CN114685681A

CN114685681A - Application of HrpNECh protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal pathways thereof

Info

Publication number: CN114685681A
Application number: CN202011633925.1A
Authority: CN
Inventors: 吴伯骥; 吴保珍
Original assignee: Kunming Rsd Technology Co ltd
Current assignee: Kunming Rsd Technology Co ltd
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-07-01

Abstract

The invention discloses an application of an HrpNECh 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 HrpNECh protein is shown as SEQ ID NO. 1. The HrpNECh protein is used as a ligand protein molecule with a special structure rich in a plurality of epitopes (linearity and conformation), can recognize, activate and combine various membrane receptors, membrane proteins, information channels and metabolic channels of animals in a cross-boundary way, and is a special multi-epitope ligand protein with brand new functions, brand new action mechanisms and brand new application prospects.

Description

Application of HrpNECh protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal pathways thereof

Technical Field

The invention relates to the field of biomedicine, in particular to application of an HrpNECh 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 and 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 the pharmacological actions of proteins and classified protein drugs into four major categories as follows: 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 future 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 HrpNECh Protein (Genebank Protein: AAY17519.1) is a secreted Protein of Erwinia chrysanthemi (Dickeya dadantii) strain NECHCL 006, an expression product of the hrpNECh gene (gene accession number: Genbank: nucleotide: AY 999000.1). It is composed of 339 amino acid (amino acids) residues, has one, two, three-stage structure but no four-stage structure, contains no cystine and cysteine, and is rich in glycine; theoretical isoelectric point/molecular weight (Theoretical pI/Mw) 6.07/34146.22; the conserved domain 9-334, alpha-helix structure 39-62, 105-118, 131-134, 147-163, 192-213, 233-245, 268-273; beta-sheet structures 2-7, 204-205, do-structures 1-2, 8-11, 13-40, 66-100, 131-139, 173-177, 339.

The structural domain is a region with a specific structure and an independent function in a biological macromolecule, 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-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 sheet is formed by laterally gathering two or more extended polypeptide chains (or a plurality of peptide segments of one polypeptide chain), and a zigzag sheet structure is formed by regular hydrogen bonds between N-H and C ═ O on the main chains of the adjacent polypeptide 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 HrpNEch proteins are multidomain proteins, form a special structure of multiple linear and conformational epitopes, and different domains are often associated with different functions, thereby determining their application in pharmaceuticals for recognizing and activating various types of receptors, membrane proteins, and signaling and metabolic pathways in animals and causing multifunctional cascade biological effects. However, there is no report on this.

Disclosure of Invention

The invention aims to: in view of the existing problems, the invention provides an application of the HrpNECh protein in the pharmacy for recognizing and activating various receptors and/or membrane proteins and signal paths thereof and causing cascade biological effects.

The technical scheme adopted by the invention is as follows:

the application of the HrpNECh protein in the pharmacy for recognizing and activating various receptors, membrane proteins and signal paths thereof of animals and causing multifunctional cascade biological effects, wherein the amino acid sequence of the HrpNECh protein is shown as SEQ ID NO. 1.

The HrpNECh 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 strong hydrogen bond with a cysteine recognition region Pam3CSK4 of a receptor; polar uncharged amino acid residues: serine forms a tight binding surface or complex with multiple types of receptors through polar and strong hydrogen bonds.

Further, the full sequence of the HrpNEch protein has 339 amino acid residues, wherein the critical amino acid residues are 236: 106 hydrophobic nonpolar amino acid residues, 40 polar uncharged amino acid residues, 42 amido amino acid residues, 48 acidic positively charged and basic negatively charged amino acid residues, and the key amino acid accounts for 69 percent of the total sequence; the protein sequence of the HrpNECh conserved structural region has 326 amino acid residues, 226 key amino acid residues, 99 hydrophobic nonpolar amino acid residues, 40 polar uncharged amino acid residues, 40 amido amino acid residues, 47 acidic positively charged and basic negatively charged amino acid residues, and the key amino acid accounts for 69 percent in the conserved structural region; the HrpNECh alpha-helix structural region has 7 alpha-helices, 2 beta-sheets and 7 do-structural regions, wherein the alpha-helix region has 100 amino acid residues, 71 key amino acid residues, 35 hydrophobic nonpolar amino acid residues, 12 polar uncharged amino acid residues, 13 amido amino acid residues and 11 acidic positively charged and basic negatively charged amino acid residues, and the amino acids in the alpha-helix structure account for 71 key amino acids; further, the inventors screened, cloned and prepared hypersensitive proteins such as HrpNECc, HrpNECa, HrpNECb, HrpNECh, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNECa, HrpNSam, HrpNBag, HrpNPas, HrpNECnt and the like from Erwinia and Pseudomonas, analyzed the molecular structures according to bioinformatics, and the hypersensitive proteins have similar structural characteristics, structural evolution trends and structures with a plurality of conformational epitopes and linear epitope structures similar to those of the multi-epitope HrpNECh 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, a polar uncharged amino acid residue: 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; furthermore, the amino acid residues (generally referred to as key amino acid residues) of the HrpNEch protein are not limited to the amino acid residues, and can realize 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 a complex with multiple types of receptors, cause the change of the conformation, energy, electrical property and information of the receptor molecules, and 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, protein binding, protein binding, 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 plurality of receptor proteins include GNG12 guanine nucleotide binding protein gamma-12 receptor, ANXA5 annexin a5 receptor, ANXA2 annexin a2 receptor, ANXA1 annexin a1 receptor, IGHG2 immunoglobulin heavy constant gamma 2 receptor, IGHM immunoglobulin heavy constant Mu receptor, CACNA1S calcium voltage-gated channel subunit alpha 1S receptor, ZNF185 zinc finger protein 185 receptor, and HLA-a major histocompatibility complex, class I, class a receptor, LAMP2 lysosomal associated membrane protein 2 receptor, GNB 2G guanine nucleotide binding protein subunit beta 2 receptor, KTN1 driver binding protein 1 receptor.

Preferably, the membrane proteins include DSC3 desmoglein, ANXA8/ANXA8L1 annexin a 8/annexin a 8-like protein 1, EVPL epifocal protein, POF1B actin-binding protein premature ovarian failure 1B, CTNNA1 catenin, TGM1 transglutaminase 1, BAIAP2 BAI 1-associated protein 2, RAB29 RAS oncogene family member, CLDN19 delrin 19, STXBP2 synaptic fusion protein binding protein 2, VAMP vesicle-associated membrane protein A, VCL focal adhesion protein, Ezrin ezetimibe-epitheliosis calcium adhesin, PKP3 platelet affinity protein 3, NAALAD 2N acetylated alpha linked acidic dipeptidase 2, PKP1 platelet affinity protein 1, SPRR1A proline-rich miniprotein 1A.

