CN114681591A

CN114681591A - Application of HrpZpss protein in identifying and activating multiple types of receptors and/or membrane proteins and signal pathways thereof in pharmacy

Info

Publication number: CN114681591A
Application number: CN202011644674.7A
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 HrpZpss protein in pharmacy for identifying 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 biological medicine, wherein the amino acid sequence of the HrpZpss protein is shown as SEQ ID NO. 1. The HrpZpss protein is taken as a ligand protein molecule which is rich in a plurality of epitopes (linear and conformational) and has a special structure, can recognize, activate and combine membrane receptors, membrane proteins, information pathways and metabolic pathways of various animals in a cross-boundary way, and is a special multi-epitope ligand protein with brand new functions, brand new action mechanism and brand new application prospect.

Description

Application of HrpZpss protein in identifying and activating multiple types of receptors and/or membrane proteins and signal pathways thereof in pharmacy

Technical Field

The invention relates to the field of biological medicines, in particular to application of HrpZpss protein in pharmacy for recognizing and activating multiple receptors and/or membrane proteins and signal paths thereof and causing cascade biological effects.

Background

Molecular biology is the science of studying life phenomena at the molecular level, elucidating the nature of various life phenomena by studying the structure, function and metabolism of biological macromolecules, and its content covers the entire course of life. DNA, RNA and proteins are three important biological macromolecules that are the molecular basis for life phenomena. The genome determines what life is, the proteome determines what life can do, and the metabolome determines what life actually happens. Modern life science, biotechnology and medical biotechnology, especially proteomics and metabonomics, have been developed rapidly, the ideas of understanding, diagnosing, preventing and controlling, treating and recovering diseases are updated, new understanding and new ways of novel efficient and safe medicines are created, the development of modern medicine enters a brand-new stage, and a wide application prospect is opened up.

The receptor theory of modern life science isPharmacodynamics of medicineOne of the basic theories of (1) is to be derived fromMoleculeControlled physiological processes for horizontally interpreting life andpathology of diseaseThe process, the pharmacological action mechanism of the medicine and the structural effect relationship of the medicine molecules. The ligand is a signal substance which has no other direct functions except for recognizing, binding and activating the receptor, cannot participate in metabolism to produce useful products, does not directly induce any cellular activity, and has no characteristics of enzyme.

The signal path (cell communication) is a communication mechanism for transmitting and receiving information in cells or cells of a multicellular organism with high accuracy and high efficiency, and a rapid cell physiological and biochemical reaction is caused by amplification or gene activity is started, and then a series of cell physiological and biochemical activities are generated to coordinate the activities of various tissues, so that the unified whole life can comprehensively react to changeable internal and external environments, and the coordinated joint mechanism for growth, development, defense and metabolism is built by systems, tissues, organs, cells, subcells, molecules and sub-molecules of a living organism.

The receptor is a functional protein for mediating cell signal transduction, can recognize certain trace substances in the surrounding environment (intracellular and extracellular environments), is recognized and combined with the trace substances, is activated, and triggers subsequent physiological and biochemical reactions through a signal amplification system. Receptors are biological macromolecules composed of cell membranes and intracellular proteins, nucleic acids, lipids, polysaccharides, and the like. Receptors are a broad concept in cell biology, meaning any biological macromolecule capable of binding to hormones, neurotransmitters, drugs or signaling molecules both inside and outside the cell and causing a change in cell function, in which case the signaling molecule is called a ligand. There are hundreds of different signaling molecules in multicellular organisms that transmit information between and within cells, including proteins, amino acid derivatives, nucleotides, cholesterol, fatty acid derivatives, and soluble gas molecules. Receptors present on the plasma membrane of cells are called membrane receptors, the chemical nature of which is for the most part sugar mosaics; receptors located in the cytosol and nucleus, called intracellular receptors, are all DNA binding proteins.

The ligand is a signal substance which has no other direct functions except for recognizing, binding and activating the receptor, cannot participate in metabolism to produce useful products, does not directly induce any cellular activity, and has no characteristics of enzyme.

The combination of ligand and receptor is the process of intermolecular recognition and activation, which depends on the actions of ion coordination bond, hydrogen bond, pi-pi stacking action, electrostatic action, hydrophobic action, van der waals force, etc. with the complementation and the interaction degree of the two molecular spatial structures, the distance between the interacting groups is shortened and the acting force is greatly increased, so the interactivity and the complementarity of the ligand and the receptor molecular spatial structures are the main factors of specific combination, i.e. the epitope concept adopted by the invention. The same ligand may correspond to two or more different receptors, and binding of the same ligand to different types of receptors results in different cellular responses. After the ligand is combined with the receptor, related series of physiological activities are initiated, no matter whether the ligand is endogenous or exogenous, after the ligand is combined with the receptor, the ligand and the receptor form a ligand-receptor combination surface or a compound, so that information is transmitted, and through conduction and transduction, rapid cell physiological and biochemical reactions are initiated through amplification, or gene activities are initiated, a series of cascade reactions occur later to coordinate the activities of various tissues, organs and cells, so that the unified whole life makes comprehensive reactions to changeable internal and external environments.

In 2008, Leader et al first proposed ideas classified according to protein pharmacological actions and classified protein drugs into four major classes: protein medicine for treating diseases with the enzyme activity and regulating activity of protein; ② protein drugs with special targeting activity; ③ recombinant protein vaccines; and fourthly, the recombinant protein medicine for diagnosis. Of these, the first and second classes are mainly used in basic protein therapy, and the third and fourth classes emphasize the use of proteins in vaccines and diagnostic applications. After a century of exploration and zigzag development, protein drugs have matured one step by one step and have a great significance in pharmaceutical industry and clinical application. They have important effects on almost all disease fields such as tumors, infections, autoimmune diseases, metabolic genetic diseases, various senile diseases and degenerative diseases, and are becoming important therapeutic, prophylactic and diagnostic drugs in the 21 st century. The wide application of biotechnology with recombinant DNA technology as the core is expected to give protein drugs a wider development space in the next 30 years: the recombinant protein drug will gradually replace the non-recombinant protein; the restructuring and in-vitro and in-vivo modification become conventional; products expressed with mammalian cell systems will predominate; the non-injectable route of administration of protein drugs is receiving increasing attention; biomimic drugs and biosimilar drugs will be most likely. (Zhuxun, functional classification and development trend of protein drugs, volume 5, No.1 of 2.2010, Chinese medicinal biotechnology, Chin Med Biotechnol, February 2010, Vol.5, No. 1).

It has been shown that the recognition binding of ligand to 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 the recognition of binding to the bovine IgG2 receptor, and further e.g. 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 the 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.

The HrpZpss protein is an expression product of the hrpZpss gene (Genbank ID: AAY36247.1), which consists of 343 amino acid residues, a non-enzymatic protein having a mono-, di-, and tertiary structure without a quaternary structure, is free of cystine and cysteine, is rich in glycine, and has an Mw of 34.73 kDa. The conserved domain of the HrpZpss protein consists of 343 amino acids, and the whole sequence of the conserved domain consists of 1-343; the alpha-helical structure region 37-52,61-72, 80-93, 109-.

The structural domain is a region with a specific structure and an independent function in biological macromolecules, in particular to an independent stable structural region formed by combining different secondary structures and super-secondary structures in protein, the structural domain is also a functional unit of the protein, and in multi-structural-domain protein, different structural domains are often associated with different functions; the secondary and supersecondary structures of proteins are maintained mainly by hydrogen bonds, and include alpha helices, beta sheets, beta turns, random coils, do-structures, etc., alpha helices being repetitive structures with phi and psi near-57 deg. and-47 deg. for each alpha-carbon in the helix, respectively. Each coil of helix occupies 3.6 amino acid residues, the residues rise by 0.54nm along the direction of the helical axis, each residue rotates by 100 degrees around the axis and rises by 0.15nm along the axis, hydrogen bonds are formed between adjacent coils, and the orientation of the hydrogen bonds is almost parallel to the helical axis; beta sheet: the beta-folded 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 HrpZpss proteins are multidomain proteins forming a special structure of a plurality of 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 and causing multifunctional cascade biological effects. However, there is no report on this.

