WO2022142977A1 - HrpZ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 - Google Patents
HrpZ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 Download PDFInfo
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Classifications
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
- A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
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- 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
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- 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
- C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Definitions
- the invention relates to the field of biomedicine, in particular to the application of HrpZ-type protein in the pharmacy of identifying and activating multiple types of receptors and/or membrane proteins and their signal pathways and causing 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. In the field of protection, biopesticides can safely induce plants to produce disease resistance, insect repellency, stress resistance, and promote plant growth and development and increase yield.
- the conserved domain of HrpZpss protein consists of 343 amino acids, and the entire sequence is the conserved domain 1-343; -265, 290-309, 317-336.
- HrpZpst protein (GenBank accession number: AY999001) is the expression product of hrpZpst gene (Pseudomonas syringae pv tomato Strain CSCS008), which consists of 370 amino acid residues, a non-enzymatic protein with one, two and three tertiary structures but no quaternary structure, Does not contain cystine and cysteine, rich in glycine and serine, the molecule is 36.52kDa, the HrpZpst protein conserved domain consists of 370 amino acids, the full sequence is the conserved structural region, 1-370, ⁇ -helical structure 33-49 , 58-70, 78-92, 136-155, 172-183, 192-197, 204-223, 265-277, 279-284, 314-333, 341-359, 364-366 proteins.
- 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 ( Intrinsically disordered proteins, intrinsically disordered proteins, referred to as IDPs), etc., ⁇ helix is a repetitive structure, and the ⁇ and ⁇ of each ⁇ -carbon in the helix are around -57° and -47°, respectively.
- IDPs Intrinsically disordered proteins, intrinsically disordered proteins, referred to as IDPs
- 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
- HrpZ-type proteins are a class of proteins encoded by genes in the "hyper sensitive response and pathogenicity (hrp)" gene cluster with similar properties and functions, including high 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.
- HrpZ-type protein in the pharmacy of identifying and activating multiple types of receptors and/or membrane proteins and their signaling pathways and causing cascade biological effects.
- HrpZ-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.
- HrpZ-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.
- HrpZ-type protein According to the structural characteristics of HrpZ-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 HrpZ-type polyprotein. Epitope-like ligand proteins (multi epitopic-like ligand proteins).
- HrpZ-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.
- HrpZ-type multi-epitope ligand protein in recognizing 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.
- HrpZ-type multi-epitopic 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: Valine, leucine, isoleucine, alanine, phenylalanine, methionine, polar uncharged amino acid residues: serine, amide polar uncharged amino acid residues: asparagine, glutamine Amides, acidic positively charged, basic negatively charged amino acid residues: asparagine, glutamic acid, lysine, histidine, arginine; hydrophobic non-polar amino acid residues, polar uncharged amino acid residues The polar uncharged amino acid residues,
- HrpZ-type multi-epitope ligand proteins include HrpZPsa, HrpZPsm, HrpZPss, HrpZPst, HrpZPsap, HrpZPsr, HrpZPsth, HrpZPave, HrpZPam, HrpZPcar, HrpZPcor, HrpZPcst, HrpZPcat, HrpZPcory, HrpZPcp, HrpZPsav, HrpZPvirp, HrpZPvir HrpZPade, HrpZPsac, HrpZPsg, HrpZPsc.
- these molecules are highly homologous, ranging from 60% to 99%;
- HrpZ polymimetic epitope ligand protein homology comparison is as follows:
- HrpZ-type multi-epitope ligand protein is rich in multiple linear and conformational structural groups or epitopes. It refers to a functional group composed of amino acid residues that can recognize and bind to cell membrane receptors, membrane proteins, etc.
- This functional group It is composed of the following amino acid residues, including rich proton-donating or proton-accepting amino acid residues that can recognize, bind, and activate with receptors; further, contains one or more hydrophobic non-polar amino acid residues, contains one or more Acidic positively charged, basic negatively charged amino acid residues, containing one or more amide-group polar uncharged amino acid residues, containing one or more polar uncharged amino acid residues; further, rich in proton-donating type (methionine residues) or proton-accepting (including methionine residues) amino acid residues: glutamic acid, aspartic acid, lysine, histidine, methionine, serine, threonine, tyrosine, arginine , they can recognize and activate connection with the corresponding amino acid residues of multiple types of receptor proteins by hydrogen bonding to form binding surfaces or complexes; further, hydrophobic non-polar amino acid residues: valine, leucine, isoleucine , Alan
- HrpZpss polypeptide has 343 amino acid residues, 235 key amino acid residues, 105 hydrophobic non-polar amino acid residues, 33 polar uncharged amino acid residues, 40 amide amino acid residues, and acidic amino acid residues.
- HrpZpst multi-epitope ligand protein has 370 amino acid residues, the full sequence is a conserved structural region, there are 370 amino acid residues, accounting for 100% of the full sequence, 261 key amino acid residues, hydrophobic non-hydrophobic There are 114 polar amino acid residues, 43 polar uncharged amino acid residues, 54 amide amino acid residues, 50 acidic positively charged and basic negatively charged amino acid residues, and key amino acid residues account for the total amino acid residues 70.5%, molecular weight 36.52kd, isoelectric point PI 4.01; 12 ⁇ -helices, 2 ⁇ -sheets and 7 do-structure regions; ⁇ -helix region, 164 amino acid residues, key amino acid residues 136, 66 hydrophobic non-polar amino acid residues, 12 polar uncharged amino acid residues, 29 amide amino acid residues, 29 acidic positively charged, basic negatively charged amino acid residues, key amino acid residues accounted for 82
- the complementarity , interaction, and specific recognition, activation, and binding of inter-structure and electrical properties can form tight binding surfaces or complexes with multiple types of receptors, which can cause changes in the conformation, energy, electrical properties and information of receptor molecules.
- signal transduction and transduction a series of biological effects are amplified and expressed.
- 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 HrpZpst multi-mimetic epitope ligand protein is shown in SEQ ID NO: 1; the amino acid sequence of the HrpZpst multi-mimetic epitope ligand protein is shown in SEQ ID NO: 2.
- the HrpZ-type multi-epitope ligand protein is HrpZpss multi-epitope ligand protein
- the multi-type receptors that recognize and bind include HLA-C major histocompatibility complex, I, C type receptors, free fatty acids
- HLA-C major histocompatibility complex I, C type receptors, free fatty acids
- receptor 4 tyrosine protein kinase transmembrane receptor 1
- ASGPR1 asialoglycoprotein receptor 1 adipocyte plasma membrane-associated protein receptor
- insulin-like growth factor 2 receptor insulin-like growth factor 2 receptor
- the HrpZ type multi-epitope ligand protein is HrpZpst multi-epitope ligand protein
- the multi-type receptors that recognize and bind include HLA-C major histocompatibility complex, I, C type receptors, ASGPR1 One or more of sialoglycoprotein receptor 1, adipocyte plasma membrane associated protein receptor, insulin-like growth factor 2 receptor, LDL receptor associated protein 1.
