hsp metabolism, hsp 05020: eukaryotic mRNA metabolism, and hsp 05020: metabolic pathways mediated by human albumin, Hsa05205 proteoglycan in cancer, and Hsa05206 small molecule RNA in cancer; including cell junctions, nerve junctions, blood vessels, endocrine, reproductive metabolic pathways: hsa04723: retrograde neural signaling, hsa04726: serotonin-activated synapse, hsa00900: terpenoid biosynthetic backbone, hsa04520: adherent knot, hsa05032: morphine addiction and hsa04510: focal adhesion, hsa04724: glutamatergic synapse, hsa04530: tight junction, hsa00830: retinol metabolism, hsa04114: oocyte meiosis, hsa04728: dopaminergic neural synapse, hsa00100: steroid biosynthesis, hsa04261: adrenergic signaling of cardiomyocytes, hsa04727: neuronal synapse, hsa04725: cholinergic synapse, hsa04540: gap junction, hsa04971: gastric acid secretion, hsa04713: diurnal entrainment, hsa04931: insulin resistance 931.
Preferably, the cascade biological effect includes functional pathways such as Cellular Processes (Cellular Processes), Environmental Information Processing (Environmental Information Processing), Genetic Information Processing (Genetic Information Processing), Metabolism (Metabolism), and biological Systems (organic Systems); further, the Cellular process (Cellular Processes): the multiple differential expression genes induced by the HrpNECb protein participate in cell processes such as transport and catabolism, cell population, cell activity, cell growth and death and the like; environmental Information Processing (Environmental Information Processing) wherein multiple differentially expressed genes induced by the HrpNECb protein participate in the Environmental Information Processing processes of signal molecules, interaction, signal transduction, membrane transportation and the like; genetic Information Processing (Genetic Information Processing) multiple differentially expressed genes induced by HrpNECb protein participate in biological processes such as translation, replication and repair, folding, classification and degradation; metabolism (Metabolism), wherein a plurality of differentially expressed genes induced by HrpNECb protein participate in the metabolic processes such as biodegradation and Metabolism, nucleotide Metabolism, Metabolism of other amino acids, metabolic accessory factors and vitamins, lipid Metabolism, biosynthesis and Metabolism of sugar, global and overview maps, energy Metabolism, carbohydrate Metabolism and amino acid Metabolism; multiple differential expression genes induced by HrpNECb protein participate in biological processes of sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, development and circulation system, etc.
Preferably, the formulation of the product or medicament for use in the pharmaceutical is a liquid, powder, tablet or capsule.
The pharmaceutical uses also include the formulation and administration of pharmaceutically therapeutically active compounds (HrpNEcb protein preparations and/or drugs) for HrpNEcb protein and derivatives thereof, typically in unit dosage forms or multiple dosage forms, each containing a predetermined amount of the therapeutically active compound, in association with a desired pharmaceutical carrier, vehicle or excipient sufficient to produce the desired therapeutic effect. Examples of unit dosage forms include ampoules and syringes and individually packaged tablets or capsules. The unit dosage forms may be administered in portions or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container that will be administered in separate unit dosage forms. Examples of multiple dosage forms include vials, tablets or capsules or gallon bottles. Thus, a multiple dosage form is a plurality of unit doses that are not segregated into packages. Dosage forms or compositions may be prepared containing from 0.001% to 100% of the active ingredient, with the remainder being composed of a non-toxic carrier, and for oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules, with pharmaceutically acceptable excipients such as binding agents (including, but not limited to, pregelatinized corn starch, polyvinylpyrrolidone, or propylmethylcellulose) by conventional methods; fillers (including, but not limited to, lactose, microcrystalline cellulose); lubricants (including, but not limited to, magnesium stearate, talc, or silica); disintegrants (including, but not limited to, potato starch or sodium starch glycolate); or wetting agents (including, but not limited to, sodium lauryl sulfate). The tablets may be coated by methods well known in the art. Pharmaceutical compositions may also be in liquid form, including, but not limited to, solutions, syrups or suspensions, or may be presented as a pharmaceutical product for reconstitution with water or other suitable vehicle before use. Such liquid formulations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (including, but not limited to, sorbitol syrup, cellulose derivatives or edible fats); emulsifying agents (including, but not limited to, lecithin or acacia); non-aqueous vehicles (including, but not limited to, almond oil, oily esters, or fractionated vegetable oils); and preservatives (including, but not limited to, methyl or propyl parabens or sorbic acid). Formulations suitable for rectal administration may be presented as unit dose suppositories. These can be prepared by mixing the HrpNEcb protein active compound with one or more solid carriers, such as cocoa butter, and then shaping the resulting mixture. Formulations suitable for topical application to the skin or eye include, but are not limited to, chondromains, creams, lotions, pastes, gels, sprays, aerosols, and oils. Exemplary carriers include, but are not limited to, petrolatum, lanolin, polyethylene glycols, alcohols, and combinations of two or more thereof. The topical formulation may also contain 0.001% to 15%, 20%, 25% by weight of a thickening agent selected from the group including, but not limited to, hydroxypropylmethyl cellulose, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, poly/hydroxyalkyl (meth) acrylates or poly (meth) acrylamides. The topical formulations are typically applied by instillation or as a chondrogenic agent applied to the conjunctival capsules. It can also be used to irrigate or lubricate the eye, facial sinuses and external auditory canal. It can also be injected into the anterior chamber of the eye and elsewhere. Topical formulations in the liquid state may also be present in the form of a tape or contact lens in a hydrophilic three-dimensional polymeric matrix from which the active ingredient is released. For formulations suitable for buccal (sublingual) administration, there are included, but are not limited to, lozenges comprising the active compound in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base including, but not limited to, gelatin and glycerin or sucrose and acacia. Pharmaceutical compositions of the ligand isoforms may be formulated for parenteral administration by injection, including, but not limited to, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with added additives. The compositions may be presented as suspensions, solutions or emulsions in oily or aqueous vehicles, and may include, but are not limited to, formulating agents such as suspending, stabilizing and stabilizing agents, alternatively the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvent, before use. Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for an extended period of time. Such patches suitably contain the active compound as an aqueous solution of the active compound, optionally fluke. Formulations suitable for transdermal administration may be delivered by iontophoresis and take the form of an optionally flushed aqueous solution of the active compound.
Preferably, the preparation or medicament is prepared predominantly from depolymerisation of the activated HrpNEcb protein.
The preparation method for producing the HrpNECb protein purification by depolymerizing and activating the high-polymerization-state HrpNECb protein comprises the following steps:
1. pretreatment: with glucose Na2HPO4-KH2PO4The buffer solution adjusts the volume concentration range of the high polymeric HrpNECb multi-epitope protein prepared by collection and fermentation to be 0-30%, and the concentration is preferably 0-5%; preferably at a concentration of 30% -20%; preferably at a concentration of 5% -10%; preferably at a concentration of 20% -15%; most preferably 10% -15% concentration; at normal temperature (20-30 ℃), pre-treating glucose Na2HPO4-KH2PO4Buffer solution, pH range is 1-14, glucose concentration range is 0-2500mmol, buffer system pH, preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-6; most preferably at a pH of 5-5.5. The concentration of glucose is 0-100 mmol; preferably at a concentration of 100-; preferably a concentration of 2500-; preferably at a concentration of 1000-; most preferably at a concentration of 200-300 mmol. The treatment time is 0-24h, preferably 0-2 h; the preferable time is 24-15 h; preferably for 2-4 h; preferably for a time of 15-6 h; most preferably for a period of 4-6 hours.
2. Depolymerizing the activated HrpNEcb high polymeric multiple epitope protein: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution, wherein the ultrahigh pressure range is 1000-3000MPa, preferably 3000 MPa; preferably 1500 Mpa; preferably 2500 Mpa; preferably 2000 Mpa; most preferably 2000-.
3. And (3) post-treatment: after the ultrahigh pressure depolymerization and activation operation is finished, standing for 0-24h at 35-38 ℃, preferably for 0-1 h; preferably for 24-10 h; preferably for a period of 1-2 hours; preferably for 10-4 hours; most preferably for 2-4 hours, and then collecting the depolymerized activated HrpNEcb multi-epitope protein molecules.
4. And (3) purifying the high-aggregation HrpNECb multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, and performing protein purification according to a method suggested by an NI-NTA affinity chromatography gel manufacturer to complete the purification preparation of the depolymerized and activated multi-epitope protein HrpNECb original drug.
The route of use of the HrpNEcb protein preparation of the present invention, which recognizes various types of receptors, membrane proteins and their signaling pathways that activate animals and induce multifunctional cascade biological effects, can be administered by any route known to those skilled in the art, including internal, external, oral, injection, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, nasal, oral, rectal, topical, buccal and transdermal administration or any route; the HrpNEcb multiple epitope ligand protein may be administered by any convenient route, such as by perfusion or rapid perfusion, absorption through epithelial or cutaneous mucosal linings (e.g., oral mucosa, nasal mucosa, gastric mucosa, rectal and intestinal mucosa, etc.), and may be administered sequentially, intermittently, or in the same composition with other bioactive agents; depending on the treatment site, administration may be local, topical or systemic. Topical application to the area in need of treatment can be, but is not limited to, topical infusion, topical application, by immersion, by injection, by catheter, by suppository; administration can also include controlled release systems, including controlled release formulations and devices controlled release, such as by pumps; the most suitable route in any given case will depend on the nature and severity of the disease or condition being treated and the nature of the particular composition used. A variety of delivery systems are known and can be used to administer a variety of epitope ligand proteins, which can be encapsulated in liposomes, microparticles, microcapsules. Pharmaceutical compositions of the various epitope ligand proteins can be prepared, and typically, pharmaceutically acceptable compositions will be prepared for use in patients following approval by regulatory agencies or according to generally recognized pharmacopoeias.
The multifunctional cascade biological effect and the diversity function caused by the HrpNECb multi-epitope ligand protein which can identify and activate various receptors, membrane proteins and signal channels thereof of animals and induce the multifunctional cascade biological effect widely relate to the diagnosis, prevention, treatment, rehabilitation and the application of food, apotype, makeup, mechanical and health word products or medicines for various systems, tissues, organs and cells related diseases and conditions.