Disclosure of Invention

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

The technical scheme adopted by the invention is as follows:

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

The HrpZpss 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 identified 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 identified, combined and activated with the receptors and are rich in proton-supplying amino acid residues or proton-receiving 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 various receptor proteins in a hydrogen bond mode to form a binding surface or a compound; further, hydrophobic apolar amino acid residues: valine, leucine, isoleucine, alanine and phenylalanine can form a tight combination surface or compound with various types of receptors by nonpolar hydrophobic and van der waals force; acidic positively charged, basic negatively charged amino acid residues: the aspartyl acid, the glutamic acid, the lysine and the arginine can form a tight combination surface or a compound with various types of receptors through ionic bonds; amide group polar uncharged amino acid residue: the amide groups of asparagine and glutamine can form a bonding surface or a compound with a cysteine recognition region Pam3 CSK4 of a receptor through stronger hydrogen bonds; polar uncharged amino acid residues: serine forms a tight binding surface or complex with multiple types of receptors through polar and strong hydrogen bonds.

Furthermore, the whole sequence of the HrpZpss polypeptide has 343 amino acid residues, 235 key amino acid residues, 105 hydrophobic nonpolar amino acid residues, 33 polar uncharged amino acid residues, 40 amido amino acid residues, 57 acidic, basic positively charged and negatively charged amino acid residues, the molecular weight is 34.73kd, the isoelectric point PI is 4.6, and the key amino acid accounts for 68.5 percent; the polypeptide sequence of the conserved structural region has 343 amino acid residues, which account for 100 percent of the whole sequence, 235 key amino acid residues, 105 hydrophobic nonpolar amino acid residues, 33 polar uncharged amino acid residues, 40 amido amino acid residues, 57 acidic positively charged and basic negatively charged amino acid residues, and the key amino acid accounts for 68.5 percent; the alpha-helix structural region has 12 alpha-helices, 1 beta-sheet and 8 do-structural regions, wherein the alpha-helix region has 162 amino acid residues, 127 key amino acid residues, 66 hydrophobic nonpolar amino acid residues, 6 polar uncharged amino acid residues, 19 amido amino acid residues, 36 acidic positively charged and basic negatively charged amino acid residues, and the proportion of the key amino acid residues is 78.4%; further, we screened, cloned and prepared the hypersensitive proteins HrpZPsa, HrpZPsm, HrpZPss, HrpZPst, HrpZPsap, HrpZPsr, HrpZPsth, HrpZPave, HrpZPam, HrpZPcar, HrpZPcor, HrpZPcst, HrpZPcat, HrpZCory, HrpZPcp, HrpZPsav, HrpZPvir, HrpZPspe, Hrpam, rpHZPade, HzPsac, HrpZPsg, HrpZPsc and the like from Erwinia and Pseudomonas, and the molecular structure analysis shows that they have similar structural characteristics, structural trend and have multiple epitope conformational and linear epitope structures similar to those of the above polyepitope HzPss ligand protein according to bioinformatics analysis: contains one or more hydrophobic non-polar amino acid residues, contains one or more polar uncharged amino acid residues, contains one or more amide polar uncharged amino acid residues, contains one or more acidic positively charged and basic negatively charged amino acid residues; further, hydrophobic apolar amino acid residues: valine, leucine, isoleucine, alanine, phenylalanine, methionine, polar uncharged amino acid residues: serine, amido polar uncharged amino acid residues: asparagine, glutamine, acidic positively charged, basic negatively charged amino acid residues: aspartyl acid, glutamic acid, lysine, histidine, arginine; further, the above-mentioned key amino acid residues account for 72.97% -70.65% of the entire sequence of these protein molecules, 72.97% -71.82% of the conserved domain, and 85.98% -81.48% of the α -helical structure; further, the above amino acid residues (generally referred to as key amino acid residues) of the HrpZpss protein, without limitation, can achieve complementarity, interactivity and specific recognition, activation and combination of the spatial structure and the electrical property of the ligand and receptor molecules through hydrogen bonds, ionic bonds, hydrophobicity, non-polarity, polarity and van der waals force, form a tight binding surface or a complex with multiple types of receptors, can cause the change of the conformation, energy, electrical property and information of the receptor molecules, and can amplify and express a series of biological effects through signal transduction 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 multiple classes of receptors include HLA-C major histocompatibility complex, class I, C receptor, free fatty acid receptor 4, tyrosine protein kinase transmembrane receptor 1, ASGPR1 asialoglycoprotein receptor 1, adipose cytoplasmic membrane associated protein receptor (APMAP), insulin-like growth factor 2 receptor (IGF 2R).

Preferably, the membrane proteins include solute carrier family 5 (sodium dependent vitamin transporter), member 6, solute carrier family 26, member 4, solute carrier family 38, member 2(SLC38a2), DNM2 dynein 2, CAP1 adenylate cyclase-associated protein 1, ICAM1 intercellular adhesion molecule 1 LanC-like protein 1 (lanthionine synthase C) (LANCL1), MLEC stress protein-androgen like receptor kinase, TJP2 tight junction protein 2, ZYX macular synprotein.

Preferably, the signaling pathways include the role of the hsa04933 age-anger signaling pathway in diabetic complications, the hsa04064 NF-kappa B signaling pathway, the hsa04072 phospholipase D signaling pathway.

Preferably, the signaling pathway further comprises a metabolic signaling pathway comprising an antiviral, antibacterial, anti-foreign, anti-inflammatory associated metabolic pathway: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa04666 Fc gamma r-mediated phagocytosis, hsa04210 apoptosis, hsa04218 cell senescence, hsa05130 pathogenic E.coli infection, hsa04612 antigen processing and presentation, hsa05100 bacterial invasion of epithelial cells, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05164 influenza A, hsa05150 Staphylococcus aureus infection, hsa05167 Kaposi sarcoma-associated herpesvirus infection, hsa04916 bactericidal effect, hsa04650 natural killer cell-mediated cytotoxicity, hsa05169 Epstein Barr virus infection, hsa05416 viral myocarditis, hsa05110 Vibrio infection, hsa05144 malaria, hsa05163 human cytomegalovirus infection, hsa05170 human immunodeficiency virus type 1 infection, hsa rheumatoid arthritis, 05323 viral leukocyte migration; including important neurological metabolic pathways: not detected; including the related pathways of nucleic acid, protein, amino acid, sugar and fat metabolism: hsa03050 proteasome, hsa04974 protein digestion and absorption, and hsa04120 ubiquitin-mediated proteolysis; including cell junctions, nerve junctions, blood vessels, endocrine, reproductive metabolic pathways: hsa04510 focal adhesions, hsa04724 glutamatergic synapses, hsa04530 tight junctions, hsa04261, adrenergic signaling of cardiomyocytes, hsa04940 type I diabetes, hsa04924, renin secretion, hsa04721 synaptic vesicle cycling, reabsorption of calcium mediated by factors such as hsa04961 endocrine, hsa04970, salivary secretion, hsa04979 cholesterol metabolism, hsa04918 thyroid hormone synthesis, hsa04514 Cell Adhesion Molecules (CAMs), hsa04727 neurons, hsa05320 autoimmune synaptic thyroid disease, hsa05418 fluid shear stress, and atherosclerosis.

Preferably, the cascade biological effect includes functional pathways such as Cellular Processes (Cellular Processes), Environmental Information Processing (Environmental Information Processing), Genetic Information Processing (Genetic Information Processing), Metabolism (Metabolism), and biological Systems (organic Systems); further, the Cellular process (Cellular Processes): multiple differential expression genes induced by HrpZpss protein participate in cell processes such as transportation, catabolism, cell population, cell activity, cell growth and death and the like; processing Environmental Information (Environmental Information Processing) that multiple differentially expressed genes induced by HrpZpss protein participate in the Processing process of Environmental Information such as signal molecule interaction, signal transduction, membrane transportation and the like; processing Genetic Information (Genetic Information Processing) multiple differentially expressed genes induced by HrpZpss 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 HrpZpss protein participate in the metabolic processes of 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 differentially expressed genes induced by HrpZpss protein participate in biological processes of sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, developmental circulation system and the like.