- the HrpZ type multi-epitope ligand protein is HrpZpss multi-mimetic epitope ligand protein
- the membrane proteins that recognize and bind include solute carrier family 5, member 6, solute carrier family 26, member 4, solute carrier family 38, member 2.
- DNM2 dynein 2 CAP1 adenylyl cyclase-related protein 1, ICAM1 intercellular adhesion molecule 1, LanC-like protein 1, MLEC stress protein-androgen-like receptor kinase, TJP2 tight junction protein 2, ZYX plaques one or more of catenin.
- the HrpZ-type multi-epitope ligand protein is HrpZpst multi-epitope ligand protein
- the membrane proteins that recognize and bind include solute carrier family 38, member 2, DNM2 dynein 2, SPTAN1 non-erythrocyte 1 ⁇ spectrin, ⁇ - Mutual protein 1, CAP1 adenylate cyclase-related protein 1, ICAM1 intercellular adhesion molecule 1, LanC-like protein 1, MLEC stress protein-androgen-like receptor kinase, TJP2 claudin 2, ZYX plaques one or more of catenin.
- the HrpZ-type multi-epitope ligand protein is HrpZpss multi-epitope ligand protein
- the receptors that recognize the binding and the signaling pathways involved in membrane proteins include hsa04933 The role of age-anger signaling pathway in diabetic complications, hsa04064 One or more of NF-kappa B signaling pathway and hsa04072 phospholipase D signaling pathway.
- the HrpZ-type multi-epitope ligand protein is HrpZpst multi-epitope ligand protein
- the receptors that recognize the binding and the signaling pathways involved in membrane proteins include hsa04933 The role of age-anger signaling pathway in diabetic complications, hsa04064 One or more of NF-kappa B signaling pathway, hsa04072 phospholipase D signaling pathway, and hsa04668TNF signaling pathway.
- the signaling pathways involved in the recognition of activated receptors and membrane proteins include metabolic signaling pathways, and the metabolic signaling pathways include antiviral, antibacterial, anti-foreign body, and anti-inflammatory metabolic pathways; including nucleic acid, protein, amino acid, sugar, and fat metabolism Pathways; including cell junctions, neural junctions, blood vessels, endocrine, reproductive system metabolic pathways.
- the HrpZ-type multi-epitope ligand protein is HrpZpss multi-epitope ligand protein, and the anti-viral, anti-bacterial, anti-foreign body, and anti-inflammatory metabolic pathways involved in the recognition of activated membrane proteins: hsa04144 endocytosis , hsa04145 phagosome, hsa04142 lysosome, hsa04666Fc gamma r-mediated phagocytosis, hsa04210 apoptosis, hsa04218 cellular senescence, hsa05130: pathogenic E.
- coli infection hsa04612 antigen processing and presentation, hsa05100 bacterial invasion of epithelial cells, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05164 influenza A, hsa05150 staphylococcus aureus infection, hsa05167 Kaposi sarcoma with herpes virus infection, hsa04916: bactericidal effect, hsa04650 natural killer cell-mediated cytotoxicity, hsa05169 Epstein-Barr virus infection, hsa05416 viral myocarditis, hsa05110 Vibrio cholerae infection, hsa05144 malaria, hsa05163 human cytomegalovirus infection, hsa05170 human immunodeficiency virus type 1 infection, hsa05323 rheumatoid arthritis, hsa04670 leukocyte transcellular migration;
- the HrpZ-type multi-epitope ligand protein is the HrpZpst protein, which recognizes the antiviral, antibacterial, anti-foreign and anti-inflammatory metabolic pathways involved in the activated membrane protein: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome , hsa04666Fc gamma r-mediated phagocytosis, hsa04210 apoptosis, hsa04218 cellular senescence, hsa04612 antigen processing and presentation, hsa05100 bacterial invasion of epithelial cells, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05164 influenza A, hsa05150 Staphylococcus aureus infection, hsa05143 African trypanosomiasis, hsa04650 Natural killer cell-mediated cyto
- the HrpZ-type multi-epitope ligand protein is HrpZpss 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 HrpZpss protein are involved in transport and catabolism, cell population, cell activity , cell processes such as cell growth and death; 2Environmental Information Processing: Multiple differentially expressed genes induced by HrpZpss multi-epitope ligand protein are involved in signaling molecules and interactions, signal transduction, membrane transport and other environments Information processing process; 3 Genetic Information Processing: Multiple differentially expressed genes induced by HrpZpss multi-epitope ligand protein are involved in biological processes such as translation, replication and repair, folding, classification and degradation; 4 Metabolism (Metabolism ): HrpZpss polyepitopic ligand protein-induced multiple
- HrpZ-type multi-epitope ligand protein is HrpZpst 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 HrpZpst multi-epitope ligand protein are involved in transport and catabolism , cell population, cell activity, cell growth and death and other cellular processes; 2Environmental Information Processing: Multiple differentially expressed genes induced by HrpZpst multi-epitope ligand protein are involved in signaling molecules and interactions, signal transduction 3 Genetic Information Processing: Multiple differentially expressed genes induced by HrpZpst multi-epitope ligand protein are involved in translation, replication and repair, folding, classification and degradation of biological Process; 4Metabolism: Multiple differentially expressed genes induced by HrpZpst multi-epitope ligand protein are involved
- the HrpZ-type multi-epitope ligand protein is HrpZpss multi-epitope ligand protein
- the cascade biological effect also includes the results of the significant differential expression of gene functional groups induced by the HrpZpss 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 enclosed cavity, complex
- the HrpZ-type multi-epitope ligand protein is HrpZpst multi-epitope ligand protein
- the cascade biological effect also includes the results of the significant differential expression of gene functional groups induced by the HrpZpst 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
- 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 (HrpZpss polyepitope ligand protein preparations and/or drugs) and derivatives thereof for HrpZpss polyepitope 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 HrpZpss polyepitope ligand protein preparations and/or drugs
- derivatives thereof for HrpZpss polyepitope 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 HrpZpss 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 further includes active compounds (HrpZpst poly-epitope ligand protein preparations and/or drugs) and derivatives thereof for HrpZpst poly-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.
- 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.
- 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 HrpZpst 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 HrpZ type polymimetic epitope ligand protein is HrpZpss polymimetic epitope ligand protein
- the product or medicine is mainly prepared from purified HrpZEcb polymimetic epitope ligand protein, and the mass content is 0.001%-100%.
- the HrpZ-type polymimetic epitope ligand protein is HrpZpst polymimetic epitope ligand protein
- the product or medicine is mainly prepared from purified HrpZEch polymimetic epitope ligand protein, and the mass content is 0.001%-100%.
- the HrpZ-type multi-epitope ligand protein is a purified HrpZ-type protein.
