WO2023185442A1 - 葡萄糖调控基因表达环路控制系统及其调控血糖的应用 - Google Patents

葡萄糖调控基因表达环路控制系统及其调控血糖的应用 Download PDF

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WO2023185442A1
WO2023185442A1 PCT/CN2023/081232 CN2023081232W WO2023185442A1 WO 2023185442 A1 WO2023185442 A1 WO 2023185442A1 CN 2023081232 W CN2023081232 W CN 2023081232W WO 2023185442 A1 WO2023185442 A1 WO 2023185442A1
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glucose
gene expression
control system
expression
loop control
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French (fr)
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叶海峰
管宁子
高纤云
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华东师范大学
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Definitions

  • the invention relates to the fields of synthetic biology and cell therapy, which combines microbial synthetic biology with disease treatment. Specifically, it relates to a glucose-regulated gene expression loop control system and its application in regulating blood sugar.
  • Biosensors can directly use intact microbial cells or cell fragments as sensitive materials, and use the enzymes and metabolic systems in their bodies to identify and detect various substances. In recent years, the development of synthetic biology has greatly promoted the development of biosensor functions. In biomedicine, they can well detect various physiological indicators or disease signals, including stimuli and external signals in their host environment. Probiotics can specifically target the intestine and serve as intestinal-directed drug delivery carriers, meeting almost all requirements as biotherapeutics. Using probiotics to develop biosensors to achieve integrated diagnosis and treatment is an important means to achieve precision medicine.
  • impaired wound healing process is a growing medical problem closely related to metabolic diseases and aging. Open wounds in the skin can cause severe discomfort and provide an entry point for bacteria to invade. During the inflammatory phase of wound healing, immune cells accumulate under the action of alarm signals, cytokines, and chemokines released by injured or activated cells, thereby playing a crucial role in wound healing. Macrophages and neutrophils are the main immune cell groups at trauma sites. While they prevent microbial invasion, they can also promote the healing process by secreting additional chemokines, growth factors, and matrix digestive enzymes.
  • Chronic wounds are often associated with underlying pathological processes that increase wound susceptibility or reduce healing capacity, such as arterial/venous insufficiency, diabetes, or ongoing systemic steroid therapy. Take diabetic foot ulcers as an example. As one of the common complications of diabetes, it can lead to long-term pain, decreased energy, and may even require amputation. Standard treatments for chronic wounds such as diabetic foot ulcers include surgical or chemical removal of dead tissue, Repeated dressing changes and the use of antibiotics for infection control yield inconsistent results and are associated with adverse side effects. In addition, different types of chronic wounds are also treated clinically through local application of growth factors or in combination with different biological materials, but the effect is not significant.
  • the invention proposes a glucose-regulated gene expression loop control system, which includes a transcription repressor HexR, a glucose-inducible strong promoter and a sequence to be transcribed.
  • the transcription repressor HexR can homodimerize and combine with a specific DNA sequence, and the specific DNA sequence is selected from the sequence SEQ ID NO. 78-80.
  • the glucose-inducible strong promoter is a combination of a constitutive strong promoter and the specific DNA sequence; which includes but is not limited to any nucleotide sequence such as SEQ ID NO. 1-10.
  • the transcription repressor can bind to the strong glucose-inducible promoter and thereby inhibit the expression of downstream sequences to be transcribed.
  • the sequence to be transcribed includes a single coding expression reporter protein or a functional protein, or the tandem expression of multiple proteins.
  • the transcription repressor, strong glucose-inducible promoter and sequence to be transcribed can be constructed on one or two plasmid vectors.
  • the invention also proposes a glucose-induced gene expression regulation method, which is regulated by the glucose-regulated gene expression loop control system.
  • the transcription repressor when glucose is absent, the transcription repressor can bind to the glucose-inducible strong promoter, thereby repressing the expression of downstream sequences to be transcribed; when glucose is present, glucose passes through the Entner-Doudoroff pathway Metabolism generates the metabolic intermediate 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDPG blocks its binding, dissociating the transcription repressor from the strong glucose-inducible promoter and initiating downstream transcription. Sequence expression.
  • the regulation method of inducing gene expression is shown in Figure 1.
  • the glucose-regulated gene expression loop control system starts expression and secretion of genes precisely regulated by glucose, and consists of three parts: the transcription repressor HexR, a glucose-inducible strong promoter, and a sequence to be transcribed.
  • the transcription repressor HexR is the repressor protein HexR derived from the HexR operon system of Pseudomonas putida (its amino acid sequence Genbank accession number: AE015451), which is continuously expressed by a strong promoter.
  • the transcription repressor HexR can be expressed by different types of strong promoters, including but not limited to PBBA23100 (its nucleotide sequence Genbank accession number: MG649435), P Lac (its nucleotide sequence Genebank accession number: LC652750), P Tac (its nucleotide sequence Genebank accession number: MN913428), etc.
  • PBBA23100 its nucleotide sequence Genbank accession number: MG649435
  • P Lac its nucleotide sequence Genebank accession number: LC652750
  • P Tac its nucleotide sequence Genebank accession number: MN913428
  • the glucose-inducible strong promoter is formed by inserting different copy numbers of HexR operon binding sites between the strong promoter and the RBS site; the HexR operon binding sites are derived from the manipulation of the HexR operon system Subcomponent HexO.
  • the strong glucose-inducible promoter can drive the expression of downstream genes.
  • the HexR operon system is composed of the repressor protein HexR and the operator element HexO, which was originally isolated from Pseudomonas putida and is affected by the glucose metabolite 2-keto-3-deoxy-6-phosphogluconate (KDPG) strict regulation.
  • the glucose-inducible strong promoter can be composed of different types of fusion strong promoters according to the type of the strong promoter and the different copy numbers of the operator HexO, including: a) as shown in SEQ ID NO.1 Glucose-inducible strong promoter P HexR1 nucleotide sequence P HexR1 (P Tac -HexO); b) Glucose-inducible strong promoter P HexR2 nucleotide sequence P HexR2 (P Tac -HexO) as shown in SEQ ID NO.2 2 ⁇ HexO); c) Glucose-inducible strong promoter P HexR3 nucleotide sequence P HexR3 (P Tac -3 ⁇ HexO) as shown in SEQ ID NO.3; d) As shown in SEQ ID NO.4 Glucose-inducible strong promoter P HexR4 nucleotide sequence P HexR4 (P Tac -4 ⁇ H
  • sequence to be transcribed can be a single-encoded expression reporter protein such as sfGFP (amino acid sequence Genbank accession number: AB971579), LuxCDABE (amino acid sequence Genbank accession number: EF173694) or a functional protein such as the pancreatic pancreatic protein shown in SEQ ID NO. 11 Glucagon-like peptide GLP-1 amino acid sequence, glucagon-like peptide EK-GLP-1 amino acid sequence shown in SEQ ID NO. 12, glucagon-like peptide GLP shown in SEQ ID NO.
  • mouse pancreatic islet duodenal homeobox-1 (mPDX-1) amino acid sequence as shown in SEQ ID NO.61, human as shown in SEQ ID NO.66
  • Source pancreatic islet duodenum homology box-1 (hPDX-1) amino acid sequence, etc.