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 (HrpZpss protein preparations and/or drugs) for the HrpZpss protein and derivatives thereof, typically in unit dosage form or multiple dosage forms, each unit dosage 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 HrpZpss 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, chondromas, creams, lotions, pastes, gels, sprays, aerosols, and oils. Exemplary carriers include, but are not limited to, petrolatum, lanolin, polyethylene glycols, alcohols, and combinations of two or more thereof. The topical formulation may also contain 0.001% to 15%, 20%, 25% by weight of a thickening agent selected from the group including, but not limited to, hydroxypropylmethyl cellulose, methyl cellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, poly/hydroxyalkyl (meth) acrylates or poly (meth) acrylamides. The topical formulations are typically applied by instillation or as a chondrogenic agent applied to the conjunctival capsules. It can also be used to irrigate or lubricate the eye, facial sinuses and external auditory canal. It can also be injected into the anterior chamber of the eye and elsewhere. Topical formulations in the liquid state may also be present in the form of a tape or contact lens in a hydrophilic three-dimensional polymeric matrix from which the active ingredient is released. For formulations suitable for buccal (sublingual) administration, there are included, but are not limited to, lozenges comprising the active compound in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base including, but not limited to, gelatin and glycerin or sucrose and acacia. Pharmaceutical compositions of the ligand isoforms may be formulated for parenteral administration by injection, including, but not limited to, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added additive. 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 agents, stabilizing agents, and alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water or other solvent, before use. Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for an extended period of time. Such patches suitably contain the active compound as an aqueous solution of the active compound, optionally fluke. Formulations suitable for transdermal administration may be delivered by iontophoresis and take the form of an optionally flushed aqueous solution of the active compound.

Preferably, the preparation or medicament is prepared predominantly from depolymerised activated HrpZpss proteins.

The preparation method for producing the purified HrpZpss protein by depolymerizing and activating the high-polymerization-state HrpZpss protein, which is adopted by the invention, comprises the following steps:

1. pretreatment: with glucose Na₂HPO₄-KH₂PO₄The buffer solution regulates and collects the volume concentration range of the high polymeric HrpZpss multiple epitope proteins 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 10% -15% concentration; at normal temperature (20-30 ℃), pre-treating glucose Na₂HPO₄-KH₂PO₄Buffer solution, pH range is 1-14, glucose concentration range is 0-2500mmol, buffer system pH, preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-6; most preferably at a pH of 5-5.5. The concentration of glucose is 0-100 mmol; preferably at a concentration of 100-200 mmol; preferably a concentration of 2500-; preferably at a concentration of 1000-300 mmol; 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 15-6 h; most preferably for a period of 4-6 hours.

2. Depolymerizing and activating HrpZpss high-polymerization-state multi-epitope proteins: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution, wherein the ultrahigh pressure range is 1000-3000MPa, preferably 3000 MPa; preferably 1500 Mpa; preferably 2500 Mpa; preferably 2000 Mpa; most preferably 2000-.

3. And (3) post-treatment: after the ultrahigh pressure depolymerization and activation operation is finished, standing for 0-24h at 35-38 ℃, preferably for 0-1 h; preferably for 24-10 h; preferably for a period of 1-2 hours; preferably for 10-4 hours; most preferably for 2-4 hours, and then collecting the disaggregated activated HrpZpss multi-epitope protein molecules.

4. And (3) purifying the multiple epitope proteins of the high-aggregation HrpZpss, namely 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 and preparation of the original drug of the depolymerized and activated multiple epitope proteins HrpZpss.

The route of use of the HrpZpss protein preparation identifying the various classes of receptors, membrane proteins and their signaling pathways that activate the animal and induce multifunctional cascade biological effects described herein may 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 HrpZpss multiple epitope ligand proteins may be administered by any convenient route, such as by perfusion or rapid perfusion, absorbed 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 the other bioactive agent; 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 on the nature of the specific 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, or 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 HrpZpss 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 application of products or medicines of diagnosis, prevention, treatment, rehabilitation, food type, apotype, cosmetic type, mechanical type and health type of diseases and conditions related to multiple systems, tissues, organs and cells.

The invention relates to a preparation or a medicament for identifying and activating multiple receptors, membrane proteins and signal channels thereof of animals and inducing multi-functional cascade biological effects of multiple epitope ligand proteins of HrpZpss, which is applied to the pharmacy, and the application of the preparation or the medicament in diagnosing, preventing, treating or rehabilitating diseases and conditions of the nervous system, the digestive system, the motor system, the circulatory system, the respiratory system, the endocrine system, the immune system, the urinary system and the reproductive system:

use of a preparation or medicament of a ligand protein of said plurality of epitopes of the invention for the diagnosis, or and prevention, or and treatment, or and rehabilitation of diseases of nervous linkages, dementia, parkinson's disease, central nervous system diseases, neuromuscular diseases, epilepsy, headache and neuralgia, peripheral neuropathies, attention deficit hyperactivity disorder and tic disorders, insomnia, depression, anxiety disorders, bipolar disorder, psychotic disorders, neurodermatitis-associated nervous system diseases and conditions;

the application of the product or the medicine of the ligand protein of the multiple epitopes in diagnosing, or preventing, or treating, or recovering the abnormal secretion of gastric acid, gastrointestinal neurosis, gastrointestinal motility, gastrointestinal mucositis, liver diseases and digestive system diseases and conditions related to microecological disorder;

use of a preparation or medicament of a ligand protein of said plurality of epitopes of the invention for the diagnosis, or and prevention, or and treatment, or and rehabilitation of arthritis, muscle spasms, pain, muscular dystrophy, muscle nerve injury, dehydration-related motor system diseases and conditions;

the use of a preparation or a medicament of a ligand protein of the plurality of epitopes of the invention for the diagnosis, or and prevention, or and treatment, or and rehabilitation of heart failure, arrhythmia, hypertension, myocardial injury, ischemia, angina pectoris, hyperlipidemia, calcium channel blockade, vasospasm, blood coagulation, abnormal hemogram, diseases and conditions of the circulatory system associated with myocardial infarction;

the use of a preparation or medicament of a ligand protein of the plurality of epitopes of the invention in the diagnosis, or and prevention, or and treatment, or and rehabilitation of asthma, chronic obstructive pulmonary disease, bronchiectasis, allergen immunity, allergy, pneumonia, acute or chronic bronchitis, bronchial asthma, gastroesophageal reflux, rhinitis-related respiratory diseases and conditions;

the application of the products or medicines of the ligand proteins of the epitopes in the invention in diagnosis, or prevention, or treatment, or rehabilitation of diabetes, thyroid diseases, pituitary diseases, hyperprolactinemia, diabetes insipidus, adrenal diseases, parathyroid diseases, diseases and conditions of endocrine systems related to osteoporosis;

the invention relates to the application of the products or medicines of ligand proteins of a plurality of epitopes in diagnosing, or preventing, or treating, or recovering immune hypofunction, rheumatoid arthritis and lupus erythematosus related immune system diseases and conditions;

the product or the medicine of the ligand protein with multiple epitopes is applied to diagnosing, or preventing, or treating, or rehabilitating the urogenital system diseases and conditions such as nephrotic syndrome, interstitial nephritis, renal failure, urinary infection, genital system infection, pyelonephritis, cystitis, prostatitis, urethritis, epididymitis or orchitis, prostatic hyperplasia, overactive bladder, sexual dysfunction, various andrological and gynecological infectious inflammations and functional diseases.

The product or the medicament of the ligand protein of the multiple epitopes is applied to diagnosis, or and prevention, or and treatment, or recovery of whole body skin cell nutrition, activation, regeneration, repair, removal, fine and smooth, ultraviolet melanin deposition, eczema, roughness, cracks, dark lines, dry skin, hard skin, erythema, allergy, neurodermatitis, injury, whelk, pimples, scars, dark skin, mites, oily skin, inflammatory dermatosis, autoimmune dermatosis, pigmentary dermatosis, skin atrophy, thinning, dryness, pigmentation, wrinkle hyperplasia, epidermal keratosis, xeroderma, contact dermatitis, skin aging resistance, skin function improvement, whitening and freckle removal, and prevention and treatment of skin disease and related skin system diseases and conditions.

The invention relates to a method for preparing HrpNECb multi-epitope ligand protein which can identify and activate various receptors, membrane proteins and signal channels thereof of animals and induce multifunctional cascade biological effect, comprising the following steps:

1. the preparation of the HrpZpss multi-epitope ligand protein can separate and purify the HrpZpss protein from secretory protein of a strain of 'Pseudomonas syringae pv. strain 301D hrpZpss' collected from the Israwa of Deyang, Quitang, China, can be carried out by adopting a conventional protein separation and purification method according to the specific molecular weight of the HrpZpss protein, and collects a depolymerized and activated HrpZpss purified protein product by the established depolymerization and activation technology of the high-polymerization-state HrpZpss multi-epitope protein molecules.

