WO2022142976A1 - Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals - Google Patents

Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals Download PDF

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WO2022142976A1
WO2022142976A1 PCT/CN2021/134714 CN2021134714W WO2022142976A1 WO 2022142976 A1 WO2022142976 A1 WO 2022142976A1 CN 2021134714 W CN2021134714 W CN 2021134714W WO 2022142976 A1 WO2022142976 A1 WO 2022142976A1
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protein
ligand protein
epitope
hrpn
metabolism
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PCT/CN2021/134714
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French (fr)
Chinese (zh)
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吴伯骥
吴保珍
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昆明锐斯得科技有限公司
吴伯骥
吴保珍
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Priority claimed from CN202011633925.1A external-priority patent/CN114685681A/en
Priority claimed from CN202011633933.6A external-priority patent/CN112675293A/en
Application filed by 昆明锐斯得科技有限公司, 吴伯骥, 吴保珍 filed Critical 昆明锐斯得科技有限公司
Priority to AU2021412922A priority Critical patent/AU2021412922A1/en
Publication of WO2022142976A1 publication Critical patent/WO2022142976A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria

Definitions

  • the present invention relates to the field of biomedicine, in particular to the application of HrpN-type protein in the pharmacy that recognizes and activates multiple types of receptors and/or membrane proteins and their signal pathways and causes cascade biological effects.
  • Molecular biology is a science that studies life phenomena at the molecular level. It clarifies the nature of various life phenomena by studying the structure, function and metabolism of biological macromolecules, and its research content covers the whole process of life.
  • DNA, RNA and protein are three important biological macromolecules, which are the molecular basis of life phenomena.
  • the genome determines what life has, the proteome determines what life can do, and the metabolome determines what actually happens to life.
  • Modern life sciences, biotechnology and medical biotechnology, especially proteomics and metabolomics have developed by leaps and bounds, updating the concepts of disease understanding, diagnosis, prevention and control, treatment and rehabilitation, creating new and efficient The new understanding and new approach of safe drugs have brought the development of modern medicine into a new stage and opened up broad application prospects.
  • Ligand is a kind of signal substance. Except for recognizing, binding and activating the receptor, it has no other direct functions. It cannot participate in metabolism to produce useful products, nor does it directly induce any cell activity, and it has no enzyme characteristics. Its only function is to transmit special signals or information that exist in the internal and external environment to cells through the recognition, binding and activation of receptors.
  • Signaling pathway refers to the communication mechanism that sends and receives information with high precision and efficiency between cells or within cells of multicellular organisms, and causes rapid cellular physiological and biochemical reactions through amplification, or initiates gene activity, and then a series of events occur.
  • the physiological and biochemical activities of cells coordinate the activities of various organizations, and promote the unified whole of life to make a comprehensive response to the changing internal and external environment.
  • Receptors refer to a class of functional proteins that mediate cell signal transduction. They can recognize certain trace substances in the surrounding environment (internal and external environment of cells), recognize, bind to, and be activated to trigger subsequent physiological processes through the signal amplification system. biochemical reaction. Receptors are biological macromolecules composed of cell membranes and intracellular proteins, nucleic acids, lipids, and polysaccharides. Receptor is a very broad concept in cell biology, which refers to any biological macromolecules that can bind to hormones, neurotransmitters, drugs or signal molecules inside and outside cells and can cause changes in cell function. called ligands. There are hundreds of different signaling molecules in multicellular organisms that transmit information between and within cells.
  • These signaling molecules include proteins, amino acid derivatives, nucleotides, cholesterol, fatty acid derivatives, and soluble gas molecules.
  • the receptors present on the cytoplasmic membrane are called membrane receptors, and most of the chemical essences are sugar mosaic proteins; the receptors located in the cytosol and nucleus are called intracellular receptors, and they are all DNA-binding proteins.
  • Ligand is a kind of signal substance. Except for recognizing, binding and activating the receptor, it has no other direct functions. It cannot participate in metabolism to produce useful products, nor does it directly induce any cell activity, and it has no enzyme characteristics. Its only function is to transmit special signals or information that exist in the internal and external environment to cells through the recognition, binding and activation of receptors.
  • the binding of ligands and receptors is an intermolecular recognition and activation process, which relies on ionic coordination bonds, hydrogen bonds, ⁇ - ⁇ stacking, electrostatic interaction, hydrophobic interaction, van der Waals force, etc., with the complementarity of the two molecular spatial structures.
  • the degree of interaction increases, the distance between the interacting groups will be shortened, and the interaction force will be greatly increased. Therefore, the interaction and complementarity of the spatial structure of the ligand and receptor molecules are the main factors for specific binding, that is, the The concept of "structural group" or "epitope" employed in the invention.
  • the same ligand may correspond to two or more different receptors, and the binding of the same ligand to different types of receptors will produce different cellular responses.
  • the ligand After the ligand binds to the receptor, it will trigger a series of physiological activities, no matter whether the ligand is endogenous or exogenous, after binding to the receptor, the two form a ligand-receptor binding surface or complex, thereby Transmission of information, through conduction and transduction, and through amplification to cause rapid cellular physiological and biochemical reactions, or to initiate gene activity, and then a series of cascade reactions occur to coordinate the activities of various tissues, organs, and cells, and contribute to the unity of life. Make a comprehensive response to the changing internal and external environment.
  • leader et al. first proposed the idea of classification according to the pharmacological effects of proteins, and divided protein drugs into the following four categories: (1) protein drugs using the enzymatic activity and regulating activity of proteins for treatment; (2) proteins with special targeting activities Drugs; 3 recombinant protein vaccines; 4 recombinant protein drugs for diagnosis.
  • the first and second categories are mainly used for basic protein therapy, and the third and fourth categories emphasize the application of proteins in vaccines and diagnostic drugs.
  • protein drugs After more than a century of exploration and tortuous development, protein drugs have matured step by step and play an important role in the pharmaceutical industry and clinical applications.
  • the phenylalanine (Phe 82), isoleucine (Ile83), and pyrimidine (Val 85) of the binding epitope are the key amino acid residues for recognizing binding to bovine IgG2 receptor, for another example, positions 142-149 of boFc ⁇ RI Threonine (Thr 142), asparagine (Asn 143), leucine (Leu 144), glycine (Gly 148) and isoleucine (Ile 149) of the linear ligand-binding epitope of the polypeptide TNLSHNGI are recognized binding
  • Harpin is a class of proteins with similar properties and functions encoded by genes in the "hyper sensitive response and pathogenicity (hrp)" gene cluster of Gram-negative bacteria, rich in glycine and not containing cystine , Sensitive to protein enzymes, thermostable, and can cause allergic reactions in non-host plants.
  • Hypersensitive reaction (HR) is characterized by rapid and local atrophy and necrosis of infected tissues of non-host plants, which limits the spread of pathogenic bacteria and induces systemic resistance, which is a common manifestation of plant resistance to pathogenic infection. and effective ways. After nearly 30 years of research, these encoded proteins have been recognized by biologists, phytopathologists and applied researchers in the field.
  • Harpin hypersensitivity proteins belong to the class of anti-inducing proteins that induce plant systemic resistance.
  • biopesticides that can safely induce plants to produce disease resistance, insect repellency, stress resistance and promote plant growth and development and increase yield, such as patent publication number CN1687420, are named as a kind of encoding plant multifunctional activity and The gene hrpNEccs of broad-spectrum resistance cell signaling factor and its expression product HrpNEccs protein report content.
  • HrpNEcb protein (GenBank ID: ABD22989.1) is the expression product of hrpNEcb gene (Gene Accession No.: DQ355519.1), which consists of 370 amino acid residues, and is a non-enzymatic non-enzymatic protein with primary, secondary and tertiary structures without quaternary structure. Protein, free of cystine and cysteine, rich in glycine and serine, isoelectric point pI 5.43, molecular weight Mw 36636.21Da, GenBank ID: ABD22989.1.
  • HrpNEcb protein The conserved domain of HrpNEcb protein consists of 201 amino acids, located at the C-terminus of the protein, 170-370; 262-274, 276-279, 284-285, 298-303, 313-330, 347-349, 353-368; ⁇ -sheet structure 10-15, 255-256; IDPs structure (intrinsically disordered protein, intrinsically disordered proteins, referred to as IDPs) 1-11, 13-43, 67-95, 99-139, 157-174, 197-216, 340-341, 364-370.
  • HrpNEch protein (GenBank Protein: AAY17519.1) is the secreted protein of Erwinia "Erwinia chrysanthemi (Dickya dadantii) NEchCSCL006 strain", the expression product of hrpNEch gene (Gene Registration No.: GenBank: AY999000.1).
  • Structural domains are regions of biological macromolecules with specific structures and independent functions, especially the independent stable structural regions in proteins composed of different secondary structures and super-secondary structures. Domains are also protein functional units. In domain proteins, different domains are often associated with different functions; the secondary and super-secondary structures of proteins are mainly maintained by hydrogen bonds, including ⁇ -helix, ⁇ -sheet, ⁇ -turn, random coil and IDPs structure, etc.
  • the alpha helix is a repetitive structure with ⁇ and ⁇ around -57° and -47°, respectively, for each ⁇ -carbon in the helix.
  • Each turn of the helix occupies 3.6 amino acid residues, rising 0.54 nm along the helix axis, each residue rotates 100° around the axis, rising 0.15 nm along the axis, and hydrogen bonds are formed between adjacent turns, and the orientation of the hydrogen bonds is almost the same as
  • the helical axes are parallel;
  • IDPs-structure is the structural region of intrinsically disordered proteins (IDPs), which has a wide range of allosteric effects. It is widely involved in and regulates transcription, translation, cell division, protein aggregation and cell signal transduction with high repeatability, chargeability, easy binding, spatial superiority and high coordination, and is particularly involved in the process of self-assembly regulation .
  • IDPs intrinsically disordered proteins
  • HrpN-type protein is a class of proteins encoded by genes in the "hyper sensitive response and pathogenicity (hrp)" gene cluster with similar properties and functions, including higher homology and close evolutionary relationship.
  • hrp hyper sensitive response and pathogenicity
  • the application prospects of various types of receptors, membrane proteins, and signaling pathways and metabolic pathways in animal (including human) cells can cause multifunctional cascade biological effects. However, there are no reports and applications of this kind of protein cross-border recognition, especially in animals and humans.
  • HrpN-type protein in pharmaceutics that recognizes and activates multiple types of receptors and/or membrane proteins and their signaling pathways and causes cascade biological effects.
  • HrpN-type proteins as a class of ligand protein molecules with special structures rich in multiple linear and conformational epitopes, can recognize, activate, and bind membrane receptors, membrane proteins, information pathways and metabolic pathways across various types of animals.
  • HrpN-type proteins are a class of ligand proteins with special multiple epitope structures, new functions, new mechanisms of action and new application prospects. They induce multi-directional, multi-level and multi-faceted biological effects and functions.
  • HrpN-type protein According to the structural characteristics of HrpN-type protein and its ability to recognize and bind membrane receptors and membrane proteins of various types of animals across borders, thereby activating multiple information pathways and metabolic pathways, we call it HrpN-type polyprotein. Epitope-like ligand proteins (multi epitopic-like ligand proteins).
  • HrpN-type multi-epitope ligand proteins in food, cosmetics, health care products or pharmaceuticals that recognize and activate various types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects.
  • HrpN-type multi-epitope ligand protein in identifying food, disinfectant, cosmetic, health care product or medicine that activates various types of receptors and/or membrane proteins and their signaling pathways and causes cascade biological effects.
  • HrpN-type polymimetic epitope ligand proteins contain structural groups or epitopes of one or more hydrophobic non-polar amino acid residues, structural groups or epitopes containing one or more polar uncharged amino acid residues, contain one or more A structural group or epitope of an amide polar uncharged amino acid residue, a structural group or epitope containing one or more acidic positively charged, basic negatively charged amino acid residues; hydrophobic nonpolar amino acid residues include valine, leucine, isoleucine, alanine, phenylalanine, methionine residues; polar uncharged amino acid residues include serine residues; amide group polar uncharged amino acid residues include aspartic acid residues Amide, glutamine residues; acidic positively charged, basic negatively charged amino acid residues including asparagine, glutamic acid, lysine, histidine, arginine residues; hydrophobic non-polar amino acid residues Base, polar uncharged amino acid
  • HrpN-type multi-epitopic ligand proteins include HrpNEcc, HrpNEca, HrpNEcb, HrpNEch, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNEa, HrpNSam, HrpNBag, HrpNPas, HrpNEnt.
  • these molecules are highly homologous, ranging from 60% to 99%;
  • HrpN-type multi-epitope ligand proteins are rich in multiple linear and conformational structural groups or epitopes, which refer to functional groups composed of key amino acid residues that can recognize and bind to cell membrane receptors, membrane proteins, etc.
  • the group is composed of the following amino acid residues, including proton-donating or proton-accepting amino acid residues that can recognize, bind, and activate with receptors; further, structural groups containing one or more hydrophobic non-polar amino acid residues Groups or epitopes, structural groups or epitopes containing one or more acidic positively charged, basic negatively charged amino acid residues, structural groups or epitopes containing one or more amide polar uncharged amino acid residues, Structural groups or epitopes containing one or more polar uncharged amino acid residues; further, rich in proton-donating (except methionine residues) or proton-accepting (including methionine residues) amino acid residues: glutamic acid , asparagine,
  • HrpNEcb multi-epitope ligand protein has 370 amino acid residues, of which 226 are key amino acid residues: including 94 hydrophobic non-polar amino acid residues, 41 polar uncharged amino acid residues, amide There are 44 base amino acid residues, 47 acidic positively charged and basic negatively charged amino acid residues, and key amino acids account for 61% of the entire sequence;
  • HrpNEcb multi-epitope ligand protein conserved structural region has 200 amino acid residues: among which the key There are 138 amino acid residues, including 51 hydrophobic non-polar amino acid residues, 19 polar uncharged amino acid residues, 28 amide amino acid residues, 40 acidic positively charged and basic negatively charged amino acid residues, Key amino acids account for 69% of the conserved domain; HrpNEcb protein ⁇ -helix region, which has 71 amino acid residues, 52 key amino acid residues: including 27 hydrophobic non-polar amino acid residues, polar uncharged amino acid residues
  • HrpNEch multi-epitope ligand protein has 339 amino acid residues, including 236 key amino acid residues: including 106 hydrophobic non-polar amino acid residues, 40 polar uncharged amino acid residues, amide There are 42 base amino acid residues, 48 acidic positively charged and basic negatively charged amino acid residues, and key amino acids account for 69% of the entire sequence; HrpNEch conserved structural region, the protein sequence has 326 amino acid residues, and 226 key amino acid residues , including 99 hydrophobic non-polar amino acid residues, 40 polar uncharged amino acid residues, 40 amide amino acid residues, 47 acidic positively charged, basic negatively charged amino acid residues, with amino acids in the conservative In the structural region, key amino acids account for 69%; the HrpNEch ⁇ -helix structure region has 7 ⁇ -helices, 2 ⁇ -sheets and 7 IDPs-structure regions, of which the ⁇ -helix region has 100 amino acid residues , 71 key amino acid residue
  • the above-mentioned key amino acid residues of the HrpNEch multi-epitope ligand protein can pass hydrogen Bonds, ionic bonds, hydrophobic, non-polar, polar, van der Waals forces, realize the complementarity, interaction, specific recognition, activation, and binding of ligands and receptors in terms of spatial structure and electrical properties, and form with multiple types of receptors Tight binding surfaces or complexes can cause changes in the conformation, energy, electrical properties and information of receptor molecules, and through signal transduction and transduction, lead to amplified cascade biological effects.
  • Multifunctional cascade biological effects refer to the significant differences in the expression of related functional gene groups at three levels of cellular components, molecular functions and biological processes in different organs and tissues, including cellular components (including cells, cell nodes, and cell parts). , extracellular matrix, extracellular matrix components, extracellular region, extracellular region part, macromolecular complex, membrane, membrane part, membrane-enclosed cavity, organelle, organelle part, supramolecular fiber, synapse, synaptic part, Antioxidant activity, etc.), molecular functions (including binding, catalytic activity, chemoattractant activity, chemorepellent activity, electron carrier activity, metal chaperone protein activity, molecular function regulators, active molecular sensors, nucleic acid binding transcription factors) activity, signal sensor activity, structural molecular activity, transcription factor activity protein binding, transport activity, etc.), biological processes (including behavior, bioadhesion, bioregulation, cell aggregation, cell death, cellular component organization or biogenesis, cellular processes, Detoxification, processes of
  • the amino acid sequence of the HrpNEcb multi-mimetic epitope ligand protein is shown in SEQ ID NO: 1; the amino acid sequence of the HrpNEch multi-mimetic epitope ligand protein is shown in SEQ ID NO: 2.
  • the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein
  • the multiple types of receptors that recognize and bind include LRRC1515-leucine repeat membrane protein receptor, HLA-A major histocompatibility Complex, one of class I, A receptors, LGALS3BP galactose 3 binding protein (receptor), LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein subunit Beta 2 receptor or more.
  • the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein
  • the multi-type receptors of mouse liver culture cells that recognize and bind include GNG12 guanine nucleotide binding protein ⁇ -12 receptor, ANXA5 Annexin A5 receptor, ANXA2 annexin A2 receptor, ANXA1 annexin A1 receptor, IGHG2 immunoglobulin weight constant ⁇ 2 receptor, IGHM immunoglobulin weight constant Mu receptor, CACNA1S calcium voltage-gated channel sub Unit ⁇ 1S receptor, ZNF185 zinc finger protein 185 receptor and HLA-A major histocompatibility complex, I, A receptors, LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein One or more of subunit ⁇ 2 receptor, KTN1 kinesin 1 receptor.
  • the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein
  • the recognized and bound mouse liver culture cell membrane proteins include DSG4 desmocollin, ANXA4 annexin A4, CAPRIN1 cyclin, 1UTRN Dystrophin, pinin desmosomal protein, VAMP-associated protein A, VCL focal adhesion protein, Ezrin epithelial-type cadherin, PKP3 platelet avidin 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD2N acetylated alpha linkage One or more of acid dipeptidase 2.
  • the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein
  • the recognized and bound mouse liver culture cell membrane proteins include DSC3 desmocollin, ANXA8/ANXA8L1 annexin A8/annexin A8 Similar protein 1, EVPL coat protein, POF1B actin binding protein premature ovarian failure 1B, CTNNA1 catenin, TGM1 transglutaminase 1, BAIAP2BAI1 associated protein 2, RAB29RAS oncogene family member, CLDN19 docking protein 19, STXBP2 Syntaxin-binding protein 2, VAMP vesicle-associated membrane protein-associated protein A, VCL focal adhesion protein, Ezrin epithelial cadherin, PKP3 platelet avidin 3, NAALAD2N acetylated ⁇ -linked acid dipeptidase 2, One or more of PKP1 platelet affinity protein 1, SPRR1A small
  • the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein
  • the signal pathways involved in the recognition and binding of mouse liver culture cell membrane proteins include hsa04152: AMPK signal pathway, hsa03460: Fanconi anemia anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: Chemokine signaling pathway, hsa04015: Rap1 signaling pathway, One or more of hsa04024: camp signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, hsa04390: hippo signaling pathway.
  • the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein
  • the signal pathways involved in the recognition and binding of mouse liver culture cell membrane proteins include 22 signal pathways hsa03320: PPAR signal pathway, hsa05120: pylorus Epithelial cell signal transduction in Helicobacter infection, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04070: phosphatidylinositol signaling system, hsa04010: MAPK signaling pathway, hsa04310: Wnt Signaling Pathways, hsa04062: Chemokine Signaling Pathway, hsa04015: Rap1 Signaling Pathway, hsa04024: CAMP Signaling Pathway, hsa04915: Estrogen
  • the signaling pathways include metabolic signaling pathways, and the metabolic signaling pathways include antiviral, antibacterial, anti-foreign body, and anti-inflammatory metabolic pathways, including important neurological disease metabolic pathways; including nucleic acid, protein, amino acid, sugar, and fat metabolic pathways; including Cell junctions, neural junctions, blood vessels, endocrine, reproductive system metabolic pathways.
  • the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein
  • the anti-viral, anti-bacterial, anti-foreign body, and anti-inflammatory metabolic pathways involved in recognizing bound mouse liver culture cell membrane proteins include: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa01130: biosynthesis of antibiotics, hsa05131: shigellosis, hsa04612: antigen processing and presentation, hsa05130: pathogenic E.
  • coli infection hsa05100: epithelial cell Bacterial invasion, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05168: Herpes simplex virus 1 infection, hsa05203: Viral carcinogenesis, hsa05166: HTLV-I infection, hsa05164: Influenza A, hsa05134: Legionnaires' disease, hsa05160: Type C hepatitis, hsa05162: measles, hsa05133: pertussis, hsa05322: systemic lupus erythematosus, hsa04670: transepithelial migration of leukocytes, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: pathway in cancer; Important neurological disease metabolic pathways: hsa05012: Parkinson's disease, hsa05016:
  • the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, which recognizes 29 anti-virus, anti-bacterial, anti-foreign body, anti-inflammatory related metabolisms involved in the bound mouse liver culture cell membrane protein Pathways include: hsa04144: endocytosis, hsa04145: phagosome, hsa04142: lysosome, hsa04666: Fc-r-mediated phagocytosis, hsa01130: biosynthesis of antibiotics, hsa05131: shigellosis, hsa04612: antigen processing and presentation, hsa05130: Pathogenic E.
  • coli infection hsa05100: Bacterial invasion of epithelial cells, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05203: Viral carcinogenesis, hsa05134: Legionnaires’ disease, hsa05160: Hepatitis C, hsa05162: measles, hsa05133: pertussis, hsa05322: systemic lupus erythematosus, hsa04670: transendothelial migration of leukocytes, hsa05152: tuberculosis, hsa05150: staphylococcus aureus infection, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: in Pathways in Cancer, hsa05143: African trypanosomiasis, hsa04750: Inflammatory mediator regulation of TRP channels, h
  • the biological effects of multifunctional cascades include significant differences in the expression of related functional gene groups at three levels of cellular components, molecular functions and biological processes that can induce different organs and tissues, including cellular components (including cells, cell nodes, cell parts, extracellular matrix, extracellular matrix components, extracellular region, extracellular region part, macromolecular complex, membrane, membrane part, membrane-enclosed cavity, organelle, part of organelle, supramolecular fiber, synapse, synaptic part, anti- oxidative activity, etc.), molecular functions (including binding, catalytic activity, chemoattractant activity, chemorepellent activity, electron carrier activity, metal chaperone protein activity, molecular function regulators, active molecular sensors, nucleic acid binding transcription factor activity , signal sensor activity, structural molecular activity, transcription factor activity protein binding, transport activity, etc.), biological processes (including behavior, bioadhesion, bioregulation, cell aggregation, cell death, cellular component organization or biogenesis, cellular processes, detoxification ,
  • the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein, and the cascade biological effects include Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism and Organismal Systems and other functional pathways; further, 1 Cellular Processes: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand proteins are involved in transport and catabolism, and cellular processes Population, cell activity, cell growth and death and other cellular processes; 2Environmental Information Processing: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand protein are involved in signaling molecules and interactions, signal transduction, Environmental information processing processes such as membrane transport; 3 Genetic Information Processing: Multiple differentially expressed genes induced by HrpNEcb protein participate in biological processes such as translation, replication and repair, folding, classification and degradation; 4 Metabolism: HrpNEcb protein-induced multiple differentially expressed genes involved in biodegradation and metabolism, nucleotide
  • HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, and the cascade biological effects include Cellular Processes, Environmental Information Processing, Genetic Information Processing, Functional pathways such as Metabolism and Organismal Systems; further, 1 Cellular Processes: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in transport and catabolism, and cellular processes Population, cell activity, cell growth and death and other cellular processes; 2Environmental Information Processing: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in signaling molecules and interactions, signal transduction, Environmental information processing processes such as membrane transport; 3 Genetic Information Processing: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in biological processes such as translation, replication and repair, folding, classification and degradation; 4Metabolism: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins
  • the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein
  • the cascade biological effect also includes the results of the significant differential expression of gene functional groups induced by the HrpNEcb multi-epitope ligand protein, including: 1Differentially expressed genes related to biological processes: including reproduction, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, movement, single Organizing processes, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, stimulus response, localization, biological regulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic
  • the process involves synaptic transmission; 2 Differentially expressed genes related to cellular components: covering cells and extracellular regions, nucleoids, membranes, virions, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecule
  • the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein
  • the cascade biological effect also includes the significant differential expression results of gene functional groups induced by HrpNEch multi-epitope ligand protein, including: 1Differentially expressed genes related to biological processes: including reproduction, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, movement, single Organizing processes, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, stimulus response, localization, biological regulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic
  • the process involves synaptic transmission; 2 Differentially expressed genes related to cellular components: covering cells and extracellular regions, nucleoids, membranes, virions, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules, organelles,
  • the dosage form of the product or drug used in the pharmacy is liquid, powder, tablet or capsule.
  • the pharmaceutical application further includes active compounds (HrpNEcb multi-epitope ligand protein preparations and/or drugs) and derivatives thereof for HrpNEcb multi-epitope ligand protein according to pharmaceutical treatment, usually in unit dosage form or Multiple dosage forms are formulated and administered, each unit dosage containing a predetermined quantity of the therapeutically active compound in association with the required pharmaceutical carrier, vehicle or excipient sufficient to produce the desired therapeutic effect.
  • active compounds HrpNEcb multi-epitope ligand protein preparations and/or drugs
  • derivatives thereof for HrpNEcb multi-epitope ligand protein according to pharmaceutical treatment usually in unit dosage form or Multiple dosage forms are formulated and administered, each unit dosage containing a predetermined quantity of the therapeutically active compound in association with the required pharmaceutical carrier, vehicle or excipient sufficient to produce the desired therapeutic effect.
  • unit dosage forms include ampoules and syringes and individually packaged tablets or capsules.
  • the unit dosage form can be administered in fractions or multiples thereof.
  • Examples of multiple dosage forms include vials, bottles of tablets or capsules, or gallon bottles.
  • a multiple-dose form is a number of unit doses that are not separated in packaging.
  • Dosage forms or compositions can be prepared containing from 0.001% to 100% of the active ingredient, the remainder consisting of a non-toxic carrier, for oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules, which are prepared by conventional methods of pharmacy Acceptable excipients such as binders (including, but not limited to, pregelatinized cornstarch, polyvinylpyrrolidone, or propylmethylcellulose); fillers (including, but not limited to, lactose, microcrystalline cellulose lubricants (including, but not limited to, magnesium stearate, talc, or silicon dioxide); disintegrants (including, but not limited to, potato starch or sodium starch glycolate); or wetting agents (including, But not limited to, sodium lauryl sulfate) preparation.
  • binders including, but
  • compositions can also be in liquid form, including, but not limited to, solutions, syrups or suspensions, or can be presented as a pharmaceutical product for reconstitution with water or other suitable vehicle before use.
  • Such liquid formulations can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (including, but not limited to, sorbitol syrup, cellulose derivatives, or edible fats); emulsifiers (including, but not limited to, edible fats); without limitation, 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, methylparaben) or propyl ester or sorbic acid).
  • suspending agents including, but not limited to, sorbitol syrup, cellulose derivatives, or edible fats
  • emulsifiers including, but not limited to, edible fats
  • non-aqueous vehicles including, but not limited to, almond oil, oily esters, or fraction
  • Formulations suitable for rectal administration may be presented as unit dose suppositories. These can be prepared by mixing the HrpNEcb polymimetic ligand protein active compound with one or more solid carriers, such as cocoa butter, and shaping the resulting mixture.
  • Formulations suitable for topical application to the skin or eye include, but are not limited to, cartilage agents, 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 from 0.001% to 15%, 20%, 25% by weight of a thickening agent selected from the group consisting of, but not limited to, hydroxypropyl methylcellulose, methylcellulose, polyvinylpyrrolidone, Polyvinyl alcohol, polyethylene glycol, poly/hydroxyalkyl(meth)acrylates or poly(meth)acrylamides.
  • Topical formulations are typically applied by instillation or as a cartilaginous agent into the conjunctival capsule. It can also be used to flush or lubricate the eyes, facial sinuses and external auditory canal. It can also be injected into the anterior chamber and elsewhere.
  • Topical formulations in liquid form can also be presented in the form of tapes or contact lenses in a hydrophilic three-dimensional polymer matrix from which the active ingredient is released.
  • Formulations suitable for buccal (sublingual) administration include, but are not limited to, lozenges containing the active compound in a flavored base (usually sucrose and acacia or tragacanth); and lozenges in an inert base include, but are not limited to , pastilles containing the compound in gelatin and glycerin or sucrose and acacia.
  • Pharmaceutical compositions of ligand isoforms can 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, eg, in ampoules or in multi-dose containers, with added additives.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may include, but are not limited to, formulatory agents such as suspending agents, stabilizing agents, alternatively, the active ingredient may be in powder form for use.
  • a suitable carrier such as sterile pyrogen-free water or other solvent.
  • Formulations suitable for transdermal administration may be presented as discrete patches suitable for maintaining intimate contact with the epidermis of the recipient for extended periods of time. Such patches suitably contain the active compound as an optionally buffered aqueous solution of the active compound.
  • Formulations suitable for transdermal administration may be delivered by iontophoresis and take the form of an optionally buffered aqueous solution of the active compound.
  • the pharmaceutical application also includes active compounds (HrpNEch polyepitope ligand protein preparations and/or drugs) and derivatives thereof according to pharmaceutical treatment for HrpNEch polyepitope ligand proteins, usually in unit dosage form or Multiple dosage forms are formulated and administered, each unit dosage containing a predetermined quantity of the therapeutically active compound in association with the required pharmaceutical carrier, vehicle or excipient sufficient to produce the desired therapeutic effect.
  • active compounds HrpNEch polyepitope ligand protein preparations and/or drugs
  • derivatives thereof according to pharmaceutical treatment for HrpNEch polyepitope ligand proteins, usually in unit dosage form or Multiple dosage forms are formulated and administered, each unit dosage containing a predetermined quantity of the therapeutically active compound in association with the required pharmaceutical carrier, vehicle or excipient sufficient to produce the desired therapeutic effect.
  • unit dosage forms include ampoules and syringes and individually packaged tablets or capsules.
  • the unit dosage form can be administered in fractions or multiples thereof.
  • a multiple dosage form is
  • Examples of multiple dosage forms include vials, bottles of tablets or capsules, or gallon bottles.
  • a multiple-dose form is a number of unit doses that are not separated in packaging.
  • Dosage forms or compositions can be prepared containing from 0.001% to 100% of the active ingredient, the remainder consisting of a non-toxic carrier, for oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules, which are prepared by conventional methods of pharmacy Acceptable excipients such as binders (including, but not limited to, pregelatinized cornstarch, polyvinylpyrrolidone, or propylmethylcellulose); fillers (including, but not limited to, lactose, microcrystalline cellulose lubricants (including, but not limited to, magnesium stearate, talc, or silicon dioxide); disintegrants (including, but not limited to, potato starch or sodium starch glycolate); or wetting agents (including, But not limited to, sodium lauryl sulfate) preparation.
  • binders including, but
  • compositions can also be in liquid form, including, but not limited to, solutions, syrups or suspensions, or can be presented as a pharmaceutical product for reconstitution with water or other suitable vehicle before use.
  • Such liquid formulations can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (including, but not limited to, sorbitol syrup, cellulose derivatives, or edible fats); emulsifiers (including, but not limited to, edible fats); without limitation, 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, methylparaben) or propyl ester or sorbic acid).
  • suspending agents including, but not limited to, sorbitol syrup, cellulose derivatives, or edible fats
  • emulsifiers including, but not limited to, edible fats
  • non-aqueous vehicles including, but not limited to, almond oil, oily esters, or fraction
  • Formulations suitable for rectal administration may be presented as unit dose suppositories. These can be prepared by mixing the HrpNEch poly-epitope ligand protein active compound with one or more solid carriers, such as cocoa butter, and shaping the resulting mixture.
  • Formulations suitable for topical application to the skin or eye include, but are not limited to, cartilage agents, 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 from 0.001% to 15%, 20%, 25% by weight of a thickening agent selected from the group consisting of, but not limited to, hydroxypropyl methylcellulose, methylcellulose, polyvinylpyrrolidone, Polyvinyl alcohol, polyethylene glycol, poly/hydroxyalkyl(meth)acrylates or poly(meth)acrylamides.
  • Topical formulations are typically applied by instillation or as a cartilaginous agent into the conjunctival capsule. It can also be used to flush or lubricate the eyes, facial sinuses and external auditory canal. It can also be injected into the anterior chamber and elsewhere.
  • Topical formulations in liquid form can also be presented in the form of tapes or contact lenses in a hydrophilic three-dimensional polymer matrix from which the active ingredient is released.
  • Formulations suitable for buccal (sublingual) administration include, but are not limited to, lozenges containing the active compound in a flavored base (usually sucrose and acacia or tragacanth); and lozenges in an inert base include, but are not limited to , pastilles containing the compound in gelatin and glycerin or sucrose and acacia.
  • Pharmaceutical compositions of ligand isoforms can 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, eg, in ampoules or in multi-dose containers, with added additives.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may include, but are not limited to, formulatory agents such as suspending agents, stabilizing agents, alternatively, the active ingredient may be in powder form for use.
  • a suitable carrier such as sterile pyrogen-free water or other solvent.
  • Formulations suitable for transdermal administration may be presented as discrete patches suitable for maintaining intimate contact with the epidermis of the recipient for extended periods of time. Such patches suitably contain the active compound as an optionally buffered aqueous solution of the active compound.
  • Formulations suitable for transdermal administration may be delivered by iontophoresis and take the form of an optionally buffered aqueous solution of the active compound.
  • the HrpN-type polymimetic epitope ligand protein is HrpNEcb polymimetic epitope ligand protein, and the products or medicines are mainly prepared from purified HrpNEcb protein, and the mass content is 0.001%-100%.
  • the HrpN type polymimetic epitope ligand protein is the HrpNEch polymimetic epitope ligand protein, and the products or medicines are mainly prepared from the purified HrpNEch protein, and the mass content is 0.001%-100%.
  • the HrpN-type polymimetic epitope ligand protein is a purified HrpN-type protein.
  • the method for purifying HrpN-type protein includes the following steps:
  • Step 1 The high pressure crusher crushes the engineering bacteria, and the crushed bacteria liquid is passed into the butterfly continuous flow centrifuge to remove the cell wall, and the high pressure range is 800-1000Mpa;
  • Step 2 Purify the HrpN-type polymimetic epitope ligand protein-His recombinant protein with a Ni-NTA agarose column to obtain a purified HrpN-type polymimetic epitope ligand protein original drug.
  • the high-efficiency expression of HrpNEcb protein adopted in the present invention comprises the following steps:
  • HrpNEcb protein The engineering bacteria fermentation preparation of HrpNEcb protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes and similar genes and their genes) encoding HrpNEcb polymimetic ligand proteins are used.
  • Modified) plasmid engineering bacteria E.coli
  • the expression product HrpNEcb protein was analyzed by 10% SDS-PAGE polyacrylamide gel electrophoresis, and a 36.64kda band appeared on the sample lane of the electrophoresis gel plate, which was the expression product HrpNEcb protein of the gene hrpNEcb.
  • the high-pressure crusher crushes engineering bacteria, and continuously uses 800-1000Mpa pressure to crush engineering bacteria.
  • centrifugal force range 1000-8000g, preferably centrifugal force 1000-2000g; preferably centrifugal force 2000-3500g; preferably centrifugal force 8000-6000g; preferably centrifugal force 6000-4500g; most preferably The centrifugal force is 3500-4500g.
  • the HrpNEcb multi-epitope ligand protein was present in the supernatant.
  • the application route of the HrpNEcb protein preparation that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multifunctional cascade biological effects according to the present invention can be administered by any route known to those skilled in the art, Such routes include internal, topical, oral, injection, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, nasal, oral, rectal, topical, buccal and transdermal administration or any route; by any convenient HrpNEcb polyepitope ligand proteins are administered by routes such as by perfusion or rapid perfusion, absorption through epithelia or mucocutaneous linings (eg, oral mucosa, nasal mucosa, gastric mucosa, rectal and intestinal mucosa, etc.), and can be combined with other
  • the biologically active agents are administered sequentially, intermittently, or in the same composition; depending on the site of treatment, administration can be topical, topical, or systemic.
  • Topical administration to the area in need of treatment can be, but is not limited to, local 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 Pump; the most appropriate route in any given situation will depend on the nature and severity of the disease or condition being treated and the nature of the particular composition used.
  • Various delivery systems are known and can be used to administer the multi-epitope ligand protein, which can be encapsulated in liposomes, microparticles, microcapsules.
  • Pharmaceutical compositions of the polymimetic ligand protein can be prepared, typically, as approved by a regulatory agency or as a pharmaceutically acceptable composition for use in a patient.
  • the preparations or medicines involving HrpNEcb multi-epitope ligand proteins that recognize and activate various types of receptors, membrane proteins and their signaling pathways in animals and induce multifunctional cascade biological effects and are used in the pharmacy of the present invention are used in diagnosis, or and application in the prevention, or treatment, or rehabilitation of diseases and conditions of the nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system:
  • the products or medicines of the multi-epitope ligand protein described in the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of neural connection diseases, dementia, Parkinson's disease, central nervous system disease, neuromuscular disease, epilepsy, Use in headache and neuralgia, peripheral neuropathy, attention deficit hyperactivity disorder and tic disorder, insomnia, depression, anxiety disorders, bipolar disorder, psychotic disorders, neurodermatitis-related neurological diseases and conditions;
  • the products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or recovery of gastric acid secretion disorder, gastrointestinal neurosis, gastrointestinal motility, gastrointestinal mucositis, liver Use in diseases and disorders of the digestive system associated with diseases and micro-ecological disorders;
  • the product or drug of the polyepitope ligand protein of the present invention is useful in the diagnosis, or prevention, or treatment, or rehabilitation of arthritis, muscle spasm, pain, muscular dystrophy, muscle and nerve injury, and dehydration-related motor systems Applications in diseases and conditions;
  • the products or medicines of the multi-epitope ligand protein described in the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of heart failure, arrhythmia, hypertension, myocardial injury, ischemia, angina pectoris, hyperlipidemia , calcium channel blockade, vasospasm, blood coagulation, abnormal blood picture, myocardial infarction-related circulatory system diseases and conditions;
  • the products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or 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 products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of diabetes, thyroid disease, pituitary disease, hyperprolactinemia, diabetes insipidus, adrenal disease, Parathyroid disease, osteoporosis-related endocrine system diseases and conditions;
  • the application of the product or medicine of the multi-epitope ligand protein of the present invention in diagnosis, or prevention, or treatment, or rehabilitation of immune system diseases and conditions related to immunosuppression, rheumatoid arthritis, and lupus erythematosus;
  • the products or medicines of the multi-epitope ligand protein described in the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of nephrotic syndrome, interstitial nephritis, renal failure, urinary and reproductive system infections, pyelonephritis, Cystitis, prostatitis, urethritis, epididymitis or orchitis, benign prostatic hyperplasia, overactive bladder, sexual dysfunction-related, and urogenital diseases and conditions related to various male and gynecological infectious inflammatory and functional diseases Applications.
  • the products or medicines of the multi-epitope ligand protein of the present invention can be used in diagnosis, or prevention, or treatment, or rehabilitation of whole body skin cell nutrition, activation, regeneration, repair, clearing, delicate and smooth, ultraviolet melanin deposition, eczema , rough, cracked, dark lines, dry, crusty, erythema, allergies, neurodermatitis, lesions, pimples, acne, scars, dullness, mites, oily skin, inflammatory skin diseases, autoimmune skin diseases, pigmentation Skin diseases related to skin diseases, skin atrophy, thinning, dryness, hyperpigmentation, wrinkle hyperplasia, dyskeratosis, xeroderma, contact dermatitis, anti-aging, improving skin function, whitening and freckle, preventing and treating skin diseases Applications in Diseases and Conditions.
  • the preparation of the HrpNEcb multi-epitope ligand protein that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multi-functional cascade biological effects according to the present invention includes the following methods: 1.
  • the HrpNEcb poly For the preparation of the epitope ligand protein, the engineering bacteria containing the EcbCSL101HrpNEcb gene (cloned into the high-efficiency expression vector PET28a(+)) were used to ferment and purify the HrpNEcb polymimetic epitope ligand protein:
  • HrpNEcb protein The engineering bacteria fermentation preparation of HrpNEcb protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes, and similar genes and their genes) encoding HrpNEcb polymimetic epitope ligand proteins are used.
  • IPTG isopropyl thiogalactoside, Isopropyl ⁇ -D-Thiogalactosid
  • the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
  • the fermentation temperature range is 0-60°C.
  • the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
  • Fermentation proliferation liquid medium glucose concentration range 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%;
  • Fermentation induction liquid medium glucose concentration range 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%;
  • Fermentation induction liquid medium lactose concentration range 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 time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
  • the HrpNEcb polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column.
  • the protein purification was implemented according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEcb polymimetic epitope ligand protein. preparation.
  • HrpNEcb polymimetic epitope ligand protein further, the HrpNEcb protein can also be prepared by the expression protein of an "artificially synthesized gene", and the HrpNEcb polymimetic epitope ligand protein can be prepared by fermentation and purification, specifically including the following step:
  • the artificial gene synthesis was entrusted to the GeneArt Gene Synthesis and Services Division of Thermo Fisher Scientific.
  • the advantages of artificially synthesized protein genes are: a) the synthesis cycle is short, and the sequence can be guaranteed to be 100% correct; b) the codons can be optimized to improve the expression efficiency of the gene; due to the different coding codons preferred by each species, When heterologous proteins are expressed in E. coli, some proteins are difficult to express at high levels. If the codon of the heterologous protein is changed to the codon preferred by Escherichia coli, the high-efficiency expression of the protein gene can be achieved, the expression level of the gene can be improved, and it is suitable for large-scale industrial production; c) The gene can be designated as needed. Mutations are used to modify genes and improve the efficiency of proteins; d) researchers can design genes that are difficult to obtain or even do not exist in nature according to their own wishes.
  • the HrpNEcb protein will show a 36.64kda band on the sample lane of the electrophoresis gel plate, which is the HrpNEcb multi-epito
  • the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
  • the fermentation temperature range is 0-60°C.
  • the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
  • Fermentation proliferation liquid medium glucose concentration range 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%;
  • Fermentation induction liquid medium glucose concentration range 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%;
  • Fermentation induction liquid medium lactose concentration range 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 time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
  • the HrpNEcb polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column.
  • the protein purification was implemented according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEcb polymimetic epitope ligand protein. preparation.
  • the use route of the HrpNEch protein preparation that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multifunctional cascade biological effects according to the present invention can be administered by any route known to those skilled in the art, Such routes include internal, topical, oral, injection, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, nasal, oral, rectal, topical, buccal and transdermal administration or any route; by any convenient Routes of administration of HrpNEch polyepitope ligand proteins, such as by perfusion or rapid perfusion, absorption through epithelial or mucocutaneous linings (eg, oral mucosa, nasal mucosa, gastric mucosa, rectal and intestinal mucosa, etc.), and can be combined with other
  • the biologically active agents are administered sequentially, intermittently, or in the same composition; depending on the site of treatment, administration can be topical, topical, or systemic.
  • Topical administration to the area in need of treatment can be, but is not limited to, local 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 Pump; the most appropriate route in any given situation will depend on the nature and severity of the disease or condition being treated and the nature of the particular composition used.
  • Various delivery systems are known and can be used to administer the multi-epitope ligand protein, which can be encapsulated in liposomes, microparticles, microcapsules.
  • Pharmaceutical compositions of the polymimetic ligand protein can be prepared, typically, as approved by a regulatory agency or as a pharmaceutically acceptable composition for use in a patient.
  • the preparations or medicines involving HrpNEch multi-epitope ligand proteins that recognize and activate various types of receptors, membrane proteins and their signaling pathways in animals and induce multifunctional cascade biological effects and are used in the pharmacy of the present invention are used in diagnosis, or and application in the prevention, or treatment, or rehabilitation of diseases and conditions of the nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system:
  • the products or medicines of the multi-epitope ligand proteins of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of neural connection diseases, dementia, Parkinson's disease, central nervous system diseases, neuromuscular diseases, epilepsy, Use in headache and neuralgia, peripheral neuropathy, attention deficit hyperactivity disorder and tic disorder, insomnia, depression, anxiety disorders, bipolar disorder, psychotic disorders, neurodermatitis-related neurological diseases and conditions;
  • the product or drug of the multi-epitope ligand protein of the present invention is used for diagnosis, or prevention, or treatment, or recovery of gastric acid secretion disorder, gastrointestinal neurosis, gastrointestinal motility, gastrointestinal mucositis, liver Use in diseases and disorders of the digestive system associated with diseases and micro-ecological disorders;
  • the preparations or drugs of the multi-epitope ligand proteins of the present invention are useful in the diagnosis, or prevention, or treatment, or rehabilitation of arthritis, muscle spasm, pain, muscular dystrophy, muscle and nerve damage, and dehydration-related motor systems Applications in diseases and conditions;
  • the products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of heart failure, arrhythmia, hypertension, myocardial injury, ischemia, angina pectoris, hyperlipidemia , calcium channel blockade, vasospasm, blood coagulation, abnormal blood picture, myocardial infarction-related circulatory system diseases and conditions;
  • the products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or 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 products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of diabetes, thyroid disease, pituitary disease, hyperprolactinemia, diabetes insipidus, adrenal disease, Parathyroid disease, osteoporosis-related endocrine system diseases and conditions;
  • the products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of nephrotic syndrome, interstitial nephritis, renal failure, urinary and reproductive system infections, pyelonephritis, Cystitis, prostatitis, urethritis, epididymitis or orchitis, benign prostatic hyperplasia, overactive bladder, sexual dysfunction-related, as well as various male and gynecological infectious inflammatory and functional diseases and other urogenital diseases and conditions Applications.
  • the products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of whole body skin cell nutrition, activation, regeneration, repair, clearing, delicate and smooth, ultraviolet melanin deposition, eczema , rough, cracked, dark lines, dry, crusty, erythema, allergies, neurodermatitis, lesions, pimples, acne, scars, dullness, mites, oily skin, inflammatory skin diseases, autoimmune skin diseases, pigmentation Skin diseases related to skin diseases, skin atrophy, thinning, dryness, hyperpigmentation, wrinkle hyperplasia, dyskeratosis, xeroderma, contact dermatitis, anti-aging, improving skin function, whitening and freckle, preventing and treating skin diseases Applications in Diseases and Conditions.
  • the preparation of the HrpNEch polymimetic epitope ligand protein that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multifunctional cascade biological effects:
  • the HrpNEch multi-epitope ligand protein was prepared by using the gene containing our registered HrpNEch protein (GenBank Protein: AAY17519.1) (Gene Registration No.: GenBank: nucleotide: AY999000.1) (cloned into a high-efficiency expression The engineered bacteria of the carrier PET28a(+)) are fermented and purified to prepare the HrpNEch polymimetic epitope ligand protein:
  • HrpNEch multi-epitope ligand protein The engineering bacteria fermentation preparation of HrpNEch multi-epitope ligand protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes) encoding HrpNEch multi-epitope ligand proteins are prepared.
  • the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
  • the fermentation temperature range is 0-60°C.
  • the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
  • Fermentation proliferation liquid medium glucose concentration range 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%;
  • Fermentation induction liquid medium glucose concentration range 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%;
  • Fermentation induction liquid medium lactose concentration range 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 time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
  • HrpNEch polymimetic epitope ligand protein genetically engineered bacteria after production and fermentation 1 Sterilize the fermentation broth at 80°C for 30 minutes, and quickly cool down to below 30°C; 2 Clean with Glucose Na 2 HPO 4 -KH 2 PO 4 buffer (pH range is 1-14, glucose concentration range is 0-2500 mmol, buffer system pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably 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, wash the engineering bacteria five to eight times in the butterfly continuous flow centrifuge; 3.
  • the HrpNEch polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column.
  • the protein purification was carried out according to the method suggested by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEch polymimetic epitope ligand protein. preparation.
  • HrpNEch multi-epitope ligand protein further, the HrpNEch protein can also be prepared by the expression protein of "artificially synthesized gene", which specifically includes the following steps:
  • the artificial gene synthesis was entrusted to the GeneArt Gene Synthesis and Services Division of Thermo Fisher Scientific.
  • the advantages of artificially synthesized protein genes are: a) the synthesis cycle is short, and the sequence can be guaranteed to be 100% correct; b) the codons can be optimized to improve the expression efficiency of the gene; due to the different coding codons preferred by each species, When heterologous proteins are expressed in E. coli, some proteins are difficult to express at high levels. If the codon of the heterologous protein is changed to the codon preferred by Escherichia coli, the high-efficiency expression of the protein gene can be achieved, the expression level of the gene can be improved, and it is suitable for large-scale industrial production; c) The gene can be designated as needed. Mutations are used to modify genes and improve the efficiency of proteins; d) researchers can design genes that are difficult to obtain or even do not exist in nature according to their own wishes.
  • HrpNEch multi-epitope ligand protein shows a 34.15kda band on the sample lane of the electrophoresis gel plate, which is the HrpNEch multi-epito
  • the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
  • the fermentation temperature range is 0-60°C.
  • the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
  • Fermentation proliferation liquid medium glucose concentration range 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 pH0.1%-0.05%;
  • Fermentation induction liquid medium glucose concentration range 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%;
  • Fermentation induction liquid medium lactose concentration range 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 time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
  • HrpNEch polymimetic epitope ligand protein genetically engineered bacteria Post-treatment of HrpNEch polymimetic epitope ligand protein genetically engineered bacteria after production and fermentation: 1 Sterilize fermentation broth at 80°C for 30 minutes to complete the sterilization treatment, and quickly cool down to below 30°C; 2 Clean with Glucose Na 2 HPO 4 -KH 2 PO 4 buffer (pH range is 1-14, glucose concentration range is 0-2500 mmol, buffer system pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably 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, wash the engineering bacteria five to eight times in the butterfly continuous flow centrifuge; 3.
  • the HrpNEch polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column.
  • the protein purification was carried out according to the method suggested by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEch polymimetic epitope ligand protein. preparation.
  • HrpN-type multi-epitope ligand proteins as a class of ligand protein molecules rich in multiple linear and conformational epitopes with special structures, can recognize, activate and bind membrane receptors, membrane proteins, Information pathways and metabolic pathways, HrpN-type multi-epitope ligand proteins are a class of ligand proteins with special multiple epitope structures, new functions, new mechanisms of action and new application prospects. They induce multi-directional, multi-level and Multi-faceted biological effects and functions, widely involved in the diagnosis, or prevention, or treatment, or recovery of multi-system, multi-tissue, multi-organ, and multi-cell related diseases and conditions, and related diseases and conditions. , eliminate font size, makeup font size, mechanical font size and health font size products or drugs in the pharmaceutical application.
  • Figure 1 shows the electrophoresis detection of the HrpNEcb polymimetic epitope ligand protein before and after purification: the left side is the molecular weight marker band, of which 1: the highly expressed HrpNEcb polymimetic epitope ligand protein band (before purification); 2: the polymimetic epitope after purification The ligand protein HrpNEcb band.
  • Fig. 2 is that HrpNEcb multi-epitope ligand protein liquid injection induces the allergic reaction diagram of tobacco leaves: wherein the focal spot is formed through the treatment of HarpinEcb protein liquid about 24hr, B, D: H 2 O injection; A, C: HarpinEcb protein solution (250 ⁇ g/ml) injection, namely the hypersensitivity reaction of HarpinEcb protein on tobacco leaves, B and D are controls, A and C are treatments.
  • Figure 3 is a volcano plot of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the liver, from left to right: oral administration for 6 hours, oral administration for 24 hours;
  • Figure 4 is a volcano diagram of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the thalamus, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
  • Figure 5 is a volcano plot of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the heart, from left to right: oral administration for 6h, oral administration for 24h; smear for 6h, smear for 12h;
  • Figure 6 is a volcano diagram of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the cerebral cortex, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours, application for 12 hours;
  • Figure 7 is a volcano diagram of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differential gene expression in the hippocampus of the brain, from left to right: oral administration for 6h, oral administration for 24h;
  • Figure 8 is a cluster heat map of HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differentially expressed gene sets in the liver, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
  • Figure 9 is a clustering heat map of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differentially expressed gene sets in the thalamus, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
  • Figure 10 is a cluster heat map of the HrpNEcb multi-epitope ligand protein of the present invention orally and smearing experimental mice to induce differential gene set expression in the hippocampus, from left to right are oral administration for 6h, oral administration for 24h; smear for 6h;
  • Figure 11 is a clustering heat map of the differentially expressed gene sets in the cerebral cortex induced by oral administration of HrpNEcb multi-epitope ligand protein and smearing in experimental mice, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
  • Fig. 12 is KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally 6 hours to deal with the comparison of experimental mouse liver and control (total gene);
  • Figure 13 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein orally for 24 hours to treat the experimental mouse liver and control (total gene);
  • Figure 14 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat experimental mouse liver and control (total gene);
  • Figure 15 is a comparison of the KEGG Pathway:HrpEcb multi-epitope ligand protein of the present invention orally treating the experimental mouse liver for 6 hours and the control (up-regulated gene);
  • Figure 16 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein orally treated experimental mouse liver for 24 hours (up-regulated gene);
  • Figure 17 is a comparison of the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the experimental mouse liver and the control (up-regulated gene);
  • Figure 18 is a comparison of the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein orally for 6 hours to treat the experimental mouse liver and the control (down-regulated gene);
  • Figure 19 is a comparison of the KEGG Pathway:HrpEcb multi-epitope ligand protein of the present invention orally treating the experimental mouse liver for 24 hours and the control (down-regulated gene);
  • Figure 20 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the experimental mouse liver and control (down-regulated gene);
  • Fig. 21 is KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours and compared with the control (total gene) of experimental mouse heart;
  • Figure 22 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours and compared with the control (total gene);
  • Figure 23 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 6 hours to treat the experimental mouse heart and control comparison (total gene);
  • Figure 24 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 12 hours to treat the heart of the experimental mouse compared with the control (total gene);
  • Figure 25 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the heart of experimental mice and the control (up-regulated genes);
  • Figure 26 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours and compared with the control (up-regulated gene);
  • Figure 27 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 6 hours to treat the heart of experimental mice and control (up-regulated genes);
  • Figure 28 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours to treat the heart of experimental mice and control (up-regulated genes);
  • Figure 29 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in experimental mouse heart and control (down-regulated genes);
  • Figure 30 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours and compared with the control (down-regulated gene);
  • Figure 31 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the heart of experimental mice and control (down-regulated genes);
  • Figure 32 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 12 hours to treat the heart of experimental mice and control (down-regulated genes);
  • Figure 33 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the brain hippocampus of experimental mice and the control (total genes);
  • Figure 34 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the brain hippocampus of experimental mice and the control (total genes);
  • Figure 35 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the brain hippocampus of experimental mice and control (total genes);
  • Figure 36 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 12 hours to treat the rat brain hippocampus and the control comparison (total genes);
  • Figure 37 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the brain hippocampus of experimental mice and the control (up-regulated genes);
  • Figure 38 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the brain hippocampus of experimental mice and controls (up-regulated genes);
  • Figure 39 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the brain hippocampus of experimental mice and control (up-regulated genes);
  • Figure 40 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the brain hippocampus of experimental mice and control (up-regulated genes);
  • Figure 41 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the brain hippocampus of experimental mice and the control (down-regulated genes);
  • Figure 42 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the brain hippocampus of experimental mice and the control (down-regulated genes);
  • Figure 43 is a comparison of the KEGG Pathway of the present invention: HrpEcb polymimetic epitope ligand protein smearing for 6 hours in the brain hippocampus of the experimental mice and the control (down-regulated genes);
  • Figure 44 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the brain hippocampus of experimental mice and control (down-regulated genes);
  • Figure 45 is a comparison of the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours between the cerebral cortex of the experimental mouse and the control (total genes);
  • Figure 46 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral cortex of experimental mice compared with the control (total genes);
  • Figure 47 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral cortex of experimental mice and control (total genes);
  • Figure 48 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the cerebral cortex of experimental mice and control (total genes);
  • Figure 49 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in experimental mouse cerebral cortex and control (up-regulated gene);
  • Figure 50 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral cortex of experimental mice and controls (up-regulated genes);
  • Figure 51 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral cortex of experimental mice and control (up-regulated genes);
  • Figure 52 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the cerebral cortex of experimental mice and control (up-regulated genes);
  • Figure 53 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the cerebral cortex of experimental mice and controls (down-regulated genes);
  • Figure 54 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours between the cerebral cortex of experimental mice and the control (down-regulated genes);
  • Figure 55 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the cerebral cortex of experimental mice and control (down-regulated genes);
  • Figure 56 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours between the cerebral thalamus of experimental mice and the control (total genes);
  • Figure 57 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours between the cerebral thalamus of experimental mice and the control (total genes);
  • Figure 58 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral thalamus of experimental mice and control (total genes);
  • Figure 59 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the cerebral thalamus of experimental mice and the control (up-regulated genes);
  • Figure 60 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral thalamus of experimental mice and the control (up-regulated genes);
  • Figure 61 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral thalamus of experimental mice and control (up-regulated genes);
  • Figure 62 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the cerebral thalamus of experimental mice and the control (down-regulated genes);
  • Figure 63 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral thalamus of experimental mice and the control (down-regulated genes);
  • Figure 64 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral thalamus of experimental mice and control (down-regulated genes);
  • Figure 65 is a flow chart of the mRNA (RNA-Seq) sequencing experiment of the present invention.