Preferably, the signaling pathways include 22 signaling pathways hsa03320: PPAR signaling pathway, hsa05120: signal transduction of helicobacter pylori-infected epithelial cells, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04070: phosphatidylinositol signaling system, hsa04010: MAPK signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: camp signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, hsa04390: river horse signaling pathway, hsa04922: glucagon signaling pathway, hsa04912: gonadotropin signaling pathway, hsa04022: PKMP-MP-G signaling pathway, hsa04921: oxytocin signaling pathway, hsa 0404040404723: generation signaling pathway, reverse nerve growth signaling pathway, cGA 04066: cGA 0401: cGA 04020 signal pathway.

Preferably, the metabolic pathways comprise 29 antiviral, antibacterial, anti-foreign, anti-inflammatory related metabolic pathways: hsa04144: endocytosis, hsa04145: phagosome, hsa04142: lysosome, hsa04666: fc-r mediated phagocytosis, 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: bal virus infection, hsa05203: viral carcinogenesis, hsa05134: legionnaire's disease, hsa05160: hepatitis c, hsa05162: measles, hsa05133: pertussis, hsa05322: systemic lupus erythematosus, hsa04670: migration of leukocytes across endothelium, hsa05152: tuberculosis, hsa05150: staphylococcus aureus infection, hsa05146: amebiasis, hsa05142: trypanosomiasis nana, hsa05200: in cancer pathways, hsa05143: african trypanosomiasis, hsa04750: inflammatory mediator modulation of TRP channels, hsa04916: bactericidal action, hsa05230: central carbon metabolism in cancer, hsa05214: glioma, hsa05212: pancreatic cancer; comprises 3 important metabolic channels for neurological diseases: hsa05010 Alzheimer's disease, hsa05012 Parkinson's disease, hsa05016 Huntington's chorea; comprises 39 related pathways of nucleic acid, protein, amino acid, sugar and fat metabolism: hsa03013 RNA transport, hsa03018 RNA degradation, hsa03040 splice, hsa03010 ribosome, hsa04141 endoplasmin processing, hsa04810 actin-mediated regulation of the actin skeleton, hsa03050 proteasome, hsa01230 amino acid biosynthesis, hsa00190 oxidative phosphorylation, hsa00230 purine metabolism, hsa04932 non-alcoholic fatty liver, hsa00020 citric acid cycle, hsa03008 eukaryotic ribosome biogenesis, hsa00240 pyrimidine metabolism, hsa 50 Butanoate metabolism, hsa01200 carbon metabolism, hsa00020 amino sugar and nucleotide sugar metabolism, hsa 0434 alcoholism, hsa00071 fatty acid degradation, hsa04120 protein hydrolysis mediated by hsa 030726, hsa05205 proteoglycan in cancer, small molecule nucleic acid 00505206 pillar nucleic acid in cancer, 00400410 a 00310 alanine, histidine 00310, histidine 000450 histidine 00310, threonine synthase 00000450, histidine phagostimulation, hsp 000450 and histidine 003900, hsa04610 complement and coagulation cascade, hsa00330 arginine and proline metabolism, hsa04520 adherent nodes, hsa00860 porphyrin and chlorophyll metabolism, hsa00010 glycolysis and glyconeogenesis, hsa00982 drug metabolism-cytochrome P450, hsa00980 cytochrome P450 metabolism to exogenous drugs, hsa04962 vasopressin regulated water reabsorption, hsa00983 drug metabolism-other enzymes; and 34 cell junctions, nerve junctions, vascular, endocrine, reproductive metabolic pathways: hsa04510 focal adhesion, hsa 04724 glutamatergic synapses, hsa04530 tight junctions, hsa00830 retinol metabolism, hsa04114 oocyte meiosis, hsa04728 dopaminergic synapses, hsa00140 steroid hormone biosynthesis, hsa04261 adrenergic signaling of cardiomyocytes, hsa04727 gamma-aminobutyric synapses, hsa04725 cholinergic synapses, hsa04540 gap junctions, hsa04971 gastric acid secretion, hsa04713 diurnal entrainment, hsa04931 insulin resistance, hsa05031 amphetamine addiction, hsa04924 renin secretion, hsa04925 aldosterone synthesis and secretion, hsa00590 arachidonic acid metabolism, hsa04270 vascular smooth muscle contraction, hsa00760 nicotinic acid and nicotinamide metabolism, hsa04740 olfactory transduction, hsa04260 olfactory contraction, hsa04720 olfactory dysgenosis, hsa04720 photoperiodic tone 05744, right ventricular hypertrophy, right ventricular end tract 05410 cardiac arrhythmia 056, right ventricular hypertrophy collection, right ventricular hypertrophy and cardiac arrhythmia 05410, hsa04146 peroxisome, hsa05414 dilated cardiomyopathy, hsa04970 salivary secretion, hsa04611 platelet activation, hsa05204 chemocarcinogenesis, and hsa04721 synaptic vesicle cycling.

Preferably, the cascade biological effect includes functional pathways such as Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism, and biological Systems; further, the Cellular process (Cellular Processes): multiple differential expression genes induced by the HrpNECh protein participate in cell processes such as transportation, catabolism, cell population, cell activity, cell growth and death and the like; environmental Information Processing (Environmental Information Processing) that multiple differentially expressed genes induced by the HrpNECh 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 the HrpNECh 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 the HrpNECh protein participate in the metabolic processes such as biodegradation and Metabolism, nucleotide Metabolism, Metabolism of other amino acids, metabolic auxiliary factors and vitamins, lipid Metabolism, biosynthesis and Metabolism of sugar, global and overview maps, energy Metabolism, carbohydrate Metabolism, amino acid Metabolism and the like; multiple differential expression genes induced by HrpNECh 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 (HrpNEch protein preparations and/or drugs) for HrpNEch 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 HrpNEch 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 include, 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 . 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 mainly prepared from depolymerized and activated HrpNECh protein, and the mass content is 0.001-100%.

The preparation method for producing and purifying the HrpNECh protein by depolymerizing and activating the high-polymerization-state HrpNECh protein comprises the following steps:

1. pretreatment: with glucose Na₂HPO₄-KH₂PO₄The buffer solution is used for regulating and collecting the volume concentration range of the high polymeric HrpNECh multi-epitope protein prepared by 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 at a concentration of 10-15%; 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 2500-1000 mmol; 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 operations of ultrahigh pressure depolymerization and activation are 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 depolymerized activated HrpNEch multi-epitope protein molecules.

4. And (3) purifying the high-aggregation HrpNECh 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 HrpNECh original drug.

The route of use of the HrpNEch 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 HrpNEch multi-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 may be prepared, typically, to patients prepared as approved by regulatory agencies or according to generally recognized pharmacopoeias.