The invention relates to a preparation or a medicament for recognizing and activating multiple types of receptors, membrane proteins and signal channels thereof of animals and inducing multi-functional cascade biological effects, which is applied to the pharmacy, and the application of the preparation or the medicament in diagnosing, preventing, treating or rehabilitating diseases and conditions of the nervous system, the digestive system, the motor system, the circulatory system, the respiratory system, the endocrine system, the immune system, the urinary system and the reproductive system comprises the following components:
use of a preparation or medicament of a ligand protein of said plurality of epitopes of the invention for the diagnosis, or and prevention, or and treatment, or and rehabilitation of diseases of nervous linkages, dementia, parkinson's disease, central nervous system diseases, neuromuscular diseases, epilepsy, headache and neuralgia, peripheral neuropathies, attention deficit hyperactivity disorder and tic disorders, insomnia, depression, anxiety disorders, bipolar disorder, psychotic disorders, neurodermatitis-associated nervous system diseases and conditions;
the application of the product or the medicine of the ligand protein of the multiple epitopes in diagnosing, or preventing, or treating, or recovering the abnormal secretion of gastric acid, gastrointestinal neurosis, gastrointestinal motility, gastrointestinal mucositis, liver diseases and digestive system diseases and conditions related to microecological disorder;
use of a preparation or medicament of a ligand protein of said plurality of epitopes of the invention for the diagnosis, or and prevention, or and treatment, or and rehabilitation of arthritis, muscle spasms, pain, muscular dystrophy, muscle nerve injury, dehydration-related motor system diseases and conditions;
the use of a preparation or a medicament of a ligand protein of the plurality of epitopes of the invention for the diagnosis, or and prevention, or and treatment, or and rehabilitation of heart failure, arrhythmia, hypertension, myocardial injury, ischemia, angina pectoris, hyperlipidemia, calcium channel blockade, vasospasm, blood coagulation, abnormal hemogram, diseases and conditions of the circulatory system associated with myocardial infarction;
the use of a preparation or medicament of a ligand protein of the plurality of epitopes of the invention in the diagnosis, or and prevention, or and treatment, or and rehabilitation of asthma, chronic obstructive pulmonary disease, bronchiectasis, allergen immunity, allergy, pneumonia, acute or chronic bronchitis, bronchial asthma, gastroesophageal reflux, rhinitis-related respiratory diseases and conditions;
the application of the products or medicines of the ligand proteins of the epitopes in the invention in diagnosis, or prevention, or treatment, or rehabilitation of diabetes, thyroid diseases, pituitary diseases, hyperprolactinemia, diabetes insipidus, adrenal diseases, parathyroid diseases, diseases and conditions of endocrine systems related to osteoporosis;
the invention relates to the application of the products or medicines of ligand proteins of a plurality of epitopes in diagnosing, or preventing, or treating, or recovering immune hypofunction, rheumatoid arthritis and lupus erythematosus related immune system diseases and conditions;
the product or the medicine of the ligand protein with multiple epitopes is applied to diagnosing, or preventing, or treating, or rehabilitating the urogenital system diseases and conditions such as nephrotic syndrome, interstitial nephritis, renal failure, urinary infection, genital system infection, pyelonephritis, cystitis, prostatitis, urethritis, epididymitis or orchitis, prostatic hyperplasia, overactive bladder, sexual dysfunction, various andrological and gynecological infectious inflammations and functional diseases.
The product or the medicament of the ligand protein of the multiple epitopes is applied to diagnosis, or and prevention, or and treatment, or recovery of whole body skin cell nutrition, activation, regeneration, repair, removal, fine and smooth, ultraviolet melanin deposition, eczema, roughness, cracks, dark lines, dry skin, hard skin, erythema, allergy, neurodermatitis, injury, whelk, pimples, scars, dark skin, mites, oily skin, inflammatory dermatosis, autoimmune dermatosis, pigmentary dermatosis, skin atrophy, thinning, dryness, pigmentation, wrinkle hyperplasia, epidermal keratosis, xeroderma, contact dermatitis, skin aging resistance, skin function improvement, whitening and freckle removal, and prevention and treatment of skin disease and related skin system diseases and conditions.
The invention relates to a method for preparing HrpNECb multi-epitope ligand protein which can identify and activate various receptors, membrane proteins and signal channels thereof of animals and induce multifunctional cascade biological effect, comprising the following steps:
1. the preparation of the HrpNECb multi-epitope ligand protein can separate and purify the HrpNECb protein from secretory protein of EcbCSL101 strain of Pectobacterium betavacuorum collected from Sword Jichang, Sichuan Deyang, 37025, China, and can adopt a conventional protein separation and purification method according to the specific molecular weight of the HrpNECb protein, and then collect a depolymerized and activated HrpNECb purified protein product through the established depolymerization and activation technology of the HrpNECb multi-epitope protein molecules with high polymerization state.
2. The preparation of the HrpNECb multi-epitope ligand protein can also adopt the registered engineering bacteria of EcbCSL101HrpNECb gene to prepare and collect depolymerized activated HrpNECb protein by fermentation and purification:
1) and (3) engineering bacteria fermentation preparation of the HrpNECb protein: engineering bacteria (E.coli) of genes (including but not limited to natural genes, chemically synthesized genes, transgenic genetic recombinant genes and similar genes of biological samples and gene modifications thereof) for encoding HrpNECb proteins, wherein the production line of related proteins is specially modified derivative bacteria JY-01(DE3) of K-12 original bacteria, IPTG (Isopropyl thiogalactoside, Isopropypyl beta-D-thiogalactoside) (final concentration of 1mMol) is added when the bacteria are cultured in LB liquid culture medium (containing 50 micrograms per liter of kanamycin) under the condition of certain temperature until OD600 is 0.7, and bacteria are collected by centrifugation after the bacteria are continuously cultured. The expression product of the HrpNECb protein was analyzed by 10% SDS-PAGE polyacrylamide gel electrophoresis, and an 36.64kda band was shown in the sample lane of the electrophoresis gel plate, which is the expression product of the gene hrpNECb protein;
wherein the fermentation medium is Na2HPO4-KH2PO4A buffer system, the pH of the buffer system is in the range of 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-7; most preferablyPreferably pH 6.5-5.5;
the fermentation temperature is 0-60 ℃. Preferably at a temperature of 0-20 ℃; preferably at a temperature of 20-35 ℃; preferably at a temperature of 60-50 ℃; preferably at a temperature of 50-45 ℃; most preferably at a temperature of 37-38 ℃;
the glucose concentration range of the fermentation proliferation liquid culture medium is 3.00-0.00%; preferably 3.00% -1.00%; preferably 0.00% -0.01%; preferably 1.00% -0.3%; most preferably 0.01% -0.05%; most preferably 0.1% -0.05%;
the glucose concentration range of the fermentation induction liquid culture medium is 3.00-0.00%; preferably 3.00% -1.00%; preferably 1.00% -0.3%; preferably 0.3% -0.1%; preferably 0.1% -0.05%; most preferably 0.05% -0.00%;
the lactose concentration range of the fermentation induction liquid culture medium is 10.00-0.00%; preferably 10.00% -1.00%; preferably 0.00% -0.1%; preferably 1.00% -0.6%; preferably 0.1% -0.3%; most preferably 0.5% -0.4%;
the fermentation induction liquid culture time range is 0-24 h; preferably for a time of 0-2 h; preferably for 24-15 h; preferably for 2-6 h; preferably for 15-10 h; most preferably for a period of 7-9 hours.
2) The engineering bacteria production system is post-treatment after the production and fermentation of a plurality of epitope proteins are finished: sterilizing: the fermentation liquor is sterilized at 80 ℃ for 30 minutes, and is rapidly cooled to below 30 ℃; cleaning: with glucose Na2HPO4-KH2PO4Buffer solution (pH range is 1-14, glucose concentration range is 0-2500mmol, buffer system pH is 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-6; most preferably pH 5-5.5. glucose concentration is 0-100 mmol; preferably concentration is 100-200 mmol; preferably concentration is 2500-1000 mmol; preferably concentration is 1000-300 mmol; most preferably concentration is 200-300 mmol), engineering bacteria are washed five to eight times in a continuous flow of a butterfly centrifuge; engineering bacteria are crushed and cell walls are cleared, Na with pH 5-5.5 and glucose concentration of 200-300mmol is used for clearing cell walls2HPO4-KH2PO4Diluting the thallus with buffer solution, and regulating the fresh weight of the thallus to 20-30% of the diluted solutionIntroducing the strain into a high-pressure crusher, continuously crushing the engineering bacteria by using the pressure of 800-;
3) depolymerization and activation of high polymeric HrpNECb multi-epitope protein molecules
(I) Pretreatment: with glucose Na2HPO4-KH2PO4The buffer solution regulates the volume concentration range of high polymeric HrpNECb multi-epitope protein collected by fermentation to be 0-30%, preferably 0-5%; preferably at a concentration of 30% -20%; preferably at a concentration of 5% -10%; preferably at a concentration of 20% -15%; most preferably at a concentration of 10% to 15%. At normal temperature (20-30 ℃), pre-treating glucose Na2HPO4-KH2PO4Buffer solution, pH range is 1-14, glucose concentration range is 0-2500mmol, buffer system pH, preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-6; most preferably at a pH of 5-5.5. The concentration of glucose is 0-100 mmol; preferably at a concentration of 100-; preferably a concentration of 2500-; preferably at a concentration of 1000-; most preferably at a concentration of 200-300 mmol. The treatment time is 0-24h, preferably 0-2 h; the preferable time is 24-15 h; preferably for 2-4 h; preferably for a time of 15-6 h; most preferably for a period of 4-6 hours.
(II) depolymerisation of activated HrpNEcb high polymeric polyepitopic protein: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution, wherein the ultrahigh pressure range is 1000-3000MPa, preferably 3000 MPa; preferably 1500 Mpa; preferably 2500 Mpa; preferably 2000 Mpa; most preferably 2000-;
(III) post-treatment: after the ultrahigh pressure depolymerization and activation operation is finished, standing for 0-24h at 35-38 ℃, preferably for 0-1 h; preferably for 24-10 h; preferably for a period of 1-2 hours; preferably for 10-4 hours; most preferably for 2-4h, and then collecting the de-polymerized activated HrpNEcb polyepitope protein molecules.
(IV) purifying the high polymeric multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, wherein the protein purification is carried out according to the method suggested by NI-NTA affinity chromatography gel manufacturers, and the purification preparation of the depolymerized and activated HrpNECb protein is completed.