  • Figure 66 is a flow chart of mRNA sequencing data analysis of the present invention.
  • Figure 67 shows the electrophoresis detection of HrpNEch protein before and after purification: the left side is the molecular weight marker band, 1: HrpNEch polymimetic epitope ligand protein band before purification; 2: HrpNEch polymimetic epitope ligand protein band after purification;
  • Figure 68 is a graph of HrpNEch multi-mimetic epitope ligand protein liquid injection to induce allergic reactions in tobacco leaves: where the focal spots seen are formed through the treatment of HrpNEch multi-mimetic epitope ligand protein liquid for about 24hr, the upper row of leaves: H2O injection, For the control; the lower row of leaves: HrpNEch multi-epitope ligand protein solution (250 ⁇ g/ml) injection, for the treatment, that is, the hypersensitivity reaction of HrpNEch multi-epitope ligand protein on tobacco leaves;
  • Figure 69 is the HrpNEch multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differential gene expression in the kidney, and from left to right are oral administration for 6h, oral administration for 24h; smear for 6h;
  • Figure 70 is a volcano plot of the HrpNEch multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in testis, from left to right: oral administration for 6h, oral administration for 24h; smear for 6h;
  • Figure 71 is a cluster heat map of the differentially expressed genes in the kidneys induced by the oral administration of HrpNEch multi-epitope ligand protein and smearing experimental mice, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
  • Figure 72 is a cluster heat map of the differentially expressed genes in testis induced by oral administration of HrpNEch multi-epitope ligand protein and smearing in experimental mice, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
  • Figure 73 shows the comparison of the KEGG Pathway (total gene) of the experimental mouse kidney treated with the HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
  • Figure 74 shows the comparison of KEGG Pathway (up-regulated gene) in the kidney of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention (up-regulated gene), from left to right, oral administration for 6 hours, oral administration for 24 hours; smearing for 6 hours;
  • Figure 75 shows the comparison of the KEGG Pathway (down-regulated gene) in the kidneys of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right: oral administration for 6 hours, oral administration for 24 hours;
  • Figure 76 shows the comparison of KEGG Pathway (total gene) in the testis of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
  • Figure 77 shows the comparison of KEGG Pathway (up-regulated gene) in the testis of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention (up-regulated gene), from left to right: oral administration for 6h, oral administration for 24h; smear for 6h;
  • Figure 78 shows the comparison of KEGG Pathway (down-regulated gene) in the testis of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
  • Figure 79 is a flow chart of the mRNA (RNA-Seq) sequencing experiment of the present invention.
  • Figure 80 is a flow chart of mRNA sequencing data analysis of the present invention.
  • test methods used in the following examples are conventional methods unless otherwise specified.
  • the HrpNEcb multi-epitope ligand protein was fermented with the registered EcbCSL101hrpNEcb gene (GenBank: ABD22989.1) (cloned into the high-efficiency expression vector PET28a(+)), and the HrpNEcb multi-epitope ligand protein was purified, prepared and collected. Specifically include the following steps:
  • the engineering bacteria fermentation preparation of HrpNEcb protein the engineering bacteria (E. coli), the production line of the related protein is a specially modified derivative of the original K-12 bacteria JY-01 (DE3), in LB liquid medium (containing 50 micrograms of kanamycin per liter) at a certain temperature
  • LB liquid medium containing 50 micrograms of kanamycin per liter
  • IPTG isopropylthiogalactoside, Isopropyl ⁇ -D-Thiogalactosid
  • final concentration 1 mMol was added, and the cells were collected by centrifugation after continuing the culture.
  • the pH of the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system is 6.5-5.5; the fermentation temperature is 37-38° C.; the glucose concentration of the fermentation and proliferation liquid medium is 0.01%-0.05%; 0.05%-0.00%; the lactose concentration of the fermentation induction liquid medium is 0.5%-0.4%; the fermentation induction liquid culture time is 7-9h.
  • the HrpNEcb multi-epitope ligand protein was present in the supernatant.
  • the HrpNEcb protein is prepared by the expression protein of "artificially synthesized gene", which specifically includes the following steps:
  • the first step artificial synthesis of the hrpNEcb gene encoding the HrpNEcb protein
  • Step 2 2) According to the above DNA sequence, when artificially synthesizing protein gene, add BamHI and HindIII enzyme cleavage sites to the 5' and 3' of the gene respectively to facilitate protein gene cloning;
  • the third step artificial gene synthesis is entrusted to the GeneArt gene synthesis and service department of Thermo Fisher Scientific. 3) The synthesized DNA fragments encoding the HrpNEcb protein gene were cloned into the BamHI-HindIII site of the high-efficiency protein expression vector PET28a(+) (containing His-Tag label) one by one, and the accuracy of the clone was ensured through DNA sequencing;
  • the fermentation medium is Na 2 HPO 4 -KH 2 PO 4 buffer system
  • the pH of the buffer system is 6.5-5.5
  • the concentration of glucose in the fermentation and proliferation liquid medium is 0.01%-0.05%
  • the concentration of lactose in the fermentation induction liquid medium is 0.5%- 0.4%;
  • Step 5 Suspend the collected cells into Na 2 HPO 4 -KH 2 PO 4 buffer, complete the sterilization treatment at 80°C for 30 minutes, quickly cool down to 30°C, and place in a butterfly continuous flow centrifuge. Wash the engineered bacteria for five to eight times, import them into a high-pressure crusher, and continuously use 800-1000Mpa pressure to break the engineered bacteria. Pass the broken bacteria liquid into a butterfly continuous flow centrifuge to remove the cell wall and collect HrpNEcb polymimetic epitope ligands. protein molecule, HrpNEcb multi-epitope ligand protein is present in the supernatant;
  • NI-NTA agarose column Use NI-NTA agarose column to purify the polymimetic epitope ligand protein-His recombinant protein.
  • the protein purification is carried out according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification and preparation of the HrpNEcb polymimetic epitope ligand protein. .
  • the highly expressed purified protein-His recombinant band was detected by 10% SDS polyacrylamide gel electrophoresis, see Figure 1 for details.
  • the left side is the molecular weight identification band
  • lane 1 is the electrophoresis band before purification, and there are more bands in the corresponding molecular weight region, including the 36.64kda band
  • lane 2 is the purified HrpNEcb protein band , the molecular weight is 36.64kda, which is in the corresponding molecular weight region of the protein, indicating that the corresponding purified HrpNEcb polymimetic epitope ligand protein has been obtained.
  • the allergy test detection of the purified polymimetic epitope ligand protein: HrpNEcb polymimetic epitope ligand protein preparation and the reaction results of tobacco leaves 24hr after sterile water treatment are shown in Figure 2, wherein, A, C The point is the injection of 300 ⁇ g ⁇ mL -1 of HrpNEcb multi-epitope ligand protein solution of 100 ⁇ L; the points B and D are the injection of 100 ⁇ L of sterile water as the control treatment. 300 ⁇ g ⁇ mL -1 of HrpEcb poly-epitope ligand protein solution treatment for about 12hrs caused tobacco leaves to shrink and collapse, and 24hrs to die; the water control treatment of tobacco leaves had no allergic reaction.
  • the purified multi-epitope ligand protein can generally induce hypersensitivity reactions in various plant leaves.
  • the tested plant species can be: tobacco, pepper, eggplant, tomato, potato, strawberry, cucumber, spinach, celosia, glass begonia, September chrysanthemum, pansy, nopal, petunia, grape, rose, locust tree, pea, peach tree, bunch of red, loofah, green beans, cauliflower, spinach, rape, yam, cowpea, broad bean, corn, 36 kinds of different plants such as rice, soybean, cyclamen, mulberry, pumpkin, loquat, toon tree, etc.
  • the research object of mRNA sequencing is all RNAs with a poly-A tail that can be transcribed by a specific cell in a certain functional state, mainly mRNA.
  • Cell-generated mRNA is converted into DNA (cDNA, complementation, and library construction of the obtained cDNA) by a process of reverse transcription.
  • the resulting DNA is then sequenced and from the observed abundance of specific DNA, the original amount of mRNA in the cell can be inferred from it to find genes or transcripts whose transcription levels change under the experimental conditions, i.e. differentially expressed.
  • HrpNEcb multi-epitope ligand protein treatment groups including four treatments of oral administration for 6 hours, 24 hours, and application for 6 hours and 12 hours. There were 3 experimental mice for each treatment, a total of 12 mice; the blank control group had 4 experimental mice; the buffer without HrpNEcb multi-epitope ligand protein controlled the sham-operated group, including oral administration for 6 hours, 24 hours and application for 6 hours, 12 hours.
  • mice in the experimental treatment group were fed with HrpNEcb polymimetic ligand protein buffer containing 600 mg ⁇ L -1 concentration.
  • the mice in the buffer control sham-operated group were fed and smeared with buffer, and the mice in the blank control group did not receive any treatment.
  • the cerebral cortex, thalamus, cerebral hippocampus, liver, heart and other tissues of mice were divided into groups for RNA-Seq sequencing and analysis.
  • RNA extraction of the samples was performed using the miRNeasy Micro Kit (Cat#1071023Qiagen) and according to the standard operating procedure provided by the manufacturer.
  • the total RNA was quality-checked by NanoDrop ND-2000 spectrophotometer and Agilent Bioanalyzer 4200 (Agilent technologies, Santa Clara, CA, US), and the qualified RNA was used for subsequent sequencing experiments.
  • Oligo(dT) can be used to enrich mRNAs with polyA tails.
  • the enriched mRNA is then subjected to fragmentation, double-stranded cDNA synthesis, end repair, addition of A at the 3' end, ligation of adapters, and amplification.
  • the constructed library uses 2.0 Fluorometer detects concentration, Agilent2100 detects size.
  • Illumina sequencing is performed on the library, and the sequencer captures the fluorescent signal and converts the light signal into a sequencing peak through computer software to obtain the sequence information of the fragment to be detected.
  • the differential gene volcano plot was used to display the overall distribution of genes with significant differential expression induced by HrpNEcb multi-epitope ligand protein.
  • Horizontal axis fold change of gene expression in different samples (log2Fold-Change); vertical axis: significant level of gene expression difference (-log10p-value); the expression of the right point is significantly up-regulated; the left point is significantly down-regulated Genes; genes with no significant changes in expression at lower points.
  • Figures 3-7 are the differential gene volcano plots of HrpNEcb multi-epitope ligand protein induced by oral administration and smearing in the liver, thalamus, heart, cerebral cortex and cerebral hippocampus of mice, respectively. HrpNEcb is abbreviated as N1 in the figure.
  • FIG. 8-11 is a clustering heat map of differentially expressed genes in liver, thalamus, hippocampus, and cerebral cortex.
  • HrpNEcb is abbreviated as N1.
  • Gene Ontology is an ontology widely used in the field of bioinformatics.
  • Gene Ontology is a description of genes in different dimensions and levels, covering biological processes (biological_process), cellular components (cellular_component) and molecular functions (molecular_function).
  • biological processes are describing which biological processes the gene is involved in; cellular components explain where the gene is present, including whether the gene is in the cytoplasm or in the nucleus? If cytoplasm is present in which organelle? If it is in the mitochondria, is it on the mitochondrial membrane or in the matrix of the mitochondria, etc., this information belongs to the cell group; the molecular function explains what is the function of the gene at the molecular level?
  • Gene Ontology database is a structured standard biological model constructed by the GO organization (Gene Ontology Consortium) in 2000. It aims to establish a standard vocabulary system for knowledge of genes and their products, covering the biological process of genes. process), cellular component, molecular function.
  • Term is the basic description unit in GO. GO Terms are used to describe the function of gene products. By performing GO enrichment analysis on differential genes, genes can be classified according to different functions, so as to achieve the purpose of annotating and classifying genes.
  • HrpNEcb multi-epitope ligand protein we carried out GO term enrichment analysis on the differentially expressed genes induced by HrpNEcb multi-epitope ligand protein, and the results proved and confirmed that HrpNEcb multi-epitope ligand protein, as a class of special structures with multiple epitopes, new functions , a ligand protein with a new mechanism of action and a new application prospect, induced the differential expression of multiple genes in multiple organs (liver, thalamus, heart, cerebral cortex and brain hippocampus, etc.) of the tested mice, and these differentially expressed genes covered biological processes. , cellular components and molecular functions. Differentially expressed genes were analyzed by GO using Fisher's exact test. Fisher's exact test was calculated to obtain the p-value, and multiple hypothesis test correction was performed to obtain the q-value. GO entries with p-value less than 0.05 were screened as significantly enriched GO entries.
  • the results of GO enrichment analysis of differentially expressed genes induced by HrpNEcb multi-epitope ligand proteins are further described as follows: 1
  • the biological process (biological_process) related differentially expressed genes include reproduction, cell death, immune system processes, behavior, metabolic processes, and cellular processes. , reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, motility, processes in individual tissues, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, Stimulatory responses, localization, bioregulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic processes involve synaptic transmission.
  • the results of GO enrichment analysis of biological processes are shown in Tables 1 to 6.
  • differentially expressed genes related to cellular components cover cells and extracellular regions, nucleoids, membranes, virus particles, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules, organelles, extracellular matrix components, cells Parts of the outer region, organelle parts, virion parts, membrane parts, synaptic parts, cell parts, synapses, and cellular supramolecular fibers, etc.
  • the results of GO enrichment analysis of cell components are shown in Table 1 to Table 6.
  • Molecular function-related differentially expressed genes cover transcription factor activity, protein binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, and morphogen activity , Antioxidant activity, metal chaperone protein activity, protein labeling, chemoattractant activity, translation regulation, chemorepellent activity, active molecular sensor, molecular function regulation, etc.
  • the molecular function GO enrichment analysis results are shown in Table 1 to Table 6.
  • HrpNEcb in Table 1-6 is abbreviated as N1.
  • N1 HrpNEcb in Table 1-6
  • blanks indicate that the corresponding data that does not meet the standard of p-value less than 0.05 have not been collected.
  • the blanks in the following and all tables have the same meaning.
  • Table 1 The biological process, cellular components and molecular function-related functional groups of the heart induced by HrpNEcb multi-epitope ligand protein were significantly up-regulated and expressed GO terms classification gene number statistics (6, 24 hours after oral administration and 6 hours after application, 12 hours after application )
  • HrpNEcb multi-epitope ligand protein induces the biological process, cellular components and molecular function-related functional groups of the thalamus and hippocampus to significantly up-regulate the expression of GO terms. 12 hours
  • Table 4 The biological process, cellular components and molecular function-related functional groups of the heart induced by HrpNEcb multi-epitope ligand protein were significantly down-regulated and expressed GO terms classification. )
  • Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database for systematic analysis of gene function and genomic information. The process of gene and expression information is studied holistically as a network.
  • KEGG The main feature of KEGG is to link genes with various biochemical reactions, providing integrated metabolic pathways.
  • KEGG currently contains a total of 19 sub-databases, which are classified into three categories: systematic information, genomic information and chemical information. In organisms, different gene products coordinate with each other to perform biological functions.
  • Pathway annotation analysis of differentially expressed genes is helpful for further interpretation of gene functions.
  • the KEGG pathway enrichment analysis was performed on the differentially expressed genes induced by HrpNEcb protein to obtain the roles (upstream and downstream relationships) and biological functions of these differential genes in the signaling pathway, and to deeply understand the relationship between genes and functions.
  • Pathway analysis of differentially expressed genes was performed using Fisher's exact test. Fisher's exact test was calculated to obtain the p-value, and multiple hypothesis test correction was performed to obtain the q-value. Pathways with p-value less than 0.05 were screened as significantly enriched Pathways.
  • HrpNEcb multi-epitope ligand protein as a kind of ligand protein with multi-epitope special structure, new function, new mechanism of action and new application prospect, induces multiple organs (liver, thalamus) of mice. , heart, cerebral cortex and brain hippocampus, etc.) differential expression of multiple genes, these differentially expressed genes are involved in cellular processes (Cellular Processes), environmental information processing (Environmental Information Processing), genetic information processing (Genetic Information Processing), metabolism Functional pathways such as Metabolism and Organismal Systems.
  • Cellular Processes Cellular Processes
  • environmental information processing Environmental Information Processing
  • genetic information processing Genetic Information Processing
  • metabolism Functional pathways such as Metabolism and Organismal Systems.
  • HrpNEcb multi-epitope ligand proteins are involved in biological processes such as translation, replication and repair, folding, classification and degradation (see Figure 12 to Figure 64 for details) .
  • 4Metabolism Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand protein are involved in biodegradation and metabolism, nucleotide metabolism, metabolism of other amino acids, metabolic cofactors and vitamins, lipid metabolism, sugar Biosynthesis and metabolism, global and overview maps, metabolic processes such as energy metabolism, carbohydrate metabolism and amino acid metabolism (see Figure 12 to Figure 64 for details).
  • the HrpNEcb polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column. The protein purification was performed according to the method recommended by the NI-NTA agarose column manufacturer. The prepared HrpNEcb polymimetic epitope ligand protein was used for later use. (hereinafter referred to as capture protein or target protein).
  • bait protein Extraction of total protein from cultured hepatocytes (hereinafter referred to as bait protein) for experiments
  • Biotin blocking 1Add 250 ⁇ l biotin blocking solution to the Spin Column tube. Tighten the top cover and bottom stopper, and gently invert 4 times to mix; 2Incubate at room temperature for 5 min. Remove the top cover, put the Spin Column tube in the collection tube, and centrifuge at 1250 ⁇ g for 50s; 3 Repeat steps 1 and 2 once; 4 Add 250 ⁇ l of TBS to the Spin Column tube. Tighten the top cap and invert it upside down 4 times to mix it up; 5Remove the top cap, put it in a collection tube, and centrifuge at 1250 ⁇ g for 50s; 6Repeat steps 3 and 4 twice, and put the Spin Column tube back into the collection tube Tube.
  • HrpNEcb multi-epitope ligand protein recognizes bound cell membrane receptors: recognizes and binds to 6 membrane receptors, including HLA-A major histocompatibility complex, class I, A receptors, LGALS3BP galactose 3 binding protein (receptor), LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein subunit Beta 2 receptor, LRRC1515-leucine repeat membrane protein receptor, KTN1 kinesin 1 receptor .
  • HLA-A major histocompatibility complex class I
  • a receptors LGALS3BP galactose 3 binding protein (receptor)
  • LAMP2 lysosome-associated membrane protein 2 receptor GNB2G guanine nucleotide binding protein subunit Beta 2 receptor
  • LRRC1515-leucine repeat membrane protein receptor KTN1 kinesin 1 receptor .
  • HrpNEcb multi-epitope ligand protein recognizes bound cell membrane proteins: recognizes and binds to 11 membrane proteins, including DSG4 desmocore protein, ANXA4 annexin A4, CAPRIN1 cyclin, 1UTRN dystrophin protein, pinin bridge Granulin, VAMP-associated protein A, VCL focal adhesion protein, Ezrin epithelial cadherin, PKP3 platelet affinity protein 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD2N acetylated ⁇ -linked acid dipeptidase 2 one or more.
  • HrpNEcb multi-epitope ligand protein recognizes and binds to 13 membrane proteins involved in signaling pathways: including hsa04152: AMPK signaling pathway, hsa03460: Fanconi anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: Sphingolipid signaling pathway , hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: Chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: Camp signaling pathway, hsa04915: Estrogen signaling pathway, hsa04910: Insulin signaling pathway, hsa04390: Hippo signaling pathway.
  • hsa04152 AMPK signaling pathway
  • hsa03460 Fanconi anemia pathway
  • HrpNEcb multi-epitope ligand protein recognizes and binds to membrane proteins: including hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome , hsa01130: Biosynthesis of antibiotics, hsa05131: Shigellosis, hsa04612: Antigen processing and presentation, hsa05130: Pathogenic E.
  • coli infection hsa05100: Bacterial invasion of epithelial cells, hsa05132: Salmonella infection, hsa05169: Barr virus infection , hsa05168: Herpes simplex virus 1 infection, hsa05203: Viral carcinogenesis, hsa05166: HTLV-I infection, hsa05164: Influenza A, hsa05134: Legionnaires' disease, hsa05160: Hepatitis C, hsa05162: Measles, hsa05133: Pertussis, hsa05322: System lupus erythematosus, hsa04670: transepithelial migration of leukocytes, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: pathways in cancer.
  • HrpNEcb multi-epitope ligand protein recognizes and binds membrane proteins involved in 3 important neurological disease metabolic pathways: including hsa05012: Parkinson's disease, hsa05016: Huntington's disease, hsa05010: Alzheimer's disease.
  • HrpNEcb multi-epitope ligand protein recognizes and binds membrane proteins involved in 30 nucleic acid, protein, amino acid, sugar, and fat metabolism-related pathways: including hsa03420: nucleotide excision repair, hsa00970: aminoacyl biosynthesis, hsa03430: Mismatch repair, hsa01210: 2-oxocarboxylic acid metabolism, hsa03440: homologous recombination, hsa04360: axon guidance, hsa00051: fructose and mannose metabolism, hsa00565: ether lipid metabolism, hsa00510: N-glycan biosynthesis, and hsa04110 : cell cycle, hsa03030: DNA replication, hsa03013: RNA trafficking, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: rib
  • HrpNEcb multi-epitope ligand protein recognizes and binds membrane proteins involved in 19 metabolic pathways such as cell junction, neural junction, blood vessel, endocrine, reproductive system, etc.: hsa04723: retrograde neural signal, hsa04726: serotonin-activated synapse , hsa00900: terpenoid biosynthetic pillars, hsa04520: adhesion knots, hsa05032: morphine addiction and hsa04510: focal adhesions, hsa04724: glutamatergic synapses, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114: eggs blast meiosis, hsa04728: dopaminergic synapses, hsa00100: steroid biosynthesis, hsa04261: adrenergic signaling in cardiomy
  • HrpNEcb multi-epitopic ligand protein as a class of ligand protein molecules rich in special multiple linear and conformational epitope structures, can recognize and bind various types of membrane receptors, membrane proteins, information pathways and metabolism across borders Pathways, and further analyze the location, structure, properties, mechanism of action and function of these membrane receptors, membrane proteins, information pathways and metabolic pathways, which are widely involved in and affect the growth, development, metabolism, defense and life of programmed cell death.
  • HrpNEcb multi-epitope ligand protein is a kind of special multi-epitope ligand protein with new function, new mechanism of action and new application prospect.
  • the HrpNEch polymimetic epitope ligand protein is fermented, highly expressed, purified and prepared by engineering bacteria with registered genes, which specifically includes the following steps:
  • HrpNEch multi-epitope ligand protein The engineering bacteria fermentation preparation of HrpNEch multi-epitope ligand protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes) encoding HrpNEch multi-epitope ligand proteins are prepared.
  • Mimic epitope ligand protein wherein, the pH of the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system is 6.5-5.5; the fermentation temperature is 37-38° C.; the concentration of glucose in the fermentation and proliferation liquid medium is 0.01-0.05%; The concentration of glucose in the induction liquid medium is 0.05-0.00%; the concentration of lactose in the fermentation-induction liquid medium is 0.5-0.4%; the incubation time of the fermentation-induction liquid medium is 7-9 hours.
  • HrpNEch polymimetic epitope ligand protein molecule Purify HrpNEch polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column, and the protein purification is according to the NI-NTA agarose column manufacturer's suggestion The method is implemented, and the preparation of purified HrpNEch polymimetic epitope ligand protein is completed.
  • the HrpNEch multi-epitope ligand protein is prepared by expressing the protein of "artificially synthesized gene", which specifically includes the following steps:
  • the first step artificial synthesis of the hrpNEch gene encoding the HrpNEch protein
  • Step 2 2) According to the above DNA sequence, when artificially synthesizing protein gene, add BamHI and HindIII enzyme cleavage sites to the 5' and 3' of the gene respectively to facilitate protein gene cloning;
  • the third step artificial gene synthesis is entrusted to the GeneArt gene synthesis and service department of Thermo Fisher Scientific. 3) The synthesized DNA fragments encoding the HrpNEch protein gene are cloned into the BamHI-HindIII site of the high-efficiency protein expression vector PET28a(+) (containing His-Tag label) one by one, and the accuracy of the clone is ensured through DNA sequencing;
  • the fourth step clone the gene encoding HrpNEch protein from 1) to 3) and transfer it into Escherichia coli engineering bacteria (E.coli), and the production line (E.coli) of the related protein is the original K-12 bacteria after special transformation.
  • the fermentation medium is Na 2 HPO 4 -KH 2 PO 4 buffer system, the pH of the buffer system is 6.5-5.5; the concentration of glucose in the fermentation and proliferation liquid medium is 0.01-0.05%; the concentration of lactose in the fermentation induction liquid medium is 0.5-0.4% ;
  • Step 5 Suspend the collected cells into Na 2 HPO 4 -KH 2 PO 4 buffer, complete the sterilization treatment at 80°C for 30 minutes, quickly cool down to 30°C, and place in a butterfly continuous flow centrifuge. Wash the engineered bacteria for five to eight times, import them into a high-pressure crusher, and continuously use 800-1000Mpa pressure to break the engineered bacteria. Pass the broken bacteria liquid into a butterfly continuous flow centrifuge to remove the cell wall and collect HrpNEch polymimetic epitope ligands. Protein molecules, HrpNEch multi-epitope ligand proteins are present in the supernatant;
  • Step 6 Purify the HrpNEch polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column.
  • the protein purification is carried out according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification of HrpNEch polymimetic epitopes. Preparation of ligand proteins.
  • the highly expressed purified protein-His recombinant band was detected by 10% SDS polyacrylamide gel electrophoresis, see Figure 67 for details.
  • the left side is the molecular weight identification band
  • lane 1 is the electrophoresis band before purification, and there are more bands in the corresponding molecular weight region, including the 34.15kda band
  • lane 2 is the purified HrpNEch polymimetic epitope ligand
  • the body protein band, with a molecular weight of 34.15kda, is in the corresponding molecular weight region of the protein, indicating that the corresponding HrpNEch purified protein has been obtained.
  • the allergy test of HrpNEch poly-epitope ligand protein the focal spots seen are formed by HrpNEch poly-epitope ligand protein solution treatment for about 24hrs, the upper row of leaves: H 2 O injection, For the control; the lower row of leaves: HrpNEch poly-epitope ligand protein solution (250 ⁇ g/ml) injection, for the treatment, that is, the hypersensitivity reaction of HrpNEch poly-epitope ligand protein on tobacco leaves.
  • the purified HrpNEch multi-epitope ligand protein can generally induce hypersensitivity reactions in the leaves of various plants.
  • the tested plant species can be: tobacco, pepper, eggplant, tomato, potato, strawberry, cucumber, water spinach, celosia, glass begonia , September chrysanthemum, pansy, nopal, petunia, grape, rose, locust tree, pea, peach tree, bunch of red, loofah, green beans, cauliflower, spinach, rapeseed, yam, cowpea, broad bean, corn , rice, soybean, cyclamen, mulberry, pumpkin, loquat, toon tree and other 36 kinds of different plants.
  • the research object of mRNA sequencing is all RNAs with a poly-A tail that can be transcribed by a specific cell in a certain functional state, mainly mRNA.
  • Cell-generated mRNA is converted into DNA (cDNA, complementation, and library construction of the obtained cDNA) by a process of reverse transcription.
  • the resulting DNA is then sequenced and from the observed abundance of specific DNA, the original amount of mRNA in the cell can be inferred from it to find genes or transcripts whose transcription levels change under the experimental conditions, i.e. differentially expressed.
  • mice 8-week-old balb/C mice were selected for the experiment and divided into HrpNEch multi-epitope ligand protein treatment groups, including four treatments of oral administration for 6 hours, 24 hours, and application for 6 hours and 12 hours.
  • the mice in the buffer control sham-operated group were fed and smeared with buffer, and the mice in the blank control group did not receive any treatment.
  • RNA-Seq mRNA sequencing experimental flow chart
  • RNA extraction of the samples was performed using the miRNeasy Micro Kit (Cat#1071023Qiagen) and according to the standard operating procedure provided by the manufacturer.
  • the total RNA was quality-checked by NanoDrop ND-2000 spectrophotometer and Agilent Bioanalyzer 4200 (Agilent technologies, Santa Clara, CA, US), and the qualified RNA was used for subsequent sequencing experiments.
  • Oligo(dT) can be used to enrich mRNAs with polyA tails.
  • the enriched mRNA is then subjected to fragmentation, double-stranded cDNA synthesis, end repair, 3' end addition, ligation of adapters, and amplification.
  • the constructed library uses 2.0 Fluorometer detects concentration, Agilent2100 detects size.
  • Illumina sequencing is performed on the library that has passed the quality inspection.
  • the sequencer captures the fluorescent signal and converts the optical signal into a sequencing peak through computer software to obtain the sequence information of the fragment to be tested.
  • the differential gene volcano plot was used to display the overall distribution of genes with significant differential expression induced by HrpNEch protein.
  • Horizontal axis fold change of gene expression in different samples (log2 Fold-Change); vertical axis: significant level of gene expression difference (-log10 p-value); the expression of the right point is significantly up-regulated gene; the expression of the left point Significantly down-regulated genes; genes with no significant changes in expression at lower points.
  • Figures 69-70 are the differential gene volcano plots of mouse testis and kidney HrpNEch multi-epitope ligand protein induced by oral administration and smearing, respectively, HrpNEch is abbreviated as N2 in the figure.
  • FIGS 71-72 are cluster heatmaps of differentially expressed gene sets in testis and kidney, respectively, in which HrpNEch is abbreviated as N2.
  • Gene Ontology is an ontology widely used in the field of bioinformatics.
  • Gene Ontology is a description of genes in different dimensions and levels, covering biological processes (biological_process), cellular components (cellular_component) and molecular functions (molecular_function).
  • biological processes are describing which biological processes the gene is involved in; cellular components explain where the gene is present, including whether the gene is in the cytoplasm or in the nucleus? If cytoplasm is present in which organelle? If it is in the mitochondria, is it on the mitochondrial membrane or in the matrix of the mitochondria, etc., this information belongs to the cell group; the molecular function explains what is the function of the gene at the molecular level?
  • Gene Ontology database is a structured standard biological model constructed by the GO organization (Gene Ontology Consortium) in 2000. It aims to establish a standard vocabulary system for knowledge of genes and their products, covering the biological process of genes. process), cellular component, molecular function.
  • Term is the basic description unit in GO. GO Terms are used to describe the function of gene products. By performing GO enrichment analysis on differential genes, genes can be classified according to different functions, so as to achieve the purpose of annotating and classifying genes.
  • HrpNEch multi-epitope ligand protein we carried out GO term enrichment analysis on the differentially expressed genes induced by HrpNEch multi-epitope ligand protein, and the results proved and confirmed that HrpNEch multi-epitope ligand protein, as a class of special structures with multiple epitopes, new functions , a ligand protein with a new mechanism of action and a new application prospect, induced the differential expression of multiple genes in multiple organs (testis, kidney) of the tested mice, and these differentially expressed genes covered biological processes, cellular components and molecular functions. Differentially expressed genes were analyzed by GO using Fisher's exact test. Fisher's exact test was calculated to obtain the p-value, and multiple hypothesis test correction was performed to obtain the q-value.
  • Biological_process-related differentially expressed genes include reproduction, cell death, immune system processes, behaviors, metabolic processes, and cellular processes , reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, motility, processes in individual tissues, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, Stimulatory responses, localization, bioregulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic processes involve synaptic transmission.
  • the results of GO enrichment analysis of biological processes are shown in Table 8 to Table 11.
  • 2 The differentially expressed genes related to cellular components cover cells and extracellular regions, nucleoids, membranes, virus particles, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules, organelles, extracellular matrix components, cells Parts of the outer region, organelle parts, virion parts, membrane parts, synaptic parts, cell parts, synapses, and cellular supramolecular fibers, etc.
  • the results of GO enrichment analysis of cell components are shown in Table 8 to Table 11.
  • Molecular function-related differentially expressed genes cover transcription factor activity, protein binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, and morphogen activity , antioxidant activity, metal chaperone protein activity, protein labeling, chemoattractant activity, translation regulation, chemorepellent activity, active molecular sensors, molecular function regulation, etc.
  • the molecular function GO enrichment analysis results are shown in Table 8 to Table 11.
  • Table 8 The biological process, cellular components and molecular function-related functional groups of HrpNEch multi-epitope ligand protein-induced kidney were significantly up-regulated and expressed GO terms classification gene number statistics table (6, 24 hours after oral administration and 6 hours after application)
  • Table 9 The biological process, cellular components and molecular function-related functional groups of testis induced by HrpNEch multi-epitope ligand protein were significantly up-regulated and expressed GO terms classification gene number statistics table (6, 24 hours after oral administration and 6 hours after application)
  • Table 11 The biological process, cellular components and molecular function-related functional groups of testis induced by HrpNEch multi-epitope ligand protein were significantly down-regulated and expressed GO terms. Statistical table of the number of classified genes (6, 24 hours after oral administration and 6 hours after application)
  • Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database for systematic analysis of gene function and genomic information. The process of gene and expression information is studied holistically as a network.
  • KEGG The main feature of KEGG is to link genes with various biochemical reactions, providing integrated metabolic pathways.
  • KEGG currently contains a total of 19 sub-databases, which are classified into three categories: systematic information, genomic information and chemical information. In organisms, different gene products coordinate with each other to perform biological functions. Pathway annotation analysis of differentially expressed genes is helpful for further interpretation of gene functions.
  • the KEGG pathway enrichment analysis was performed on the differentially expressed genes induced by HrpNEch multi-epitopic ligand proteins, and the roles (upstream and downstream relationships) and biological functions of these differential genes in the signaling pathway were obtained, and the relationship between genes and functions was deeply understood.
  • HrpNEch multi-epitope ligand protein as a kind of ligand protein with multi-epitope special structure, new function, new mechanism of action and new application prospect, induces multiple organs (testis, kidney) of mice. ) differential expression of multiple genes involved in cellular processes, environmental information processing, genetic information processing, metabolism (Metabolism) and biological systems (Organismal) Systems) and other functional pathways.
  • the HrpNEch polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column.
  • the protein purification was carried out according to the method recommended by the NI-NTA agarose column manufacturer.
  • the prepared HrpNEch polymimetic epitope ligand protein was purified. Standby (hereinafter referred to as capture protein or target protein).
  • bait protein Extraction of total protein from cultured hepatocytes (hereinafter referred to as bait protein) for experiments
  • Biotin blocking 1Add 250 ⁇ l biotin blocking solution to the Spin Column tube. Tighten the top cover and bottom stopper, and gently invert 4 times to mix; 2Incubate at room temperature for 5 min. Remove the top cover, put the Spin Column tube in the collection tube, and centrifuge at 1250 ⁇ g for 50s; 3 Repeat steps 1 and 2 once; 4 Add 250 ⁇ l of TBS to the Spin Column tube. Tighten the top cap and invert it upside down 4 times to mix it up; 5Remove the top cap, put it in a collection tube, and centrifuge at 1250 ⁇ g for 50s; 6Repeat steps 3 and 4 twice, and put the Spin Column tube back into the collection tube. Tube.
  • HrpNEch multi-epitope ligand protein recognizes and binds to cell membrane receptors: recognizes and binds to 12 membrane receptors, GNG12 guanine nucleotide binding protein ⁇ -12 receptor, ANXA5 annexin A5 receptor, ANXA2 membrane linker Protein A2 receptor, ANXA1 annexin A1 receptor, IGHG2 immunoglobulin weight constant ⁇ 2 receptor, IGHM immunoglobulin weight constant Mu receptor, CACNA1S calcium voltage-gated channel subunit ⁇ 1S receptor, ZNF185 zinc finger protein 185 Receptors and HLA-A major histocompatibility complex, class I, A receptors, LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein subunit ⁇ 2 receptor, KTN1 kinesin binding protein 1 receptor.
  • HrpNEch multi-epitope ligand protein recognizes bound cell membrane proteins: recognizes and binds to 16 membrane proteins, DSC3 desmocollin, ANXA8/ANXA8L1 annexin A8/annexin A8 similar protein 1, EVPL coat protein , POF1B actin-binding protein premature ovarian failure 1B, CTNNA1-catenin, TGM1 transglutaminase 1, BAIAP2BAI1-associated protein 2, RAB29RAS oncogene family members, CLDN19 docking protein 19, STXBP2 syntaxin-binding protein 2, VAMP Vesicle-associated membrane protein-associated protein A, VCL focal adhesion protein, Ezrin epithelial cadherin, PKP3 platelet affinity protein 3, NAALAD2N acetylated ⁇ -linked acid dipeptidase 2, PKP1 platelet affinity protein 1, SPRR1A rich Proline-containing small protein 1A.
  • hsa03320 PPAR signaling pathway
  • hsa05120 Helicobacter pylori infection epithelial cell signal transduction
  • hsa04071 Sphingolipid signaling pathway
  • hsa04014 Ras signaling pathway
  • hsa04151 PI3K-Akt signaling pathway
  • hsa04070 Phosphatidylinositol signaling system
  • hsa04010 MAPK signaling pathway
  • hsa04310 Wnt signaling pathway
  • hsa04062 Chemokine signaling pathway
  • hsa04015 Rap1 signaling pathway
  • hsa04024 camp signaling pathway
  • hsa04915 estrogen signaling pathway
  • hsa04910 insulin signaling pathway
  • hsa04390 hippo signaling pathway
  • HrpNEch multi-epitope ligand protein recognizes and binds to participate in: hsa04144: endocytosis, hsa04145: phagosome, hsa04142: lysis Enzymes, hsa04666: Fc-r-mediated phagocytosis, hsa01130: Biosynthesis of antibiotics, hsa05131: Shigellosis, hsa04612: Antigen processing and presentation, hsa05130: Pathogenic E.
  • coli infection hsa05100: Epithelial Bacterial invasion, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05203: Viral carcinogenesis, hsa05134: Legionnaires' disease, hsa05160: Hepatitis C, hsa05162: Measles, hsa05133: Pertussis, hsa05322: Systemic lupus erythematosus, hsa04670: Leukocytosis Endothelial migration, hsa05152: Tuberculosis, hsa05150: Staphylococcus aureus infection, hsa05146: Amoebiasis, hsa05142: Chagas disease, hsa05200: Pathways in cancer, hsa05143: African trypanosomiasis, hsa04750: Inflammation of TRP channels Mediator regulation, h
  • HrpNEch multi-epitope ligand protein recognizes and binds membrane proteins involved in 3 important neurological disease metabolic pathways: including hsa05012: Parkinson's disease, hsa05016: Huntington's disease, hsa05010: Alzheimer's disease.
  • HrpNEch multi-epitope ligand protein recognizes and binds to 39 nucleic acid, protein, amino acid, sugar, and fat metabolism-related pathways involved in membrane proteins: including hsa03013: RNA transport, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: Ribosome, hsa04141: endoplasmic reticulum protein processing, hsa04810: regulation of the actin backbone hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa00230: purine metabolism, hsa04932: nonalcoholic fatty liver disease, hsa00020 : Citric acid cycle, hsa03008: Biogenesis of eukaryotic ribosomes, hsa00240: Pyrimidine metabolism, hsa00650: Meth
  • HrpNEch multi-epitope ligand protein recognizes and binds membrane proteins involved in 34 metabolic pathways including cell junctions, neural junctions, blood vessels, endocrine, and reproductive systems: including hsa04510: focal adhesions, hsa 04724: glutamatergic spikes haptic, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114: oocyte meiosis, hsa04728: dopamine synapses, hsa00140: steroid hormone biosynthesis, hsa04261: adrenergic signaling in cardiomyocytes, hsa04727: gamma - GABAergic synapses, hsa04725: cholinergic synapses, hsa04540: gap junctions, hsa04971: gastric acid secretion, hsa04713: circadian en
  • HrpNEch multi-epitopic ligand protein as a class of ligand protein molecules rich in special multiple linear and conformational epitope structures, can recognize and bind various types of membrane receptors, membrane proteins, information pathways and metabolism across borders Pathways, and further analyze the location, structure, properties, mechanism of action and function of these membrane receptors, membrane proteins, information pathways and metabolic pathways, which widely affect the growth, development, metabolism, defense and the basic properties of life of programmed cell death. , and is widely involved in the diagnosis, prevention, treatment, rehabilitation of diseases and conditions of the nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system, and skin system.
  • HrpNEch multi-epitope ligand protein is a kind of special multi-epitope ligand protein with new function, new mechanism of action and new application prospect.
  • HrpN type multi-epitope ligand protein of the present invention in food, cosmetics, health care products and medicines also involves identifying and activating various types of receptors, membrane proteins and their signaling pathways in animals (including humans) and inducing multifunctionality.
  • HrpNEcb, HrpNEch and other multi-epitope ligands HrpN-type multi-epitope ligand protein products can be widely used in food, cosmetics, and health care products.
  • the auxiliary conditioning functions of HrpN-type multi-epitope ligand protein mainly include 1 , Enhance immune function; 2. Assist blood lipid lowering function; 3. Assist blood sugar lowering function; 4. Antioxidant function; 5. Assist in improving memory function; 6. Relieve visual fatigue function; Pharyngeal function; 9. Auxiliary blood pressure lowering function; 10. Improve sleep function; 11. Promote lactation function; 12. Relieve physical fatigue function; 13. Improve hypoxia tolerance function; 14. Have auxiliary protective function against radiation hazards; 15 , weight loss function; 16, improve growth and development function; 17, increase bone Density function; 18. Improve nutritional anemia function; 19. Auxiliary protection function against chemical liver damage; 20. Remove acne function; 21. Remove melasma function; 22. Improve skin moisture function; 23. Improve skin oil content 24. Regulate the function of intestinal flora; 25. Promote digestive function; 26. Laxative function; 27. Have auxiliary protective function for gastric mucosal damage.
  • the preparations of the HrpN-type multi-epitope ligand protein of the present invention can be used in foods that assist in regulating the expression and regulation of the body's growth, development, defense, metabolism, and programmed cell death functions, including various human Finished products and raw materials for eating or drinking;
  • the preparations of the HrpN-type multi-epitope ligand protein of the present invention can be used in cosmetics that assist in regulating the expression and regulation of the growth, development, defense, metabolism, and programmed cell death functions of the body, including rubbing, Spraying or other similar methods, spread on any part of the human body surface, including skin, hair, nails, lips, etc., to achieve the purpose of cleaning, eliminating bad odor, skin care, beauty and grooming biotechnology products, or including ordinary cosmetics and special purposes cosmetic;
  • the preparation of the HrpN-type multi-epitope ligand protein of the present invention can be used in health care products that assist in regulating the growth, development, defense, metabolism of the body and the expression and regulation of programmed cell death functions, including health care functional foods , is a type of food, has the commonality of general food, can regulate the functions of the human body, and is suitable for consumption by specific groups of people, but not for the purpose of curing diseases.

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Abstract

Provided is the use of an HrpN-type multi-mimotope ligand protein in foods, cosmetics, health care products or pharmaceuticals. The HrpN-type multi-mimotope ligand protein comprises HrpNEcc, HrpNEca, HrpNEcb, HrpNEch, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNEa, HrpNSam, HrpNBag, HrpNPas and HrpNEnt. The HrpN-type multi-mimotope ligand protein serves as a ligand protein molecule having a special structure and which is rich in multiple epitopes (linear and conformation), and can identify, activate and bind to various types of membrane receptors, membrane proteins, information pathways and metabolic pathways of animals in a transboundary mode.

Description

HrpN型多拟表位配体蛋白在食品、化妆品、保健品或制药中的应用Application of HrpN-type multi-epitope ligand protein in food, cosmetics, health care products or pharmaceuticals 技术领域technical field
本发明涉及生物医药领域，具体的涉及HrpN型蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的制药中的应用。The present invention relates to the field of biomedicine, in particular to the application of HrpN-type protein in the pharmacy that recognizes and activates multiple types of receptors and/or membrane proteins and their signal pathways and causes cascade biological effects.
背景技术Background technique
分子生物学是在分子水平上研究生命现象的科学，通过研究生物大分子的结构、功能和代谢来阐明各种生命现象的本质，其研究内容涵盖了生命的全过程。DNA，RNA和蛋白质是三种重要的生物大分子，是生命现象的分子基础。基因组决定了生命有什么，蛋白质组决定了生命能做什么，代谢组决定了生命实际发生了什么。现代生命科学、生物技术和医药生物技术，特别是蛋白质组学和代谢组学，得到了突飞猛进的发展，更新了对疾病的认识、诊断、防控、治疗和康复的理念，开创了对新型高效安全药物的新认识和新途径，使现代医学的发展进入到了一个崭新阶段，开辟了广阔的应用前景。Molecular biology is a science that studies life phenomena at the molecular level. It clarifies the nature of various life phenomena by studying the structure, function and metabolism of biological macromolecules, and its research content covers the whole process of life. DNA, RNA and protein are three important biological macromolecules, which are the molecular basis of life phenomena. The genome determines what life has, the proteome determines what life can do, and the metabolome determines what actually happens to life. Modern life sciences, biotechnology and medical biotechnology, especially proteomics and metabolomics, have developed by leaps and bounds, updating the concepts of disease understanding, diagnosis, prevention and control, treatment and rehabilitation, creating new and efficient The new understanding and new approach of safe drugs have brought the development of modern medicine into a new stage and opened up broad application prospects.
现代生命科学的受体理论是药效学的基本理论之一，是从分子水平解释生命的可控的生理过程和病理过程、药物的药理作用机制、药物分子的结构效应关系的一个重要依据。配体是一类信号物质，除了与受体识别、结合和激活受体外，本身并无其他直接功能，它不能参加代谢产生有用产物，也不直接诱导任何细胞活性，更无酶的特点，它唯一的功能就是通过对受体的识别、结合和激活，向细胞传导在内外环境中存在的特殊信号或信息。The receptor theory of modern life science is one of the basic theories of pharmacodynamics, and it is an important basis for explaining the controllable physiological and pathological processes of life, the pharmacological mechanism of drugs, and the structure-effect relationship of drug molecules at the molecular level. Ligand is a kind of signal substance. Except for recognizing, binding and activating the receptor, it has no other direct functions. It cannot participate in metabolism to produce useful products, nor does it directly induce any cell activity, and it has no enzyme characteristics. Its only function is to transmit special signals or information that exist in the internal and external environment to cells through the recognition, binding and activation of receptors.
信号通路(细胞通讯)是指在多细胞生物的细胞间或细胞内通过高度精确和高效发送与接收信息的通讯机制，并通过放大引起快速的细胞生理生化反应，或启动基因活动，尔后发生一系列的细胞生理、生化活动来协调各组织活动，促使生命的统一整体对多变的内外界环境作出综合反应，这是生命机体的系统、组织、器官、细胞、亚细胞、分子、亚分子建造一个协调的生长、发育、防御和代谢的联结机制。Signaling pathway (cellular communication) refers to the communication mechanism that sends and receives information with high precision and efficiency between cells or within cells of multicellular organisms, and causes rapid cellular physiological and biochemical reactions through amplification, or initiates gene activity, and then a series of events occur. The physiological and biochemical activities of cells coordinate the activities of various organizations, and promote the unified whole of life to make a comprehensive response to the changing internal and external environment. Linked mechanisms for coordinated growth, development, defense, and metabolism.
受体是指一类介导细胞信号转导的功能蛋白，能识别周围环境(细胞内外环境)中的某些微量物质，并与之识别、结合，被激活，通过信号放大系统触发后续的生理生化反应。受体是由细胞膜和细胞内的蛋白质、核酸、脂质、多糖等组成的生物大分子。受体在细胞生物学中是一个很泛的概念，意指任何能够同激素、神经递质、药物或细胞内外的信号分子结合并能引起细胞功能变化的生物大分子，此时的信号分子被称为配体。多细胞生物中有几百种不同的信号分子在细胞间和细胞内传递信息，这些信号分子中有蛋白质、氨基酸衍生物、核苷酸、胆固醇、脂肪酸衍生物以及可溶解的气体分子等。存在于细胞质膜上的受体称为膜受体，化学本质绝大部分是糖镶嵌蛋白；位于胞液和细胞核中的受体称为胞内受体，它们全部为DNA结合蛋白。Receptors refer to a class of functional proteins that mediate cell signal transduction. They can recognize certain trace substances in the surrounding environment (internal and external environment of cells), recognize, bind to, and be activated to trigger subsequent physiological processes through the signal amplification system. biochemical reaction. Receptors are biological macromolecules composed of cell membranes and intracellular proteins, nucleic acids, lipids, and polysaccharides. Receptor is a very broad concept in cell biology, which refers to any biological macromolecules that can bind to hormones, neurotransmitters, drugs or signal molecules inside and outside cells and can cause changes in cell function. called ligands. There are hundreds of different signaling molecules in multicellular organisms that transmit information between and within cells. These signaling molecules include proteins, amino acid derivatives, nucleotides, cholesterol, fatty acid derivatives, and soluble gas molecules. The receptors present on the cytoplasmic membrane are called membrane receptors, and most of the chemical essences are sugar mosaic proteins; the receptors located in the cytosol and nucleus are called intracellular receptors, and they are all DNA-binding proteins.
配体是一类信号物质，除了与受体识别、结合和激活受体外，本身并无其他直接功能，它不能参加代谢产生有用产物，也不直接诱导任何细胞活性，更无酶的特点，它唯一的功能就是通过对受体的识别、结合和激活，向细胞传导在内外环境中存在的特殊信号或信息。Ligand is a kind of signal substance. Except for recognizing, binding and activating the receptor, it has no other direct functions. It cannot participate in metabolism to produce useful products, nor does it directly induce any cell activity, and it has no enzyme characteristics. Its only function is to transmit special signals or information that exist in the internal and external environment to cells through the recognition, binding and activation of receptors.
配体与受体的结合是分子间识别激活过程，它靠离子配位键、氢键、π-π堆积作用、静电作用、疏水作用、范德华力等的作用，随着两种分子空间结构互补和互作程度增加，相互作用基团之间距离就会缩短，作用力就会大大增加，因此配体和受体分子空间结构的互作性和互补性是特异结合的主要因素，也即本发明所采用的“结构基团”或“表位”概念。同一配体可能对应两种或两种以上的不同受体，同一配体与不同类型受体结合会产生不同的细胞反应。配体与受体结合后，将引发有关系列生理活性，无论配体是 内源性的还是外源性的，与受体结合后，二者形成配体-受体结合面或复合物，从而传递信息，通过传导和转导，并通过放大引起快速的细胞生理、生化反应，或者启动基因活动，尔后发生一系列的级联反应来协调各组织、器官、细胞的活动，促成生命的统一整体对多变的内外界环境作出综合反应。The binding of ligands and receptors is an intermolecular recognition and activation process, which relies on ionic coordination bonds, hydrogen bonds, π-π stacking, electrostatic interaction, hydrophobic interaction, van der Waals force, etc., with the complementarity of the two molecular spatial structures. As the degree of interaction increases, the distance between the interacting groups will be shortened, and the interaction force will be greatly increased. Therefore, the interaction and complementarity of the spatial structure of the ligand and receptor molecules are the main factors for specific binding, that is, the The concept of "structural group" or "epitope" employed in the invention. The same ligand may correspond to two or more different receptors, and the binding of the same ligand to different types of receptors will produce different cellular responses. After the ligand binds to the receptor, it will trigger a series of physiological activities, no matter whether the ligand is endogenous or exogenous, after binding to the receptor, the two form a ligand-receptor binding surface or complex, thereby Transmission of information, through conduction and transduction, and through amplification to cause rapid cellular physiological and biochemical reactions, or to initiate gene activity, and then a series of cascade reactions occur to coordinate the activities of various tissues, organs, and cells, and contribute to the unity of life. Make a comprehensive response to the changing internal and external environment.
2008年，Leader等首次提出根据蛋白质药理学作用分类的思路，并将蛋白质药物分为如下四大类：①应用蛋白质的酶活性及调节活性进行治疗的蛋白质药物；②有特殊靶向活性的蛋白质药物；③重组蛋白质疫苗；④用于诊断的重组蛋白质药物。其中第一类和第二类主要用于基础蛋白质疗法，第三类和第四类重点强调蛋白质在疫苗及诊断用药中的应用。经过一个多世纪的摸索和曲折发展，蛋白质药物已经一步一步的成熟起来，在制药工业及临床应用中均占有举足轻重的地位。它们对肿瘤、感染、自身免疫性疾病、代谢性遗传病、各种老年病及退行性疾病等几乎所有疾病领域均具有重要影响，正在成为21世纪重要的治疗、预防和诊断用药。展望未来30年，以重组DNA技术为核心的生物技术的广泛应用必将赋予蛋白质药物更为广阔的发展空间：重组蛋白质药物将逐步取代非重组蛋白；重组改构、体内外修饰将成为常规；用哺乳动物细胞体系表达的产品将占主导地位；蛋白质药物非注射给药途径将受到越来越多的关注；生物仿制药和生物相似药将大有可为。(朱迅，蛋白质药物的功能分类及发展趋势，中国医药生物技术2010年2月第5卷第1期，China Med Biotechnol，February 2010，Vol.5，No.1)。In 2008, Leader et al. first proposed the idea of classification according to the pharmacological effects of proteins, and divided protein drugs into the following four categories: (1) protein drugs using the enzymatic activity and regulating activity of proteins for treatment; (2) proteins with special targeting activities Drugs; ③ recombinant protein vaccines; ④ recombinant protein drugs for diagnosis. The first and second categories are mainly used for basic protein therapy, and the third and fourth categories emphasize the application of proteins in vaccines and diagnostic drugs. After more than a century of exploration and tortuous development, protein drugs have matured step by step and play an important role in the pharmaceutical industry and clinical applications. They have an important impact on almost all disease fields such as tumors, infections, autoimmune diseases, metabolic genetic diseases, various geriatric diseases and degenerative diseases, and are becoming important therapeutic, preventive and diagnostic drugs in the 21st century. Looking forward to the next 30 years, the wide application of biotechnology with recombinant DNA technology as the core will give protein drugs a broader space for development: recombinant protein drugs will gradually replace non-recombinant proteins; reorganization, in vitro and in vivo modification will become routine; Products expressed in mammalian cell systems will dominate; non-injectable delivery routes for protein drugs will receive increasing attention; and biosimilars and biosimilars will be promising. (Zhu Xun, Functional Classification and Development Trend of Protein Drugs, China Med Biotechnol, February 2010, Vol.5, No.1, China Med Biotechnol, February 2010, Vol.5, No.1).