The multifunctional cascade biological effect and the diversity function caused by the HrpNECh multi-epitope ligand proteins which are used for identifying and activating various receptors, membrane proteins and signal paths thereof of animals and inducing the multifunctional cascade biological effect widely relate to the diagnosis, prevention, treatment, rehabilitation and the application of food, apotype, cosmetic, mechanical and health word products or medicines for various systems, tissues, organs and cells related diseases and conditions.

The invention relates to the application of products or medicines of HrpNECh multi-epitope ligand proteins which are used in pharmacy and are used for identifying and activating various receptors, membrane proteins and signal paths thereof of animals and inducing multifunctional cascade biological effects in diagnosing, preventing, treating or rehabilitating diseases and conditions of nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system and reproductive system:

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.

Preferably, the preparation of the HrpNECh multi-epitope ligand protein which can identify and activate various types of receptors, membrane proteins and signal paths thereof of animals and induce multifunctional cascade biological effects comprises the following steps:

1. the preparation of the HrpNECh multi-epitope ligand protein can separate and purify the HrpNECh protein from secreted protein of Erwinia chrysanthemi (Dickeya dadantii) NECCSCL 006 strain collected from the Sword Jitsu village in Sichuan, can be carried out by adopting a conventional protein separation and purification method according to the specific molecular weight of the HrpNECh protein, and collects a depolymerized and activated HrpNECh purified protein product by the established depolymerization and activation technology of the high polymeric HrpNECh multi-epitope protein molecule.

2. The preparation of the HrpNECh multi-epitope ligand Protein can also adopt engineering bacteria of our registered HrpNECh Protein (Genebank Protein: AAY17519.1) gene (gene registration number: Genbank: nucleotide: AY999000.1), and depolymerize the activated HrpNECh Protein through fermentation, purification and collection:

1) and (3) fermentation preparation of the engineering bacteria of the HrpNECh 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 HrpNECh 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. Analyzing the expression product of the HrpNECh protein by using 10% SDS-PAGE polyacrylamide gel electrophoresis, wherein a 34.15kda strip is shown on a sample lane of an electrophoresis gel plate and is the expression product of the gene hrpNECh protein;

wherein the fermentation medium is Na₂HPO₄-KH₂PO₄A buffer system, wherein the pH range of the buffer system is 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 culture time of fermentation induction liquid 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 production line of the HrpNECh protein genetic engineering strain is post-treated after the production and fermentation of the multi-epitope protein are finished: firstly, sterilizing fermentation liquor at 80 ℃ for 30 minutes, and rapidly cooling to below 30 ℃; ② glucose Na for cleaning₂HPO₄-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 a buffer solution, adjusting the fresh weight of the thallus to be 20% -30% of the diluent, introducing into a high-pressure crusher, continuously crushing the engineering bacteria with the pressure of 800-;

3) depolymerization and activation of high polymeric HrpNECh multi-epitope protein molecules (I) pretreatment with glucose Na₂HPO₄-KH₂PO₄Buffer solution adjustment fermentation collected high polymerization state HrpNECh multi-tableThe volume concentration range of the metaprotein 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 10-15%. 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 and activating the HrpNECb high polymeric multi-epitope protein to carry out ultrahigh pressure depolymerization and activation on the pretreated high polymeric protein pretreatment solution, wherein the ultrahigh pressure range is 1000-3000MPa, and preferably 3000 MPa; preferably 1500 Mpa; preferably 2500 Mpa; preferably 2000 Mpa; most preferably 2000-2500 Mpa;

(III) after finishing the operation of the post-treatment ultrahigh pressure depolymerization and activation, standing for 0-24h at 35-38 ℃, preferably for 0-1 h; preferably for 24-10 h; preferably for a time of 1-2 hours; preferably for 10-4 hours; most preferably for 2-4h, and then collecting the deagglomerated activated HrpNEch polyepitopic protein molecules.

(IV) purifying the high polymeric HrpNECh multi-epitope protein-His-Tag recombinant protein by 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 HrpNECh protein is completed.

3. The preparation of the HrpNECh multi-epitope ligand protein, further, the HrpNECh protein can also be prepared by expression protein of 'artificially synthesized gene', and the preparation method specifically comprises the following steps:

artificial synthesis of hrpNECh gene for coding HrpNECh protein and preparation of its expression protein

1) The hrpNECh gene nucleotide sequence of the coded HrpNECh protein published to GenBank according to modern bioinformatics is used as the artificial synthetic HrpNECh multi-epitope protein gene, and the DNA sequence of the HrpNECh multi-epitope protein gene is from Genbank: nucleotide AY 999000.1.

Cloning of gene encoding HrpNEch protein:

according to the DNA sequence of hrpNECh gene hrpNECh, the DNA sequence is as follows:

cloning the entire gene design and using primers (BamHI and HindIII sites underlined, respectively):

5’-tgcggatccatgcaaattacgatcaaagcgcac-3’

5’-tgcaagctttcaggcgttggcagctaccagcga-3’

amplifying a DNA fragment of a required test coding HrpNECh protein hologene by using high-fidelity Taq enzyme, and carrying out PCR amplification according to a method suggested by a high-fidelity Taq enzyme manufacturer;

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 HrpNECh 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) fermentation preparation of the engineering bacteria of the HrpNECh 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 HrpNECh proteins 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 HrpNECh protein, and a 34.15kda band appears on a sample lane of the electrophoresis gel plate, which is the expression product HrpNECh protein of the gene hrpNECh.

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 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 production line of the HrpNECh protein engineering strain is post-treated after the production and fermentation of the multi-epitope protein are finished: firstly, sterilizing fermentation liquor at 80 ℃ for 30 minutes, and rapidly cooling to below 30 ℃; ② glucose Na for cleaning₂HPO₄-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, 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 HrpNECh multi-epitope protein molecules.

6) Depolymerization and activation of high polymeric HrpNECh polyepitope protein molecule (I) pretreatment with glucose Na₂HPO₄-KH₂PO₄The buffer solution adjusts the volume concentration range of the high polymeric HrpNECh 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) depolymerizing and activating the HrpNECh high-polymerization-state multi-epitope protein, and carrying out ultrahigh pressure depolymerization and activation on the pretreated high-polymerization-state protein pretreatment solution within the ultrahigh pressure range of 1000-3000MPa, preferably 3000 MPa; preferably 1500 Mpa; preferably 2500 Mpa; preferably 2000 Mpa; most preferably 2000-;

(III) after finishing the operation of the post-treatment ultrahigh pressure depolymerization and activation, 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 deagglomerated activated HrpNEch polyepitopic protein molecules.