3. The preparation of the HrpNECb multi-epitope ligand protein, further, the HrpNECb protein can also be prepared by expression protein of 'artificial synthetic gene', and the depolymerized and activated HrpNECb protein is prepared and collected by fermentation and purification, and the method specifically comprises the following steps:
artificial synthesis of hrpNECb gene for coding HrpNECb protein and preparation of its expression protein
1) The nucleotide sequence of hrpNECb gene for coding HrpNECb protein published to GenBank according to modern bioinformatics is used for artificially synthesizing HrpNECb multi-epitope protein gene, and the DNA sequence of the HRpNECb multi-epitope protein gene is obtained from GenBank: ABD 22989.1:
cloning of the gene encoding the HrpNEcb protein:
according to the DNA sequence of hrpNECb of the gene encoding the HrpNECb protein, the DNA sequence is as follows:
Figure BDA0002877782140000071
primers were designed and used (BamHI and HindIII sites underlined, respectively):
5’-tgcggatccatgcttaattctcttggtggcggt
5’-tgcaagctttaagctggagagcttctgcagccc
2) according to the DNA sequence, when the protein gene is artificially synthesized, BamHI enzyme cutting sites and HindIII enzyme cutting sites are respectively added on the 5 'and 3' of the gene, so that the protein gene can be conveniently cloned;
artificial gene synthesis was entrusted to the GeneArt Gene Synthesis and service department of Thermo Fisher Scientific, Inc. The advantages of the artificial synthetic protein gene are mainly that: a) the synthesis period is short, and 100% of sequences can be ensured to be correct; b) codons can be optimized to improve the expression efficiency of the gene; since the preferred codons differ for each species, some proteins are difficult to highly express when heterologous proteins are expressed in E.coli. If the codon of the heterologous protein is changed into the codon preferred by escherichia coli, the high-efficiency expression of the gene of the protein can be realized, the expression level of the gene is improved, and the method is suitable for large-scale industrial production; c) the site-directed mutagenesis of the gene can be carried out according to the needs to modify the gene, so as to improve the action efficiency of the protein; d) researchers can design genes which are difficult to obtain or even do not exist in nature according to own wishes.
3) The synthesized DNA fragment for coding the HrpNECb protein gene is cloned to the BamHI-HindIII site of the constructed high-efficiency protein expression vector JY-01 (containing His-Tag label) one by one, and the cloning accuracy is ensured by DNA sequencing;
4) and (3) engineering bacteria fermentation preparation of the HrpNECb protein: cloning genes (including but not limited to natural genes, chemically synthesized genes, transgenic genetic recombinant genes, similar genes and gene modifications thereof) of the HrpNECb protein coded in the genes from 1) to 3) into an engineering bacterium (E.coli), wherein a production line (E.coli) of related proteins is a derivative bacterium JY-01(DE3) of a K-12 original bacterium after special modification; when cultured in LB liquid medium (50. mu.g of kanamycin per liter) at a certain temperature until OD600 is 0.7, IPTG (Isopropyl thiogalactoside, Isopropyl beta-D-thiogalactoside) (final concentration: 1mMol) is added, the culture is continued, and then the thalli are centrifugally collected, 10% SDS-PAGE polyacrylamide gel electrophoresis is used for analyzing and coding the HrpNECb protein, and a 36.64kda band appears on a sample lane of an electrophoresis gel plate, which is the expression product HrpNECb protein of the gene hrpNECb.
Wherein the fermentation medium is Na2HPO4-KH2PO4A buffer system, the pH of the buffer system is in the range of 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-7; most preferably pH 6.5-5.5;
the fermentation temperature is 0-60 ℃. Preferably at a temperature of 0-20 ℃; preferably at a temperature of 20-35 ℃; preferably at a temperature of 60-50 ℃; preferably at a temperature of 50-45 ℃; most preferably at a temperature of 37-38 ℃;
the glucose concentration range of the fermentation proliferation liquid culture medium is 3.00-0.00%; preferably 3.00% -1.00%; preferably 0.00% -0.01%; preferably 1.00% -0.3%; most preferably 0.01% -0.05%; most preferably 0.1% -0.05%;
the glucose concentration range of the fermentation induction liquid culture medium is 3.00-0.00%; preferably 3.00% -1.00%; preferably 1.00% -0.3%; preferably 0.3% -0.1%; preferably 0.1% -0.05%; most preferably 0.05% -0.00%;
the lactose concentration range of the fermentation induction liquid culture medium is 10.00-0.00%; preferably 10.00% -1.00%; preferably 0.00% -0.1%; preferably 1.00% -0.6%; preferably 0.1% -0.3%; most preferably 0.5% -0.4%;
the fermentation induction liquid culture time range is 0-24 h; preferably for a time of 0-2 h; preferably for 24-15 h; preferably for 2-6 h; preferably for 15-10 h; most preferably for a period of 7-9 hours.
5) The engineering bacteria production system is post-treatment after the production and fermentation of a plurality of epitope proteins are finished: sterilizing: the fermentation liquor is sterilized at 80 ℃ for 30 minutes, and is rapidly cooled to below 30 ℃; cleaning: with glucose Na2HPO4-KH2PO4Buffer solution (pH range is 1-14, glucose concentration range is 0-2500mmol, buffer system pH is 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-6; most preferably pH 5-5.5. glucose concentration is 0-100 mmol; preferably concentration is 100-200 mmol; preferably concentration is 2500-1000 mmol; preferably concentration is 1000-300 mmol; most preferably concentration is 200-300 mmol), engineering bacteria are washed five to eight times in a continuous flow of a butterfly centrifuge; engineering bacteria are crushed and cell walls are cleared, Na with pH 5-5.5 and glucose concentration of 200-300mmol is used for clearing cell walls2HPO4-KH2PO4Diluting the thallus with buffer solution, adjusting the fresh weight of the thallus to 20-30% of the diluent, introducing into a high-pressure crusher, continuously crushing the engineering bacteria with the pressure of 800-1000MPa, introducing the crushed bacteria liquid into a butterfly continuous flow centrifuge, removing cell walls, and collecting multiple epitope protein molecules of high polymeric HrpNECb.
6) Depolymerization and activation of high polymeric HrpNECb multi-epitope protein molecules
(I) Pretreatment: with glucose Na2HPO4-KH2PO4Buffer solution adjustment of high polymeric HrpNECb multi-epitope protein collected by fermentationThe volume concentration range is 0-30%, preferably 0-5%; preferably at a concentration of 30% -20%; preferably at a concentration of 5% -10%; preferably at a concentration of 20% -15%; most preferably at a concentration of 10% to 15%. At normal temperature (20-30 ℃), pre-treating glucose Na2HPO4-KH2PO4Buffer solution, pH range is 1-14, glucose concentration range is 0-2500mmol, buffer system pH, preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-6; most preferably at a pH of 5-5.5. The concentration of glucose is 0-100 mmol; preferably at a concentration of 100-; preferably a concentration of 2500-; preferably at a concentration of 1000-; most preferably at a concentration of 200-300 mmol. The treatment time is 0-24h, preferably 0-2 h; the preferable time is 24-15 h; preferably for 2-4 h; preferably for a time of 15-6 h; most preferably for a period of 4-6 hours.
(II) depolymerizing the activated HrpNEcb high polymeric form multiple epitope protein: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution, wherein the ultrahigh pressure range is 1000-3000MPa, preferably 3000 MPa; preferably 1500 Mpa; preferably 2500 Mpa; preferably 2000 Mpa; most preferably 2000-;
(III) post-treatment: after the ultrahigh pressure depolymerization and activation operation is finished, standing for 0-24h at 35-38 ℃, preferably for 0-1 h; preferably for 24-10 h; preferably for a period of 1-2 hours; preferably for 10-4 hours; most preferably for 2-4 hours, and then collecting the depolymerized activated HrpNEcb multi-epitope protein molecules.
(IV) purifying the high-polymeric multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, wherein the protein purification is carried out according to the method suggested by NI-NTA affinity chromatography gel manufacturers, and the purification preparation of the depolymerized and activated HrpNECb protein is completed.
Compared with the prior art, the invention has the beneficial effects that:
the HrpNECb protein is a ligand protein with special structure rich in multiple linear and conformational epitopes, can cross-border recognize, activate and combine multiple types of animal membrane receptors, membrane proteins, information channels and metabolic channels, is a ligand protein with special multiple epitope structures, brand-new functions, brand-new action mechanisms and brand-new application prospects, induces multidirectional, multilevel and multifaceted biological effects and functions, and widely relates to the diagnosis, prevention, treatment or rehabilitation of diseases and conditions related to multiple systems, multiple tissues, multiple organs and multiple cells, and the application of food, word, cosmetic, mechanical word and health word products or medicines related to the diseases and conditions.
Drawings
FIG. 1 shows electrophoretic detection before and after disaggregation of HrpNECb protein: the molecular weight marker band is on the left, wherein 1: depolymerizing multiple epitope ligand protein HrpNECb bands before activation and purification; 2: multiple bands of epitope ligand protein HrpNECb after depolymerization activation and purification.
FIG. 2 is a graph of tobacco leaf allergy induced by HrpNECb protein solution injection, wherein the focal spot is formed by HarpinEcb protein solution treatment for about 24hr, B, D: h2O injection; A. c: HarpinEcb protein solution (250. mu.g/ml) was injected, i.e., harpinEcb protein hypersensitivity reaction on tobacco leaves, B, D as control, A, C as treatment.