已有研究表明，配体与受体的识别结合，是由组成配体线性或和构象结构基团或表位的关键氨基酸残基决定的，比如，boFcγ2R的82-85位多肽FIGV线性配体结合表位的苯丙氨酸(Phe 82)、异亮氨酸(Ile83)和颉氨酸(Val 85)是识别结合牛IgG2受体的关键氨基酸残基，再比如，boFcγRⅠ的142-149位多肽TNLSHNGI线性配体结合表位的苏氨酸(Thr 142)、天门冬酰胺(Asn 143)、亮氨酸(Leu 144)、甘氨酸(Gly 148)和异亮氨酸(Ile 149)是识别结合牛IgG1受体的关键氨基酸残基；又比如，boFcγRⅢ的98-103位短肽AQRVVN线性配体结合表位的丙氨酸(Ala 98)、谷氨酸(Gln 99)、颉氨酸(Val 101)、颉氨酸(Val102)和天门冬酰胺(Asn 103)是识别结合牛IgG1受体的关键氨基酸残基。Studies have shown that the recognition and binding of ligands and receptors is determined by the key amino acid residues that constitute the linear or conformational structural groups or epitopes of the ligand, for example, the 82-85 position of the boFcγ2R polypeptide FIGV linear ligand The phenylalanine (Phe 82), isoleucine (Ile83), and pyrimidine (Val 85) of the binding epitope are the key amino acid residues for recognizing binding to bovine IgG2 receptor, for another example, positions 142-149 of boFcγRI Threonine (Thr 142), asparagine (Asn 143), leucine (Leu 144), glycine (Gly 148) and isoleucine (Ile 149) of the linear ligand-binding epitope of the polypeptide TNLSHNGI are recognized binding The key amino acid residues of bovine IgG1 receptor; another example, the alanine (Ala 98), glutamic acid (Gln 99), glutamic acid (Val) of the 98-103 short peptide AQRVVN linear ligand binding epitope of boFcγRIII 101), amino acid (Val102), and asparagine (Asn 103) are key amino acid residues that recognize binding to bovine IgG1 receptor.
Harpin是由革兰氏阴性细菌“过敏反应与致病性(hyper sensitive response and pathogenicity，hrp)”基因簇中的基因编码的性质和功能相似的一类蛋白质，富含甘氨酸、不含胱氨酸、对蛋白质酶敏感、热稳定，能在非寄主植物上引起过敏反应。过敏性反应(hypersensitive reaction,HR)表现为非寄主植物受侵染组织快速、局部的萎缩和坏死，从而限制了病原菌的扩散，进而诱导系统抗性，这是植物抵抗病原侵染的普遍表现形式和有效途径。经过近三十年来的研究，这些编码蛋白已获本领域生物学家、植物病理学家及应用研究者公认，属于诱发植物系统抗性的诱抗蛋白类的Harpin超敏蛋白，现已成为植物保护领域的能安全的能诱导植物产生抗病性、驱虫性、抗逆性并促进植物生长发育和提高产量的生物农药，如专利公开号为CN1687420，名称为一种编码植物多功能活性和广谱抗性细胞信号因子的基因hrpNEccs及其表达产物HrpNEccs蛋白质报道的内容。Harpin is a class of proteins with similar properties and functions encoded by genes in the "hyper sensitive response and pathogenicity (hrp)" gene cluster of Gram-negative bacteria, rich in glycine and not containing cystine , Sensitive to protein enzymes, thermostable, and can cause allergic reactions in non-host plants. Hypersensitive reaction (HR) is characterized by rapid and local atrophy and necrosis of infected tissues of non-host plants, which limits the spread of pathogenic bacteria and induces systemic resistance, which is a common manifestation of plant resistance to pathogenic infection. and effective ways. After nearly 30 years of research, these encoded proteins have been recognized by biologists, phytopathologists and applied researchers in the field. Harpin hypersensitivity proteins belong to the class of anti-inducing proteins that induce plant systemic resistance. In the field of protection, biopesticides that can safely induce plants to produce disease resistance, insect repellency, stress resistance and promote plant growth and development and increase yield, such as patent publication number CN1687420, are named as a kind of encoding plant multifunctional activity and The gene hrpNEccs of broad-spectrum resistance cell signaling factor and its expression product HrpNEccs protein report content.
HrpNEcb蛋白(GenBank ID:ABD22989.1)是hrpNEcb基因(基因注册号：DQ355519.1)的表达产物，其由370个氨基酸残基组成，有一、二、三级结构而无四级结构的非酶蛋白质，不含胱氨酸和半胱氨酸，富含甘氨酸和丝氨酸，等电点pI 5.43，分子量Mw 36636.21Da，GenBank ID:ABD22989.1。HrpNEcb蛋白保守结构域由201个氨基酸组成，位于蛋白的C-端，170-370；α-螺旋结构44-64、110-118、139-155、174-192、221-243、259-260、262-274、276-279、284-285、298-303、313-330、347-349、353-368；β-折叠结构10-15、255-256；IDPs结构(固有无序蛋白质，intrinsically disordered proteins，简称IDPs)1-11、13-43、67-95、99-139、157-174、197-216、340-341、364-370。HrpNEcb protein (GenBank ID: ABD22989.1) is the expression product of hrpNEcb gene (Gene Accession No.: DQ355519.1), which consists of 370 amino acid residues, and is a non-enzymatic non-enzymatic protein with primary, secondary and tertiary structures without quaternary structure. Protein, free of cystine and cysteine, rich in glycine and serine, isoelectric point pI 5.43, molecular weight Mw 36636.21Da, GenBank ID: ABD22989.1. The conserved domain of HrpNEcb protein consists of 201 amino acids, located at the C-terminus of the protein, 170-370; 262-274, 276-279, 284-285, 298-303, 313-330, 347-349, 353-368; β-sheet structure 10-15, 255-256; IDPs structure (intrinsically disordered protein, intrinsically disordered proteins, referred to as IDPs) 1-11, 13-43, 67-95, 99-139, 157-174, 197-216, 340-341, 364-370.
HrpNEch蛋白(GenBank Protein:AAY17519.1)是欧文氏“Erwinia chrysanthemi(Dickeya dadantii)NEchCSCL006菌株”的分泌蛋白，hrpNEch基因(基因注册号：GenBank:AY999000.1)的表达产物。其 由339个氨基酸(aminoacids)残基组成，有一、二、三级结构而无四级结构的非酶蛋白质，不含胱氨酸和半胱氨酸，富含甘氨酸和丝氨酸；理论等电点/分子量(Theoretical pI/Mw):6.07/34146.22；保守区结构域9-334，α-螺旋结构39-62，105-118，131-134，147-163，192-213，233-245，268-273；β-折叠结构2-7、204-205，IDPs结构1--2、8-11、13-40、66-100、131-139、173-177、339。HrpNEch protein (GenBank Protein: AAY17519.1) is the secreted protein of Erwinia "Erwinia chrysanthemi (Dickya dadantii) NEchCSCL006 strain", the expression product of hrpNEch gene (Gene Registration No.: GenBank: AY999000.1). It is composed of 339 amino acids (aminoacids) residues, a non-enzymatic protein with one, two and three tertiary structures but no quaternary structure, does not contain cystine and cysteine, and is rich in glycine and serine; theoretical isoelectric point /Molecular weight (Theoretical pI/Mw): 6.07/34146.22; Conserved region 9-334, α-helical structure 39-62, 105-118, 131-134, 147-163, 192-213, 233-245, 268 -273; β-sheet structures 2-7, 204-205, IDPs structures 1--2, 8-11, 13-40, 66-100, 131-139, 173-177, 339.
结构域是生物大分子中具有特异结构和独立功能的区域，特别是指蛋白质中由不同二级结构和超二级结构组合而成的独立的稳定结构区域，结构域也是蛋白质功能单元，在多结构域蛋白质中，不同的结构域常与不同的功能相联系；蛋白质的二级和超二级结构主要由氢键维持，包括α螺旋、β折叠、β转角、无规卷以及IDPs结构等，α螺旋是一种重复性结构，螺旋中每个α-碳的Φ和Ψ分别为-57°和-47°附近。每圈螺旋占3.6个氨基酸残基，沿螺旋轴方向上升0.54nm，每个残基绕轴旋转100°，沿轴上升0.15nm，相邻螺圈之间形成氢键，氢键的取向几乎与螺旋轴平行；β折叠：β－折叠片由两条或多条伸展的多肽链(或一条多肽链的若干肽段)侧向聚集，通过相邻肽链主链上的N-H与C＝O之间有规则的氢键，形成锯齿状片层结构；IDPs-结构是固有无序蛋白质(intrinsically disordered proteins,简称IDPs)结构区域，具广泛的变构效应，作为柔性的连接区域，内存多种构象和运动状态，并以重复性高、带电性、易结合、以及空间优越性和高度协调性，广泛参与和调控转录、翻译、细胞分裂、蛋白质聚集和细胞信号转导，特别参与自组装调控过程。Structural domains are regions of biological macromolecules with specific structures and independent functions, especially the independent stable structural regions in proteins composed of different secondary structures and super-secondary structures. Domains are also protein functional units. In domain proteins, different domains are often associated with different functions; the secondary and super-secondary structures of proteins are mainly maintained by hydrogen bonds, including α-helix, β-sheet, β-turn, random coil and IDPs structure, etc. The alpha helix is a repetitive structure with Φ and Ψ around -57° and -47°, respectively, for each α-carbon in the helix. Each turn of the helix occupies 3.6 amino acid residues, rising 0.54 nm along the helix axis, each residue rotates 100° around the axis, rising 0.15 nm along the axis, and hydrogen bonds are formed between adjacent turns, and the orientation of the hydrogen bonds is almost the same as The helical axes are parallel; β-sheets: β-sheets are aggregated laterally by two or more stretched polypeptide chains (or several peptide segments of a polypeptide chain), through the relationship between N-H and C=O on the main chain of adjacent peptide chains. There are regular hydrogen bonds between them to form a zigzag lamellar structure; IDPs-structure is the structural region of intrinsically disordered proteins (IDPs), which has a wide range of allosteric effects. It is widely involved in and regulates transcription, translation, cell division, protein aggregation and cell signal transduction with high repeatability, chargeability, easy binding, spatial superiority and high coordination, and is particularly involved in the process of self-assembly regulation .
HrpN型蛋白是由“过敏反应与致病性(hyper sensitive response and pathogenicity，hrp)”基因簇中的基因编码的性质和功能相似的一类蛋白质，包括同源性较高，进化上亲缘关系相近的一类多结构域蛋白质，具有由多个关键氨基酸残基结合的线性和构象结构基团或表位，不同的结构域常与不同的功能相联系，从而决定了它们的在跨界识别激活动物(包括人类)细胞多类受体、膜蛋白以及信号通路和代谢通路并能引起多功能级联生物学效应的制药中的应用前景。但是，目前还未见这类蛋白跨界识别特别是在动物和人类上的相关报道及应用。HrpN-type protein is a class of proteins encoded by genes in the "hyper sensitive response and pathogenicity (hrp)" gene cluster with similar properties and functions, including higher homology and close evolutionary relationship. A class of multi-domain proteins with linear and conformational structural groups or epitopes bound by multiple key amino acid residues, different domains are often associated with different functions, thus determining their activation in transboundary recognition The application prospects of various types of receptors, membrane proteins, and signaling pathways and metabolic pathways in animal (including human) cells can cause multifunctional cascade biological effects. However, there are no reports and applications of this kind of protein cross-border recognition, especially in animals and humans.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于：针对上述存在的问题，本发明提供HrpN型蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的制药中的应用。HrpN型蛋白作为一类富含多个线性和构象表位特殊结构的配体蛋白分子，能够跨界识别、激活、结合多种类型的动物的膜受体、膜蛋白、信息通路和代谢通路，HrpN型蛋白是一类具有特殊多个表位结构、全新功能、全新作用机制和全新应用前景的配体蛋白，它们诱导的多方向、多层次和多方面的生物学效应和功能。根据HrpN型蛋白的结构特点及其能够跨界识别、结合多种类型的动物的膜受体、膜蛋白，进而激活多个信息通路和代谢通路的功能特性，我们将其称之为HrpN型多拟表位配体蛋白(multi epitopic-like ligand proteins)。The purpose of the present invention is: in view of the above-mentioned problems, the present invention provides the application of HrpN-type protein in pharmaceutics that recognizes and activates multiple types of receptors and/or membrane proteins and their signaling pathways and causes cascade biological effects. HrpN-type proteins, as a class of ligand protein molecules with special structures rich in multiple linear and conformational epitopes, can recognize, activate, and bind membrane receptors, membrane proteins, information pathways and metabolic pathways across various types of animals. HrpN-type proteins are a class of ligand proteins with special multiple epitope structures, new functions, new mechanisms of action and new application prospects. They induce multi-directional, multi-level and multi-faceted biological effects and functions. According to the structural characteristics of HrpN-type protein and its ability to recognize and bind membrane receptors and membrane proteins of various types of animals across borders, thereby activating multiple information pathways and metabolic pathways, we call it HrpN-type polyprotein. Epitope-like ligand proteins (multi epitopic-like ligand proteins).
本发明采用的技术方案如下：The technical scheme adopted in the present invention is as follows:
HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用。Application of HrpN-type multi-epitope ligand proteins in food, cosmetics, health care products or pharmaceuticals that recognize and activate various types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects.
HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、消毒品、化妆品、保健品或药物中的应用。Application of HrpN-type multi-epitope ligand protein in identifying food, disinfectant, cosmetic, health care product or medicine that activates various types of receptors and/or membrane proteins and their signaling pathways and causes cascade biological effects.
HrpN型多拟表位配体蛋白含有一至多个疏水非极性氨基酸残基的结构基团或表位、含有一至多个极性不带电氨基酸残基的结构基团或表位、含有一至多个酰胺基极性不带电氨基酸残基的结构基团或表位、含有一至多个酸性带正电、碱性带负电氨基酸残基的结构基团或表位；疏水非极性氨基酸残基包括缬氨酸、亮氨酸、异亮氨酸、丙氨酸、苯丙氨酸、蛋氨酸残基；极性不带电氨基酸残基包括丝氨酸残基；酰胺基极性不带电氨基酸残基包括天冬酰胺、谷氨酰胺残基；酸性带正电、碱性带负电氨基酸残基包括天 冬酰酸、谷氨酸、赖氨酸、组氨酸、精氨酸残基；疏水非极性氨基酸残基，极性不带电氨基酸残基，酰胺基极性不带电氨基酸残基，酸性带正电、碱性带负电氨基酸残基在HrpN型多拟表位配体蛋白分子的全序列中占比62.3％-73.7％，在保守结构域中占比61％-74％，在α-螺旋结构中占比66.2％-79％；疏水非极性氨基酸残基的结构基团或表位，极性不带电氨基酸残基的结构基团或表位，酰胺基极性不带电氨基酸残基的结构基团或表位和酸性带正电、碱性带负电氨基酸残基的结构基团或表位通过氢键、离子键、疏水、非极性、极性、范德华力，实现配体和受体分子空间结构和电性的互补性、互作性以及特异识别、激活、结合，与多类型受体形成紧密结合面或复合物，能引起受体分子的构象、能量、电性和信息的变化，经信号传导和转导，引发放大的级联生物学效应。HrpN-type polymimetic epitope ligand proteins contain structural groups or epitopes of one or more hydrophobic non-polar amino acid residues, structural groups or epitopes containing one or more polar uncharged amino acid residues, contain one or more A structural group or epitope of an amide polar uncharged amino acid residue, a structural group or epitope containing one or more acidic positively charged, basic negatively charged amino acid residues; hydrophobic nonpolar amino acid residues include valine, leucine, isoleucine, alanine, phenylalanine, methionine residues; polar uncharged amino acid residues include serine residues; amide group polar uncharged amino acid residues include aspartic acid residues Amide, glutamine residues; acidic positively charged, basic negatively charged amino acid residues including asparagine, glutamic acid, lysine, histidine, arginine residues; hydrophobic non-polar amino acid residues Base, polar uncharged amino acid residues, amide polar uncharged amino acid residues, acidic positively charged, basic negatively charged amino acid residues accounted for 62.3 in the total sequence of HrpN-type multi-epitope ligand protein molecules %-73.7%, accounting for 61%-74% in conserved domains, and 66.2%-79% in α-helical structures; structural groups or epitopes of hydrophobic non-polar amino acid residues, with different polarity. Structural groups or epitopes of charged amino acid residues, structural groups or epitopes of amide polar uncharged amino acid residues and structural groups or epitopes of acidic positively charged, basic negatively charged amino acid residues through hydrogen Bonds, ionic bonds, hydrophobic, non-polar, polar, van der Waals forces, realize the complementarity, interaction, specific recognition, activation, and binding of ligands and receptors in terms of spatial structure and electrical properties, and form with multiple types of receptors Tight binding surfaces or complexes can cause changes in the conformation, energy, electrical properties and information of receptor molecules, and through signal transduction and transduction, lead to amplified cascade biological effects.
HrpN型多拟表位配体蛋白包括HrpNEcc、HrpNEca、HrpNEcb、HrpNEch、HrpNDaz、HrpNDada、HrpNDasp、HrpNad、HrpNDaf、HrpNEa、HrpNSam、HrpNBag、HrpNPas、HrpNEnt。HrpN-type multi-epitopic ligand proteins include HrpNEcc, HrpNEca, HrpNEcb, HrpNEch, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNEa, HrpNSam, HrpNBag, HrpNPas, HrpNEnt.
依据生物信息学分析，这类分子同源性很高，从60％到99％；According to bioinformatics analysis, these molecules are highly homologous, ranging from 60% to 99%;
HrpN同源性比较如下：The HrpN homology comparison is as follows:
HrpN同源性比较HrpN homology comparison
CLUSTAL O(1.2.4)multiple sequence alignmentCLUSTAL O(1.2.4) multiple sequence alignment
Figure PCTCN2021134714-appb-000001
Figure PCTCN2021134714-appb-000001
Figure PCTCN2021134714-appb-000002
Figure PCTCN2021134714-appb-000002
Figure PCTCN2021134714-appb-000003
Figure PCTCN2021134714-appb-000003
Figure PCTCN2021134714-appb-000004
Figure PCTCN2021134714-appb-000004
HrpN Phylogenetic Tree(进化系统树):HrpN Phylogenetic Tree:
Figure PCTCN2021134714-appb-000005
Figure PCTCN2021134714-appb-000005
HrpN型多拟表位配体蛋白富含多个线性和构象结构基团或表位，是指能与细胞膜受体、膜蛋白等识别结合的关键氨基酸残基组成的功能基团，这个功能基团是由以下氨基酸残基组成，包括能与受体识别、结合、激活的富含供质子型或接受质子型氨基酸残基；进一步的，含有一个至多个疏水非极性氨基酸残基的结构基团或表位，含有一个至多个酸性带正电、碱性带负电氨基酸残基的结构基团或表位，含有一个至多个酰胺基极性不带电氨基酸残基的结构基团或表位，含有一个至多个极性不带电氨基酸残基的结构基团或表位；进一步的，富含供质子型(蛋氨酸残基除外)或接受质子型(包括蛋氨酸残基)氨基酸残基：谷氨酸、天门冬酰酸、赖氨酸、组氨酸、蛋氨酸、丝氨酸、苏氨酸、酪氨酸、精氨酸，它们能与多类型受体蛋白对应氨基酸残基以氢键方式识别激活连接形成结合面或复合物；进一步的，疏水非极性氨基酸残基：缬氨酸、亮氨酸、异亮氨酸、丙氨酸、苯丙氨酸能以非极性疏水、范德华力与多类型受体形成紧密的结合面或复合物；酸性带正电、碱性带负电氨基酸残基：天冬酰酸、谷氨酸、赖氨酸、精氨酸能通过离子键与多类型受体形成紧密的结合面或复合物；酰胺基极性不带电氨基酸残基：天冬酰胺、谷氨酰胺的酰胺基能与受体的半胱氨酸识别区Pam3CSK4以较强氢键形成结合面或复合物；极性不带电氨基酸残基：丝氨酸通过极性以较强氢键与多类型受体形成紧密的结合面或复合物。HrpN-type multi-epitope ligand proteins are rich in multiple linear and conformational structural groups or epitopes, which refer to functional groups composed of key amino acid residues that can recognize and bind to cell membrane receptors, membrane proteins, etc. The group is composed of the following amino acid residues, including proton-donating or proton-accepting amino acid residues that can recognize, bind, and activate with receptors; further, structural groups containing one or more hydrophobic non-polar amino acid residues Groups or epitopes, structural groups or epitopes containing one or more acidic positively charged, basic negatively charged amino acid residues, structural groups or epitopes containing one or more amide polar uncharged amino acid residues, Structural groups or epitopes containing one or more polar uncharged amino acid residues; further, rich in proton-donating (except methionine residues) or proton-accepting (including methionine residues) amino acid residues: glutamic acid , asparagine, lysine, histidine, methionine, serine, threonine, tyrosine, arginine, which can recognize and activate connection with the corresponding amino acid residues of multiple types of receptor proteins by hydrogen bonding Binding surface or complex; further, hydrophobic non-polar amino acid residues: valine, leucine, isoleucine, alanine, phenylalanine can be combined with non-polar hydrophobic, van der Waals and multi-type Receptors form tight binding surfaces or complexes; acidic positively charged, basic negatively charged amino acid residues: aspartic acid, glutamic acid, lysine, arginine can form ionic bonds with multiple types of receptors Tight binding surfaces or complexes; amide polar uncharged amino acid residues: the amide groups of asparagine and glutamine can form binding surfaces or complexes with the cysteine recognition region Pam3CSK4 of the receptor through strong hydrogen bonds Compounds; polar uncharged amino acid residues: serine forms tight binding surfaces or complexes with multiple types of receptors through polar and strong hydrogen bonds.
进一步的，HrpNEcb多拟表位配体蛋白全序列有370个氨基酸残基，其中关键氨基酸残基226个：包括疏水非极性氨基酸残基94个，极性不带电氨基酸残基41个，酰胺基氨基酸残基44个，酸性带正电、碱性带负电氨基酸残基47个，关键氨基酸占全序列61％；HrpNEcb多拟表位配体蛋白保守结构区域有200个氨基酸残基：其中关键氨基酸残基138个，包括疏水非极性氨基酸残基51个，极性不带电氨基酸残基19个，酰胺基氨基酸残基28个，酸性带正电、碱性带负电氨基酸残基40个，关键氨基酸占保守结构域的69％；HrpNEcb蛋白α-螺旋区，其中有71个氨基酸残基，关键氨基酸残基52个：包括疏水非极性氨基酸残基27个，极性不带电氨基酸残基7个，酰胺基氨基酸残基8个，酸性带正电、碱性带负电氨基酸残基10个，关键氨基酸占α-螺旋结构的73％；进一步的，我们从多个欧文氏菌属及其他菌属细菌中筛选、克隆、制备了HrpNEcc、HrpNEca、HrpNEcb、HrpNEch、HrpNDaz、HrpNDada、HrpNDasp、HrpNad、HrpNDaf、HrpNEa、HrpNSam、HrpNBag、HrpNPas、HrpNEnt等超敏蛋白，依据生物信息学分析这些分子结构表明，它们具有类似于上述多表位HrpNEcb配体蛋白的相似结构特征、结构进化趋势、同源性较高以及具有多个构象表位和线性表位结构：含有一至多个疏水非极性氨基酸残基的结构基团或表位、含有一至多个极性不带电氨基酸残基的结构基团或表位、含有一至多个酰胺基极性不带电氨基酸残基的结构基团或表位、含有一至多个酸性带正电、碱性带负电氨基酸残基的结构基团或表位；进一步的，疏水非极性氨基酸残基：缬氨酸、亮氨酸、异亮氨酸、丙氨酸、苯丙氨酸、蛋氨酸，极性不带电氨基酸残基：丝氨酸，酰胺基极性不带电氨基酸残基：天冬酰胺、谷氨酰胺，酸性带正电、碱性带负电氨基酸残基：天冬酰酸、谷氨酸、赖氨酸、组氨酸、精氨酸；进一步的，以上所述关键氨基酸残基在这些蛋白分子的全序列中占比62.3％-73.7％，在保守结构域中占比61％-74％，在α-螺旋结构中占比66.2％-79％；进一步的，HrpNEcb多拟表位配体蛋白的上述关键氨基酸残基，又不限于这些氨基酸残基，能通过氢键、离子键、疏水、非极性、极性、范德华力，实现配体和受体分子空间结构和电性的互补性、互作性以及特异识别、激活、结合，与多类型受体形成紧密结合面或复合物，能引起受体分子的构象、能量、电性和信息的变化，经信号传导和转导，引发放大的级联生物学效应。Further, the full sequence of HrpNEcb multi-epitope ligand protein has 370 amino acid residues, of which 226 are key amino acid residues: including 94 hydrophobic non-polar amino acid residues, 41 polar uncharged amino acid residues, amide There are 44 base amino acid residues, 47 acidic positively charged and basic negatively charged amino acid residues, and key amino acids account for 61% of the entire sequence; HrpNEcb multi-epitope ligand protein conserved structural region has 200 amino acid residues: among which the key There are 138 amino acid residues, including 51 hydrophobic non-polar amino acid residues, 19 polar uncharged amino acid residues, 28 amide amino acid residues, 40 acidic positively charged and basic negatively charged amino acid residues, Key amino acids account for 69% of the conserved domain; HrpNEcb protein α-helix region, which has 71 amino acid residues, 52 key amino acid residues: including 27 hydrophobic non-polar amino acid residues, polar uncharged amino acid residues 7 amino acid residues, 8 amide amino acid residues, 10 acidic positively charged and basic negatively charged amino acid residues, and key amino acids account for 73% of the α-helix structure; HrpNEcc, HrpNEca, HrpNEcb, HrpNEch, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNEa, HrpNSam, HrpNBag, HrpNPas, HrpNEnt and other hypersensitive proteins were screened, cloned, and prepared from the genus Bacteria, and their molecular structures were analyzed based on bioinformatics showed that they have similar structural features, structural evolution trends, high homology, and multiple conformational epitopes and linear epitope structures similar to the above-mentioned multi-epitopic HrpNEcb ligand proteins: containing one or more hydrophobic non-polar amino acids A structural group or epitope of a residue, a structural group or epitope containing one or more polar uncharged amino acid residues, a structural group or epitope containing one or more amide polar uncharged amino acid residues, Structural groups or epitopes containing one or more acidic positively charged, basic negatively charged amino acid residues; further, hydrophobic non-polar amino acid residues: valine, leucine, isoleucine, alanine Acid, phenylalanine, methionine, polar uncharged amino acid residues: serine, amide group polar uncharged amino acid residues: asparagine, glutamine, acidic positively charged, basic negatively charged amino acid residues: Asparagine, glutamic acid, lysine, histidine, arginine; further, the above-mentioned key amino acid residues account for 62.3%-73.7% of the entire sequences of these protein molecules, which are in the conservative structure It accounts for 61%-74% in the domain and 66.2%-79% in the α-helical structure; further, the above-mentioned key amino acid residues of the HrpNEcb multi-epitope ligand protein are not limited to these amino acid residues, Through hydrogen bonding, ionic bonding, hydrophobicity, non-polarity, polarity, van der Waals force, it can realize the complementarity, interaction, specific recognition, activation, and binding of ligand and receptor molecular spatial structure and electricity. Combined, it forms a tight binding surface or complex with multiple types of receptors, which can cause changes in the conformation, energy, electrical properties and information of the receptor molecule, and through signal transduction and transduction, trigger an amplified cascade of biological effects.
进一步的，HrpNEch多拟表位配体蛋白全序列有339个氨基酸残基，其中关键氨基酸残基236个：包括疏水非极性氨基酸残基106个，极性不带电氨基酸残基40个，酰胺基氨基酸残基42个，酸性带正电、碱性带负电氨基酸残基48个，关键氨基酸占全序列69％；HrpNEch保守结构区域，蛋白质序列有 326个氨基酸残基，关键氨基酸残基226个，包括疏水非极性氨基酸残基99个，极性不带电氨基酸残基40个，酰胺基氨基酸残基40个，酸性带正电、碱性带负电氨基酸残基47个，具有的氨基酸在保守结构区域中，关键氨基酸占69％；HrpNEchα-螺旋结构区域，有7个α-螺旋，有2个β-折叠以及7个IDPs-结构区域，其中，α-螺旋区，有100个氨基酸残基，关键氨基酸残基71个：包括疏水非极性氨基酸残基35个，极性不带电氨基酸残基12个，酰胺基氨基酸残基13个，酸性带正电、碱性带负电氨基酸残基11个，具有的氨基酸在α-螺旋结构中，关键氨基酸占71％；进一步的，发明人从多个欧文氏菌属及其他菌属细菌中筛选、克隆、制备了HrpNEcc、HrpNEca、HrpNEcb、HrpNEch、HrpNDaz、HrpNDada、HrpNDasp、HrpNad、HrpNDaf、HrpNEa、HrpNSam、HrpNBag、HrpNPas、HrpNEnt等超敏蛋白，依据生物信息学分析这些分子结构表明，它们具有类似于上述多表位HrpNEch配体蛋白的相似结构特征、结构进化趋势、同源性较高以及具有多个构象表位和线性表位结构：含有一至多个疏水非极性氨基酸残基的结构基团或表位、含有一至多个极性不带电氨基酸残基的结构基团或表位、含有一至多个酰胺基极性不带电氨基酸残基的结构基团或表位、含有一至多个酸性带正电、碱性带负电氨基酸残基的结构基团或表位；进一步的，疏水非极性氨基酸残基：缬氨酸、亮氨酸、异亮氨酸、丙氨酸、苯丙氨酸、蛋氨酸，极性不带电氨基酸残基：丝氨酸，酰胺基极性不带电氨基酸残基：天冬酰胺、谷氨酰胺，酸性带正电、碱性带负电氨基酸残基：天冬酰酸、谷氨酸、赖氨酸、组氨酸、精氨酸；进一步的，以上所述关键氨基酸残基在这些蛋白分子的全序列中占比62.3％-73.7％，在保守结构域中占比61％-74％，在α-螺旋结构中占比66.2％-79％；进一步的，HrpNEch多拟表位配体蛋白的上述关键氨基酸残基，又不限于这些氨基酸残基，能通过氢键、离子键、疏水、非极性、极性、范德华力，实现配体和受体分子空间结构和电性的互补性、互作性以及特异识别、激活、结合，与多类型受体形成紧密结合面或复合物，能引起受体分子的构象、能量、电性和信息的变化，经信号传导和转导，引发放大的级联生物学效应。Further, the full sequence of HrpNEch multi-epitope ligand protein has 339 amino acid residues, including 236 key amino acid residues: including 106 hydrophobic non-polar amino acid residues, 40 polar uncharged amino acid residues, amide There are 42 base amino acid residues, 48 acidic positively charged and basic negatively charged amino acid residues, and key amino acids account for 69% of the entire sequence; HrpNEch conserved structural region, the protein sequence has 326 amino acid residues, and 226 key amino acid residues , including 99 hydrophobic non-polar amino acid residues, 40 polar uncharged amino acid residues, 40 amide amino acid residues, 47 acidic positively charged, basic negatively charged amino acid residues, with amino acids in the conservative In the structural region, key amino acids account for 69%; the HrpNEchα-helix structure region has 7 α-helices, 2 β-sheets and 7 IDPs-structure regions, of which the α-helix region has 100 amino acid residues , 71 key amino acid residues: including 35 hydrophobic non-polar amino acid residues, 12 polar uncharged amino acid residues, 13 amide amino acid residues, 11 acidic positively charged, basic negatively charged amino acid residues 71% of the amino acids are in the α-helix structure; further, the inventors screened, cloned and prepared HrpNEcc, HrpNEca, HrpNEcb, HrpNEch, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNEa, HrpNSam, HrpNBag, HrpNPas, HrpNEnt and other hypersensitive proteins, and bioinformatics analysis of these molecular structures shows that they have similar structural features to the above-mentioned multi-epitopic HrpNEch ligand proteins , structural evolution trend, high homology, and multiple conformational epitopes and linear epitope structures: structural groups or epitopes containing one or more hydrophobic non-polar amino acid residues, containing one or more polar uncharged Structural groups or epitopes of amino acid residues, structural groups or epitopes containing one or more amide-group polar uncharged amino acid residues, structures containing one or more acidic positively charged, basic negatively charged amino acid residues group or epitope; further, hydrophobic non-polar amino acid residues: valine, leucine, isoleucine, alanine, phenylalanine, methionine, polar uncharged amino acid residues: serine , amide polar uncharged amino acid residues: asparagine, glutamine, acidic positively charged, basic negatively charged amino acid residues: asparagine, glutamic acid, lysine, histidine, sperm amino acid; further, the above-mentioned key amino acid residues account for 62.3%-73.7% of the full sequences of these protein molecules, 61%-74% of the conserved domains, and α-helical structures. 66.2%-79%; further, the above-mentioned key amino acid residues of the HrpNEch multi-epitope ligand protein, but not limited to these amino acid residues, can pass hydrogen Bonds, ionic bonds, hydrophobic, non-polar, polar, van der Waals forces, realize the complementarity, interaction, specific recognition, activation, and binding of ligands and receptors in terms of spatial structure and electrical properties, and form with multiple types of receptors Tight binding surfaces or complexes can cause changes in the conformation, energy, electrical properties and information of receptor molecules, and through signal transduction and transduction, lead to amplified cascade biological effects.
多功能级联生物学效应是指能诱导不同器官、组织的细胞组分、分子功能和生物学过程三个层次相关功能基因群显著表达差异，包括细胞组分(包括细胞、细胞结、细胞部分、细胞外基质、细胞外基质成分、细胞外区域、细胞外区域部分、大分子复合物、膜、膜部分、膜封闭腔、细胞器、细胞器的部分、超分子纤维、突触、突触部分、抗氧化活性等)，分子功能(包括绑定、催化活性、化学引诱物的活动、化学排斥物活动、电子载体活动、金属伴侣蛋白活动、分子功能监管机构、活动分子传感器、核酸结合转录因子的活性、信号传感器活动、结构分子活动、转录因子活性蛋白质结合、运输活动等)，生物过程(包括行为、生物粘附、生物调节、细胞聚集、细胞死亡、细胞成分组织或生物发生、细胞过程、解毒、发展的过程、增长、免疫系统的过程、本地化、运动、代谢过程、多生物过程、多细胞生物的过程、生物过程负调控、生物过程的正调控、突触前过程涉及突触传递、生物过程调节、繁殖、生殖过程、刺激反应、有节奏的过程、信号、单一生物过程等)的相关功能基因群表达差异发生显著性变化。Multifunctional cascade biological effects refer to the significant differences in the expression of related functional gene groups at three levels of cellular components, molecular functions and biological processes in different organs and tissues, including cellular components (including cells, cell nodes, and cell parts). , extracellular matrix, extracellular matrix components, extracellular region, extracellular region part, macromolecular complex, membrane, membrane part, membrane-enclosed cavity, organelle, organelle part, supramolecular fiber, synapse, synaptic part, Antioxidant activity, etc.), molecular functions (including binding, catalytic activity, chemoattractant activity, chemorepellent activity, electron carrier activity, metal chaperone protein activity, molecular function regulators, active molecular sensors, nucleic acid binding transcription factors) activity, signal sensor activity, structural molecular activity, transcription factor activity protein binding, transport 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, movement, metabolic processes, multibiological processes, processes of multicellular organisms, negative regulation of biological processes, positive regulation of biological processes, presynaptic processes involving synaptic transmission , biological process regulation, reproduction, reproductive process, stimulus response, rhythmic process, signal, single biological process, etc.) related functional gene group expression differences changed significantly.
HrpNEcb多拟表位配体蛋白的氨基酸序列如SEQ ID NO:1所示；HrpNEch多拟表位配体蛋白的氨基酸序列如SEQ ID NO:2所示。The amino acid sequence of the HrpNEcb multi-mimetic epitope ligand protein is shown in SEQ ID NO: 1; the amino acid sequence of the HrpNEch multi-mimetic epitope ligand protein is shown in SEQ ID NO: 2.
优选的，HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，识别结合的多类受体包括LRRC1515-亮氨酸的重复膜蛋白受体、HLA-A主要组织相容性复合体，I,A类受体、LGALS3BP半乳糖3结合蛋白(受体)、LAMP2溶酶体关联膜蛋白2受体、GNB2G鸟嘌呤核苷酸结合蛋白亚单位Beta 2受体中的一种或多种。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein, and the multiple types of receptors that recognize and bind include LRRC1515-leucine repeat membrane protein receptor, HLA-A major histocompatibility Complex, one of class I, A receptors, LGALS3BP galactose 3 binding protein (receptor), LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein subunit Beta 2 receptor or more.
优选的，HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，识别结合的小鼠肝培养细胞多类受体包括GNG12鸟嘌呤核苷酸结合蛋白γ-12受体、ANXA5膜联蛋白A5受体、ANXA2膜联蛋白A2受体、ANXA1膜联蛋白A1受体、IGHG2免疫球蛋白重常数γ2受体、IGHM免疫球蛋白重常数Mu受 体、CACNA1S钙电压门控通道亚单位α1S受体、ZNF185锌指蛋白185受体以及HLA-A主要组织相容性复合体，I，A类受体、LAMP2溶酶体关联膜蛋白2受体、GNB2G鸟嘌呤核苷酸结合蛋白亚单位β2受体、KTN1驱动结合蛋白1受体中的一种或多种。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, and the multi-type receptors of mouse liver culture cells that recognize and bind include GNG12 guanine nucleotide binding protein γ-12 receptor, ANXA5 Annexin A5 receptor, ANXA2 annexin A2 receptor, ANXA1 annexin A1 receptor, IGHG2 immunoglobulin weight constant γ2 receptor, IGHM immunoglobulin weight constant Mu receptor, CACNA1S calcium voltage-gated channel sub Unit α1S receptor, ZNF185 zinc finger protein 185 receptor and HLA-A major histocompatibility complex, I, A receptors, LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein One or more of subunit β2 receptor, KTN1 kinesin 1 receptor.
优选的，HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，识别结合的小鼠肝培养细胞膜蛋白包括DSG4桥粒芯蛋白、ANXA4膜联蛋白A4、CAPRIN1细胞周期蛋白、1UTRN肌营养不良蛋白、pinin桥粒蛋白、VAMP关联蛋白A、VCL黏着斑蛋白、Ezrin埃兹上皮型钙黏附素、PKP3血小板亲和蛋白3、TM9SF2跨膜9超家族成员2、NAALAD2N乙酰化α连接酸性二肽酶2中的一种或多种。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein, and the recognized and bound mouse liver culture cell membrane proteins include DSG4 desmocollin, ANXA4 annexin A4, CAPRIN1 cyclin, 1UTRN Dystrophin, pinin desmosomal protein, VAMP-associated protein A, VCL focal adhesion protein, Ezrin epithelial-type cadherin, PKP3 platelet avidin 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD2N acetylated alpha linkage One or more of acid dipeptidase 2.
优选的，HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，识别结合的小鼠肝培养细胞膜蛋白包括DSC3桥粒胶蛋白、ANXA8/ANXA8L1膜联蛋白A8/膜联蛋白A8相似蛋白1、EVPL外被斑蛋白、POF1B肌动蛋白结合蛋白卵巢早衰1B、CTNNA1连环蛋白、TGM1转谷氨酰胺酶1、BAIAP2BAI1关联蛋白2、RAB29RAS致癌基因家族成员、CLDN19靠停蛋白19、STXBP2突触融合蛋白结合蛋白2、VAMP囊泡相关膜蛋白关联蛋白A、VCL黏着斑蛋白、Ezrin埃兹上皮型钙黏附素、PKP3血小板亲和蛋白3、NAALAD2N乙酰化α连接酸性二肽酶2、PKP1血小板亲和蛋白1、SPRR1A富含脯氨酸小蛋白1A中的一种或多种。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, and the recognized and bound mouse liver culture cell membrane proteins include DSC3 desmocollin, ANXA8/ANXA8L1 annexin A8/annexin A8 Similar protein 1, EVPL coat protein, POF1B actin binding protein premature ovarian failure 1B, CTNNA1 catenin, TGM1 transglutaminase 1, BAIAP2BAI1 associated protein 2, RAB29RAS oncogene family member, CLDN19 docking protein 19, STXBP2 Syntaxin-binding protein 2, VAMP vesicle-associated membrane protein-associated protein A, VCL focal adhesion protein, Ezrin epithelial cadherin, PKP3 platelet avidin 3, NAALAD2N acetylated α-linked acid dipeptidase 2, One or more of PKP1 platelet affinity protein 1, SPRR1A small proline-rich protein 1A.
优选的，HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，其识别结合的小鼠肝培养细胞膜蛋白参与的信号通路包括hsa04152:AMPK信号通路、hsa03460:范可尼贫血贫血通路、hsa03320:PPAR信号通路、hsa04071:鞘脂类信号通路、hsa04014:Ras信号通路、hsa04151:PI3K-Akt信号通路、hsa04310:Wnt信号通路、hsa04062:趋化因子信号通路、hsa04015:Rap1信号通路、hsa04024:阵营信号通路、hsa04915:雌激素信号通路、hsa04910:胰岛素信号通路、hsa04390:河马信号通路中的一种或多种。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein, and the signal pathways involved in the recognition and binding of mouse liver culture cell membrane proteins include hsa04152: AMPK signal pathway, hsa03460: Fanconi anemia anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: Chemokine signaling pathway, hsa04015: Rap1 signaling pathway, One or more of hsa04024: camp signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, hsa04390: hippo signaling pathway.
优选的，HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，其识别结合的小鼠肝培养细胞膜蛋白参与的信号通路包括22条信号通路hsa03320:PPAR信号通路、hsa05120:幽门螺杆菌感染的上皮细胞信号转导、hsa04071:鞘脂类信号通路、hsa04014:Ras信号通路、hsa04151:PI3K-Akt信号通路、hsa04070:磷脂酰肌醇信号系统、hsa04010:MAPK信号通路、hsa04310:Wnt信号通路、hsa04062:趋化因子信号通路、hsa04015:Rap1信号通路、hsa04024:阵营信号通路、hsa04915:雌激素信号通路、hsa04910:胰岛素信号通路、hsa04390:河马信号通路、hsa04922:胰高糖素信号通路、hsa04912:促性腺激素信号通路、hsa04022:cGMP-PKG信号通路、hsa04921:催产素信号通路、hsa04722:生成信号通路、hsa04723:逆行神经信号、hsa04066:HIF-1信号通路、hsa04020:钙信号通路的一种或多种。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, and the signal pathways involved in the recognition and binding of mouse liver culture cell membrane proteins include 22 signal pathways hsa03320: PPAR signal pathway, hsa05120: pylorus Epithelial cell signal transduction in Helicobacter infection, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04070: phosphatidylinositol signaling system, hsa04010: MAPK signaling pathway, hsa04310: Wnt Signaling Pathways, hsa04062: Chemokine Signaling Pathway, hsa04015: Rap1 Signaling Pathway, hsa04024: CAMP Signaling Pathway, hsa04915: Estrogen Signaling Pathway, hsa04910: Insulin Signaling Pathway, hsa04390: Hippo Signaling Pathway, hsa04922: Glucagon Signaling Pathway , hsa04912: gonadotropin signaling pathway, hsa04022: cGMP-PKG signaling pathway, hsa04921: oxytocin signaling pathway, hsa04722: generative signaling pathway, hsa04723: retrograde neural signaling, hsa04066: HIF-1 signaling pathway, hsa04020: calcium signaling pathway one or more.
优选的，信号通路包括代谢信号通路，代谢信号通路包括抗病毒、抗细菌、抗异物、抗炎性代谢通路，包括重要神经疾病代谢通道；包括核酸、蛋白质、氨基酸、糖、脂肪代谢通路；包括细胞联结、神经连结、血管、内分泌、生殖系统代谢通路。Preferably, the signaling pathways include metabolic signaling pathways, and the metabolic signaling pathways include antiviral, antibacterial, anti-foreign body, and anti-inflammatory metabolic pathways, including important neurological disease metabolic pathways; including nucleic acid, protein, amino acid, sugar, and fat metabolic pathways; including Cell junctions, neural junctions, blood vessels, endocrine, reproductive system metabolic pathways.
优选的，HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，其识别结合的小鼠肝培养细胞膜蛋白参与的抗病毒、抗细菌、抗异物、抗炎性代谢通路包括：hsa04144内吞作用、hsa04145吞噬体、hsa04142溶酶体、hsa01130:抗生素的生物合成、hsa05131:志贺氏菌病、hsa04612:抗原处理和呈递、hsa05130:致病性大肠杆菌感染、hsa05100:上皮细胞的细菌侵袭、hsa05132:沙门氏菌感染、hsa05169:巴尔病毒感染、hsa05168:单纯疱疹病毒1感染、hsa05203:病毒致癌作用、hsa05166:HTLV-I感染、hsa05164:甲型流感、hsa05134:军团病、hsa05160:丙型肝炎、hsa05162:麻疹、hsa05133:百日咳、hsa05322:系统性红斑狼疮、hsa04670:白细胞经上皮的迁移、hsa05146:阿米巴病、hsa05142:南美锥虫病、hsa05200:在癌症中通路；所述识别结合的重要神经疾病代谢通道：hsa05012:帕金森病、hsa05016:亨廷顿氏舞蹈症、hsa05010:阿尔茨海默氏症；所述识别结合的核酸、蛋白质、氨基酸、糖、脂肪代谢通路：hsa03420:核苷酸切除修复、hsa00970: 氨酰生物合成、hsa03430:错配修复、hsa01210:2-氧代羧酸代谢、hsa03440:同源重组、hsa04360:轴突引导、hsa00051:果糖和甘露糖代谢、hsa00565:醚脂质代谢、hsa00510:N-多糖生物合成以及hsa04110:细胞周期、hsa03030:DNA复制、hsa03013:RNA运输、hsa03018:RNA降解、hsa03040:剪接体、hsa03010:核糖体、hsa04141:内质网蛋白加工、hsa04810:肌动蛋白骨架的调控、hsa03050:蛋白酶体、hsa01230:氨基酸生物合成、hsa00190:氧化磷酸化、hsa04932:非酒精性脂肪肝(NAFLD)、hsa00020:柠檬酸循环、hsa00564:甘油磷脂新陈代谢、hsa03008:真核生物核糖体的生物发生、hsa03015:mRNA监测通路、hsa01200:碳代谢、hsa00520:氨基糖和核苷酸糖代谢、hsa05034:酗酒、hsa04120:泛素介导的蛋白水解作用、hsa05205:蛋白聚糖在癌症、hsa05206:小分子核糖核酸在癌症；所述识别结合的细胞联结、神经连结、血管、内分泌、生殖系统代谢通路：hsa04723:逆行神经信号、hsa04726:血清素激活的突触、hsa00900:萜类化合物生物合成支柱、hsa04520:黏着结、hsa05032:吗啡上瘾以及hsa04510:粘着斑、hsa04724:谷氨酸能的突触、hsa04530:紧密连接、hsa00830:视黄醇新陈代谢、hsa04114:卵母细胞减数分裂、hsa04728:多巴胺能神经突触、hsa00100:类固醇生物合成、hsa04261:心肌细胞的肾上腺素能信号、hsa04727:神经元突触、hsa04725:胆碱能突触、hsa04540:缝隙连接、hsa04971:胃酸分泌、hsa04713:昼夜夹带、hsa04931:胰岛素抵抗。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein, and the anti-viral, anti-bacterial, anti-foreign body, and anti-inflammatory metabolic pathways involved in recognizing bound mouse liver culture cell membrane proteins include: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa01130: biosynthesis of antibiotics, hsa05131: shigellosis, hsa04612: antigen processing and presentation, hsa05130: pathogenic E. coli infection, hsa05100: epithelial cell Bacterial invasion, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05168: Herpes simplex virus 1 infection, hsa05203: Viral carcinogenesis, hsa05166: HTLV-I infection, hsa05164: Influenza A, hsa05134: Legionnaires' disease, hsa05160: Type C hepatitis, hsa05162: measles, hsa05133: pertussis, hsa05322: systemic lupus erythematosus, hsa04670: transepithelial migration of leukocytes, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: pathway in cancer; Important neurological disease metabolic pathways: hsa05012: Parkinson's disease, hsa05016: Huntington's disease, hsa05010: Alzheimer's disease; The identification of binding nucleic acid, protein, amino acid, sugar, fat metabolic pathway: hsa03420: Nucleoside acid excision repair, hsa00970: aminoacyl biosynthesis, hsa03430: mismatch repair, hsa01210: 2-oxocarboxylic acid metabolism, hsa03440: homologous recombination, hsa04360: axon guidance, hsa00051: fructose and mannose metabolism, hsa00565: ether Lipid metabolism, hsa00510: N-glycan biosynthesis and hsa04110: cell cycle, hsa03030: DNA replication, hsa03013: RNA transport, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome, hsa04141: endoplasmic reticulum protein processing, hsa04810: regulation of the actin backbone, hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa04932: nonalcoholic fatty liver disease (NAFLD), hsa00020: citric acid cycle, hsa00564: glycerophospholipid metabolism, hsa03008 : Biogenesis of the eukaryotic ribosome, hsa03015: mRNA surveillance pathways, hsa01200: carbon metabolism, hsa00520: amino sugar and nucleotide sugar metabolism, hsa05034: alcoholism, hsa04120: ubiquitin-mediated proteolysis , hsa05205: proteoglycans in cancer, hsa05206: small ribonucleic acid in cancer; the identification of binding cell junctions, neural junctions, blood vessels, endocrine, reproductive system metabolic pathways: hsa04723: retrograde neural signaling, hsa04726: serotonin-activated synapses, hsa00900: terpenoid biosynthetic pillars, hsa04520: adhesion junctions, hsa05032: morphine addiction and hsa04510: focal adhesions, hsa04724: glutamatergic synapses, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114 : oocyte meiosis, hsa04728: dopaminergic synapses, hsa00100: steroid biosynthesis, hsa04261: adrenergic signaling in cardiomyocytes, hsa04727: neuronal synapses, hsa04725: cholinergic synapses, hsa04540: slits Connection, hsa04971: gastric acid secretion, hsa04713: diurnal entrainment, hsa04931: insulin resistance.
优选的，HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，其识别结合的小鼠肝培养细胞膜蛋白参与的29条抗病毒、抗细菌、抗异物、抗炎性相关代谢通路包括：hsa04144:内吞作用、hsa04145:吞噬体、hsa04142:溶酶体、hsa04666:Fc-r介导的吞噬作用、hsa01130:抗生素的生物合成、hsa05131:志贺氏菌病、hsa04612:抗原处理和呈递、hsa05130:致病性大肠杆菌感染、hsa05100:上皮细胞的细菌侵袭、hsa05132:沙门氏菌感染、hsa05169:巴尔病毒感染、hsa05203:病毒致癌作用、hsa05134:军团病、hsa05160:丙型肝炎、hsa05162:麻疹、hsa05133:百日咳、hsa05322:系统性红斑狼疮、hsa04670:白细胞跨内皮的迁移、hsa05152:肺结核、hsa05150:金黄色葡萄球菌感染、hsa05146:阿米巴病、hsa05142:南美锥虫病、hsa05200:在癌症中通路、hsa05143:非洲锥虫病、hsa04750:TRP通道的炎症介质调节、hsa04916:杀菌作用、hsa05230:癌症中的中心碳代谢、hsa05214:神经胶质瘤、hsa05212:胰腺癌；包括识别结合的3条重要神经疾病代谢通道：hsa05010:阿尔茨海默氏症、hsa05012:帕金森病、hsa05016:亨廷顿氏舞蹈症；包括识别结合的39条核酸、蛋白质、氨基酸、糖、脂肪代谢相关通路：hsa03013:RNA运输、hsa03018:RNA降解、hsa03040:剪接体、hsa03010:核糖体、hsa04141:内质网蛋白加工、hsa04810:肌动蛋白骨架的调hsa03050:蛋白酶体、hsa01230:氨基酸生物合成、hsa00190:氧化磷酸化、hsa00230:嘌呤代谢、hsa04932:非酒精性脂肪肝、hsa00020:柠檬酸循环、hsa03008:真核生物核糖体的生物发生、hsa00240:嘧啶代谢、hsa00650:Butanoate新陈代谢、hsa01200:碳代谢、hsa00520:氨基糖和核苷酸糖代谢、hsa05034:酗酒、hsa00071:脂肪酸降解、hsa04120:泛素介导的蛋白水解作用、hsa05205:蛋白聚糖在癌症、hsa05206:小分子核糖核酸在癌症、hsa00410:hsa00410:丙胺酸新陈代谢、hsa00340:组氨酸代谢、hsa00910:氮代谢、hsa00250:丙氨酸、天冬氨酸、谷氨酸代谢、hsa00350:酪氨酸代谢、hsa04726:血清素激活的突触、hsa00900:萜类化合物生物合成支柱、hsa04610:补体和凝血级联、hsa00330:精氨酸和脯氨酸代谢、hsa04520:粘附的结、hsa00860:卟啉与叶绿素代谢、hsa00010:糖酵解和糖质新生、hsa00982:药物代谢-细胞色素P450、hsa00980:细胞色素P450对外源性药物的代谢作用、hsa04962:加压素调节水重吸收、hsa00983:药物代谢-其他酶；以及包括识别结合的34条细胞联结、神经连结、血管、内分泌、生殖系统代谢通路：hsa04510:粘着斑、hsa 04724:谷氨酸能的突触、hsa04530:紧密连接、hsa00830:视黄醇新陈代谢、hsa04114:卵母细胞减数分裂、hsa04728:多巴胺神经突触、hsa00140:类固醇激素生物合成、hsa04261:心肌细胞的肾上腺素能信号、hsa04727:γ-氨基丁酸能的突触、hsa04725:胆碱能突触、hsa04540:缝隙连接、hsa04971:胃酸分泌、hsa04713:昼夜夹带、hsa04931:胰岛素抵抗、hsa05031:安非他命上瘾、hsa04924:肾素分泌、hsa04925:醛固酮的合成与分泌、hsa00590:花生四烯酸代谢、hsa04270: 血管平滑肌收缩、hsa00760:烟酸和烟酰胺代谢、hsa04740:嗅觉传导、hsa04260:心肌收缩、hsa04720:长期势差现象、hsa04744:光传导、hsa04966:收集管道酸性分泌物、hsa05412:致心律失常性右心室心肌病、hsa05410:肥厚性心肌病、hsa04146:过氧物酶体、hsa05414:扩张型心肌病、hsa04970:唾液分泌、hsa04611:血小板激活、hsa05204:化学致癌作用、hsa04721:突触囊泡循环。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, which recognizes 29 anti-virus, anti-bacterial, anti-foreign body, anti-inflammatory related metabolisms involved in the bound mouse liver culture cell membrane protein Pathways include: hsa04144: endocytosis, hsa04145: phagosome, hsa04142: lysosome, hsa04666: Fc-r-mediated phagocytosis, hsa01130: biosynthesis of antibiotics, hsa05131: shigellosis, hsa04612: antigen processing and presentation, hsa05130: Pathogenic E. coli infection, hsa05100: Bacterial invasion of epithelial cells, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05203: Viral carcinogenesis, hsa05134: Legionnaires’ disease, hsa05160: Hepatitis C, hsa05162: measles, hsa05133: pertussis, hsa05322: systemic lupus erythematosus, hsa04670: transendothelial migration of leukocytes, hsa05152: tuberculosis, hsa05150: staphylococcus aureus infection, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: in Pathways in Cancer, hsa05143: African trypanosomiasis, hsa04750: Inflammatory mediator regulation of TRP channels, hsa04916: Bactericidal effects, hsa05230: Central carbon metabolism in cancer, hsa05214: Glioma, hsa05212: Pancreatic cancer; includes recognition of bound 3 important metabolic pathways for neurological diseases: hsa05010: Alzheimer's disease, hsa05012: Parkinson's disease, hsa05016: Huntington's disease; including 39 nucleic acid, protein, amino acid, sugar, and fat metabolism-related pathways that recognize and bind: hsa03013 : RNA trafficking, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome, hsa04141: endoplasmic reticulum protein processing, hsa04810: regulation of the actin backbone hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphate Chemistry, hsa00230: Purine metabolism, hsa04932: Nonalcoholic fatty liver disease, hsa00020: Citric acid cycle, hsa03008: Biogenesis of eukaryotic ribosomes, hsa00240: Pyrimidine metabolism, hsa00650: Butanoate metabolism, hsa01200: Carbon metabolism, hsa00520: Amino Sugar and Nucleotide Sugar Metabolism, hsa05034: Alcoholism, hsa00071: Fatty Acid Degradation, hsa04120: Ubiquitin-Mediated Proteolysis, hsa05205: Proteoglycans in Cancer, hsa05206: Small RNAs in Cancer, h sa00410: hsa00410: Alanine metabolism, hsa00340: Histidine metabolism, hsa00910: Nitrogen metabolism, hsa00250: Alanine, aspartate, glutamate metabolism, hsa00350: Tyrosine metabolism, hsa04726: Serotonin-activated touch, hsa00900: terpenoid biosynthetic pillar, hsa04610: complement and coagulation cascade, hsa00330: arginine and proline metabolism, hsa04520: adherent knot, hsa00860: porphyrin and chlorophyll metabolism, hsa00010: glycolysis and Glycogenesis, hsa00982: Drug Metabolism - Cytochrome P450, hsa00980: Metabolism of Cytochrome P450 on Exogenous Drugs, hsa04962: Vasopressin Regulates Water Reabsorption, hsa00983: Drug Metabolism - Other Enzymes; 34 cell-junction, neural-junction, vascular, endocrine, reproductive system metabolic pathways: hsa04510: focal adhesions, hsa 04724: glutamatergic synapses, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114: oocytes meiosis, hsa04728: dopaminergic synapses, hsa00140: steroid hormone biosynthesis, hsa04261: adrenergic signaling in cardiomyocytes, hsa04727: GABAergic synapses, hsa04725: cholinergic synapses, hsa04540: Gap junction, hsa04971: gastric acid secretion, hsa04713: diurnal entrainment, hsa04931: insulin resistance, hsa05031: amphetamine addiction, hsa04924: renin secretion, hsa04925: synthesis and secretion of aldosterone, hsa00590: arachidonic acid metabolism, hsa04270: vascular smooth muscle contraction , hsa00760: niacin and nicotinamide metabolism, hsa04740: olfactory conduction, hsa04260: myocardial contraction, hsa04720: long-term potential difference phenomenon, hsa04744: phototransduction, hsa04966: collection duct acid secretion, hsa05412: arrhythmogenic right ventricular cardiomyopathy , hsa05410: hypertrophic cardiomyopathy, hsa04146: peroxisomes, hsa05414: dilated cardiomyopathy, hsa04970: salivation, hsa04611: platelet activation, hsa05204: chemical carcinogenesis, hsa04721: synaptic vesicle recycling.