Compared with the prior art, the invention has the beneficial effects that:

the HrpNECb protein is a ligand protein molecule which is rich in a plurality of linear and conformational epitope special structures and can cross-boundary recognize, activate and combine a plurality of types of animal membrane receptors, membrane proteins, information channels and metabolic channels, the HrpNECh protein is a ligand protein with a special multi-epitope structure, brand-new functions, brand-new action mechanisms and brand-new application prospects, and can induce multidirectional, multilevel and multifaceted biological effects and functions, and is widely applied to diagnosis, prevention, treatment, rehabilitation and pharmaceutical application of related diseases and conditions such as multisystem, multiorgan and multicellular related diseases and conditions, food, word elimination, makeup, mechanical word and health word products or medicines.

Drawings

FIG. 1 shows the electrophoretic detection before and after disaggregation of the HrpNECh protein: the molecular weight marker band is on the left, 1: depolymerizing, activating and purifying a polyepitope ligand protein HrpNECh band; 2: depolymerizing, activating and purifying the purified multi-epitope ligand protein HrpNECh band;

FIG. 2 is a graph of allergic reaction of tobacco leaves induced by HrpNECh protein solution injection, wherein the focal spot is formed by Harpinech protein solution treatment for about 24hr, and the leaves are arranged on the upper row: h₂O injection, control; blade lower row: harpinech protein solution (250 μ g/ml) was injected for treatment, i.e., hypersensitivity reaction of Harpinech protein on tobacco leaves;

FIG. 3 shows that the HrpNECh protein of the present invention is orally administered and smeared to induce the expression of the kidney differential gene volcano, and the administration is carried out for 6h and 24h from left to right; smearing for 6 h;

fig. 4 is a diagram of the expression differential gene volcano of testis induced by oral administration and smearing of the hrpech protein of the invention, which is orally administered for 6h and orally administered for 24h from left to right; smearing for 6 h;

FIG. 5 is a cluster chart of the gene cluster of the difference of kidney expression induced by oral administration of the HrpNECh protein and smearing of the experimental mice, wherein the oral administration is carried out for 6 hours and the oral administration is carried out for 24 hours from left to right; smearing for 6 h;

FIG. 6 is a cluster chart of the gene cluster of the difference of the oral administration of the HrpNECh protein and the testis expression induced by smearing on experimental mice, wherein the oral administration is carried out for 6 hours and the oral administration is carried out for 24 hours from left to right; smearing for 6 h;

FIG. 7 shows a comparison of the HrpNECh protein-treated kidney of the present invention with a control KEGG Pathway (total gene) which is orally administered for 6 hours and orally administered for 24 hours from left to right; smearing for 6 h;

FIG. 8 shows a comparison of the HrpNECh protein-treated kidney of the present invention with a control KEGG Pathway (up-regulated gene) which is orally administered for 6 hours and orally administered for 24 hours from left to right; smearing for 6 h;

FIG. 9 shows a comparison of the HrpNECh protein-treated kidney of the present invention with a control KEGG Pathway (downregulated gene), which is orally administered for 6h and orally administered for 24h from left to right; smearing for 6 h;

FIG. 10 shows a comparison of the HrpNECh protein-treated testis of the present invention with a control KEGG Pathway (total gene) which is orally administered for 6 hours and orally administered for 24 hours from left to right; smearing for 6 h;

FIG. 11 shows a comparison of the HrpNECh protein-treated testis of the present invention with a control KEGG Pathway (up-regulated gene) which is orally administered for 6 hours and orally administered for 24 hours from left to right; smearing for 6 h;

FIG. 12 shows a comparison of the HrpNECh protein-treated testis of the present invention with a control KEGG Pathway (downregulated gene), which is orally administered for 6h and orally administered for 24h from left to right; smearing for 6 h;

FIG. 13 is a flowchart of the mRNA (RNA-Seq) sequencing experiment according to the present invention;

FIG. 14 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 method comprises the following steps of fermenting the HrpNECh multi-epitope ligand protein by adopting engineering bacteria of registered genes, purifying, preparing and collecting depolymerized and activated HrpNECh protein:

1) and (3) fermentation preparation of the engineering bacteria of the HrpNECh 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 HrpNECh proteins, wherein the production line of related proteins is special modified derivative bacteria JY-01(DE3) of K-12 original bacteria, IPTG (Isopropyl thiogalactoside, isopropy beta-D-thiogalactoside) is added when the bacteria are cultured in LB liquid medium (containing 50 micrograms of kanamycin per liter) under the condition of certain temperature until OD600 is 0.7) (final concentration: 1mMol), further culturing, and then centrifuging to collect the cells. Analyzing the expression product of the HrpNECh protein by using 10% SDS-PAGE polyacrylamide gel electrophoresis, wherein a 34.15kda strip is shown on a sample lane of an electrophoresis gel plate and is the expression product of the gene hrpNECh protein; 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, 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 high-polymerization-state HrpNECh multi-epitope protein molecules.

3) Depolymerization and activation of high polymeric HrpNECh multi-epitope protein molecules: preprocessing: with glucose Na₂HPO₄-KH₂PO₄Adjusting the volume concentration of the high-polymerization-state HrpNECh multi-epitope protein collected by fermentation to 10-15% by using buffer solution, and pretreating glucose Na at normal temperature (20-30 deg.C)₂HPO₄-KH₂PO₄Buffer solution, pH 5-5.5, glucose concentration 200-. (ii) depolymerizing and activating the HrpNECh 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; ③ ultra-high pressure decompositionStanding for 2-4h at 35-38 ℃ after the polymerization and activation operations are completed, and then collecting the depolymerized and activated HrpNECb multi-epitope protein molecules; and fourthly, purifying the high-aggregation multi-epitope protein-His-Tag recombinant protein by NI-NTA affinity chromatography gel, and performing protein purification according to a method suggested by NI-NTA affinity chromatography gel manufacturers to finish the preparation of the depolymerized and activated purified HrpNECh protein.

Example 2

The HrpNECh 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 hrpNECh gene coding the HrpNECh protein;

1) the hrpNECh gene nucleotide sequence of encoding the HrpNECh protein published to GenBank according to modern bioinformatics is used as artificially synthesized HrpNECh multi-epitope protein gene, and the DNA sequence of the hrpNECh multi-epitope protein gene is from Genbank: nucleotide AY 999000.1.

Cloning of the gene encoding the HrpNEch protein:

clone the entire gene design and use primers (BamHI and HindIII sites underlined, respectively):

5’-tgcggatccatgcaaattacgatcaaagcgcac-3’

5’-tgcaagctttcaggcgttggcagctaccagcga-3’

the second step: 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 HrpNECh 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 through DNA sequencing;

the fourth step: transferring the gene clone of the HrpNECh protein coded in the steps 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-thiogalactoside) (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 an expression product HrpNECh protein, a 34.15kda band appears on a sample lane of an electrophoresis gel plate, and the HrpNECh protein is the expression product of the gene hrpNECh and is shown in figure 1 in detail;

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 HrpNECh polyepitope protein molecule (I) pretreatment with glucose Na₂HPO₄-KH₂PO₄The volume concentration of the collected and purified high polymeric HrpNECh polyepitope protein is adjusted by buffer solution and is 10-15%. Under the condition of normal temperature (20-30 ℃), the method is simple and convenientBy pretreating 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 HrpNECh 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 finishing the operation of the post-treatment ultrahigh pressure depolymerization and activation, standing for 2-4h at 35-38 ℃, and then collecting the depolymerized and activated HrpNECh multi-epitope protein molecules.