  • the glucose-regulated gene expression loop control system can accurately regulate the expression of one or multiple reporter proteins and functional proteins in series in the form of polycistrons.
  • glucose-regulated gene expression loop control system is loaded by an artificially designed and synthesized dual plasmid system.
  • the sequences involved in the dual plasmid system are detailed in Table 1.
  • the present invention also proposes an expression vector, engineered cell, sensor or recombinant probiotic, which contains the glucose-regulated gene expression loop control system.
  • the glucose-regulated gene expression loop control system is located in any one of the chromosomes or plasmids of the engineered cells, sensors, or recombinant probiotics.
  • the present invention also proposes an application, that is, the glucose-regulated gene expression loop control system.
  • the expression vector, engineered cells, sensors or recombinant probiotics are used in the preparation of drugs for treating and/or preventing diabetes and the preparation of drugs for regulating blood sugar. or application in the product.
  • the drugs or products include, but are not limited to, various dosage forms such as oral agents, lubricants, and injections.
  • oral administration is used.
  • recombinant probiotics containing the glucose-regulated gene expression loop control system i.e., an intelligent and controllable microbial drug factory
  • glucose-induced gene expression regulation is used to express hypoglycemic peptides, thereby regulating blood sugar.
  • the method of inducing the production of glucagon-like peptides through blood sugar can accurately regulate GLP-1 or/and The expression of PDX-1 achieves the effect of treating diabetes.
  • blood sugar is regulated and blood sugar levels are controlled by regulating the release of glucagon-like peptide and islet-duodenal homeobox-1 hypoglycemic protein.
  • the present invention also proposes a method for regulating blood sugar, that is, using the glucose-regulated gene expression loop control system, and/or using the expression vector, engineered cells, sensors or recombinant probiotics to regulate blood sugar.
  • the method achieves the effect of regulating blood sugar by regulating the expression of GLP-1 and PDX-1 hypoglycemic proteins.
  • the method for regulating blood sugar includes the following steps:
  • step b) Transform the expression vector constructed in step a) or the functional module for chromosome integration into microbial cells or integrate into the chromosomes of microbial cells to prepare engineered cells containing a glucose-induced GLP-1/PDX-1 expression control system;
  • step c) transplant the engineered cells prepared in step b) into diabetic model mice by intragastric administration;
  • mice Glucose closed loop in mice induces the engineered cells to express and secrete GLP-1/PDX-1, which is absorbed into the blood to lower blood sugar.
  • the method for regulating blood sugar can be in the form of oral probiotics.
  • the present invention also provides a new method for treating diabetes.
  • oral probiotics can be used to reduce the risk of diabetes. Blood sugar effects.
  • the present invention provides a new strategy for treating diabetes that is safe, reliable, and can accurately regulate the release of glucagon-like peptide or/and islet-duodenal homeobox-1.
  • the present invention provides new methods and strategies for treating diabetes.
  • the system can regulate the expression of glucagon-like peptide GLP-1 and islet duodenal homeobox-1PDX-1.
  • the expression of the glucagon-like peptide GLP-1 includes EK-GLP-1, GLP-1-Fc, etc.
  • the expression of islet duodenal homeobox-1PDX-1 includes mPDX-1, hPDX-1, etc.
  • the glucose-regulated gene expression loop control system of the present invention can quickly regulate gene expression through glucose metabolites, and has the characteristics of accurately controlling the amount of gene expression and regulating high gene expression multiples.
  • the glucose-regulated gene expression loop control system of the present invention has the characteristics of fine-tuning and reversible expression dynamics.
  • the fine-tunability means that downstream gene expression is precisely regulated by glucose and shows a dose-dependent relationship; the reversibility means that the entire process of glucose regulating gene expression is reversible, and can be controlled by controlling the presence or absence of glucose. Turn gene expression on or off.
  • the expression kinetic characteristics of fine-tuning and reversibility are also reflected in the glucose-induced gene expression control method, the blood sugar control method, as well as the expression vector, engineered cells, sensors or recombinant probiotics, etc.
  • the invention also proposes a plasmid selected from the group consisting of plasmids pGN11, pGN69, pGN89, pGN90, pGN227, pGN12, pGN228, pGN229, pGN220, pGN221, pGN222, pGN223, pGN224, pGN231, pGN232, pGN233, pGN65, pGN237, pGN238, One or more of pGN239, pGN241, pGN242, pGN243, pGN288, pGN308, pGN306, pGN307, and pXG58.
  • the plasmids are detailed in Table 1.
  • the present invention also proposes a nucleotide sequence, which is one of glucose-inducible strong promoters P HexR1 , P HexR2 , P HexR3, P HexR4 , P HexR5 , P HexR6 , P HexR7 , P HexR8 , P HexR9 and P HexR10 .
  • a nucleotide sequence which is one of glucose-inducible strong promoters P HexR1 , P HexR2 , P HexR3, P HexR4 , P HexR5 , P HexR6 , P HexR7 , P HexR8 , P HexR9 and P HexR10 .
  • One kind whose nucleotide sequence is shown in one of SEQ ID NO. 1 to 10 respectively.
  • the present invention also provides a primer, the sequence of which is one or more of the ones shown in SEQ ID NO. 16-60, 62-65, 67-74, 76-77.
  • the present invention also proposes a primer pair, the sequence of which is shown as No. 1-30 One or more primer pairs. As shown in Table 1 and Table 2.
  • the present invention also proposes an intelligent and controllable microbial drug factory, which includes a chassis microorganism and a gene expression loop control system uploaded to the chassis microorganism.
  • the gene expression loop control system is located in any one of the chromosomes or plasmids of the chassis microorganism.
  • the intelligent and controllable microbial drug factory of the present invention is preferably a glucose-regulated probiotic drug factory, which is an engineered cell containing a glucose-regulated gene expression loop control system. It can also be a microbial drug factory with other control methods, that is, engineered cells containing gene expression loop control systems with other control methods.
  • the engineered cells can be any probiotics, such as Escherichia coli Nissle 1917 (EcN), etc.
  • the gene expression loop control system includes but is not limited to a glucose-regulated gene expression loop control system, and gene expression loop control systems with other regulatory methods, such as xylose-induced gene expression system, arabinose-induced gene expression system, uric acid-induced gene expression system, etc.
  • the glucose-regulated gene expression loop control system includes three parts: a transcription repressor HexR, a glucose-inducible strong promoter and a sequence to be transcribed.
  • the transcription repressor HexR can bind to the strong glucose-inducible promoter and thereby inhibit the expression of downstream sequences to be transcribed;
  • the strong glucose-inducible promoter is composed of a constitutive strong promoter It is composed of DNA sequences that specifically bind to HexR;
  • the sequence to be transcribed includes a single-coding expression reporter protein such as LuxCDABE (amino acid sequence Genbank accession number: EF173694) or a functional protein such as human acidic fibroblasts shown in SEQ ID NO.14 Growth factor rhaFGF135 amino acid sequence, chemokine CXCL12 amino acid sequence as shown in SEQ ID NO.