多功能级联生物学效应包括能诱导不同器官、组织的细胞组分、分子功能和生物学过程三个层次相关功能基因群显著表达差异，包括细胞组分(包括细胞、细胞结、细胞部分、细胞外基质、细胞外基质成分、细胞外区域、细胞外区域部分、大分子复合物、膜、膜部分、膜封闭腔、细胞器、细胞器的部分、超分子纤维、突触、突触部分、抗氧化活性等)，分子功能(包括绑定、催化活性、化学引诱物的活动、化学排斥物活动、电子载体活动、金属伴侣蛋白活动、分子功能监管机构、活动分子传感器、核酸结合转录因子的活性、信号传感器活动、结构分子活动、转录因子活性蛋白质结合、运输活动等)，生物过程(包括行为、生物粘附、生物调节、细胞聚集、细胞死亡、细胞成分组织或生物发生、细胞过程、解毒、发展的过程、增长、免疫系统的过程、本地化、运动、代谢过程、多生物过程、多细胞生物的过程、生物过程负调控、生物过程的正调控、突触前过程涉及突触传递、生物过程调节、繁殖、生殖过程、刺激反应、有节奏的过程、信号、单一生物过程等)的相关功能基因群表达差异发生显著性变化。The biological effects of multifunctional cascades include significant differences in the expression of related functional gene groups at three levels of cellular components, molecular functions and biological processes that can induce different organs and tissues, including cellular components (including cells, cell nodes, cell parts, extracellular matrix, extracellular matrix components, extracellular region, extracellular region part, macromolecular complex, membrane, membrane part, membrane-enclosed cavity, organelle, part of organelle, supramolecular fiber, synapse, synaptic part, anti- oxidative activity, etc.), molecular functions (including binding, catalytic activity, chemoattractant activity, chemorepellent activity, electron carrier activity, metal chaperone protein activity, molecular function regulators, active molecular sensors, nucleic acid binding transcription factor activity , signal sensor activity, structural molecular activity, transcription factor activity protein binding, transport 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, movement, metabolic processes, multibiological processes, processes in multicellular organisms, negative regulation of biological processes, positive regulation of biological processes, presynaptic processes involving synaptic transmission, There were significant changes in the expression differences of related functional gene groups related to biological process regulation, reproduction, reproductive process, stimulus response, rhythmic process, signal, single biological process, etc.
HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，级联生物学效应包括细胞过程(Cellular Processes)、环境信息处理(Environmental Information Processing)、遗传信息处理(Genetic Information Processing)、新陈代谢(Metabolism)和生物体系统(Organismal Systems)等功能途径；进一步地，①细胞过程(Cellular Processes)：HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了运输和分解代谢，细胞群体，细胞活性，细胞生长与死亡等细胞过程；②环境信息处理(Environmental Information Processing):HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了信号分子与相互作用，信号转导，膜运输等环境信息处理过程；③遗传信息处理(Genetic Information Processing):HrpNEcb蛋白诱导的多个差异表达基因参与了翻译，复制和修复，折叠、分类和降解等生物过程；④新陈代谢(Metabolism):HrpNEcb蛋白诱导的多个差异表达基因参与了生物降解和代谢，核苷酸代谢，其他氨基酸的代谢，代谢的辅助因子和维生素，脂质代谢，糖的生物合成和代谢，全局和概览地图，能量代谢，碳水化合物代谢及氨基酸代谢等代谢过程；⑤生物体系统(Organismal Systems):HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了感觉系统，神经系统，免疫系统，排泄系统，环境适应，内分泌系统，消化系统，发育循环系统等生物过程。The HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein, and the cascade biological effects include Cellular Processes, Environmental Information Processing, Genetic Information Processing, Metabolism and Organismal Systems and other functional pathways; further, ① Cellular Processes: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand proteins are involved in transport and catabolism, and cellular processes Population, cell activity, cell growth and death and other cellular processes; ②Environmental Information Processing: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand protein are involved in signaling molecules and interactions, signal transduction, Environmental information processing processes such as membrane transport; ③ Genetic Information Processing: Multiple differentially expressed genes induced by HrpNEcb protein participate in biological processes such as translation, replication and repair, folding, classification and degradation; ④ Metabolism: HrpNEcb protein-induced multiple differentially expressed genes involved in biodegradation and metabolism, nucleotide metabolism, metabolism of other amino acids, metabolic cofactors and vitamins, lipid metabolism, sugar biosynthesis and metabolism, global and overview maps, energy Metabolism, carbohydrate metabolism and amino acid metabolism and other metabolic processes; ⑤Organismal Systems: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand protein are involved in the sensory system, nervous system, immune system, excretory system, Biological processes such as environmental adaptation, endocrine system, digestive system, developmental circulatory system, etc.
HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，级联生物学效应包括细胞过程(Cellular Processes)、环境信息处理(Environmental Information Processing)、遗传信息处理(Genetic Information Processing)、新陈代谢(Metabolism)和生物体系统(Organismal Systems)等功能途径；进一步地，①细胞过程(Cellular Processes)：HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了运输和分解代谢，细胞群体，细胞活性，细胞生长与死亡等细胞过程；②环境信息处理(Environmental Information Processing):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了信号分子与相互作用，信号转导，膜运输等环境信息处理过程；③遗传信息处理(Genetic Information Processing):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了翻译，复制和修复，折叠、分类和降解等生物过程；④新陈代谢(Metabolism):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了生物降解和代谢，核苷酸代谢，其他氨基酸的代谢，代谢的辅助因子和维生素，脂质代谢，糖的生物合成和代谢，全局和概览地图，能量代谢，碳水化合物代谢及氨基酸代谢等代谢过程；⑤生物体系统(Organismal Systems):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了感觉系统，神经系统，免疫系统，排泄系统，环境适 应，内分泌系统，消化系统，发育循环系统等生物过程。HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, and the cascade biological effects include Cellular Processes, Environmental Information Processing, Genetic Information Processing, Functional pathways such as Metabolism and Organismal Systems; further, ① Cellular Processes: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in transport and catabolism, and cellular processes Population, cell activity, cell growth and death and other cellular processes; ②Environmental Information Processing: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in signaling molecules and interactions, signal transduction, Environmental information processing processes such as membrane transport; ③ Genetic Information Processing: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in biological processes such as translation, replication and repair, folding, classification and degradation; ④Metabolism: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in biodegradation and metabolism, nucleotide metabolism, metabolism of other amino acids, metabolic cofactors and vitamins, lipid metabolism, sugar Biosynthesis and metabolism, global and overview maps, metabolic processes such as energy metabolism, carbohydrate metabolism and amino acid metabolism; ⑤Organismal Systems: Multiple differentially expressed genes induced by HrpNEch multi-epitopic ligand proteins are involved Sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, developmental circulatory system and other biological processes.
优选的，HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，级联生物学效应还包括HrpNEcb多拟表位配体蛋白诱导的基因功能群显著差异表达结果，其中包括：①生物过程相关差异表达基因：包括了繁殖，细胞死亡，免疫系统的过程，行为，代谢过程，细胞过程，生殖过程，生物粘附，信号，多细胞生物过程，发育过程，增长，运动，单个组织的过程，生物相，有节奏的过程，生物过程的正调控，生物过程负调控，生物过程调节，刺激反应，定位，生物调节，细胞成分组织或生物发生，细胞聚集，解毒以及突触前过程涉及突触传递；②细胞组分相关差异表达基因：涵盖了细胞及细胞外区域，类核，膜，病毒粒子，细胞结，细胞外基质，细胞膜封闭腔，复杂大分子，细胞器，细胞外基质成分，细胞外区域部分，细胞器部件，病毒粒子部件，膜部件，突触部分，细胞部件，突触，以及细胞超分子纤维等；③分子功能相关差异表达基因：涵盖了转录因子活性，蛋白质结合，核酸结合转录因子活性，催化活性，信号传感器活动，结构分子活动，运输活动，绑定，电子载体活动，成形素活动，抗氧化活性，金属伴侣蛋白活性，蛋白质标记，化学引诱物的活动，转译调控，化学排斥物活性，活动分子传感器，分子功能调控等。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEcb multi-epitope ligand protein, and the cascade biological effect also includes the results of the significant differential expression of gene functional groups induced by the HrpNEcb multi-epitope ligand protein, including: ①Differentially expressed genes related to biological processes: including reproduction, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, movement, single Organizing processes, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, stimulus response, localization, biological regulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic The process involves synaptic transmission; ② Differentially expressed genes related to cellular components: covering cells and extracellular regions, nucleoids, membranes, virions, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules, organelles, extracellular Matrix components, extracellular regions, organelle components, virus particle components, membrane components, synaptic components, cell components, synapses, and cellular supramolecular fibers, etc.; ③ Molecular function-related differentially expressed genes: covering transcription factor activity, protein Binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, morphogen activity, antioxidant activity, metal chaperone activity, protein labeling, chemoattractant activity , translational regulation, chemorepellent activity, active molecular sensors, molecular function regulation, etc.
优选的，HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，级联生物学效应还包括HrpNEch多拟表位配体蛋白诱导的基因功能群显著差异表达结果，其中包括：①生物过程相关差异表达基因：包括了繁殖，细胞死亡，免疫系统的过程，行为，代谢过程，细胞过程，生殖过程，生物粘附，信号，多细胞生物过程，发育过程，增长，运动，单个组织的过程，生物相，有节奏的过程，生物过程的正调控，生物过程负调控，生物过程调节，刺激反应，定位，生物调节，细胞成分组织或生物发生，细胞聚集，解毒以及突触前过程涉及突触传递；②细胞组分相关差异表达基因：涵盖了细胞及细胞外区域，类核，膜，病毒粒子，细胞结，细胞外基质，细胞膜封闭腔，复杂大分子，细胞器，细胞外基质成分，细胞外区域部分，细胞器部件，病毒粒子部件，膜部件，突触部分，细胞部件，突触，以及细胞超分子纤维等；③分子功能相关差异表达基因：涵盖了转录因子活性，蛋白质结合，核酸结合转录因子活性，催化活性，信号传感器活动，结构分子活动，运输活动，绑定，电子载体活动，成形素活动，抗氧化活性，金属伴侣蛋白活性，蛋白质标记，化学引诱物的活动，转译调控，化学排斥物活性，活动分子传感器，分子功能调控等。Preferably, the HrpN-type multi-epitope ligand protein is HrpNEch multi-epitope ligand protein, and the cascade biological effect also includes the significant differential expression results of gene functional groups induced by HrpNEch multi-epitope ligand protein, including: ①Differentially expressed genes related to biological processes: including reproduction, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, movement, single Organizing processes, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, stimulus response, localization, biological regulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic The process involves synaptic transmission; ② Differentially expressed genes related to cellular components: covering cells and extracellular regions, nucleoids, membranes, virions, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules, organelles, extracellular Matrix components, extracellular regions, organelle components, virus particle components, membrane components, synaptic components, cell components, synapses, and cellular supramolecular fibers, etc.; ③ Molecular function-related differentially expressed genes: covering transcription factor activity, protein Binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, morphogen activity, antioxidant activity, metal chaperone activity, protein labeling, chemoattractant activity , translational regulation, chemorepellent activity, active molecular sensors, molecular function regulation, etc.
优选的，所述制药中的应用的制品或药物的剂型为液剂、粉剂、片剂或胶囊剂。Preferably, the dosage form of the product or drug used in the pharmacy is liquid, powder, tablet or capsule.
优选的，所述制药中的应用还包括为HrpNEcb多拟表位配体蛋白依据药学治疗的活性化合物(HrpNEcb多拟表位配体蛋白制剂和/或药物)和其衍生物通常以单位剂型或多次剂型的配制和施用，每个单位剂量含有预定量的治疗活性化合物，其与所需的药物载体、运载体或赋形剂结合足够产生希望的治疗效果。单位剂型的实例包括安瓿和注射器和单独包装的片剂或胶囊剂。可以以部分或其倍数施用单位剂型。多次剂型是在单个容器中包装的多个相同的单位剂型，其将以分开的单位剂型施用。多次剂型的实例包括小瓶、片剂或胶囊瓶或加仑瓶。所以，多次剂型是在包装中不分开的多个单位剂量。可以制备含有0.001％到100％的活性成分的剂型或组合物，剩余部分由无毒载体组成，对于口服施用，药物组合物可以采取例如片剂或胶嚢剂的形式，其通过常规方法用药学上可接受的赋形剂如黏合剂(包括，但不限于，预胶化的玉米淀粉、聚乙烯吡咯烷酮或者丙基甲基纤维素)；填充剂(包括，但不限于，乳糖、微晶纤维素)；润滑剂(包括，但不限于，硬脂酸镁、滑石粉或二氧化硅)；崩解剂(包括，但不限于，马铃薯淀粉或淀粉羟乙酸钠)；或湿润剂(包括，但不限于，十二烷基硫酸钠)制备。可以通过本领域公知的方法包衣片剂。药物组合物也可以为液体形式，包括，但不限于，溶液剂、糖浆剂或混悬剂，或者可以以药物产品给出，其在使用前用水或其他合适的运载体重构。此类液体制剂可以通过常规方法用药学上可接受的 添加剂制备，所述添加剂为如悬浮剂(包括，但不限于，山梨醇糖浆、纤维素衍生物或食用脂肪)；乳化剂(包括，但不限于，卵磷脂或阿拉伯胶)；非水性运载体(包括，但不限于，杏仁油、油性酯，或者分级分离的植物油)；和防腐剂(包括，但不限于，对羟基苯甲酸甲酯或丙酯或山梨酸)。适于直肠施用的制剂可以作为单位剂量栓剂提供。这些可以通过将HrpNEcb多拟表位配体蛋白活性化合物与一种或多种固体载体，如可可脂混合，然后将所得混合物成型来制备。适于局部应用于皮肤或眼睛的制剂包括，但不限于，软骨剂、霜剂、洗剂、糊剂、凝胶剂、喷雾剂、气雾剂和油。示例性载体包括，但不限于，凡士林、羊毛脂、聚乙二醇、醇类，和其两种或多种的组合。局部制剂也可以含有按重量计0.001％到15％、20％、25％的增稠剂，其选自包括，但不限于，羟丙基甲基纤维素、甲基纤维素、聚乙烯吡咯烷酮、聚乙烯醇、聚乙二醇、聚/羟基烷基(甲基)丙烯酸酯或聚(甲基)丙烯酰胺类。通常通过滴注或作为软骨剂应用到结膜嚢中来应用局部制剂。它也可以用于冲洗或润滑眼、面部窦和外耳道。也可以将它注射到眼前房和其他地方。液体状态的局部制剂也可以以带或者隐形眼镜的形式存在于亲水的三维聚合物基质中，活性成分从所述基质释放。对于适于含服(舌下)施用的制剂包括，但不限于，在调味基质(通常蔗糖和阿拉伯胶或西黄蓍胶)中含有活性化合物的锭剂；和在惰性基质包括，但不限于，明胶和甘油或蔗糖和阿拉伯胶中含有化合物的软锭剂。可以配制配体同种型的药物组合物用于通过注射，包括，但不限于，通过快速浓注或连续灌注进行肠胃外施用。用于注射的制剂可以为单位剂型，例如，在安瓿或多剂量容器中，具有加入的添加剂。组合物可以为油性或水性载体中的混悬剂、溶液剂或乳剂，并且可以包括，但不限于，配制试剂，如悬浮剂、稳定剂，备选地，活性成分可以为粉末形式，在使用前用合适的载体如无菌无致热原的水或其他溶剂重构。适于经皮施用的制剂可以以离散的贴剂给出，适于在长时间内与接受者的表皮保持密切接触。此类贴剂合适地含有活性化合物作为活性化合物的任选緩冲的水溶液。适于经皮施用的制剂可以通过离子电渗疗法递送并采取活性化合物的任选緩沖的水溶液形式。Preferably, the pharmaceutical application further includes active compounds (HrpNEcb multi-epitope ligand protein preparations and/or drugs) and derivatives thereof for HrpNEcb multi-epitope ligand protein according to pharmaceutical treatment, usually in unit dosage form or Multiple dosage forms are formulated and administered, each unit dosage containing a predetermined quantity of the therapeutically active compound in association with the required 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 form can be administered in fractions or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container, which are to be administered in separate unit dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or gallon bottles. Thus, a multiple-dose form is a number of unit doses that are not separated in packaging. Dosage forms or compositions can be prepared containing from 0.001% to 100% of the active ingredient, the remainder consisting of a non-toxic carrier, for oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules, which are prepared by conventional methods of pharmacy Acceptable excipients such as binders (including, but not limited to, pregelatinized cornstarch, polyvinylpyrrolidone, or propylmethylcellulose); fillers (including, but not limited to, lactose, microcrystalline cellulose lubricants (including, but not limited to, magnesium stearate, talc, or silicon dioxide); disintegrants (including, but not limited to, potato starch or sodium starch glycolate); or wetting agents (including, But not limited to, sodium lauryl sulfate) preparation. Tablets may be coated by methods known in the art. Pharmaceutical compositions can also be in liquid form, including, but not limited to, solutions, syrups or suspensions, or can be presented as a pharmaceutical product for reconstitution with water or other suitable vehicle before use. Such liquid formulations can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (including, but not limited to, sorbitol syrup, cellulose derivatives, or edible fats); emulsifiers (including, but not limited to, edible fats); without limitation, 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, methylparaben) or propyl ester or sorbic acid). Formulations suitable for rectal administration may be presented as unit dose suppositories. These can be prepared by mixing the HrpNEcb polymimetic ligand protein active compound with one or more solid carriers, such as cocoa butter, and shaping the resulting mixture. Formulations suitable for topical application to the skin or eye include, but are not limited to, cartilage agents, 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 from 0.001% to 15%, 20%, 25% by weight of a thickening agent selected from the group consisting of, but not limited to, hydroxypropyl methylcellulose, methylcellulose, polyvinylpyrrolidone, Polyvinyl alcohol, polyethylene glycol, poly/hydroxyalkyl(meth)acrylates or poly(meth)acrylamides. Topical formulations are typically applied by instillation or as a cartilaginous agent into the conjunctival capsule. It can also be used to flush or lubricate the eyes, facial sinuses and external auditory canal. It can also be injected into the anterior chamber and elsewhere. Topical formulations in liquid form can also be presented in the form of tapes or contact lenses in a hydrophilic three-dimensional polymer matrix from which the active ingredient is released. Formulations suitable for buccal (sublingual) administration include, but are not limited to, lozenges containing the active compound in a flavored base (usually sucrose and acacia or tragacanth); and lozenges in an inert base include, but are not limited to , pastilles containing the compound in gelatin and glycerin or sucrose and acacia. Pharmaceutical compositions of ligand isoforms can 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, eg, in ampoules or in multi-dose containers, with added additives. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may include, but are not limited to, formulatory agents such as suspending agents, stabilizing agents, alternatively, the active ingredient may be in powder form for use. Before reconstitution with a suitable carrier such as sterile pyrogen-free water or other solvent. Formulations suitable for transdermal administration may be presented as discrete patches suitable for maintaining intimate contact with the epidermis of the recipient for extended periods of time. Such patches suitably contain the active compound as an optionally buffered aqueous solution of the active compound. Formulations suitable for transdermal administration may be delivered by iontophoresis and take the form of an optionally buffered aqueous solution of the active compound.
优选的，所述制药中的应用还包括为HrpNEch多拟表位配体蛋白质依据药学治疗的活性化合物(HrpNEch多拟表位配体蛋白制剂和/或药物)和其衍生物通常以单位剂型或多次剂型的配制和施用，每个单位剂量含有预定量的治疗活性化合物，其与所需的药物载体、运载体或赋形剂结合足够产生希望的治疗效果。单位剂型的实例包括安瓿和注射器和单独包装的片剂或胶囊剂。可以以部分或其倍数施用单位剂型。多次剂型是在单个容器中包装的多个相同的单位剂型，其将以分开的单位剂型施用。多次剂型的实例包括小瓶、片剂或胶囊瓶或加仑瓶。所以，多次剂型是在包装中不分开的多个单位剂量。可以制备含有0.001％到100％的活性成分的剂型或组合物，剩余部分由无毒载体组成，对于口服施用，药物组合物可以采取例如片剂或胶嚢剂的形式，其通过常规方法用药学上可接受的赋形剂如黏合剂(包括，但不限于，预胶化的玉米淀粉、聚乙烯吡咯烷酮或者丙基甲基纤维素)；填充剂(包括，但不限于，乳糖、微晶纤维素)；润滑剂(包括，但不限于，硬脂酸镁、滑石粉或二氧化硅)；崩解剂(包括，但不限于，马铃薯淀粉或淀粉羟乙酸钠)；或湿润剂(包括，但不限于，十二烷基硫酸钠)制备。可以通过本领域公知的方法包衣片剂。药物组合物也可以为液体形式，包括，但不限于，溶液剂、糖浆剂或混悬剂，或者可以以药物产品给出，其在使用前用水或其他合适的运载体重构。此类液体制剂可以通过常规方法用药学上可接受的添加剂制备，所述添加剂为如悬浮剂(包括，但不限于，山梨醇糖浆、纤维素衍生物或食用脂肪)；乳化剂(包括，但不限于，卵磷脂或阿拉伯胶)；非水性运载体(包括，但不限于，杏仁油、油性酯，或者分级分离的植物油)；和防腐剂(包括，但不限于，对羟基苯甲酸甲酯或丙酯或山梨酸)。适于直肠施用的制剂可以作为单位剂量栓剂提供。这些可以通过将HrpNEch多拟表位配体蛋白活性化合物与一种或多种固体载体，如可可脂混合，然后将所得混合物成型来制备。适于局部应用于皮肤或眼睛的制剂包括，但不限于，软骨剂、霜剂、洗剂、糊剂、凝胶剂、喷雾剂、气雾剂和油。示例性载体包括，但不限于，凡士林、羊 毛脂、聚乙二醇、醇类，和其两种或多种的组合。局部制剂也可以含有按重量计0.001％到15％、20％、25％的增稠剂，其选自包括，但不限于，羟丙基甲基纤维素、甲基纤维素、聚乙烯吡咯烷酮、聚乙烯醇、聚乙二醇、聚/羟基烷基(甲基)丙烯酸酯或聚(甲基)丙烯酰胺类。通常通过滴注或作为软骨剂应用到结膜嚢中来应用局部制剂。它也可以用于冲洗或润滑眼、面部窦和外耳道。也可以将它注射到眼前房和其他地方。液体状态的局部制剂也可以以带或者隐形眼镜的形式存在于亲水的三维聚合物基质中，活性成分从所述基质释放。对于适于含服(舌下)施用的制剂包括，但不限于，在调味基质(通常蔗糖和阿拉伯胶或西黄蓍胶)中含有活性化合物的锭剂；和在惰性基质包括，但不限于，明胶和甘油或蔗糖和阿拉伯胶中含有化合物的软锭剂。可以配制配体同种型的药物组合物用于通过注射，包括，但不限于，通过快速浓注或连续灌注进行肠胃外施用。用于注射的制剂可以为单位剂型，例如，在安瓿或多剂量容器中，具有加入的添加剂。组合物可以为油性或水性载体中的混悬剂、溶液剂或乳剂，并且可以包括，但不限于，配制试剂，如悬浮剂、稳定剂，备选地，活性成分可以为粉末形式，在使用前用合适的载体如无菌无致热原的水或其他溶剂重构。适于经皮施用的制剂可以以离散的贴剂给出，适于在长时间内与接受者的表皮保持密切接触。此类贴剂合适地含有活性化合物作为活性化合物的任选緩冲的水溶液。适于经皮施用的制剂可以通过离子电渗疗法递送并采取活性化合物的任选緩沖的水溶液形式。Preferably, the pharmaceutical application also includes active compounds (HrpNEch polyepitope ligand protein preparations and/or drugs) and derivatives thereof according to pharmaceutical treatment for HrpNEch polyepitope ligand proteins, usually in unit dosage form or Multiple dosage forms are formulated and administered, each unit dosage containing a predetermined quantity of the therapeutically active compound in association with the required 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 form can be administered in fractions or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container, which are to be administered in separate unit dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or gallon bottles. Thus, a multiple-dose form is a number of unit doses that are not separated in packaging. Dosage forms or compositions can be prepared containing from 0.001% to 100% of the active ingredient, the remainder consisting of a non-toxic carrier, for oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules, which are prepared by conventional methods of pharmacy Acceptable excipients such as binders (including, but not limited to, pregelatinized cornstarch, polyvinylpyrrolidone, or propylmethylcellulose); fillers (including, but not limited to, lactose, microcrystalline cellulose lubricants (including, but not limited to, magnesium stearate, talc, or silicon dioxide); disintegrants (including, but not limited to, potato starch or sodium starch glycolate); or wetting agents (including, But not limited to, sodium lauryl sulfate) preparation. Tablets may be coated by methods known in the art. Pharmaceutical compositions can also be in liquid form, including, but not limited to, solutions, syrups or suspensions, or can be presented as a pharmaceutical product for reconstitution with water or other suitable vehicle before use. Such liquid formulations can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (including, but not limited to, sorbitol syrup, cellulose derivatives, or edible fats); emulsifiers (including, but not limited to, edible fats); without limitation, 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, methylparaben) or propyl ester or sorbic acid). Formulations suitable for rectal administration may be presented as unit dose suppositories. These can be prepared by mixing the HrpNEch poly-epitope ligand protein active compound with one or more solid carriers, such as cocoa butter, and shaping the resulting mixture. Formulations suitable for topical application to the skin or eye include, but are not limited to, cartilage agents, 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 from 0.001% to 15%, 20%, 25% by weight of a thickening agent selected from the group consisting of, but not limited to, hydroxypropyl methylcellulose, methylcellulose, polyvinylpyrrolidone, Polyvinyl alcohol, polyethylene glycol, poly/hydroxyalkyl(meth)acrylates or poly(meth)acrylamides. Topical formulations are typically applied by instillation or as a cartilaginous agent into the conjunctival capsule. It can also be used to flush or lubricate the eyes, facial sinuses and external auditory canal. It can also be injected into the anterior chamber and elsewhere. Topical formulations in liquid form can also be presented in the form of tapes or contact lenses in a hydrophilic three-dimensional polymer matrix from which the active ingredient is released. Formulations suitable for buccal (sublingual) administration include, but are not limited to, lozenges containing the active compound in a flavored base (usually sucrose and acacia or tragacanth); and lozenges in an inert base include, but are not limited to , pastilles containing the compound in gelatin and glycerin or sucrose and acacia. Pharmaceutical compositions of ligand isoforms can 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, eg, in ampoules or in multi-dose containers, with added additives. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may include, but are not limited to, formulatory agents such as suspending agents, stabilizing agents, alternatively, the active ingredient may be in powder form for use. Before reconstitution with a suitable carrier such as sterile pyrogen-free water or other solvent. Formulations suitable for transdermal administration may be presented as discrete patches suitable for maintaining intimate contact with the epidermis of the recipient for extended periods of time. Such patches suitably contain the active compound as an optionally buffered aqueous solution of the active compound. Formulations suitable for transdermal administration may be delivered by iontophoresis and take the form of an optionally buffered aqueous solution of the active compound.
HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，制品或药物主要由纯化的HrpNEcb蛋白制备得到，质量含量为0.001％-100％。The HrpN-type polymimetic epitope ligand protein is HrpNEcb polymimetic epitope ligand protein, and the products or medicines are mainly prepared from purified HrpNEcb protein, and the mass content is 0.001%-100%.
HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，制品或药物主要由纯化的HrpNEch蛋白制备得到，质量含量为0.001％-100％。The HrpN type polymimetic epitope ligand protein is the HrpNEch polymimetic epitope ligand protein, and the products or medicines are mainly prepared from the purified HrpNEch protein, and the mass content is 0.001%-100%.
HrpN型多拟表位配体蛋白为纯化后的HrpN型蛋白。The HrpN-type polymimetic epitope ligand protein is a purified HrpN-type protein.
HrpN型蛋白的纯化的方法，包括以下步骤：The method for purifying HrpN-type protein includes the following steps:
步骤1：高压破碎机破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁,高压范围800-1000Mpa；Step 1: The high pressure crusher crushes the engineering bacteria, and the crushed bacteria liquid is passed into the butterfly continuous flow centrifuge to remove the cell wall, and the high pressure range is 800-1000Mpa;
步骤2：用Ni-NTA琼脂糖凝胶柱纯化HrpN型多拟表位配体蛋白-His重组蛋白，得到纯化的HrpN型多拟表位配体蛋白原药。Step 2: Purify the HrpN-type polymimetic epitope ligand protein-His recombinant protein with a Ni-NTA agarose column to obtain a purified HrpN-type polymimetic epitope ligand protein original drug.
本发明采用的HrpNEcb蛋白的高效表达、纯化生产HrpNEcb蛋白的制备方法，包括以下步骤：The high-efficiency expression of HrpNEcb protein adopted in the present invention, the preparation method of purifying and producing HrpNEcb protein, comprises the following steps:
1.HrpNEcb蛋白的工程菌发酵制备：将带有编码HrpNEcb多拟表位配体蛋白的基因(包括，但不限于生物样品天然基因、化学合成基因、转基因遗传重组体基因以及相似基因及其基因修饰)质粒的工程菌(E.coli)，相关蛋白的生产系是K-12原菌经特殊改造后的衍生菌JY-01(DE3)，在LB液体培养基(每升含卡那霉素50微克)中，在一定的温度条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体。用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析表达产物HrpNEcb蛋白，在电泳胶板的样品泳道上，会呈现一条36.64kda条带，它就是基因hrpNEcb的表达产物HrpNEcb蛋白。1. The engineering bacteria fermentation preparation of HrpNEcb protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes and similar genes and their genes) encoding HrpNEcb polymimetic ligand proteins are used. Modified) plasmid engineering bacteria (E.coli), the production line of the related protein is a special modified derivative of K-12 original bacteria JY-01 (DE3), in LB liquid medium (containing kanamycin per liter) 50 μg), under certain temperature conditions, when cultured to OD600=0.7, IPTG (isopropyl thiogalactoside, Isopropylβ-D-Thiogalactosid) (final concentration 1mMol) was added, and the cells were collected by centrifugation after continuing to culture. . The expression product HrpNEcb protein was analyzed by 10% SDS-PAGE polyacrylamide gel electrophoresis, and a 36.64kda band appeared on the sample lane of the electrophoresis gel plate, which was the expression product HrpNEcb protein of the gene hrpNEcb.
2.高压破碎机破碎工程菌，连续用800-1000Mpa压力，破碎工程菌。2. The high-pressure crusher crushes engineering bacteria, and continuously uses 800-1000Mpa pressure to crush engineering bacteria.
3.离心收集HrpNEcb多拟表位配体蛋白分子，离心力范围1000-8000g，优选地离心力1000-2000g；优选地离心力2000-3500g；优选地离心力8000-6000g；优选地离心力6000-4500g；最优选地离心力3500-4500g。HrpNEcb多拟表位配体蛋白存在于上清液中。3. Centrifugal collection of HrpNEcb polymimetic epitope ligand protein molecules, centrifugal force range 1000-8000g, preferably centrifugal force 1000-2000g; preferably centrifugal force 2000-3500g; preferably centrifugal force 8000-6000g; preferably centrifugal force 6000-4500g; most preferably The centrifugal force is 3500-4500g. The HrpNEcb multi-epitope ligand protein was present in the supernatant.
4.用Ni-NTA琼脂糖凝胶柱纯化HrpNEcb多拟表位配体蛋白-His重组蛋白，蛋白纯化按Ni-NTA琼脂糖凝胶柱厂家建议方法实施，得到纯化的HrpN型多拟表位配体蛋白原药。4. Purify the HrpNEcb polymimetic epitope ligand protein-His recombinant protein with Ni-NTA agarose column. The protein purification is carried out according to the method recommended by the manufacturer of the Ni-NTA agarose column, and the purified HrpN type polymimetic epitope is obtained. Ligand protein prodrug.
本发明所述识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效应的 HrpNEcb蛋白制剂的使用途径，可通过本领域技术人员已知的任一途径施用，所述途径包括内用、外用、口服、注射、肌内、静脉内、皮内、腹膜内、皮下、鼻、口、直肠、局部、含服和经皮施用或任何途径；可以通过任何方便的途径施用HrpNEcb多拟表位配体蛋白，例如通过灌注或快速灌注，通过上皮或皮肤粘膜内层(例如，口粘膜、鼻腔黏膜、胃黏膜、直肠和肠粘膜等等)吸收，并且可以与其它生物活性剂顺序、间歇地或在相同组合物中施用；依据治疗部位，施用可以是局部的、表面的或者全身的。局部施用于需要治疗的区域可以，但不限于，局部灌注、表面应用，通过浸泡，通过注射，通过导管，通过栓剂；施用还可以包括控释系统，包括控释制剂和装置控释，如通过泵；在任一给定情况下最合适的途径将取决于被治疗的疾病或状况的性质和严重性和使用的具体组合物的性质。多种递送系统是已知的并且可以用于施用多拟表位配体蛋白，可以在脂质体、微粒、微嚢中包裹。可以制备多拟表位配体蛋白的药物组合物，通常，将按照管理机构的批准制备或者根据公认的药典制备药学上可接受的组合物用于患者。The application route of the HrpNEcb protein preparation that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multifunctional cascade biological effects according to the present invention can be administered by any route known to those skilled in the art, Such routes include internal, topical, oral, injection, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, nasal, oral, rectal, topical, buccal and transdermal administration or any route; by any convenient HrpNEcb polyepitope ligand proteins are administered by routes such as by perfusion or rapid perfusion, absorption through epithelia or mucocutaneous linings (eg, oral mucosa, nasal mucosa, gastric mucosa, rectal and intestinal mucosa, etc.), and can be combined with other The biologically active agents are administered sequentially, intermittently, or in the same composition; depending on the site of treatment, administration can be topical, topical, or systemic. Topical administration to the area in need of treatment can be, but is not limited to, local 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 Pump; the most appropriate route in any given situation will depend on the nature and severity of the disease or condition being treated and the nature of the particular composition used. Various delivery systems are known and can be used to administer the multi-epitope ligand protein, which can be encapsulated in liposomes, microparticles, microcapsules. Pharmaceutical compositions of the polymimetic ligand protein can be prepared, typically, as approved by a regulatory agency or as a pharmaceutically acceptable composition for use in a patient.
本发明所述识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效的HrpNEcb多拟表位配体蛋白引起的多功能级联生物学效应和多样性功能广泛涉及多系统、组织、器官、细胞相关疾病和状况的诊断、或和预防、或和治疗、或和康复以及广泛涉及有关疾病和状况的食字号、消字号、妆字号、械字号和健字号制品或药物的制药中的应用。The multi-functional cascade biological effects and diverse functions caused by the HrpNEcb multi-mimetic epitope ligand protein that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multi-functional cascade biological effects Widely involved in the diagnosis, or prevention, or treatment, or recovery of multi-system, tissue, organ, and cell-related diseases and conditions, as well as related diseases and conditions. Pharmaceutical applications of products or drugs.
本发明所述制药中应用的涉及识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效应的HrpNEcb多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复神经系统、消化系统、运动系统、循环系统、呼吸系统、内分泌系统、免疫系统、泌尿系统、生殖系统疾病和状况中的应用：The preparations or medicines involving HrpNEcb multi-epitope ligand proteins that recognize and activate various types of receptors, membrane proteins and their signaling pathways in animals and induce multifunctional cascade biological effects and are used in the pharmacy of the present invention are used in diagnosis, or and application in the prevention, or treatment, or rehabilitation of diseases and conditions of the nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system:
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复神经连结疾病、痴呆、帕金森病、中枢神经系统疾病、神经肌肉病、癫痫、头痛和神经痛、周围神经病、注意缺陷多动障碍和抽动障碍、失眠症、抑郁症、焦虑障碍、双相情感障碍、精神病性障碍、神经性皮炎相关的神经系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein described in the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of neural connection diseases, dementia, Parkinson's disease, central nervous system disease, neuromuscular disease, epilepsy, Use in headache and neuralgia, peripheral neuropathy, attention deficit hyperactivity disorder and tic disorder, insomnia, depression, anxiety disorders, bipolar disorder, psychotic disorders, neurodermatitis-related neurological diseases and conditions;
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复胃酸分泌不调、胃肠神经官能症、胃肠动力、胃肠黏膜炎、肝脏疾病、微生态障碍相关的消化系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or recovery of gastric acid secretion disorder, gastrointestinal neurosis, gastrointestinal motility, gastrointestinal mucositis, liver Use in diseases and disorders of the digestive system associated with diseases and micro-ecological disorders;
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复关节炎、肌肉痉挛、疼痛、肌营养不良、肌肉神经损伤、脱水相关的运动系统疾病和状况中的应用；The product or drug of the polyepitope ligand protein of the present invention is useful in the diagnosis, or prevention, or treatment, or rehabilitation of arthritis, muscle spasm, pain, muscular dystrophy, muscle and nerve injury, and dehydration-related motor systems Applications in diseases and conditions;
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复心力衰竭、心律失常、高血压、心肌损伤、缺血、心绞痛、高脂血症、钙通道阻滞、血管痉挛、血凝、血象异常、心肌梗死相关的循环系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein described in the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of heart failure, arrhythmia, hypertension, myocardial injury, ischemia, angina pectoris, hyperlipidemia , calcium channel blockade, vasospasm, blood coagulation, abnormal blood picture, myocardial infarction-related circulatory system diseases and conditions;
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复哮喘、慢性阻塞性肺疾病、支气管扩张、过敏原免疫、变态反应、肺炎、急性或慢性支气管炎、支气管哮喘、胃食管反流、鼻炎相关的呼吸系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or 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;
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复糖尿病、甲状腺疾病、垂体疾病、高催乳素血症、尿崩症、肾上腺疾病、甲状旁腺疾病、骨质疏松症相关的内分泌系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of diabetes, thyroid disease, pituitary disease, hyperprolactinemia, diabetes insipidus, adrenal disease, Parathyroid disease, osteoporosis-related endocrine system diseases and conditions;
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复免疫低下、类风湿关节炎、红斑狼疮相关的免疫系统疾病和状况中的应用；The application of the product or medicine of the multi-epitope ligand protein of the present invention in diagnosis, or prevention, or treatment, or rehabilitation of immune system diseases and conditions related to immunosuppression, rheumatoid arthritis, and lupus erythematosus;
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复肾病综合征、间质性肾炎、肾衰竭、泌尿、生殖系统感染、肾盂肾炎、膀胱炎、前列腺炎、尿道炎、附睾炎或睾丸炎、 前列腺增生、膀胱过度活动症、性功能障碍相关、以及各类男科、妇科感染性炎症和功能性疾病等的泌尿生殖系统疾病和状况中的应用。The products or medicines of the multi-epitope ligand protein described in the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of nephrotic syndrome, interstitial nephritis, renal failure, urinary and reproductive system infections, pyelonephritis, Cystitis, prostatitis, urethritis, epididymitis or orchitis, benign prostatic hyperplasia, overactive bladder, sexual dysfunction-related, and urogenital diseases and conditions related to various male and gynecological infectious inflammatory and functional diseases Applications.
本发明所述多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复全身肌肤细胞营养、激活、再生、修复、清除、细腻光洁、紫外黑色素沉积、湿疹、粗糙、裂纹、暗纹、干枯、硬皮、红斑、过敏、神经性皮炎、损伤、青春痘、暗疮、疤痕、暗沉、螨虫、油性皮肤、炎症性皮肤病、自身免疫性皮肤病、色素性皮肤病、皮肤萎缩、变薄、干燥、色素沉着、皱纹增生、表皮角化不良、干皮症、接触性皮炎、抗皮肤衰老、改善皮肤功能、美白祛斑、防治皮肤疾病的相关的皮肤系统疾病和状况中的应用。The products or medicines of the multi-epitope ligand protein of the present invention can be used in diagnosis, or prevention, or treatment, or rehabilitation of whole body skin cell nutrition, activation, regeneration, repair, clearing, delicate and smooth, ultraviolet melanin deposition, eczema , rough, cracked, dark lines, dry, crusty, erythema, allergies, neurodermatitis, lesions, pimples, acne, scars, dullness, mites, oily skin, inflammatory skin diseases, autoimmune skin diseases, pigmentation Skin diseases related to skin diseases, skin atrophy, thinning, dryness, hyperpigmentation, wrinkle hyperplasia, dyskeratosis, xeroderma, contact dermatitis, anti-aging, improving skin function, whitening and freckle, preventing and treating skin diseases Applications in Diseases and Conditions.
本发明所述识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效应的HrpNEcb多拟表位配体蛋白的制备，包括以下方法：1、所述HrpNEcb多拟表位配体蛋白的制备，采用含EcbCSL101HrpNEcb基因(克隆到高效表达载体PET28a(+))的工程菌，通过发酵，纯化制备HrpNEcb多拟表位配体蛋白：The preparation of the HrpNEcb multi-epitope ligand protein that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multi-functional cascade biological effects according to the present invention includes the following methods: 1. The HrpNEcb poly For the preparation of the epitope ligand protein, the engineering bacteria containing the EcbCSL101HrpNEcb gene (cloned into the high-efficiency expression vector PET28a(+)) were used to ferment and purify the HrpNEcb polymimetic epitope ligand protein:
1)HrpNEcb蛋白的工程菌发酵制备：将带有编码HrpNEcb多拟表位配体蛋白的基因(包括，但不限于生物样品天然基因、化学合成基因、转基因遗传重组体基因以及相似基因及其基因修饰)质粒的工程菌相关蛋白的生产系是K-12原菌经特殊改造后的衍生菌JY-01(DE3)，在LB液体培养基(每升含卡那霉素50微克)中，在一定的温度下条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体。用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析表达产物HrpNEcb多拟表位配体蛋白，在电泳胶板的样品泳道上，会呈现一条36.64kda条带，它就是基因hrpNEcb的表达产物HrpNEcb多拟表位配体蛋白；1) The engineering bacteria fermentation preparation of HrpNEcb protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes, and similar genes and their genes) encoding HrpNEcb polymimetic epitope ligand proteins are used. The production line of the engineered bacteria-related protein of the modified) plasmid is a specially modified derivative of the original K-12 bacteria JY-01 (DE3), in LB liquid medium (containing 50 micrograms of kanamycin per liter), in Under certain temperature conditions, when cultured to OD600=0.7, IPTG (isopropyl thiogalactoside, Isopropylβ-D-Thiogalactosid) (final concentration 1mMol) was added, and the cells were collected by centrifugation after continuing to culture. Using 10% SDS-PAGE polyacrylamide gel electrophoresis to analyze the expression product HrpNEcb multi-epitope ligand protein, a 36.64kda band will appear on the sample lane of the electrophoresis gel plate, which is the expression product of the gene hrpNEcb HrpNEcb polyprotein. Epitope-like ligand proteins;
其中，发酵用培养基Na 2HPO 4-KH 2PO 4缓冲系统，缓冲系统的pH范围是1-14；优选地pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-7；最优选地pH 6.5-5.5； Wherein, the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system, the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
发酵温度范围0-60℃。优选地温度为0-20℃；优选地温度为20-35℃；优选地温度为60-50℃；优选地温度为50-45℃；最优选地温度为37-38℃；The fermentation temperature range is 0-60℃. Preferably the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
发酵增殖液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地0.00％-0.01％；优选地1.00％-0.3％；最优选地0.01％-0.05％；最优选地0.1％-0.05％；Fermentation proliferation liquid medium glucose concentration range 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%;
发酵诱导液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地1.00％-0.3％；优选地0.3％-0.1％；优选地0.1％-0.05％；最优选地0.05％-0.00％；Fermentation induction liquid medium glucose concentration range 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%;
发酵诱导液体培养基乳糖浓度范围10.00％-0.00％；优选地10.00％-1.00％；优选地0.00％-0.1％；优选地1.00％-0.6％；优选地0.1％-0.3％；最优选地0.5％-0.4％；Fermentation induction liquid medium lactose concentration range 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%;
发酵诱导液体培养时间范围0-24h；优选地时间0-2h；优选地时间为24-15h；优选地时间为2-6h；优选地时间为15-10h；最优选地时间为7-9h。The time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
2)工程菌生产HrpNEcb多拟表位配体蛋白生产发酵结束后的后处理：①灭菌：发酵液在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃以下；②清洗：用葡萄糖Na 2HPO 4-KH 2PO 4缓冲液(pH范围是1-14，葡萄糖浓度范围是0-2500mmol，缓冲系统pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-6；最优选地pH 5-5.5。葡萄糖浓度为0-100mmol；优选地浓度为100-200mmol；优选地浓度为2500-1000mmol；优选地浓度为1000-300mmol；最优选地浓度为200-300mmol，在蝶式连续流离心机里清洗工程菌体五至八次；③破碎工程菌并清除细胞壁，再用pH 5-5.5、葡萄糖浓度200-300mmol的Na 2HPO 4-KH 2PO 4缓冲液稀释菌体，调节菌体鲜重为稀释液的20％-30％，导入高压破碎机，连续用800-1000Mpa压力，破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁，HrpNEcb多拟表位 配体蛋白存在于上清液中。 2) Post-processing of HrpNEcb polymimetic ligand protein production by engineering bacteria after fermentation: ① Sterilization: The fermentation broth is sterilized at a temperature of 80 °C for 30 minutes, and then rapidly cooled to below 30 °C; ② Cleaning: With glucose Na 2 HPO 4 -KH 2 PO 4 buffer (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 For 200-300mmol, in the butterfly continuous flow centrifuge, clean the engineering bacteria five to eight times; 3. break the engineering bacteria and remove the cell wall, then use the Na 2 HPO 4 -KH 2 of pH 5-5.5, glucose concentration 200-300 mmol Dilute the bacterial cells with PO 4 buffer, adjust the fresh weight of the bacterial cells to 20%-30% of the diluent, introduce into a high-pressure crusher, continuously use 800-1000Mpa pressure to break the engineering bacteria, and pass the broken bacteria liquid into the butterfly continuous flow centrifuge machine, to clear the cell wall, the HrpNEcb multi-epitope ligand protein was present in the supernatant.
3)HrpNEcb多拟表位配体蛋白分子的纯化3) Purification of HrpNEcb polymimetic epitope ligand protein molecule
用NI-NTA琼脂糖凝胶柱纯化HrpNEcb多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，完成HrpNEcb多拟表位配体蛋白的纯化制备。The HrpNEcb polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column. The protein purification was implemented according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEcb polymimetic epitope ligand protein. preparation.
3、所述HrpNEcb多拟表位配体蛋白的制备，进一步，HrpNEcb蛋白也可以通过“人工合成基因”的表达蛋白进行制备，通过发酵，纯化制备HrpNEcb多拟表位配体蛋白，具体包括以下步骤：3. The preparation of the HrpNEcb polymimetic epitope ligand protein, further, the HrpNEcb protein can also be prepared by the expression protein of an "artificially synthesized gene", and the HrpNEcb polymimetic epitope ligand protein can be prepared by fermentation and purification, specifically including the following step:
编码HrpNEcb蛋白的hrpNEcb基因的人工合成及其表达蛋白制备Artificial synthesis of hrpNEcb gene encoding HrpNEcb protein and preparation of its expressed protein
1)按照GenBank公布的编码HrpNEcb蛋白的hrpNEcb基因(GenBank:DQ355519.1)核苷酸序列,人工合成hrpNEcb基因，其DNA序列为：1) According to the nucleotide sequence of the hrpNEcb gene (GenBank: DQ355519.1) encoding the HrpNEcb protein published by GenBank, artificially synthesize the hrpNEcb gene, and its DNA sequence is:
Figure PCTCN2021134714-appb-000006
Figure PCTCN2021134714-appb-000006
2)根据以上DNA序列，人工合成蛋白基因时，在基因的5′和3′分别加上BamHI和HindIII酶切位点，方便蛋白基因克隆；2) According to the above DNA sequence, when artificially synthesizing the protein gene, add BamHI and HindIII enzyme cleavage sites to the 5' and 3' of the gene respectively to facilitate the cloning of the protein gene;
人工基因合成委托Thermo Fisher Scientific公司的GeneArt基因合成与服务部门完成。人工合成蛋白基因的优点主要是：a)合成周期短，可以保证序列的100％正确无误；b)可以对密码子进行优化，以提高基因的表达效率；由于每个物种偏爱的编码子不同，当异源蛋白在大肠杆菌里表达时，有些蛋白很难得到高表达。如果将异源蛋白的密码子改为大肠杆菌偏爱的密码子，就可以实现蛋白的基因的高效表达，提高该基因的表达水平，适于大规模工业生产；c)可根据需要进行基因的定点突变以改造基因，提高蛋白的作用效率；d)研究人员可根据自己的意愿设计得到自然界中很难获得甚至不存在的基因。The artificial gene synthesis was entrusted to the GeneArt Gene Synthesis and Services Division of Thermo Fisher Scientific. The advantages of artificially synthesized protein genes are: a) the synthesis cycle is short, and the sequence can be guaranteed to be 100% correct; b) the codons can be optimized to improve the expression efficiency of the gene; due to the different coding codons preferred by each species, When heterologous proteins are expressed in E. coli, some proteins are difficult to express at high levels. If the codon of the heterologous protein is changed to the codon preferred by Escherichia coli, the high-efficiency expression of the protein gene can be achieved, the expression level of the gene can be improved, and it is suitable for large-scale industrial production; c) The gene can be designated as needed. Mutations are used to modify genes and improve the efficiency of proteins; d) Researchers can design genes that are difficult to obtain or even do not exist in nature according to their own wishes.
3)将合成的编码HrpNEcb蛋白基因的DNA片段，逐一克隆到高效蛋白表达载体PET28a(+)(含His-Tag标签)BamHI-HindIII位点，经DNA测序确保克隆的准确性；3) The synthesized DNA fragments encoding the HrpNEcb protein gene were cloned into the high-efficiency protein expression vector PET28a(+) (containing His-Tag label) BamHI-HindIII site one by one, and the accuracy of the clone was ensured through DNA sequencing;
4)HrpNEcb多拟表位配体蛋白的工程菌发酵制备：将1)至3)编码HrpNEcb蛋白的基因克隆转入大肠杆菌工程菌(E.coli)中，相关蛋白的生产系(E.coli)是K-12原菌经特殊改造后的衍生菌JY-01(DE3)；在LB液体培养基(每升含卡那霉素50微克)中，在一定的温度下条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体，用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析编码HrpNEcb蛋白，在电泳胶板的样品泳道上，会呈现一条36.64kda条带，它就是基因hrpNEcb的表达产物HrpNEcb多拟表位配体蛋白。4) Fermentation preparation of HrpNEcb poly-epitope ligand protein by engineering bacteria: clone the gene encoding HrpNEcb protein from 1) to 3) and transfer it into E. coli engineering bacteria (E.coli), and the production line of the related protein (E.coli) ) is a specially modified derivative of the original K-12 bacteria JY-01 (DE3); in LB liquid medium (containing 50 micrograms of kanamycin per liter), under certain temperature conditions, cultivate to OD600 = 0.7, add IPTG (isopropyl thiogalactoside, Isopropylβ-D-Thiogalactosid) (final concentration 1mMol), continue to culture, centrifuge to collect bacteria, use 10% SDS-PAGE polyacrylamide gel electrophoresis to analyze the code The HrpNEcb protein will show a 36.64kda band on the sample lane of the electrophoresis gel plate, which is the HrpNEcb multi-epitope ligand protein, the expression product of the gene hrpNEcb.
其中，发酵用培养基Na 2HPO 4-KH 2PO 4缓冲系统，缓冲系统的pH范围是1-14；优选地pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-7；最优选地pH 6.5-5.5； Wherein, the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system, the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
发酵温度范围0-60℃。优选地温度为0-20℃；优选地温度为20-35℃；优选地温度为60-50℃；优选地温度为50-45℃；最优选地温度为37-38℃；The fermentation temperature range is 0-60℃. Preferably the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
发酵增殖液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地0.00％-0.01％；优选地1.00％-0.3％；最优选地0.01％-0.05％；最优选地0.1％-0.05％；Fermentation proliferation liquid medium glucose concentration range 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%;
发酵诱导液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地1.00％-0.3％；优选地0.3％-0.1％；优选地0.1％-0.05％；最优选地0.05％-0.00％；Fermentation induction liquid medium glucose concentration range 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%;
发酵诱导液体培养基乳糖浓度范围10.00％-0.00％；优选地10.00％-1.00％；优选地0.00％-0.1％；优选地1.00％-0.6％；优选地0.1％-0.3％；最优选地0.5％-0.4％；Fermentation induction liquid medium lactose concentration range 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%;
发酵诱导液体培养时间范围0-24h；优选地时间0-2h；优选地时间为24-15h；优选地时间为2-6h；优选地时间为15-10h；最优选地时间为7-9h。The time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
5)工程菌生产HrpNEcb多拟表位配体蛋白生产发酵结束后的后处理：①灭菌：发酵液在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃以下；②清洗：用葡萄糖Na 2HPO 4-KH 2PO 4缓冲液(pH范围是1-14，葡萄糖浓度范围是0-2500mmol，缓冲系统pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-6；最优选地pH 5-5.5。葡萄糖浓度为0-100mmol；优选地浓度为100-200mmol；优选地浓度为2500-1000mmol；优选地浓度为1000-300mmol；最优选地浓度为200-300mmol，在蝶式连续流离心机里清洗工程菌体五至八次；③破碎工程菌并清除细胞壁，再用pH 5-5.5、葡萄糖浓度200-300mmol的Na 2HPO 4-KH 2PO 4缓冲液稀释菌体，调节菌体鲜重为稀释液的20％-30％，导入高压破碎机，连续用800-1000Mpa压力，破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁，HrpNEcb多拟表位配体蛋白存在于上清液中。 5) Production of HrpNEcb polymimetic epitope ligand protein by engineering bacteria Post-processing after fermentation: ① Sterilization: The fermentation broth is sterilized at 80°C for 30 minutes, and rapidly cooled to below 30°C; ②Cleaning: With glucose Na 2 HPO 4 -KH 2 PO 4 buffer (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 For 200-300mmol, in the butterfly continuous flow centrifuge, clean the engineering bacteria five to eight times; 3. break the engineering bacteria and remove the cell wall, then use the Na 2 HPO 4 -KH 2 of pH 5-5.5, glucose concentration 200-300 mmol Dilute the bacterial cells with PO 4 buffer, adjust the fresh weight of the bacterial cells to 20%-30% of the diluent, introduce into a high-pressure crusher, continuously use 800-1000Mpa pressure to break the engineering bacteria, and pass the broken bacteria liquid into the butterfly continuous flow centrifuge machine, to clear the cell wall, the HrpNEcb multi-epitope ligand protein was present in the supernatant.