2. The preparation of the HrpZpss multi-epitope ligand protein can also adopt engineering bacteria of registered hrpZpss genes (Genbank ID: AAY36247.1) to prepare and collect depolymerized and activated HrpZpss protein through fermentation and purification:

1) and (3) engineering bacteria fermentation preparation of the HrpZpss 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 HrpZpss proteins and related proteins are produced by specially modified K-12 original bacteria JY-01(DE3), when the bacteria are cultured in LB liquid medium (containing 50 micrograms per liter of kanamycin) under the condition of certain temperature until OD600 is 0.7, IPTG (Isopropyl thiogalactoside, Isopropypyl beta-D-Thiogalactosid) (the final concentration is 1mMol) is added, and the bacteria are collected by centrifugation after the continuous culture. Analyzing the HrpZpss protein of the expression product by using 10% SDS-PAGE polyacrylamide gel electrophoresis, wherein a 34.73kd strip is shown on a sample lane of an electrophoresis gel plate, and the strip is the HrpZpss protein of the expression product of the gene hrpZpss;

wherein the fermentation medium is Na₂HPO₄-KH₂PO₄A buffer system, the pH of the buffer system is in the range of 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-7; most preferably pH 6.5-5.5;

the fermentation temperature is 0-60 ℃. Preferably at a temperature of 0-20 ℃; preferably at a temperature of 20-35 ℃; preferably at a temperature of 60-50 ℃; preferably at a temperature of 50-45 ℃; most preferably at a temperature of 37-38 ℃;

the glucose concentration range of the fermentation proliferation liquid culture medium is 3.00-0.00%; preferably 3.00% -1.00%; preferably 0.00% -0.01%; preferably 1.00% -0.3%; most preferably 0.01% -0.05%; most preferably 0.1% -0.05%;

the glucose concentration range of the fermentation induction liquid culture medium is 3.00-0.00%; preferably 3.00% -1.00%; preferably 1.00% -0.3%; preferably 0.3% -0.1%; preferably 0.1% -0.05%; most preferably 0.05% -0.00%;

the lactose concentration range of the fermentation induction liquid culture medium is 10.00-0.00%; preferably 10.00% -1.00%; preferably 0.00% -0.1%; preferably 1.00% -0.6%; preferably 0.1% -0.3%; most preferably 0.5% -0.4%;

the fermentation induction liquid culture time range is 0-24 h; preferably for a time of 0-2 h; preferably for 24-15 h; preferably for 2-6 h; preferably for 15-10 h; most preferably for a period of 7-9 hours.

2) The engineering bacteria production system is post-treatment after the production and fermentation of a plurality of epitope proteins are finished: sterilizing: the fermentation liquor is sterilized at 80 ℃ for 30 minutes, and is rapidly cooled to below 30 ℃; cleaning: with glucose Na₂HPO₄-KH₂PO₄Buffer solution (pH range is 1-14, glucose concentration range is 0-2500mmol, buffer system pH is 1-3; preferably pH 14-10; preferably pH 4-5; preferably pH 9-6; most preferably pH 5-5.5. glucose concentration is 0-100 mmol; preferably concentration is 100-200 mmol; preferably concentration is 2500-1000 mmol; preferably concentration is 1000-300 mmol; most preferably concentration is 200-300 mmol), engineering bacteria are washed five to eight times in a continuous flow of a butterfly centrifuge; engineering bacteria are crushed and cell walls are cleared, Na with pH 5-5.5 and glucose concentration of 200-300mmol is used for clearing cell walls₂HPO₄-KH₂PO₄Diluting thallus with buffer solution, adjusting fresh weight of thallus to 20-30% of the diluent, introducing into high-pressure crusher, continuously crushing engineering bacteria with pressure of 800-1000MPa, introducing the crushed bacteria into butterfly continuous flow centrifuge, removing cell wall, and collecting high-pressure bacteriaPoly-state HrpZpss multi-epitope protein molecules;

3) depolymerization and activation of multiple epitope protein molecules of high polymeric HrpZpss (I) pretreatment: with glucose Na₂HPO₄-KH₂PO₄The buffer solution regulates the volume concentration range of the high polymeric HrpZpss multi-epitope protein collected by fermentation to be 0-30%, and preferably the concentration is 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 HrpZpss 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 range is 1000-3000MPa, preferably 3000 MPa; preferably 1500 Mpa; preferably 2500 Mpa; preferably 2000 Mpa; most preferably 2000-;

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

(IV) purifying the high polymeric multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, wherein the protein purification is carried out according to the method suggested by NI-NTA affinity chromatography gel manufacturers, and the purification preparation of the depolymerized and activated HrpZpss protein is completed.

3. The preparation of the HrpZpss multi-epitope ligand protein, further the HrpZpss protein can also be prepared by expression protein of 'artificially synthesized gene', and the depolymerized and activated HrpZpss protein is prepared and collected by fermentation and purification, and specifically comprises the following steps:

artificial synthesis of HrpZpss gene for coding HrpZpss protein and preparation of expression protein thereof 1) nucleotide sequence of hrpZpss gene (Genbank ID: AAY36247.1) for coding HrpZpss protein published to GenBank according to modern bioinformatics is used as artificial synthesis of multiple epitope protein genes of HrpZpss, and DNA sequence of the gene is from GenBank: AAY36247.1

Cloning of the gene encoding the HrpZpss protein:

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

primers were designed and used (BamHI and HindIII sites underlined, respectively):

5’-tgcggatccatgcagagtctcagtcttaacagc-3’

5’-tgcaagctttcaggccgcggcctgaatgcgggt-3’

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 as follows: 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 HrpZpss protein gene is cloned to the BamHI-HindIII site of the constructed high-efficiency protein expression vector JY-01 (containing His-Tag label) one by one, and the cloning accuracy is ensured by DNA sequencing;

4) and (3) engineering bacteria fermentation preparation of the HrpZpss 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 HrpZpss proteins coded in the steps 1) to 3) into an engineering bacterium (E.coli), wherein a production line (E.coli) of related proteins is a derivative bacterium JY-01(DE3) of a K-12 original bacterium after special modification; when cultured in LB liquid medium (50. mu.g of kanamycin per liter) at a certain temperature until OD600 is 0.7, IPTG (Isopropyl thiogalactoside, Isopropyl beta-D-thiogalactoside) (final concentration: 1mMol) is added, the culture is continued, and then the thalli are centrifugally collected, 10% SDS-PAGE polyacrylamide gel electrophoresis is used for analyzing and coding the HrpNECb protein, and a 34.73kda band appears on a sample lane of an electrophoresis gel plate, which is the expression product HrpZpss protein of the gene hrpZpss.

5) The engineering bacteria production system is post-treatment 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 1-3, preferably pH 14-10, preferably pH 4-5, preferably pH 9-6, most preferably pH 5-5.5, glucose concentration is 0-100mmol, preferably concentration is 100-200mmol, preferably concentration is 2500-1000mmol, preferably concentration is 1000-300mmol, most preferably concentration is 200-300mmol, engineering bacteria are cleaned five to eight times in a butterfly continuous flow centrifuge, engineering bacteria are crushed and cell walls are cleaned, Na with pH 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 HrpZpss multi-epitope protein molecules.

6) Depolymerization and activation of high polymeric HrpZpss multi-epitope protein molecules (I) pretreatment with glucose Na₂HPO₄-KH₂PO₄The buffer solution regulates the volume concentration range of the high polymeric HrpZpss polyepitope protein collected 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% 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 1000-300 mmol; 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 HrpZpss high-polymerization-state multi-epitope protein, and carrying out ultrahigh pressure depolymerization and activation on the pretreated high-polymerization-state 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-;

(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 and activated HrpZpss polyepitopic protein molecules.