  • the control method is that when glucose is not present, the transcription repressor can be bound to the glucose-inducible strong promoter, thereby inhibiting the expression of the downstream sequence to be transcribed; when glucose is present, the glucose is passed through Entner -Doudoroff pathway metabolism generates the metabolic intermediate 2-keto-3-deoxy-6-phosphogluconate (KDPG). KDPG blocks its binding, causing the transcription repressor to dissociate from the glucose-inducible strong promoter and start Expression of downstream sequences to be transcribed.
  • KDPG 2-keto-3-deoxy-6-phosphogluconate
  • the gene expression loop control system includes a transcription repressor, an inducible strong promoter and a sequence to be transcribed.
  • the transcription repressor, the strong inducible promoter and the sequence to be transcribed can be constructed on one or two plasmid vectors.
  • the intelligently controllable microbial drug factory can express the target gene in the presence of an inducer.
  • the inducer is added exogenously or is produced in vivo.
  • the chassis microorganisms include probiotics.
  • the probiotics include but are not limited to Escherichia coli Nissle 1917, Lactococcus lactis, Lactobacillus plantarum, Bacillus subtilis, etc.
  • the intelligent and controllable microbial drug factory is recombinant probiotics.
  • the intelligent and controllable microbial drug factories of the present invention include, but are not limited to, those that can be in liquid, or freeze-dried or spray-dried.
  • the present invention also proposes a method for constructing the intelligent controllable microbial drug factory, which includes the following steps:
  • the method for constructing an intelligent and controllable microbial drug factory proposed by the present invention involves transforming the plasmid containing the functional modules and reporting modules constructed in steps b) and c) into probiotic competent cells, or transforming the step b ) and the functional module and reporter module constructed in step c) are integrated into the probiotic chromosome through the RED recombinase system or the CRISPR system to prepare inducible engineered cells, that is, the intelligent and controllable microbial drug factory is prepared.
  • the present invention also proposes expression vectors and engineered cells, which contain the intelligent and controllable microbial drug factory.
  • a prokaryotic expression vector containing a glucose-regulated gene expression loop control system is provided.
  • the present invention also proposes the application of the intelligent controllable microbial drug factory in preparing drugs or products for preventing and/or treating diseases.
  • the drugs or products include but are not limited to drugs that promote skin wound healing, drugs that prevent and/or treat metabolic diseases, drugs that prevent and/or treat other diseases, etc.
  • the skin wounds include, but are not limited to, chronic skin wounds, diabetic skin wounds, diabetic foot ulcers, and the like.
  • the present invention also proposes the application of intelligent and controllable microbial (preferably probiotic) drug factories in promoting diabetic skin wound healing.
  • the application to promote diabetic skin wound healing is through in situ dripping into the wound.
  • the method of applying an intelligent and controllable probiotic drug factory is realized by constructing an intelligent and controllable probiotic drug factory to induce the production of growth factors, cytokines and chemokines through blood sugar, that is, the treatment method It uses intelligent and controllable probiotic drug factories to precisely regulate the expression and secretion of growth factors, cytokines and chemokines to promote wound healing.
  • the present invention also proposes a method of utilizing the intelligently controllable microbial (probiotic) drug factory to promote skin wound healing.
  • the method includes the following steps:
  • step b) The engineered cells prepared in step b) are transplanted into the skin wound of the diabetic model mouse in the form of in-situ dripping onto the wound;
  • Glucose closed loop in mouse skin wounds induces engineered cells to express and secrete growth factors, macrophage M2 polarizing cytokines and chemokines to achieve the effect of promoting wound healing.
  • the growth factors are rhaFGF135, FGF2, FGF7, TGF- ⁇ , TGF- ⁇ , PDGF, EGF, VEGF, IGF-1, IGF-2, PDGF, HGF;
  • the macrophage M2 polarizing cytokine is IL4, IL-10, IL-13, CSFl, and IL34;
  • the chemokines are CXCL12, CXCL13, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL10.
  • the present invention also proposes a plasmid, which is one or more of plasmids pGN233, pGN65, pGN299, pGN300, and pGN314. See Table 1 for details.
  • the present invention also proposes a nucleotide sequence, which is one of glucose-inducible strong promoters P HexR4 and P HexR9 , and its sequences are shown in SEQ ID NO. 4 and SEQ ID NO. 9 respectively.
  • the beneficial effects of the present invention include: the present invention provides a new method for promoting the healing of chronic skin wounds.
  • the wound is In-situ application of probiotics can accelerate wound healing.
  • the present invention provides a new strategy for accelerating the healing of chronic skin wounds, diabetic foot ulcers and other chronic skin wounds that is safe, reliable, and can accurately regulate the release of therapeutic factors.
  • the system can regulate the expression and secretion of growth factors, macrophage M2 polarizing cytokines and chemokines.
  • the present invention innovatively develops an optimized technology for delivering growth factors, macrophage M2 polarizing cytokines and chemokines directly to injured skin.
  • Probiotics are used as carriers, and plasmids or chromosomal genomes are used to encode growth factors, macrophages.
  • the beneficial effects of the present invention include providing an intelligent and controllable probiotic drug factory, including a gene expression loop control system and chassis probiotics.
  • the glucose-regulated gene expression loop control system of the present invention has the characteristics of fine-tuning and reversible expression dynamics.
  • the present invention also provides the intelligent and controllable probiotic drug factory or the prokaryotic expression vector containing the glucose-regulated gene expression loop control system or the engineered cells containing the glucose-regulated gene expression loop control system in treating diabetes and promoting chronic skin wounds. Application in medicines for healing, promoting the healing of chronic skin wounds such as diabetic foot ulcers, and preventing and/or treating metabolic diseases.
  • Figure 1 is a schematic diagram of the principle of the glucose regulation gene expression loop control system and regulation method of the present invention.
  • Figure 2 is an optimization study of the glucose-regulated gene expression loop control system of the present invention, that is, the experimental results of using different promoters to promote the expression vector of HexR and combining them with the glucose-inducible strong promoter P HexR1 .
  • Figure 3 is an optimization study of the glucose-regulated gene expression loop control system of the present invention, that is, the experimental results of using P Tac to activate the expression vector of HexR and combining it with five different response elements.
  • Figure 4 is an optimization study of the glucose-regulated gene expression loop control system of the present invention, that is, the experimental results of combining the expression vector of RBS-regulated HexR with the glucose-inducible strong promoter P HexR4 respectively.
  • Figure 5 is an experimental result of the fine-tuned expression kinetic characteristics of the glucose-regulated gene expression loop control system of the present invention.
  • Figure 6 is an experimental result of the reversible expression kinetic characteristics of the glucose-regulated gene expression loop control system of the present invention.
  • Figure 7 is an experimental result of the effects of different glucose induction times on gene expression in the glucose-regulated gene expression loop control system of the present invention.
  • Figure 8 is the experimental results of the glucose-regulated gene expression loop control system of the present invention regulating the expression of the reporter gene luxCDABE in wild-type mice and type 1 diabetic mice.
  • Figure 9 is the experimental results of treating type 1 diabetic mice with the probiotic drug factory of the present invention that regulates the expression and secretion of glucagon-like peptide.
  • Figure 10 is the experimental results of long-term control of blood sugar in type 1 diabetic mice by the probiotic drug factory of the present invention that regulates the expression and secretion of glucagon-like peptide.