(IV) purifying the high polymeric HrpNECh multi-epitope protein-His-Tag recombinant protein by NI-NTA affinity chromatography gel, wherein the protein purification is implemented according to the method suggested by NI-NTA affinity chromatography gel manufacturers, and the purification preparation of the depolymerized and activated multi-epitope protein HrpNECh 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, including a 34.15kda band; the band at position 2 is a band of depolymeration-activated purified HrpNECh protein, which has a molecular weight of 34.15kda and is located in the region of the corresponding molecular weight of the ligand protein, indicating that the corresponding depolymeration-activated purified HrpNECh protein has been obtained.

As shown in fig. 2, allergy assay detection of deagglomerated activated polyepitope ligand protein HrpNEch: wherein the focal spot is formed by processing Harpinech protein liquid for about 24hr, and the blades are arranged in rows: h₂O injection, control; blade lower row: harpinech protein solution (250. mu.g/ml) was injected as a treatment, i.e., a hypersensitivity reaction of Harpinech protein on tobacco leaves.

The depolymerized and activated polyepitope ligand protein can generally trigger hypersensitive reaction 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 experiment selects 8-week-old balb/C experimental mice, which are divided into HrpNECh protein treatment groups, wherein the HrpNECh protein treatment groups comprise 4 treatments of 6 hours and 24 hours of oral administration and 6 hours and 12 hours of smearing, and 3 experimental mice are treated in each treatment group, and the total number of the experimental mice is 12; blank control group 4 experimental mice; the buffer solution control sham operation group without the HrpNECh protein comprises 4 treatments of oral administration for 6 hours and 24 hours and smearing for 6 hours and 12 hours, wherein each treatment comprises 4 experimental mice, 16 experimental mice are counted, and the three treatments are repeated; 600 mg.L for experimental treatment group mice^-1Feeding and smearing HrpNECh protein buffer solution with concentration, feeding and smearing buffer solution on mice in a buffer control sham operation group, and not performing any treatment on mice in a blank control group. Under the same feeding condition, tissues such as mouse testis, kidney and the like are respectively taken in groups according to different time, and RNA-Seq sequencing and analysis are carried out.

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. 13.

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 Bioanalyzer4200(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

2.0Fluorometer assay concentration, Agilent2100 assay size.

Computer sequencing

And performing Illumina sequencing on the library qualified by quality inspection, and acquiring sequence information of the fragment to be tested by capturing a fluorescent signal and converting an optical signal into a sequencing peak through computer software by using a sequencer.

Mrna sequencing data analysis was performed according to the data analysis flow of fig. 14.

3. Analysis of results

1) Screening of HrpNECh protein-induced differential genes

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) HrpNECh protein-induced differential gene volcano plot

And (3) displaying the overall distribution condition of the HrpNECh protein-induced expression difference significant genes by using a difference gene volcanic chart. 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-point expression significantly up-regulated genes; left lateral point expression significantly down-regulated genes; lower spots expressed genes that did not significantly change. FIGS. 3-4 are graphs of the differential gene volcanic patterns of mouse testis, kidney HrpNECh protein induced by oral administration and smearing, respectively, wherein HarpinNECh is abbreviated as N2.

3) HrpNECh 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 (scale number) and clustered, with red indicating high expression and blue indicating low expression in the heatmap. FIGS. 5-6 are testis and kidney expression differential gene clustering heatmaps, respectively, in which HarpinNECh is abbreviated as N2.

4) Enrichment analysis of differential gene GO induced by HrpNECh protein

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 HrpNECh protein proves that the HrpNECh protein has a plurality of epitope special structures, brand new functions, brand new action mechanisms and ligand proteins with brand new application prospects, and induces differential expression of multiple genes of multiple organs (testis and kidney) of a mouse, and the differential expression genes cover biological processes, cell components and molecular functions. The results of the enrichment analysis of the HrpNEch 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 4.② 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 analysis results are detailed in tables 1 to 4. (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 4.

Gene Ontology (GO) is an Ontology widely used in bioinformatics to cover the Goterms taxonomic Gene statistics tables 1-4 with p-value less than 0.05 for three levels of biological cellular components, molecular functions, and biological processes. Harpinnech multi-epitope ligand protein induced biological processes, cellular components and molecular function related functional groups of kidney and testis to significantly express GO terms classifier gene statistics (oral 6, 24 hours and smeared 6 hours).

In all tables 1-4, blank space indicates that no relevant data is collected, and the following and all tables have the same blank meaning.

TABLE 1 HarpinNECh protein induces biological processes, cellular components and functional groups related to molecular function of kidney significantly up-regulated expression of GO terms classifier gene statistics (6, 24 hours oral and 6 hours smeared)

TABLE 2 Harpinnecb protein induces biological processes, cellular components and molecular function-related functional groups in testis significantly up-regulated expression GO terms classifier gene statistics (6, 24 hours for oral administration and 6 hours for smearing)

TABLE 3 Harpinnecb protein induces biological processes, cellular components and molecular function related functional groups of kidneys significantly downregulate the expression of GO terms classifier gene statistics (6, 24 hours oral and 6 hours smeared)

TABLE 4 Harpinnecb protein induces biological processes, cellular components and functional groups related to molecular function of testis to significantly downregulate expression of GO terms classifier gene statistics (6, 24 hours for oral administration and 6 hours for smearing)

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 HrpNECh 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 HrpNECh 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 (testis and kidney) of a mouse, and the differential expression genes participate in functional pathways belonging to Cellular Processes (Cellular Processes), Environmental Information Processing (Environmental Information Processing), Genetic Information Processing (Genetic Information Processing), Metabolism (Metabolism) and organism Systems (organic Systems). The results of the enrichment analysis of the HrpNEch protein-induced differential gene GO are further described as follows: (ii) Cellular Processes (Cellular Processes): the multiple differentially expressed genes induced by the HrpNEch protein are involved in cellular processes such as trafficking and catabolism, cell population, cell activity, cell growth and death (see fig. 7-12 for details). Environmental Information Processing (Environmental Information Processing) multiple differentially expressed genes induced by the HrpNECh protein participate in the Environmental Information Processing processes such as signal molecule interaction, signal transduction, membrane transport and the like (see figures 7 to 12 for details). Genetic Information Processing (Genetic Information Processing) several differentially expressed genes induced by the HrpNEch protein are involved in the biological processes of translation, replication and repair, folding, classification and degradation (see fig. 7 to 12 for details). Metabolism (Metabolism) the various differentially expressed genes induced by the HrpNEch 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. 7 to 12 for details). Organism Systems (organic Systems) multiple differentially expressed genes induced by the HrpNECh protein are involved in cell processes such as sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, and developmental circulatory system (see FIGS. 7-12).