6)HrpNEcb多拟表位配体蛋白分子的纯化6) Purification of HrpNEcb polymimetic epitope ligand protein molecule
用NI-NTA琼脂糖凝胶柱纯化HrpNEcb多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，完成HrpNEcb多拟表位配体蛋白的纯化制备。The HrpNEcb polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column. The protein purification was implemented according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEcb polymimetic epitope ligand protein. preparation.
本发明所述识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效应的HrpNEch蛋白制剂的使用途径，可通过本领域技术人员已知的任一途径施用，所述途径包括内用、外用、口服、注射、肌内、静脉内、皮内、腹膜内、皮下、鼻、口、直肠、局部、含服和经皮施用或任何途径；可以通过任何方便的途径施用HrpNEch多拟表位配体蛋白，例如通过灌注或快速灌注，通过上皮或皮肤粘膜内层(例如，口粘膜、鼻腔黏膜、胃黏膜、直肠和肠粘膜等等)吸收，并且可以与其它生物活性剂顺序、间歇地或在相同组合物中施用；依据治疗部位，施用可以是局部的、表面的或者全身的。局部施用于需要治疗的区域可以，但不限于，局部灌注、表面应用，通过浸泡，通过注射，通过导管，通过栓剂；施用还可以包括控释系统，包括控释制剂和装置控释，如通过泵；在任一给定情况下最合适的途径将取决于被治疗的疾病或状况的性质和严重性和使用的具体组合物的性质。多种递送系统是已知的并且可以用于施用多拟表位配体蛋白，可以在脂质体、微粒、微嚢中包裹。可以制备多拟表位配体蛋白的药物组合物，通常，将按照管理机构的批准制备或者根据公认的药典制备药学上可接受的组合物用于患者。The use route of the HrpNEch protein preparation that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multifunctional cascade biological effects according to the present invention can be administered by any route known to those skilled in the art, Such routes include internal, topical, oral, injection, intramuscular, intravenous, intradermal, intraperitoneal, subcutaneous, nasal, oral, rectal, topical, buccal and transdermal administration or any route; by any convenient Routes of administration of HrpNEch polyepitope ligand proteins, such as by perfusion or rapid perfusion, absorption through epithelial or mucocutaneous linings (eg, oral mucosa, nasal mucosa, gastric mucosa, rectal and intestinal mucosa, etc.), and can be combined with other The biologically active agents are administered sequentially, intermittently, or in the same composition; depending on the site of treatment, administration can be topical, topical, or systemic. Topical administration to the area in need of treatment can be, but is not limited to, local 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 Pump; the most appropriate route in any given situation will depend on the nature and severity of the disease or condition being treated and the nature of the particular composition used. Various delivery systems are known and can be used to administer the multi-epitope ligand protein, which can be encapsulated in liposomes, microparticles, microcapsules. Pharmaceutical compositions of the polymimetic ligand protein can be prepared, typically, as approved by a regulatory agency or as a pharmaceutically acceptable composition for use in a patient.
本发明所述识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效的HrpNEch多拟表位配体蛋白引起的多功能级联生物学效应和多样性功能广泛涉及多系统、组织、器官、细胞相关疾病和状况的诊断、或和预防、或和治疗、或和康复以及广泛涉及有关疾病和状况的食字号、消字号、妆字号、械字号和健字号制品或药物的制药中的应用。The multi-functional cascade biological effects and diverse functions caused by the HrpNEch multi-mimetic epitope ligand protein that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multi-functional cascade biological effects Widely involved in the diagnosis, or prevention, or treatment, or recovery of multi-system, tissue, organ, and cell-related diseases and conditions, as well as related diseases and conditions. Pharmaceutical applications of products or drugs.
本发明所述制药中应用的涉及识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效应的HrpNEch多拟表位配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复神经系统、消化系统、运动系统、循环系统、呼吸系统、内分泌系统、免疫系统、泌尿系统、生殖系统疾病和状况中的应用：The preparations or medicines involving HrpNEch multi-epitope ligand proteins that recognize and activate various types of receptors, membrane proteins and their signaling pathways in animals and induce multifunctional cascade biological effects and are used in the pharmacy of the present invention are used in diagnosis, or and application in the prevention, or treatment, or rehabilitation of diseases and conditions of the nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system:
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复神经连结疾病、痴呆、帕金森病、中枢神经系统疾病、神经肌肉病、癫痫、头痛和神经痛、周围神经病、注意缺陷多动障碍和抽动障碍、失眠症、抑郁症、焦虑障碍、双相情感障碍、精神病性障碍、神经性皮炎相关的神经系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand proteins of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of neural connection diseases, dementia, Parkinson's disease, central nervous system diseases, neuromuscular diseases, epilepsy, Use in headache and neuralgia, peripheral neuropathy, attention deficit hyperactivity disorder and tic disorder, insomnia, depression, anxiety disorders, bipolar disorder, psychotic disorders, neurodermatitis-related neurological diseases and conditions;
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复胃酸分泌不调、胃肠神经官能症、胃肠动力、胃肠黏膜炎、肝脏疾病、微生态障碍相关的消化系统疾病和状况中的应用；The product or drug of the multi-epitope ligand protein of the present invention is used for diagnosis, or prevention, or treatment, or recovery of gastric acid secretion disorder, gastrointestinal neurosis, gastrointestinal motility, gastrointestinal mucositis, liver Use in diseases and disorders of the digestive system associated with diseases and micro-ecological disorders;
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复关节炎、肌肉痉挛、疼痛、肌营养不良、肌肉神经损伤、脱水相关的运动系统疾病和状况中的应用；The preparations or drugs of the multi-epitope ligand proteins of the present invention are useful in the diagnosis, or prevention, or treatment, or rehabilitation of arthritis, muscle spasm, pain, muscular dystrophy, muscle and nerve damage, and dehydration-related motor systems Applications in diseases and conditions;
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复心力衰竭、心律失常、高血压、心肌损伤、缺血、心绞痛、高脂血症、钙通道阻滞、血管痉挛、血凝、血象异常、心肌梗死相关的循环系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of heart failure, arrhythmia, hypertension, myocardial injury, ischemia, angina pectoris, hyperlipidemia , calcium channel blockade, vasospasm, blood coagulation, abnormal blood picture, myocardial infarction-related circulatory system diseases and conditions;
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复哮喘、慢性阻塞性肺疾病、支气管扩张、过敏原免疫、变态反应、肺炎、急性或慢性支气管炎、支气管哮喘、胃食管反流、鼻炎相关的呼吸系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or 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;
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复糖尿病、甲状腺疾病、垂体疾病、高催乳素血症、尿崩症、肾上腺疾病、甲状旁腺疾病、骨质疏松症相关的内分泌系统疾病和状况中的应用；The products or medicines of the multi-epitope ligand protein of the present invention are used in the diagnosis, or prevention, or treatment, or rehabilitation of diabetes, thyroid disease, pituitary disease, hyperprolactinemia, diabetes insipidus, adrenal disease, Parathyroid disease, osteoporosis-related endocrine system diseases and conditions;
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复免疫低下、类风湿关节炎、红斑狼疮相关的免疫系统疾病和状况中的应用；Use of the product or drug of the multi-epitope ligand protein of the present invention in diagnosing, or preventing, or treating, or recovering from immunosuppression, rheumatoid arthritis, and lupus erythematosus-related immune system diseases and conditions;
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复肾病综合征、间质性肾炎、肾衰竭、泌尿、生殖系统感染、肾盂肾炎、膀胱炎、前列腺炎、尿道炎、附睾炎或睾丸炎、前列腺增生、膀胱过度活动症、性功能障碍相关、以及各类男科、妇科感染性炎症和功能性疾病等的泌尿生殖系统疾病和状况中的应用。The products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of nephrotic syndrome, interstitial nephritis, renal failure, urinary and reproductive system infections, pyelonephritis, Cystitis, prostatitis, urethritis, epididymitis or orchitis, benign prostatic hyperplasia, overactive bladder, sexual dysfunction-related, as well as various male and gynecological infectious inflammatory and functional diseases and other urogenital diseases and conditions Applications.
本发明所述多表位的配体蛋白的制品或药物在诊断、或和预防、或和治疗、或和康复全身肌肤细胞营养、激活、再生、修复、清除、细腻光洁、紫外黑色素沉积、湿疹、粗糙、裂纹、暗纹、干枯、硬皮、红斑、过敏、神经性皮炎、损伤、青春痘、暗疮、疤痕、暗沉、螨虫、油性皮肤、炎症性皮肤病、自身免疫性皮肤病、色素性皮肤病、皮肤萎缩、变薄、干燥、色素沉着、皱纹增生、表皮角化不良、干皮症、接触性皮炎、抗皮肤衰老、改善皮肤功能、美白祛斑、防治皮肤疾病的相关的皮肤系统疾病和状况中的应用。The products or medicines of the multi-epitope ligand protein of the present invention are used in diagnosis, or prevention, or treatment, or rehabilitation of whole body skin cell nutrition, activation, regeneration, repair, clearing, delicate and smooth, ultraviolet melanin deposition, eczema , rough, cracked, dark lines, dry, crusty, erythema, allergies, neurodermatitis, lesions, pimples, acne, scars, dullness, mites, oily skin, inflammatory skin diseases, autoimmune skin diseases, pigmentation Skin diseases related to skin diseases, skin atrophy, thinning, dryness, hyperpigmentation, wrinkle hyperplasia, dyskeratosis, xeroderma, contact dermatitis, anti-aging, improving skin function, whitening and freckle, preventing and treating skin diseases Applications in Diseases and Conditions.
优选地，所述识别激活动物的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效应的HrpNEch多拟表位配体蛋白的制备：Preferably, the preparation of the HrpNEch polymimetic epitope ligand protein that recognizes and activates multiple types of receptors, membrane proteins and their signaling pathways in animals and induces multifunctional cascade biological effects:
1、所述HrpNEch多拟表位配体蛋白的制备，采用含我们已注册的HrpNEch蛋白(GenBank Protein:AAY17519.1)基因(基因注册号：GenBank:nucleotide:AY999000.1)(克隆到高效表达载体PET28a(+))的工程菌，通过发酵，纯化制备HrpNEch多拟表位配体蛋白：1. The HrpNEch multi-epitope ligand protein was prepared by using the gene containing our registered HrpNEch protein (GenBank Protein: AAY17519.1) (Gene Registration No.: GenBank: nucleotide: AY999000.1) (cloned into a high-efficiency expression The engineered bacteria of the carrier PET28a(+)) are fermented and purified to prepare the HrpNEch polymimetic epitope ligand protein:
1)HrpNEch多拟表位配体蛋白的工程菌发酵制备：将带有编码HrpNEch多拟表位配体蛋白的基因(包 括，但不限于生物样品天然基因、化学合成基因、转基因遗传重组体基因以及相似基因及其基因修饰)质粒的工程菌(E.coli)，相关蛋白的生产系是K-12原菌经特殊改造后的衍生菌JY-01(DE3)，在LB液体培养基(每升含卡那霉素50微克)中，在一定的温度下条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体。用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析表达产物HrpNEch多拟表位配体蛋白，在电泳胶板的样品泳道上，会呈现一条34.15kda条带，它就是基因hrpNEch的表达产物HrpNEch多拟表位配体蛋白；1) The engineering bacteria fermentation preparation of HrpNEch multi-epitope ligand protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes) encoding HrpNEch multi-epitope ligand proteins are prepared. and engineering bacteria (E.coli) with similar genes and their genetic modifications) plasmids, and the production line of related proteins is a specially modified derivative of the original K-12 bacteria JY-01 (DE3), in LB liquid medium (every liter containing kanamycin 50 μg), at a certain temperature, when cultured to OD600=0.7, add IPTG (isopropyl thiogalactoside, Isopropylβ-D-Thiogalactosid) (final concentration 1mMol), After culturing, the cells were collected by centrifugation. 10% SDS-PAGE polyacrylamide gel electrophoresis was used to analyze the expression product HrpNEch multi-epitope ligand protein. On the sample lane of the electrophoresis gel plate, there will be a 34.15kda band, which is the expression product of the gene hrpNEch HrpNEch polyprotein. Epitope-like ligand proteins;
其中，发酵用培养基Na 2HPO 4-KH 2PO 4缓冲系统，缓冲系统的pH范围是1-14；优选地pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-7；最优选地pH 6.5-5.5； Wherein, the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system, the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
发酵温度范围0-60℃。优选地温度为0-20℃；优选地温度为20-35℃；优选地温度为60-50℃；优选地温度为50-45℃；最优选地温度为37-38℃；The fermentation temperature range is 0-60℃. Preferably the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
发酵增殖液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地0.00％-0.01％；优选地1.00％-0.3％；最优选地0.01％-0.05％；最优选地0.1％-0.05％；Fermentation proliferation liquid medium glucose concentration range 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%;
发酵诱导液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地1.00％-0.3％；优选地0.3％-0.1％；优选地0.1％-0.05％；最优选地0.05％-0.00％；Fermentation induction liquid medium glucose concentration range 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%;
发酵诱导液体培养基乳糖浓度范围10.00％-0.00％；优选地10.00％-1.00％；优选地0.00％-0.1％；优选地1.00％-0.6％；优选地0.1％-0.3％；最优选地0.5％-0.4％；Fermentation induction liquid medium lactose concentration range 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%;
发酵诱导液体培养时间范围0-24h；优选地时间0-2h；优选地时间为24-15h；优选地时间为2-6h；优选地时间为15-10h；最优选地时间为7-9h。The time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
2)HrpNEch多拟表位配体蛋白基因工程菌在生产发酵结束后的后处理：①灭菌发酵液在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃以下；②清洗用葡萄糖Na 2HPO 4-KH 2PO 4缓冲液(pH范围是1-14，葡萄糖浓度范围是0-2500mmol，缓冲系统pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-6；最优选地pH 5-5.5。葡萄糖浓度为0-100mmol；优选地浓度为100-200mmol；优选地浓度为2500-1000mmol；优选地浓度为1000-300mmol；最优选地浓度为200-300mmol，在蝶式连续流离心机里清洗工程菌体五至八次；③破碎工程菌并清除细胞壁，再用pH 5-5.5、葡萄糖浓度200-300mmol的Na 2HPO 4-KH 2PO 4缓冲液稀释菌体，调节菌体鲜重为稀释液的20％-30％，导入高压破碎机，连续用800-1000Mpa压力，破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁，收集HrpNEch多表位蛋白分子，HrpNEch多拟表位配体蛋白存在于上清液中。 2) Post-processing of HrpNEch polymimetic epitope ligand protein genetically engineered bacteria after production and fermentation: ① Sterilize the fermentation broth at 80°C for 30 minutes, and quickly cool down to below 30°C; ② Clean with Glucose Na 2 HPO 4 -KH 2 PO 4 buffer (pH range is 1-14, glucose concentration range is 0-2500 mmol, buffer system pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably 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, wash the engineering bacteria five to eight times in the butterfly continuous flow centrifuge; 3. break the engineering bacteria and remove the cell wall, and then use the Na 2 HPO 4 -KH 2 PO of pH 5-5.5, glucose concentration 200-300mmol 4. Dilute the bacteria with buffer solution, adjust the fresh weight of the bacteria to 20%-30% of the diluted solution, import it into a high-pressure crusher, use a continuous pressure of 800-1000Mpa to crush engineering bacteria, and pass the crushed bacteria liquid into a butterfly continuous flow centrifuge , to clear the cell wall and collect HrpNEch polyepitope protein molecules, HrpNEch polyepitope ligand proteins are present in the supernatant.
3)HrpNEch多拟表位配体蛋白分子的纯化3) Purification of HrpNEch polymimetic epitope ligand protein molecules
用NI-NTA琼脂糖凝胶柱纯化HrpNEch多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，完成HrpNEch多拟表位配体蛋白的纯化制备。The HrpNEch polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column. The protein purification was carried out according to the method suggested by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEch polymimetic epitope ligand protein. preparation.
2、所述HrpNEch多拟表位配体蛋白的制备，进一步，HrpNEch蛋白也可以通过“人工合成基因”的表达蛋白进行制备，具体包括以下步骤：2. The preparation of the HrpNEch multi-epitope ligand protein, further, the HrpNEch protein can also be prepared by the expression protein of "artificially synthesized gene", which specifically includes the following steps:
编码HrpNEch蛋白的hrpNEch基因的人工合成及其表达蛋白制备Artificial synthesis of hrpNEch gene encoding HrpNEch protein and preparation of its expressed protein
1)按照GeneBank公布的编码HrpNEch蛋白的hrpNEch基因(GenBank:AY999000.1)核苷酸序列,人工合成hrpNEch基因，其DNA序列为：1) According to the nucleotide sequence of the hrpNEch gene (GenBank: AY999000.1) encoding the HrpNEch protein published by GeneBank, artificially synthesize the hrpNEch gene, and its DNA sequence is:
Figure PCTCN2021134714-appb-000007
Figure PCTCN2021134714-appb-000007
Figure PCTCN2021134714-appb-000008
Figure PCTCN2021134714-appb-000008
2)根据以上DNA序列，人工合成蛋白基因时，在基因的5′和3′分别加上BamHI和HindIII酶切位点，方便蛋白基因克隆；2) According to the above DNA sequence, when artificially synthesizing the protein gene, add BamHI and HindIII enzyme cleavage sites to the 5' and 3' of the gene respectively to facilitate the cloning of the protein gene;
人工基因合成委托Thermo Fisher Scientific公司的GeneArt基因合成与服务部门完成。人工合成蛋白基因的优点主要是：a)合成周期短，可以保证序列的100％正确无误；b)可以对密码子进行优化，以提高基因的表达效率；由于每个物种偏爱的编码子不同，当异源蛋白在大肠杆菌里表达时，有些蛋白很难得到高表达。如果将异源蛋白的密码子改为大肠杆菌偏爱的密码子，就可以实现蛋白的基因的高效表达，提高该基因的表达水平，适于大规模工业生产；c)可根据需要进行基因的定点突变以改造基因，提高蛋白的作用效率；d)研究人员可根据自己的意愿设计得到自然界中很难获得甚至不存在的基因。The artificial gene synthesis was entrusted to the GeneArt Gene Synthesis and Services Division of Thermo Fisher Scientific. The advantages of artificially synthesized protein genes are: a) the synthesis cycle is short, and the sequence can be guaranteed to be 100% correct; b) the codons can be optimized to improve the expression efficiency of the gene; due to the different coding codons preferred by each species, When heterologous proteins are expressed in E. coli, some proteins are difficult to express at high levels. If the codon of the heterologous protein is changed to the codon preferred by Escherichia coli, the high-efficiency expression of the protein gene can be achieved, the expression level of the gene can be improved, and it is suitable for large-scale industrial production; c) The gene can be designated as needed. Mutations are used to modify genes and improve the efficiency of proteins; d) Researchers can design genes that are difficult to obtain or even do not exist in nature according to their own wishes.
3)将合成的编码HrpNEch蛋白基因的DNA片段，逐一克隆到高效蛋白表达载体PET28a(+)(含His-Tag标签)BamHI-HindIII位点，经DNA测序确保克隆的准确性；3) The synthesized DNA fragments encoding the HrpNEch protein gene are cloned into the high-efficiency protein expression vector PET28a(+) (containing His-Tag label) BamHI-HindIII site one by one, and the accuracy of the clone is ensured through DNA sequencing;
4)HrpNEch多拟表位配体蛋白的工程菌发酵制备：将1)至3)编码HrpNEch蛋白的基因克隆转入大肠杆菌工程菌(E.coli)中，相关蛋白的生产系(E.coli)是K-12原菌经特殊改造后的衍生菌JY-01(DE3)；在LB液体培养基(每升含卡那霉素50微克)中，在一定的温度下条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体，用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析编码HrpNEch多拟表位配体蛋白，在电泳胶板的样品泳道上，会呈现一条34.15kda条带，它就是基因hrpNEch的表达产物HrpNEch多拟表位配体蛋白。4) Fermentative preparation of HrpNEch multi-epitope ligand protein by engineering bacteria: clones 1) to 3) encoding HrpNEch protein were transferred into Escherichia coli engineering bacteria (E.coli), and the production line of related proteins (E.coli ) is a specially modified derivative of the original K-12 bacteria JY-01 (DE3); in LB liquid medium (containing 50 micrograms of kanamycin per liter), under certain temperature conditions, cultivate to OD600 = 0.7, add IPTG (isopropyl thiogalactoside, Isopropylβ-D-Thiogalactosid) (final concentration 1mMol), continue to culture, centrifuge to collect bacteria, use 10% SDS-PAGE polyacrylamide gel electrophoresis to analyze the code The HrpNEch multi-epitope ligand protein shows a 34.15kda band on the sample lane of the electrophoresis gel plate, which is the HrpNEch multi-epitope ligand protein, the expression product of the gene hrpNEch.
其中，发酵用培养基Na 2HPO 4-KH 2PO 4缓冲系统，缓冲系统的pH范围是1-14；优选地pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-7；最优选地pH 6.5-5.5； Wherein, the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system, the pH range of the buffer system is 1-14; preferably pH 1-3; preferably pH 14-10; preferably pH 4-5; pH 9-7; most preferably pH 6.5-5.5;
发酵温度范围0-60℃。优选地温度为0-20℃；优选地温度为20-35℃；优选地温度为60-50℃；优选地温度为50-45℃；最优选地温度为37-38℃；The fermentation temperature range is 0-60℃. Preferably the temperature is 0-20°C; preferably the temperature is 20-35°C; preferably the temperature is 60-50°C; preferably the temperature is 50-45°C; most preferably the temperature is 37-38°C;
发酵增殖液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地0.00％-0.01％；优选地1.00％-0.3％；最优选地0.01％-0.05％；最优选地pH0.1％-0.05％；Fermentation proliferation liquid medium glucose concentration range 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 pH0.1%-0.05%;
发酵诱导液体培养基葡萄糖浓度范围3.00％-0.00％；优选地3.00％-1.00％；优选地1.00％-0.3％；优选地0.3％-0.1％；优选地0.1％-0.05％；最优选地0.05％-0.00％；Fermentation induction liquid medium glucose concentration range 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%;
发酵诱导液体培养基乳糖浓度范围10.00％-0.00％；优选地10.00％-1.00％；优选地0.00％-0.1％；优选地1.00％-0.6％；优选地0.1％-0.3％；最优选地0.5％-0.4％；Fermentation induction liquid medium lactose concentration range 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%;
发酵诱导液体培养时间范围0-24h；优选地时间0-2h；优选地时间为24-15h；优选地时间为2-6h；优选地时间为15-10h；最优选地时间为7-9h。The time range of fermentation induction liquid culture is 0-24h; preferably time is 0-2h; preferably time is 24-15h; preferably time is 2-6h; preferably time is 15-10h; most preferably time is 7-9h.
5)HrpNEch多拟表位配体蛋白基因工程菌在生产发酵结束后的后处理：①灭菌发酵液在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃以下；②清洗用葡萄糖Na 2HPO 4-KH 2PO 4缓冲液(pH范围是1-14， 葡萄糖浓度范围是0-2500mmol，缓冲系统pH 1-3；优选地pH 14-10；优选地pH 4-5；优选地pH 9-6；最优选地pH 5-5.5。葡萄糖浓度为0-100mmol；优选地浓度为100-200mmol；优选地浓度为2500-1000mmol；优选地浓度为1000-300mmol；最优选地浓度为200-300mmol，在蝶式连续流离心机里清洗工程菌体五至八次；③破碎工程菌并清除细胞壁，再用pH 5-5.5、葡萄糖浓度200-300mmol的Na 2HPO 4-KH 2PO 4缓冲液稀释菌体，调节菌体鲜重为稀释液的20％-30％，导入高压破碎机，连续用800-1000Mpa压力，破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁，收集HrpNEch多拟表位配体蛋白分子，HrpNEch多拟表位配体蛋白存在于上清液中。 5) Post-treatment of HrpNEch polymimetic epitope ligand protein genetically engineered bacteria after production and fermentation: ① Sterilize fermentation broth at 80°C for 30 minutes to complete the sterilization treatment, and quickly cool down to below 30°C; ② Clean with Glucose Na 2 HPO 4 -KH 2 PO 4 buffer (pH range is 1-14, glucose concentration range is 0-2500 mmol, buffer system pH 1-3; preferably pH 14-10; preferably pH 4-5; preferably 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, wash the engineering bacteria five to eight times in the butterfly continuous flow centrifuge; 3. break the engineering bacteria and remove the cell wall, and then use the Na 2 HPO 4 -KH 2 PO of pH 5-5.5, glucose concentration 200-300mmol 4. Dilute the bacteria with buffer solution, adjust the fresh weight of the bacteria to 20%-30% of the diluted solution, import it into a high-pressure crusher, use a continuous pressure of 800-1000Mpa to crush engineering bacteria, and pass the crushed bacteria liquid into a butterfly continuous flow centrifuge , remove the cell wall, and collect the HrpNEch polyepitope ligand protein molecules, which are present in the supernatant.
6)HrpNEch多拟表位配体蛋白分子的纯化6) Purification of HrpNEch polymimetic epitope ligand protein molecules
用NI-NTA琼脂糖凝胶柱纯化HrpNEch多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，完成HrpNEch多拟表位配体蛋白的纯化制备。The HrpNEch polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column. The protein purification was carried out according to the method suggested by the NI-NTA agarose column manufacturer to complete the purification of the HrpNEch polymimetic epitope ligand protein. preparation.
与现有的技术相比本发明的有益效果是：Compared with the prior art, the beneficial effects of the present invention are:
HrpN型多拟表位配体蛋白作为一类富含多个线性和构象表位特殊结构的配体蛋白分子，能够跨界识别、激活、结合多种类型的动物的膜受体、膜蛋白、信息通路和代谢通路，HrpN型多拟表位配体蛋白是一类具有特殊多个表位结构、全新功能、全新作用机制和全新应用前景的配体蛋白，它们诱导的多方向、多层次和多方面的生物学效应和功能，广泛涉及多系统、多组织、多器官、多细胞相关疾病和状况的诊断、或和预防、或和治疗、或和康复以及广泛涉及有关疾病和状况的食字号、消字号、妆字号、械字号和健字号制品或药物的制药中的应用。HrpN-type multi-epitope ligand proteins, as a class of ligand protein molecules rich in multiple linear and conformational epitopes with special structures, can recognize, activate and bind membrane receptors, membrane proteins, Information pathways and metabolic pathways, HrpN-type multi-epitope ligand proteins are a class of ligand proteins with special multiple epitope structures, new functions, new mechanisms of action and new application prospects. They induce multi-directional, multi-level and Multi-faceted biological effects and functions, widely involved in the diagnosis, or prevention, or treatment, or recovery of multi-system, multi-tissue, multi-organ, and multi-cell related diseases and conditions, and related diseases and conditions. , eliminate font size, makeup font size, mechanical font size and health font size products or drugs in the pharmaceutical application.
附图说明Description of drawings
图1为HrpNEcb多拟表位配体蛋白纯化前后的电泳检测：左边为分子量标识带，其中1：高效表达HrpNEcb多拟表位配体蛋白条带(纯化前)；2：纯化后多拟表位配体蛋白HrpNEcb条带。Figure 1 shows the electrophoresis detection of the HrpNEcb polymimetic epitope ligand protein before and after purification: the left side is the molecular weight marker band, of which 1: the highly expressed HrpNEcb polymimetic epitope ligand protein band (before purification); 2: the polymimetic epitope after purification The ligand protein HrpNEcb band.
图2为HrpNEcb多拟表位配体蛋白液注射诱导烟草叶片过敏反应图:其中所见焦斑处是经过HarpinEcb蛋白液处理24hr左右所形成，B、D：H 2O注射；A、C：HarpinEcb蛋白液(250μg/ml)注射，即HarpinEcb蛋白在烟草叶片上的超敏反应，B、D为对照，A、C为处理。 Fig. 2 is that HrpNEcb multi-epitope ligand protein liquid injection induces the allergic reaction diagram of tobacco leaves: wherein the focal spot is formed through the treatment of HarpinEcb protein liquid about 24hr, B, D: H 2 O injection; A, C: HarpinEcb protein solution (250μg/ml) injection, namely the hypersensitivity reaction of HarpinEcb protein on tobacco leaves, B and D are controls, A and C are treatments.
图3为本发明的HrpNEcb多拟表位配体蛋白通过口服和涂抹实验鼠诱导肝脏表达差异基因火山图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 3 is a volcano plot of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the liver, from left to right: oral administration for 6 hours, oral administration for 24 hours;
图4为本发明的HrpNEcb多拟表位配体蛋白通过口服和涂抹实验鼠诱导丘脑表达差异基因火山图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 4 is a volcano diagram of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the thalamus, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
图5为本发明的HrpNEcb多拟表位配体蛋白通过口服和涂抹实验鼠诱导心脏表达差异基因火山图，自左至右依次为口服6h、口服24h；涂抹6h、涂抹12h；Figure 5 is a volcano plot of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the heart, from left to right: oral administration for 6h, oral administration for 24h; smear for 6h, smear for 12h;
图6为本发明的HrpNEcb多拟表位配体蛋白通过口服和涂抹实验鼠诱导大脑皮层表达差异基因火山图，自左至右依次为口服6h、口服24h；涂抹6h、涂抹12h；Figure 6 is a volcano diagram of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the cerebral cortex, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours, application for 12 hours;
图7为本发明的HrpNEcb多拟表位配体蛋白口服和涂抹实验鼠诱导大脑海马表达差异基因火山图，自左至右依次为口服6h、口服24h；涂抹6h、涂抹12h；Figure 7 is a volcano diagram of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differential gene expression in the hippocampus of the brain, from left to right: oral administration for 6h, oral administration for 24h;
图8为本发明的HrpNEcb多拟表位配体蛋白口服和涂抹实验鼠诱导肝脏表达差异基因集聚类热图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 8 is a cluster heat map of HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differentially expressed gene sets in the liver, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
图9为本发明的HrpNEcb多拟表位配体蛋白口服和涂抹实验鼠诱导丘脑表达差异基因集聚类热图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 9 is a clustering heat map of the HrpNEcb multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differentially expressed gene sets in the thalamus, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
图10为本发明的HrpNEcb多拟表位配体蛋白口服和涂抹实验鼠诱导海马表达差异基因集聚类热图，自左 至右依次为口服6h、口服24h；涂抹6h；Figure 10 is a cluster heat map of the HrpNEcb multi-epitope ligand protein of the present invention orally and smearing experimental mice to induce differential gene set expression in the hippocampus, from left to right are oral administration for 6h, oral administration for 24h; smear for 6h;
图11为本发明的HrpNEcb多拟表位配体蛋白口服和涂抹实验鼠诱导大脑皮层表达差异基因集聚类热图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 11 is a clustering heat map of the differentially expressed gene sets in the cerebral cortex induced by oral administration of HrpNEcb multi-epitope ligand protein and smearing in experimental mice, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
图12为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白口服6小时处理实验鼠肝脏与对照比较(总基因)；Fig. 12 is KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally 6 hours to deal with the comparison of experimental mouse liver and control (total gene);
图13为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白口服24小时处理实验鼠肝脏与对照比较(总基因)；Figure 13 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein orally for 24 hours to treat the experimental mouse liver and control (total gene);
图14为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠肝脏与对照比较(总基因)；Figure 14 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat experimental mouse liver and control (total gene);
图15为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白口服6小时处理实验鼠肝脏与对照比较(上调基因)；Figure 15 is a comparison of the KEGG Pathway:HrpEcb multi-epitope ligand protein of the present invention orally treating the experimental mouse liver for 6 hours and the control (up-regulated gene);
图16为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白口服24小时处理实验鼠肝脏与对照比较(上调基因)；Figure 16 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein orally treated experimental mouse liver for 24 hours (up-regulated gene);
图17为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠肝脏与对照比较(上调基因)；Figure 17 is a comparison of the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the experimental mouse liver and the control (up-regulated gene);
图18为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白口服6小时处理实验鼠肝脏与对照比较(下调基因)；Figure 18 is a comparison of the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein orally for 6 hours to treat the experimental mouse liver and the control (down-regulated gene);
图19为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白口服24小时处理实验鼠肝脏与对照比较(下调基因)；Figure 19 is a comparison of the KEGG Pathway:HrpEcb multi-epitope ligand protein of the present invention orally treating the experimental mouse liver for 24 hours and the control (down-regulated gene);
图20为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠肝脏与对照比较(下调基因)；Figure 20 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the experimental mouse liver and control (down-regulated gene);
图21为本发明的KEGG Pathway:HrpEcb多拟表位配体蛋白口服6小时处理实验鼠心脏与对照比较(总基因)；Fig. 21 is KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours and compared with the control (total gene) of experimental mouse heart;
图22为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠心脏与对照比较(总基因)；Figure 22 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours and compared with the control (total gene);
图23为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠心脏与对照比较(总基因)；Figure 23 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 6 hours to treat the experimental mouse heart and control comparison (total gene);
图24为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠心脏与对照比较(总基因)；Figure 24 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 12 hours to treat the heart of the experimental mouse compared with the control (total gene);
图25为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠心脏与对照比较(上调基因)；Figure 25 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the heart of experimental mice and the control (up-regulated genes);
图26为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠心脏与对照比较(上调基因)；Figure 26 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours and compared with the control (up-regulated gene);
图27为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠心脏与对照比较(上调基因)；Figure 27 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 6 hours to treat the heart of experimental mice and control (up-regulated genes);
图28为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠心脏与对照比较(上调基因)；Figure 28 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours to treat the heart of experimental mice and control (up-regulated genes);
图29为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠心脏与对照比较(下调基因)；Figure 29 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in experimental mouse heart and control (down-regulated genes);
图30为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠心脏与对照比较(下调基因)；Figure 30 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours and compared with the control (down-regulated gene);
图31为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠心脏与对照比较(下调基因)；Figure 31 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the heart of experimental mice and control (down-regulated genes);
图32为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠心脏与对照比较(下调基因)；Figure 32 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 12 hours to treat the heart of experimental mice and control (down-regulated genes);
图33为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑海马与对照比较(总基因)；Figure 33 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the brain hippocampus of experimental mice and the control (total genes);
图34为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑海马与对照比较(总基因)；Figure 34 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the brain hippocampus of experimental mice and the control (total genes);
图35为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑海马与对照比较(总基因)；Figure 35 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours to treat the brain hippocampus of experimental mice and control (total genes);
图36为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠大脑海马与对照比较(总基因)；Figure 36 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smeared for 12 hours to treat the rat brain hippocampus and the control comparison (total genes);
图37为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑海马与对照比较(上调基因)；Figure 37 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the brain hippocampus of experimental mice and the control (up-regulated genes);
图38为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑海马与对照比较(上调基因)；Figure 38 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the brain hippocampus of experimental mice and controls (up-regulated genes);
图39为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑海马与对照比较(上调基因)；Figure 39 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the brain hippocampus of experimental mice and control (up-regulated genes);
图40为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠大脑海马与对照比较(上调基因)；Figure 40 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the brain hippocampus of experimental mice and control (up-regulated genes);
图41为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑海马与对照比较(下调基因)；Figure 41 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the brain hippocampus of experimental mice and the control (down-regulated genes);
图42为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑海马与对照比较(下调基因)；Figure 42 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the brain hippocampus of experimental mice and the control (down-regulated genes);
图43为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑海马与对照比较(下调基因)；Figure 43 is a comparison of the KEGG Pathway of the present invention: HrpEcb polymimetic epitope ligand protein smearing for 6 hours in the brain hippocampus of the experimental mice and the control (down-regulated genes);
图44为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠大脑海马与对照比较(下调基因)；Figure 44 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the brain hippocampus of experimental mice and control (down-regulated genes);
图45为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑皮层与对照比较(总基因)；Figure 45 is a comparison of the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours between the cerebral cortex of the experimental mouse and the control (total genes);
图46为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑皮层与对照比较(总基因)；Figure 46 is the KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral cortex of experimental mice compared with the control (total genes);
图47为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑皮层与对照比较(总基因)；Figure 47 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral cortex of experimental mice and control (total genes);
图48为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠大脑皮层与对照比较(总基因)；Figure 48 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the cerebral cortex of experimental mice and control (total genes);
图49为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑皮层与对照比 较(上调基因)；Figure 49 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in experimental mouse cerebral cortex and control (up-regulated gene);
图50为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑皮层与对照比较(上调基因)；Figure 50 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral cortex of experimental mice and controls (up-regulated genes);
图51为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑皮层与对照比较(上调基因)；Figure 51 shows the comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral cortex of experimental mice and control (up-regulated genes);
图52为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠大脑皮层与对照比较(上调基因)；Figure 52 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the cerebral cortex of experimental mice and control (up-regulated genes);
图53为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑皮层与对照比较(下调基因)；Figure 53 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the cerebral cortex of experimental mice and controls (down-regulated genes);
图54为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑皮层与对照比较(下调基因)；Figure 54 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours between the cerebral cortex of experimental mice and the control (down-regulated genes);
图55为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹12小时处理实验鼠大脑皮层与对照比较(下调基因)；Figure 55 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 12 hours in the cerebral cortex of experimental mice and control (down-regulated genes);
图56为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑丘脑与对照比较(总基因)；Figure 56 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours between the cerebral thalamus of experimental mice and the control (total genes);
图57为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑丘脑与对照比较(总基因)；Figure 57 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours between the cerebral thalamus of experimental mice and the control (total genes);
图58为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑丘脑与对照比较(总基因)；Figure 58 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral thalamus of experimental mice and control (total genes);
图59为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑丘脑与对照比较(上调基因)；Figure 59 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the cerebral thalamus of experimental mice and the control (up-regulated genes);
图60为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑丘脑与对照比较(上调基因)；Figure 60 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral thalamus of experimental mice and the control (up-regulated genes);
图61为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑丘脑与对照比较(上调基因)；Figure 61 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral thalamus of experimental mice and control (up-regulated genes);
图62为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服6小时处理实验鼠大脑丘脑与对照比较(下调基因)；Figure 62 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 6 hours in the cerebral thalamus of experimental mice and the control (down-regulated genes);
图63为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白口服24小时处理实验鼠大脑丘脑与对照比较(下调基因)；Figure 63 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein is orally treated for 24 hours in the cerebral thalamus of experimental mice and the control (down-regulated genes);
图64为本发明的KEGG Pathway：HrpEcb多拟表位配体蛋白涂抹6小时处理实验鼠大脑丘脑与对照比较(下调基因)；Figure 64 is a comparison of KEGG Pathway of the present invention: HrpEcb multi-epitope ligand protein smearing for 6 hours in the cerebral thalamus of experimental mice and control (down-regulated genes);
图65为本发明的mRNA(RNA-Seq)测序实验流程图；Figure 65 is a flow chart of the mRNA (RNA-Seq) sequencing experiment of the present invention;
图66为本发明的mRNA测序数据分析流程图；Figure 66 is a flow chart of mRNA sequencing data analysis of the present invention;
图67为HrpNEch蛋白纯化前后的电泳检测：左边为分子量标识带，1：纯化前HrpNEch多拟表位配体蛋白条带；2：纯化后HrpNEch多拟表位配体蛋白条带；Figure 67 shows the electrophoresis detection of HrpNEch protein before and after purification: the left side is the molecular weight marker band, 1: HrpNEch polymimetic epitope ligand protein band before purification; 2: HrpNEch polymimetic epitope ligand protein band after purification;
图68为HrpNEch多拟表位配体蛋白液注射诱导烟草叶片过敏反应图:其中所见焦斑处是经过HrpNEch多拟表位配体蛋白液处理24hr左右所形成，叶片上排：H2O注射，为对照；叶片下排：HrpNEch多拟表位配体蛋白液(250μg/ml)注射，为处理，即HrpNEch多拟表位配体蛋白在烟草叶片上的超敏反应；Figure 68 is a graph of HrpNEch multi-mimetic epitope ligand protein liquid injection to induce allergic reactions in tobacco leaves: where the focal spots seen are formed through the treatment of HrpNEch multi-mimetic epitope ligand protein liquid for about 24hr, the upper row of leaves: H2O injection, For the control; the lower row of leaves: HrpNEch multi-epitope ligand protein solution (250μg/ml) injection, for the treatment, that is, the hypersensitivity reaction of HrpNEch multi-epitope ligand protein on tobacco leaves;
图69为本发明的HrpNEch多拟表位配体蛋白通过口服和涂抹实验鼠诱导肾脏表达差异基因火山图，自左 至右依次为口服6h、口服24h；涂抹6h；Figure 69 is the HrpNEch multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differential gene expression in the kidney, and from left to right are oral administration for 6h, oral administration for 24h; smear for 6h;
图70为本发明的HrpNEch多拟表位配体蛋白通过口服和涂抹实验鼠诱导睾丸表达差异基因火山图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 70 is a volcano plot of the HrpNEch multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in testis, from left to right: oral administration for 6h, oral administration for 24h; smear for 6h;
图71为本发明的HrpNEch多拟表位配体蛋白口服和涂抹实验鼠诱导肾脏表达差异基因集聚类热图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 71 is a cluster heat map of the differentially expressed genes in the kidneys induced by the oral administration of HrpNEch multi-epitope ligand protein and smearing experimental mice, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
图72为本发明的HrpNEch多拟表位配体蛋白口服和涂抹实验鼠诱导睾丸表达差异基因集聚类热图，自左至右依次为口服6h、口服24h；涂抹6h；Figure 72 is a cluster heat map of the differentially expressed genes in testis induced by oral administration of HrpNEch multi-epitope ligand protein and smearing in experimental mice, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
图73为本发明的HrpNEch多拟表位配体蛋白处理实验鼠肾脏与对照比较KEGG Pathway(总基因)，自左至右依次为口服6h、口服24h；涂抹6h；Figure 73 shows the comparison of the KEGG Pathway (total gene) of the experimental mouse kidney treated with the HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
图74为本发明的HrpNEch多拟表位配体蛋白处理实验鼠肾脏与对照比较KEGG Pathway(上调基因)，自左至右依次为口服6h、口服24h；涂抹6h；Figure 74 shows the comparison of KEGG Pathway (up-regulated gene) in the kidney of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention (up-regulated gene), from left to right, oral administration for 6 hours, oral administration for 24 hours; smearing for 6 hours;
图75为本发明的HrpNEch多拟表位配体蛋白处理实验鼠肾脏与对照比较KEGG Pathway(下调基因)，自左至右依次为口服6h、口服24h；涂抹6h；Figure 75 shows the comparison of the KEGG Pathway (down-regulated gene) in the kidneys of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right: oral administration for 6 hours, oral administration for 24 hours;
图76为本发明的HrpNEch多拟表位配体蛋白处理实验鼠睾丸与对照比较KEGG Pathway(总基因)，自左至右依次为口服6h、口服24h；涂抹6h；Figure 76 shows the comparison of KEGG Pathway (total gene) in the testis of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
图77为本发明的HrpNEch多拟表位配体蛋白处理实验鼠睾丸与对照比较KEGG Pathway(上调基因)，自左至右依次为口服6h、口服24h；涂抹6h；Figure 77 shows the comparison of KEGG Pathway (up-regulated gene) in the testis of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention (up-regulated gene), from left to right: oral administration for 6h, oral administration for 24h; smear for 6h;
图78为本发明的HrpNEch多拟表位配体蛋白处理实验鼠睾丸与对照比较KEGG Pathway(下调基因)，自左至右依次为口服6h、口服24h；涂抹6h；Figure 78 shows the comparison of KEGG Pathway (down-regulated gene) in the testis of experimental mice treated with HrpNEch multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
图79为本发明的mRNA(RNA-Seq)测序实验流程图；Figure 79 is a flow chart of the mRNA (RNA-Seq) sequencing experiment of the present invention;
图80为本发明的mRNA测序数据分析流程图。Figure 80 is a flow chart of mRNA sequencing data analysis of the present invention.
具体实施方式Detailed ways
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅用以解释本发明，并不用于限定本发明。In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.
实施例1Example 1
HrpN同源性比较如说明书所述，此处不再重复。The HrpN homology comparison is as described in the specification and will not be repeated here.
下述实施例中所使用的试验方法如无特殊，均为常规方法。The test methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊的说明，均可从商业途径得到。The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例2Example 2
HrpNEcb多拟表位配体蛋白采用已注册的EcbCSL101hrpNEcb基因(GenBank:ABD22989.1)(克隆到高效表达载体PET28a(+))的工程菌发酵，纯化制备和收集HrpNEcb多拟表位配体蛋白，具体包括以下步骤：The HrpNEcb multi-epitope ligand protein was fermented with the registered EcbCSL101hrpNEcb gene (GenBank: ABD22989.1) (cloned into the high-efficiency expression vector PET28a(+)), and the HrpNEcb multi-epitope ligand protein was purified, prepared and collected. Specifically include the following steps:
1)HrpNEcb蛋白的工程菌发酵制备：将编码HrpNEcb蛋白的基因(包括，但不限于生物样品天然基因、化学合成基因、转基因遗传重组体基因以及相似基因及其基因修饰)的工程菌(E.coli)，相关蛋白的生产系是K-12原菌经特殊改造后的衍生菌JY-01(DE3)，在LB液体培养基(每升含卡那霉素50微克)中，在一定的温度下条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体。用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析表达产物HrpNEcb多拟表位配体蛋白，在电泳胶板的样品泳道上，会呈现一条36.64kda条带，它就是基因hrpNEcb的表达产物HrpNEcb多拟表位配体蛋白。其中，发酵用培养基Na 2HPO 4-KH 2PO 4缓 冲系统pH 6.5-5.5；发酵温度为37-38℃；发酵增殖液体培养基葡萄糖浓度0.01％-0.05％；发酵诱导液体培养基葡萄糖浓度0.05％-0.00％；发酵诱导液体培养基乳糖浓度0.5％-0.4％；发酵诱导液体培养时间为7-9h。 1) The engineering bacteria fermentation preparation of HrpNEcb protein: the engineering bacteria (E. coli), the production line of the related protein is a specially modified derivative of the original K-12 bacteria JY-01 (DE3), in LB liquid medium (containing 50 micrograms of kanamycin per liter) at a certain temperature Under the following conditions, when cultured to OD600=0.7, IPTG (isopropylthiogalactoside, Isopropylβ-D-Thiogalactosid) (final concentration 1 mMol) was added, and the cells were collected by centrifugation after continuing the culture. Using 10% SDS-PAGE polyacrylamide gel electrophoresis to analyze the expression product HrpNEcb multi-epitope ligand protein, a 36.64kda band will appear on the sample lane of the electrophoresis gel plate, which is the expression product of the gene hrpNEcb HrpNEcb polyprotein. Epitope-like ligand proteins. Among them, the pH of the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system is 6.5-5.5; the fermentation temperature is 37-38° C.; the glucose concentration of the fermentation and proliferation liquid medium is 0.01%-0.05%; 0.05%-0.00%; the lactose concentration of the fermentation induction liquid medium is 0.5%-0.4%; the fermentation induction liquid culture time is 7-9h.
2)工程菌生产系在多拟表位配体蛋白生产发酵结束后的后处理：①灭菌：发酵液在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃以下；②清洗：用葡萄糖Na 2HPO 4-KH 2PO 4缓冲液，pH 5-5.5，葡萄糖浓度200-300mmol，在蝶式连续流离心机里清洗工程菌体五至八次；③破碎工程菌并清除细胞壁，再用pH 5-5.5、葡萄糖浓度200-300mmol的Na 2HPO 4-KH 2PO 4缓冲液稀释菌体，调节菌体鲜重为稀释液的20％-30％，导入高压破碎机，连续用800-1000Mpa压力，破碎工程菌。 2) The post-processing of the engineering bacteria production system after the fermentation of the polymimetic epitope ligand protein production is completed: ① Sterilization: the fermentation broth is sterilized at a temperature of 80 °C for 30 minutes, and then rapidly cooled to below 30 °C; ② Cleaning : Use glucose Na 2 HPO 4 -KH 2 PO 4 buffer, pH 5-5.5, glucose concentration 200-300 mmol, wash the engineering bacteria five to eight times in a butterfly continuous flow centrifuge; ③ break the engineering bacteria and remove the cell wall , and then dilute the cells with a Na 2 HPO 4 -KH 2 PO 4 buffer solution with pH 5-5.5 and a glucose concentration of 200-300 mmol, adjust the fresh weight of the cells to 20%-30% of the diluted solution, and introduce them into a high pressure crusher for continuous Use 800-1000Mpa pressure to break engineering bacteria.
3)离心收集HrpNEcb多拟表位配体蛋白分子，离心力范围1000-8000g，优选地离心力1000-2000g；优选地离心力2000-3500g；优选地离心力8000-6000g；优选地离心力6000-4500g；最优选地离心力3500-4500g。HrpNEcb多拟表位配体蛋白存在于上清液中。3) Centrifugal collection of HrpNEcb polymimetic epitope ligand protein molecules, centrifugal force range 1000-8000g, preferably centrifugal force 1000-2000g; preferably centrifugal force 2000-3500g; preferably centrifugal force 8000-6000g; preferably centrifugal force 6000-4500g; most preferably The centrifugal force is 3500-4500g. The HrpNEcb multi-epitope ligand protein was present in the supernatant.
4)用Ni-NTA琼脂糖凝胶柱纯化HrpNEcb多拟表位配体蛋白-His重组蛋白，蛋白纯化按Ni-NTA琼脂糖凝胶柱厂家建议方法实施，得到纯化的HrpNEcb多拟表位配体蛋白原药。4) Purify the HrpNEcb polymimetic epitope ligand protein-His recombinant protein with Ni-NTA agarose column. body protein drug.
实施例3Example 3
HrpNEcb蛋白通过“人工合成基因”的表达蛋白进行制备，具体包括以下步骤：The HrpNEcb protein is prepared by the expression protein of "artificially synthesized gene", which specifically includes the following steps:
第一步：编码HrpNEcb蛋白的hrpNEcb基因的人工合成；The first step: artificial synthesis of the hrpNEcb gene encoding the HrpNEcb protein;
1)按照GenBank公布的编码HrpNEcb蛋白的hrpNEcb基因(GenBank:DQ355519.1)核苷酸序列,人工合成hrpNEcb基因，其DNA序列为：1) According to the nucleotide sequence of the hrpNEcb gene (GenBank: DQ355519.1) encoding the HrpNEcb protein published by GenBank, artificially synthesize the hrpNEcb gene, and its DNA sequence is:
Figure PCTCN2021134714-appb-000009
Figure PCTCN2021134714-appb-000009
第二步：2)根据以上DNA序列，人工合成蛋白基因时，在基因的5′和3′分别加上BamHI和HindIII酶切位点，方便蛋白基因克隆；Step 2: 2) According to the above DNA sequence, when artificially synthesizing protein gene, add BamHI and HindIII enzyme cleavage sites to the 5' and 3' of the gene respectively to facilitate protein gene cloning;
第三步：人工基因合成委托Thermo Fisher Scientific公司的GeneArt基因合成与服务部门完成。3)将合成的编码HrpNEcb蛋白基因的DNA片段，逐一克隆到高效蛋白表达载体PET28a(+)(含His-Tag标签)的BamHI-HindIII位点，经DNA测序确保克隆的准确性；The third step: artificial gene synthesis is entrusted to the GeneArt gene synthesis and service department of Thermo Fisher Scientific. 3) The synthesized DNA fragments encoding the HrpNEcb protein gene were cloned into the BamHI-HindIII site of the high-efficiency protein expression vector PET28a(+) (containing His-Tag label) one by one, and the accuracy of the clone was ensured through DNA sequencing;
第四步：将1)至3)编码HrpNEcb蛋白的基因克隆转入大肠杆菌工程菌(E.coli)中，相关蛋白的 生产系(E.coli)是K-12原菌经特殊改造后的衍生菌JY-01(DE3)；在LB液体培养基(每升含卡那霉素50微克)中，37℃条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体，用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析表达产物HrpNEcb多拟表位配体蛋白，在电泳胶板的样品泳道上，会呈现一条36.64kda条带，它就是基因hrpNEcb的表达产物HrpNEcb多拟表位配体蛋白，详见图1；The fourth step: clone the gene encoding HrpNEcb protein from 1) to 3) and transfer it into Escherichia coli engineering bacteria (E. Derivative bacteria JY-01 (DE3); in LB liquid medium (containing 50 micrograms of kanamycin per liter), under the condition of 37 °C, when cultured to OD600=0.7, IPTG (isopropyl thiogalactoside) was added. , Isopropylβ-D-Thiogalactosid) (final concentration 1 mMol), continue to culture, collect the bacteria by centrifugation, and analyze the expression product HrpNEcb polymimetic ligand protein by 10% SDS-PAGE polyacrylamide gel electrophoresis. On the sample lane, there will be a 36.64kda band, which is the HrpNEcb multi-epitope ligand protein, the expression product of the gene hrpNEcb, see Figure 1 for details;
其中发酵用培养基Na 2HPO 4-KH 2PO 4缓冲系统，缓冲系统的pH6.5-5.5；发酵增殖液体培养基葡萄糖浓度为0.01％-0.05％；发酵诱导液体培养基乳糖浓度0.5％-0.4％； Among them, the fermentation medium is Na 2 HPO 4 -KH 2 PO 4 buffer system, the pH of the buffer system is 6.5-5.5; the concentration of glucose in the fermentation and proliferation liquid medium is 0.01%-0.05%; the concentration of lactose in the fermentation induction liquid medium is 0.5%- 0.4%;
第五步：将收集菌体悬浮到Na 2HPO 4-KH 2PO 4缓冲液中，在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃，在蝶式连续流离心机里清洗工程菌体五至八次，导入高压破碎机，连续用800-1000Mpa压力下，破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁，收集HrpNEcb多拟表位配体蛋白分子，HrpNEcb多拟表位配体蛋白存在于上清液中； Step 5: Suspend the collected cells into Na 2 HPO 4 -KH 2 PO 4 buffer, complete the sterilization treatment at 80°C for 30 minutes, quickly cool down to 30°C, and place in a butterfly continuous flow centrifuge. Wash the engineered bacteria for five to eight times, import them into a high-pressure crusher, and continuously use 800-1000Mpa pressure to break the engineered bacteria. Pass the broken bacteria liquid into a butterfly continuous flow centrifuge to remove the cell wall and collect HrpNEcb polymimetic epitope ligands. protein molecule, HrpNEcb multi-epitope ligand protein is present in the supernatant;
第六步：HrpNEcb多拟表位配体蛋白分子的纯化Step 6: Purification of HrpNEcb Multi-Epitope Ligand Protein Molecules
用NI-NTA琼脂糖凝胶柱纯化多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，完成HrpNEcb多拟表位配体蛋白的纯化制备。Use NI-NTA agarose column to purify the polymimetic epitope ligand protein-His recombinant protein. The protein purification is carried out according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification and preparation of the HrpNEcb polymimetic epitope ligand protein. .
10％SDS聚丙酰胺凝胶电泳检测高效表达的纯化蛋白-His重组条带，详见图1。The highly expressed purified protein-His recombinant band was detected by 10% SDS polyacrylamide gel electrophoresis, see Figure 1 for details.
如图1所示，左面为分子量标识带；泳道1为纯化前的电泳谱带，对应分子量区聚集了较多的条带，也包括36.64kda条带；泳道2为纯化后的HrpNEcb蛋白条带，分子量36.64kda，在蛋白对应分子量区，表明已经得到相应的纯化的HrpNEcb多拟表位配体蛋白。As shown in Figure 1, the left side is the molecular weight identification band; lane 1 is the electrophoresis band before purification, and there are more bands in the corresponding molecular weight region, including the 36.64kda band; lane 2 is the purified HrpNEcb protein band , the molecular weight is 36.64kda, which is in the corresponding molecular weight region of the protein, indicating that the corresponding purified HrpNEcb polymimetic epitope ligand protein has been obtained.