  • Figure 11 is the experimental results of glucose tolerance during the treatment of type 1 diabetes using the glucose-regulated probiotic drug factory of the present invention.
  • Figure 12 is an experimental result of the glucose-regulated gene expression loop control system of the present invention regulating the expression of the reporter gene luxCDABE at the skin wound of diabetic mice.
  • Figure 13 is the experimental result of the probiotic drug factory of the present invention inducing the expression and secretion of human acidic fibroblast growth factor rhaFGF135 under the regulation of glucose.
  • Figure 14 is an experimental result of the probiotic drug factory of the present invention inducing the expression and secretion of chemokine CXCL12 under glucose regulation.
  • Figure 15 is the experimental result of activating the probiotic drug factory to express and secrete human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12 in the wound of diabetic mice of the present invention.
  • Figure 16 is the experimental results of the glucose-regulated probiotic drug factory of the present invention promoting skin wound healing in diabetic mice.
  • Figure 17 shows the blood indicators of mice after the probiotic drug factory was applied in situ to the wound of the present invention.
  • Figure 18 shows the residues of recombinant probiotics in various organs and blood of mice after the probiotic drug factory was dropped in situ on the wound of the present invention.
  • All primers used for PCR were synthesized by Jinweizhi Biotechnology Co., Ltd.
  • the expression plasmids constructed in the examples of the present invention have all undergone sequence determination, and the sequence determination was completed by Jinweizhi Biotechnology Co., Ltd.
  • Phanta Max Super-Fidelity DNA polymerase used in the examples of the present invention was purchased from Nanjing Novezan Biotechnology Co., Ltd.
  • Endonuclease and T4DNA ligase were purchased from TaKaRa Company.
  • Homologous recombinase was purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.
  • PhantaMax Super-Fidelity DNA Polymerase comes with the corresponding polymerase buffer and dNTPs when purchased. Endonuclease, T4DNA ligase, Homologous recombinase comes with corresponding buffer when purchased. Yeast Extract, trypton, agar powder, M9 medium, glucose, ampicillin (Amp), and kanamycin (Kan) were purchased from Shanghai Sangon Bioengineering Technology Co., Ltd.
  • the DNA fragments were gel recovered and purified according to the operating instructions of the DNA gel recovery kit and PCR product purification kit (Kangwei Century Biotechnology Co., Ltd.); the plasmid extraction steps were carried out according to the plasmid extract (Tiangen Biochemical Technology (Beijing) ) Co., Ltd.) extraction kit instructions.
  • the following bacterial chassis cells and electroconversion are used as examples to illustrate the working conditions of the glucose-regulated gene expression loop control system in prokaryotic cells and animals, but this does not limit the scope of protection of the present invention.
  • Bacterial culture Escherichia coli EcN is cultured in LB medium, and 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin solution are added to the culture medium; bacteria are cultured in a shaker at 37°C and 210 rpm.
  • E. coli EcN competent cells All solutions and consumables used for preparation of competent cells have been sterilized by high temperature and high pressure. Streak the E. coli EcN strain on a plate without antibiotics, and incubate it upside down at 37°C for 12 to 16 hours; pick a single colony into a 2 mL LB shaking tube without antibiotics, and culture overnight at 37°C and 210 rpm with shaking. Absorb 1 mL of bacterial liquid and transfer it to 100 mL of fresh LB medium. Shake and culture at 37°C and 210 rpm until the OD 600 is between 0.4 and 0.6.
  • the conversion of EcN uses an optimized electroconversion method. Simply put, add the pre-cooled plasmid (300-500ng) and competent (100 ⁇ l) mixture to the bottom of the electroporation cup, set the electroporation parameters to 2mm, 2500V, and shock once. After the electroshock, add 900 ⁇ l of culture medium into the electroporation cup, mix well, aspirate and place in a sterilized EP tube, incubate at 37°C and 210 rpm for 1 hour, and spread the bacteria onto a resistant plate (100 ⁇ g/ml Amp and 50 ⁇ g/ml). ml of Kan), incubate at 37°C for 16-20h.
  • sfGFP reporter gene green fluorescent protein
  • Green fluorescent protein sfGFP has fluorescence visible to the naked eye under blue light, and its fluorescence intensity can be measured with a microplate reader Synergy H1. Take 100 ⁇ l of bacterial suspension into a black 96-well microplate, place it in a microplate reader, and detect the emission reading of the bacterial suspension at 520nm under the action of excitation light with a wavelength of 480nm, which is the green fluorescence intensity. At the same time, draw 100 ⁇ l of bacterial suspension into a transparent 96-well microplate, and use a microplate reader to detect the absorbance value of the bacterial culture at 600 nm, which is the bacterial cell density. Fluorescence intensity/bacterial cell density was used to characterize the expression efficiency of the reporter gene.
  • This embodiment includes the construction method of the plasmid vector involved in the glucose-regulated gene expression loop control system. See Appendix 1 for detailed design plans and steps.
  • Example 2 Optimization study of the glucose-regulated gene expression loop control system in EcN, that is, expression vectors using different promoters to promote the expression of HexR were optimized in combination with the glucose-inducible strong promoter P HexR1 .
  • the first step is plasmid construction.
  • the details of plasmid construction in this example are shown in Appendix Table 1.
  • the second step is transformation.
  • the transformation system in this example can be divided into 4 groups, including pGN11, pGN11 and pGN69, pGN11 and pGN89, and pGN11 and pGN90. Each set of plasmids above was electroporated into EcN electroporation competent cells.
  • the third step is to cultivate. Pick positive single clones and transfer them to liquid LB medium, and culture them at 37°C and 210 rpm for 12 hours.
  • the fourth step is to detect the expression of the reporter gene sfGFP.
  • Example 3 Optimization study of the glucose-regulated gene expression loop control system in EcN, that is, using P Tac to activate the expression vector of HexR for combination optimization with 5 different response elements.
  • the first step is plasmid construction.
  • the details of plasmid construction in this example are shown in Appendix Table 1.
  • the second step is transformation.
  • the transformation system in this example can be divided into 5 groups, including pGN89 and pGN11, pGN89 and pGN227, pGN89 and pGN12, pGN89 and pGN228, and pGN89 and pGN229. Each set of plasmids above was electroporated into EcN electroporation competent cells.
  • the third step is culturing (the specific steps are the same as in Example 2).
  • the fourth step is induction. Centrifuge at 5000 rpm for 5 minutes, pipet an equal amount of M9 culture medium evenly with a pipette tip, and distribute it into a 48-well plate according to a culture volume of 500 ⁇ L. Different experimental groups were induced by adding sterile glucose solution of corresponding concentrations and cultured with shaking at 37°C and 150 rpm for 12 h.
  • the fifth step is to detect the expression level of the reporter gene sfGFP (the specific steps are the same as Example 2 of the present invention).
  • Example 4 Optimization study of the glucose-regulated gene expression loop control system in EcN, that is, the expression vector using RBS to regulate HexR was optimized in combination with the glucose-inducible strong promoter P HexR4 .
  • the first step is plasmid construction.