(IV) purifying the high-polymerization-state multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, wherein the protein purification is implemented according to a method suggested by an NI-NTA affinity chromatography gel manufacturer, and the purification preparation of the depolymerized and activated HrpZpss protein is completed;

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

the HrpZpss protein is a ligand protein with a special multi-epitope structure, brand-new functions, brand-new action mechanism and brand-new application prospect, can induce multidirectional, multilevel and multifaceted biological effects and functions, and widely relates to the diagnosis, prevention, treatment, rehabilitation and the application of food, word elimination, word making up, mechanical and word strengthening products and medicines of diseases and conditions.

Drawings

FIG. 1 shows electrophoretic detection before and after disaggregation of HrpZpss proteins: the molecular weight marker band is on the left, 1: depolymerizing, activating and purifying the pre-multi-epitope ligand protein HrpZpss bands; 2: depolymerizing, activating and purifying the bands of the multi-epitope ligand protein HrpZpss;

FIG. 2 is a graph showing the allergic reaction induced in tobacco leaves by protein solution injection of HrpZpss, wherein the focal spot is formed by treating the tobacco leaves with the protein solution of HrpZpss for about 24hr, and the two sides are as follows: h₂O injection; 3. 4: injecting HrpZpss protein solution (300 μ g/ml), namely hypersensitive reaction of the HrpZpss protein on tobacco leaves, wherein the two sides are provided with controls, and the 3 and 4 are provided with treatments;

fig. 3 is a volcano graph of kidney-induced differential gene expression of the HrpHrpZpss protein of the invention induced by oral administration and smearing on experimental mice, wherein the oral administration lasts for 6 hours and the oral administration lasts for 24 hours from left to right; smearing for 6 h;

FIG. 4 shows that the HrpHrpZpss protein of the invention is orally taken for 6h and orally taken for 24h from left to right by inducing testis expression differential gene volcano diagram of experimental mice through oral administration and smearing; smearing for 6 h;

FIG. 5 is a clustering chart of the induced kidney expression differential gene set of HrpHrpZpss protein oral administration and smearing experimental mice, which is orally administered for 6 hours and orally administered for 24 hours from left to right; smearing for 6 h;

FIG. 6 is a cluster chart of the gene set of difference in testis expression induced by oral administration of HrpHrpZpss protein and smearing of experimental mice, which is orally administered for 6h and orally administered for 24h from left to right; smearing for 6 h;

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

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

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

FIG. 12 shows a comparison of the HrpZpss protein-treated testis and a control KEGG Pathway (down-regulated gene) of the present invention, 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 preparation of the HrpZpss multi-epitope ligand protein can adopt engineering bacteria of registered hrpZpss genes (Genbank ID: AAY36247.1) to prepare and collect depolymerized and activated HrpZpss protein through fermentation and purification, and specifically comprises the following steps:

1) and (3) engineering bacteria fermentation preparation of the HrpZpss 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 modification thereof) for encoding HrpZpss proteins, wherein the production line of related proteins is derived from K-12 original bacteria after special modificationThe bacterium JY-01(DE3) was cultured in LB liquid medium (containing 50. mu.g of kanamycin per liter) at a constant temperature until OD600 became 0.7, IPTG (Isopropyl thiogalactoside, IsopropyL. beta. -D-Thiogalactosid) (final concentration: 1mMol) was added, and the cells were collected by centrifugation after continuing the culture. The expression product of the HrpZpss protein is analyzed by 10% SDS-PAGE polyacrylamide gel electrophoresis, and a 34.73kd band is shown on a sample lane of an electrophoresis gel plate, and is the expression product of the gene hrpZpss HrpZpss 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 of 5-5.5 and glucose concentration of 200-300mmol, and washing the engineering bacteria in a butterfly continuous flow centrifuge for five to eight times; thirdly, the engineering bacteria are broken and the cell wall is removed, then Na with pH of 5-5.5 and glucose concentration of 200-300mmol is used₂HPO₄-KH₂PO₄Diluting the thallus with buffer solution, regulating the fresh weight of the thallus to 20-30% of the diluent, introducing into a high-pressure crusher, continuously crushing the engineering bacteria with the pressure of 800-1000MPa, introducing the crushed bacteria liquid into a butterfly continuous flow centrifuge, removing cell walls, and collecting high-polymerization-state HrpNECb multi-epitope protein molecules.

3) Depolymerization and activation of high polymeric HrpZpss multi-epitope protein molecules: preprocessing: with glucose Na₂HPO₄-KH₂PO₄Adjusting the volume concentration of the high-polymerization-state HrpNECb multi-epitope protein collected by fermentation to 10-15% by using buffer solution, and pretreating glucose Na under the condition of normal temperature (20-30 ℃)₂HPO₄-KH₂PO₄Buffer solution, pH 5-5.5, glucose concentration 200-And (4) mmol, pretreatment and treatment time is 4-6 h. ② depolymerizing and activating HrpZpss high-polymerization-state multi-epitope protein: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution, wherein the ultrahigh pressure is 2000-2500 Mpa; thirdly, after the operations of ultrahigh pressure depolymerization and activation are finished, standing for 2-4h at the temperature of 35-38 ℃, and then collecting the depolymerized and activated HrpZpss multi-epitope protein molecules; and fourthly, purifying the high-aggregation multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, and performing protein purification according to a method suggested by an NI-NTA affinity chromatography gel manufacturer to complete the preparation of the depolymerized and activated purified HrpZpss protein.

Example 2

The HrpZpss 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 HrpZpss gene encoding HrpZpss protein;

1) the nucleotide sequence of hrpZpss gene (Genbank ID: AAY36247.1) for encoding the HrpZpss protein published to GenBank according to modern bioinformatics is used as an artificially synthesized HrpZpss multi-epitope protein gene, and the DNA sequence of the hrpZpss gene is derived from GenBank: AAY36247.1

Cloning of the gene encoding the HrpZpss protein:

5’-tgcggatccatgcagagtctcagtcttaacagc-3’

5’-tgcaagctttcaggccgcggcctgaatgcgggt-3’

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

the second step is that: 2) according to the DNA sequence, when the protein gene is artificially synthesized, BamHI enzyme cutting sites and HindIII enzyme cutting sites are respectively added on the 5 'and 3' of the gene, so that the protein gene can be conveniently cloned;

the third step: artificial gene synthesis was delegated to the GeneArt Gene Synthesis and service department of Thermo Fisher Scientific. 3) The synthesized DNA fragment for coding the HrpNECb protein gene is cloned to the BamHI-HindIII site of the constructed high-efficiency protein expression vector JY-01 (containing His-Tag label) one by one, and the cloning accuracy is ensured by DNA sequencing;

the fourth step: transferring the gene clone of the HrpNECb protein coded in the 1) to 3) into an escherichia coli engineering bacterium (E.coli), wherein a production line (E.coli) of related protein is a derivative bacterium JY-01(DE3) of K-12 original bacterium after special modification; when cultured in LB liquid medium (50. mu.g of kanamycin per liter) at 37 ℃ until OD600 is 0.7, IPTG (Isopropyl thiogalactoside, IsopropyL beta-D-Thiogalactosid) (final concentration is 1mMol) is added, the culture is continued, then the thalli are centrifugally collected, 10% SDS-PAGE polyacrylamide gel electrophoresis is used for analyzing the expression product HrpNECb protein, and an 36.64kda band appears on a sample lane of an electrophoresis gel plate, which is the expression product HrpZpss protein of the hrpZpss, and is shown in detail in figure 1;

wherein the culture medium Na for fermentation₂HPO₄-KH₂PO₄A buffer system, the pH of the buffer system is 6.5-5.5; the glucose concentration of the fermentation proliferation liquid culture medium is 0.01-0.05%; the lactose concentration of the fermentation induction liquid culture medium is 0.5-0.4%;

the fifth step: suspending the collected cells in Na₂HPO₄-KH₂PO₄In a buffer solution, finishing sterilization treatment at the temperature of 80 ℃ for 30 minutes, rapidly cooling to 30 ℃, cleaning engineering bacteria for five to eight times in a butterfly continuous flow centrifuge, introducing into a high-pressure crusher, continuously crushing the engineering bacteria under the pressure of 800-;

and a sixth step: depolymerization and activation of high polymeric HrpNECb multi-epitope protein molecules (I) pretreatment with glucose Na₂HPO₄-KH₂PO₄Buffer solution adjustment for collecting purified high-polymerized HrpZpss multi-epitope proteinThe volume concentration is 10-15%. At normal temperature (20-30 ℃), pre-treating glucose Na₂HPO₄-KH₂PO₄Buffer, pH 5-5.5. The glucose concentration is 200-300mmol, and pretreatment is carried out; the treatment time is 4-6 h.