FIG. 3 shows that the HrpNECb protein of the present invention is orally administered and smeared to induce liver to express differential gene volcano, and the administration time is 6h and 24h from left to right; smearing for 6 h;
FIG. 4 shows that the HrpNECb protein of the present invention induces the expression of the differential gene volcano pattern of thalamus by oral administration and smearing on experimental mice, wherein the oral administration is 6 hours and the oral administration is 24 hours from left to right; smearing for 6 h;
FIG. 5 shows that the HrpNECb protein of the present invention is orally administered for 6h and orally administered for 24h from left to right by inducing heart to express the differential gene volcano pattern through oral administration and smearing on experimental mice; smearing for 6h and smearing for 12 h;
FIG. 6 shows that the HrpNECb protein of the present invention induces the expression of the differential gene volcano in the cerebral cortex by oral administration and smearing on the experimental mouse, and the oral administration is 6 hours and 24 hours from left to right; smearing for 6h and smearing for 12 h;
FIG. 7 shows the volcano chart of the differential gene expression of hippocampus cerebri induced by oral administration of HrpNECb protein and smearing of experimental mice, which is orally administered for 6h and 24h from left to right; smearing for 6h and smearing for 12 h;
FIG. 8 is a cluster chart of the induced liver expression differential gene cluster of HrpNECb protein oral administration and smearing experimental mice of the present invention, which is orally administered for 6h and orally administered for 24h from left to right; smearing for 6 h;
FIG. 9 is a cluster chart of the difference gene cluster of the oral administration and smearing of the HrpNECb protein of the present invention for inducing thalamus expression, which is orally administered for 6h and 24h from left to right; smearing for 6 h;
FIG. 10 is a clustering chart of Hippocampus expression differential gene sets induced by oral administration of HrpNECb protein and smearing of experimental mice, which is orally administered for 6h and 24h from left to right; smearing for 6 h;
FIG. 11 shows a cluster chart of the gene set of differences in the expression of cerebral cortex induced by oral administration and smearing of HrpNECb protein of the present invention, which is orally administered for 6 hours and orally administered for 24 hours from left to right; smearing for 6 h;
FIG. 12 is a comparison of KEGG Pathway HrpEcb orally administered 6 hour treated livers of the present invention with controls (total genes);
FIG. 13 shows a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated livers of the present invention with controls (total genes);
FIG. 14 is a comparison of 6 hour treated liver smeared with KEGG Pathway HrpEcb of the present invention versus control (total genes);
FIG. 15 is a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated liver of the present invention with a control (upregulated genes);
FIG. 16 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated livers of the present invention with controls (upregulated genes);
FIG. 17 shows a comparison of 6 hour smeared treated livers with the KEGG Pathway HrpEcb of the present invention versus a control (up-regulated gene);
FIG. 18 shows a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated liver of the present invention with a control (down-regulated gene);
FIG. 19 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated livers of the present invention with controls (down-regulated genes);
FIG. 20 shows a comparison of 6 hour smeared treated livers with the KEGG Pathway HrpEcb of the present invention versus a control (downregulated genes);
FIG. 21 shows a comparison of KEGG Pathway HrpEcb orally administered 6 hour treated hearts of the present invention with controls (total genes);
FIG. 22 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated hearts of the invention with controls (total genes);
FIG. 23 shows a comparison of 6 hour treated hearts smeared with KEGG Pathway HrpEcb of the present invention with controls (total genes);
FIG. 24 shows a comparison of 12 hour treated hearts coated with KEGG Pathway HrpEcb of the present invention with controls (total genes);
FIG. 25 is a comparison of KEGG Pathway HrpEcb orally administered 6 hour treated hearts of the invention with controls (upregulated genes);
FIG. 26 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated hearts of the invention with controls (upregulated genes);
FIG. 27 shows a comparison of 6 hour treated hearts smeared with KEGG Pathway HrpEcb of the present invention with controls (upregulated genes);
FIG. 28 is a comparison of KEGG Pathway HrpEcb smeared 12 hours treated hearts of the invention versus controls (upregulated genes);
FIG. 29 is a comparison of KEGG Pathway HrpEcb orally administered 6 hour treated hearts of the invention with controls (downregulated genes);
FIG. 30 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated hearts of the invention with controls (downregulated genes);
FIG. 31 is a comparison of KEGG Pathway HrpEcb smeared 6 hours treated hearts of the invention versus controls (downregulated genes);
FIG. 32 is a comparison of KEGG Pathway HrpEcb smeared 12 hours treated hearts of the invention versus controls (downregulated genes);
FIG. 33 is a comparison of KEGG Pathway HrpEcb orally administered 6 hour treated hippocampus of brain versus control (total genes) of the present invention;
FIG. 34 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated hippocampus of brain versus control (total genes) of the present invention;
FIG. 35 is a comparison of the KEGG Pathway HrpEcb smeared for 6 hours treated hippocampus of brain versus control (total genes) of the present invention;
FIG. 36 is a comparison of the KEGG Pathway HrpEcb smeared for 12 hours treated hippocampus of brain versus control (total genes) of the present invention;
FIG. 37 is a comparison of KEGG Pathway HrpEcb orally administered 6 hour treated hippocampus of brain versus control (upregulated genes) of the present invention;
FIG. 38 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated hippocampus of brain versus control (upregulated genes) of the present invention;
FIG. 39 is a comparison of the KEGG Pathway HrpEcb smeared for 6 hours treated hippocampus of brain versus control (upregulated genes) of the present invention;
FIG. 40 is a comparison of the 12 hour treatment of hippocampus of brain treated with KEGG Pathway HrpEcb of the present invention with a control (upregulated genes);
FIG. 41 is a comparison of KEGG Pathway HrpEcb orally administered 6 hour treated hippocampus of brain versus control (down-regulated genes) of the present invention;
FIG. 42 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated hippocampus of brain versus control (down-regulated genes) of the present invention;
FIG. 43 is a comparison of KEGG Pathway HrpEcb smeared for 6 hours treated hippocampus of brain versus control (downregulated genes) of the present invention;
FIG. 44 is a comparison of the treatment of hippocampus of the brain with a control (down-regulated genes) with a KEGG Pathway HrpEcb of the present invention applied for 12 hours;
FIG. 45 is a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated cerebral cortex of the present invention with a control (total genes);
FIG. 46 is a comparison of KEGG Pathway HrpEcb orally administered 24 hours treated cerebral cortex of the present invention with a control (total genes);
FIG. 47 shows a comparison of the 6 hour treated cortex of KEGG Pathway HrpEcb smeared with the control (total gene) of the present invention;
FIG. 48 is a comparison of KEGG Pathway HrpEcb smeared for 12 hours treated cortex compared to control (total genes) of the present invention;
FIG. 49 shows a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated cerebral cortex of the present invention with a control (upregulated genes);
FIG. 50 is a comparison of KEGG Pathway HrpEcb orally administered 24 hours treated cerebral cortex of the present invention with a control (upregulated genes);
FIG. 51 shows a comparison of the 6 hour treated cortex of KEGG Pathway HrpEcb smeared with the control (upregulated genes) of the present invention;
FIG. 52 is a comparison of KEGG Pathway HrpEcb smeared for 12 hours treated cortex compared to a control (upregulated genes);
FIG. 53 is a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated cerebral cortex of the present invention with a control (down-regulated gene);
FIG. 54 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated cerebral cortex of the present invention with a control (down-regulated gene);
FIG. 55 shows a 12 hour treatment of cerebral cortex treated by applying KEGG Pathway HrpEcb of the present invention to a control (down-regulated gene);
FIG. 56 is a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated cerebral thalamus of the present invention with controls (total genes);
FIG. 57 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated cerebral thalamus of the present invention with controls (total genes);
FIG. 58 is a comparison of KEGG Pathway HrpEcb smeared for 6 hours treated cerebral thalamus of the present invention with controls (total genes);
FIG. 59 is a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated cerebral thalamus of the present invention with a control (upregulated genes);
FIG. 60 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated cerebral thalamus of the present invention with a control (upregulated genes);
FIG. 61 is a comparison of KEGG Pathway HrpEcb smeared for 6 hours treated cerebral thalamus of the present invention with controls (upregulated genes);
FIG. 62 is a comparison of KEGG Pathway HrpEcb orally administered 6 hours treated cerebral thalamus of the present invention with a control (down-regulated gene);
FIG. 63 is a comparison of KEGG Pathway HrpEcb orally administered 24 hour treated cerebral thalamus of the present invention with a control (down-regulated gene);
FIG. 64 is a comparison of KEGG Pathway HrpEcb smeared for 6 hours treated cerebral thalamus of the present invention with a control (down-regulated gene);
FIG. 65 is a flowchart of the mRNA (RNA-Seq) sequencing experiment according to the present invention;
FIG. 66 is a flow chart of mRNA sequencing data analysis according to the present invention.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It is to be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention, i.e., the described embodiments are a subset of the embodiments of the invention rather than a full set of embodiments.
The test methods used in the examples below are all conventional methods, unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
The HrpNECb multi-epitope ligand protein is prepared by adopting the engineering bacteria fermentation of registered EcbCSL101hrpNECb gene (GenBank: ABD22989.1), purifying and collecting depolymerized activated HrpNECb protein, and the method specifically comprises the following steps:
1) and (3) engineering bacteria fermentation preparation of the HrpNECb protein: engineering bacteria (E.coli) of genes (including but not limited to natural genes, chemically synthesized genes, transgenic genetic recombinant genes and similar genes of biological samples and gene modifications thereof) for encoding HrpNECb proteins, wherein the production line of related proteins is specially modified derivative bacteria JY-01(DE3) of K-12 original bacteria, IPTG (Isopropyl thiogalactoside, Isopropypyl beta-D-thiogalactoside) (final concentration of 1mMol) is added when the bacteria are cultured in LB liquid culture medium (containing 50 micrograms per liter of kanamycin) under the condition of certain temperature until OD600 is 0.7, and bacteria are collected by centrifugation after the bacteria are continuously cultured. The expression product of HrpNECb protein was analyzed by 10% SDS-PAGE polyacrylamide gel electrophoresis, and an 36.64kda band was shown in the sample lane of the electrophoresis gel plate, which is the expression product of HrpNECb protein of gene hrpNECb. Wherein the fermentation medium is Na2HPO4-KH2PO4The pH value of the buffer system is 6.5-5.5; the fermentation temperature is 37-38 ℃; the glucose concentration of the fermentation proliferation liquid culture medium is 0.01-0.05%; the glucose concentration of the fermentation induction liquid culture medium is 0.05-0.00%; the lactose concentration of the fermentation induction liquid culture medium is 0.5-0.4%; the culture time of fermentation induction liquid is 7-9 h.
2) The engineering bacteria production system is post-treatment after the production and fermentation of the multi-epitope protein are finished: sterilizing: the fermentation liquor is sterilized at 80 ℃ for 30 minutes, and is rapidly cooled to below 30 ℃; cleaning: with glucose Na2HPO4-KH2PO4Buffer solution with pH 5-5.5 and glucose concentration 200-; thirdly, the engineering bacteria are broken and the cell wall is removed, then Na with pH of 5-5.5 and glucose concentration of 200-300mmol is used2HPO4-KH2PO4Diluting the thallus with buffer solution, regulating the fresh weight of the thallus to 20-30% of the diluent, introducing into a high-pressure crusher, continuously crushing the engineering bacteria with the pressure of 800-1000MPa, introducing the crushed bacteria liquid into a butterfly continuous flow centrifuge, removing cell walls, and collecting high-polymerization-state HrpNECb multi-epitope protein molecules.
3) Depolymerization and activation of high polymeric HrpNECb multi-epitope protein molecules: preprocessing: with glucose Na2HPO4-KH2PO4Adjusting the volume concentration of the high-polymerization-state HrpNECb multi-epitope protein collected by fermentation to 10-15% by using buffer solution, and pretreating glucose Na under the condition of normal temperature (20-30 ℃)2HPO4-KH2PO4Buffer solution, pH 5-5.5, glucose concentration 200-. (ii) depolymerizing the activated HrpNECb high-polymerization-state multi-epitope protein: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution, wherein the ultrahigh pressure is 2000-2500 Mpa; thirdly, after the operations of ultrahigh pressure depolymerization and activation are finished, standing for 2-4h at the temperature of 35-38 ℃, and then collecting depolymerized and activated HrpNECb multi-epitope protein molecules; and fourthly, purifying the high-aggregation multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, and performing protein purification according to a method suggested by an NI-NTA affinity chromatography gel manufacturer to complete the preparation of the depolymerzation activated purified HrpNECb protein.