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. 7-12.

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 HrpNECh protein

1. Sample preparation and processing

1) Purification of HrpNECh protein

The high polymeric HrpNECh multi-epitope protein-His-Tag recombinant protein is purified by NI-NTA affinity chromatography gel, 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 HrpNECh is completed for standby (hereinafter referred to as capture protein or target protein).

2) Extraction of total protein (bait protein) from cultured liver cells 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 5 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 incubate Spin Column tubes for 1 minute at room temperature. The top cap and bottom plug were removed, and the Spin Column tubes were placed on collection tubes and centrifuged at 1250 Xg 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 of1 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 HrpNEch protein recognizes bound cell membrane receptors: recognizing the binding of 12 membrane receptors, GNG12 guanine nucleotide binding protein gamma-12 receptor, ANXA5 annexin a5 receptor, ANXA2 annexin a2 receptor, ANXA1 annexin a1 receptor, IGHG2 immunoglobulin heavy constant gamma 2 receptor, IGHM immunoglobulin heavy constant Mu receptor, CACNA1S calcium voltage-gated channel subunit alpha 1S receptor, ZNF185 zinc finger protein 185 receptor, and HLA-a major histocompatibility complex, class I, class a receptor, LAMP2 lysosomal associated membrane protein 2 receptor, GNB 2G guanine nucleotide binding protein subunit beta 2 receptor, KTN1 driver binding protein 1 receptor.

2) The HrpNEch protein recognizes the bound cell membrane protein: recognizes binding to 16 membrane proteins, DSC3 desmoglein, ANXA8/ANXA8L1 annexin a 8/annexin a8 analogous protein 1, EVPL coat plaque protein, POF1B actin binding protein premature ovarian failure 1B, CTNNA1 catenin, TGM1 transglutaminase 1, BAIAP2 BAI1 associated protein 2, RAB29 RAS oncogene family member, CLDN19 arrestin 19, STXBP2 synapsin binding protein 2, VAMP vesicle associated membrane protein associated protein A, VCL focal adhesion protein, Ezrin ezetizotzin, PKP3 platelet affinity protein 3, NAALAD 2N acetylated alpha linked acidic dipeptidase 2, PKP1 platelet affinity protein 1, SPRR1A proline-rich miniprotein 1A.

3) The HrpNEch protein recognizes the bound signaling pathway: the recognition binding 22 strips include hsa03320: PPAR signaling pathway, hsa05120: signal transduction of helicobacter pylori-infected epithelial cells, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04070: phosphatidylinositol signaling system, hsa04010: MAPK signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: chemokine signaling pathway, hsa 1 signaling pathway, hsa04024: fastigial signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, hsa04390: hippopotamus signaling pathway, hsa04922: glucagon signaling pathway, hsa 04: gonadotropin signaling pathway, hsa04022: cGMP-PKG signaling pathway, 912: oxytocin signaling pathway, 04a 04921: 722: 04a signaling pathway, 723: generation signaling pathway, 723: reverse nerve signaling pathway, hsa 0511: HSa04066: signaling pathway, HIF 04020: signaling pathway, 04020: cGMG signaling pathway, and the like.

4) The HrpNEch protein recognizes the associated metabolic pathways of antiviral, antibacterial, anti-foreign body, anti-inflammatory binding: recognition of binding to 29, hsa04144 endocytosis, hsa04145 phagosomes, hsa04142 lysosomes, hsa04666 Fc-r mediated phagocytosis, hsa01130 biosynthesis of antibiotics, hsa05131 Shigellasis, hsa04612 antigen processing and presentation, hsa05130 pathogenic E.coli infection, bacterial invasion of hsa05100 epithelial cells, hsa05132 Salmonella infection, hsa05169 Barr virus infection, hsa05203 viral carcinogenesis, hsa05134 Legionella disease, hsa05160 hepatitis C, hsa05162 measles, hsa05133 pertussis, hsa05322 systemic lupus erythematosus, hsa04670 leukocyte transendothelial migration, hsa05152 pulmonary tuberculosis, hsa 50 Hsa Staphylococcus aureus infection, hsa 46 Ammi disease, hsa 42 Mediterranean disease, 05100 Mediterranean pathway 05143, 051 pathway 051, 051 glioma 051, 051 750 Trypanosoma 05114, and central nervous system 05114 TRPA 051 pathway 051 receptor metabolism, hsa05212 pancreatic cancer.

5) The HrpNEch protein recognizes the important neurological 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 HrpNECh protein recognizes the related pathways of nucleic acid, protein, amino acid, sugar and fat metabolism: recognition of 39 binding events including hsa03013 RNA transport, hsa03018 RNA degradation, hsa03040 spliceosome, hsa03010 ribosome, hsa04141 endoplasmic reticulum protein processing, hsa04810 actin scaffold modulation of hsa03050 proteasome, hsa01230 amino acid biosynthesis, hsa00190 oxidative phosphorylation, hsa00230 purine metabolism, hsa04932 non-alcoholic fatty liver, hsa00020 citric acid cycle, hsa03008 eukaryotic ribosome biogenesis, hsa00240 pyrimidine metabolism, hsa00650 methyl butyrate metabolism, hsa01200 carbon metabolism, hsa00520 amino sugar and nucleotide sugar metabolism, hsa05034 alcoholism, hsa00071 fatty acid degradation, hsa04120 mediated proteolysis, hsa05205 proteoglycan in cancer, hsa 06 small molecule RNA in cancer, 00410 histidine 00310 alanine, histidine 00340 tyrosine metabolism, histidine tyrosine metabolism, 0004910, and histidine metabolism, Hsa00900: terpenoid biosynthesis pillars, hsa04610: complement and coagulation cascade, hsa00330: arginine and proline metabolism, hsa04520: adhered knots, hsa00860: porphyrin and chlorophyll metabolism, hsa00010: glycolysis and carbohydrate neogenesis, hsa00982: drug metabolism-cytochrome P450, and hsa00980: the metabolic action of cytochrome P450 on exogenous drugs, hsa04962: vasopressin regulation of water reabsorption, and hsa00983: drug metabolism-other enzymes.