(II) depolymerizing and activating the HrpNECb high-polymerization-state multi-epitope protein to perform ultrahigh-pressure depolymerization and activation on the high-polymerization-state protein pretreatment solution obtained by the pretreatment, wherein the ultrahigh-pressure range is 2000-2500 Mpa;

(III) after the operation of ultrahigh pressure depolymerization and activation is finished, standing for 2-4h at 35-38 ℃, and then collecting the depolymerized and activated HrpZpss multi-epitope protein molecules.

(IV) purifying the high-aggregation multi-epitope protein-His-Tag recombinant protein by using 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 HrpZpss 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 electrophoresis band before depolymerization and activation is 1, and more bands are gathered in the corresponding molecular weight region, and a band of 34.73kd is also included; at 2 is a band of depolyated and activated purified HrpZpss proteins with a molecular weight of 34.73kd in the corresponding molecular weight region of the ligand protein, indicating that the corresponding depolyated and activated purified HrpZpss proteins have been obtained.

As shown in fig. 2, allergy assay tests for disaggregating activated multiple epitope ligand proteins: the result of tobacco leaf reaction 24hr after the HrpZpss protein preparation and the sterile water treatment is shown in FIG. 2, wherein point A, C is injection of 300. mu.g.mL^-1100 μ L of the HrpZpss protein solution; B. point D is a control treatment of 100. mu.L of sterile water injected. 300. mu.g/mL^-1Treating with HrpZpss protein solution for about 12hr to cause tobacco leaf atrophy and collapse, and dying at 24 hr; the water control treated tobacco leaves had no allergic reactions.

The depolymerized and activated multi-epitope ligand protein can generally trigger hypersensitivity of various plant leaves, and the types of the test plants can be as follows: tobacco, pepper, eggplant, tomato, potato, strawberry, cucumber, water spinach, cockscomb, begonia glauca, chamomile, pansy, annatto, petunia, grape, Chinese rose, locust tree, pea, peach, sage, luffa, kidney bean, cauliflower, spinach, rape, yam, cowpea, broad bean, corn, rice, soybean, cyclamen, mulberry, pumpkin, loquat, and toona sinensis.

Example 3

Sequencing of animal Experimental mRNA (RNA-Seq)

mRNA-seq is the conversion of RNA produced by cells into DNA by a reverse transcription process (cDNA, complementation, and library construction of the obtained cDNA). The resulting DNA is then sequenced and the original amount of mRNA in the cell is inferred from the observed abundance of the particular DNA, thereby finding genes or transcripts whose transcription levels vary under the experimental conditions, i.e., differential expression. By finding these differentially expressed genes and transcripts, functional characteristics of the different conditions were deduced. We used RNA-seq technology to study and demonstrate that the HrpNECb protein induces differential expression of multiple genes in multiple organs of mice.

1. Laboratory animal sample treatment

The experiment is carried out by entrusting a protein mass spectrum technology platform of Shanghai Huaying biological medicine science and technology Limited company.

Treatment of experimental samples: the experimental selection of 8-week-old balb/C experimental mice is divided into HrpZpss protein treatment groups, which comprise 4 treatments of oral administration for 6 hours and 24 hours and smearing for 6 hours and 12 hours, wherein 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 HrpZpss 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 HrpZpss protein buffer solution with the 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 kidney, testis 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 Bioanalyzer 4200(Agilent technologies, Santa Clara, Calif., US), and RNA that was qualified for quality testing was subjected to subsequent sequencing experiments.

Library construction and quality control

Use of the constructed library

2.0Fluorometer assay concentration, Agilent2100 assay size.

Computer sequencing

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

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

3. Analysis of results

1) HrpZpss protein-induced differential gene screening

The method comprises the steps of firstly normalizing fragment counts, then calculating p-value according to a hypothesis test model, and finally carrying out test correction on p-value multiple hypotheses to obtain an FDR value. FP KM values were calculated as Fold-change differential expression using the edgeR software. The differential gene screening conditions were as follows: p-value <0.05 and | Fold-change | > 2.

2) HrpZpss protein-induced differential gene volcano pattern

The volcano plot of the difference gene is adopted to show the overall distribution of the HrpNECb protein-induced expression difference significant genes. The abscissa: fold change in gene expression in different samples (log2 Fold-Chan ge); the ordinate is: 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 volcanoes induced by oral and smear administration of mouse kidney and testis HrpZpss proteins, respectively, wherein HrpZpss is abbreviated as Z2.

3) HrpZpss protein-induced differential gene clustering map

And carrying out cluster analysis on the differential gene set, gathering the genes with similar expression modes together, and displaying that the genes have common functions or participate in a common signal path. Log10(FPKM +1) values were normalized (sca le number) and clustered, with red indicating high expression and blue indicating low expression in the heatmap. FIGS. 5-6 are cluster heatmaps of differential gene cluster for kidney and testis expression, respectively, where HrpZpss are abbreviated as Z2.

4) Enrichment analysis of differential gene GO induced by HrpZpss 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 to indicate which biological processes the gene is involved in; 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, it is 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 differential expression genes induced by the HrpNECb protein are subjected to GO term enrichment analysis, and the results prove that the HrpNECb protein has a plurality of epitope special structures, brand-new functions, brand-new action mechanisms and brand-new ligand proteins with application prospects, and induces the differential expression of multiple genes of multiple organs (kidney and testis) of a mouse, and the differential expression genes cover biological processes, cell components and molecular functions. The results of the enrichment analysis of the HrpNEcb protein-induced differential gene GO are further described as follows: biologically-process-related differentially expressed genes include reproductive, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesive, signaling, multicellular biological processes, developmental processes, growth, movement, single tissue processes, biological, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, stimulatory responses, localization, bioregulation, cell component organization or biogenesis, cell aggregation, detoxification, and presynaptic processes involving synaptic transmission. The results of the bioprocess GO enrichment analysis are detailed in tables 1 to 2.② 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 1-2. (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 the molecular function GO are detailed in tables 1 to 2.

Gene Ontology (GO) is an Ontology widely used in bioinformatics to cover the statistical Table 1-2 of Goterms classification genes with p-values less than 0.05 for three levels of biological cellular components, molecular functions, and biological processes.

Wherein HrpZpss is abbreviated as HrpZ2 in tables 1-2, and blank spaces in all tables indicate that no corresponding related data not reaching the p-va l ue less than 0.05 standard is collected, and the following and all tables have the same blank meaning.

TABLE 1 biological Process, cellular Components and related functional groups of molecular function of HrpZpss protein induced testis and kidney significantly up-regulated expression GO terms Classification gene statistical table (oral 6, 24 hours and smearing 6 hours)

TABLE 2 biological Process, cellular Components and functional groups related to molecular function of HrpZpss protein-induced testis and kidney-significantly downregulated expression of GO terms classifier gene statistics (oral 6, 24 hours and 6 hours after 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 HrpZpss protein-induced differential expression genes, the roles (upstream and downstream relationship) 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 HrpZpss 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 (kidney, testis and the like) 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 HrpZpss protein-induced differential gene GO are further described as follows: (ii) Cellular Processes (Cellular Processes): the HrpZpss protein induces a plurality of differentially expressed genes which are involved in cellular processes such as trafficking and catabolism, cell population, cell activity, cell growth and death (see FIGS. 7 to 12 for details). (Environmental Information Processing) multiple differentially expressed genes induced by the HrpZpss protein participate in the Environmental Information Processing processes such as signal molecule interaction, signal transduction, membrane transport and the like (see detailed FIGS. 7 to 12). (iii) Genetic Information Processing (Genetic Information Processing) multiple differentially expressed genes induced by the HrpZpss protein are involved in the biological processes of translation, replication and repair, folding, classification and degradation (see FIGS. 7 to 12 for details). Metabolism (Metabolism) the various differentially expressed genes induced by the HrpZpss proteins 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 FIGS. 7 to 12 for details). Multiple differentially expressed genes induced by HrpZpss protein are involved in cell processes of sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, developmental circulatory system, etc. (detailed in FIGS. 7 to 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, informational 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; the ordinate is: 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 identifying and binding specific protein by HrpZpss protein

1. Sample preparation and processing

1) HrpZpss protein purification

And (3) purifying the high-polymeric HrpZpss multi-epitope protein-His-Tag recombinant protein by using NI-NTA affinity chromatography gel, wherein the protein purification is carried out according to the method suggested by NI-NTA affinity chromatography gel manufacturers, and the purification preparation of the depolymerized and activated multi-epitope protein HrpZpss 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 3BCA assay for protein concentration

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) Fixing biotin-labeled bait protein and biotin, namely adding the biotin and the biotin-labeled bait protein into a Spin Column tube respectively, and screwing a top cover and a bottom plug; gently shaking the rotary platform rotating platform, and incubating for 60min at 4 ℃; thirdly, after the incubation is finished, removing the top cover and the bottom plug of the Spin Column tube, and putting the Spin Column tube into a collecting tube; 1250 Xg, after centrifugation for 60s, the Spin Column tube was replaced in the collection tube.