  • the plasmid construction in this example is detailed in Appendix Table 1.
  • the second step is transformation.
  • the transformation system in this example can be divided into 4 groups, including pGN89 and pGN228, pGN231 and pGN228, pGN232 and pGN228, and pGN233 and pGN228.
  • Each set of plasmids above was electroporated into EcN electroporation competent cells.
  • the third step is culturing (the specific steps are the same as Example 2 of the present invention).
  • the fourth step is induction (the specific steps are the same as Example 3 of the present invention).
  • the fifth step is to detect the expression level of the reporter gene sfGFP (the specific steps are the same as Example 2 of the present invention).
  • Example 5 Study on the fine-tuned expression kinetic characteristics of the glucose-regulated gene expression loop control system.
  • Example 4 of the present invention In the first step, inoculate and culture the engineering bacteria with the best induction multiple obtained in Example 4 of the present invention, centrifuge at 5000 rpm for 5 minutes, use an equal amount of M9 culture medium to pipette evenly with a pipette tip, and distribute it into a 48-well plate according to a culture volume of 500 ⁇ L. .
  • Different experimental groups were induced by adding sterile glucose solutions with different final concentrations, and cultured with shaking at 37°C and 150 rpm for 12 h.
  • the expression level of the reporter gene sfGFP is detected (the specific steps are the same as Example 2 of the present invention).
  • Example 6 Study on the reversible expression kinetic characteristics of the glucose-regulated gene expression loop control system.
  • Example 4 of the present invention In the first step, inoculate and culture the engineering bacteria with the best induction multiple obtained in Example 4 of the present invention, centrifuge at 5000 rpm for 5 minutes, use an equal amount of M9 culture medium to pipette evenly with a pipette tip, and distribute it into a 48-well plate according to a culture volume of 500 ⁇ L. .
  • the "ON-OFF-ON" experimental group was induced by adding a sterile glucose solution with a final concentration of 20mM at 0h; at 2h, the original culture medium was washed away by centrifugation, and glucose-free M9 culture medium was added to resuspend the culture; At 7 h, the culture medium was washed away by centrifugation, and M9 medium containing a final concentration of 20 mM glucose was added to resuspend the culture.
  • Example 7 in the glucose-regulated gene expression loop control system, a study on the control of gene expression by different induction times of glucose.
  • Example 4 of the present invention In the first step, inoculate and culture the engineering bacteria with the best induction multiple obtained in Example 4 of the present invention, centrifuge at 5000 rpm for 5 minutes, use an equal amount of M9 culture medium to pipette evenly with a pipette tip, and distribute it into a 48-well plate according to a culture volume of 500 ⁇ L. .
  • Different experimental groups added sterile glucose solution with a final concentration of 20mM, and cultured with shaking at 37°C and 150rpm for different times (0h, 2h, 4h, 6h, 8h, 10h, 12h).
  • the expression level of the reporter gene sfGFP is detected (the specific steps are the same as Example 2 of the present invention).
  • Example 8 Using streptozotocin (STZ) modeling method to construct type 1 diabetes model mice.
  • the first step is to fast. Before administration, 40 male C57BL/6J mice weighing about 25g were selected and fasted for 16 hours.
  • the second step is to administer the drug.
  • STZ was dissolved in citric acid buffer (0.1 mol/L, pH 4.5), and then intraperitoneally injected into mice at a dosage of 40-50 mg/kg for 5 consecutive days. Since STZ is easily degraded, the entire process needs to ensure that the drug is kept at low temperature and protected from light, and the injection process needs to be fast.
  • the third step is to measure the blood sugar level. On the 9th day, the blood glucose level of the mice was measured after 4 hours of starvation. Mice with a blood glucose level higher than 16.7mM were considered as successful modeling.
  • Example 9 Study on the regulation of the expression of the reporter gene luxCDABE by the glucose-regulated gene expression loop control system in type 1 diabetic mice.
  • the first step is plasmid construction.
  • the details of plasmid construction in this example are shown in Appendix Table 1.
  • the second step is transformation.
  • pGN65 and pGN233 were electroporated into EcN electroporation competent cells.
  • the third step is culturing (the specific steps are the same as Example 2 of the present invention).
  • the fourth step is cell collection. Centrifuge at 5000 rpm for 5 min, wash 3 times with sterile PBS and resuspend (10 9 CFU/100 ⁇ l).
  • the fifth step is to give it to the stomach.
  • the bacterial suspension was orally administered into wild-type mice and type 1 diabetic mice (fasted for 4 hours), with 100 ⁇ l (10 9 CFU of engineered bacteria) per mouse.
  • the sixth step is to detect the expression of luxCDABE in mice. After 6 hours of intragastric administration, the bioluminescence signal was detected using a small animal live imager.
  • the experimental results show that the glucose-regulated gene expression loop control system can activate gene expression in type 1 diabetic mice, but cannot activate gene expression in wild-type mice.
  • Example 10 Construction and determination of engineering bacteria that regulate the expression and secretion of glucagon-like peptides with glucose.
  • the first step is plasmid construction.
  • the details of plasmid construction in this example are shown in Appendix Table 1.
  • the second step is transformation (the specific steps are the same as Example 2 of the present invention).
  • the transformation system in this example can be divided into 8 groups, including pGN237 and pGN233, pGN238 and pGN233, pGN239 and pGN233, pGN241 and pGN233, pGN242 and pGN233, pGN243 and pGN233, pGN288 and pGN233, pGN308 and pGN233.
  • Each set of plasmids above was electroporated into EcN electroporation competent cells.
  • the third step is to identify the expression and secretion of glucagon-like peptides. After inoculating the amplified engineering bacteria, 20mM glucose was added for induction. After 12 hours, the intracellular and intracellular glucagon-like peptide content was detected. The engineered bacteria with good secretion-inducing effect are amplified and preserved.
  • Example 11 Study on the treatment of type 2 diabetes (dbdb) mice with probiotics that regulate the expression and secretion of glucagon-like peptide.
  • This embodiment uses type 2 diabetic mice as an example to demonstrate the closed-loop treatment function of probiotic glucose sensors for diabetes, but does not limit the scope of the present invention. Specific steps are as follows:
  • the first step is cell collection.
  • the effect of inducing glucagon-like peptide secretion obtained by screening in Example 10
  • the optimal engineering bacteria and the engineering bacteria that regulate the expression of the reporter gene luxCDABE screened in Example 9 of the present invention are amplified and cultured, centrifuged at 5000 rpm for 5 minutes, washed three times with sterile PBS and resuspended (10 9 CFU/100 ⁇ l).
  • the second step is oral administration.
  • PBS and the two engineered bacterial suspensions amplified in step 1 were administered into type 2 diabetic mice (fasted for 4 hours), with 100 ⁇ l (10 9 CFU of engineered bacteria) per mouse.
  • the third step is to detect the blood sugar of mice. After 24 hours of intragastric administration, blood glucose test strips were used to detect the blood glucose of the mice (the mice were fasted for 4 hours). The blood glucose of wild-type mice was also measured as a control.
  • Example 12 Study on the long-term effect of probiotics that regulate the expression and secretion of glucagon-like peptide in treating type 2 diabetic mice.