如图2所示，纯化的多拟表位配体蛋白的过敏实验检测：HrpNEcb多拟表位配体蛋白制剂以及无菌水处理后24hr的烟草叶片反应结果见图2，其中，A、C点为注射300μg·mL -1的HrpNEcb多拟表位配体蛋白液100μL；B、D点为注射无菌水100μL为对照处理。300μg·mL -1的HrpEcb多拟表位配体蛋白液处理12hr左右引起烟草叶片萎缩、塌陷，24hr该处枯死；水对照处理烟草叶片无过敏反应。 As shown in Figure 2, the allergy test detection of the purified polymimetic epitope ligand protein: HrpNEcb polymimetic epitope ligand protein preparation and the reaction results of tobacco leaves 24hr after sterile water treatment are shown in Figure 2, wherein, A, C The point is the injection of 300μg·mL -1 of HrpNEcb multi-epitope ligand protein solution of 100μL; the points B and D are the injection of 100μL of sterile water as the control treatment. 300μg·mL -1 of HrpEcb poly-epitope ligand protein solution treatment for about 12hrs caused tobacco leaves to shrink and collapse, and 24hrs to die; the water control treatment of tobacco leaves had no allergic reaction.
纯化的多拟表位配体蛋白能普遍引发多种植物叶片发生超敏反应，供试植物种类可以是：烟草、辣椒、茄子、西红柿、土豆、草莓、黄瓜、空心菜、鸡冠花、玻璃海棠、九月菊、三色堇、胭脂花、矮牵牛、葡萄、月季、槐树、豌豆、桃树、一串红、丝瓜、四季豆、花椰菜、菠菜、油菜、薯蓣、豇豆、蚕豆、玉米、水稻、大豆、仙客来、桑树、南瓜、枇杷、椿树等36个种类的不同植物。The purified multi-epitope ligand protein can generally induce hypersensitivity reactions in various plant leaves. The tested plant species can be: tobacco, pepper, eggplant, tomato, potato, strawberry, cucumber, spinach, celosia, glass begonia, September chrysanthemum, pansy, nopal, petunia, grape, rose, locust tree, pea, peach tree, bunch of red, loofah, green beans, cauliflower, spinach, rape, yam, cowpea, broad bean, corn, 36 kinds of different plants such as rice, soybean, cyclamen, mulberry, pumpkin, loquat, toon tree, etc.
实施例4Example 4
动物实验mRNA(mRNA-Seq)测序Animal experimental mRNA (mRNA-Seq) sequencing
mRNA测序的研究对象为特定细胞在某一功能状态下所能转录出来的所有带ploy-A尾的RNA，主要为mRNA。通过逆转录过程将细胞产生的mRNA转化为DNA(cDNA，互补，并对获得的cDNA进行文库构建)。然后对得到的DNA进行测序，并从观察到的特定DNA丰度中，从中推断细胞中mRNA的原始量，从而找到在实验条件下转录水平发生变化的基因或转录本，即差异表达。通过找到这些差异表达的基因和转录本，能够全面快速地获得某一物种特定组织或器官在某一状态下的几乎所有mRNA表达丰度，已广泛应用于基础研究和药物研发等多个领域。我们采用mRNA-seq技术，证明了HrpNEcb多拟表位配体蛋白诱导小鼠多个器官中多基因的差异表达。The research object of mRNA sequencing is all RNAs with a poly-A tail that can be transcribed by a specific cell in a certain functional state, mainly mRNA. Cell-generated mRNA is converted into DNA (cDNA, complementation, and library construction of the obtained cDNA) by a process of reverse transcription. The resulting DNA is then sequenced and from the observed abundance of specific DNA, the original amount of mRNA in the cell can be inferred from it to find genes or transcripts whose transcription levels change under the experimental conditions, i.e. differentially expressed. By finding these differentially expressed genes and transcripts, it is possible to comprehensively and quickly obtain almost all mRNA expression abundances of a specific tissue or organ of a species in a certain state, which has been widely used in basic research and drug development and other fields. Using mRNA-seq technology, we demonstrate that the HrpNEcb polyepitopic ligand protein induces differential expression of multiple genes in multiple organs in mice.
1.实验动物样品处理1. Experimental animal sample processing
本实验委托上海华盈生物医药科技有限公司蛋白质谱技术平台实施。This experiment was entrusted to Shanghai Huaying Biomedical Technology Co., Ltd. to implement the protein profiling technology platform.
实验样品处理：实验选用8周龄的balb/C的实验小鼠，分为HrpNEcb多拟表位配体蛋白处理组，包 括口服6小时、24小时和涂抹6小时、12小时共4种处理，每种处理3只实验鼠，共计12只；空白对照组4只实验鼠；无HrpNEcb多拟表位配体蛋白的缓冲液对照假手术组，包括口服6小时、24小时和涂抹6小时、12小时共4种处理，每种处理4只实验鼠，共计16只实验鼠，三次重复；对实验处理组鼠用含600mg·L -1浓度的HrpNEcb多拟表位配体蛋白缓冲液饲喂和涂抹处理，缓冲液对照假手术组小鼠用缓冲液饲喂和涂抹处理，空白对照组小鼠不做任何处理。在相同饲养条件下，按不同时间，分别分组取小鼠大脑皮层、丘脑、大脑海马、肝脏、心脏等组织，进行RNA-Seq的测序及分析。 Treatment of experimental samples: 8-week-old balb/C mice were selected for the experiment and divided into HrpNEcb multi-epitope ligand protein treatment groups, including four treatments of oral administration for 6 hours, 24 hours, and application for 6 hours and 12 hours. There were 3 experimental mice for each treatment, a total of 12 mice; the blank control group had 4 experimental mice; the buffer without HrpNEcb multi-epitope ligand protein controlled the sham-operated group, including oral administration for 6 hours, 24 hours and application for 6 hours, 12 hours. There were 4 kinds of treatments in 4 hours, 4 experimental mice in each treatment, 16 experimental mice in total, and repeated three times; the mice in the experimental treatment group were fed with HrpNEcb polymimetic ligand protein buffer containing 600 mg·L -1 concentration. For the smearing treatment, the mice in the buffer control sham-operated group were fed and smeared with buffer, and the mice in the blank control group did not receive any treatment. Under the same feeding conditions and at different times, the cerebral cortex, thalamus, cerebral hippocampus, liver, heart and other tissues of mice were divided into groups for RNA-Seq sequencing and analysis.
2.mRNA(RNA-Seq)测序2. mRNA (RNA-Seq) sequencing
见图65mRNA(RNA-Seq)测序实验流程图。See Figure 65 mRNA (RNA-Seq) sequencing experiment flow chart.
I.RNA抽提与质检I. RNA extraction and quality inspection
采用miRNeasy Micro Kit(Cat#1071023Qiagen)并且根据生产厂商提供的标准操作流程进行样品的总RNA抽提。总RNA经NanoDrop ND-2000分光光度计及Agilent Bioanalyzer 4200(Agilent technologies,Santa Clara,CA,US)进行质检，质检合格的RNA用于后续的测序实验。Total RNA extraction of the samples was performed using the miRNeasy Micro Kit (Cat#1071023Qiagen) and according to the standard operating procedure provided by the manufacturer. The total RNA was quality-checked by NanoDrop ND-2000 spectrophotometer and Agilent Bioanalyzer 4200 (Agilent technologies, Santa Clara, CA, US), and the qualified RNA was used for subsequent sequencing experiments.
II.文库构建与质检II. Library Construction and Quality Control
由于真核生物大部分mRNA都带有polyA尾，利用Oligo(dT)可以富集带有polyA尾的mRNA。接下来对富集mRNA进行片段化、双链cDNA合成、末端修复、3’末端加A、连接接头、扩增。Since most mRNAs in eukaryotes have polyA tails, Oligo(dT) can be used to enrich mRNAs with polyA tails. The enriched mRNA is then subjected to fragmentation, double-stranded cDNA synthesis, end repair, addition of A at the 3' end, ligation of adapters, and amplification.
构建的文库使用
Figure PCTCN2021134714-appb-000010
2.0 Fluorometer检测浓度，Agilent2100检测大小。 The constructed library uses
Figure PCTCN2021134714-appb-000010
2.0 Fluorometer detects concentration, Agilent2100 detects size.
III.上机测序III. On-board sequencing
对文库进行Illumina测序，测序仪通过捕获荧光信号，并通过计算机软件将光信号转化为测序峰，从而获得待测片段的序列信息。Illumina sequencing is performed on the library, and the sequencer captures the fluorescent signal and converts the light signal into a sequencing peak through computer software to obtain the sequence information of the fragment to be detected.
IV.mRNA测序数据分析按图66数据分析流程进行。IV. Analysis of mRNA sequencing data was performed according to the data analysis flow of Figure 66.
3.结果分析3. Analysis of results
1)HrpNEcb多拟表位配体蛋白诱导的差异基因筛选1) Differential gene screening induced by HrpNEcb multi-epitope ligand proteins
首先对fragment counts进行归一化，再根据假设检验模型计算p-value，最后对p-value多重假设检验校正，得到FDR值。FP KM值计算差异表达倍数(Fold-change)，使用edgeR软件。差异基因筛选条件如下：p-value<0.05且|Fold-change|>＝2First normalize the fragment counts, then calculate the p-value according to the hypothesis testing model, and finally correct the p-value for multiple hypothesis testing to obtain the FDR value. The FP KM value was used to calculate the differential expression fold (Fold-change) using edgeR software. Differential gene screening conditions are as follows: p-value<0.05 and |Fold-change|>=2
2)HrpNEcb多拟表位配体蛋白诱导的差异基因火山图2) Volcano plot of differential genes induced by HrpNEcb multi-epitope ligand proteins
采用差异基因火山图显示HrpNEcb多拟表位配体蛋白诱导的表达差异显著性基因的整体分布情况。横坐标：基因在不同样本中的表达倍数变化(log2Fold-Chan ge)；纵坐标：基因表达差异的显著性水平(-log10p-value)；右侧点表达显著上调基因；左侧点表达显著下调基因；下部点表达变化不显著基因。图3-7分别为小鼠的肝脏、大脑丘脑、心脏、大脑皮层及大脑海马HrpNEcb多拟表位配体蛋白通过口服和涂抹诱导的差异基因火山图，图中HrpNEcb缩写为N1。The differential gene volcano plot was used to display the overall distribution of genes with significant differential expression induced by HrpNEcb multi-epitope ligand protein. Horizontal axis: fold change of gene expression in different samples (log2Fold-Change); vertical axis: significant level of gene expression difference (-log10p-value); the expression of the right point is significantly up-regulated; the left point is significantly down-regulated Genes; genes with no significant changes in expression at lower points. Figures 3-7 are the differential gene volcano plots of HrpNEcb multi-epitope ligand protein induced by oral administration and smearing in the liver, thalamus, heart, cerebral cortex and cerebral hippocampus of mice, respectively. HrpNEcb is abbreviated as N1 in the figure.
3)HrpNEcb蛋白诱导的差异基因聚类图3) Cluster map of differential genes induced by HrpNEcb protein
差异基因集进行聚类分析，将表达模式相近的基因聚在一起，显示基因具有共同功能或参与到共同信号通路。将log10(FPKM+1)值进行归一化转换(scale number)并进行聚类，图8-11分别为肝脏、丘脑、海马、大脑皮层表达差异基因集聚类热图，图中HrpNEcb缩写为N1。Cluster analysis was performed on differential gene sets, and genes with similar expression patterns were clustered together, indicating that genes have common functions or participate in common signaling pathways. The log10 (FPKM+1) value is normalized and transformed (scale number) and clustered. Figure 8-11 is a clustering heat map of differentially expressed genes in liver, thalamus, hippocampus, and cerebral cortex. In the figure, HrpNEcb is abbreviated as N1.
4)HrpNEcb蛋白诱导的差异基因GO富集分析4) GO enrichment analysis of differential genes induced by HrpNEcb protein
基因本体(Gene Ontology，GO)是一个在生物信息学领域中广泛使用的本体。基因本体论是对基因在不同维度和不同层次上的描述，涵盖了生物学的生物过程(biological_process)，细胞组分 (cellular_component)及分子功能(molecular_function)。生物过程是在说明该基因参与了哪些生物学过程；细胞组分解释的是基因存在在哪里，包括该基因在细胞质还是在细胞核？如果存在细胞质那在哪个细胞器上？如果是在线粒体中那是存在线粒体膜上还是在线粒体的基质当中等等，这些信息都属于细胞组；分子功能解释的是该基因在分子层面的功能是什么？描述其在个体分子生物学上的活性，如催化活性或结合活性。基因本体数据库(Gene Ontology)是GO组织(Gene Ontology Consortium)在2000年构建的一个结构化的标准生物模型，旨在建立基因及其产物知识的标准词汇体系，涵盖了基因的生物学过程(biological process)，细胞组分(cellular component)、分子功能(molecular function)。Term是GO里面的基本描述单元。GO Terms用于描述基因产物的功能。通过将差异基因做GO富集分析，可以把基因按照不同的功能进行归类，达到对基因进行注释和分类的目的。我们对HrpNEcb多拟表位配体蛋白诱导的差异表达基因进行了GO term富集分析,其结果证明、证实了HrpNEcb多拟表位配体蛋白作为一类具有多个表位特殊结构，全新功能，全新作用机制和全新应用前景的配体蛋白，诱导了受试小鼠多个器官(肝脏、丘脑、心脏、大脑皮层及大脑海马等)多基因的差异表达，这些差异表达基因涵盖了生物过程、细胞组分及分子功能。差异表达基因利用Fisher精确检验进行GO分析。Fisher精确检验计算得到p-value，并进行多重假设检验校正得到q-value。筛选p-value小于0.05的GO条目作为显著富集的GO条目。Gene Ontology (GO) is an ontology widely used in the field of bioinformatics. Gene Ontology is a description of genes in different dimensions and levels, covering biological processes (biological_process), cellular components (cellular_component) and molecular functions (molecular_function). Biological processes are describing which biological processes the gene is involved in; cellular components explain where the gene is present, including whether the gene is in the cytoplasm or in the nucleus? If cytoplasm is present in which organelle? If it is in the mitochondria, is it on the mitochondrial membrane or in the matrix of the mitochondria, etc., this information belongs to the cell group; the molecular function explains what is the function of the gene at the molecular level? Describe its activity in individual molecular biology, such as catalytic activity or binding activity. Gene Ontology database (Gene Ontology) is a structured standard biological model constructed by the GO organization (Gene Ontology Consortium) in 2000. It aims to establish a standard vocabulary system for knowledge of genes and their products, covering the biological process of genes. process), cellular component, molecular function. Term is the basic description unit in GO. GO Terms are used to describe the function of gene products. By performing GO enrichment analysis on differential genes, genes can be classified according to different functions, so as to achieve the purpose of annotating and classifying genes. We carried out GO term enrichment analysis on the differentially expressed genes induced by HrpNEcb multi-epitope ligand protein, and the results proved and confirmed that HrpNEcb multi-epitope ligand protein, as a class of special structures with multiple epitopes, new functions , a ligand protein with a new mechanism of action and a new application prospect, induced the differential expression of multiple genes in multiple organs (liver, thalamus, heart, cerebral cortex and brain hippocampus, etc.) of the tested mice, and these differentially expressed genes covered biological processes. , cellular components and molecular functions. Differentially expressed genes were analyzed by GO using Fisher's exact test. Fisher's exact test was calculated to obtain the p-value, and multiple hypothesis test correction was performed to obtain the q-value. GO entries with p-value less than 0.05 were screened as significantly enriched GO entries.
HrpNEcb多拟表位配体蛋白诱导的差异基因GO富集分析结果进一步表述如下：①生物过程(biological_process)相关差异表达基因包括了繁殖,细胞死亡,免疫系统的过程,行为,代谢过程,细胞过程,生殖过程,生物粘附,信号,多细胞生物过程,发育过程，增长，运动，单个组织的过程，生物相，有节奏的过程，生物过程的正调控，生物过程负调控，生物过程调节，刺激反应，定位，生物调节，细胞成分组织或生物发生，细胞聚集，解毒以及突触前过程涉及突触传递。生物过程GO富集分析结果详见表1至表6。②细胞组分(cellular_component)相关差异表达基因涵盖了细胞及细胞外区域，类核，膜，病毒粒子，细胞结，细胞外基质，细胞膜封闭腔，复杂大分子，细胞器，细胞外基质成分，细胞外区域部分，细胞器部件，病毒粒子部件，膜部件，突触部分，细胞部件，突触，以及细胞超分子纤维等。细胞组分GO富集分析结果详见表1至表6。③分子功能(molecular function)相关差异表达基因涵盖了转录因子活性，蛋白质结合,核酸结合转录因子活性,催化活性,信号传感器活动,结构分子活动,运输活动,绑定,电子载体活动,成形素活动,抗氧化活性,金属伴侣蛋白活性,蛋白质标记,化学引诱物的活动,转译调控，化学排斥物活性，活动分子传感器，分子功能调控等。分子功能GO富集分析结果详见表1至表6。The results of GO enrichment analysis of differentially expressed genes induced by HrpNEcb multi-epitope ligand proteins are further described as follows: ① The biological process (biological_process) related differentially expressed genes include reproduction, cell death, immune system processes, behavior, metabolic processes, and cellular processes. , reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, motility, processes in individual tissues, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, Stimulatory responses, localization, bioregulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic processes involve synaptic transmission. The results of GO enrichment analysis of biological processes are shown in Tables 1 to 6. ②The differentially expressed genes related to cellular components cover cells and extracellular regions, nucleoids, membranes, virus particles, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules, organelles, extracellular matrix components, cells Parts of the outer region, organelle parts, virion parts, membrane parts, synaptic parts, cell parts, synapses, and cellular supramolecular fibers, etc. The results of GO enrichment analysis of cell components are shown in Table 1 to Table 6. ③ Molecular function-related differentially expressed genes cover transcription factor activity, protein binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, and morphogen activity , Antioxidant activity, metal chaperone protein activity, protein labeling, chemoattractant activity, translation regulation, chemorepellent activity, active molecular sensor, molecular function regulation, etc. The molecular function GO enrichment analysis results are shown in Table 1 to Table 6.
其中，表1-6的HrpNEcb缩写为N1，所有表格中，空格表示没有收集到对应的未达到p-value小于0.05标准的相关数据，以下及其所有表格空白含义与此相同。Among them, HrpNEcb in Table 1-6 is abbreviated as N1. In all tables, blanks indicate that the corresponding data that does not meet the standard of p-value less than 0.05 have not been collected. The blanks in the following and all tables have the same meaning.
表1 HrpNEcb多拟表位配体蛋白诱导心脏的生物学过程、细胞组分和分子功能相关功能群显著上调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时、涂抹12小时)Table 1 The biological process, cellular components and molecular function-related functional groups of the heart induced by HrpNEcb multi-epitope ligand protein were significantly up-regulated and expressed GO terms classification gene number statistics (6, 24 hours after oral administration and 6 hours after application, 12 hours after application )
Figure PCTCN2021134714-appb-000011
Figure PCTCN2021134714-appb-000011
Figure PCTCN2021134714-appb-000012
Figure PCTCN2021134714-appb-000012
Figure PCTCN2021134714-appb-000013
Figure PCTCN2021134714-appb-000013
Figure PCTCN2021134714-appb-000014
Figure PCTCN2021134714-appb-000014
表2 HrpNEcb多拟表位配体蛋白诱导肝脏、皮层的生物学过程、细胞组分和分子功能相关功能群显著上调表达GO terms分类基因数统计表(口服6、24小时和涂抹6、12小时)Table 2 HrpNEcb multi-epitope ligand protein induced significant up-regulation of biological processes, cellular components and molecular function-related functional groups in liver and cortex )
Figure PCTCN2021134714-appb-000015
Figure PCTCN2021134714-appb-000015
Figure PCTCN2021134714-appb-000016
Figure PCTCN2021134714-appb-000016
Figure PCTCN2021134714-appb-000017
Figure PCTCN2021134714-appb-000017
表3 HrpNEcb多拟表位配体蛋白诱导丘脑、海马的生物学过程、细胞组分和分子功能相关功能群显著上调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时、涂抹12小时)Table 3 HrpNEcb multi-epitope ligand protein induces the biological process, cellular components and molecular function-related functional groups of the thalamus and hippocampus to significantly up-regulate the expression of GO terms. 12 hours)
Figure PCTCN2021134714-appb-000018
Figure PCTCN2021134714-appb-000018
Figure PCTCN2021134714-appb-000019
Figure PCTCN2021134714-appb-000019
Figure PCTCN2021134714-appb-000020
Figure PCTCN2021134714-appb-000020
表4 HrpNEcb多拟表位配体蛋白诱导心脏的生物学过程、细胞组分和分子功能相关功能群显著下调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时、涂抹12小时)Table 4 The biological process, cellular components and molecular function-related functional groups of the heart induced by HrpNEcb multi-epitope ligand protein were significantly down-regulated and expressed GO terms classification. )
Figure PCTCN2021134714-appb-000021
Figure PCTCN2021134714-appb-000021
Figure PCTCN2021134714-appb-000022
Figure PCTCN2021134714-appb-000022
Figure PCTCN2021134714-appb-000023
Figure PCTCN2021134714-appb-000023
Figure PCTCN2021134714-appb-000024
Figure PCTCN2021134714-appb-000024
表5 HrpNEcb多拟表位配体蛋白诱导肝脏和皮层的生物学过程、细胞组分和分子功能相关功能群显著下调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时、涂抹12小时)Table 5 The biological process, cellular components and molecular function-related functional groups of the liver and cortex induced by HrpNEcb multi-epitope ligand protein were significantly down-regulated and expressed GO terms. 12 hours)
Figure PCTCN2021134714-appb-000025
Figure PCTCN2021134714-appb-000025
Figure PCTCN2021134714-appb-000026
Figure PCTCN2021134714-appb-000026
Figure PCTCN2021134714-appb-000027
Figure PCTCN2021134714-appb-000027
Figure PCTCN2021134714-appb-000028
Figure PCTCN2021134714-appb-000028
表6 HrpNEcb多拟表位配体蛋白诱导丘脑和海马的生物学过程、细胞组分和分子功能相关功能群显著下调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时、涂抹12小时)Table 6 The biological process, cellular components and molecular function-related functional groups of the thalamus and hippocampus induced by HrpNEcb multi-epitope ligand protein were significantly down-regulated. 12 hours)
Figure PCTCN2021134714-appb-000029
Figure PCTCN2021134714-appb-000029
Figure PCTCN2021134714-appb-000030
Figure PCTCN2021134714-appb-000030
Figure PCTCN2021134714-appb-000031
Figure PCTCN2021134714-appb-000031
Figure PCTCN2021134714-appb-000032
Figure PCTCN2021134714-appb-000032
5.差异表达基因的KEGG pathway富集分析5. KEGG pathway enrichment analysis of differentially expressed genes
京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)，是系统分析基因功能与基因组信息的数据库，它整合了基因组学、生物化学和系统功能组学的信息，有助于研究者把基因及表达信息的过程作为一个网络进行整体研究。Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database for systematic analysis of gene function and genomic information. The process of gene and expression information is studied holistically as a network.
KEGG主要的特点是将基因与各种生化反应联系在了一起，提供整合的代谢途径。KEGG目前共包含了19个子数据库，它们被分类为系统信息、基因组信息和化学信息三个类别。在生物体内，不同的基因产物相互协调来行使生物学功能，对差异表达基因的通路(Pathway)注释分析有助于进一步解读基因的功能。对HrpNEcb蛋白诱导的差异表达基因进行了KEGG pathway富集分析，获得这些差异基因在信号通路中的角色(上下游关系)和生物学功能，深入理解基因与功能的关系。差异表达基因利用Fisher精确检验进行Pathway分析。Fisher精确检验计算得到p-value，并进行多重假设检验校正得到q-value。筛选p-value小于0.05的Pathway作为显著富集的Pathway。The main feature of KEGG is to link genes with various biochemical reactions, providing integrated metabolic pathways. KEGG currently contains a total of 19 sub-databases, which are classified into three categories: systematic information, genomic information and chemical information. In organisms, different gene products coordinate with each other to perform biological functions. Pathway annotation analysis of differentially expressed genes is helpful for further interpretation of gene functions. The KEGG pathway enrichment analysis was performed on the differentially expressed genes induced by HrpNEcb protein to obtain the roles (upstream and downstream relationships) and biological functions of these differential genes in the signaling pathway, and to deeply understand the relationship between genes and functions. Pathway analysis of differentially expressed genes was performed using Fisher's exact test. Fisher's exact test was calculated to obtain the p-value, and multiple hypothesis test correction was performed to obtain the q-value. Pathways with p-value less than 0.05 were screened as significantly enriched Pathways.
研究结果证明、证实了HrpNEcb多拟表位配体蛋白作为一类具有多表位特殊结构，全新功能，全新作用机制和全新应用前景的配体蛋白，诱导了小鼠多个器官(肝脏、丘脑、心脏、大脑皮层及大脑海马等)多基因的差异表达，这些差异表达基因参与了归属于细胞过程(Cellular Processes)、环境信息处理(Environmental Information Processing)、遗传信息处理(Genetic Information Processing)、新陈代谢(Metabolism)和生物体系统(Organismal Systems)等功能途径。HrpNEcb多拟表位配体蛋白诱导的差异基因GO富集分析结果进一步表述如下：①细胞过程(Cellular Processes)：HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了运输和分解代谢，细胞群体，细胞活性，细胞生长与死亡等细胞过程(详见图12至图64)。②环境信息处理(Environmental Information Processing):HrpNEcb蛋白诱导的多个差异表达基因参与了信号分子与相互作用,信号转导,膜运输等环境信息处理过程(详见图12至图64)。③遗传信息处理(Genetic Information Processing):HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了翻译,复制和修复,折叠、分类和降解等生物过程(详见图12至图64)。④新陈代谢(Metabolism):HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了生物降解和代谢,核苷酸代谢,其他氨基酸的代谢,代谢的辅助因子和维生素,脂质代谢,糖的生物合成和代谢,全局和概览地图,能量代谢,碳水化合物代谢及氨基酸代谢等代谢过程(详见图12至图64)。⑤生物体系统(Organismal Systems):HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了感觉系统,神经系统,免疫系统,排泄系统,环境适应,内分泌系统,消化系统,发育循环系统等细胞过程(详见图12至图64)。The research results proved and confirmed that HrpNEcb multi-epitope ligand protein, as a kind of ligand protein with multi-epitope special structure, new function, new mechanism of action and new application prospect, induces multiple organs (liver, thalamus) of mice. , heart, cerebral cortex and brain hippocampus, etc.) differential expression of multiple genes, these differentially expressed genes are involved in cellular processes (Cellular Processes), environmental information processing (Environmental Information Processing), genetic information processing (Genetic Information Processing), metabolism Functional pathways such as Metabolism and Organismal Systems. The results of GO enrichment analysis of differentially expressed genes induced by HrpNEcb multi-epitope ligand protein are further described as follows: ① Cellular Processes: Multiple differentially expressed genes induced by HrpNEcb multi-epitopic ligand protein are involved in transport and catabolism , cell population, cell viability, cell growth and death and other cellular processes (see Figure 12 to Figure 64 for details). ②Environmental Information Processing: Multiple differentially expressed genes induced by HrpNEcb protein are involved in environmental information processing processes such as signal molecules and interactions, signal transduction, and membrane transport (see Figure 12 to Figure 64 for details). ③ Genetic Information Processing: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand proteins are involved in biological processes such as translation, replication and repair, folding, classification and degradation (see Figure 12 to Figure 64 for details) . ④Metabolism: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand protein are involved in biodegradation and metabolism, nucleotide metabolism, metabolism of other amino acids, metabolic cofactors and vitamins, lipid metabolism, sugar Biosynthesis and metabolism, global and overview maps, metabolic processes such as energy metabolism, carbohydrate metabolism and amino acid metabolism (see Figure 12 to Figure 64 for details). ⑤Organismal Systems: Multiple differentially expressed genes induced by HrpNEcb multi-epitope ligand protein are involved in sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, developmental circulatory system isocellular processes (see Figures 12 to 64 for details).
与GO分类统计类似，对KEGG各个生物学途径(pathway)上的差异表达基因数目进行统计并以图形 展示如图12至图64所示。Similar to GO classification statistics, the number of differentially expressed genes on each biological pathway of KEGG was counted and displayed graphically as shown in Figure 12 to Figure 64.
说明：图形右边示图，从上到下中文译名：细胞过程、信息过程、遗传信息过程、代谢过程、组织系统发育过程；横坐标：涉及表达差异的各功能基因群的基因数目；纵坐标：涉及表达差异的细胞过程、信息过程、遗传信息过程、代谢过程、组织系统发育过程的功能基因群。Description: The diagram on the right side of the figure, from top to bottom, Chinese translations: cellular process, information process, genetic information process, metabolic process, tissue phylogeny; abscissa: the number of genes involved in different functional gene groups; ordinate: Functional gene groups involved in differentially expressed cellular processes, information processes, genetic information processes, metabolic processes, and tissue phylogeny.
实施例5Example 5
HrpNEcb蛋白识别结合特异蛋白的pull-down实验Pull-down experiment of HrpNEcb protein recognition and binding specific protein
1.样品准备和处理1. Sample Preparation and Handling
1)HrpNEcb多拟表位配体蛋白纯化1) HrpNEcb polymimetic epitope ligand protein purification
用NI-NTA琼脂糖凝胶柱纯化HrpNEcb多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，制备的HrpNEcb多拟表位配体蛋白备用(以下称为捕获蛋白或目的蛋白)。The HrpNEcb polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column. The protein purification was performed according to the method recommended by the NI-NTA agarose column manufacturer. The prepared HrpNEcb polymimetic epitope ligand protein was used for later use. (hereinafter referred to as capture protein or target protein).
2)实验用培养肝细胞总蛋白(以下称为钓饵蛋白)抽提2) Extraction of total protein from cultured hepatocytes (hereinafter referred to as bait protein) for experiments
I.培养肝细胞总蛋白的抽提：①用移液枪吸取裂解液(IP专用裂解液，加入1×cocktail蛋白酶抑制剂)加入到细胞。超声，冰上静置2h以上；②使用超声细胞破碎仪，冰上超声2s，停5s，共1min，冰上裂解总时长2h以上(间隔30min振荡器震荡混匀)；③将细胞裂解产物4℃下13000rpm离心15min，吸取上清转移至新的1.5mLEP管，冰上放置待用；④将蛋白抽提液再次于4℃下13000rpm离心5min，小心地吸取中间层的溶液，转移至新的1.5mL EP管中，4℃冰箱放置待用，并取部分稀释后测浓度(稀释10倍)，采用BCA蛋白浓度测定法测定浓度。I. Extraction of total protein from cultured hepatocytes: ①Aspirate lysate (IP-specific lysate, add 1×cocktail protease inhibitor) with a pipette and add it to the cells. Ultrasonic, let stand on ice for more than 2h; ②Using an ultrasonic cell disruptor, ultrasonicate on ice for 2s, stop for 5s, a total of 1min, the total lysis time on ice is more than 2h (30min interval shaker and mix); ③The cell lysate 4 Centrifuge at 13000rpm for 15min at ℃, transfer the supernatant to a new 1.5mL EP tube, and put it on ice for use; ④ Centrifuge the protein extract again at 13000rpm for 5min at 4℃, carefully aspirate the solution in the middle layer, and transfer it to a new Put it in a 1.5mL EP tube and place it in a refrigerator at 4°C for later use, and take a part of it to measure the concentration after dilution (10 times dilution), and use the BCA protein concentration assay to measure the concentration.
II.蛋白浓度测定：参照BCA试剂盒的方法，对提取的蛋白溶液进行浓度测定。II. Determination of protein concentration: according to the method of the BCA kit, the concentration of the extracted protein solution was determined.
表7 BCA法测定蛋白浓度Table 7 Determination of protein concentration by BCA method
NO.NO. 样本名称sample name 实验编号Experiment number 浓度(μg/μL)Concentration (μg/μL) 体积(μL)Volume (μL) 总量(μg)Total (μg)
11 HEPG2HEPG2 HEPG2HEPG2 8.348.34 25002500 20861.3020861.30
2.pull-down实验流程2. Pull-down experimental process
1)平衡固定链霉亲和素凝胶:①准备Pierce TM Spin Column管；②上下颠倒重悬凝胶液，吸取50μl悬液于标记好的Spin Column管，塞好底塞并放置于收集管中；③再向Spin Column管中加入250μl TBS，拧紧顶盖，轻轻上下颠倒4次混匀液体；④去掉顶盖和底塞，1250×g离心50s，弃掉收集管中的清洗液，把SpinColumn管重新插入收集管中；⑤重复步骤3和步骤4两次。再在Spin Column管底部塞上管底塞。1) Equilibrate and fix the streptavidin gel: ① Prepare a Pierce TM Spin Column tube; ② Resuspend the gel by inverting it upside down, pipette 50 μl of the suspension into the labeled Spin Column tube, plug the bottom stopper and place it in the collection tube ③Add 250μl of TBS to the Spin Column tube, tighten the top cap, and gently invert 4 times to mix the liquid; ④Remove the top cap and bottom stopper, centrifuge at 1250×g for 50s, discard the cleaning solution in the collection tube, Reinsert the SpinColumn tube into the collection tube; ⑤ Repeat steps 3 and 4 twice. Then put the bottom plug on the bottom of the Spin Column tube.
2)生物素标记生物素标记钓饵蛋白和生物素的固定:①向Spin Column管中分别加入生物素和生物素标记钓饵蛋白，拧紧顶盖和底塞；②在旋转平台rotating platform上温和地摇动，4℃孵育60min；③孵育结束后，移去Spin Column管顶盖和底塞，放入收集管中；④1250×g，60s离心后，将Spin Column管重新放入收集管中。2) Fixation of biotin-labeled biotin-labeled bait protein and biotin: ①Add biotin and biotin-labeled bait protein to Spin Column tubes, respectively, and tighten the top cap and bottom plug; ② Gently shake on the rotating platform , incubate at 4°C for 60 min; ③ After the incubation, remove the top cap and bottom plug of the Spin Column tube and put it into the collection tube; ④ After centrifugation at 1250 × g for 60 s, put the Spin Column tube back into the collection tube.
3)生物素的封闭:①向Spin Column管中加入250μl生物素封闭溶液。拧紧顶盖和底塞，轻轻上下颠倒4次使之混匀；②室温下孵育5min。移去顶盖，将Spin Column管放于收集管中，1250×g离心50s；③重复步骤1和步骤2一次；④再向Spin Column管中加入TBS 250μl。拧紧顶盖，轻轻上下颠倒4次使之混匀；⑤移去顶盖，放于收集管中，1250×g离心50s；⑥重复步骤3和步骤4两次，Spin Column管重新放入收集管。3) Biotin blocking: ①Add 250 μl biotin blocking solution to the Spin Column tube. Tighten the top cover and bottom stopper, and gently invert 4 times to mix; ②Incubate at room temperature for 5 min. Remove the top cover, put the Spin Column tube in the collection tube, and centrifuge at 1250×g for 50s; ③ Repeat steps 1 and 2 once; ④ Add 250 μl of TBS to the Spin Column tube. Tighten the top cap and invert it upside down 4 times to mix it up; ⑤Remove the top cap, put it in a collection tube, and centrifuge at 1250×g for 50s; ⑥Repeat steps 3 and 4 twice, and put the Spin Column tube back into the collection tube Tube.
4)生物素标记生物素标记蛋白的捕获:①向Spin Column管中加入300μL(1mg蛋白)的捕获蛋白(目的蛋白)样品溶液，拧紧顶盖；②在旋转平台rotating platform上温和地摇动，4℃孵育过夜； ③孵育结束后，移去顶盖和底塞。将Spin Column管放入准备好的收集管中；④将收集管，1250×g，60s，离心，收集管标记“prey flow-through(B)”；⑤移去收集管中Spin Column管，盖好收集管的盖子，冰上放置以便后续分析；⑥将Spin Column管放入新的收集管，准备洗脱。4) Capture of biotin-labeled biotin-labeled protein: ① Add 300 μL (1 mg protein) of the captured protein (target protein) sample solution to the Spin Column tube, and tighten the top cover; ② Gently shake on the rotating platform, 4 Incubate overnight at ℃; ③ After the incubation, remove the top cover and bottom plug. Put the Spin Column tube into the prepared collection tube; ④ Centrifuge the collection tube, 1250×g, 60s, mark the collection tube as "prey flow-through(B)"; ⑤ Remove the Spin Column tube in the collection tube, cover Secure the lid of the collection tube and put it on ice for subsequent analysis; ⑥Put the Spin Column tube into a new collection tube to prepare for elution.
5)Spin Column管的诱饵蛋白和靶蛋白复合物的洗脱:①在每个Spin Column管中加入250μl Wash Buffer。拧紧顶盖和底塞，轻轻颠倒6次使之混匀；②将Spin Column管在室温下孵育1分钟。将顶盖底塞去掉，将Spin Column管置于收集管上，1250×g离心50s。另外重复步骤1-2，3次；③在冲洗的过程中，在收集管上写上标签“Wash1，……，Wash3”；④最后一次冲洗时，加入200μl Wash Buffer后，将管内液体连同珠子一起转移至1.5mL；⑤新的离心管中，离心后弃170μl上清，该步骤重复3次。5) Elution of the bait protein and target protein complexes in the Spin Column tube: ①Add 250 μl Wash Buffer to each Spin Column tube. Tighten the top cap and bottom stopper, and gently invert 6 times to mix; ②Incubate the Spin Column tube at room temperature for 1 minute. Remove the top cap and bottom stopper, place the Spin Column tube on the collection tube, and centrifuge at 1250×g for 50s. In addition, repeat steps 1-2, 3 times; ③ During the rinsing process, write the label "Wash1, ..., Wash3" on the collection tube; ④ During the last rinsing, add 200 μl of Wash Buffer, add the liquid in the tube together with the beads Transfer to 1.5mL together; ⑤In a new centrifuge tube, discard 170μl of supernatant after centrifugation, and repeat this step 3 times.
6)检测：①吸干Sepharose上面的液体后，加入20μl 1×蛋白电泳上样缓冲液，沸水浴5min，放入-20℃冰箱备用；②通过SDS-PAGE和Western blot进行检测。6) Detection: ① After blotting up the liquid on Sepharose, add 20 μl of 1× protein electrophoresis loading buffer, take a boiling water bath for 5 min, and put it in a -20°C refrigerator for later use; ② Detect by SDS-PAGE and Western blot.
3.结果分析3. Analysis of results
1)HrpNEcb多拟表位配体蛋白识别结合的细胞膜受体：识别结合6个膜受体，包括HLA-A主要组织相容性复合体，I,A类受体，LGALS3BP半乳糖3结合蛋白(受体)，LAMP2溶酶体关联膜蛋白2受体，GNB2G鸟嘌呤核苷酸结合蛋白亚单位Beta 2受体，LRRC1515-亮氨酸的重复膜蛋白受体，KTN1驱动结合蛋白1受体。1) HrpNEcb multi-epitope ligand protein recognizes bound cell membrane receptors: recognizes and binds to 6 membrane receptors, including HLA-A major histocompatibility complex, class I, A receptors, LGALS3BP galactose 3 binding protein (receptor), LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein subunit Beta 2 receptor, LRRC1515-leucine repeat membrane protein receptor, KTN1 kinesin 1 receptor .
2)HrpNEcb多拟表位配体蛋白识别结合的细胞膜蛋白：识别结合11个膜蛋白，包括DSG4桥粒芯蛋白、ANXA4膜联蛋白A4、CAPRIN1细胞周期蛋白、1UTRN肌营养不良蛋白蛋白、pinin桥粒蛋白、VAMP关联蛋白A、VCL黏着斑蛋白、Ezrin埃兹上皮型钙黏附素、PKP3血小板亲和蛋白3、TM9SF2跨膜9超家族成员2、NAALAD2N乙酰化α连接酸性二肽酶2中的一种或多种。2) HrpNEcb multi-epitope ligand protein recognizes bound cell membrane proteins: recognizes and binds to 11 membrane proteins, including DSG4 desmocore protein, ANXA4 annexin A4, CAPRIN1 cyclin, 1UTRN dystrophin protein, pinin bridge Granulin, VAMP-associated protein A, VCL focal adhesion protein, Ezrin epithelial cadherin, PKP3 platelet affinity protein 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD2N acetylated α-linked acid dipeptidase 2 one or more.
3)HrpNEcb多拟表位配体蛋白识别结合的膜蛋白参与的信号通路13条：包括hsa04152:AMPK信号通路、hsa03460:范可尼贫血通路、hsa03320:PPAR信号通路、hsa04071:鞘脂类信号通路、hsa04014:Ras信号通路、hsa04151:PI3K-Akt信号通路、hsa04310:Wnt信号通路、hsa04062:趋化因子信号通路、hsa04015:Rap1信号通路、hsa04024:阵营信号通路、hsa04915:雌激素信号通路、hsa04910:胰岛素信号通路、hsa04390:河马信号通路。3) HrpNEcb multi-epitope ligand protein recognizes and binds to 13 membrane proteins involved in signaling pathways: including hsa04152: AMPK signaling pathway, hsa03460: Fanconi anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: Sphingolipid signaling pathway , hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: Chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: Camp signaling pathway, hsa04915: Estrogen signaling pathway, hsa04910: Insulin signaling pathway, hsa04390: Hippo signaling pathway.
4)HrpNEcb多拟表位配体蛋白识别结合的膜蛋白参与的与抗病毒、抗细菌、抗异物、抗炎性相关代谢通路23条：包括hsa04144内吞作用、hsa04145吞噬体、hsa04142溶酶体、hsa01130:抗生素的生物合成、hsa05131:志贺氏菌病、hsa04612:抗原处理和呈递、hsa05130:致病性大肠杆菌感染、hsa05100:上皮细胞的细菌侵袭、hsa05132:沙门氏菌感染、hsa05169:巴尔病毒感染、hsa05168:单纯疱疹病毒1感染、hsa05203:病毒致癌作用、hsa05166:HTLV-I感染、hsa05164:甲型流感、hsa05134:军团病、hsa05160:丙型肝炎、hsa05162:麻疹、hsa05133:百日咳、hsa05322:系统性红斑狼疮、hsa04670:白细胞经上皮的迁移、hsa05146:阿米巴病、hsa05142:南美锥虫病、hsa05200:在癌症中通路。4) 23 metabolic pathways involved in antiviral, antibacterial, anti-foreign body, and anti-inflammatory activities that HrpNEcb multi-epitope ligand protein recognizes and binds to membrane proteins: including hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome , hsa01130: Biosynthesis of antibiotics, hsa05131: Shigellosis, hsa04612: Antigen processing and presentation, hsa05130: Pathogenic E. coli infection, hsa05100: Bacterial invasion of epithelial cells, hsa05132: Salmonella infection, hsa05169: Barr virus infection , hsa05168: Herpes simplex virus 1 infection, hsa05203: Viral carcinogenesis, hsa05166: HTLV-I infection, hsa05164: Influenza A, hsa05134: Legionnaires' disease, hsa05160: Hepatitis C, hsa05162: Measles, hsa05133: Pertussis, hsa05322: System lupus erythematosus, hsa04670: transepithelial migration of leukocytes, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: pathways in cancer.
5)HrpNEcb多拟表位配体蛋白识别结合的膜蛋白参与的重要神经疾病代谢通道3条：包括hsa05012:帕金森病，hsa05016:亨廷顿氏舞蹈症，hsa05010:阿尔茨海默氏症。5) HrpNEcb multi-epitope ligand protein recognizes and binds membrane proteins involved in 3 important neurological disease metabolic pathways: including hsa05012: Parkinson's disease, hsa05016: Huntington's disease, hsa05010: Alzheimer's disease.
6)HrpNEcb多拟表位配体蛋白识别结合的膜蛋白参与的核酸、蛋白质、氨基酸、糖、脂肪代谢相关通路30条：包括hsa03420:核苷酸切除修复、hsa00970:氨酰生物合成、hsa03430:错配修复、hsa01210:2-氧代羧酸代谢、hsa03440:同源重组、hsa04360:轴突引导、hsa00051:果糖和甘露糖代谢、hsa00565:醚脂质代谢、hsa00510:N-多糖生物合成以及hsa04110:细胞周期、hsa03030:DNA复制、hsa03013:RNA运输、 hsa03018:RNA降解、hsa03040:剪接体、hsa03010:核糖体、hsa04141:内质网蛋白加工、hsa04810:肌动蛋白骨架的调控、hsa03050:蛋白酶体、hsa01230:氨基酸生物合成、hsa00190:氧化磷酸化、hsa04932:非酒精性脂肪肝(NAFLD)、hsa00020:柠檬酸循环、hsa00564:甘油磷脂新陈代谢、hsa03008:真核生物核糖体的生物发生、hsa03015:mRNA监测通路、hsa01200:碳代谢、hsa00520:氨基糖和核苷酸糖代谢、hsa05034:酗酒、hsa04120:泛素介导的蛋白水解作用、hsa05205:蛋白聚糖在癌症、hsa05206:小分子核糖核酸在癌症。6) HrpNEcb multi-epitope ligand protein recognizes and binds membrane proteins involved in 30 nucleic acid, protein, amino acid, sugar, and fat metabolism-related pathways: including hsa03420: nucleotide excision repair, hsa00970: aminoacyl biosynthesis, hsa03430: Mismatch repair, hsa01210: 2-oxocarboxylic acid metabolism, hsa03440: homologous recombination, hsa04360: axon guidance, hsa00051: fructose and mannose metabolism, hsa00565: ether lipid metabolism, hsa00510: N-glycan biosynthesis, and hsa04110 : cell cycle, hsa03030: DNA replication, hsa03013: RNA trafficking, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome, hsa04141: endoplasmic reticulum protein processing, hsa04810: regulation of the actin backbone, hsa03050: proteasome , hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa04932: nonalcoholic fatty liver disease (NAFLD), hsa00020: citric acid cycle, hsa00564: glycerophospholipid metabolism, hsa03008: eukaryotic ribosome biogenesis, hsa03015: mRNA Monitoring pathways, hsa01200: Carbon metabolism, hsa00520: Amino sugar and nucleotide sugar metabolism, hsa05034: Alcoholism, hsa04120: Ubiquitin-mediated proteolysis, hsa05205: Proteoglycans in cancer, hsa05206: Small RNAs in cancer .
7)HrpNEcb多拟表位配体蛋白识别结合的的膜蛋白参与的细胞联结、神经连结、血管、内分泌、生殖系统等代谢通路19条：hsa04723:逆行神经信号、hsa04726:血清素激活的突触、hsa00900:萜类化合物生物合成支柱、hsa04520:黏着结、hsa05032:吗啡上瘾以及hsa04510:粘着斑、hsa04724:谷氨酸能的突触、hsa04530:紧密连接、hsa00830:视黄醇新陈代谢、hsa04114:卵母细胞减数分裂、hsa04728:多巴胺能神经突触、hsa00100:类固醇生物合成、hsa04261:心肌细胞的肾上腺素能信号、hsa04727:神经元突触、hsa04725:胆碱能突触、hsa04540:缝隙连接、hsa04971:胃酸分泌、hsa04713:昼夜夹带、hsa04931:胰岛素抵抗。7) HrpNEcb multi-epitope ligand protein recognizes and binds membrane proteins involved in 19 metabolic pathways such as cell junction, neural junction, blood vessel, endocrine, reproductive system, etc.: hsa04723: retrograde neural signal, hsa04726: serotonin-activated synapse , hsa00900: terpenoid biosynthetic pillars, hsa04520: adhesion knots, hsa05032: morphine addiction and hsa04510: focal adhesions, hsa04724: glutamatergic synapses, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114: eggs blast meiosis, hsa04728: dopaminergic synapses, hsa00100: steroid biosynthesis, hsa04261: adrenergic signaling in cardiomyocytes, hsa04727: neuronal synapses, hsa04725: cholinergic synapses, hsa04540: gap junctions, hsa04971: gastric acid secretion, hsa04713: diurnal entrainment, hsa04931: insulin resistance.
HrpNEcb多拟表位配体蛋白，作为一类富含特殊多个线性和构象表位结构的配体蛋白分子，能够跨界识别、结合多种类型的膜受体、膜蛋白、信息通路和代谢通路，进一步分析这些膜受体、膜蛋白、信息通路和代谢通路的位置、结构、特性、作用机制和功能，它们广泛涉及和影响机体的生长、发育、代谢、防御和细胞程序性死亡的生命基本属性，并广泛涉及诊断、预防、治疗、康复神经系统、消化系统、运动系统、循环系统、呼吸系统、内分泌系统、免疫系统、泌尿系统、生殖系统、皮肤系统疾病和状况。HrpNEcb多拟表位配体蛋白是一类具有全新功能，全新作用机制和全新应用前景的特殊多拟表位配体蛋白。HrpNEcb multi-epitopic ligand protein, as a class of ligand protein molecules rich in special multiple linear and conformational epitope structures, can recognize and bind various types of membrane receptors, membrane proteins, information pathways and metabolism across borders Pathways, and further analyze the location, structure, properties, mechanism of action and function of these membrane receptors, membrane proteins, information pathways and metabolic pathways, which are widely involved in and affect the growth, development, metabolism, defense and life of programmed cell death. Essential attributes and broadly relate to the diagnosis, prevention, treatment, rehabilitation of diseases and conditions of the nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system, and skin system. HrpNEcb multi-epitope ligand protein is a kind of special multi-epitope ligand protein with new function, new mechanism of action and new application prospect.
实施例6Example 6
HrpNEch多拟表位配体蛋白采用已注册的基因的工程菌发酵，高效表达，纯化制备，具体包括以下步骤：The HrpNEch polymimetic epitope ligand protein is fermented, highly expressed, purified and prepared by engineering bacteria with registered genes, which specifically includes the following steps:
1)HrpNEch多拟表位配体蛋白的工程菌发酵制备：将带有编码HrpNEch多拟表位配体蛋白的基因(包括，但不限于生物样品天然基因、化学合成基因、转基因遗传重组体基因以及相似基因及其基因修饰)质粒的工程菌(E.coli)，相关蛋白的生产系是K-12原菌经特殊改造后的衍生菌JY-01(DE3)，在LB液体培养基(每升含卡那霉素50微克)中，在一定的温度下条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体。用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析表达产物HrpNEch多拟表位配体蛋白，在电泳胶板的样品泳道上，会呈现一条34.15kda条带，它就是基因hrpNEch的表达产物HrpNEch多拟表位配体蛋白；其中，发酵用培养基Na 2HPO 4-KH 2PO 4缓冲系统pH 6.5-5.5；发酵温度为37-38℃；发酵增殖液体培养基葡萄糖浓度0.01-0.05％；发酵诱导液体培养基葡萄糖浓度0.05-0.00％；发酵诱导液体培养基乳糖浓度0.5-0.4％；发酵诱导液体培养时间为7-9h。 1) The engineering bacteria fermentation preparation of HrpNEch multi-epitope ligand protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes) encoding HrpNEch multi-epitope ligand proteins are prepared. and engineering bacteria (E.coli) with similar genes and their genetic modifications) plasmids, and the production line of related proteins is a specially modified derivative of the original K-12 bacteria JY-01 (DE3), in LB liquid medium (every liter containing kanamycin 50 μg), at a certain temperature, when cultured to OD600=0.7, add IPTG (isopropyl thiogalactoside, Isopropylβ-D-Thiogalactosid) (final concentration 1mMol), After culturing, the cells were collected by centrifugation. 10% SDS-PAGE polyacrylamide gel electrophoresis was used to analyze the expression product HrpNEch multi-epitope ligand protein. On the sample lane of the electrophoresis gel plate, there will be a 34.15kda band, which is the expression product of the gene hrpNEch HrpNEch polyprotein. Mimic epitope ligand protein; wherein, the pH of the fermentation medium Na 2 HPO 4 -KH 2 PO 4 buffer system is 6.5-5.5; the fermentation temperature is 37-38° C.; the concentration of glucose in the fermentation and proliferation liquid medium is 0.01-0.05%; The concentration of glucose in the induction liquid medium is 0.05-0.00%; the concentration of lactose in the fermentation-induction liquid medium is 0.5-0.4%; the incubation time of the fermentation-induction liquid medium is 7-9 hours.
2)工程菌生产系在HrpNEch多拟表位配体蛋白生产发酵结束后的后处理：①灭菌：发酵液在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃以下；②清洗：用葡萄糖Na 2HPO 4-KH 2PO 4缓冲液，pH5-5.5，葡萄糖浓度200-300mmol，在蝶式连续流离心机里清洗工程菌体五至八次；③破碎工程菌并清除细胞壁，再用pH 5-5.5、葡萄糖浓度200-300mmol的Na 2HPO 4-KH 2PO 4缓冲液稀释菌体，调节菌体鲜重为稀释液的20-30％，导入高压破碎机，连续用800-1000Mpa压力，破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁。 2) The post-processing of the engineering bacteria production system after the HrpNEch polymimetic epitope ligand protein production and fermentation is completed: ① Sterilization: the fermentation broth is sterilized at a temperature of 80 °C for 30 minutes, and then rapidly cooled to below 30 °C; ② Cleaning: Use glucose Na 2 HPO 4 -KH 2 PO 4 buffer, pH5-5.5, glucose concentration 200-300mmol, wash the engineering bacteria in a butterfly continuous flow centrifuge five to eight times; ③ break the engineering bacteria and remove the cell wall , and then dilute the bacterial cells with a Na 2 HPO 4 -KH 2 PO 4 buffer solution of pH 5-5.5 and a glucose concentration of 200-300 mmol, adjust the fresh weight of the bacterial cells to 20-30% of the diluted solution, introduce into a high-pressure crusher, and continuously use 800-1000Mpa pressure, crush the engineering bacteria, pass the crushed bacteria liquid into the butterfly continuous flow centrifuge to remove the cell wall.
3)HrpNEch多拟表位配体蛋白分子的纯化：用NI-NTA琼脂糖凝胶柱纯化HrpNEch多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，完成纯化HrpNEch多拟表位配 体蛋白制备。3) Purification of HrpNEch polymimetic epitope ligand protein molecule: Purify HrpNEch polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column, and the protein purification is according to the NI-NTA agarose column manufacturer's suggestion The method is implemented, and the preparation of purified HrpNEch polymimetic epitope ligand protein is completed.