- the method for purifying HrpZ-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 HrpZ polymimetic epitope ligand protein-His recombinant protein with a Ni-NTA agarose column to obtain a purified HrpZ-type polymimetic epitope ligand protein original drug.
- the high-efficiency expression, purification and production of the HrpZpss polymimetic epitope ligand protein used in the present invention comprises the following steps:
- HrpZpss multi-mimetic epitope ligand protein the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes) encoding HrpZpss multi-mimetic epitope ligand proteins are prepared.
- 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 HrpZpss multi-epitope ligand protein was present in the supernatant.
- the application route of the HrpZpss 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 HrpZpss polyepitopic ligand proteins, 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 functions are 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 food, elimination, makeup, machinery, and health related diseases and conditions. Pharmacy applications of products or drugs.
- HrpZpss 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, 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 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 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 includes the following methods:
- HrpZpss polymimetic epitope ligand protein using the engineering bacteria containing the hrpZpss gene (GenBank: AAY36247.1) (cloned into the high-efficiency expression vector PET28a(+)), through fermentation, purification and preparation of HrpZpss polymimetic epitope Ligand protein:
- the engineering bacteria fermentation preparation of HrpZpss 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 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.
- HrpZpss polymimetic epitope ligand protein Purify HrpZpss polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column. Purification and preparation of HrpZpss polymimetic epitope ligand protein.
- the preparation of the HrpZpss polymimetic epitope ligand protein can also be prepared by the expression protein of an "artificially synthesized gene", and the HrpZpss polymimetic epitope ligand protein can be prepared by fermentation and purification. Somatic protein, including 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.
- 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.
- HrpZpss polymimetic epitope ligand protein Purify HrpZpss polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column. Purification and preparation of HrpZpss polymimetic epitope ligand protein.
- the high-efficiency expression of HrpZpst protein adopted in the present invention comprises the following steps:
- HrpZpst protein The engineering bacteria fermentation preparation of HrpZpst 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 HrpZpst polymimetic epitope ligand proteins are used.
- Modified) plasmid engineering bacteria E.coli
- IPTG isopropyl thiogalactoside, Isopropyl ⁇ -D-Thiogalactosid
- the expression product HrpZpst protein was analyzed by 10% SDS-PAGE polyacrylamide gel electrophoresis.
- 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 HrpZpst poly-epitope ligand protein was present in the supernatant.
- the application route of the HrpZpst 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 HrpZpst polyepitopic 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 bioactive agents are administered sequentially, intermittently, or in the same composition; the administration may be topical, topical, or systemic, depending on the site of treatment.
- Topical application 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 polymimetic ligand proteins, 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 HrpZpst multi-epitope ligand proteins that recognize and activate various types of receptors, membrane proteins and their signaling pathways in animals and induce multi-functional cascade biological effects 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, 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 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 HrpZpst 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 includes the following methods:
- 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 broth 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.
- HrpZpst polymimetic epitope ligand protein Purify HrpZpst polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column. Purification and preparation of HrpZpst polymimetic epitope ligand protein.
- the preparation of the HrpZpst polymimetic epitope ligand protein can also be prepared by the expression protein of "artificially synthesized gene", and the HrpZpst 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 HrpZpst protein encodes a 36.52kda band on the sample lane of the electrophoresis gel plate, which is the HrpZps
- 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.
- HrpZpst polymimetic epitope ligand protein Purify HrpZpst polymimetic epitope ligand protein-His recombinant protein with NI-NTA agarose column. Purification and preparation of HrpZpst polymimetic epitope ligand protein.
- HrpZ-type multi-epitope ligand proteins as a class of ligand proteins rich in multiple linear and conformational epitopes with special structures, can recognize, activate and bind membrane receptors, membrane proteins, Information pathways and metabolic pathways, HrpZ-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 HrpZpst polymimetic epitope ligand protein before and after purification: the left side is the molecular weight marker band, of which 1: the highly expressed HrpZpst polymimetic epitope ligand protein band (before purification); 2: the polymimetic epitope protein after purification The ligand protein HrpZpst band;
- Fig. 2 is that HrpZpst multi-epitope ligand protein liquid injection induces allergic reaction diagram of tobacco leaves: the focal spot is formed through HrpZpst protein liquid processing about 24hr, both sides: H 2 O injection; 1, 2: HrpZpst Protein solution (300 ⁇ g/ml) injection, that is, the hypersensitivity reaction of HrpZpst protein on tobacco leaves, both sides are controls, 1 and 2 are treatments;
- Figure 3 is a volcano plot of the HrpZpst 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 HrpZpst polyepitopic ligand protein of the present invention induced by oral administration and smearing of experimental mice to induce differential gene expression in the thalamus, from left to right: oral administration for 6h, oral administration for 24h; smear for 6h;
- Figure 5 is a volcano plot of the HrpZpst 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 HrpZpst 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 HrpZpst 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 the differentially expressed genes in the liver induced by oral administration of HrpZpst multi-epitope ligand protein and smearing experimental mice, from left to right: oral administration for 6h, oral administration for 24h; application for 6h; (treatment group left 3 Lane, control group, lane 4 from the right);
- Figure 9 is a clustering heat map of the differentially expressed gene sets in the thalamus induced by oral administration of HrpZpst multi-epitope ligand protein and smearing experimental mice, from left to right: oral administration for 6h, oral administration for 24h; application for 6h; (treatment group left 3 Lane, control group, lane 4 from the right);
- Figure 10 is a cluster heat map of HrpZpst 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 hippocampus, from left to right: oral administration for 6h, oral administration for 24h; 3 lanes from the left of the group, 3 lanes from the right of the treatment group);
- Figure 11 is a clustering heat map of the differentially expressed gene sets in the cerebral cortex induced by oral administration of HrpZpst multi-epitope ligand protein and smearing of experimental mice, from left to right: oral administration for 6h, oral administration for 24h; application for 6h, application for 12h; ( 3 lanes on the left in the control group, 3 lanes on the right in the treatment group);
- Figure 12 shows the comparison of the KEGG Pathway (total gene) in the liver of the experimental mice treated with the HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right: oral administration for 6h, oral administration for 24h; smear for 6h;
- Figure 13 shows the comparison of KEGG Pathway (up-regulated gene) in the liver of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention and the control.