  • the first step is to collect cells (the specific steps are the same as in Example 11 of the present invention).
  • the second step is oral administration.
  • the third step is to detect the blood sugar of mice (the specific steps are the same as in Example 11 of the present invention).
  • Example 13 Glucose tolerance study of glucose-regulated probiotic drug factory in the treatment of type 2 diabetes.
  • Example 12 This example is developed after type 2 diabetes model mice were treated in Example 12 of the present invention.
  • the specific experimental method of glucose tolerance is as follows:
  • the model mice were fasted for 16 hours.
  • the mouse's 0-point blood glucose was measured and intraperitoneally injected at a glucose dose of 1.25g/kg. Then, the blood glucose values of the mice were measured at 30, 60, 90, and 120 min.
  • Example 14 Skin wound modeling in diabetic mice.
  • the first step is anesthesia. Forty type 1 diabetes model mice were selected and anesthetized with gas using inhalation anesthesia. Anesthetic isoflurane anesthesia.
  • the second step is hair removal. Use small animal electric clippers and hair removal cream to remove the hair on the mouse's back, and wipe it clean with alcohol cotton.
  • the third step is full-thickness skin loss.
  • Example 15 Study on the regulation of the expression of the reporter gene luxCDABE in skin wounds of type 1 diabetic mice by a glucose-regulated probiotic drug factory.
  • the first step is transformation.
  • pGN65 and pGN233 were electroporated into EcN electroporation competent cells.
  • the second step is to cultivate. Pick positive single clones and transfer them to liquid LB medium, and culture them at 37°C and 210 rpm for 12 hours.
  • the third step is cell collection. Centrifuge at 5000 rpm for 5 min, wash 3 times with sterile PBS and resuspend (10 9 CFU/10 ⁇ l).
  • the fourth step is to apply it on the skin in situ.
  • the bacterial suspension was dropped onto the skin wounds of type 1 diabetic mice, 10 ⁇ l (10 9 CFU of engineered bacteria) for each mouse.
  • the fifth step is to detect the expression of luxCDABE in mouse skin wounds. After the engineered bacteria are drop-coated, the bioluminescent signal is detected by a small animal live imager.
  • Example 16 Construction and determination of a probiotic drug factory in which glucose regulates the expression and secretion of human acidic fibroblast growth factor rhaFGF135.
  • the first step is plasmid construction.
  • the details of plasmid construction in this example are shown in Appendix Table 1.
  • the second step is transformation.
  • pGN299 and pGN233 were electroporated into EcN electroporation competent cells.
  • the third step is to identify the expression and secretion of rhaFGF135.
  • 20mM glucose was added for induction. After 12 hours, centrifuge the supernatant, extract the protein in the supernatant, and detect the content of rhaFGF135 through western blotting.
  • the engineered bacteria with good secretion-inducing effect are amplified and preserved.
  • Example 17 Construction and determination of a probiotic drug factory that regulates the expression and secretion of chemokine CXCL12 by glucose.
  • the first step is plasmid construction.
  • the details of plasmid construction in this example are shown in Appendix Table 1.
  • the second step is transformation.
  • pXG22 and pGN233 were electroporated into EcN electroporation competent cells.
  • the third step is to identify the expression and secretion of CXCL12.
  • 20mM glucose was added for induction. After 12 hours, centrifuge the supernatant, extract the protein in the supernatant, and detect the CXCL12 content through western blotting.
  • the engineered bacteria with good secretion-inducing effect are amplified and preserved.
  • Example 18 verified that the wounds of diabetic mice activated the probiotic drug factory to express and secrete human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12.
  • the first step is bacterial culture.
  • Probiotics and wild-type EcN which regulate the expression and secretion of human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12, and wild-type EcN were inoculated into liquid LB medium and cultured at 37°C and 210 rpm for 12 hours.
  • the second step is cell collection (the specific steps are the same as Example 4 of the present invention).
  • the third step is to drop-coat the skin in situ (the specific steps are the same as Example 4 of the present invention).
  • the fourth step is skin tissue sampling. After 6 hours, the mice were sacrificed, and the skin of the mouse wound was cut.
  • the fifth step is tissue sectioning.
  • the skin tissue was fixed in 4% paraformaldehyde for 24 hours, rinsed with running water overnight, and the skin was sequentially passed through 30%, 50%, 75%, 85%, 95%, 100% ethanol, 50% ethanol and 50% ethanol. Soak in the xylene mixture for 1 hour each, and then pass through 100% xylene and new 100% xylene for 5 minutes each. Then soak the wax in 50% xylene and 50% paraffin for 1 hour, soak in 100% paraffin overnight, and transfer to new 100% paraffin for 1 hour the next day. Use an embedding machine to embed and freeze overnight at -20°C. Use a paraffin microtome for tissue sectioning.
  • the sixth step is immunofluorescence staining.
  • the tissue sections were placed in a repair box filled with EDTA antigen retrieval buffer (pH 8.0) and antigen retrieval was performed in a microwave oven. Bring to a boil over medium heat for 8 minutes, stop the heat for 8 minutes, then turn to medium-low heat for 7 minutes. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker for 5 minutes each time.
  • DAPI UV excitation wavelength is 330-380nm, emission wavelength is 420nm, and emits blue light;
  • CY3 excitation wavelength is 510-560, emission wavelength is 590nm, and emits red light).
  • Example 19 Research on the probiotic drug factory that regulates the expression and secretion of human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12 to promote skin wound healing in diabetic mice.
  • This example takes skin wounds of type 1 diabetic mice as an example to prove that the glucose-regulated probiotic drug factory can promote the healing of mouse skin wounds, but it does not limit the scope of the present invention. Specific steps are as follows:
  • the first step is bacterial culture (the specific steps are the same as Example 8 of the present invention).
  • the second step is cell collection. Amplify and culture the recombinant probiotics and wild-type EcN obtained through screening in Examples 5-7 that are effective in inducing the secretion of human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12, centrifuge at 5000 rpm for 5 minutes, and use sterile PBS (pH 6.35) Wash three times and resuspend (10 9 CFU/10 ⁇ l).
  • the third step is to apply bacteria in situ.
  • PBS human acidic fibroblast growth factor rhaFGF135, wild-type EcN, probiotics that regulate the expression and secretion of human acidic fibroblast growth factor rhaFGF135, and probiotic suspensions that regulate the expression and secretion of chemokine CXCL12 were dripped onto diabetic mice in situ.
  • the fourth step is to detect the size of the mouse skin wound. Take pictures of mouse skin wounds every 24 hours, and use Image J software to count the wound area.
  • Example 20 safety verification of probiotic drug factory to promote skin wound healing.
  • the first step is bacterial culture (the specific steps are the same as Example 7 of the present invention).
  • the second step is cell collection (the specific steps are the same as Example 4 of the present invention).
  • the third step is to drop-coat bacteria in situ (the specific steps are the same as Example 4 of the present invention).
  • the fourth step is sampling. 24 hours after the inoculation, the blood of the mice was taken, and then the mice were killed. The hearts, livers, spleens, lungs, and kidneys were taken respectively, and PBS buffer was added and then ground.