3) Blocking of biotin firstly, adding 250 mu l of biotin blocking solution into a Spin Column tube. Screwing down the top cover and the bottom plug, and slightly reversing the top cover and the bottom plug for 4 times to uniformly mix the mixture; ② incubating for 5min at room temperature. Removing the top cover, placing Spin Column tubes into the collection tube, and centrifuging at 1250 Xg for 50 s; thirdly, repeating the step 1 and the step 2 once; fourthly, 250 mul of TBS is added into the Spin Column tube. Screwing down the top cover, and slightly reversing the top cover and the bottom cover for 4 times to uniformly mix the mixture; fifthly, removing the top cover, placing the top cover in a collecting pipe, and centrifuging for 50s at 1250 Xg; sixthly, repeating the step 3 and the step 4 twice, and putting the Spin Column tube into the collecting tube again.

4) Capture of biotin-labeled protein (i.e., adding 300. mu.L (1mg protein) of capture protein (target protein) sample solution into Spin Column tube, and screwing down the cap; gently shaking the rotary platform rotating platform, and incubating overnight at 4 ℃; and thirdly, removing the top cover and the bottom plug after the incubation is finished. Putting the Spin Column tube into a prepared collecting tube; fourthly, collecting the tube, 1250 Xg, 60s, centrifuging, and marking the collecting tube with "prey flow-through (B)"; fifthly, removing the Spin Column tube in the collecting tube, covering the cover of the collecting tube, and placing on ice for subsequent analysis; sixthly, putting the Spin Column tube into a new collecting tube to prepare for elution.

5) Elution of complexes of bait protein and target protein from Spin columns (i.e., 250. mu.l of Wash Buffer was added to each Spin Column). Screwing down the top cover and the bottom plug, and slightly reversing for 6 times to uniformly mix the mixture; ② the Spin Column tube was incubated at room temperature for 1 minute. The top and bottom plugs were removed, Spin columns were placed on collection tubes, and centrifuged at 1250 × g for 50 s. Repeating the steps for 1-2 and 3 times; ③ during the flushing process, the label 'Wash 1, … …, Wash 3' is written on the collecting tube; fourthly, when the last washing is carried out, 200 mul of Wash Buffer is added, and then the liquid in the tube together with the beads is transferred to 1.5 mL; fifthly, in a new centrifuge tube, after centrifugation, 170 mu l of supernatant is discarded, and the step is repeated for 3 times.

6) And (3) detection: sucking up the liquid on Sepharose, adding 20 mul of 1 Xprotein electrophoresis sample buffer solution, boiling water bath for 5min, and placing in a refrigerator at-20 deg.C for later use; and secondly, detecting through SDS-PAGE and Western blot.

3. Analysis of results

1) The HrpZpss protein recognizes bound cell membrane receptors: HLA-C major histocompatibility complex, class I, class C receptor, free fatty acid receptor 4, tyrosine protein kinase transmembrane receptor 1, ASGPR1 asialoglycoprotein receptor 1, adipose cytoplasmic membrane associated protein receptor (APMAP), insulin-like growth factor 2 receptor (IGF 2R).

2) The HrpZpss protein recognizes the bound cell membrane protein: solute carrier family 5 (sodium-dependent vitamin transporter), member 6, solute carrier family 26, member 4, solute carrier family 38, member 2(SLC38a2), DNM2 dynein 2, CAP1 adenylate cyclase-associated protein 1, ICAM1 intercellular adhesion molecule 1 LanC-like protein 1 (lanthionine synthase C) (LANCL1), MLEC stress protein-androgen-like receptor kinase, TJP2 claudin 2, ZYX zebra-catenin.

3) The HrpZpss protein recognizes the bound signaling pathway: the role of the hsa04933 age-anger signaling pathway in diabetic complications, the hsa04064 NF-kappa B signaling pathway, the hsa04072 phospholipase D signaling pathway.

4) The HrpZpss protein recognizes the associated metabolic pathways of antiviral, antibacterial, anti-foreign, anti-inflammatory activity in combination: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa04666 Fc gamma r-mediated phagocytosis, hsa04210 apoptosis, hsa04218 cell senescence, hsa05130 pathogenic E.coli infection, hsa04612 antigen processing and presentation, hsa05100 bacterial invasion of epithelial cells, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05164 influenza A, hsa05150 Staphylococcus aureus infection, hsa05167 Kaposi sarcoma-associated herpesvirus infection, hsa04916 bactericidal effect, hsa04650 natural killer cell-mediated cytotoxicity, hsa05169 Epstein Barr virus infection, hsa05416 viral myocarditis, hsa05110 Vibrio infection, hsa05144 malaria, hsa05163 human cytomegalovirus infection, hsa05170 human immunodeficiency virus type 1 infection, hsa rheumatoid arthritis, 05323 viral leukocyte migration across cells.

5) The HrpZpss protein recognizes the combined important neurological disease metabolic pathways: it was not detected.

6) The HrpZpss protein recognizes the associated pathways for nucleic acid, protein, amino acid, sugar, fat metabolism: hsa03050 proteasome, protein digestion and absorption of hsa04974, and hsa04120 ubiquitin mediated proteolysis.

7) The HrpZpss protein recognizes the combined metabolic pathways of cell junctions, nerve junctions, blood vessels, endocrine, reproductive systems and the like: hsa04510 focal adhesions, hsa04724 glutamatergic synapses, hsa04530 tight junctions, hsa04261, adrenergic signaling of cardiomyocytes, hsa04940 type I diabetes, hsa04924, renin secretion, hsa04721 synaptic vesicle cycling, reabsorption of calcium mediated by factors such as hsa04961 endocrine, hsa04970, salivary secretion, hsa04979 cholesterol metabolism, hsa04918 thyroid hormone synthesis, hsa04514 Cell Adhesion Molecules (CAMs), hsa04727 neurons, hsa05320 autoimmune synaptic thyroid disease, hsa05418 fluid shear stress, and atherosclerosis.

The HrpZpss 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 analyze the positions, structures, characteristics, action mechanisms and functions of the membrane receptors, the membrane proteins, the information channels and the metabolic channels, widely influence the basic life attributes of the organism such as growth, development, metabolism, defense and programmed cell death, and widely relate 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 HrpZpss protein is a special multi-epitope ligand protein with brand new functions, brand new action mechanism and brand new application prospect.

The above-mentioned embodiments only express the specific embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, without departing from the technical idea of the present application, several changes and modifications can be made, which are all within the protection scope of the present application.