- Figure 14 shows the comparison of the KEGG Pathway (down-regulated gene) in the liver of the experimental mice treated with the HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h and oral administration for 24h;
- Figure 15 shows the comparison of the KEGG Pathway (total gene) of the experimental mouse heart treated with the HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h; smearing for 6h, smearing for 12h;
- Figure 16 shows the comparison of KEGG Pathway (up-regulated gene) in the heart of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h; smear for 6h, smear for 12h;
- Figure 17 shows the comparison of the KEGG Pathway (down-regulated gene) in the hearts of experimental mice treated with the HrpZpst 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 18 is the comparison of KEGG Pathway (total gene) in the hippocampus of experimental mice treated with HrpZpst poly-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h; smearing for 6h, smearing for 12h;
- Figure 19 shows the comparison of KEGG Pathway (up-regulated gene) in the hippocampus of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention (up-regulated genes), from left to right, oral administration for 6h, oral administration for 24h; smearing 6h, smearing 12h;
- Figure 20 is a comparison of KEGG Pathway (down-regulated gene) in the hippocampus of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h; smear for 6h, smear for 12h;
- Figure 21 shows the comparison of KEGG Pathway (total gene) in the cerebral cortex of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h; smear for 6h, smear for 12h;
- Figure 22 shows the comparison of KEGG Pathway (up-regulated gene) in the cerebral cortex of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention (up-regulated genes), from left to right, oral administration for 6h, oral administration for 24h; smear for 6h, smear for 12h;
- Figure 23 shows the comparison of KEGG Pathway (down-regulated gene) in the cerebral cortex of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h; smear for 6h, smear for 12h;
- Figure 24 is a comparison of the KEGG Pathway (total gene) of the rat cerebral thalamus treated with the HrpZpst multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h; smear for 6h;
- Figure 25 shows the comparison of KEGG Pathway (up-regulated gene) in the cerebral thalamus of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention (up-regulated gene), from left to right, oral administration for 6h, oral administration for 24h;
- Figure 26 shows the comparison of KEGG Pathway (down-regulated gene) in the cerebral thalamus of experimental mice treated with HrpZpst multi-epitope ligand protein of the present invention (down-regulated gene), from left to right, oral administration for 6h, oral administration for 24h; smearing for 6h;
- Figure 27 is a flow chart of the mRNA (RNA-Seq) sequencing experiment of the present invention.
- Figure 28 is a flow chart of mRNA sequencing data analysis of the present invention.
- Figure 29 shows the electrophoresis detection of HrpZpss protein before and after purification: the left side is the molecular weight marker band, 1: HrpZpss multi-mimetic epitope ligand protein band before purification; 2: HrpZpss multi-mimetic epitope ligand protein band after purification;
- Figure 30 is a graph of the allergic reaction of tobacco leaves induced by HrpZpss multi-mimetic epitope ligand protein liquid injection: the focal spots seen are formed by HrpZpss multi-mimetic epitope ligand protein liquid treatment for about 24hr, both sides: H 2 O injection ; 3, 4: HrpZpss multi-epitope ligand protein solution (300 ⁇ g/ml) injection, namely the hypersensitivity reaction of HrpZpss multi-epitope ligand protein on tobacco leaves, both sides are controls, 3 and 4 are treatments;
- Figure 31 is a volcano plot of the HrpHrpZpss multi-epitope ligand protein of the present invention induced by oral administration and smearing of experimental mice to express differentially expressed genes in the kidneys, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
- Figure 32 is a volcano diagram of the HrpHrpZpss 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 33 is a cluster heat map of the differentially expressed gene sets in the kidneys induced by oral administration of the HrpHrpZpss multi-epitope ligand protein of the present invention and smearing in experimental mice, from left to right: oral administration for 6 hours, oral administration for 24 hours; application for 6 hours;
- Figure 34 is a cluster heat map of the differentially expressed genes in testis induced by oral administration of HrpHrpZpss 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 35 shows the comparison of the KEGG Pathway (total gene) of the experimental mouse kidney treated with the HrpZpss 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 36 shows the comparison of the KEGG Pathway (up-regulated gene) in the kidneys of experimental mice treated with HrpZpss multi-epitope ligand protein of the present invention (up-regulated genes), from left to right, oral administration for 6h, oral administration for 24h; smearing for 6h;
- Figure 37 shows the comparison of the KEGG Pathway (down-regulated gene) in the kidneys of experimental mice treated with HrpZpss multi-epitope ligand protein of the present invention and the control, from left to right: oral administration for 6h, oral administration for 24h; smear for 6h;
- Figure 38 shows the comparison of KEGG Pathway (total gene) in the testis of experimental mice treated with HrpZpss multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
- Figure 39 shows the comparison of KEGG Pathway (up-regulated gene) in the testis of experimental mice treated with HrpZpss multi-epitope ligand protein of the present invention (up-regulated gene), from left to right, oral administration for 6h, oral administration for 24h; smearing for 6h;
- Figure 40 shows the comparison of KEGG Pathway (down-regulated genes) in the testis of experimental mice treated with HrpZpss multi-epitope ligand protein of the present invention and the control, from left to right, oral administration for 6h, oral administration for 24h;
- Figure 41 is a flow chart of the mRNA (RNA-Seq) sequencing experiment of the present invention.
- Figure 42 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 HrpZpst multi-epitope ligand protein is fermented, purified, prepared and collected using the engineered bacteria with the registered (Pseudomonas syringae pv tomato Strain CSCS008) hrpZpst gene (GenBank: AY999001) (cloned into the high-efficiency expression vector PET28a(+)) plasmid
- the HrpZpst polymimetic epitope ligand protein specifically includes the following steps:
- E.coli engineering bacteria fermentation preparation of HrpZpst multi-epitope ligand protein
- 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 HrpZpst poly-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 hrpZpst gene encoding the HrpZpst 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 synthetic DNA fragments encoding the HrpZpst 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 HrpZpst protein from 1) to 3) and transfer it into Escherichia coli engineering bacteria (E.coli), and the production line (E.coli) of the relevant 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 engineering bacteria five to eight times, import it into a high-pressure crusher, and continuously use 800-1000Mpa pressure to crush the engineering bacteria, pass the crushed bacteria liquid into a butterfly continuous flow centrifuge, remove the cell wall, and collect HrpZpst polymimetic epitope ligands The protein molecule, HrpZpst multi-epitope ligand protein, was present in the supernatant;
- the 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 manufacturer of the NI-NTA agarose column to complete the purification and preparation of the HrpZpst 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 36.52kda band
- lane 2 is the purified HrpZpst protein band
- the molecular weight is 36.52kda, which is in the corresponding molecular weight region of the protein, indicating that the corresponding purified HrpZpst polymimetic epitope ligand protein has been obtained.
- the allergy test detection of the purified polymimetic epitope ligand protein HrpZpst polymimetic epitope ligand protein preparation and the reaction results of tobacco leaves after 24hr of sterile water treatment are shown in Figure 2, wherein, A, C The point is the injection of 300 ⁇ g ⁇ mL -1 of HrpZpst polymimetic 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.
- mice 8-week-old balb/C mice were selected for the experiment and divided into HrpZpst multi-epitope ligand protein treatment groups, including oral administration for 6 hours, 24 hours, and application for 6 hours and 12 hours, a total of 4 treatments. There were 3 experimental mice for each treatment, a total of 12 mice; the blank control group had 4 experimental mice; the buffer without HrpZpst 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 and smeared with HrpZpst polymimetic ligand protein buffer at a concentration of 600 mg ⁇ L-1
- 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 mice were grouped into liver, thalamus, heart, cerebral cortex, cerebral hippocampus and other tissues for RNA-Seq sequencing and analysis.