7) The HrpNEch protein recognizes the combined metabolic pathways of cell junction, nerve junction, blood vessel, endocrine, reproductive system and the like: identifying the combination of strips 34, comprising hsa04510: focal adhesion, hsa 04724: glutamatergic synapse, hsa04530: tight junction, hsa00830: retinol metabolism, hsa04114: oocyte meiosis, hsa04728: dopamine synapse, hsa00140: steroid hormone biosynthesis, hsa04261: adrenergic signaling of cardiomyocytes, hsa04727: synapse of γ -aminobutyric acid, hsa04725: cholinergic synapse, hsa04540: gap junction, hsa04971: gastric acid secretion, hsa04713: diurnal entrainment, hsa04931: insulin resistance, hsa05031: amphetamine addiction, hsa04924: renin secretion, hsa04925: aldosterone synthesis and secretion, hsa 0490: arachidonic acid metabolism, hsa04270: vascular smooth muscle contraction, hsa00760: niacin metabolism, 04a 04740: olfactory transduction 925: contractility 925: addict, hsa04720: atrial right-stimulated arrhythmia 04412, right-ventricular arrhythmia collection 04966: myocardial infarction 048: atrial premature ventricular arrhythmia 048: atrial arrhythmia induction, hsa05410 hypertrophic cardiomyopathy, hsa04146 peroxisome, hsa05414 dilated cardiomyopathy, hsa04970 salivary secretion, hsa04611 platelet activation, hsa05204 chemocarcinogenesis, hsa04721 synaptic vesicle cycling.

The HrpNECh 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 basic life attributes of the organism 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 HrpNECh protein is a special multi-epitope ligand protein with brand new functions, brand new action mechanism and brand new application prospect.

The above embodiments only express specific embodiments of the present application, and the description is 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> HrpNECh protein in pharmacy for recognizing and activating multiple types of receptors and/or membrane proteins and signal paths thereof

<160> 1

<170> Patent In Version 2.1

<210> 1

<211> 339

<212> PRT

<213>HrpNEch（Dickeya dadantii CSCL006(hrpNEch）gene（SEQ ID NO1）

<220> 1

<221> DOMAIN

<222> conserved region domains (9) - (334); a-helical domains (39) - (62), (105) - (118), (131) - (134), (147) - (163), (192) - (213), (233) - (245), (268) - (273); β -sheet knob domains (2) - (7), (204) - (205); do-domains (1) - (2), (8) - (11), (13) - (40), (66) - (100), (131) - (139), (173) - (177), (339).