Example 2
The HrpNECb protein is prepared by expression protein of 'artificially synthesized gene', and specifically comprises the following steps:
the first step is as follows: artificial synthesis of HrpNEcb gene encoding HrpNEcb protein;
1) the hrpNECb gene nucleotide sequence of the encoding HrpNECb protein published to GenBank according to modern bioinformatics is used as a gene for artificially synthesizing multiple epitope proteins of HrpNECb,
its DNA sequence is from GenBank: ABD 22989.1:
cloning of the gene encoding the HrpNEcb protein:
according to the DNA sequence of hrpNECb of the gene encoding the HrpNECb protein, the DNA sequence is as follows:
Figure BDA0002877782140000111
primers were designed and used (BamHI and HindIII sites underlined, respectively):
5’-tgcggatccatgcttaattctcttggtggcggt
5’-tgcaagctttaagctggagagcttctgcagccc
amplifying a DNA fragment of a required test coding HrpNECb protein whole gene by using high-fidelity Taq enzyme, and carrying out PCR amplification according to a method suggested by a high-fidelity Taq enzyme manufacturer;
the second step is that: 2) according to the DNA sequence, when the protein gene is artificially synthesized, BamHI enzyme cutting sites and HindIII enzyme cutting sites are respectively added on the 5 'and 3' of the gene, so that the protein gene can be conveniently cloned;
the third step: artificial gene synthesis was entrusted to the GeneArt Gene Synthesis and service department of Thermo Fisher Scientific, Inc. 3) The synthesized DNA fragment for coding the HrpNECb protein gene is cloned to the BamHI-HindIII site of the constructed high-efficiency protein expression vector JY-01 (containing His-Tag label) one by one, and the cloning accuracy is ensured by DNA sequencing;
the fourth step: transferring the gene clone of the HrpNECb protein coded in the 1) to 3) into an escherichia coli engineering bacterium (E.coli), wherein a production line (E.coli) of related protein is a derivative bacterium JY-01(DE3) of K-12 original bacterium after special modification; when cultured in LB liquid medium (50. mu.g of kanamycin per liter) at 37 ℃ until OD600 is 0.7, IPTG (Isopropyl thiogalactoside, IsopropyL beta-D-Thiogalactosid) (final concentration is 1mMol) is added, the culture is continued, then the thalli are centrifugally collected, 10% SDS-PAGE polyacrylamide gel electrophoresis is used for analyzing the expression product HrpNECb protein, and an 36.64kda band appears on a sample lane of an electrophoresis gel plate, which is the expression product HrpNECb protein of the gene hrpNECb, and is shown in detail in FIG. 1;
wherein the culture medium Na for fermentation2HPO4-KH2PO4A buffer system, the pH of the buffer system is 6.5-5.5; the glucose concentration of the fermentation proliferation liquid culture medium is 0.01-0.05%; the lactose concentration of the fermentation induction liquid culture medium is 0.5-0.4%;
the fifth step: suspending the collected cells in Na2HPO4-KH2PO4In a buffer solution, finishing sterilization treatment at the temperature of 80 ℃ for 30 minutes, rapidly cooling to 30 ℃, cleaning engineering bacteria for five to eight times in a butterfly continuous flow centrifuge, introducing into a high-pressure crusher, continuously crushing the engineering bacteria under the pressure of 800-;
and a sixth step: depolymerization and activation of high polymeric HrpNECb multi-epitope protein molecules
(I) Glucose Na for pretreatment2HPO4-KH2PO4The volume concentration of the collected and purified high polymeric HrpNECb polyepitopic protein is adjusted by a buffer solution and is 10-15%. At normal temperature (20-30 ℃), pre-treating glucose Na2HPO4-KH2PO4Buffer, pH 5-5.5. The glucose concentration is 200-300mmol, and pretreatment is carried out; the treatment time is 4-6 h.
(II) depolymerizing and activating the HrpNECb high-polymerization-state multi-epitope protein, and carrying out ultrahigh-pressure depolymerization and activation operation on the pretreated high-polymerization-state protein pretreatment solution within the ultrahigh-pressure range of 2000-2500 Mpa;
(III) after the operation of the post-treatment ultrahigh pressure depolymerization and activation is finished, standing for 2-4h at the temperature of 35-38 ℃, and then collecting the depolymerized and activated HrpNECb multi-epitope protein molecules.
(IV) purifying the high polymeric multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, wherein the protein purification is carried out according to the method suggested by NI-NTA affinity chromatography gel manufacturers, and the purification preparation of the depolymerized activated multi-epitope protein HrpNECb is completed.
The 10% SDS polyacrylamide gel electrophoresis detects the highly expressed depolymerized activated protein-His-Tag recombinant band, which is shown in figure 1 in detail.
As shown in fig. 1, the molecular weight marker band is on the left; the part 1 is an electrophoretic band before depolymerization and activation, and more bands are gathered in a corresponding molecular weight region, and 36.64kda bands are also included; the band at 2 is the band of depolyactivated purified HrpNEcb protein, molecular weight 36.64kda, in the region of the corresponding molecular weight of the ligand protein, indicating that the corresponding depolyactivated purified HrpNEcb protein has been obtained.
As shown in fig. 2, allergy assay tests for disaggregating activated multiple epitope ligand proteins: the tobacco leaf reaction results 24hr after the HrpNECb protein preparation and sterile water treatment are shown in FIG. 2, wherein point A, C is injection of 300. mu.g.mL-1100 μ L of the HrpNECb protein solution; B. point D is a control treatment of 100. mu.L of sterile water injected. 300. mu.g/mL-1Treating with the protein solution of HrpEcb for about 12hr to cause tobacco leaf atrophy and collapse, and withering and death for 24 hr; the water control treated tobacco leaves had no allergic reactions.
The depolymerized and activated multi-epitope ligand protein can generally trigger hypersensitivity of various plant leaves, and the types of the test plants can be as follows: tobacco, pepper, eggplant, tomato, potato, strawberry, cucumber, water spinach, cockscomb, begonia glauca, chamomile, pansy, annatto, petunia, grape, Chinese rose, locust tree, pea, peach, sage, luffa, kidney bean, cauliflower, spinach, rape, yam, cowpea, broad bean, corn, rice, soybean, cyclamen, mulberry, pumpkin, loquat, and toona sinensis.
Example 3
Sequencing of animal Experimental mRNA (RNA-Seq)
mRNA-seq is the conversion of RNA produced by cells into DNA by a reverse transcription process (cDNA, complementation, and library construction of the obtained cDNA). The resulting DNA is then sequenced and the original amount of mRNA in the cell is inferred from the observed abundance of the particular DNA, thereby finding genes or transcripts whose transcription levels vary under the experimental conditions, i.e., differential expression. By finding these differentially expressed genes and transcripts, functional characteristics of the different conditions were deduced. We used RNA-seq technology to study and demonstrate that the HrpNECb protein induces differential expression of multiple genes in multiple organs of mice.
1. Laboratory animal sample treatment
The experiment is carried out by entrusting a protein mass spectrum technology platform of Shanghai Huaying biological medicine science and technology Limited company.
Treatment of experimental samples: the experimental selection of the balb/C experimental mice of 8 weeks of age, divided into HrpNECb protein treatment groups, including oral 6 hours, 24 hours and smearing 6 hours, 12 hours of 4 treatments, each treatment of 3 experimental mice, total 12; blank control group 4 experimental mice; buffer control sham group without HrpNEcb protein, including 4 treatments of 6 hours, 24 hours oral administration and 6 hours, 12 hours smearing, each treatment of 4 mice, 16 mice in total, three replicates; 600 mg.L for experimental treatment group mice-1The HrpNECb protein buffer solution with the concentration is fed and smeared, the buffer solution control sham operation group mice are fed and smeared with the buffer solution, and blank control group mice are not treated. Under the same breeding condition, according to different time, respectively grouping mouse cerebral cortex, thalamus, cerebral hippocampus, liver, heart and other tissues, and carrying out RNA-Seq sequencing and analysis.
Sequencing of mRNA (RNA-Seq)
Almost all the mRNA expression abundance of a specific tissue or organ of a certain species in a certain state can be comprehensively and rapidly obtained through next generation sequencing, and the mRNA (RNA-Seq) sequencing experiment flow chart is shown in FIG. 65.
Quality control of RNA
Total RNA extraction of the samples was performed using the miRNeasy Micro Kit (Cat #1071023Qiagen) and according to the standard protocol provided by the manufacturer. Total RNA was quality-tested using a NanoDrop ND-2000 spectrophotometer and an Agilent Bioanalyzer 4200(Agilent technologies, Santa Clara, Calif., US), and RNA that was qualified for quality testing was subjected to subsequent sequencing experiments.
Library construction and quality control
Use of the constructed library
Figure BDA0002877782140000131
2.0Fluorometer assay concentration, Agilent2100 assay size.
Computer sequencing
And carrying out Illumina sequencing on the library qualified by quality inspection, and acquiring sequence information of the fragment to be detected by a sequencer through capturing a fluorescent signal and converting an optical signal into a sequencing peak through computer software.
Mrna sequencing data analysis was performed according to the data analysis flow of fig. 66.
3. Analysis of results
1) HrpNECb protein-induced differential gene screening
The method comprises the steps of firstly normalizing fragment counts, then calculating p-value according to a hypothesis test model, and finally carrying out test correction on p-value multiple hypotheses to obtain an FDR value. FP KM values were calculated as Fold-change differential expression using the edgeR software. The differential gene screening conditions were as follows: p-value <0.05 and | Fold-change | > 2
2) HrpNECb protein-induced differential gene volcano plot
The volcano plot of the difference gene is adopted to show the overall distribution of the HrpNECb protein-induced expression difference significant genes. The abscissa: fold change in gene expression in different samples (log2 Fold-Chan ge); ordinate: the level of significance of the difference in gene expression (-log10 p-value); right-hand dots express significant up-regulated genes; left lateral point expression significantly down-regulated genes; lower spots expressed genes that did not significantly change. FIGS. 3-7 are graphs of the mouse liver, cerebral thalamus, heart, cerebral cortex and hippocampus HrpNECb protein, which are orally and smear-induced differential gene volcanoes, respectively, wherein HrpNECb is abbreviated as N1.
3) HrpNECb protein-induced differential gene clustering map
And carrying out cluster analysis on the differential gene set, gathering the genes with similar expression modes together, and displaying that the genes have common functions or participate in a common signal path. Log10(FPKM +1) values were normalized (sca le number) and clustered, with red indicating high expression and blue indicating low expression in the heatmap. FIGS. 8-11 are cluster heatmaps of liver, thalamus, hippocampus, and cerebral cortex expression differential gene sets, respectively, in which HrpNECb is abbreviated as N1.