  • the fifth step is security verification. Carry out routine blood analysis on the mouse blood; absorb the tissue grinding fluid and coat it on an LB plate, incubate at 37°C for 12 hours and then count the colonies.

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Abstract

提供了葡萄糖调控基因表达环路控制系统及其在调控血糖中的应用，该控制系统包括转录抑制子HexR，葡萄糖诱导型强启动子和待转录序列。还提供了包括该控制系统的表达载体、工程化细胞、传感器或重组益生菌，及其在制备治疗糖尿病的药物中的应用。还提供了该控制系统所涉及的质粒、葡萄糖诱导型强启动子和引物对。

Description

葡萄糖调控基因表达环路控制系统及其调控血糖的应用 技术领域
本发明涉及合成生物学、细胞治疗领域，是将微生物合成生物学与疾病治疗相结合，具体涉及葡萄糖调控基因表达环路控制系统及其调控血糖的应用。
背景技术
生物传感器可以直接使用完整的微生物细胞或细胞碎片作为敏感材料，利用其体内的酶及代谢系统来识别与检测各类物质。近年来合成生物学的发展极大促进了生物传感器功能的开发，在生物医学中，可以很好地检测各种生理指标或者疾病信号，包括其宿主环境中的刺激和外部信号。益生菌能特异性靶向肠道作为肠道定向给药载体，满足作为生物治疗剂的几乎所有要求，利用益生菌开发生物传感器实现诊疗一体化是实现精准医学的重要手段。
目前糖尿病的治疗方式主要包括注射胰岛素，服用药物和控制饮食等，但目前的医疗水平仍不能将糖尿病彻底治愈，糖尿病患者需每天口服降血糖药物或注射胰岛素来维持血糖稳定。采用口服或注射给药，当前应用的大多数临床药物的有效成分会失活，只有很少一部分能够作用到预期的靶点部位。如果因为药物作用效率不够甚至过低，而导致需要的药物剂量激增，通常损害患者健康甚至出现众多副作用。且注射胰岛素无法达到可控地释放胰岛素，极易造成低血糖风险。能够实时自动监控体内血糖浓度并且能够准确的对血糖浓度及时进行调整对于治疗糖尿病非常重要。因此，亟需开发新的治疗方式来提高治疗效果，降低治疗风险，提高治疗的便捷性。
另一方面，伤口愈合过程受损是一个与代谢性疾病和衰老密切相关的日益严重的医学问题。皮肤上的开放性伤口会引起严重的不适，并为细菌的入侵提供了入口。在伤口愈合炎症阶段，免疫细胞在受伤或激活的细胞释放的警报信号、细胞因子和趋化因子的作用下积累，从而对伤口的愈合起到至关重要的作用。巨噬细胞和中性粒细胞是创伤部位的主要免疫细胞群，它们在阻止微生物入侵的同时也可以通过分泌额外的趋化因子、生长因子和基质消化酶等来促进愈合过程。
慢性创伤通常与潜在的能提高伤口易感性或降低愈合能力的病理过程有关，如动/静脉功能不足、糖尿病或正在接受全身类固醇治疗等。以糖尿病足溃疡为例，作为糖尿病的常见并发症之一，会导致长期疼痛、活力下降，甚至可能需要截肢。对于糖尿病足溃疡等慢性创伤的标准治疗包括手术或化学清除坏死组织、 反复更换敷料、使用抗生素进行感染控制等，结果不稳定，并伴有不良副作用。此外，临床上也通过局部应用生长因子或与不同生物材料联合应用对不同类型的慢性创伤进行治疗，但效果并不显著。其中，伤口的蛋白水解特性限制了药物的利用度，是慢性伤口药物治疗的主要障碍。因此，亟需开发新的治疗方式对难以愈合的慢性皮肤伤口进行稳定、安全的治疗，提高治疗效果和治疗的便捷性。
发明内容
本发明提出了一种葡萄糖调控基因表达环路控制系统，其包括转录抑制子HexR，葡萄糖诱导型强启动子和待转录序列。其中，所述转录抑制子HexR可同源二聚化并与特异的DNA序列相结合，所述特异的DNA序列选自序列SEQ ID NO.78～80。其中，所述葡萄糖诱导型强启动子是由组成型强启动子与所述特异的DNA序列组合而成；其包括但不限于如SEQ ID NO.1-10之任一种核苷酸序列。
其中，所述转录抑制子为可结合至所述葡萄糖诱导型强启动子上并进而阻遏下游待转录序列的表达。所述待转录序列包括单一编码表达报告蛋白或功能型蛋白，或串联表达多种蛋白。所述转录抑制子、葡萄糖诱导型强启动子和待转录序列可构建在一个或两个质粒载体上。
本发明还提出了一种葡萄糖诱导基因表达调控方法，由所述葡萄糖调控基因表达环路控制系统进行调控。在具体实施方案中，当葡萄糖不存在时，所述转录抑制子可结合至所述葡萄糖诱导型强启动子上，进而阻遏下游待转录序列的表达；当葡萄糖存在时，葡萄糖经Entner-Doudoroff途径代谢生成代谢中间物2-keto-3-deoxy-6-phosphogluconate(KDPG)，KDPG阻断其结合，使所述转录抑制子从所述葡萄糖诱导型强启动子上解离下来，启动下游待转录序列的表达。在具体实施方案中，其诱导基因表达的调控方式如图1所示。
本发明中，所述葡萄糖调控基因表达环路控制系统即由葡萄糖精确调控基因的启动表达和分泌，由转录抑制子HexR，葡萄糖诱导型强启动子和待转录序列三部分组成。
其中，所述转录抑制子HexR为来源于恶臭假单胞菌Pseudomonas putida的HexR操纵子系统的阻遏蛋白HexR(其氨基酸序列Genbank登录号：AE015451)，由强启动子持续表达。
优选地，所述的转录抑制子HexR可由不同种类的强启动子启动表达，所述的强启动子包括但不限于PBBA23100(其核苷酸序列Genbank登录号：MG649435)、 PLac(其核苷酸序列Genebank登录号：LC652750)、PTac(其核苷酸序列Genebank登录号：MN913428)等。
其中，所述葡萄糖诱导型强启动子是通过强启动子与RBS位点之间插入不同拷贝数的HexR操纵子结合位点构成；所述HexR操纵子结合位点来源于HexR操纵子系统的操纵子元件HexO。所述的葡萄糖诱导型强启动子可驱动下游基因的表达。所述HexR操纵子系统是由阻遏蛋白HexR和操纵子元件HexO组成，其最初是从恶臭假单胞菌Pseudomonas putida中分离得到，并受到葡萄糖的代谢产物2-keto-3-deoxy-6-phosphogluconate(KDPG)的严格调控。