- RNA-Seq mRNA sequencing experimental flow chart
- RNA extraction of the samples was performed using the miRNeasy Micro Kit (Cat#1071023 Qiagen) and according to the standard operating procedure provided by the manufacturer.
- Total RNA was quality-checked by NanoDrop ND-2000 spectrophotometer and Agilent Bioanalyzer 4200 (Agilent technologies, Santa Clara, CA, US), and RNA that passed the quality check 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 HrpZpst multi-epitope ligand 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 3-7 show the differential gene volcano map of HrpZpst multi-epitope ligand protein induced by oral administration and smearing in the liver, thalamus, heart, cerebral cortex and cerebral hippocampus of mice, respectively. HrpZpst is abbreviated as Z1 in the figure.
- FIGS. 8-11 are cluster heatmaps of differentially expressed gene sets in liver, thalamus, cerebral cortex, and hippocampus, respectively. HrpZpst is abbreviated as Z1 in the figure.
- 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.
- HrpZpst multi-epitope ligand protein we carried out GO term enrichment analysis on the differentially expressed genes induced by HrpZpst multi-epitope ligand protein, and the results proved and confirmed that HrpZpst 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 differential genes induced by HrpZpst protein are further expressed as follows: 1 biological process (biological_process) related differentially expressed genes include reproduction, cell death, immune system process, behavior, metabolic process, cellular process, reproductive process, biological adhesion attachment, signaling, multicellular biological processes, developmental processes, growth, movement, processes in a single tissue, biological phases, rhythmic processes, positive regulation of biological processes, negative regulation of biological processes, regulation of biological processes, stimulus response, localization, biology Regulation, organization or biogenesis of cellular components, cell 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 sensors, molecular function regulation, etc.
- the molecular function GO enrichment analysis results are shown in Table 1 to Table 6.
- HrpZpst in Tables 1-6 is abbreviated as HrpZ1.
- HrpZ1 HrpZ1
- 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 HrpZpst multi-epitope ligand protein induces the biological process, cellular components and molecular function-related functional groups of the liver and the heart to significantly up-regulate the expression of GO terms. 12 hours)
- HrpZpst 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
- HrpZpst multi-epitope ligand protein induces the biological process, cellular components and molecular function-related functional groups of the thalamus and hippocampus to significantly down-regulate the expression of GO terms. 12 hours
- 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 HrpZpst protein to obtain the roles (upstream and downstream relationship) and biological functions of these differentially expressed 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.
- HrpZpst 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.
- HrpZpst multi-epitope ligand protein are involved in biological processes such as translation, replication and repair, folding, classification and degradation (see Figure 12 to Figure 26 for details) .
- 4Metabolism Multiple differentially expressed genes induced by HrpZpst polyepitopic ligand protein 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, energy metabolism, carbohydrate metabolism and amino acid metabolism and other metabolic processes (see Figure 12 to Figure 26 for details).
- the right side of the figure shows the cellular process, information process, genetic information process, metabolic process, and tissue phylogenetic process; abscissa: the number of genes in each functional gene group involved in expression differences; ordinate: cellular processes involved in expression differences, Functional gene groups for information processes, genetic information processes, metabolic processes, and tissue phylogeny.
- the HrpZpst 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 HrpZpst 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.
- HrpZpst multi-epitope ligand protein recognizes bound cell membrane receptors: recognizes and binds to 5 membrane receptors, including HLA-C major histocompatibility complex, class I, C receptors, ASGPR1 asialoglycoprotein Receptor 1, adipocyte plasma membrane-associated protein receptor (APMAP), insulin-like growth factor 2 receptor (IGF2R), LDL receptor-associated protein 1 (LRPAP1).
- HLA-C major histocompatibility complex class I, C receptors, ASGPR1 asialoglycoprotein Receptor 1, adipocyte plasma membrane-associated protein receptor (APMAP), insulin-like growth factor 2 receptor (IGF2R), LDL receptor-associated protein 1 (LRPAP1).
- HrpZpst multi-epitope ligand protein recognizes bound cell membrane proteins: recognizes and binds to 10 membrane proteins, including solute carrier family 38, member 2 (SLC38A2), DNM2 dynein 2, SPTAN1 non-erythrocyte 1 ⁇ spectrin, ⁇ - Mutual protein 1 (SNTB1), CAP1 adenylate cyclase-related protein 1, ICAM1 intercellular adhesion molecule 1, LanC-like protein 1 (lanthionine synthase C) (LANCL1), MLEC stress protein-androgen Hormone-like receptor kinase, TJP2 claudin 2, ZYX patchin.
- HrpZpst multi-epitope ligand protein recognition and binding of membrane proteins involved in signaling pathways recognition and binding of 4, including hsa04933 age-anger signaling pathway in diabetes complications, hsa04064 NF-kappa B signaling pathway, hsa04072 phospholipid Enzyme D signaling pathway, hsa04668TNF signaling pathway.
- HrpZpst multi-epitope ligand protein recognizes and binds membrane proteins and participates in antiviral, antibacterial, anti-foreign body, and anti-inflammatory related metabolic pathways: recognizes and binds 27, hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysis Enzyme, hsa04666Fc gamma r-mediated phagocytosis, hsa04210 apoptosis, hsa04218 cellular senescence, hsa04612 antigen processing and presentation, hsa05100 bacterial invasion of epithelial cells, hsa05168 herpes simplex virus 1 infection, hsa05203 virus carcinogenesis, hsa05164 influenza A , hsa05150 Staphylococcus aureus infection, hsa05143 African trypanosomiasis, hsa04650 Natural killer cell-mediated mediated
- HrpZpst multi-epitope ligand protein recognizes and binds membrane proteins involved in important neurological disease metabolic pathways: not detected.
- HrpZpst multi-epitope ligand protein recognizes and binds membrane proteins involved in nucleic acid, protein, amino acid, sugar, fat metabolism-related pathways: recognizes and binds one, including hsa04974 protein digestion and absorption.
- HrpZpst multi-epitope ligand protein recognizes and binds membrane proteins involved in cell junction, neural junction, blood vessel, endocrine, reproductive system and other metabolic pathways: recognizes and binds 12, hsa04510 focal adhesions, hsa04724 glutamatergic synapses , hsa04530 tight junctions, hsa04940 type I diabetes, hsa04721 synaptic vesicle cycle, hsa04961 endocrine and other factors regulate calcium reabsorption, hsa04979 cholesterol metabolism, hsa04918 thyroid hormone synthesis, hsa04514 cell adhesion molecules (CAMs), hsa04727 neuronal synapses, hsa05320 Autoimmune thyroid disease, hsa05418 Fluid shear stress and atherosclerosis.