4) Enrichment analysis of HrpNECb protein-induced differential gene GO
Gene Ontology (GO) is an Ontology widely used in the field of bioinformatics. Gene ontology is the description of genes in different dimensions and at different levels, and covers biological processes, cellular components and molecular functions. The biological process is used for explaining which biological processes are involved in the gene; cellular components explain where a gene is present, including whether the gene is in the cytoplasm or the nucleus? Which organelle if cytoplasm is present? If it is in mitochondria, on the mitochondrial membrane or in the matrix of mitochondria, etc., these information belong to the group of cells; what explains the molecular function is what is the function of the gene at the molecular level? Describes its activity, such as catalytic activity or binding activity, in the individual molecular biology. The Gene Ontology database (Gene Ontology) is a structured standard biological model constructed in 2000 by the GO organization (Gene Ontology Consortium), aims to establish a standard vocabulary system of Gene and product knowledge, and covers biological processes (biological processes), cell components (cellular components) and molecular functions (molecular functions) of genes. Term is the basic description unit inside GO. GO terminals are used to describe the function of gene products. By carrying out GO enrichment analysis on the differential genes, the genes can be classified according to different functions, and the purpose of annotating and classifying the genes is achieved. The result of GO term enrichment analysis of differential expression genes induced by HrpNECb protein proves that HrpNECb protein has a plurality of epitope special structures, brand new functions, brand new action mechanisms and ligand protein with brand new application prospects, induces differential expression of multiple genes of a plurality of organs (such as liver, thalamus, heart, cerebral cortex, cerebral hippocampus and the like) of a mouse, and the differential expression genes cover biological processes, cell components and molecular functions. The results of the enrichment analysis of the HrpNEcb protein-induced differential gene GO are further described as follows: biologically-process-related differentially expressed genes include reproductive, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesive, signaling, multicellular biological processes, developmental processes, growth, movement, single tissue processes, biological, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, stimulatory responses, localization, bioregulation, cell component organization or biogenesis, cell aggregation, detoxification, and presynaptic processes involving synaptic transmission. The results of the bioprocess GO enrichment analysis are detailed in tables 1 to 6.② cell component (cellular _ component) -related differentially expressed genes encompass cells and extracellular domains, nuclei-like, membranes, virions, cell junctions, extracellular matrix, cell membrane-enclosed cavities, complex macromolecules, organelles, extracellular matrix components, extracellular domain portions, organelle components, virion components, membrane components, synapse components, cellular components, synapses, and cellular supramolecular fibers, and the like. The cell component GO enrichment assay results are detailed in tables 1 to 6. (iii) molecular function (molecular function) -related differentially expressed genes encompass transcription factor activity, protein binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecule activity, transport activity, binding, electron carrier activity, morphogen activity, antioxidant activity, chaperone activity, protein labeling, activity of chemoattractants, translational regulation, chemical repulsor activity, mobile molecule sensors, molecular function regulation, and the like. The results of the enrichment analysis of molecular functional GO are detailed in tables 1 to 6.
The Gene Ontology (GO) is an Ontology widely used in the field of bioinformatics to cover the GO terms taxonomic Gene statistics tables 1-6 with p-values less than 0.05 corresponding to three levels of biological cellular components, molecular functions, and biological processes.
Wherein HarpinNECb of tables 1-6 is abbreviated as N1, and in all tables, blank spaces indicate that no corresponding related data not reaching p-va l ue less than 0.05 standard is collected, and the following and all tables have the same blank meaning.
TABLE 1 Harpinnecb protein induces biological processes, cellular components and functional groups related to molecular function of heart significantly up-regulated expression of GO terms classifier gene statistics (6, 24 hours oral and 6 hours smeared, 12 hours smeared)
Figure BDA0002877782140000141
Figure BDA0002877782140000151
Figure BDA0002877782140000161
TABLE 2 Harpinnecb protein induces biological processes, cell composition and molecular function related functional groups of liver and cortex to significantly up-regulate expression of GO terms classifier gene statistical tables (oral 6, 24 hours and smear 6, 12 hours)
Figure BDA0002877782140000162
Figure BDA0002877782140000171
Figure BDA0002877782140000181
TABLE 3 Harpinnecb protein induces biological processes, cell composition and molecular function related functional groups of thalamus and hippocampus to significantly up-regulate expression of GO terms classifier gene statistics (6, 24 hours oral and 6 hours, 12 hours smearing)
Figure BDA0002877782140000182
Figure BDA0002877782140000191
Figure BDA0002877782140000201
Figure BDA0002877782140000211
TABLE 4 Harpinnecb protein induces biological processes, cellular components and functional groups related to molecular function of heart significantly downregulate expression of GO terms classifier gene statistics (6, 24 hours oral and 6 hours smeared, 12 hours smeared)
Figure BDA0002877782140000212
Figure BDA0002877782140000221
Figure BDA0002877782140000231
TABLE 5 Harpinnecb protein induces biological processes, cell composition and molecular function related functional groups of liver and cortex significantly downregulate expression of GO terms classifier gene statistics (6, 24 hours oral and 6 hours smeared, 12 hours smeared)
Figure BDA0002877782140000232
Figure BDA0002877782140000241
Figure BDA0002877782140000251
Figure BDA0002877782140000261
TABLE 6 Harpinnecb protein induces biological processes, cellular components and functional groups related to molecular function in thalamus and hippocampus significantly downregulating expression of GO terms classifier gene statistics (6, 24 hours oral and 6 hours, 12 hours smeared)
Figure BDA0002877782140000262
Figure BDA0002877782140000271
Figure BDA0002877782140000281
Figure BDA0002877782140000291
5. KEGG pathway enrichment analysis of differentially expressed genes
Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database for systematically analyzing gene functions and genome information, integrates information of genomics, biochemistry and systematic functional omics, and is helpful for researchers to integrally research the process of gene and expression information as a network.
The key feature of KEGG is to link genes with various biochemical reactions to provide an integrated metabolic pathway. KEGG currently contains a total of 19 sub-databases that are classified into three categories, systematic, genomic, and chemical. In organisms where different gene products coordinate to perform biological functions, Pathway annotation analysis of differentially expressed genes helps to further decipher gene function. KEGG pathway enrichment analysis is carried out on the HrpNECb protein-induced differential expression genes, the roles (upstream and downstream relation) and the biological functions of the differential genes in a signal path are obtained, and the relationship between the genes and the functions is deeply understood. Research results prove that the HrpNECb protein, as a ligand protein with multi-epitope special structure, brand-new function, brand-new action mechanism and brand-new application prospect, induces differential expression of multiple genes of multiple organs (such as liver, thalamus, heart, cerebral cortex, cerebral hippocampus and the like) of a mouse, and the differential expression genes participate in functional pathways such as Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism and biological Systems. The results of the enrichment analysis of the HrpNEcb protein-induced differential gene GO are further described as follows: (ii) Cellular Processes (Cellular Processes): the HrpNECb protein induces a plurality of differentially expressed genes involved in cellular processes such as trafficking and catabolism, cell population, cell activity, cell growth and death (see FIGS. 12 to 64 for details). (Environmental Information Processing) multiple differentially expressed genes induced by the HrpNECb protein participate in the Environmental Information Processing processes such as signal molecule interaction, signal transduction, membrane transport and the like (see FIG. 12 to FIG. 64 for details). (iii) Genetic Information Processing (Genetic Information Processing) multiple differentially expressed genes induced by the HrpNECb protein are involved in biological processes such as translation, replication and repair, folding, classification and degradation (see FIGS. 12 to 64 for details). Metabolism (Metabolism) the various differentially expressed genes induced by the HrpNEcb protein are involved in the metabolic processes of biodegradation and Metabolism, nucleotide Metabolism, Metabolism of other amino acids, metabolic cofactors and vitamins, lipid Metabolism, biosynthesis and Metabolism of sugars, global and overview maps, energy Metabolism, carbohydrate Metabolism and amino acid Metabolism (see fig. 12 to 64 for details). Multiple differentially expressed genes induced by HrpNECb protein are involved in cell processes of sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, developmental circulatory system, etc. (detailed in FIGS. 12 to 64).
Similar to GO classification statistics, the number of differentially expressed genes on each biological pathway (pathway) of KEGG was counted and graphically displayed as shown in fig. 12-64.
Description of the drawings: the diagram on the right side shows the Chinese translation from top to bottom: cellular processes, information processes, genetic information processes, metabolic processes, tissue system development processes; the abscissa: the number of genes of each functional gene group involved in expression difference; ordinate: functional gene groups involved in cellular processes, information processes, genetic information processes, metabolic processes, tissue phylogenetic processes of differential expression.
Example 4
Pull-down experiment for recognition of binding specific protein by HrpNECb protein
1. Sample preparation and processing
1) HrpNECb protein purification
And (3) purifying the high polymeric HrpNECb multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, wherein the protein purification is carried out according to the method suggested by NI-NTA affinity chromatography gel manufacturers, and the purification preparation of the depolymerized and activated multi-epitope protein HrpNECb is completed for standby (hereinafter referred to as capture protein or target protein).
2) Total protein (bait protein) extraction of cultured liver cell for experiment
I. Extraction of total cell protein: firstly, a lysate (a lysate special for IP, and 1 Xcocktail protease inhibitor is added) is absorbed by a pipette gun and added into cells. Performing ultrasonic treatment, and standing for more than 2 hours on ice; secondly, using an ultrasonic cell disruptor to carry out ultrasonic treatment on ice for 2s and stop for 5s for 1min, wherein the total time of cracking on ice is more than 2h (shaking and mixing by an oscillator at intervals of 30 min); ③ centrifuging the cell lysate for 15min at 13000rpm at 4 ℃, sucking the supernatant, transferring the supernatant to a new 1.5mLEP tube, and placing the tube on ice for standby; fourthly, centrifuging the protein extract again at 13000rpm for 5min at 4 ℃, carefully absorbing the solution in the middle layer, transferring the solution into a new 1.5mL EP tube, standing the tube in a refrigerator at 4 ℃ for standby, taking part of the diluted solution, measuring the concentration (10 times of the diluted solution), and measuring the concentration by using a BCA method.
Protein concentration determination: the extracted protein solution was subjected to concentration measurement with reference to the method of the BCA kit.
TABLE 7 BCA assay for protein concentration
NO. Sample name Experiment number Concentration (μ g/. mu.L) Volume (μ L) Total amount (μ g)
1 HEPG2 HEPG2 8.34 2500 20861.30
Pull-down experiment process
1) Equilibrium fixing streptavidin gel, namely preparing a Pierce TM Spin Column tube; secondly, the resuspension gel solution is inverted up and down, 50 mul of suspension is sucked into a marked Spin Column tube, a bottom plug is plugged, and the suspension is placed in a collecting tube; thirdly, adding 250 mul TBS into the Spin Column tube, screwing down the top cover, and slightly reversing the top and the bottom for 4 times to mix the liquid uniformly; fourthly, removing the top cover and the bottom plug, centrifuging at 1250 Xg for 50s, discarding the cleaning solution in the collecting pipe, and reinserting the SpinColumn pipe into the collecting pipe; repeating step 3 and step 4 twice. And then plugging the tube bottom plug at the bottom of the Spin Column tube.