实施例7Example 7
HrpNEch多拟表位配体蛋白通过“人工合成基因”的表达蛋白进行制备，具体包括以下步骤：The HrpNEch multi-epitope ligand protein is prepared by expressing the protein of "artificially synthesized gene", which specifically includes the following steps:
第一步：编码HrpNEch蛋白的hrpNEch基因的人工合成；The first step: artificial synthesis of the hrpNEch gene encoding the HrpNEch protein;
1)按照GenBank公布的编码HrpNEch蛋白的hrpNEch基因(GenBank:AY999000.1)核苷酸序列,人工合成hrpNEch基因，其DNA序列为：1) According to the nucleotide sequence of the hrpNEch gene (GenBank: AY999000.1) encoding the HrpNEch protein published by GenBank, artificially synthesize the hrpNEch gene, and its DNA sequence is:
Figure PCTCN2021134714-appb-000033
Figure PCTCN2021134714-appb-000033
第二步：2)根据以上DNA序列，人工合成蛋白基因时，在基因的5′和3′分别加上BamHI和HindIII酶切位点，方便蛋白基因克隆；Step 2: 2) According to the above DNA sequence, when artificially synthesizing protein gene, add BamHI and HindIII enzyme cleavage sites to the 5' and 3' of the gene respectively to facilitate protein gene cloning;
第三步：人工基因合成委托Thermo Fisher Scientific公司的GeneArt基因合成与服务部门完成。3)将合成的编码HrpNEch蛋白基因的DNA片段，逐一克隆到高效蛋白表达载体PET28a(+)(含His-Tag标签)的BamHI-HindIII位点，经DNA测序确保克隆的准确性；The third step: artificial gene synthesis is entrusted to the GeneArt gene synthesis and service department of Thermo Fisher Scientific. 3) The synthesized DNA fragments encoding the HrpNEch protein gene are cloned into the BamHI-HindIII site of the high-efficiency protein expression vector PET28a(+) (containing His-Tag label) one by one, and the accuracy of the clone is ensured through DNA sequencing;
第四步：将1)至3)编码HrpNEch蛋白的基因克隆转入大肠杆菌工程菌(E.coli)中，相关蛋白的生产系(E.coli)是K-12原菌经特殊改造后的衍生菌JY-01(DE3)；在LB液体培养基(每升含卡那霉素50微克)中，37℃条件下，培养至OD600＝0.7时，加入IPTG(异丙基硫代半乳糖苷，Isopropylβ-D-Thiogalactosid)(终浓度1mMol)，继续培养后离心收集菌体，用10％SDS－PAGE聚丙烯酰胺凝胶电泳分析表达产物HrpNEch蛋白，在电泳胶板的样品泳道上，会呈现一条34.15kda条带，它就是基因hrpNEch的表达产物HrpNEch多拟表位配体蛋白，详见图67；The fourth step: clone the gene encoding HrpNEch protein from 1) to 3) and transfer it into Escherichia coli engineering bacteria (E.coli), and the production line (E.coli) of the related protein is the original K-12 bacteria after special transformation. Derivative bacteria JY-01 (DE3); in LB liquid medium (containing 50 micrograms of kanamycin per liter), under the condition of 37 °C, when cultured to OD600=0.7, IPTG (isopropyl thiogalactoside) was added. , Isopropylβ-D-Thiogalactosid) (final concentration 1 mMol), continue to culture, collect the bacteria by centrifugation, and analyze the expression product HrpNEch protein by 10% SDS-PAGE polyacrylamide gel electrophoresis. On the sample lane of the electrophoresis gel plate, it will appear A 34.15kda band, which is the HrpNEch polymimetic ligand protein, the expression product of the gene hrpNEch, see Figure 67 for details;
其中发酵用培养基Na 2HPO 4-KH 2PO 4缓冲系统，缓冲系统的pH6.5-5.5；发酵增殖液体培养基葡萄糖浓度为0.01-0.05％；发酵诱导液体培养基乳糖浓度0.5-0.4％； The fermentation medium is Na 2 HPO 4 -KH 2 PO 4 buffer system, the pH of the buffer system is 6.5-5.5; the concentration of glucose in the fermentation and proliferation liquid medium is 0.01-0.05%; the concentration of lactose in the fermentation induction liquid medium is 0.5-0.4% ;
第五步：将收集菌体悬浮到Na 2HPO 4-KH 2PO 4缓冲液中，在80℃温度下，30分钟完成灭菌处理，迅速降温至30℃，在蝶式连续流离心机里清洗工程菌体五至八次，导入高压破碎机，连续用800-1000Mpa压力下，破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁，收集HrpNEch多拟表位配体蛋白分子，HrpNEch多拟表位配体蛋白存在于上清液中； Step 5: Suspend the collected cells into Na 2 HPO 4 -KH 2 PO 4 buffer, complete the sterilization treatment at 80°C for 30 minutes, quickly cool down to 30°C, and place in a butterfly continuous flow centrifuge. Wash the engineered bacteria for five to eight times, import them into a high-pressure crusher, and continuously use 800-1000Mpa pressure to break the engineered bacteria. Pass the broken bacteria liquid into a butterfly continuous flow centrifuge to remove the cell wall and collect HrpNEch polymimetic epitope ligands. Protein molecules, HrpNEch multi-epitope ligand proteins are present in the supernatant;
第六步：用NI-NTA琼脂糖凝胶柱纯化HrpNEch多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，完成纯化HrpNEch多拟表位配体蛋白的制备。Step 6: Purify the HrpNEch polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column. The protein purification is carried out according to the method recommended by the NI-NTA agarose column manufacturer to complete the purification of HrpNEch polymimetic epitopes. Preparation of ligand proteins.
10％SDS聚丙酰胺凝胶电泳检测高效表达的纯化蛋白-His重组条带，详见图67。The highly expressed purified protein-His recombinant band was detected by 10% SDS polyacrylamide gel electrophoresis, see Figure 67 for details.
如图67所示，左面为分子量标识带；泳道1为纯化前的电泳谱带，对应分子量区聚集了较多的条带，也包括34.15kda条带；泳道2为纯化HrpNEch多拟表位配体蛋白条带，分子量34.15kda，在蛋白对应分子量区，表明已经得到相应的HrpNEch纯化蛋白。As shown in Figure 67, the left side is the molecular weight identification band; lane 1 is the electrophoresis band before purification, and there are more bands in the corresponding molecular weight region, including the 34.15kda band; lane 2 is the purified HrpNEch polymimetic epitope ligand The body protein band, with a molecular weight of 34.15kda, is in the corresponding molecular weight region of the protein, indicating that the corresponding HrpNEch purified protein has been obtained.
如图68所示，HrpNEch多拟表位配体蛋白的过敏实验检测：其中所见焦斑是经过HrpNEch多拟表位配体蛋白液处理24hr左右所形成，叶片上排：H 2O注射，为对照；叶片下排：HrpNEch多拟表位配体蛋白液(250μg/ml)注射，为处理，即HrpNEch多拟表位配体蛋白在烟草叶片上的超敏反应。 As shown in Figure 68, the allergy test of HrpNEch poly-epitope ligand protein: the focal spots seen are formed by HrpNEch poly-epitope ligand protein solution treatment for about 24hrs, the upper row of leaves: H 2 O injection, For the control; the lower row of leaves: HrpNEch poly-epitope ligand protein solution (250μg/ml) injection, for the treatment, that is, the hypersensitivity reaction of HrpNEch poly-epitope ligand protein on tobacco leaves.
纯化的HrpNEch多拟表位配体蛋白能普遍引发多种植物叶片发生超敏反应，供试植物种类可以是：烟草、辣椒、茄子、西红柿、土豆、草莓、黄瓜、空心菜、鸡冠花、玻璃海棠、九月菊、三色堇、胭脂花、矮牵牛、葡萄、月季、槐树、豌豆、桃树、一串红、丝瓜、四季豆、花椰菜、菠菜、油菜、薯蓣、豇豆、蚕豆、玉米、水稻、大豆、仙客来、桑树、南瓜、枇杷、椿树等36个种类的不同植物。The purified HrpNEch multi-epitope ligand protein can generally induce hypersensitivity reactions in the leaves of various plants. The tested plant species can be: tobacco, pepper, eggplant, tomato, potato, strawberry, cucumber, water spinach, celosia, glass begonia , September chrysanthemum, pansy, nopal, petunia, grape, rose, locust tree, pea, peach tree, bunch of red, loofah, green beans, cauliflower, spinach, rapeseed, yam, cowpea, broad bean, corn , rice, soybean, cyclamen, mulberry, pumpkin, loquat, toon tree and other 36 kinds of different plants.
实施例8Example 8
动物实验mRNA(RNA-Seq)测序mRNA (RNA-Seq) sequencing in animal experiments
mRNA测序的研究对象为特定细胞在某一功能状态下所能转录出来的所有带ploy-A尾的RNA，主要为mRNA。通过逆转录过程将细胞产生的mRNA转化为DNA(cDNA，互补，并对获得的cDNA进行文库构建)。然后对得到的DNA进行测序，并从观察到的特定DNA丰度中，从中推断细胞中mRNA的原始量，从而找到在实验条件下转录水平发生变化的基因或转录本，即差异表达。通过找到这些差异表达的基因和转录本，能够全面快速地获得某一物种特定组织或器官在某一状态下的几乎所有mRNA表达丰度，已广泛应用于基础研究和药物研发等多个领域。我们采用mRNA-seq技术，证明了HrpNEcb多拟表位配体蛋白诱导小鼠多个器官中多基因的差异表达。The research object of mRNA sequencing is all RNAs with a poly-A tail that can be transcribed by a specific cell in a certain functional state, mainly mRNA. Cell-generated mRNA is converted into DNA (cDNA, complementation, and library construction of the obtained cDNA) by a process of reverse transcription. The resulting DNA is then sequenced and from the observed abundance of specific DNA, the original amount of mRNA in the cell can be inferred from it to find genes or transcripts whose transcription levels change under the experimental conditions, i.e. differentially expressed. By finding these differentially expressed genes and transcripts, it is possible to comprehensively and quickly obtain almost all mRNA expression abundances of a specific tissue or organ of a species in a certain state, which has been widely used in basic research and drug development and other fields. Using mRNA-seq technology, we demonstrate that the HrpNEcb polyepitopic ligand protein induces differential expression of multiple genes in multiple organs in mice.
1.实验动物样品处理1. Experimental animal sample processing
本实验委托上海华盈生物医药科技有限公司蛋白质谱技术平台实施。This experiment was entrusted to Shanghai Huaying Biomedical Technology Co., Ltd. to implement the protein profiling technology platform.
实验样品处理：实验选用8周龄的balb/C的实验小鼠，分为HrpNEch多拟表位配体蛋白处理组，包括口服6小时、24小时和涂抹6小时、12小时共4种处理，每种处理3只实验鼠，共计12只；空白对照组4只实验鼠；不含HrpNEch多拟表位配体蛋白的缓冲液对照假手术组，包括口服6小时、24小时和涂抹6小时、12小时共4种处理，每种处理4只实验鼠，共计16只实验鼠，三次重复；对实验处理组鼠用含600mg·L -1HrpNEch多拟表位配体蛋白的缓冲液饲喂和涂抹处理，缓冲液对照假手术组小鼠用缓冲液饲喂和涂抹处理，空白对照组小鼠不做任何处理。在相同饲养条件下，按不同时间，分别分组取小鼠睾丸、肾脏等组织，进行RNA-Seq的测序及分析。 Experimental sample treatment: 8-week-old balb/C mice were selected for the experiment and divided into HrpNEch multi-epitope ligand protein treatment groups, including four treatments of oral administration for 6 hours, 24 hours, and application for 6 hours and 12 hours. There were 3 experimental mice for each treatment, a total of 12 mice; 4 experimental mice in the blank control group; the buffer control sham-operated group without HrpNEch multi-epitope ligand protein, including oral administration for 6 hours, 24 hours and application for 6 hours, There were 4 kinds of treatments in 12 hours, 4 experimental mice in each treatment, 16 experimental mice in total, and repeated three times; the mice in the experimental treatment group were fed with buffer containing 600 mg·L -1 HrpNEch polymimetic ligand protein and For the smearing treatment, the mice in the buffer control sham-operated group were fed and smeared with buffer, and the mice in the blank control group did not receive any treatment. Under the same feeding conditions, at different times, the mouse testis, kidney and other tissues were grouped and collected for RNA-Seq sequencing and analysis.
2.mRNA(RNA-Seq)测序2. mRNA (RNA-Seq) sequencing
通过新一代测序，能够全面快速地获得某一物种特定组织或器官在某一状态下的几乎所有mRNA表达丰度，见图79mRNA(RNA-Seq)测序实验流程图。Through next-generation sequencing, the expression abundance of almost all mRNAs in a specific tissue or organ of a species in a certain state can be obtained comprehensively and rapidly, as shown in Figure 79 mRNA (RNA-Seq) sequencing experimental flow chart.
I.RNA抽提与质检I. RNA extraction and quality inspection
采用miRNeasy Micro Kit(Cat#1071023Qiagen)并且根据生产厂商提供的标准操作流程进行样品的总RNA抽提。总RNA经NanoDrop ND-2000分光光度计及Agilent Bioanalyzer 4200(Agilent technologies,Santa Clara,CA,US)进行质检，质检合格的RNA用于后续的测序实验。Total RNA extraction of the samples was performed using the miRNeasy Micro Kit (Cat#1071023Qiagen) and according to the standard operating procedure provided by the manufacturer. The total RNA was quality-checked by NanoDrop ND-2000 spectrophotometer and Agilent Bioanalyzer 4200 (Agilent technologies, Santa Clara, CA, US), and the qualified RNA was used for subsequent sequencing experiments.
II.文库构建与质检II. Library Construction and Quality Control
由于真核生物大部分mRNA都带有polyA尾，利用Oligo(dT)可以富集带有polyA尾的mRNA。接下来对富集mRNA进行片段化、双链cDNA合成、末端修复、3’末端加A、连接接头、扩增。构建的文库使用
Figure PCTCN2021134714-appb-000034
2.0Fluorometer检测浓度，Agilent2100检测大小。 Since most mRNAs in eukaryotes have polyA tails, Oligo(dT) can be used to enrich mRNAs with polyA tails. The enriched mRNA is then subjected to fragmentation, double-stranded cDNA synthesis, end repair, 3' end addition, ligation of adapters, and amplification. The constructed library uses
Figure PCTCN2021134714-appb-000034
2.0 Fluorometer detects concentration, Agilent2100 detects size.
III.上机测序III. On-board sequencing
对质检合格的文库进行Illumina测序，测序仪通过捕获荧光信号，并通过计算机软件将光信号转化为测序峰，从而获得待测片段的序列信息。Illumina sequencing is performed on the library that has passed the quality inspection. The sequencer captures the fluorescent signal and converts the optical signal into a sequencing peak through computer software to obtain the sequence information of the fragment to be tested.
IV.mRNA测序数据分析按图80数据分析流程进行。IV. Analysis of mRNA sequencing data is performed according to the data analysis process shown in FIG. 80 .
3.结果分析3. Analysis of results
1)HrpNEch多拟表位配体蛋白诱导的差异基因筛选1) Differential gene screening induced by HrpNEch multi-epitopic ligand proteins
首先对fragment counts进行归一化，再根据假设检验模型计算p-value，最后对p-value多重假设检验校正，得到FDR值。FP KM值计算差异表达倍数(Fold-change)，使用edgeR软件。差异基因筛选条件如下：p-value<0.05且|Fold-change|>＝2。First normalize the fragment counts, then calculate the p-value according to the hypothesis testing model, and finally correct the p-value for multiple hypothesis testing to obtain the FDR value. The FP KM value was used to calculate the differential expression fold (Fold-change) using edgeR software. Differential gene screening conditions are as follows: p-value<0.05 and |Fold-change|>=2.
2)HrpNEch多拟表位配体蛋白诱导的差异基因火山图2) Differential gene volcano plots induced by HrpNEch multi-epitopic ligand proteins
采用差异基因火山图显示HrpNEch蛋白诱导的表达差异显著性基因的整体分布情况。横坐标：基因在不同样本中的表达倍数变化(log2 Fold-Chan ge)；纵坐标：基因表达差异的显著性水平(-log10 p-value)；右侧点表达显著上调基因；左侧点表达显著下调基因；下部点表达变化不显著基因。图69-70分别为小鼠的睾丸、肾脏HrpNEch多拟表位配体蛋白通过口服和涂抹诱导的差异基因火山图,图中HrpNEch缩写为N2。The differential gene volcano plot was used to display the overall distribution of genes with significant differential expression induced by HrpNEch protein. Horizontal axis: fold change of gene expression in different samples (log2 Fold-Change); vertical axis: significant level of gene expression difference (-log10 p-value); the expression of the right point is significantly up-regulated gene; the expression of the left point Significantly down-regulated genes; genes with no significant changes in expression at lower points. Figures 69-70 are the differential gene volcano plots of mouse testis and kidney HrpNEch multi-epitope ligand protein induced by oral administration and smearing, respectively, HrpNEch is abbreviated as N2 in the figure.
3)HrpNEch多拟表位配体蛋白诱导的差异基因聚类图3) Clustering map of differential genes induced by HrpNEch multi-epitope ligand proteins
差异基因集进行聚类分析，将表达模式相近的基因聚在一起，显示基因具有共同功能或参与到共同信号通路。将log10(FPKM+1)值进行归一化转换(scale number)并进行聚类。图71-72分别为睾丸、肾脏表达差异基因集聚类热图，图中HrpNEch缩写为N2。Cluster analysis was performed on differential gene sets, and genes with similar expression patterns were clustered together, indicating that genes have common functions or participate in common signaling pathways. The log10(FPKM+1) values were normalized (scale number) and clustered. Figures 71-72 are cluster heatmaps of differentially expressed gene sets in testis and kidney, respectively, in which HrpNEch is abbreviated as N2.
4)HrpNEch多拟表位配体蛋白诱导的差异基因GO富集分析4) GO enrichment analysis of differential genes induced by HrpNEch multi-epitope ligand proteins
基因本体(Gene Ontology，GO)是一个在生物信息学领域中广泛使用的本体。基因本体论是对基因在不同维度和不同层次上的描述，涵盖了生物学的生物过程(biological_process)，细胞组分(cellular_component)及分子功能(molecular_function)。生物过程是在说明该基因参与了哪些生物学过程；细胞组分解释的是基因存在在哪里，包括该基因在细胞质还是在细胞核？如果存在细胞质那在哪个细胞器上？如果是在线粒体中那是存在线粒体膜上还是在线粒体的基质当中等等，这些信息都属于细胞组；分子功能解释的是该基因在分子层面的功能是什么？描述其在个体分子生物学上的活性，如催化活性或结合活性。基因本体数据库(Gene Ontology)是GO组织(Gene Ontology Consortium)在2000年构建的一个结构化的标准生物模型，旨在建立基因及其产物知识的标准词汇体系，涵盖了基因的生物学过程(biological process)，细胞组分(cellular component)、分子功能(molecular function)。Term是GO里面的基本描述单元。GO Terms用于描述基因产物的功能。通过将差异基因做GO富集分析，可以把基因按照不同的功能进行归类，达到对基因进行注释和分类的目的。我们对HrpNEch多拟表位配体蛋白诱导的差异表达基因进行了GO term富集分析，其结果证明、证实了HrpNEch多拟表位配体蛋白作为一类具有多个表位特殊结构，全新功能，全新作用机制和全新应用前景的配体蛋白，诱导了受试小鼠多个器官(睾丸、肾脏)多基因的差异表达，这些差异表达基因涵盖了生物过程、细胞组分及分子功能。差异表达基因利用Fisher精确检验进行GO分析。Fisher精确检验计算得到p-value，并进行多重假设检验校正得到q-value。筛选p-value小于0.05的GO条目作为显著富集的GO条目。HrpNEch多拟表位配体蛋白诱导的 差异基因GO富集分析结果进一步表述如下：①生物过程(biological_process)相关差异表达基因包括了繁殖，细胞死亡，免疫系统的过程，行为，代谢过程，细胞过程，生殖过程，生物粘附，信号，多细胞生物过程，发育过程，增长，运动，单个组织的过程，生物相，有节奏的过程，生物过程的正调控，生物过程负调控，生物过程调节，刺激反应，定位，生物调节，细胞成分组织或生物发生，细胞聚集，解毒以及突触前过程涉及突触传递。生物过程GO富集分析结果详见表8至表11。②细胞组分(cellular_component)相关差异表达基因涵盖了细胞及细胞外区域，类核，膜，病毒粒子，细胞结，细胞外基质，细胞膜封闭腔，复杂大分子，细胞器，细胞外基质成分，细胞外区域部分，细胞器部件，病毒粒子部件，膜部件，突触部分，细胞部件，突触，以及细胞超分子纤维等。细胞组分GO富集分析结果详见表8至表11。③分子功能(molecular function)相关差异表达基因涵盖了转录因子活性，蛋白质结合，核酸结合转录因子活性，催化活性，信号传感器活动，结构分子活动，运输活动，绑定，电子载体活动，成形素活动，抗氧化活性，金属伴侣蛋白活性，蛋白质标记，化学引诱物的活动，转译调控，化学排斥物活性，活动分子传感器，分子功能调控等。分子功能GO富集分析结果详见表8至表11。Gene Ontology (GO) is an ontology widely used in the field of bioinformatics. Gene Ontology is a description of genes in different dimensions and levels, covering biological processes (biological_process), cellular components (cellular_component) and molecular functions (molecular_function). Biological processes are describing which biological processes the gene is involved in; cellular components explain where the gene is present, including whether the gene is in the cytoplasm or in the nucleus? If cytoplasm is present in which organelle? If it is in the mitochondria, is it on the mitochondrial membrane or in the matrix of the mitochondria, etc., this information belongs to the cell group; the molecular function explains what is the function of the gene at the molecular level? Describe its activity in individual molecular biology, such as catalytic activity or binding activity. Gene Ontology database (Gene Ontology) is a structured standard biological model constructed by the GO organization (Gene Ontology Consortium) in 2000. It aims to establish a standard vocabulary system for knowledge of genes and their products, covering the biological process of genes. process), cellular component, molecular function. Term is the basic description unit in GO. GO Terms are used to describe the function of gene products. By performing GO enrichment analysis on differential genes, genes can be classified according to different functions, so as to achieve the purpose of annotating and classifying genes. We carried out GO term enrichment analysis on the differentially expressed genes induced by HrpNEch multi-epitope ligand protein, and the results proved and confirmed that HrpNEch multi-epitope ligand protein, as a class of special structures with multiple epitopes, new functions , a ligand protein with a new mechanism of action and a new application prospect, induced the differential expression of multiple genes in multiple organs (testis, kidney) of the tested mice, and these differentially expressed genes covered biological processes, cellular components and molecular functions. Differentially expressed genes were analyzed by GO using Fisher's exact test. Fisher's exact test was calculated to obtain the p-value, and multiple hypothesis test correction was performed to obtain the q-value. GO entries with p-value less than 0.05 were screened as significantly enriched GO entries. The results of GO enrichment analysis of differential genes induced by HrpNEch multi-epitope ligand proteins are further expressed as follows: ① Biological_process-related differentially expressed genes include reproduction, cell death, immune system processes, behaviors, metabolic processes, and cellular processes , reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes, growth, motility, processes in individual tissues, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, Stimulatory responses, localization, bioregulation, cellular component organization or biogenesis, cellular aggregation, detoxification, and presynaptic processes involve synaptic transmission. The results of GO enrichment analysis of biological processes are shown in Table 8 to Table 11. ②The differentially expressed genes related to cellular components cover cells and extracellular regions, nucleoids, membranes, virus particles, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules, organelles, extracellular matrix components, cells Parts of the outer region, organelle parts, virion parts, membrane parts, synaptic parts, cell parts, synapses, and cellular supramolecular fibers, etc. The results of GO enrichment analysis of cell components are shown in Table 8 to Table 11. ③ Molecular function-related differentially expressed genes cover transcription factor activity, protein binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, and morphogen activity , antioxidant activity, metal chaperone protein activity, protein labeling, chemoattractant activity, translation regulation, chemorepellent activity, active molecular sensors, molecular function regulation, etc. The molecular function GO enrichment analysis results are shown in Table 8 to Table 11.
其中，所有表格8-11中，空格表示没有收集到对应的未达到p-value小于0.05标准的相关数据，以下及其所有表格空白含义与此相同。Among them, in all Tables 8-11, blanks indicate that the corresponding data that does not meet the standard of p-value less than 0.05 have not been collected, and the blanks in the following and all tables have the same meaning.
表8 HrpNEch多拟表位配体蛋白诱导肾脏的生物学过程、细胞组分和分子功能相关功能群显著上调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时)Table 8 The biological process, cellular components and molecular function-related functional groups of HrpNEch multi-epitope ligand protein-induced kidney were significantly up-regulated and expressed GO terms classification gene number statistics table (6, 24 hours after oral administration and 6 hours after application)
Figure PCTCN2021134714-appb-000035
Figure PCTCN2021134714-appb-000035
Figure PCTCN2021134714-appb-000036
Figure PCTCN2021134714-appb-000036
Figure PCTCN2021134714-appb-000037
Figure PCTCN2021134714-appb-000037
表9 HrpNEch多拟表位配体蛋白诱导睾丸的生物学过程、细胞组分和分子功能相关功能群显著上调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时)Table 9 The biological process, cellular components and molecular function-related functional groups of testis induced by HrpNEch multi-epitope ligand protein were significantly up-regulated and expressed GO terms classification gene number statistics table (6, 24 hours after oral administration and 6 hours after application)
Figure PCTCN2021134714-appb-000038
Figure PCTCN2021134714-appb-000038
Figure PCTCN2021134714-appb-000039
Figure PCTCN2021134714-appb-000039
Figure PCTCN2021134714-appb-000040
Figure PCTCN2021134714-appb-000040
Figure PCTCN2021134714-appb-000041
Figure PCTCN2021134714-appb-000041
表10 HrpNEch多拟表位配体蛋白诱导肾脏的生物学过程、细胞组分和分子功能相关功能群显著下调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时)Table 10 The biological process, cellular components and molecular function-related functional groups of HrpNEch multi-epitope ligand protein-induced kidney were significantly down-regulated.
Figure PCTCN2021134714-appb-000042
Figure PCTCN2021134714-appb-000042
Figure PCTCN2021134714-appb-000043
Figure PCTCN2021134714-appb-000043
Figure PCTCN2021134714-appb-000044
Figure PCTCN2021134714-appb-000044
Figure PCTCN2021134714-appb-000045
Figure PCTCN2021134714-appb-000045
表11 HrpNEch多拟表位配体蛋白诱导睾丸的生物学过程、细胞组分和分子功能相关功能群显著下调表达GO terms分类基因数统计表(口服6、24小时和涂抹6小时)Table 11. The biological process, cellular components and molecular function-related functional groups of testis induced by HrpNEch multi-epitope ligand protein were significantly down-regulated and expressed GO terms. Statistical table of the number of classified genes (6, 24 hours after oral administration and 6 hours after application)
Figure PCTCN2021134714-appb-000046
Figure PCTCN2021134714-appb-000046
Figure PCTCN2021134714-appb-000047
Figure PCTCN2021134714-appb-000047
Figure PCTCN2021134714-appb-000048
Figure PCTCN2021134714-appb-000048
5.差异表达基因的KEGG pathway富集分析5. KEGG pathway enrichment analysis of differentially expressed genes
京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes，KEGG)，是系统分析基因功能与基因组信息的数据库，它整合了基因组学、生物化学和系统功能组学的信息，有助于研究者把基因及表达信息的过程作为一个网络进行整体研究。Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database for systematic analysis of gene function and genomic information. The process of gene and expression information is studied holistically as a network.
KEGG主要的特点是将基因与各种生化反应联系在了一起，提供整合的代谢途径。KEGG目前共包含了19个子数据库，它们被分类为系统信息、基因组信息和化学信息三个类别。在生物体内，不同的基因产物相互协调来行使生物学功能，对差异表达基因的通路(Pathway)注释分析有助于进一步解读基因的 功能。对HrpNEch多拟表位配体蛋白诱导的差异表达基因进行了KEGG pathway富集分析，获得这些差异基因在信号通路中的角色(上下游关系)和生物学功能，深入理解基因与功能的关系。研究结果证明、证实了HrpNEch多拟表位配体蛋白作为一类具有多表位特殊结构，全新功能，全新作用机制和全新应用前景的配体蛋白，诱导了小鼠多个器官(睾丸、肾脏)多基因的差异表达，这些差异表达基因参与了归属于细胞过程(Cellular Processes)、环境信息处理(Environmental Information Processing)、遗传信息处理(Genetic Information Processing)、新陈代谢(Metabolism)和生物体系统(Organismal Systems)等功能途径。HrpNEch多拟表位配体蛋白诱导的差异基因GO富集分析结果进一步表述如下：①细胞过程(Cellular Processes)：HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了运输和分解代谢，细胞群体，细胞活性，细胞生长与死亡等细胞过程(详见图7至图12)。②环境信息处理(Environmental Information Processing):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了信号分子与相互作用，信号转导，膜运输等环境信息处理过程(详见图7至图12)。③遗传信息处理(Genetic Information Processing):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了翻译，复制和修复，折叠、分类和降解等生物过程(详见图73至图78)。④新陈代谢(Metabolism):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了生物降解和代谢，核苷酸代谢，其他氨基酸的代谢，代谢的辅助因子和维生素，脂质代谢，糖的生物合成和代谢，全局和概览地图，能量代谢，碳水化合物代谢及氨基酸代谢等代谢过程(详见图7至图12)。⑤生物体系统(Organismal Systems):HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了感觉系统，神经系统，免疫系统，排泄系统，环境适应，内分泌系统，消化系统，发育循环系统等细胞过程(详见图7至图12)。The main feature of KEGG is to link genes with various biochemical reactions, providing integrated metabolic pathways. KEGG currently contains a total of 19 sub-databases, which are classified into three categories: systematic information, genomic information and chemical information. In organisms, different gene products coordinate with each other to perform biological functions. Pathway annotation analysis of differentially expressed genes is helpful for further interpretation of gene functions. The KEGG pathway enrichment analysis was performed on the differentially expressed genes induced by HrpNEch multi-epitopic ligand proteins, and the roles (upstream and downstream relationships) and biological functions of these differential genes in the signaling pathway were obtained, and the relationship between genes and functions was deeply understood. The research results proved and confirmed that HrpNEch multi-epitope ligand protein, as a kind of ligand protein with multi-epitope special structure, new function, new mechanism of action and new application prospect, induces multiple organs (testis, kidney) of mice. ) differential expression of multiple genes involved in cellular processes, environmental information processing, genetic information processing, metabolism (Metabolism) and biological systems (Organismal) Systems) and other functional pathways. The results of GO enrichment analysis of differential genes induced by HrpNEch multi-epitope ligand proteins are further described as follows: ① Cellular Processes: Multiple differentially expressed genes induced by HrpNEch multi-epitopic ligand proteins are involved in transport and catabolism , cell population, cell activity, cell growth and death and other cellular processes (see Figure 7 to Figure 12 for details). ②Environmental Information Processing: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in environmental information processing processes such as signal molecules and interactions, signal transduction, and membrane transport (see Figures 7 to 7 for details). Figure 12). ③ Genetic Information Processing: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in biological processes such as translation, replication and repair, folding, classification and degradation (see Figure 73 to Figure 78 for details) . ④Metabolism: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in biodegradation and metabolism, nucleotide metabolism, metabolism of other amino acids, metabolic cofactors and vitamins, lipid metabolism, sugar Global and overview maps of biosynthesis and metabolism, metabolic processes such as energy metabolism, carbohydrate metabolism and amino acid metabolism (see Figures 7 to 12 for details). ⑤Organismal Systems: Multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins are involved in sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, developmental circulatory system isocellular process (see Figures 7 to 12 for details).
与GO分类统计类似，对KEGG各个生物学途径(pathway)上的差异表达基因数目进行统计并以图形展示如图73至图78所示。Similar to GO classification statistics, the number of differentially expressed genes on each biological pathway of KEGG was counted and displayed graphically as shown in Figure 73 to Figure 78 .
说明：图形右边示图，从上到下中文译名：细胞过程、信息过程、遗传信息过程、代谢过程、组织系统发育过程；横坐标：涉及表达差异的各功能基因群的基因数目；纵坐标：涉及表达差异的细胞过程、信息过程、遗传信息过程、代谢过程、组织系统发育过程的功能基因群。Description: The diagram on the right side of the figure, from top to bottom, Chinese translations: cellular process, information process, genetic information process, metabolic process, tissue phylogeny; abscissa: the number of genes involved in different functional gene groups; ordinate: Functional gene groups involved in differentially expressed cellular processes, information processes, genetic information processes, metabolic processes, and tissue phylogeny.
实施例9Example 9
HrpNEch多拟表位配体蛋白识别结合特异蛋白的pull-down实验Pull-down experiment of HrpNEch multi-epitope ligand protein recognition and binding specific protein
1.样品准备和处理1. Sample Preparation and Handling
1)HrpNEch蛋白纯化1) HrpNEch protein purification
用NI-NTA琼脂糖凝胶柱纯化HrpNEch多拟表位配体蛋白-His重组蛋白，蛋白纯化按NI-NTA琼脂糖凝胶柱厂家建议方法实施，纯化制备的HrpNEch多拟表位配体蛋白备用(以下称为捕获蛋白或目的蛋白)。The HrpNEch polymimetic epitope ligand protein-His recombinant protein was purified by NI-NTA agarose column. The protein purification was carried out according to the method recommended by the NI-NTA agarose column manufacturer. The prepared HrpNEch polymimetic epitope ligand protein was purified. Standby (hereinafter referred to as capture protein or target protein).
2)实验用培养肝细胞总蛋白(以下称为钓饵蛋白)抽提2) Extraction of total protein from cultured hepatocytes (hereinafter referred to as bait protein) for experiments
I.培养肝细胞总蛋白的抽提：①用移液枪吸取裂解液(IP专用裂解液，加入1×cocktail蛋白酶抑制剂)加入到细胞。超声，冰上静置2h以上；②使用超声细胞破碎仪，冰上超声2s，停5s，共1min，冰上裂解总时长2h以上(间隔30min振荡器震荡混匀)；③将细胞裂解产物4℃下13000rpm离心15min，吸取上清转移至新的1.5mLEP管，冰上放置待用；④将蛋白抽提液再次于4℃下13000rpm离心5min，小心地吸取中间层的溶液，转移至新的1.5mL EP管中，4℃冰箱放置待用，并取部分稀释后测浓度(稀释10倍)，采用BCA法测定浓度。I. Extraction of total protein from cultured hepatocytes: ①Aspirate lysate (IP-specific lysate, add 1×cocktail protease inhibitor) with a pipette and add it to the cells. Ultrasonic, let stand on ice for more than 2h; ②Using an ultrasonic cell disruptor, ultrasonicate on ice for 2s, stop for 5s, a total of 1min, the total lysis time on ice is more than 2h (30min interval shaker and mix); ③The cell lysate 4 Centrifuge at 13000rpm for 15min at ℃, transfer the supernatant to a new 1.5mL EP tube, and put it on ice for use; ④ Centrifuge the protein extract again at 13000rpm for 5min at 4℃, carefully aspirate the solution in the middle layer, and transfer it to a new Put it in a 1.5mL EP tube and place it in a refrigerator at 4°C for later use, and take a part of it to measure the concentration after dilution (10 times dilution), and use the BCA method to measure the concentration.
II.蛋白浓度测定：参照BCA试剂盒的方法，对提取的蛋白溶液进行浓度测定。II. Determination of protein concentration: according to the method of the BCA kit, the concentration of the extracted protein solution was determined.
表12 BCA法测定蛋白浓度Table 12 Determination of protein concentration by BCA method
NO.NO. 样本名称sample name 实验编号Experiment number 浓度(μg/μL)Concentration (μg/μL) 体积(μL)Volume (μL) 总量(μg)Total (μg)
11 HEPG2HEPG2 HEPG2HEPG2 8.348.34 25002500 20861.3020861.30
2.pull-down实验流程2. Pull-down experimental process
1)平衡固定链霉亲和素凝胶:①准备Pierce TM Spin Column管；②上下颠倒重悬凝胶液，吸取50μl悬液于标记好的Spin Column管，塞好底塞并放置于收集管中；③再向Spin Column管中加入250μl TBS，拧紧顶盖，轻轻上下颠倒4次混匀液体；④去掉顶盖和底塞，1250×g离心50s，弃掉收集管中的清洗液，把SpinColumn管重新插入收集管中；⑤重复步骤3和步骤4两次。再在Spin Column管底部塞上管底塞。1) Equilibrate and fix the streptavidin gel: ① Prepare a Pierce TM Spin Column tube; ② Resuspend the gel by inverting it upside down, pipette 50 μl of the suspension into the labeled Spin Column tube, plug the bottom stopper and place it in the collection tube ③Add 250μl of TBS to the Spin Column tube, tighten the top cap, and gently invert 4 times to mix the liquid; ④Remove the top cap and bottom stopper, centrifuge at 1250×g for 50s, discard the cleaning solution in the collection tube, Reinsert the SpinColumn tube into the collection tube; ⑤ Repeat steps 3 and 4 twice. Then put the bottom plug on the bottom of the Spin Column tube.
2)生物素标记生物素标记钓饵蛋白和生物素的固定:①向Spin Column管中分别加入生物素和生物素标记钓饵蛋白，拧紧顶盖和底塞；②在旋转平台rotating platform上温和地摇动，4℃孵育60min；③孵育结束后，移去Spin Column管顶盖和底塞，放入收集管中；④1250×g，60s离心后，将Spin Column管重新放入收集管中。2) Fixation of biotin-labeled biotin-labeled bait protein and biotin: ①Add biotin and biotin-labeled bait protein to Spin Column tubes, respectively, and tighten the top cap and bottom plug; ② Gently shake on the rotating platform , incubate at 4°C for 60 min; ③ After the incubation, remove the top cap and bottom stopper of the Spin Column tube and put it into the collection tube; ④ After centrifugation at 1250 × g for 60 s, put the Spin Column tube back into the collection tube.
3)生物素的封闭:①向Spin Column管中加入250μl生物素封闭溶液。拧紧顶盖和底塞，轻轻上下颠倒4次使之混匀；②室温下孵育5min。移去顶盖，将Spin Column管放于收集管中，1250×g离心50s；③重复步骤1和步骤2一次；④再向Spin Column管中加入TBS 250μl。拧紧顶盖，轻轻上下颠倒4次使之混匀；⑤移去顶盖，放于收集管中，1250×g离心50s；⑥重复步骤3和步骤4两次，Spin Column管重新放入收集管。3) Biotin blocking: ①Add 250 μl biotin blocking solution to the Spin Column tube. Tighten the top cover and bottom stopper, and gently invert 4 times to mix; ②Incubate at room temperature for 5 min. Remove the top cover, put the Spin Column tube in the collection tube, and centrifuge at 1250×g for 50s; ③ Repeat steps 1 and 2 once; ④ Add 250 μl of TBS to the Spin Column tube. Tighten the top cap and invert it upside down 4 times to mix it up; ⑤Remove the top cap, put it in a collection tube, and centrifuge at 1250×g for 50s; ⑥Repeat steps 3 and 4 twice, and put the Spin Column tube back into the collection tube. Tube.
4)生物素标记生物素标记蛋白的捕获:①向Spin Column管中加入300μL(1mg蛋白)的捕获蛋白(目的蛋白)样品溶液，拧紧顶盖；②在旋转平台rotating platform上温和地摇动，4℃孵育过夜；③孵育结束后，移去顶盖和底塞。将Spin Column管放入准备好的收集管中；④将收集管，1250×g，60s，离心，收集管标记“prey flow-through(B)”；⑤移去收集管中Spin Column管，盖好收集管的盖子，冰上放置以便后续分析；⑥将Spin Column管放入新的收集管，准备洗脱。4) Capture of biotin-labeled biotin-labeled protein: ① Add 300 μL (1 mg protein) of the captured protein (target protein) sample solution to the Spin Column tube, and tighten the top cover; ② Gently shake on the rotating platform, 4 Incubate overnight at ℃; ③ After the incubation, remove the top cover and bottom plug. Put the Spin Column tube into the prepared collection tube; ④ Centrifuge the collection tube, 1250×g, 60s, mark the collection tube as "prey flow-through(B)"; ⑤ Remove the Spin Column tube in the collection tube, cover Secure the lid of the collection tube and put it on ice for subsequent analysis; ⑥Put the Spin Column tube into a new collection tube to prepare for elution.
5)Spin Column管的诱饵蛋白和靶蛋白复合物的洗脱:①在每个Spin Column管中加入250μl Wash Buffer。拧紧顶盖和底塞，轻轻颠倒6次使之混匀；②将Spin Column管在室温下孵育1分钟。将顶盖底塞去掉，将Spin Column管置于收集管上，1250×g离心50s。另外重复步骤1-2，3次；③在冲洗的过程中，在收集管上写上标签“Wash1，……，Wash3”；④最后一次冲洗时，加入200μl Wash Buffer后，将管内液体连同珠子一起转移至1.5mL；⑤新的离心管中，离心后弃170μl上清，该步骤重复3次。5) Elution of the bait protein and target protein complexes in the Spin Column tube: ①Add 250 μl Wash Buffer to each Spin Column tube. Tighten the top cap and bottom stopper, and gently invert 6 times to mix; ②Incubate the Spin Column tube at room temperature for 1 minute. Remove the top cap and bottom stopper, place the Spin Column tube on the collection tube, and centrifuge at 1250×g for 50s. In addition, repeat steps 1-2, 3 times; ③ During the rinsing process, write the label "Wash1, ..., Wash3" on the collection tube; ④ During the last rinsing, add 200 μl of Wash Buffer, add the liquid in the tube together with the beads Transfer to 1.5mL together; ⑤In a new centrifuge tube, discard 170μl of supernatant after centrifugation, and repeat this step 3 times.
6)检测：①吸干Sepharose上面的液体后，加入20μl 1×蛋白电泳上样缓冲液，沸水浴5min，放入-20℃冰箱备用；②通过SDS-PAGE和Western blot进行检测。6) Detection: ① After blotting up the liquid on Sepharose, add 20 μl of 1× protein electrophoresis loading buffer, take a boiling water bath for 5 min, and put it in a -20°C refrigerator for later use; ② Detect by SDS-PAGE and Western blot.
3.结果分析3. Analysis of results
1)HrpNEch多拟表位配体蛋白识别结合的细胞膜受体：识别结合12个膜受体，GNG12鸟嘌呤核苷酸结合蛋白γ-12受体、ANXA5膜联蛋白A5受体、ANXA2膜联蛋白A2受体、ANXA1膜联蛋白A1受体、IGHG2免疫球蛋白重常数γ2受体、IGHM免疫球蛋白重常数Mu受体、CACNA1S钙电压门控通道亚单位α1S受体、ZNF185锌指蛋白185受体以及HLA-A主要组织相容性复合体，I，A类受体、LAMP2溶酶体关联膜蛋白2受体、GNB2G鸟嘌呤核苷酸结合蛋白亚单位β2受体、KTN1驱动结合蛋白1受体。1) HrpNEch multi-epitope ligand protein recognizes and binds to cell membrane receptors: recognizes and binds to 12 membrane receptors, GNG12 guanine nucleotide binding protein γ-12 receptor, ANXA5 annexin A5 receptor, ANXA2 membrane linker Protein A2 receptor, ANXA1 annexin A1 receptor, IGHG2 immunoglobulin weight constant γ2 receptor, IGHM immunoglobulin weight constant Mu receptor, CACNA1S calcium voltage-gated channel subunit α1S receptor, ZNF185 zinc finger protein 185 Receptors and HLA-A major histocompatibility complex, class I, A receptors, LAMP2 lysosome-associated membrane protein 2 receptor, GNB2G guanine nucleotide binding protein subunit β2 receptor, KTN1 kinesin binding protein 1 receptor.
2)HrpNEch多拟表位配体蛋白识别结合的细胞膜蛋白：识别结合16个膜蛋白，DSC3桥粒胶蛋白、ANXA8/ANXA8L1膜联蛋白A8/膜联蛋白A8相似蛋白1、EVPL外被斑蛋白、POF1B肌动蛋白结合蛋白卵巢早衰1B、CTNNA1连环蛋白、TGM1转谷氨酰胺酶1、BAIAP2BAI1关联蛋白2、RAB29RAS致 癌基因家族成员、CLDN19靠停蛋白19、STXBP2突触融合蛋白结合蛋白2、VAMP囊泡相关膜蛋白关联蛋白A、VCL黏着斑蛋白、Ezrin埃兹上皮型钙黏附素、PKP3血小板亲和蛋白3、NAALAD2N乙酰化α连接酸性二肽酶2、PKP1血小板亲和蛋白1、SPRR1A富含脯氨酸小蛋白1A。2) HrpNEch multi-epitope ligand protein recognizes bound cell membrane proteins: recognizes and binds to 16 membrane proteins, DSC3 desmocollin, ANXA8/ANXA8L1 annexin A8/annexin A8 similar protein 1, EVPL coat protein , POF1B actin-binding protein premature ovarian failure 1B, CTNNA1-catenin, TGM1 transglutaminase 1, BAIAP2BAI1-associated protein 2, RAB29RAS oncogene family members, CLDN19 docking protein 19, STXBP2 syntaxin-binding protein 2, VAMP Vesicle-associated membrane protein-associated protein A, VCL focal adhesion protein, Ezrin epithelial cadherin, PKP3 platelet affinity protein 3, NAALAD2N acetylated α-linked acid dipeptidase 2, PKP1 platelet affinity protein 1, SPRR1A rich Proline-containing small protein 1A.
3)HrpNEch多拟表位配体蛋白识别结合的膜蛋白参与的信号通路22条：包括hsa03320:PPAR信号通路、hsa05120:幽门螺杆菌感染的上皮细胞信号转导、hsa04071:鞘脂类信号通路、hsa04014:Ras信号通路、hsa04151:PI3K-Akt信号通路、hsa04070:磷脂酰肌醇信号系统、hsa04010:MAPK信号通路、hsa04310:Wnt信号通路、hsa04062:趋化因子信号通路、hsa04015:Rap1信号通路、hsa04024:阵营信号通路、hsa04915:雌激素信号通路、hsa04910:胰岛素信号通路、hsa04390:河马信号通路、hsa04922:胰高糖素信号通路、hsa04912:促性腺激素信号通路、hsa04022:cGMP-PKG信号通路、hsa04921:催产素信号通路、hsa04722:生成信号通路、hsa04723:逆行神经信号、hsa04066:HIF-1信号通路、hsa04020:钙信号通路等的多种类型信号通路。3) 22 signaling pathways involved in HrpNEch multi-epitope ligand protein recognition and binding of membrane proteins: including hsa03320: PPAR signaling pathway, hsa05120: Helicobacter pylori infection epithelial cell signal transduction, hsa04071: Sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04070: Phosphatidylinositol signaling system, hsa04010: MAPK signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: Chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024 : camp signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, hsa04390: hippo signaling pathway, hsa04922: glucagon signaling pathway, hsa04912: gonadotropin signaling pathway, hsa04022: cGMP-PKG signaling pathway, hsa04921 : oxytocin signaling pathway, hsa04722: generative signaling pathway, hsa04723: retrograde nerve signaling, hsa04066: HIF-1 signaling pathway, hsa04020: calcium signaling pathway and other types of signaling pathways.
4)HrpNEch多拟表位配体蛋白识别结合的膜蛋白参与的与抗病毒、抗细菌、抗异物、抗炎性相关代谢通路29条：hsa04144:内吞作用、hsa04145:吞噬体、hsa04142:溶酶体、hsa04666:Fc-r介导的吞噬作用、hsa01130:抗生素的生物合成、hsa05131:志贺氏菌病、hsa04612:抗原处理和呈递、hsa05130:致病性大肠杆菌感染、hsa05100:上皮细胞的细菌侵袭、hsa05132:沙门氏菌感染、hsa05169:巴尔病毒感染、hsa05203:病毒致癌作用、hsa05134:军团病、hsa05160:丙型肝炎、hsa05162:麻疹、hsa05133:百日咳、hsa05322:系统性红斑狼疮、hsa04670:白细胞跨内皮的迁移、hsa05152:肺结核、hsa05150:金黄色葡萄球菌感染、hsa05146:阿米巴病、hsa05142:南美锥虫病、hsa05200:在癌症中通路、hsa05143:非洲锥虫病、hsa04750:TRP通道的炎症介质调节、hsa04916:杀菌作用、hsa05230:癌症中的中心碳代谢、hsa05214:神经胶质瘤、hsa05212:胰腺癌。4) 29 metabolic pathways related to antiviral, antibacterial, anti-foreign body, and anti-inflammatory properties that HrpNEch multi-epitope ligand protein recognizes and binds to participate in: hsa04144: endocytosis, hsa04145: phagosome, hsa04142: lysis Enzymes, hsa04666: Fc-r-mediated phagocytosis, hsa01130: Biosynthesis of antibiotics, hsa05131: Shigellosis, hsa04612: Antigen processing and presentation, hsa05130: Pathogenic E. coli infection, hsa05100: Epithelial Bacterial invasion, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05203: Viral carcinogenesis, hsa05134: Legionnaires' disease, hsa05160: Hepatitis C, hsa05162: Measles, hsa05133: Pertussis, hsa05322: Systemic lupus erythematosus, hsa04670: Leukocytosis Endothelial migration, hsa05152: Tuberculosis, hsa05150: Staphylococcus aureus infection, hsa05146: Amoebiasis, hsa05142: Chagas disease, hsa05200: Pathways in cancer, hsa05143: African trypanosomiasis, hsa04750: Inflammation of TRP channels Mediator regulation, hsa04916: bactericidal effects, hsa05230: central carbon metabolism in cancer, hsa05214: glioma, hsa05212: pancreatic cancer.
5)HrpNEch多拟表位配体蛋白识别结合的膜蛋白参与的重要神经疾病代谢通道3条：包括hsa05012:帕金森病，hsa05016:亨廷顿氏舞蹈症，hsa05010:阿尔茨海默氏症。5) HrpNEch multi-epitope ligand protein recognizes and binds membrane proteins involved in 3 important neurological disease metabolic pathways: including hsa05012: Parkinson's disease, hsa05016: Huntington's disease, hsa05010: Alzheimer's disease.
6)HrpNEch多拟表位配体蛋白识别结合的膜蛋白参与的核酸、蛋白质、氨基酸、糖、脂肪代谢相关通路39条：包括hsa03013:RNA运输、hsa03018:RNA降解、hsa03040:剪接体、hsa03010:核糖体、hsa04141:内质网蛋白加工、hsa04810:肌动蛋白骨架的调hsa03050:蛋白酶体、hsa01230:氨基酸生物合成、hsa00190:氧化磷酸化、hsa00230:嘌呤代谢、hsa04932:非酒精性脂肪肝、hsa00020:柠檬酸循环、hsa03008:真核生物核糖体的生物发生、hsa00240:嘧啶代谢、hsa00650:丁酸甲酯新陈代谢、hsa01200:碳代谢、hsa00520:氨基糖和核苷酸糖代谢、hsa05034:酗酒、hsa00071:脂肪酸降解、hsa04120:泛素介导的蛋白水解作用、hsa05205:蛋白聚糖在癌症、hsa05206:小分子核糖核酸在癌症、hsa00410:丙氨酸新陈代谢、hsa00340:组氨酸代谢、hsa00910:氮代谢、hsa00250:丙氨酸、天冬氨酸、谷氨酸代谢、hsa00350:酪氨酸代谢、hsa04726:血清素激活的突触、hsa00900:萜类化合物生物合成支柱、hsa04610:补体和凝血级联、hsa00330:精氨酸和脯氨酸代谢、hsa04520:粘附的结、hsa00860:卟啉与叶绿素代谢、hsa00010:糖酵解和糖质新生、hsa00982:药物代谢-细胞色素P450、hsa00980:细胞色素P450对外源性药物的代谢作用、hsa04962:加压素调节水重吸收、hsa00983:药物代谢-其他酶。6) HrpNEch multi-epitope ligand protein recognizes and binds to 39 nucleic acid, protein, amino acid, sugar, and fat metabolism-related pathways involved in membrane proteins: including hsa03013: RNA transport, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: Ribosome, hsa04141: endoplasmic reticulum protein processing, hsa04810: regulation of the actin backbone hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa00230: purine metabolism, hsa04932: nonalcoholic fatty liver disease, hsa00020 : Citric acid cycle, hsa03008: Biogenesis of eukaryotic ribosomes, hsa00240: Pyrimidine metabolism, hsa00650: Methyl butyrate metabolism, hsa01200: Carbon metabolism, hsa00520: Amino sugar and nucleotide sugar metabolism, hsa05034: Alcoholism, hsa00071 : fatty acid degradation, hsa04120: ubiquitin-mediated proteolysis, hsa05205: proteoglycans in cancer, hsa05206: small RNAs in cancer, hsa00410: alanine metabolism, hsa00340: histidine metabolism, hsa00910: nitrogen metabolism , hsa00250: Alanine, Aspartate, Glutamate metabolism, hsa00350: Tyrosine metabolism, hsa04726: Serotonin-activated synapses, hsa00900: Terpenoid biosynthetic pillar, hsa04610: Complement and coagulation cascade, hsa00330: Arginine and Proline Metabolism, hsa04520: Adhesive Knots, hsa00860: Porphyrin and Chlorophyll Metabolism, hsa00010: Glycolysis and Glycogenesis, hsa00982: Drug Metabolism - Cytochrome P450, hsa00980: Cytochrome P450 Metabolism of Exogenous Drugs, hsa04962: Vasopressin Regulates Water Reabsorption, hsa00983: Drug Metabolism - Other Enzymes.
7)HrpNEch多拟表位配体蛋白识别结合的膜蛋白参与的细胞联结、神经连结、血管、内分泌、生殖系统等代谢通路34条：包括hsa04510:粘着斑、hsa 04724:谷氨酸能的突触、hsa04530:紧密连接、hsa00830:视黄醇新陈代谢、hsa04114:卵母细胞减数分裂、hsa04728:多巴胺神经突触、hsa00140:类固醇激素生物合成、hsa04261:心肌细胞的肾上腺素能信号、hsa04727:γ-氨基丁酸能的突触、hsa04725:胆碱能突触、hsa04540: 缝隙连接、hsa04971:胃酸分泌、hsa04713:昼夜夹带、hsa04931:胰岛素抵抗、hsa05031:安非他命上瘾、hsa04924:肾素分泌、hsa04925:醛固酮的合成与分泌、hsa00590:花生四烯酸代谢、hsa04270:血管平滑肌收缩、hsa00760:烟酸和烟酰胺代谢、hsa04740:嗅觉传导、hsa04260:心肌收缩、hsa04720:长期势差现象、hsa04744:光传导、hsa04966:收集管道酸性分泌物、hsa05412:致心律失常性右心室心肌病、hsa05410:肥厚性心肌病、hsa04146:过氧物酶体、hsa05414:扩张型心肌病、hsa04970:唾液分泌、hsa04611:血小板激活、hsa05204:化学致癌作用、hsa04721:突触囊泡循环。7) HrpNEch multi-epitope ligand protein recognizes and binds membrane proteins involved in 34 metabolic pathways including cell junctions, neural junctions, blood vessels, endocrine, and reproductive systems: including hsa04510: focal adhesions, hsa 04724: glutamatergic spikes haptic, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114: oocyte meiosis, hsa04728: dopamine synapses, hsa00140: steroid hormone biosynthesis, hsa04261: adrenergic signaling in cardiomyocytes, hsa04727: gamma - GABAergic synapses, hsa04725: cholinergic synapses, hsa04540: gap junctions, hsa04971: gastric acid secretion, hsa04713: circadian entrainment, hsa04931: insulin resistance, hsa05031: amphetamine addiction, hsa04924: renin secretion, hsa04925: Aldosterone synthesis and secretion, hsa00590: arachidonic acid metabolism, hsa04270: vascular smooth muscle contraction, hsa00760: niacin and nicotinamide metabolism, hsa04740: olfactory conduction, hsa04260: myocardial contraction, hsa04720: long-term potential difference phenomenon, hsa04744: phototransduction , hsa04966: collection duct acidic secretions, hsa05412: arrhythmogenic right ventricular cardiomyopathy, hsa05410: hypertrophic cardiomyopathy, hsa04146: peroxisomes, hsa05414: dilated cardiomyopathy, hsa04970: salivary secretion, hsa04611: platelets Activation, hsa05204: chemical carcinogenesis, hsa04721: synaptic vesicle cycling.
HrpNEch多拟表位配体蛋白，作为一类富含特殊多个线性和构象表位结构的配体蛋白分子，能够跨界识别、结合多种类型的膜受体、膜蛋白、信息通路和代谢通路，进一步分析这些膜受体、膜蛋白、信息通路和代谢通路的位置、结构、特性、作用机制和功能，它们广泛影响机体的生长、发育、代谢、防御和细胞程序性死亡的生命基本属性，并广泛涉及诊断、预防、治疗、康复神经系统、消化系统、运动系统、循环系统、呼吸系统、内分泌系统、免疫系统、泌尿系统、生殖系统、皮肤系统疾病和状况。HrpNEch多拟表位配体蛋白是一类具有全新功能，全新作用机制和全新应用前景的特殊多拟表位配体蛋白。HrpNEch multi-epitopic ligand protein, as a class of ligand protein molecules rich in special multiple linear and conformational epitope structures, can recognize and bind various types of membrane receptors, membrane proteins, information pathways and metabolism across borders Pathways, and further analyze the location, structure, properties, mechanism of action and function of these membrane receptors, membrane proteins, information pathways and metabolic pathways, which widely affect the growth, development, metabolism, defense and the basic properties of life of programmed cell death. , and is widely involved in the diagnosis, prevention, treatment, rehabilitation of diseases and conditions of the nervous system, digestive system, motor system, circulatory system, respiratory system, endocrine system, immune system, urinary system, reproductive system, and skin system. HrpNEch multi-epitope ligand protein is a kind of special multi-epitope ligand protein with new function, new mechanism of action and new application prospect.