- CAMs cell adhesion molecules
- HrpZpst multi-epitope 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.
- HrpZpst 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 HrpZpss polymimetic epitope ligand protein is fermented, highly expressed, purified and prepared by engineering bacteria with registered genes, which specifically includes the following steps:
- HrpZpss polymimetic epitope ligand protein The engineering bacteria fermentation preparation of HrpZpss polymimetic epitope ligand protein: the genes (including, but not limited to, natural genes of biological samples, chemically synthesized genes, transgenic genetic recombinant genes) encoding HrpZpss polymimetic epitope ligand proteins are prepared.
- 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.
- HrpZpss polymimetic epitope ligand protein molecule use NI-NTA agarose column to purify HrpZpss polymimetic epitope ligand protein-His recombinant protein. The method is implemented to complete the preparation of purified HrpZpss polymimetic epitope ligand protein.
- the HrpZpss polymimetic epitope ligand protein is prepared by expressing the "artificially synthesized gene", which specifically includes the following steps:
- the first step artificial synthesis of the hrpZpss gene encoding the HrpZpss 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 synthetic DNA fragment encoding HrpNEcb protein gene is cloned into the BamHI-HindIII site of the high-efficiency protein expression vector PET28a(+) (containing His-Tag label), and the accuracy of the clone is 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. Clean the engineering bacteria five to eight times, and then introduce it into a high-pressure crusher. Under the continuous pressure of 800-1000Mpa, crush the engineering bacteria, and pass the broken bacteria liquid into the butterfly continuous flow centrifuge to remove the cell wall;
- Step 6 Purify the HrpZpss 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 HrpZpss polymimetic epitopes. Preparation of ligand proteins.
- the highly expressed purified protein-His recombinant band was detected by 10% SDS polyacrylamide gel electrophoresis, see Figure 29 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 34.73kd band
- lane 2 is the purified HrpZpss polymimetic epitope ligand
- the body protein band, with a molecular weight of 34.73kd, is in the corresponding molecular weight region of the protein, indicating that the corresponding HrpZpss purified protein has been obtained.
- HrpZpss multi-epitope ligand protein preparation As shown in Figure 30, the allergy test of HrpZpss multi-epitope ligand protein: HrpZpss multi-epitope ligand protein preparation and the reaction results of tobacco leaves after 24hr of sterile water treatment are shown in Figure 31, among which, points A and C are shown in Figure 31. 100 ⁇ L of HrpZpss polymimetic epitope ligand protein solution of 300 ⁇ g ⁇ mL -1 was injected; B and D points were injected with 100 ⁇ L of sterile water as the control treatment. 300 ⁇ g ⁇ mL -1 of HrpZpss 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 HrpZpss multi-epitope ligand protein can generally induce hypersensitivity reactions in 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.
- Experimental sample treatment 8-week-old balb/C mice were selected for the experiment and divided into HrpZpss multi-epitope ligand protein treatment groups, including oral administration for 6 hours, 24 hours, and application for 6 hours and 12 hours, a total of 4 treatments.
- mice for each treatment 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 HrpZpss 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, a total of 16 experimental mice, repeated three times; the mice in the experimental treatment group were fed and smeared with a buffer containing 600 mg ⁇ L-1HrpZpss polymimetic ligand protein. 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 kidneys, testis and other tissues were grouped and collected for RNA-Seq sequencing and analysis.
- RNA-Seq mRNA sequencing experimental flow chart
- RNA extraction of the samples was performed using the miRNeasy Micro Kit (Cat#1071023 Qiagen) 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 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 HrpNEcb 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 31-32 are the differential gene volcano plots of mouse kidney and testis HrpZpss protein induced by oral administration and smearing, respectively, in which HrpZpss is abbreviated as Z2.
- FIGS. 33-34 are cluster heat maps of differentially expressed gene sets in kidney and testis, respectively, in which HrpZpss is abbreviated as Z2.
- 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 (kidney, testis) 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.
- 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 GO enrichment analysis results of biological processes are shown in Tables 8 to 9.
- 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 Tables 8 to 9.
- 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 Tables 8 to 9.
- HrpZpss in Tables 8-9 is abbreviated as HrpZ2.
- HrpZ2 HrpZ2
- 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.
- HrpZpss multi-epitope ligand protein induces the biological processes, cellular components and molecular function-related functional groups of testis and kidneys to significantly up-regulate and express GO terms.
- Statistical table of the number of classified genes (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 and kidney induced by HrpZpss multi-epitope ligand protein were significantly down-regulated.
- 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.
- KEGG pathway enrichment analysis was performed on the differentially expressed genes induced by HrpZpss multi-epitope ligand protein, and the roles (upstream and downstream relationship) and biological functions of these differentially expressed genes in the signaling pathway were obtained, and the relationship between genes and functions was deeply understood. .
- HrpZpss 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 (kidney, testis) of mice. etc.) 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.
- HrpZpss multi-epitope ligand protein are involved in biological processes such as translation, replication and repair, folding, classification and degradation (see Figure 35 to Figure 40 for details) .
- 4Metabolism Multiple differentially expressed genes induced by HrpZpss multi-epitope ligand protein 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 Figure 35 to Figure 40 for details).
- the HrpZpss 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 HrpZpss polymimetic epitope ligand protein prepared 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.
- HrpZpss multi-epitope ligand protein recognizes and binds to cell membrane receptors: HLA-C major histocompatibility complex, I, C class receptors, free fatty acid receptor 4, tyrosine protein kinase transmembrane receptors 1.
- APMAP adipocyte plasma membrane-associated protein receptor
- IGF2R insulin-like growth factor 2 receptor
- HrpZpss multi-epitope ligand proteins recognize and bind cell membrane proteins: solute carrier family 5 (sodium-dependent vitamin transporter), member 6, solute carrier family 26, member 4, solute carrier family 38, member 2 (SLC38A2), DNM2 dynein 2, CAP1 adenylate cyclase-related protein 1, ICAM1 intercellular adhesion molecule 1 LanC-like protein 1 (lanthionine synthase C) (LANCL1), MLEC stress protein-androgen-like receptor Kinase, TJP2 claudin 2, ZYX patchin.
- HrpZpss multi-epitope ligand protein recognition and binding signaling pathways hsa04933 age-anger signaling pathway in diabetes complications, hsa04064 NF-kappa B signaling pathway, hsa04072 phospholipase D signaling pathway.
- HrpZpss multi-epitope ligand protein recognizes and binds membrane proteins and participates in antiviral, antibacterial, anti-foreign body, and anti-inflammatory metabolic pathways: hsa04144 endocytosis, hsa04145 phagosome, hsa04142 lysosome, hsa04666Fc gamma r-mediated phagocytosis, hsa04210 cell apoptosis, hsa04218 cell senescence, hsa05130: pathogenic E.