2) The biotin-labeled bait protein and the biotin are fixed, namely, the biotin and the biotin-labeled bait protein are respectively added into a Spin Column tube, and a top cover and a bottom plug are screwed down; gently shaking the rotary platform rotating platform, and incubating for 60min at 4 ℃; thirdly, after the incubation is finished, removing the top cover and the bottom plug of the Spin Column tube, and putting the Spin Column tube into a collecting tube; 1250 Xg, after centrifugation for 60s, the Spin Column tube was replaced in the collection tube.
3) Blocking of biotin firstly, adding 250 mu l of biotin blocking solution into a Spin Column tube. Screwing down the top cover and the bottom plug, and slightly reversing the top cover and the bottom plug for 4 times to uniformly mix the mixture; ② incubating for 5min at room temperature. Removing the top cover, placing Spin Column tubes into the collection tube, and centrifuging at 1250 Xg for 50 s; thirdly, repeating the step 1 and the step 2 once; fourthly, 250 mul of TBS is added into the Spin Column tube. Screwing down the top cover, and slightly reversing the top cover and the bottom cover for 4 times to uniformly mix the mixture; fifthly, removing the top cover, placing the top cover in a collecting pipe, and centrifuging for 50s at 1250 Xg; sixthly, repeating the step 3 and the step 4 twice, and putting the Spin Column tube into the collecting tube again.
4) Capture of biotin-labeled protein (i.e., adding 300. mu.L (1mg protein) of capture protein (target protein) sample solution into Spin Column tube, and screwing down the cap; gently shaking the rotary platform rotating platform, and incubating overnight at 4 ℃; and thirdly, removing the top cover and the bottom plug after the incubation is finished. Putting the Spin Column tube into a prepared collecting tube; fourthly, collecting the tube, 1250 Xg, 60s, centrifuging, and marking the collecting tube with "prey flow-through (B)"; fifthly, removing the Spin Column tube in the collecting tube, covering the cover of the collecting tube, and placing on ice for subsequent analysis; sixthly, putting the Spin Column tube into a new collecting tube to prepare for elution.
5) Elution of complexes of bait protein and target protein from Spin columns (i.e., 250. mu.l of Wash Buffer was added to each Spin Column). Screwing down the top cover and the bottom plug, and slightly reversing for 6 times to uniformly mix the mixture; ② the Spin Column tube was incubated at room temperature for 1 minute. The top and bottom plugs were removed, Spin columns were placed on collection tubes, and centrifuged at 1250 × g for 50 s. Repeating the steps for 1-2 and 3 times; ③ during the flushing process, the label 'Wash 1, … …, Wash 3' is written on the collecting tube; fourthly, when the last washing is carried out, 200 mul of Wash Buffer is added, and then the liquid in the tube together with the beads is transferred to 1.5 mL; fifthly, in a new centrifuge tube, after centrifugation, 170 mu l of supernatant is discarded, and the step is repeated for 3 times.
6) And (3) detection: sucking up the liquid on Sepharose, adding 20 mul of 1 Xprotein electrophoresis sample buffer solution, boiling water bath for 5min, and placing in a refrigerator at-20 deg.C for later use; and secondly, detecting through SDS-PAGE and Western blot.
3. Analysis of results
1) The HrpNEcb protein recognizes bound cell membrane receptors: recognition binds to 6 membrane receptors including HLA-a major histocompatibility complex, class I, class a receptor, LGALS3BP galactose 3 binding protein (receptor), LAMP2 lysosomal associated membrane protein 2 receptor, GNB 2G guanine nucleotide binding protein subunit Beta 2 receptor, LRRC 1515-leucine repeat membrane protein receptor, KTN1 driver binding protein 1 receptor.
2) The HrpNEcb protein recognizes the bound cell membrane protein: recognizing and binding 11 membrane proteins including one or more of DSG4 desmoglein, ANXA4 annexin a4, CAPRIN1 cyclin, 1UTRN dystrophin protein, pinin desmoplanin, VAMP-associated protein A, VCL focal adhesion protein, Ezrin epithelial-type cadherin, PKP3 platelet avidin 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD 2N acetylated alpha linked acidic dipeptidase 2.
3) The HrpNEcb protein recognizes the bound signaling pathway: 13 recognition combinations, including hsa04152: AMPK signaling pathway, hsa03460: fanconi anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: marketing signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, and hsa04390: river horse signaling pathway.
4) The HrpNEcb protein recognizes the associated metabolic pathways of antiviral, antibacterial, anti-foreign body, anti-inflammatory binding: identifying 23, binding including endocytosis of hsa04144, phagocytosis of hsa04145, lysosomes of hsa04142, biosynthesis of antibiotics, hsa05131 Shigellasis, hsa04612 antigen processing and presentation, hsa05130 pathogenic E.coli infection, bacterial invasion of epithelial cells of hsa05100 Salmonella infection, hsa05169 Barr virus infection, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05166 HTLV-I infection, hsa05164 influenza A, hsa05134 legionnaire's disease, hepatitis C05160, hsa05162 measles, hsa05133 whooping cough, hsa05322 systemic lupus erythematosus, hsa04670 leukocyte migration through epithelium, hsa 46 amebic disease, hsa 42 trypanosomiasis, and southern 05200 cancer.
5) The HrpNEcb protein recognizes the important neurological disease metabolic channels of binding: 3 combinations were identified, including hsa05012 for Parkinson's disease, hsa05016 for Huntington's chorea, and hsa05010 for Alzheimer's disease.
6) The HrpNEcb protein recognizes the associated nucleic acid, protein, amino acid, sugar, fat metabolism related pathway: recognition of the binding of 30 strips including hsa03420: nucleotide excision repair, hsa00970: aminoacyl biosynthesis, hsa03430: mismatch repair, hsa01210: 2-oxocarboxylic acid metabolism, hsa03440: homologous recombination, hsa04360: axonal guidance, hsa00051: fructose and mannose metabolism, hsa00565: ether lipid metabolism, hsa00510: N-polysaccharide biosynthesis and hsa04110: cell cycle, hsa03030: DNA replication, hsa03013: RNA transport, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome, hsa04141: endoplasmin processing, hsa04810: regulation of actin scaffold, hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa04932: non-alcoholic fatty liver (NAFLD), hsa 20: citric acid cycle, hsa00564: glycerophospholipid, hsa 01208: amino acid biosynthesis, hsa03015: nuclear sugar metabolism monitoring, hsa 03034: hexon-sugar metabolism, hsa 01215: hexon-cholesterol metabolism, hsa 03034: eukaryotic fatty liver (NAFLD), hsa 01220: macrophage metabolism, hsa 03034: ribosome metabolism, and hsa05010: eukaryotic carbohydrate metabolism monitoring, Hsa04120 ubiquitin-mediated proteolysis, hsa05205 proteoglycans in cancer, and hsa05206 small molecules ribonucleic acids in cancer.
7) The HrpNEcb protein recognizes the combined metabolic pathways of cell junction, nerve junction, blood vessel, endocrine, reproductive system, etc.: 19 are identified for binding, hsa04723: retrograde neural signaling, hsa04726: serotonin-activated synapses, hsa00900: terpenoid biosynthetic struts, hsa04520: adhesive knots, hsa05032: morphine addiction and hsa04510: focal adhesions, hsa04724: glutamatergic synapses, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114: oocyte meiosis, hsa04728: dopaminergic neural synapses, hsa00100: steroid biosynthesis, hsa04261: adrenergic signaling of cardiomyocytes, hsa04727: neuronal synapses, hsa04725: cholinergic synapses, hsa04540: gap junctions, hsa04971: gastric acid secretion, hsa04713: diurnal entrainment, hsa04931: insulin resistance.
The HrpNECb protein, as a ligand protein molecule rich in a specific plurality of linear and conformational epitope structures, can recognize and combine various types of membrane receptors, membrane proteins, information channels and metabolic channels in a cross-boundary manner, further analyzes the positions, structures, characteristics, action mechanisms and functions of the membrane receptors, the membrane proteins, the information channels and the metabolic channels, widely influences the life basic attributes of the body such as growth, development, metabolism, defense and programmed cell death, and is widely related to diagnosis, prevention, treatment, rehabilitation, nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system and skin system diseases and conditions. The HrpNECb protein is a special multi-epitope ligand protein with brand new functions, brand new action mechanism and brand new application prospect.
The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.
<110> Wu-Bai-Ji-Wu-Bao-Zhen-Kun-Ming-Si-Sci-Tech Co Ltd
Application of <120> HrpNECb protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof
<160>
<170> Patent In Version 2.1
<210> 1
<211> 370
<212> PRT
<213>HrpNEcb（Pectobacterium betavasculorum EcbCSL101(hrpN)gene）（SEQ ID NO1）
<220>
<221> DOMAIN
<222> conserved region domains (170) - (370); a-helical domains (44) - (64), (110) - (118), (139) - (155), (174) - (192), (221) - (243), (259) - (260), (262) - (274), (276) - (279), (284) - (285), (298) - (303), (313) - (330), (347) - (349), (353) - (368); beta-sheet knob domains (10) - (15), (255) - (256); do-domains (1) - (11), (13) - (43), (67) - (95), (99) - (139), (157) - (174), (197) - (216), (340) - (341), (364) - (370).
<400> 1
Met Leu Asn Ser Leu Gly Gly Gly Thr Ser Leu Gln Ile Thr Ile Lys
5 10 15
Ala Gly Gly Asn Gly Asp Leu Phe Gln Ser Gln Ser Ser Gln Asn Gly
20 25 30
Gly Ala Pro Ser Gln Leu Gly Leu Gly Gly Gln Arg Ser Asn Ile Ala
35 40 45
Glu Gln Leu Ser Asp Ile Met Thr Thr Met Met Phe Met Gly Ser Met
50 55 60
Met Gly Gly Gly Leu Gly Gly Leu Gly Gly Met Gly Gly Gly Leu Gly
65 70 75 80
Gly Ala Leu Gly Gly Leu Gly Ser Ser Leu Gly Gly Leu Gly Gly Gly
85 90 95
Leu Leu Gly Gln Gly Leu Gly Gly Gly Leu Ala Gly Gly Leu Gly Ser
100 105 110
Ser Leu Gly Ser Gly Leu Gly Gly Ala Leu Gly Gly Gly Leu Gly Gly
115 120 125
Ala Leu Gly Ala Gly Met Asn Ala Met Asn Pro Ser Ala Met Met Gly
130 135 140
Ser Leu Leu Phe Ser Ala Leu Glu Asp Leu Leu Gly Gly Gly Met Ser
145 150 155 160
Gln Gln Gln Gly Gly Leu Phe Gly Asn Lys Gln Pro Ala Ser Pro Glu
165 170 175
Ile Ser Ala Tyr Thr Gln Gly Val Asn Asp Thr Leu Ser Ala Ile Leu
180 185 190
Gly Asn Gly Leu Ser Gln Ala Lys Gly Gln His Ser Pro Leu Gln Leu
195 200 205
Gly Asn Asn Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln
210 215 220
Leu Gly Ser Thr Leu Gly Met Gly Val Gly Gln Lys Ala Gly Leu Gln
225 230 235 240
Glu Leu Asn Asn Ile Ser Thr His Asn Gly Ser Pro Thr Arg Tyr Phe
245 250 255
Val Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met
260 265 270
Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Glu Tyr Gln Lys Asp Asn
275 280 285
Trp Gln Thr Ala Lys Gln Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser
290 295 300
Lys Pro Asp Asp Asp Gly Met Thr Lys Gly Ser Met Asp Lys Phe Met
305 310 315 320
Lys Ala Val Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly Asn
325 330 335
Thr Asn Leu Asn Ala Arg Gly Asn Gly Gly Ala Ser Leu Gly Ile Asp
340 345 350
Ala Ala Met Ile Gly Asp Arg Ile Val Asn Met Gly Leu Gln Lys Leu
355 360 365
Ser Ser
370

Claims (10)

  1. The application of the HrpNECb protein in the pharmacy for recognizing and activating various types of receptors and/or membrane proteins and signal paths thereof and causing cascade biological effects, wherein the amino acid sequence of the HrpNECb protein is shown as SEQ ID NO. 1.