<400> 1

Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser

5 10 15

Gly Leu Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala

20 25 30

Ser Ser Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp Lys

35 40 45

Leu Thr Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln

50 55 60

Gly Leu Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly

65 70 75 80

Gln Ser Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val

85 90 95

Pro Lys Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu

100 105 110

Asp Asp Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser

115 120 125

Asn Gln Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly

130 135 140

Asn Met Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser Ile

145 150 155 160

Leu Gly Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser

165 170 175

Leu Gly Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn

180 185 190

Gln Leu Gly Asn Ala Ile Gly Met Gly Val Gly Gln Asn Ala Ala Leu

195 200 205

Ser Ala Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His

210 215 220

Phe Val Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe

225 230 235 240

Met Asp Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp

245 250 255

Gly Trp Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu

260 265 270

Ser Lys Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe

275 280 285

Arg Gln Ala Met Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly

290 295 300

Asn Thr Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly Ile

305 310 315 320

Asp Ala Ala Val Val Gly Asp Lys Ile Ala Asn Met Ser Leu Val Ala

325 330 335

Ala Asn Ala

339

Claims

The application of the HrpNECh protein in the pharmacy for recognizing and activating a plurality of receptors and/or membrane proteins and signal pathways thereof and causing cascade biological effects is disclosed, wherein the amino acid sequence of the HrpNECh protein is shown as SEQ ID NO. 1.
2. The use of the HrpNEch protein of claim 1 in the manufacture of a medicament for identifying one or more of GNG12 guanine nucleotide binding protein gamma-12 receptor, ANXA5 annexin a5 receptor, ANXA2 annexin a2 receptor, ANXA1 annexin a1 receptor, IGHG2 immunoglobulin heavy constant gamma 2 receptor, IGHM immunoglobulin heavy constant Mu receptor, CACNA1S calcium voltage gated channel subunit alpha 1S receptor, f185 zinc finger protein 185 receptor, and HLA-a major histocompatibility complex, class I receptor, LAMP2 lysosomal associated membrane protein 2 receptor, GNB 2G guanine nucleotide binding protein subunit beta 2 receptor, KTN1 kinesin binding protein 1 receptor for activating multiple classes of receptors and/or membrane proteins and their signaling pathways and causing cascade biological effects.
3. The HrpNECh protein of claim 1, for use in a pharmaceutical formulation for recognizing and activating multiple classes of receptors and/or membrane proteins and their signaling pathways, and causing cascade biological effects, wherein the membrane protein comprises one or more of DSC3 desmoglein, ANXA8/ANXA8L1 annexin A8/annexin A8 similar protein 1, EVPL coat plaque protein, POF1B actin binding protein premature ovarian failure 1B, CTNNA1 catenin, TGM1 transglutaminase 1, BAIAP2 BAI1 associated protein 2, RAB29 RAS oncogene family member, CLDN19 dependent protein 19, STXBP2 synaptic fusion protein binding protein 2, VAMP vesicle associated membrane protein A, VCL adhesion plaque protein, Ezrin epithelial calcium adhesin, PKP3 platelet affinity protein 3, NAALAD 2N acetylated alpha linked acidic dipeptidase 2, PKP1 platelet affinity protein 1, RR1A proline-rich small protein 1A.
4. The use of the HrpNECh protein of claim 1 in the manufacture of a medicament for the recognition of signals activating multiple receptors and/or membrane proteins and their signaling pathways and eliciting cascade biological effects, wherein said signaling pathways comprise the hsa03320: PPAR signaling pathway, the hsa05120: signal transduction of helicobacter pylori-infected epithelial cells, the hsa04071: sphingolipid signaling pathway, the hsa04014: Ras signaling pathway, the hsa04151: PI3K-Akt signaling pathway, the hsa 04004070: phosphatidylinositol signaling system, the hsa04010: MAPK signaling pathway, the hsa04310: Wnt signaling pathway, the hsa04062: chemokine signaling pathway, the hsa04015: Rap1 signaling pathway, the hsa04024: paroxysmal signaling pathway, the hsa04915: estrogen signaling pathway, the hsa04910: insulin signaling pathway, the hsa04390: river horse signaling pathway, the hsa 0404912: pancreatic glucagon signaling pathway, the hsa04912: gonadotropin signal pathway, the hsa 040040040040040915: insulin signaling pathway, the cGmPG signaling pathway, the hsa 921: CGA 0422: CGT signaling pathway, the Hsa 04051: Wnt signal pathway, hsa04722 generation signaling pathway, hsa04723 retrograde neural signaling, hsa04066 HIF-1 signaling pathway, hsa04020 calcium signaling pathway.
5. The use of the HrpNEch 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 including 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. The use of the HrpNEch protein of claim 5 in the manufacture of a medicament for the recognition of activation of multiple classes of receptors and/or membrane proteins and their signaling pathways and eliciting cascade biological effects, wherein said antiviral, antibacterial, anti-foreign body, anti-inflammatory associated metabolic pathways: hsa04144: endocytosis, hsa04145: phagosome, hsa04142: lysosome, hsa04666: fc-r mediated phagocytosis, 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: bal virus infection, hsa05203: viral carcinogenesis, hsa05134: legionnaire's disease, hsa05160: hepatitis c, hsa05162: measles, hsa05133: pertussis, hsa05322: systemic lupus erythematosus, hsa04670: migration of leukocytes across endothelium, hsa05152: tuberculosis, hsa05150: staphylococcus aureus infection, hsa05146: amebiasis, hsa05142: trypanosomiasis nana, hsa05200: in cancer pathways, hsa05143: african trypanosomiasis, hsa04750: inflammatory mediator modulation of TRP channels, hsa04916: bactericidal action, hsa05230: central carbon metabolism in cancer, hsa05214: glioma, hsa05212: pancreatic cancer; the important metabolic pathways for neurological diseases: hsa05010 Alzheimer's disease, hsa05012 Parkinson's disease, hsa05016 Huntington's chorea; the nucleic acid, protein, amino acid, sugar and fat metabolism related pathway: hsa03013 RNA transport, hsa03018 RNA degradation, hsa03040 spliceosome, hsa03010 ribosome, hsa04141 endoplasmic reticulum processing, hsa04810 actin scaffold modulated hsa03050 proteasome, hsa01230 amino acid biosynthesis, hsa00190 oxidative phosphorylation, hsa00230 purine metabolism, hsa04932 nonalcoholic fatty liver, hsa00020 citric acid cycle, hsa03008 biogenic ribosome biogenesis, hsa00240 pyrimidine metabolism, hsa00250 Butanoate metabolism, hsa 05001200 carbon metabolism, hsa00020 amino sugar and nucleotide sugar metabolism, hsa 00534 alcoholism wine, hsa00071 fatty acid degradation, hsa04120 ubiquitin-mediated proteolysis, hs052a 052a 05 Hs proteoglycan in cancer, riboa 05206 small molecule nucleic acid in cancer, hsa00410 strut 00510 histidine, 00340 histidine, aspart, Hsa00340 histidine 041910, hsa 00310 histidine synthase, yaa 003900 histidine synthase, yaa serum threonine synthase 0050, yamaminosine 003900, 0000000050, 0000000000 histidine synthase, 00000000 a hormone, 0000 ubiquitin, 0000 histidine synthase, 0000 serum threonine synthase, and 0000 a, hsa04610 complement and coagulation cascade, hsa00330 arginine and proline metabolism, hsa04520 adherent nodes, hsa00860 porphyrin and chlorophyll metabolism, hsa00010 glycolysis and glyconeogenesis, hsa00982 drug metabolism-cytochrome P450, hsa00980 cytochrome P450 metabolism to exogenous drugs, hsa04962 vasopressin regulated water reabsorption, hsa00983 drug metabolism-other enzymes; the cell junctions, nerve junctions, blood vessels, endocrine, reproductive metabolic pathways: hsa04510 focal adhesion, hsa 04724 glutamatergic synapses, hsa04530 tight junctions, hsa00830 retinol metabolism, hsa04114 oocyte meiosis, hsa04728 dopaminergic synapses, hsa00140 steroid hormone biosynthesis, hsa04261 adrenergic signaling of cardiomyocytes, hsa04727 gamma-aminobutyric synapses, hsa04725 cholinergic synapses, hsa04540 gap junctions, hsa04971 gastric acid secretion, hsa04713 diurnal entrainment, hsa04931 insulin resistance, hsa05031 amphetamine addiction, hsa04924 renin secretion, hsa04925 aldosterone synthesis and secretion, hsa00590 arachidonic acid metabolism, hsa04270 vascular smooth muscle contraction, hsa00760 nicotinic acid and nicotinamide metabolism, hsa04740 olfactory transduction, hsa04260 olfactory contraction, hsa04720 olfactory dysgenosis, hsa04720 photoperiodic tone 05744, right ventricular hypertrophy, right ventricular end tract 05410 cardiac arrhythmia 056, right ventricular hypertrophy collection, right ventricular hypertrophy and cardiac arrhythmia 05410, hsa04146 peroxisome, hsa05414 dilated cardiomyopathy, hsa04970 salivary secretion, hsa04611 platelet activation, hsa05204 chemocarcinogenesis, and hsa04721 synaptic vesicle cycling.
7. The use of the HrpNEch protein of claim 1 in the manufacture of a medicament for the recognition of activation of multiple classes of receptors and/or membrane proteins and their signaling pathways and for eliciting cascade biological effects, wherein said cascade biological effects comprise cellular processes, environmental information processing, genetic information processing, metabolic and biological systems 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 the HrpNECh protein; the environmental information processing comprises the steps that multiple differential expression genes induced by the HrpNECh protein participate in the processing processes of signal molecule and interaction, signal transduction and membrane transportation environmental information; the genetic information processing comprises that a plurality of differentially expressed genes induced by the HrpNECh protein participate in the biological processes of translation, replication and repair, folding, classification and degradation; metabolism includes that multiple differentially expressed genes induced by the HrpNECh 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 metabolic processes; the biological system comprises a plurality of differential expression genes induced by the HrpNECh 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 and circulation system.
8. The use of the HrpNEch protein of claim 1 in the identification of drugs that activate multiple classes of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects, wherein the product of the pharmaceutical use or the dosage form of the drug is a liquid, powder, tablet or capsule.
9. The use of the HrpNEch protein of claim 7 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 HrpNEch protein in an amount of 0.001% -100% by mass.
10. The method for depolymerization activation of an HrpNEch protein according to any one of claims 1 to 9, comprising the steps of:

step 1: pretreatment: with glucose Na₂HPO₄-KH₂PO₄Regulating and collecting the volume concentration range of the high polymeric HrpNECh multi-epitope protein prepared by fermentation by using buffer solution within 0-30%, and treating for 0-24h at 20-30 ℃;

step 2: depolymerizing and activating the HrpNEch high-aggregation multi-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 the temperature of 35-38 ℃, and then collecting depolymerized and activated multiple epitope protein molecules of HrpNECb;

and 4, step 4: purifying the high-aggregation-state multiple-epitope protein HrpNECh-His-Tag recombinant protein by using chromatography gel to obtain a depolymerized and activated multiple-epitope protein HrpNECh original drug.