- coli infection hsa04612 antigen processing and presentation, hsa05100 bacterial invasion of epithelial cells, hsa05168 herpes simplex virus 1 infection, hsa05203 viral carcinogenesis, hsa05164 Influenza A, hsa05150 Staphylococcus aureus infection, hsa05167 Kaposi's sarcoma with herpes virus infection, hsa04916: bactericidal effect, hsa04650 Natural killer cell-mediated cytotoxicity, hsa05169 Epstein-Barr virus infection, hsa05416 viral myocarditis, hsa05110 Vibrio cholerae infection, hsa05144 malaria, hsa05163 human cytomegalovirus infection, hsa05170 human immunodeficiency virus type 1 infection, hsa05323 rheumatoid arthritis, hsa04670 leukocyte transcellular migration.
- HrpZpss multi-epitope ligand protein recognizes important neurological disease metabolic pathways involved in binding membrane proteins: not detected.
- HrpZpss multi-epitope ligand proteins recognize nucleic acid, protein, amino acid, sugar, and fat metabolism-related pathways involved in binding membrane proteins: hsa03050: proteasome, hsa04974 protein digestion and absorption, hsa04120: ubiquitin-mediated proteolysis.
- HrpZpss multi-epitope ligand protein recognizes the metabolic pathways involved in cell junctions, neural junctions, blood vessels, endocrine, and reproductive systems that bind to membrane proteins: hsa04510 focal adhesions, hsa04724 glutamatergic synapses, hsa04530 tight junctions, hsa04261 : Adrenergic signaling of cardiomyocytes, hsa04940 type I diabetes, hsa04924: renin secretion, hsa04721 synaptic vesicle cycle, hsa04961 endocrine and other factors regulate calcium reabsorption, hsa04970: salivary secretion, hsa04979 cholesterol metabolism, hsa04918 thyroid hormone synthesis , hsa04514 cell adhesion molecules (CAMs), hsa04727 neuronal synapses, hsa05320 autoimmune thyroid disease, h
- HrpZpss 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.
- HrpZpss multi-epitope ligand protein is a special multi-epitope ligand protein with new functions, new mechanism of action and new application prospects.
- the application of the HrpZ type multi-epitope ligand protein of the present invention in food, cosmetics, health care products and medicines also involves identifying and activating multiple types of receptors, membrane proteins and their signaling pathways in animals, and inducing multifunctional cascade biology
- the application of HrpZpst, HrpZpss and other multi-mimetic ligand protein products in food, cosmetics and health care products; further, the pull-down experiments of HrpZpst, HrpZpss and other multi-mimetic epitope ligand proteins that recognize and bind to specific proteins 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, endocrine, multi-type
- the expression and regulatory control of molecular metabolic functions and programmed cell death functions are widely involved in the regeneration, repair and clearance of different systems, organs
- HrpZpst, HrpZpss and other multi-epitope ligands HrpZ-type multi-epitope ligand protein products can be widely used in food, cosmetics, and health care products.
- the auxiliary conditioning functions of HrpZ-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. Anti-acne function; 21. Remove melasma function; 22. Improve skin moisture function; 23. Improve skin oil 24. Regulate intestinal flora function; 25. Promote digestive function; 26. Laxative function; 27. Have auxiliary protective function for gastric mucosal damage. further:
- the preparations of the HrpZ-type multi-epitope ligand protein of the present invention can be used in food supplements for 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 HrpZ-type multi-epitope ligand protein of the present invention can be used in cosmetics that assist in regulating the expression and regulation of the body's growth, development, defense, metabolism, and programmed cell death functions, including rubbing, Spraying or other similar methods, spread on any part of the human body surface, including skin, hair, nails, lips, etc., to achieve cleaning, eliminating bad odor, skin care, beauty and modification purposes Biotechnology products, or including ordinary cosmetics and special purposes cosmetic;
- the HrpZ-type polyepitope ligand protein product of the present invention can be used in health care products that assist in regulating the growth, development, defense, metabolism, and the expression and regulation of programmed cell death functions of the body, 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
Description
NO. | 样本名称 | 实验编号 | 浓度(μg/μL) | 体积(μL) | 总量(μg) |
1 | HEPG2 | HEPG2 | 8.34 | 2500 | 20861.30 |
NO. | 样本名称 | 实验编号 | 浓度(μg/μL) | 体积(μL) | 总量(μg) |
1 | HEPG2 | HEPG2 | 8.34 | 2500 | 20861.30 |
Claims (26)
- HrpZ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。
- 根据权利要求1所述的HrpZ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:HrpZ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: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;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和酸性带正电、碱性带负电氨基酸残基在HrpZ型多拟表位配体蛋白分子的全序列中占比72.97%-70.65%,在保守结构域中占比72.97%-71.82%,在α-螺旋结构中占比85.98%-81.48%;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,放大表达系列生物学效应。
- 根据权利要求1所述的HrpZ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:HrpZ型多拟表位配体蛋白包括HrpZPsa、HrpZPsm、HrpZPss、HrpZPst,HrpZPsap、HrpZPsr、HrpZPsth、HrpZPave、HrpZPam、HrpZPcar、HrpZPcor、HrpZPcst、HrpZPcat、HrpZPcory、 HrpZPcp、HrpZPsav、HrpZPsavp、HrpZPvir、HrpZPspe、HrpZPam、HrpZPade、HrpZPsac、HrpZPsg、HrpZPsc。
- 根据权利要求3所述的HrpZ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:HrpZpss多拟表位配体蛋白的氨基酸序列如SEQ ID NO:1所示。