本发明所述HrpN型多拟表位配体蛋白在食品、化妆品、保健品和药物中的应用还涉及识别激活动物(包括人类)的多类受体、膜蛋白及其信号通路并诱发多功能级联生物学效应的HrpNEcb、HrpNEch等多拟表位配体蛋白的制品在食品、化妆品、保健品中的应用；进一步的，HrpNEcb、HrpNEch等多拟表位配体蛋白识别结合特异蛋白的pull-down实验表明，它们识别结合的动物的多类受体、膜蛋白及其信号通路蛋白，从机体内到机体外，广泛涉及机体的生长功能，发育功能，防御、免疫、除菌消炎功能，内分泌、多类型分子代谢功能以及细胞程序性死亡功能的表达和调节控制，广泛参与机体的不同系统、器官、组织、细胞、亚细胞、分子、亚分子的从结构到功能的再生、修复和清除；进一步的，HrpNEcb、HrpNEch等多拟表位配体蛋白的动物实验mRNA测序结果表明，HrpNEcb、HrpNEch等多拟表位配体蛋白诱导的多功能级联生物学效应是指能诱导不同器官、组织的细胞组分、分子功能和生物学过程三个层次的相关功能基因群显著差异表达的GO Term富集分析，以及HrpNEcb、HrpNEch等多拟表位配体蛋白诱导的多功能级联生物学效应是指能诱导不同器官、组织的细胞过程、环境信息过程、遗传信息过程、新陈代谢和生物体系统过程等五个生物过程的相关功能基因群显著差异表达的KEGG Pathway富集分析,采用对实验动物进行的口服和涂抹处理，所诱导的级联生物学效应相关功能基因群显著差异表达，同样从机体内到机体外，广泛涉及机体的生长、发育、防御、代谢以及细胞程序性死亡功能的表达和调节控制，广泛参与机体的不同系统、器官、组织、细胞、亚细胞、分子、亚分子的从结构到功能的再生、修复和清除；进一步的，HrpNEcb、HrpNEch等多拟表位配体蛋白，采用口服或和涂抹处理的方式，HrpN型多拟表位配体蛋白的制品可以在食品、化妆品、保健品中广泛应用，HrpN型多拟表位配体蛋白的辅助调理功能主要包括1、增强免疫力功能；2、辅助降血脂功能；3、辅助降血糖功能；4、抗氧化功能；5、辅助改善记忆功能；6、缓解视疲劳功能；7、促进排铅功能；8、清咽功能；9、辅助降血压功能；10、改善睡眠功能；11、促进泌乳功能；12、缓解体力疲劳功能；13、提高缺氧耐受力功能；14、对辐射危害有辅助保护功能；15、减肥功能；16、改善生长发育功能；17、增加骨密度功能；18、改善营养性贫血功能；19、对化学肝损伤有辅助保护功能；20、祛痤疮功能；21、去黄褐斑功能；22、改善皮肤水份功能；23、改善皮肤油份功能；24、调节肠道菌群功能；25、促进消化功能；26、通便功能；27、对胃黏膜损伤有辅助保护功能。The application of the HrpN type multi-epitope ligand protein of the present invention in food, cosmetics, health care products and medicines also involves identifying and activating various types of receptors, membrane proteins and their signaling pathways in animals (including humans) and inducing multifunctionality. The application of HrpNEcb, HrpNEch and other multi-mimetic ligand protein products with cascade biological effects in food, cosmetics, and health care products; further, HrpNEcb, HrpNEch and other multi-mimetic epitope ligand proteins recognize pulls that bind to specific proteins -down experiments show that they recognize multiple types of receptors, membrane proteins and their signaling pathway proteins in bound animals, from inside to outside the body, and are widely involved in the body's growth function, developmental function, defense, immunity, sterilization and anti-inflammatory functions, Expression and regulatory control of endocrine, multi-type molecular metabolic functions, and programmed cell death functions, widely involved in the regeneration, repair and clearance of different systems, organs, tissues, cells, sub-cells, molecules, and sub-molecules from structure to function Further, the mRNA sequencing results of animal experiments of HrpNEcb, HrpNEch and other multi-epitope ligand proteins show that the multi-functional cascade biological effects induced by HrpNEcb, HrpNEch and other multi-mimetic epitope ligand proteins refer to the ability to induce different organs, GO Term enrichment analysis of significantly differentially expressed related functional gene groups at three levels of tissue cellular components, molecular functions and biological processes, and multi-functional cascade biology induced by multi-epitopic ligand proteins such as HrpNEcb and HrpNEch Effect refers to the KEGG Pathway enrichment analysis of the significant differential expression of related functional gene groups in five biological processes, including cellular processes, environmental information processes, genetic information processes, metabolism and organism system processes in different organs and tissues. Oral and smearing treatments in animals induce significant differential expression of functional gene groups related to cascade biological effects, which are also widely involved in the growth, development, defense, metabolism, and programmed cell death functions of the body from in vivo to in vitro. Expression and regulatory control, widely involved in the regeneration, repair and clearance of different systems, organs, tissues, cells, sub-cells, molecules, and sub-molecules from structure to function; further, HrpNEcb, HrpNEch and other multi-epitope ligands HrpN-type multi-epitope ligand protein products can be widely used in food, cosmetics, and health care products. The auxiliary conditioning functions of HrpN-type multi-epitope ligand protein mainly include 1 , Enhance immune function; 2. Assist blood lipid lowering function; 3. Assist blood sugar lowering function; 4. Antioxidant function; 5. Assist in improving memory function; 6. Relieve visual fatigue function; Pharyngeal function; 9. Auxiliary blood pressure lowering function; 10. Improve sleep function; 11. Promote lactation function; 12. Relieve physical fatigue function; 13. Improve hypoxia tolerance function; 14. Have auxiliary protective function against radiation hazards; 15 , weight loss function; 16, improve growth and development function; 17, increase bone Density function; 18. Improve nutritional anemia function; 19. Auxiliary protection function against chemical liver damage; 20. Remove acne function; 21. Remove melasma function; 22. Improve skin moisture function; 23. Improve skin oil content 24. Regulate the function of intestinal flora; 25. Promote digestive function; 26. Laxative function; 27. Have auxiliary protective function for gastric mucosal damage.
进一步的：further:
本发明所述HrpN型多拟表位配体蛋白的制品可以在辅助调理机体的生长、发育、防御、代谢以及细胞程序性死亡功能的表达和调节控制的食品中的应用，包括各种供人食用或者饮用的成品和原料；The preparations of the HrpN-type multi-epitope ligand protein of the present invention can be used in foods that assist in regulating the expression and regulation of the body's growth, development, defense, metabolism, and programmed cell death functions, including various human Finished products and raw materials for eating or drinking;
本发明所述HrpN型多拟表位配体蛋白的制品可以在辅助调理机体的生长、发育、防御、代谢以及细胞程序性死亡功能的表达和调节控制的化妆品中的应用，包括以涂擦、喷洒或者其他类似的方法，散布于人体表面任何部位，包括皮肤、毛发、指甲、口唇等，以达到清洁、消除不良气味、护肤、美容和修饰目的的生物技术产品，或包括普通化妆品和特殊用途化妆品；The preparations of the HrpN-type multi-epitope ligand protein of the present invention can be used in cosmetics that assist in regulating the expression and regulation of the growth, development, defense, metabolism, and programmed cell death functions of the body, including rubbing, Spraying or other similar methods, spread on any part of the human body surface, including skin, hair, nails, lips, etc., to achieve the purpose of cleaning, eliminating bad odor, skin care, beauty and grooming biotechnology products, or including ordinary cosmetics and special purposes cosmetic;
本发明所述HrpN型多拟表位配体蛋白的制品可以在辅助调理机体的生长、发育、防御、代谢以及细胞程序性死亡功能的表达和调节控制的保健品中的应用，包括保健功能食品，是食品的一个种类，具有一般食品的共性，能调节人体的机能，适用于特定人群食用，但不以治疗疾病为目的。The preparation of the HrpN-type multi-epitope ligand protein of the present invention can be used in health care products that assist in regulating the growth, development, defense, metabolism of the body and the expression and regulation of programmed cell death functions, including health care functional foods , is a type of food, has the commonality of general food, can regulate the functions of the human body, and is suitable for consumption by specific groups of people, but not for the purpose of curing diseases.
以上所述实施例仅表达了本申请的具体实施方式，其描述较为具体和详细，但并不能因此而理解为对本申请保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请技术方案构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。The above-mentioned embodiments only represent specific implementations of the present application, and the descriptions thereof are specific and detailed, but should not be construed as limiting the protection scope of the present application. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can be made, which all belong to the protection scope of the present application.
Figure PCTCN2021134714-appb-000049
Figure PCTCN2021134714-appb-000049
Figure PCTCN2021134714-appb-000050
Figure PCTCN2021134714-appb-000050
Figure PCTCN2021134714-appb-000051
Figure PCTCN2021134714-appb-000051

Claims (26)

  1. HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用。Application of HrpN-type multi-epitope ligand proteins in food, cosmetics, health care products or pharmaceuticals that recognize and activate various types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects.
  2. 根据权利要求1所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白含有一至多个疏水非极性氨基酸残基的结构基团或表位、含有一至多个极性不带电氨基酸残基的结构基团或表位、含有一至多个酰胺基极性不带电氨基酸残基的结构基团或表位、含有一至多个酸性带正电、碱性带负电氨基酸残基的结构基团或表位；疏水非极性氨基酸残基包括缬氨酸、亮氨酸、异亮氨酸、丙氨酸、苯丙氨酸、蛋氨酸残基；极性不带电氨基酸残基包括丝氨酸残基；酰胺基极性不带电氨基酸残基包括天冬酰胺、谷氨酰胺残基；酸性带正电、碱性带负电氨基酸残基包括天冬酰酸、谷氨酸、赖氨酸、组氨酸、精氨酸残基；疏水非极性氨基酸残基，极性不带电氨基酸残基，酰胺基极性不带电氨基酸残基，酸性带正电、碱性带负电氨基酸残基在HrpN型多拟表位配体蛋白分子的全序列中占比62.3％-73.7％，在保守结构域中占比61％-74％，在α-螺旋结构中占比66.2％-79％；疏水非极性氨基酸残基的结构基团或表位，极性不带电氨基酸残基的结构基团或表位，酰胺基极性不带电氨基酸残基的结构基团或表位和酸性带正电、碱性带负电氨基酸残基的结构基团或表位通过氢键、离子键、疏水、非极性、极性、范德华力，实现配体和受体分子空间结构和电性的互补性、互作性以及特异识别、激活、结合，与多类型受体形成紧密结合面或复合物，能引起受体分子的构象、能量、电性和信息的变化，经信号传导和转导，引发放大的级联生物学效应。The HrpN type multi-epitope ligand protein according to claim 1 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects It is characterized in that: the HrpN-type multi-mimetic epitope ligand protein contains a structural group or epitope of one or more hydrophobic non-polar amino acid residues, a structural group containing one or more polar uncharged amino acid residues or Epitopes, structural groups or epitopes containing one or more amide polar uncharged amino acid residues, structural groups or epitopes containing one or more acidic positively charged, basic negatively charged amino acid residues; hydrophobic non-charged Polar amino acid residues include valine, leucine, isoleucine, alanine, phenylalanine, methionine residues; polar uncharged amino acid residues include serine residues; amide groups are polar uncharged Amino acid residues include asparagine, glutamine residues; acidic positively charged, basic negatively charged amino acid residues include asparagine, glutamic acid, lysine, histidine, arginine residues; Hydrophobic non-polar amino acid residues, polar uncharged amino acid residues, amide polar uncharged amino acid residues, acidic positively charged, basic negatively charged amino acid residues in HrpN-type polymimetic epitope ligand protein molecules. It accounts for 62.3%-73.7% in the whole sequence, 61%-74% in the conserved domain, and 66.2%-79% in the α-helical structure; the structural group of hydrophobic non-polar amino acid residues or Epitopes, structural groups or epitopes of polar uncharged amino acid residues, structural groups or epitopes of amide groups of polar uncharged amino acid residues and structural groups of acidic positively charged, basic negatively charged amino acid residues Through hydrogen bonding, ionic bonding, hydrophobicity, non-polarity, polarity, and van der Waals force, the group or epitope realizes the complementarity, interaction, specific recognition, activation, and binding of ligand and receptor molecular spatial structure and electrical properties. The formation of tight binding surfaces or complexes with multiple types of receptors can cause changes in the conformation, energy, electrical properties and information of receptor molecules, and through signal transduction and transduction, lead to amplified cascade biological effects.
  3. 根据权利要求1所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白包括HrpNEcc、HrpNEca、HrpNEcb、HrpNEch、HrpNDaz、 HrpNDada、HrpNDasp、HrpNad、HrpNDaf、HrpNEa、HrpNSam、HrpNBag、HrpNPas、HrpNEnt。The HrpN type multi-epitope ligand protein according to claim 1 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects The application is characterized in that: HrpN type multi-epitope ligand proteins include HrpNEcc, HrpNEca, HrpNEcb, HrpNEch, HrpNDaz, HrpNDada, HrpNDasp, HrpNad, HrpNDaf, HrpNEa, HrpNSam, HrpNBag, HrpNPas, HrpNEnt.
  4. 根据权利要求3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpNEcb蛋白的氨基酸序列如SEQ ID NO:1所示。The HrpN type multi-epitope ligand protein according to claim 3 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects Application, it is characterized in that: the amino acid sequence of HrpNEcb protein is as shown in SEQ ID NO:1.
  5. 根据权利要求3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpNEch多拟表位配体蛋白的氨基酸序列如SEQ ID NO:2所示。The HrpN type multi-epitope ligand protein according to claim 3 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects Application, it is characterized in that: the amino acid sequence of HrpNEch multi-mimetic epitope ligand protein is as shown in SEQ ID NO:2.
  6. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，识别结合的多类受体包括LRRC1515-亮氨酸的重复膜蛋白受体、HLA-A主要组织相容性复合体，I,A类受体、LGALS3BP半乳糖3结合蛋白或受体、LAMP2溶酶体关联膜蛋白2受体、GNB2 G鸟嘌呤核苷酸结合蛋白亚单位Beta 2受体中的一种或多种。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects It is characterized in that: HrpN-type multi-epitope ligand protein is HrpNEcb multi-mimetic epitope ligand protein, and the multi-type receptors that recognize and bind include LRRC1515-leucine repeat membrane protein receptor, HLA-A Major histocompatibility complex, class I,A receptors, LGALS3BP galactose 3 binding protein or receptor, LAMP2 lysosome-associated membrane protein 2 receptor, GNB2 G guanine nucleotide binding protein subunit Beta 2 receptor one or more of the body.
  7. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，识别结合的多类受体包括GNG12鸟嘌呤核苷酸结合蛋白γ-12受体、ANXA5膜联蛋白A5受体、ANXA2膜联蛋白A2受体、ANXA1膜联蛋白A1受体、IGHG2免疫球蛋白重常数γ2受体、IGHM免疫球蛋白重常数Mu受体、CACNA1S钙电压门控通道亚单位α1 S受体、ZNF185锌指蛋白185受体以及HLA-A主要组织相容性复合体，I，A类受体、LAMP2溶酶体关联膜蛋白2受体、GNB2 G鸟嘌呤核苷酸结合蛋白亚单位β2受体、KTN1驱动结合蛋白1受体中的一种或多种。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-mimetic epitope ligand protein is HrpNEch multi-mimetic epitope ligand protein, and the multi-type receptors that recognize and bind include GNG12 guanine nucleotide binding protein γ-12 receptor , ANXA5 annexin A5 receptor, ANXA2 annexin A2 receptor, ANXA1 annexin A1 receptor, IGHG2 immunoglobulin weight constant γ2 receptor, IGHM immunoglobulin weight constant Mu receptor, CACNA1S calcium voltage-gated Channel subunit α1 S receptor, ZNF185 zinc finger protein 185 receptor and HLA-A major histocompatibility complex, I, A receptors, LAMP2 lysosome-associated membrane protein 2 receptor, GNB2 G guanine nucleus One or more of the nucleotide binding protein subunit β2 receptor, KTN1 kinesin 1 receptor.
  8. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋 白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，识别结合的膜蛋白包括DSG4桥粒芯蛋白、ANXA4膜联蛋白A4、CAPRIN1细胞周期蛋白、1UTRN肌营养不良蛋白、pinin桥粒蛋白、VAMP关联蛋白A、VCL黏着斑蛋白、Ezrin埃兹上皮型钙黏附素、PKP3血小板亲和蛋白3、TM9SF2跨膜9超家族成员2、NAALAD2 N乙酰化α连接酸性二肽酶2中的一种或多种。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-mimetic epitope ligand protein is HrpNEcb multi-mimetic epitope ligand protein, and the membrane proteins that recognize and bind include DSG4 desmocollin, ANXA4 annexin A4, CAPRIN1 cell cycle protein, 1UTRN dystrophin, pinin desmosomal protein, VAMP-associated protein A, VCL focal adhesion protein, Ezrin epithelial cadherin, PKP3 platelet affinity protein 3, TM9SF2 transmembrane 9 superfamily member 2, NAALAD2 N Acetylate one or more of alpha-linked acid dipeptidase 2.
  9. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，识别结合的膜蛋白包括DSC3桥粒胶蛋白、ANXA8/ANXA8L1膜联蛋白A8/膜联蛋白A8相似蛋白1、EVPL外被斑蛋白、POF1B肌动蛋白结合蛋白卵巢早衰1B、CTNNA1连环蛋白、TGM1转谷氨酰胺酶1、BAIAP2 BAI1关联蛋白2、RAB29 RAS致癌基因家族成员、CLDN19靠停蛋白19、STXBP2突触融合蛋白结合蛋白2、VAMP囊泡相关膜蛋白关联蛋白A、VCL黏着斑蛋白、Ezrin埃兹上皮型钙黏附素、PKP3血小板亲和蛋白3、NAALAD2 N乙酰化α连接酸性二肽酶2、PKP1血小板亲和蛋白1、SPRR1A富含脯氨酸小蛋白1A中的一种或多种。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-mimetic epitope ligand protein is HrpNEch multi-mimetic epitope ligand protein, and the membrane proteins that recognize and bind include DSC3 desmocollin, ANXA8/ANXA8L1 annexin A8/membrane Catenin A8-like protein 1, EVPL coatin, POF1B actin-binding protein premature ovarian failure 1B, CTNNA1 catenin, TGM1 transglutaminase 1, BAIAP2 BAI1-associated protein 2, RAB29 RAS oncogene family member, CLDN19 dependent Arrestin 19, STXBP2 syntaxin-binding protein 2, VAMP vesicle-associated membrane protein-associated protein A, VCL focal adhesion protein, Ezrin epithelial-type cadherin, PKP3 platelet avidin 3, NAALAD2 N-acetylated α-linkage One or more of acid dipeptidase 2, PKP1 platelet affinity protein 1, SPRR1A small proline-rich protein 1A.
  10. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，识别结合的信号通路包括hsa04152:AMPK信号通路、hsa03460:范可尼贫血贫血通路、hsa03320:PPAR信号通路、hsa04071:鞘脂类信号通路、hsa04014:Ras信号通路、hsa04151:PI3K-Akt信号通路、hsa04310:Wnt信号通路、hsa04062:趋化因子信号通路、hsa04015:Rap1信号通路、hsa04024:阵营信号通路、hsa04915:雌激素信号通路、hsa04910:胰岛素信号通路、hsa04390:河马信号通路中的一种或多种。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-epitope ligand protein is HrpNEcb multi-mimetic epitope ligand protein, and the signal pathway for identification and binding includes hsa04152: AMPK signal pathway, hsa03460: Fanconi anemia anemia pathway, hsa03320: PPAR signaling pathway, hsa04071: Sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: Chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: One or more of the camp signaling pathway, hsa04915: estrogen signaling pathway, hsa04910: insulin signaling pathway, hsa04390: Hippo signaling pathway.
  11. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，识别激活的信号通路包括hsa03320:PPAR信号通路、hsa05120:幽门螺杆菌感染的上皮细胞信号转导、hsa04071:鞘脂类信号通路、hsa04014:Ras信号通路、hsa04151:PI3K-Akt信号通路、hsa04070:磷脂酰肌醇信号系统、hsa04010:MAPK信号通路、hsa04310:Wnt信号通路、hsa04062:趋化因子信号通路、hsa04015:Rap1信号通路、hsa04024:阵营信号通路、hsa04915:雌激素信号通路、hsa04910:胰岛素信号通路、hsa04390:河马信号通路、hsa04922:胰高糖素信号通路、hsa04912:促性腺激素信号通路、hsa04022:cGMP-PKG信号通路、hsa04921:催产素信号通路、hsa04722:生成信号通路、hsa04723:逆行神经信号、hsa04066:HIF-1信号通路、hsa04020:钙信号通路的一种或多种。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-mimetic epitope ligand protein is HrpNEch multi-mimetic epitope ligand protein, and the activated signal pathway includes hsa03320: PPAR signal pathway, hsa05120: Helicobacter pylori-infected epithelial cells Signal transduction, hsa04071: sphingolipid signaling pathway, hsa04014: Ras signaling pathway, hsa04151: PI3K-Akt signaling pathway, hsa04070: phosphatidylinositol signaling system, hsa04010: MAPK signaling pathway, hsa04310: Wnt signaling pathway, hsa04062: chemotaxis Chemokine signaling pathway, hsa04015: Rap1 signaling pathway, hsa04024: CAMP signaling pathway, hsa04915: Estrogen signaling pathway, hsa04910: Insulin signaling pathway, hsa04390: Hippo signaling pathway, hsa04922: Glucagon signaling pathway, hsa04912: Gonadotropin signaling pathway One or more of signaling pathway, hsa04022: cGMP-PKG signaling pathway, hsa04921: oxytocin signaling pathway, hsa04722: generative signaling pathway, hsa04723: retrograde neural signaling, hsa04066: HIF-1 signaling pathway, hsa04020: calcium signaling pathway.
  12. 根据权利要求1或3所述的HrpN型蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：识别激活的信号通路包括代谢信号通路，代谢信号通路包括抗病毒、抗细菌、抗异物、抗炎性代谢通路，包括重要神经疾病代谢通道；包括核酸、蛋白质、氨基酸、糖、脂肪代谢通路；包括细胞联结、神经连结、血管、内分泌、生殖系统代谢通路。The application of the HrpN-type protein according to claim 1 or 3 in food, cosmetics, health care products or pharmacy that recognizes and activates multiple types of receptors and/or membrane proteins and the signal pathways involved and causes cascade biological effects, It is characterized in that: the activated signal pathways include metabolic signal pathways, the metabolic signal pathways include anti-virus, anti-bacterial, anti-foreign body, anti-inflammatory metabolic pathways, including important neurological disease metabolic pathways; including nucleic acids, proteins, amino acids, sugars, fats Metabolic pathways; including cellular, neural, vascular, endocrine, and reproductive system metabolic pathways.
  13. 根据权利要求12所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，所述识别激活的膜蛋白参与的抗病毒、抗细菌、抗异物、抗炎性代谢通路：hsa04144内吞作用、hsa04145吞噬体、hsa04142溶酶体、hsa01130:抗生素的生物合成、hsa05131:志贺氏菌病、hsa04612:抗原处理和呈递、hsa05130:致病性大肠杆菌感染、hsa05100:上皮细胞的细菌侵袭、hsa05132:沙门氏菌感染、hsa05169:巴尔病毒感染、hsa05168:单纯疱疹病毒1感染、hsa05203:病毒致癌作用、 hsa05166:HTLV-I感染、hsa05164:甲型流感、hsa05134:军团病、hsa05160:丙型肝炎、hsa05162:麻疹、hsa05133:百日咳、hsa05322:系统性红斑狼疮、hsa04670:白细胞经上皮的迁移、hsa05146:阿米巴病、hsa05142:南美锥虫病、hsa05200:在癌症中通路；所述识别激活的膜蛋白参与的重要神经疾病代谢通道：hsa05012:帕金森病、hsa05016:亨廷顿氏舞蹈症、hsa05010:阿尔茨海默氏症；所述识别激活的膜蛋白参与的核酸、蛋白质、氨基酸、糖、脂肪代谢通路：hsa03420:核苷酸切除修复、hsa00970:氨酰生物合成、hsa03430:错配修复、hsa01210:2-氧代羧酸代谢、hsa03440:同源重组、hsa04360:轴突引导、hsa00051:果糖和甘露糖代谢、hsa00565:醚脂质代谢、hsa00510:N-多糖生物合成以及hsa04110:细胞周期、hsa03030:DNA复制、hsa03013:RNA运输、hsa03018:RNA降解、hsa03040:剪接体、hsa03010:核糖体、hsa04141:内质网蛋白加工、hsa04810:肌动蛋白骨架的调控、hsa03050:蛋白酶体、hsa01230:氨基酸生物合成、hsa00190:氧化磷酸化、hsa04932:非酒精性脂肪肝(NAFLD)、hsa00020:柠檬酸循环、hsa00564:甘油磷脂新陈代谢、hsa03008:真核生物核糖体的生物发生、hsa03015:mRNA监测通路、hsa01200:碳代谢、hsa00520:氨基糖和核苷酸糖代谢、hsa05034:酗酒、hsa04120:泛素介导的蛋白水解作用、hsa05205:蛋白聚糖在癌症、hsa05206:小分子核糖核酸在癌症；所述识别激活的膜蛋白参与的细胞联结、神经连结、血管、内分泌、生殖系统代谢通路：hsa04723:逆行神经信号、hsa04726:血清素激活的突触、hsa00900:萜类化合物生物合成支柱、hsa04520:黏着结、hsa05032:吗啡上瘾以及hsa04510:粘着斑、hsa04724:谷氨酸能的突触、hsa04530:紧密连接、hsa00830:视黄醇新陈代谢、hsa04114:卵母细胞减数分裂、hsa04728:多巴胺能神经突触、hsa00100:类固醇生物合成、hsa04261:心肌细胞的肾上腺素能信号、hsa04727:神经元突触、hsa04725:胆碱能突触、hsa04540:缝隙连接、hsa04971:胃酸分泌、hsa04713:昼夜夹带、hsa04931:胰岛素抵抗。The HrpN-type multi-epitope ligand protein according to claim 12 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their involved signaling pathways and cause cascade biological effects It is characterized in that: the HrpN-type multi-mimetic epitope ligand protein is HrpNEcb multi-mimetic epitope ligand protein, and the recognition activated membrane protein participates in the anti-viral, anti-bacterial, anti-foreign body, anti-inflammatory metabolic pathways : hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa01130: biosynthesis of antibiotics, hsa05131: shigellosis, hsa04612: antigen processing and presentation, hsa05130: pathogenic E. coli infection, hsa05100: epithelial cells Bacterial infestation, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05168: Herpes simplex virus 1 infection, hsa05203: Viral carcinogenesis, hsa05166: HTLV-I infection, hsa05164: Influenza A, hsa05134: Legionnaires' disease, hsa05160: C hepatitis, hsa05162: measles, hsa05133: pertussis, hsa05322: systemic lupus erythematosus, hsa04670: transepithelial migration of leukocytes, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: pathway in cancer; the identification Activated membrane proteins are involved in important neurological disease metabolic pathways: hsa05012: Parkinson's disease, hsa05016: Huntington's disease, hsa05010: Alzheimer's disease; the nucleic acids, proteins, amino acids, sugars involved in the identification of activated membrane proteins , Fat metabolism pathways: hsa03420: nucleotide excision repair, hsa00970: aminoacyl biosynthesis, hsa03430: mismatch repair, hsa01210: 2-oxocarboxylate metabolism, hsa03440: homologous recombination, hsa04360: axon guidance, hsa00051: Fructose and mannose metabolism, hsa00565: ether lipid metabolism, hsa00510: N-glycan biosynthesis and hsa04110: cell cycle, hsa03030: DNA replication, hsa03013: RNA transport, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome , hsa04141: endoplasmic reticulum protein processing, hsa04810: regulation of the actin backbone, hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation, hsa04932: non-alcoholic fatty liver disease (NAFLD), hsa00020: citric acid cycle, hsa00564: glycerophospholipid metabolism, hsa03008: biogenesis of eukaryotic ribosomes, hsa03015: mRNA surveillance pathways, hsa01200: carbon metabolism, hsa00520: amino sugar and nucleotide sugar metabolism, hsa05034: alcoholism, hsa04120: ubiquitin-mediated proteolysis, hsa05205: proteoglycans in cancer, hsa05206: small ribose sugars Nucleic acids in cancer; the identification of activated membrane proteins involved in cell junctions, neural junctions, blood vessels, endocrine, reproductive system metabolic pathways: hsa04723: retrograde neural signaling, hsa04726: serotonin-activated synapses, hsa00900: terpenoid biosynthesis Pillar, hsa04520: adhesion knots, hsa05032: morphine addiction and hsa04510: focal adhesions, hsa04724: glutamatergic synapses, hsa04530: tight junctions, hsa00830: retinol metabolism, hsa04114: oocyte meiosis, hsa04728: dopaminergic synapses, hsa00100: steroid biosynthesis, hsa04261: adrenergic signaling in cardiomyocytes, hsa04727: neuronal synapses, hsa04725: cholinergic synapses, hsa04540: gap junctions, hsa04971: gastric acid secretion, hsa04713: circadian entrainment, hsa04931: insulin resistance.
  14. 根据权利要求12所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特 征在于：HrpN型多拟表位配体蛋白为HrpNEch蛋白，所述识别激活的膜蛋白参与的抗病毒、抗细菌、抗异物、抗炎性相关代谢通路：hsa04144:内吞作用、hsa04145:吞噬体、hsa04142:溶酶体、hsa04666:Fc-r介导的吞噬作用、hsa01130:抗生素的生物合成、hsa05131:志贺氏菌病、hsa04612:抗原处理和呈递、hsa05130:致病性大肠杆菌感染、hsa05100:上皮细胞的细菌侵袭、hsa05132:沙门氏菌感染、hsa05169:巴尔病毒感染、hsa05203:病毒致癌作用、hsa05134:军团病、hsa05160:丙型肝炎、hsa05162:麻疹、hsa05133:百日咳、hsa05322:系统性红斑狼疮、hsa04670:白细胞跨内皮的迁移、hsa05152:肺结核、hsa05150:金黄色葡萄球菌感染、hsa05146:阿米巴病、hsa05142:南美锥虫病、hsa05200:在癌症中通路、hsa05143:非洲锥虫病、hsa04750:TRP通道的炎症介质调节、hsa04916:杀菌作用、hsa05230:癌症中的中心碳代谢、hsa05214:神经胶质瘤、hsa05212:胰腺癌；所述识别激活的膜蛋白参与的重要神经疾病代谢通道：hsa05010:阿尔茨海默氏症、hsa05012:帕金森病、hsa05016:亨廷顿氏舞蹈症；所述识别激活的膜蛋白参与的核酸、蛋白质、氨基酸、糖、脂肪代谢相关通路：hsa03013:RNA运输、hsa03018:RNA降解、hsa03040:剪接体、hsa03010:核糖体、hsa04141:内质网蛋白加工、hsa04810:肌动蛋白骨架的调hsa03050:蛋白酶体、hsa01230:氨基酸生物合成、hsa00190:氧化磷酸化、hsa00230:嘌呤代谢、hsa04932:非酒精性脂肪肝、hsa00020:柠檬酸循环、hsa03008:真核生物核糖体的生物发生、hsa00240:嘧啶代谢、hsa00650:Butanoate新陈代谢、hsa01200:碳代谢、hsa00520:氨基糖和核苷酸糖代谢、hsa05034:酗酒、hsa00071:脂肪酸降解、hsa04120:泛素介导的蛋白水解作用、hsa05205:蛋白聚糖在癌症、hsa05206:小分子核糖核酸在癌症、hsa00410:丙胺酸新陈代谢、hsa00340:组氨酸代谢、hsa00910:氮代谢、hsa00250:丙氨酸、天冬氨酸、谷氨酸代谢、hsa00350:酪氨酸代谢、hsa04726:血清素激活的突触、hsa00900:萜类化合物生物合成支柱、hsa04610:补体和凝血级联、hsa00330:精氨酸和脯氨酸代谢、hsa04520:粘附的结、hsa00860:卟啉与叶绿素代谢、hsa00010:糖酵解和糖质新生、hsa00982:药物代谢-细胞色素P450、hsa00980:细胞色素P450对外源性药物的代谢作用、hsa04962:加压素调节水重吸收、 hsa00983:药物代谢-其他酶；所述识别激活的膜蛋白参与的细胞联结、神经连结、血管、内分泌、生殖系统代谢通路：hsa04510:粘着斑、hsa 04724:谷氨酸能的突触、hsa04530:紧密连接、hsa00830:视黄醇新陈代谢、hsa04114:卵母细胞减数分裂、hsa04728:多巴胺神经突触、hsa00140:类固醇激素生物合成、hsa04261:心肌细胞的肾上腺素能信号、hsa04727:γ-氨基丁酸能的突触、hsa04725:胆碱能突触、hsa04540:缝隙连接、hsa04971:胃酸分泌、hsa04713:昼夜夹带、hsa04931:胰岛素抵抗、hsa05031:安非他命上瘾、hsa04924:肾素分泌、hsa04925:醛固酮的合成与分泌、hsa00590:花生四烯酸代谢、hsa04270:血管平滑肌收缩、hsa00760:烟酸和烟酰胺代谢、hsa04740:嗅觉传导、hsa04260:心肌收缩、hsa04720:长期势差现象、hsa04744:光传导、hsa04966:收集管道酸性分泌物、hsa05412:致心律失常性右心室心肌病、hsa05410:肥厚性心肌病、hsa04146:过氧物酶体、hsa05414:扩张型心肌病、hsa04970:唾液分泌、hsa04611:血小板激活、hsa05204:化学致癌作用、hsa04721:突触囊泡循环。The HrpN-type multi-epitope ligand protein according to claim 12 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their involved signaling pathways and cause cascade biological effects It is characterized in that: HrpN-type multi-epitope ligand protein is HrpNEch protein, and the anti-viral, anti-bacterial, anti-foreign body, and anti-inflammatory related metabolic pathways involved in the recognition of activated membrane proteins: hsa04144: endocytosis role, hsa04145: phagosome, hsa04142: lysosome, hsa04666: Fc-r-mediated phagocytosis, hsa01130: biosynthesis of antibiotics, hsa05131: shigellosis, hsa04612: antigen processing and presentation, hsa05130: pathogenic Escherichia coli infection, hsa05100: Bacterial invasion of epithelial cells, hsa05132: Salmonella infection, hsa05169: Barr virus infection, hsa05203: Viral carcinogenesis, hsa05134: Legionnaires' disease, hsa05160: Hepatitis C, hsa05162: Measles, hsa05133: Pertussis, hsa05322 : systemic lupus erythematosus, hsa04670: transendothelial migration of leukocytes, hsa05152: tuberculosis, hsa05150: staphylococcus aureus infection, hsa05146: amebiasis, hsa05142: Chagas disease, hsa05200: pathway in cancer, hsa05143: Africa Trypanosomiasis, hsa04750: Inflammatory Mediator Regulation of TRP Channels, hsa04916: Bactericidal Effects, hsa05230: Central Carbon Metabolism in Cancer, hsa05214: Glioma, hsa05212: Pancreatic Cancer; Disease metabolic pathways: hsa05010: Alzheimer's disease, hsa05012: Parkinson's disease, hsa05016: Huntington's disease; the identification of activated membrane proteins involved in nucleic acid, protein, amino acid, sugar, fat metabolism-related pathways: hsa03013: RNA trafficking, hsa03018: RNA degradation, hsa03040: spliceosome, hsa03010: ribosome, hsa04141: endoplasmic reticulum protein processing, hsa04810: regulation of the actin backbone hsa03050: proteasome, hsa01230: amino acid biosynthesis, hsa00190: oxidative phosphorylation , hsa00230: Purine metabolism, hsa04932: Nonalcoholic fatty liver disease, hsa00020: Citric acid cycle, hsa03008: Biogenesis of eukaryotic ribosomes, hsa00240: Pyrimidine metabolism, hsa00650: Butanoate metabolism, hsa01200: Carbon metabolism, hsa00520: Amino sugars and nucleotide sugar metabolism, hsa05034: alcoholism, hsa0 0071: Fatty acid degradation, hsa04120: Ubiquitin-mediated proteolysis, hsa05205: Proteoglycans in cancer, hsa05206: Small RNAs in cancer, hsa00410: Alanine metabolism, hsa00340: Histidine metabolism, hsa00910: Nitrogen metabolism , hsa00250: Alanine, Aspartate, Glutamate metabolism, hsa00350: Tyrosine metabolism, hsa04726: Serotonin-activated synapses, hsa00900: Terpenoid biosynthetic pillar, hsa04610: Complement and coagulation cascade, hsa00330: Arginine and Proline Metabolism, hsa04520: Adhesive Knots, hsa00860: Porphyrin and Chlorophyll Metabolism, hsa00010: Glycolysis and Glycogenesis, hsa00982: Drug Metabolism - Cytochrome P450, hsa00980: Cytochrome P450 Metabolism of exogenous drugs, hsa04962: Vasopressin regulates water reabsorption, hsa00983: Drug metabolism - other enzymes; the identification of activated membrane proteins involved in cell junctions, neural junctions, blood vessels, endocrine, reproductive system metabolic pathways: hsa04510:focal adhesions, hsa04724:glutamatergic synapses, hsa04530:tight junctions, hsa00830:retinol metabolism, hsa04114:oocyte meiosis, hsa04728:dopaminergic synapses, hsa00140:steroid hormone biosynthesis , hsa04261: Adrenergic signaling in cardiomyocytes, hsa04727: GABAergic synapses, hsa04725: Cholinergic synapses, hsa04540: Gap junctions, hsa04971: Gastric acid secretion, hsa04713: Circadian entrainment, hsa04931: Insulin resistance , hsa05031: amphetamine addiction, hsa04924: renin secretion, hsa04925: aldosterone synthesis and secretion, hsa00590: arachidonic acid metabolism, hsa04270: vascular smooth muscle contraction, hsa00760: niacin and niacinamide metabolism, hsa04740: olfactory conduction, hsa04260: Myocardial contraction, hsa04720: long-term potential difference phenomenon, hsa04744: phototransduction, hsa04966: collecting duct acidic secretions, hsa05412: arrhythmogenic right ventricular cardiomyopathy, hsa05410: hypertrophic cardiomyopathy, hsa04146: peroxisomes, hsa05414 : dilated cardiomyopathy, hsa04970: salivation, hsa04611: platelet activation, hsa05204: chemical carcinogenesis, hsa04721: synaptic vesicle cycling.
  15. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，级联生物学效应包括细胞过程、环境信息处理、遗传信息处理、新陈代谢和生物体系统功能途径；其中，细胞过程包括HrpNEcb蛋白诱导的多个差异表达基因参与了运输和分解代谢，细胞群体，细胞活性，细胞生长与死亡等细胞过程；环境信息处理包括HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了信号分子与相互作用，信号转导，膜运输环境信息处理过程；遗传信息处理包括HrpNEcb蛋白诱导的多个差异表达基因参与了翻译，复制和修复，折叠、分类和降解生物过程；新陈代谢包括HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了生物降解和代谢，核苷酸代谢，氨基酸的代谢，代谢的辅助因子和维生素，脂质代谢，糖的生物合成和代谢，全局和概览地图，能量代谢，碳水化合物代谢及氨基酸代谢过程；生物体系统包括HrpNEcb多拟表位配体蛋白诱导的多个差异表达基因参与了感觉系统，神经系 统，免疫系统，排泄系统，环境适应，内分泌系统，消化系统，发育循环系统生物过程。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-mimetic epitope ligand protein is HrpNEcb multi-mimetic epitope ligand protein, and cascade biological effects include cellular processes, environmental information processing, genetic information processing, metabolism and organisms Systemic functional pathways; among them, cellular processes include HrpNEcb protein-induced multiple differentially expressed genes involved in cellular processes such as transport and catabolism, cell population, cell activity, cell growth and death; environmental information processing includes HrpNEcb multi-epitope ligands Protein-induced multiple differentially expressed genes are involved in signal molecules and interactions, signal transduction, membrane transport and environmental information processing; genetic information processing includes HrpNEcb protein-induced multiple differentially expressed genes involved in translation, replication and repair, folding, Classification and degradation of biological processes; metabolism including HrpNEcb polyepitopic ligand protein-induced multiple differentially expressed genes involved in biodegradation and metabolism, nucleotide metabolism, amino acid metabolism, metabolic cofactors and vitamins, lipid metabolism, Global and overview maps of sugar biosynthesis and metabolism, energy metabolism, carbohydrate metabolism and amino acid metabolism processes; biological systems including HrpNEcb multi-epitope ligand protein-induced multiple differentially expressed genes involved in sensory system, nervous system, Immune system, excretory system, environmental adaptation, endocrine system, digestive system, developmental circulatory system biological processes.
  16. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，级联生物学效应包括细胞过程、环境信息处理、遗传信息处理、新陈代谢和生物体系统功能途径；其中，细胞过程包括HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了运输和分解代谢，细胞群体，细胞活性，细胞生长与死亡等细胞过程；环境信息处理包括HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了信号分子与相互作用，信号转导，膜运输环境信息处理过程；遗传信息处理包括HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了翻译，复制和修复，折叠、分类和降解生物过程；新陈代谢包括HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了生物降解和代谢，核苷酸代谢，氨基酸的代谢，代谢的辅助因子和维生素，脂质代谢，糖的生物合成和代谢，全局和概览地图，能量代谢，碳水化合物代谢及氨基酸代谢代谢过程；生物体系统包括HrpNEch多拟表位配体蛋白诱导的多个差异表达基因参与了感觉系统，神经系统，免疫系统，排泄系统，环境适应，内分泌系统，消化系统，发育循环系统生物过程。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-mimetic epitope ligand protein is HrpNEch multi-mimetic epitope ligand protein, and the cascade biological effects include cellular processes, environmental information processing, genetic information processing, metabolism and organisms Systemic functional pathways; among them, cellular processes include multiple differentially expressed genes induced by HrpNEch multi-epitope ligand proteins involved in cellular processes such as transport and catabolism, cell population, cell activity, cell growth and death; environmental information processing includes HrpNEch Multiple differentially expressed genes induced by multi-epitope ligand proteins are involved in signaling molecules and interactions, signal transduction, membrane transport and environmental information processing; genetic information processing includes multiple differences induced by HrpNEch multi-epitopic ligand proteins Expressed genes are involved in translation, replication and repair, folding, classification and degradation biological processes; metabolism includes multiple differentially expressed genes induced by HrpNEch multi-epitope ligand protein involved in biodegradation and metabolism, nucleotide metabolism, amino acid metabolism , metabolic cofactors and vitamins, lipid metabolism, sugar biosynthesis and metabolism, global and overview maps, energy metabolism, carbohydrate metabolism and amino acid metabolism metabolic processes; biological systems including HrpNEch polyepitopic ligand proteins-induced A number of differentially expressed genes are involved in the biological processes of the sensory system, nervous system, immune system, excretory system, environmental adaptation, endocrine system, digestive system, and developmental circulatory system.
  17. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，级联生物学效应还包括HrpNEcb多拟表位配体蛋白诱导的基因功能群显著差异表达结果，其中包括：生物过程相关差异表达基因：包括了繁殖，细胞死亡，免疫系统的过程，行为，代谢过程，细胞过程，生殖过程，生物粘附，信号，多细胞生物过程，发育过程，增长，运动，单个组织的过程，生物相，有节奏的过程，生物过程的正调控，生物过程负调控，生物过程调节，刺激反应，定位，生物调节，细胞成分组织或生物发生，细胞聚集，解毒以及突触前过程涉及突触传递； 细胞组分相关差异表达基因：涵盖了细胞及细胞外区域，类核，膜，病毒粒子，细胞结，细胞外基质，细胞膜封闭腔，复杂大分子，细胞器，细胞外基质成分，细胞外区域部分，细胞器部件，病毒粒子部件，膜部件，突触部分，细胞部件，突触，以及细胞超分子纤维；分子功能相关差异表达基因：涵盖了转录因子活性，蛋白质结合，核酸结合转录因子活性，催化活性，信号传感器活动，结构分子活动，运输活动，绑定，电子载体活动，成形素活动，抗氧化活性，金属伴侣蛋白活性，蛋白质标记，化学引诱物的活动，转译调控，化学排斥物活性，活动分子传感器，分子功能调控。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN type multi-mimetic epitope ligand protein is HrpNEcb multi-mimetic epitope ligand protein, and the cascade biological effect also includes HrpNEcb multi-mimetic epitope ligand protein-induced gene function group is significant Differential expression results, including: differentially expressed genes related to biological processes: including reproduction, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes , growth, movement, processes of a single tissue, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, stimulus response, localization, biological regulation, cellular component organization or biogenesis, cell aggregation , detoxification and presynaptic processes involve synaptic transmission; differentially expressed genes related to cellular components: covering cells and extracellular regions, nucleoids, membranes, virions, cell junctions, extracellular matrix, cell membrane closed cavity, complex macromolecules , organelles, extracellular matrix components, extracellular region parts, organelle parts, virion parts, membrane parts, synaptic parts, cell parts, synapses, and cellular supramolecular fibers; molecular function-related differentially expressed genes: covering transcription factors activity, protein binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, morphogen activity, antioxidant activity, metal chaperone activity, protein labeling, chemoattraction Molecular activity, translation regulation, chemorepellent activity, active molecular sensors, molecular function regulation.
  18. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，级联生物学效应还包括HrpNEch多拟表位配体蛋白诱导的基因功能群显著差异表达结果，其中包括：生物过程相关差异表达基因：包括了繁殖，细胞死亡，免疫系统的过程，行为，代谢过程，细胞过程，生殖过程，生物粘附，信号，多细胞生物过程，发育过程，增长，运动，单个组织的过程，生物相，有节奏的过程，生物过程的正调控，生物过程负调控，生物过程调节，刺激反应，定位，生物调节，细胞成分组织或生物发生，细胞聚集，解毒以及突触前过程涉及突触传递；细胞组分相关差异表达基因：涵盖了细胞及细胞外区域，类核，膜，病毒粒子，细胞结，细胞外基质，细胞膜封闭腔，复杂大分子，细胞器，细胞外基质成分，细胞外区域部分，细胞器部件，病毒粒子部件，膜部件，突触部分，细胞部件，突触，以及细胞超分子纤维；分子功能相关差异表达基因：涵盖了转录因子活性，蛋白质结合，核酸结合转录因子活性，催化活性，信号传感器活动，结构分子活动，运输活动，绑定，电子载体活动，成形素活动，抗氧化活性，金属伴侣蛋白活性，蛋白质标记，化学引诱物的活动，转译调控，化学排斥物活性，活动分子传感器，分子功能调控。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics and health care products that recognize and activate various types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. Or the application in pharmacy, it is characterized in that: HrpN-type multi-mimetic epitope ligand protein is HrpNEch multi-mimetic epitope ligand protein, and the cascade biological effect also includes HrpNEch multi-mimetic epitope ligand protein-induced gene function group significant Differential expression results, including: differentially expressed genes related to biological processes: including reproduction, cell death, immune system processes, behavior, metabolic processes, cellular processes, reproductive processes, bioadhesion, signaling, multicellular biological processes, developmental processes , growth, movement, processes of a single tissue, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, stimulus response, localization, biological regulation, cellular component organization or biogenesis, cell aggregation , detoxification and presynaptic processes involve synaptic transmission; differentially expressed genes related to cellular components: covering cells and extracellular regions, nucleoids, membranes, virions, cell junctions, extracellular matrix, cell membrane closed cavities, complex macromolecules , organelles, extracellular matrix components, extracellular region parts, organelle parts, virion parts, membrane parts, synaptic parts, cell parts, synapses, and cellular supramolecular fibers; molecular function-related differentially expressed genes: covering transcription factors activity, protein binding, nucleic acid binding transcription factor activity, catalytic activity, signal sensor activity, structural molecular activity, transport activity, binding, electron carrier activity, morphogen activity, antioxidant activity, metal chaperone activity, protein labeling, chemoattraction Molecular activity, translation regulation, chemorepellent activity, active molecular sensors, molecular function regulation.
  19. 根据权利要求1或3所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋 白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：所述制药中的应用的制品或药物的剂型为液剂、粉剂、片剂或胶囊剂。The HrpN-type multi-epitope ligand protein according to claim 1 or 3 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects It is characterized in that: the dosage form of the product or medicine used in the pharmacy is liquid, powder, tablet or capsule.
  20. 根据权利要求19所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEcb多拟表位配体蛋白，制品或药物主要由纯化的HrpNEcb多拟表位配体蛋白制备得到，质量含量为有0.001％-100％。The HrpN-type multi-epitope ligand protein according to claim 19 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate various types of receptors and/or membrane proteins and their involved signaling pathways and cause cascade biological effects It is characterized in that: the HrpN-type polymimetic epitope ligand protein is HrpNEcb polymimetic epitope ligand protein, and the product or medicine is mainly prepared from purified HrpNEcb polymimetic epitope ligand protein, and the mass content is 0.001 %-100%.
  21. 根据权利要求19所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为HrpNEch多拟表位配体蛋白，制品或药物主要由纯化的HrpNEch多拟表位配体蛋白制备得到，质量含量为0.001％-100％。The HrpN-type multi-epitope ligand protein according to claim 19 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate various types of receptors and/or membrane proteins and their involved signaling pathways and cause cascade biological effects It is characterized in that: the HrpN-type polymimetic epitope ligand protein is HrpNEch polymimetic epitope ligand protein, and the product or medicine is mainly prepared from purified HrpNEch polymimetic epitope ligand protein, and the mass content is 0.001% -100%.
  22. 根据权利要求1-21任一项所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其参与的信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白为纯化后的HrpN型多拟表位配体蛋白。The HrpN-type multi-epitope ligand protein according to any one of claims 1-21 is used in foods and cosmetics that recognize and activate multiple types of receptors and/or membrane proteins and the signal pathways involved and cause cascade biological effects. , health care products or applications in pharmacy, characterized in that: the HrpN-type multi-mimicking-epitope ligand protein is a purified HrpN-type multi-mimicking-epitope ligand protein.
  23. 根据权利要求22所述的HrpN型多拟表位配体蛋白在识别激活多类受体和/或膜蛋白及其信号通路并引起级联生物学效应的食品、化妆品、保健品或制药中的应用，其特征在于：HrpN型多拟表位配体蛋白纯化的方法，包括以下步骤：The HrpN-type multi-epitope ligand protein according to claim 22 is used in food, cosmetics, health care products or pharmaceuticals that recognize and activate multiple types of receptors and/or membrane proteins and their signaling pathways and cause cascade biological effects Application, it is characterized in that: the method for HrpN type multi-mimetic epitope ligand protein purification, comprises the following steps:
    步骤1：高压破碎机破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁,高压范围800-1000Mpa。Step 1: The high pressure crusher crushes the engineering bacteria, and the crushed bacteria liquid is passed into the butterfly continuous flow centrifuge to remove the cell wall. The high pressure range is 800-1000Mpa.
    步骤2：用Ni-NTA琼脂糖凝胶柱纯化HrpN多拟表位配体蛋白-His重组蛋白，得到纯化的HrpN型多拟表位配体蛋白原药。Step 2: Purify the HrpN polymimetic epitope ligand protein-His recombinant protein with a Ni-NTA agarose column to obtain a purified HrpN type polymimetic epitope ligand protein original drug.
  24. HrpN型蛋白纯化的方法，其特征在于，包括以下步骤：The method for HrpN-type protein purification is characterized in that, comprises the following steps:
    步骤1：高压破碎机破碎工程菌，将破碎菌液通入蝶式连续流离心机，清除细胞壁,高压范围800-1000Mpa。Step 1: The high pressure crusher crushes the engineering bacteria, and the crushed bacteria liquid is passed into the butterfly continuous flow centrifuge to remove the cell wall. The high pressure range is 800-1000Mpa.
    步骤2：用Ni-NTA琼脂糖凝胶柱纯化HrpN多拟表位配体蛋白-His重组蛋白，得到纯化的HrpN型多拟表位配体蛋白原药。Step 2: Purify the HrpN polymimetic epitope ligand protein-His recombinant protein with a Ni-NTA agarose column to obtain a purified HrpN type polymimetic epitope ligand protein original drug.
  25. 根据权利要求24所述的HrpN型多拟表位配体蛋白的纯化的方法，其特征在于，HrpN型多拟表位配体蛋白含有一至多个疏水非极性氨基酸残基的结构基团或表位、含有一至多个极性不带电氨基酸残基的结构基团或表位、含有一至多个酰胺基极性不带电氨基酸残基的结构基团或表位、含有一至多个酸性带正电、碱性带负电氨基酸残基的结构基团或表位；疏水非极性氨基酸残基包括缬氨酸、亮氨酸、异亮氨酸、丙氨酸、苯丙氨酸、蛋氨酸残基；极性不带电氨基酸残基包括丝氨酸残基；酰胺基极性不带电氨基酸残基包括天冬酰胺、谷氨酰胺残基；酸性带正电、碱性带负电氨基酸残基包括天冬酰酸、谷氨酸、赖氨酸、组氨酸、精氨酸残基；疏水非极性氨基酸残基，极性不带电氨基酸残基，酰胺基极性不带电氨基酸残基，酸性带正电、碱性带负电氨基酸残基在HrpN型多拟表位配体蛋白分子的全序列中占比62.3％-73.7％，在保守结构域中占比61％-74％，在α-螺旋结构中占比66.2％-79％；疏水非极性氨基酸残基的结构基团或表位，极性不带电氨基酸残基的结构基团或表位，酰胺基极性不带电氨基酸残基的结构基团或表位和酸性带正电、碱性带负电氨基酸残基的结构基团或表位通过氢键、离子键、疏水、非极性、极性、范德华力，实现配体和受体分子空间结构和电性的互补性、互作性以及特异识别、激活、结合，与多类型受体形成紧密结合面或复合物，能引起受体分子的构象、能量、电性和信息的变化，经信号传导和转导，引发放大的级联生物学效应。The method for purifying HrpN-type polymimetic epitope ligand protein according to claim 24, wherein the HrpN-type polymimetic epitope ligand protein contains one or more structural groups of hydrophobic non-polar amino acid residues or Epitopes, structural groups or epitopes containing one or more polar uncharged amino acid residues, structural groups or epitopes containing one or more amide polar uncharged amino acid residues, containing one or more acidic Structural groups or epitopes of charged, basic and negatively charged amino acid residues; hydrophobic non-polar amino acid residues include valine, leucine, isoleucine, alanine, phenylalanine, methionine residues ; Polar uncharged amino acid residues include serine residues; Amide polar uncharged amino acid residues include asparagine, glutamine residues; Acidic positively charged, basic negatively charged amino acid residues include asparagine , glutamic acid, lysine, histidine, arginine residues; hydrophobic non-polar amino acid residues, polar uncharged amino acid residues, amide polar uncharged amino acid residues, acidic positively charged, Basic negatively charged amino acid residues account for 62.3%-73.7% of the full sequence of HrpN-type multi-epitope ligand protein molecules, 61%-74% of conserved domains, and α-helical structures. Ratio 66.2%-79%; structural group or epitope of hydrophobic non-polar amino acid residue, structural group or epitope of polar uncharged amino acid residue, structural group or epitope of amide group polar uncharged amino acid residue Or epitopes and structural groups or epitopes of acidic positively charged, basic negatively charged amino acid residues through hydrogen bonds, ionic bonds, hydrophobic, nonpolar, polar, van der Waals forces, to achieve ligand and receptor molecular space Complementarity, interaction, and specific recognition, activation, and binding of structure and electricity, forming tight binding surfaces or complexes with multiple types of receptors, can cause changes in the conformation, energy, electricity, and information of receptor molecules. Signal transduction and transduction, triggering an amplified cascade of biological effects.
  26. 根据权利要求24所述的HrpN型多拟表位配体蛋白的纯化的方法，其特征在于，HrpN型多拟表位配体蛋白包括HrpNEcc、HrpNEca、HrpNEcb、HrpNEch、HrpNDaz、HrpNDada、HrpNDasp、HrpNad、HrpNDaf、HrpNEa、HrpNSam、HrpNBag、HrpNPas、HrpNEnt。The method for purifying HrpN-type multi-epitope ligand protein according to claim 24, wherein the HrpN-type multi-epitope ligand protein comprises HrpNEcc, HrpNEca, HrpNEcb, HrpNEch, HrpNDaz, HrpNDada, HrpNDasp, HrpNad , HrpNDaf, HrpNEa, HrpNSam, HrpNBag, HrpNPas, HrpNEnt.
PCT/CN2021/134714 2020-12-31 2021-12-01 Use of hrpn-type multi-mimotope epitope ligand protein in foods, cosmetics, health care products or pharmaceuticals WO2022142976A1 (en)

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