  2. 2. The use of the HrpNEcb protein of claim 1 in the manufacture of a medicament for identifying and activating multiple receptors and/or membrane proteins and their signaling pathways and causing cascade biological effects, wherein said multiple receptors include one or more of LRRC 1515-leucine repeat membrane protein receptor, HLA-a major histocompatibility complex, class I, class a receptor, LGALS3BP galactose 3 binding protein (receptor), LAMP2 lysosomal associated membrane protein 2 receptor, GNB 2G guanine nucleotide binding protein subunit Beta 2 receptor.
  3. 3. The use of the HrpNEcb protein of claim 1 in the manufacture of a medicament for identifying and activating multiple classes of receptors and/or membrane proteins and their signaling pathways and causing a cascade of biological effects, wherein said membrane proteins comprise one or more of DSG4 desmoglein, ANXA4 annexin a4, CAPRIN1 cyclin, 1UTRN dystrophin, pinin desmoplan, VAMP-associated protein A, VCL vinculin, Ezrin epithelioid cadherin, PKP3 thrombospondin 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD 2N acetylated alpha linked acidic dipeptidase 2.
  4. 4. The use of the HrpNECb protein of claim 1 for identifying one or more of the receptors and/or membrane proteins and their signaling pathways that activate multiple classes of receptors and/or membrane proteins and elicit cascade biological effects, wherein the signaling pathways comprise one or more of the hsa04152: AMPK signaling pathway, hsa03460: Vanconi anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: marketing signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, and hsa04390: river horse signaling pathway.
  5. 5. The use of the HrpNEcb protein of claim 1 in the manufacture of a medicament for identifying and activating multiple classes of receptors and/or membrane proteins and their signaling pathways and causing a cascade of biological effects, wherein said signaling pathways comprise metabolic signaling pathways comprising antiviral, antibacterial, anti-foreign, anti-inflammatory metabolic pathways, including important neurological disease metabolic pathways; including nucleic acid, protein, amino acid, sugar, fat metabolism pathways; including cell junctions, nerve junctions, blood vessels, endocrine, reproductive metabolic pathways.
  6. 6. Use of the HrpNECb protein of claim 5 for identifying drugs that activate multiple classes of receptors and/or membrane proteins and their signaling pathways and elicit a cascade of biological effects, wherein the antiviral, antibacterial, anti-foreign, anti-inflammatory metabolic pathways: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa01130 biosynthesis of antibiotics, hsa05131 shigellosis, hsa04612 antigen processing and presentation, hsa05130 pathogenic E.coli infection, hsa05100 bacterial invasion of epithelial cells, hsa05132 Salmonella infection, hsa05169 Barr virus infection, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05166 HTLV-I infection, hsa 05105164 influenza A, hsa05134 legionnaire's disease, hsa05160 hepatitis C, hsa05162 measles, hsa05133 whooping cough, hsa05322 systemic lupus erythematosus, 670 hsa 0404670 leukocyte epithelial migration, hsa 46 Ammi disease, hsa 42 trypanosomiasis, and hsa05200 leukocyte epithelial migration in cancer; the important metabolic pathways for neurological diseases: hsa05012 for Parkinson's disease, hsa05016 for Huntington's chorea, hsa05010 for Alzheimer's disease; the nucleic acid, protein, amino acid, sugar and fat metabolism pathways: hsa03420: nucleotide excision repair, hsa00970: aminoacyl biosynthesis, hsa03430: mismatch repair, hsa01210: 2-oxocarboxylic acid metabolism, hsa03440: homologous recombination, hsa04360: axonal guidance, hsa00051: fructose and mannose metabolism, hsa00565: ether lipid metabolism, hsa00510: N-polysaccharide biosynthesis and hsa04110: cell cycle, hsa03030: DNA replication, hsa03013: RNA transport, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome, hsa04141: endoplasmin processing, hsa04810: regulation of the actin skeleton, hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa04932: nonalcoholic fatty liver (NAFLD), hsa00020: citric acid cycle, hsa00564: glycerophospholipid, hsa 0008: cholesterol biosynthesis, hsa 05015: biogenesis monitored, hsp metabolism, hsp 05020: eukaryotic mRNA metabolism, and hsp 05020: metabolic pathways mediated by human albumin, Hsa05205 proteoglycan in cancer, and Hsa05206 small molecule RNA in cancer; the cell junctions, nerve junctions, blood vessels, endocrine, reproductive metabolic pathways: hsa04723: retrograde neural signaling, hsa04726: serotonin-activated synapse, hsa00900: terpenoid biosynthetic backbone, hsa04520: adherent knot, hsa05032: morphine addiction and hsa04510: focal adhesion, hsa04724: glutamatergic synapse, hsa04530: tight junction, hsa00830: retinol metabolism, hsa04114: oocyte meiosis, hsa04728: dopaminergic neural synapse, hsa00100: steroid biosynthesis, hsa04261: adrenergic signaling of cardiomyocytes, hsa04727: neuronal synapse, hsa04725: cholinergic synapse, hsa04540: gap junction, hsa04971: gastric acid secretion, hsa04713: diurnal entrainment, hsa04931: insulin resistance 931.
  7. 7. The HrpNECb protein of claim 1, wherein said cascade biological effect comprises cellular processes, environmental information processing, genetic information processing, metabolism and biological system functional pathways; wherein, the cell process comprises the cell processes of transportation and catabolism, cell population, cell activity, cell growth and death and the like which are involved by a plurality of differentially expressed genes induced by HrpNECb protein; the environmental information processing comprises the steps that multiple differential expression genes induced by HrpNECb protein participate in the process of signal molecule interaction, signal transduction and membrane transportation environmental information processing; the genetic information processing comprises that a plurality of differentially expressed genes induced by HrpNECb protein participate in the biological processes of translation, replication and repair, folding, classification and degradation; metabolism includes that a plurality of differentially expressed genes induced by HrpNECb protein participate in biodegradation and metabolism, nucleotide metabolism, amino acid metabolism, auxiliary factors and vitamins of metabolism, lipid metabolism, biosynthesis and metabolism of sugar, global and overview maps, energy metabolism, carbohydrate metabolism and amino acid metabolism processes; the biological system comprises a plurality of differentially expressed genes induced by HrpNECb protein and participates in biological processes of a sensory system, a nervous system, an immune system, an excretion system, environmental adaptation, an endocrine system, a digestive system and a development circulatory system.
  8. 8. The HrpNECb protein of claim 1, wherein the HrpNECb protein is used for recognizing and activating various types of receptors and/or membrane proteins and signal paths thereof, and causing cascade biological effects, and the preparation or dosage form of the HrpNECb protein used in the pharmacy is liquid, powder, tablet or capsule.
  9. 9. The use of the HrpNEcb protein of claim 9 in the manufacture of a medicament for recognizing and activating multiple classes of receptors and/or membrane proteins and their signaling pathways and causing cascade biological effects, wherein said product or medicament is prepared from depolymerizing activated HrpNEcb protein in an amount of 0.001% to 100% by weight.
  10. 10. The method for depolymerization activation of the HrpNEcb protein according to any one of claims 1 to 9, comprising the steps of:
    step 1: pretreatment: with glucose Na2HPO4-KH2PO4The buffer solution regulates and collects the volume concentration range of high polymeric HrpNECc multi-epitope protein prepared by fermentation to be 0-30%, and the processing time is 0-24h at the temperature of 20-30 ℃;
    step 2: depolymerizing the activated HrpNEcb high polymeric multiple epitope protein: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution in the step 1 within the ultrahigh pressure range of 1000-3000 MPa;
    and step 3: and (3) post-treatment: after the operations of ultrahigh pressure depolymerization and activation are finished, standing for 0-24h at 35-38 ℃, and then collecting depolymerized and activated multiple epitope protein molecules of HrpNECb;
    and 4, step 4: and purifying the high-aggregation HrpNECb multi-epitope protein-His-Tag recombinant protein by using chromatography gel to obtain a depolymerized and activated multi-epitope protein HrpNECb original drug.
CN202011633933.6A 2020-12-31 2020-12-31 Application of HrpNECb protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof Pending CN112675293A (en)

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PCT/CN2021/134714 WO2022142976A1 (en) 2020-12-31 2021-12-01 Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals
AU2021412922A AU2021412922A1 (en) 2020-12-31 2021-12-01 Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals

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WO2022142977A1 (en) * 2020-12-31 2022-07-07 吴伯骥 Use of hrpz-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals
WO2022142976A1 (en) * 2020-12-31 2022-07-07 昆明锐斯得科技有限公司 Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals

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DE10147028A1 (en) * 2001-09-25 2003-04-17 Gruenenthal Gmbh Identifying agents for treatment and diagnosis of diseases, e.g. depression or viral infections, from binding to inorganic phosphate transporters, also new agents
CN107383184A (en) * 2011-06-24 2017-11-24 哈格－斯特莱特医疗科技股份公司 photosensitive chimeric GPCR protein
CN109355269A (en) * 2018-10-21 2019-02-19 北京双因生物科技有限公司 Sphingosine kinase 1 and its fusion protein and application thereof

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CN107383184A (en) * 2011-06-24 2017-11-24 哈格－斯特莱特医疗科技股份公司 photosensitive chimeric GPCR protein
CN109355269A (en) * 2018-10-21 2019-02-19 北京双因生物科技有限公司 Sphingosine kinase 1 and its fusion protein and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022142977A1 (en) * 2020-12-31 2022-07-07 吴伯骥 Use of hrpz-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals
WO2022142976A1 (en) * 2020-12-31 2022-07-07 昆明锐斯得科技有限公司 Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals

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