<110> Wu-Bai-Ji-Wu-Bao-Zhen-Kun-Ming-Si-Sci-Tech Co Ltd

Application of <120> HrpZpss protein in pharmacy for identifying and activating multiple types of receptors and/or membrane proteins and signal paths thereof

<160> 1

<170> Patent In Version 2.1

<210> 2

<211> 343

<212> PRT

<213> HrpZpss(Pseudomonas syringae pv.syringae strain 301D hrpZ2)（SEQ ID NO1）

<220> 1

<221> DOMAIN

<222> conserved domain (1) - (343), alpha-helical domain (37) - (52), (61) - (72), (80) - (93), (109) - (128), (140) - (155), (157) - (159), (168) - (173), (180) - (199), (248) - (259), (263) - (265), (290) - (309), (317) - (336)

<400> 1

Met Gln Ser Leu Ser Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met

1 5 10 15

Ala Leu Val Leu Val Arg Pro Glu Thr Glu Thr Thr Gly Ala Ser Ser

20 25 30

Ser Ser Lys Ala Leu Gln Glu Val Val Val Lys Leu Ala Glu Glu Leu

35 40 45

Met Arg Asn Gly Gln Leu Asp Asp Ser Ser Pro Leu Gly Lys Leu Leu

50 55 60

Ala Lys Ser Met Ala Ala Asp Gly Lys Ala Gly Gly Gly Ile Glu Asp

65 70 75 80

Val Ile Ala Ala Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn

85 90 95

Phe Gly Ala Ser Ala Asp Asn Ala Ser Gly Thr Gly Gln Gln Asp Leu

100 105 110

Met Thr Gln Val Leu Asn Gly Leu Ala Lys Ser Met Leu Asp Asp Leu

115 120 125

Leu Thr Lys Gln Asp Gly Gly Thr Ser Phe Ser Glu Asp Asp Met Pro

130 135 140

Met Leu Asn Lys Ile Ala Gln Phe Met Asp Asp Asn Pro Ala Gln Phe

145 150 155 160

Pro Lys Pro Asp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn

165 170 175

Phe Leu Asp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile

180 185 190

Ile Gly Gln Gln Leu Gly Asn Gln Gln Ser Gly Thr Gly Gly Leu Ala

195 200 205

Gly Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Ser Asn Ser

210 215 220

Ser Gly Val Lys Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly

225 230 235 240

Asp Ser Gly Thr Thr Ser Gly Glu Ala Gly Gln Leu Ile Gly Glu Leu

245 250 255

Ile Asp Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu Gly Thr

260 265 270

Pro Val Asn Thr Pro Gln Thr Gly Thr Ala Ala Asn Gly Gly Gln Ser

275 280 285

Ala Gln Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu Leu Lys Gly Leu

290 295 300

Glu Ala Thr Leu Lys Asp Ala Gly Gln Thr Ala Thr Asp Val Gln Ser

305 310 315 320

Ser Ala Ala Gln Ile Ala Thr Leu Leu Val Ser Thr Leu Leu Gln Gly

325 330 335

Thr Arg Asn Gln Ala Ala Ala

340

Claims

The application of the HrpZpss protein in identifying and activating various types of receptors and/or membrane proteins and signal paths thereof and causing cascade biological effects is disclosed in the specification, wherein the amino acid sequence of the HrpZpss protein is shown in SEQ ID NO. 1.
2. The use of the HrpZpss 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 cascade biological effects, wherein the multiple classes of receptors comprise one or more of HLA-C major histocompatibility complex, class I, C receptor, free fatty acid receptor 4, tyrosine protein kinase transmembrane receptor 1, ASGPR1 asialoglycoprotein receptor 1, adipocyte plasma membrane-associated protein receptor, insulin-like growth factor 2 receptor.
3. The use of the HrpZpss protein of claim 1 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 the membrane proteins comprise one or more of solute carrier family 5, member 6, solute carrier family 26, member 4, solute carrier family 38, member 2, DNM2 dynein 2, CAP1 adenylate cyclase-associated protein 1, ICAM1 intercellular adhesion molecule 1 LanC-like protein 1, MLEC stress protein-androgen-like receptor kinase, TJP2 tight junction protein 2, ZYX macular synapterin.
4. The use of the HrpZpss protein of claim 1 in the identification of drugs that activate multiple classes of receptors and/or membrane proteins and their signaling pathways and elicit cascade biological effects, wherein the signaling pathway comprises one or more of the effects of the hsa04933 age-anger signaling pathway in diabetic complications, the hsa04064 NF-kappa B signaling pathway, the hsa04072 phospholipase D signaling pathway.
5. The use of the HrpZpss 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 the signaling pathways comprise metabolic signaling pathways comprising antiviral, antibacterial, anti-foreign, anti-inflammatory 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 HrpZpss protein of claim 5 in the identification of drugs that activate multiple classes of receptors and/or membrane proteins and their signaling pathways and elicit a cascade of biological effects, wherein the antiviral, antibacterial, anti-foreign, anti-inflammatory metabolic pathways: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa04666 Fc gamma r-mediated phagocytosis, hsa04210 apoptosis, hsa04218 cell senescence, hsa05130 pathogenic E.coli infection, hsa04612 antigen processing and presentation, hsa05100 bacterial invasion of epithelial cells, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05164 influenza A, hsa05150 Staphylococcus aureus infection, hsa05167 Kaposi sarcoma-associated herpesvirus infection, hsa04916 bactericidal effect, hsa04650 natural killer cell-mediated cytotoxicity, hsa05169 Epstein Barr virus infection, hsa05416 viral myocarditis, hsa05110 Vibrio infection, hsa05144 malaria, hsa05163 human cytomegalovirus infection, hsa05170 human immunodeficiency virus type 1 infection, hsa rheumatoid arthritis, 05323 viral leukocyte migration; the nucleic acid, protein, amino acid, sugar and fat metabolism pathways: hsa03050 proteasome, hsa04974 protein digestion and absorption, and hsa04120 ubiquitin-mediated proteolysis; the cell junctions, nerve junctions, blood vessels, endocrine, reproductive metabolic pathways: hsa04510 focal adhesions, hsa04724 glutamatergic synapses, hsa04530 tight junctions, hsa04261, adrenergic signaling of cardiomyocytes, hsa04940 type I diabetes, hsa04924, renin secretion, hsa04721 synaptic vesicle cycling, reabsorption of calcium mediated by factors such as hsa04961 endocrine, hsa04970, salivary secretion, hsa04979 cholesterol metabolism, hsa04918 thyroid hormone synthesis, hsa04514 Cell Adhesion Molecules (CAMs), hsa04727 neurons, hsa05320 autoimmune synaptic thyroid disease, hsa05418 fluid shear stress, and atherosclerosis.
7. The use of the HrpZpss protein of claim 1 in the identification of drugs that activate multiple classes of receptors and/or membrane proteins and their signaling pathways and elicit cascade biological effects, wherein the 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 HrpZpss protein; the environmental information processing comprises the steps that multiple differential expression genes induced by HrpZpss protein participate in the processes of signal molecule interaction, signal transduction and membrane transport environmental information processing; the genetic information processing comprises that a plurality of differentially expressed genes induced by HrpZpss protein participate in the biological processes of translation, replication and repair, folding, classification and degradation; metabolism includes that a plurality of differentially expressed genes induced by HrpZpss protein participate in biodegradation and metabolism, nucleotide metabolism, amino acid metabolism, metabolic accessory factors and vitamins, lipid metabolism, biosynthesis and metabolism of sugar, global and overview maps, energy metabolism, carbohydrate metabolism and amino acid metabolism processes; the biological system comprises a plurality of differentially expressed genes induced by HrpZpss protein and participates in biological processes of a sensory system, a nervous system, an immune system, an excretory system, environmental adaptation, an endocrine system, a digestive system and a development and circulation system.
8. The use of the HrpZpss protein of claim 1 for identifying drugs that activate multiple classes of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects, wherein the preparation or dosage form of the drug for use in the drug preparation is a liquid, powder, tablet or capsule.
9. The use of the HrpZpss proteins of claim 7 in the manufacture of a medicament for the recognition of and activation of multiple classes of receptors and/or membrane proteins and their signaling pathways leading to cascade biological effects, wherein the preparation or medicament is prepared predominantly from depolymerized activated HrpNECb proteins at a mass content of 0.001% to 100%.
10. The method for the depolymerization and activation of the HrpZpss 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 HrpZpss multiple epitope proteins prepared by fermentation by using buffer solution within 0-30%, and treating for 0-24h at the temperature of 20-30 ℃;

step 2: depolymerizing and activating HrpZpss high-polymerization-state multi-epitope proteins: carrying out ultrahigh pressure depolymerization and activation operation on the pretreated high polymeric protein pretreatment solution in the step 1 within the ultrahigh pressure range of 1000-3000 MPa;

and step 3: and (3) post-treatment: after the operations of ultrahigh pressure depolymerization and activation are finished, standing for 0-24h at 35-38 ℃, and then collecting depolymerized and activated multiple epitope protein molecules of HrpZpss;

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