- 根据权利要求3所述的HrpZ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:HrpZpst多拟表位配体蛋白的氨基酸序列如SEQ ID NO:2所示。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpss多拟表位配体蛋白,识别结合的多类受体包括HLA-C主要组织相容性复合体、I,C类受体、游离脂肪酸受体4、酪氨酸蛋白激酶跨膜受体1、ASGPR1去唾液酸糖蛋白受体1、脂肪细胞质膜相关蛋白受体、胰岛素样生长因子2受体中的一种或多种。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpst多拟表位配体蛋白,识别结合的多类受体包括HLA-C主要组织相容性复合体,I,C类受体、ASGPR1去唾液酸糖蛋白受体1、脂肪细胞质膜相关蛋白受体、胰岛素样生长因子2受体、LDL受体相关蛋白1中的一种或多种。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpss多拟表位配体蛋白,识别结合的膜蛋白包括溶质载体家族5、成员6、溶质载体家族26,成员4、溶质载体家族38,成员2、DNM2 动力蛋白2、CAP1腺苷酸环化酶相关蛋白1、ICAM1细胞间粘附分子1LanC-类蛋白1、MLEC应激蛋白-雄激素样受体激酶、TJP2紧密连接蛋白2、ZYX斑联蛋白中的一种或多种。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpst多拟表位配体蛋白,识别结合的膜蛋白包括溶质载体家族38,成员2、DNM2动力蛋白2、SPTAN1非红细胞1α血影蛋白、β-互生蛋白1、CAP1腺苷酸环化酶相关蛋白1、ICAM1细胞间粘附分子1、LanC-类蛋白1、MLEC应激蛋白-雄激素样受体激酶、TJP2紧密连接蛋白2、ZYX斑联蛋白中的一种或多种。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpss多拟表位配体蛋白,识别结合的信号通路包括hsa04933年龄-愤怒信号通路在糖尿病并发症中的作用、hsa04064 NF-kappa B信号通路、hsa04072磷脂酶D信号通路中的一种或多种。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpst多拟表位配体蛋白,识别结合的信号通路包括hsa04933年龄-愤怒信号通路在糖尿病并发症中的作用、hsa04064 NF-kappa B信号通路、hsa04072磷脂酶D信号通路、hsa04668 TNF信号通路中的一种或多种。
- 根据权利要求1或3所述的HrpZ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。
- 根据权利要求12所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpss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、hsa04666 Fc伽玛r介导的吞噬作用、hsa04210细胞凋亡、hsa04218细胞衰老、hsa05130:致病性大肠杆菌感染、hsa04612抗原处理及呈递、hsa05100细菌入侵上皮细胞、hsa05168单纯疱疹病毒1感染、hsa05203病毒致癌作用、hsa05164甲型流感、hsa05150金黄色葡萄球菌感染、hsa05167卡波济肉瘤伴疱疹病毒感染、hsa04916:杀菌作用、hsa04650自然杀伤细胞介导的细胞毒性、hsa05169爱泼斯坦巴尔病毒感染、hsa05416病毒性心肌炎、hsa05110霍乱弧菌感染、hsa05144疟疾、hsa05163人巨细胞病毒感染、hsa05170人类免疫缺陷病毒1型感染、hsa05323类风湿性关节炎、hsa04670白细胞跨细胞迁移;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:hsa03050:蛋白酶体、hsa04974蛋白消化吸收、hsa04120:泛素介导的蛋白水解作用;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、hsa04724谷氨酸能的突触、hsa04530紧密连接、hsa04261:心肌细胞的肾上腺素能信号、hsa04940 I型糖尿病、hsa04924:肾素分泌、hsa04721突触囊泡周期、hsa04961内分泌等因素调节钙的再吸收、hsa04970:唾液分泌、hsa04979胆固醇代谢、hsa04918甲状腺激素合成、hsa04514细胞粘附分子(CAMs)、hsa04727神经元突触、hsa05320自身免疫性甲状腺疾病、hsa05418流体剪切应力与动脉粥样硬化。
- 根据权利要求12所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpst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、 hsa04666 Fc伽玛r介导的吞噬作用、hsa04210细胞凋亡、hsa04218细胞衰老、hsa04612抗原处理及呈递、hsa05100细菌入侵上皮细胞、hsa05168单纯疱疹病毒1感染、hsa05203病毒致癌作用、hsa05164甲型流感、hsa05150金黄色葡萄球菌感染、hsa05143非洲锥虫病、hsa04650自然杀伤细胞介导的细胞毒性、hsa05166人T细胞白血病病毒1感染、hsa05167卡波济肉瘤伴疱疹病毒感染、hsa05169爱泼斯坦巴尔病毒感染、hsa05416病毒性心肌炎、hsa05110霍乱弧菌感染、hsa05144疟疾、hsa05163人巨细胞病毒感染、hsa05165人乳头瘤病毒感染、hsa05170人类免疫缺陷病毒1型感染、hsa05323类风湿性关节炎、hsa05330同种异体移植物排斥反应、hsa05332抗移植物、hsa04670白细胞跨细胞迁移;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:hsa04974蛋白消化吸收;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、hsa04724谷氨酸能的突触、hsa04530紧密连接、hsa04940 I型糖尿病、hsa04721突触囊泡周期、hsa04961内分泌等因素调节钙的再吸收、hsa04979胆固醇代谢、hsa04918甲状腺激素合成、hsa04514细胞粘附分子、hsa04727神经元突触、hsa05320自身免疫性甲状腺疾病、hsa05418流体剪切应力与动脉粥样硬化。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpss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HrpZpss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HrpZpss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HrpZpss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HrpZpss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HrpZpss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。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpst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HrpZpst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HrpZpst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HrpZpst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HrpZpst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HrpZpst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。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpss多拟表位配体蛋白,级联生物学效应还包括HrpZpss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。
- 根据权利要求1或3所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpst多拟表位配体蛋白,级联生物学效应还包括HrpZpst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。
- 根据权利要求1或3所述的HrpZ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。
- 根据权利要求19所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpss多拟表位配体蛋白,制品或药物主要由纯化的HrpZpss多拟表位配体蛋白制备得到,质量含量为有0.001%-100%。
- 根据权利要求19所述的HrpZ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:HrpZ型多拟表位配体蛋白为HrpZpst多拟表位配体蛋白,制品或药物主要由纯化的HrpZpst多拟表位配体蛋白制备得到,质量含量为0.001%-100%。
- 根据权利要求1-21任一项所述的HrpZ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:HrpZ型多拟表位配体蛋白为纯化后的HrpZ型多拟表位配体蛋白。
- 根据权利要求22所述的HrpZ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:HrpZ型多拟表位配体蛋白纯化的方法,包括以下步骤: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。步骤2:用Ni-NTA琼脂糖凝胶柱纯化HrpZ多拟表位配体蛋白-His重组蛋白,得到纯化的HrpZ型多拟表位配体蛋白原药。
- HrpZ型蛋白纯化的方法,其特征在于,包括以下步骤: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。步骤2:用Ni-NTA琼脂糖凝胶柱纯化HrpZ多拟表位配体蛋白-His重组蛋白,得到纯化的HrpZ型多拟表位配体蛋白原药。
- 根据权利要求24所述的HrpZ型多拟表位配体蛋白的纯化的方法,其特征在于,HrpZ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: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;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和酸性带正电、碱性带负电氨基酸残基在HrpZ型多拟表位配体蛋白分子的全序列中占比72.97%-70.65%,在保守结构域中占比72.97%-71.82%,在α-螺旋结构中占比85.98%-81.48%;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,放大表达系列生物学效应。
- 根据权利要求24所述的HrpZ型多拟表位配体蛋白的纯化的方法,其特征在于,HrpZ型多拟表位配体蛋白包括HrpZPsa、HrpZPsm、HrpZPss、HrpZPst,HrpZPsap、HrpZPsr、HrpZPsth、HrpZPave、HrpZPam、HrpZPcar、HrpZPcor、HrpZPcst、HrpZPcat、HrpZPcory、HrpZPcp、HrpZPsav、HrpZPsavp、HrpZPvir、HrpZPspe、HrpZPam、HrpZPade、HrpZPsac、HrpZPsg、HrpZPsc。
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