优选地，所述的葡萄糖诱导型强启动子根据强启动子的种类和操纵子HexO的不同拷贝数可组成不同类型的融合型强启动子，包括：a)如SEQ ID NO.1所示的葡萄糖诱导型强启动子PHexR1核苷酸序列PHexR1(PTac-HexO)；b)如SEQ ID NO.2所示的葡萄糖诱导型强启动子PHexR2核苷酸序列PHexR2(PTac-2×HexO)；c)如SEQ ID NO.3所示的葡萄糖诱导型强启动子PHexR3核苷酸序列PHexR3(PTac-3×HexO)；d)如SEQ ID NO.4所示的葡萄糖诱导型强启动子PHexR4核苷酸序列PHexR4(PTac-4×HexO)；e)如SEQ ID NO.5所示的葡萄糖诱导型强启动子PHexR5核苷酸序列PHexR5(PTac-5×HexO)；f)如SEQ ID NO.6所示的葡萄糖诱导型强启动子PHexR6核苷酸序列PHexR6(PLac-HexO)；g)如SEQ ID NO.7所示的葡萄糖诱导型强启动子PHexR7核苷酸序列PHexR7(PLac-2×HexO)；h)如SEQ ID NO.8所示的葡萄糖诱导型强启动子PHexR8核苷酸序列PHexR8(PLac-3×HexO)；i)如SEQ ID NO.9所示的葡萄糖诱导型强启动子PHexR9核苷酸序列PHexR9(PLac-4×HexO)；j)如SEQ ID NO.10所示的葡萄糖诱导型强启动子PHexR10核苷酸序列PHexR10(PLac-5×HexO)以及任意组成型启动子与任意拷贝数的HexO组成的葡萄糖诱导型启动子PHexRn。
其中，所述待转录序列可以是单一编码表达报告蛋白如sfGFP(氨基酸序列Genbank登录号：AB971579)、LuxCDABE(氨基酸序列Genbank登录号：EF173694)或功能型蛋白如SEQ ID NO.11所示的胰高血糖素样肽GLP-1氨基酸序列、如SEQ ID NO.12所示的胰高血糖素样肽EK-GLP-1氨基酸序列、如SEQ ID NO.13所示的胰高血糖素样肽GLP-1-Fc氨基酸序列、如SEQ ID NO.61所示的鼠源胰岛十二指肠同源盒-1(mPDX-1)氨基酸序列、如SEQ ID NO.66所示的人 源胰岛十二指肠同源盒-1(hPDX-1)氨基酸序列等。
其中，所述的葡萄糖调控基因表达环路控制系统可精确调控一种或通过多顺反子的形式串联多种报告蛋白及功能蛋白的表达。
其中，所述的葡萄糖调控基因表达环路控制系统分别由人工设计、合成的双质粒系统装载，所述双质粒系统中涉及的序列详见表1。
本发明还提出一种表达载体、工程化细胞、传感器或重组益生菌，其含有所述的葡萄糖调控基因表达环路控制系统。在具体实施方案中，所述葡萄糖调控基因表达环路控制系统位于所述工程化细胞、传感器或重组益生菌的染色体或质粒中的任意一种。
本发明还提出一种应用，即所述葡萄糖调控基因表达环路控制系统，所述表达载体、工程化细胞、传感器或重组益生菌在制备治疗和/或预防糖尿病的药物、制备调控血糖的药物或产品中的应用。
在具体实施方案中，所述药物或产品包括但不限于口服剂、涂抹剂、注射剂等各种剂型。优选，采用口服剂的方式。在具体实施方案中，通过口服含有所述葡萄糖调控基因表达环路控制系统的重组益生菌(即，智能可控的微生物药物工厂)，通过葡萄糖诱导基因表达调控，表达降血糖肽，实现调控血糖。也就是说，通过口服所述重组益生菌(经改造的智能可控的益生菌药物工厂)，使其通过血糖诱导产生胰高血糖素样肽的方法实现，可以精确调控GLP-1或/和PDX-1的表达，实现治疗糖尿病的效果。具体地，所述应用及治疗方法中，通过调控释放胰高血糖素样肽、胰岛十二指肠同源盒-1降糖蛋白来调控血糖，调控血糖水平。
本发明还提出了一种调控血糖的方法，即利用所述葡萄糖调控基因表达环路控制系统，和/或利用所述表达载体、工程化细胞、传感器或重组益生菌等来调控血糖。具体实施方案中，所述方法通过调控GLP-1、PDX-1降糖蛋白的表达来实现调控血糖的效果。具体地，所述调控血糖的方法包括以下步骤：
a)人工构建原核表达载体或用于染色体整合的功能模块；所述原核表达载体或用于染色体整合的功能模块含有葡萄糖诱导GLP-1/PDX-1表达调控系统；
b)将步骤a)构建的表达载体或用于染色体整合的功能模块转化至微生物细胞中或整合至微生物细胞染色体上，制备含有葡萄糖诱导GLP-1/PDX-1表达调控系统的工程化细胞；
c)将步骤b)制备的所述工程化细胞以灌胃形式移植至糖尿病模型鼠体内；
d)小鼠体内葡萄糖闭环诱导所述工程化细胞表达并分泌GLP-1/PDX-1，吸收至血液中以降血糖。
在具体实施方案中，所述调控血糖方法可以口服益生菌的方式。
本发明还提供了一种治疗糖尿病的新方法，通过所述智能可控的益生菌药物工厂、葡萄糖诱导GLP-1或/和PDX-1表达调控系统，采用口服益生菌的方式便可达到降血糖的效果。本发明提供了安全、可靠、可精确调控释放胰高血糖素样肽或/和胰岛十二指肠同源盒-1治疗糖尿病的新策略。本发明提供了治疗糖尿病的新方法、新策略。所述系统可调控胰高血糖素样肽GLP-1、胰岛十二指肠同源盒-1PDX-1的表达。所述胰高血糖素样肽GLP-1的表达包括EK-GLP-1、GLP-1-Fc等。所述胰岛十二指肠同源盒-1PDX-1的表达包括mPDX-1、hPDX-1等。本发明所述葡萄糖调控基因表达环路控制系统可以快速通过葡萄糖代谢物调控基因表达，具有精准控制基因表达量、调控基因表达倍数高等特点。
本发明所述葡萄糖调控基因表达环路控制系统具有微调性、可逆性的表达动力学特征。所述微调性是指下游的基因表达受葡萄糖的精准调控，并呈现出剂量依赖性的关系；所述可逆性是指葡萄糖调控基因表达的整个过程是可逆的，可以通过控制葡萄糖的存在与否实现基因表达的开启或关闭。且，具有微调性、可逆性的表达动力学特征同样体现在所述葡萄糖诱导基因表达调控方法，调控血糖方法，以及所述表达载体、工程化细胞、传感器或重组益生菌等。
本发明还提出了一种质粒，选自质粒pGN11、pGN69、pGN89、pGN90、pGN227、pGN12、pGN228、pGN229、pGN220、pGN221、pGN222、pGN223、pGN224、pGN231、pGN232、pGN233、pGN65、pGN237、pGN238、pGN239、pGN241、pGN242、pGN243、pGN288、pGN308、pGN306、pGN307、pXG58之一种或几种。所述质粒，详见表1。
本发明还提出了一种核苷酸序列，其为葡萄糖诱导型强启动子PHexR1、PHexR2、PHexR3、PHexR4、PHexR5、PHexR6、PHexR7、PHexR8、PHexR9、PHexR10之一种，其核苷酸序列分别如SEQ ID NO.1～10之一所示。
本发明还提出了一种引物，其序列如SEQ ID NO.16-60、62-65、67-74、76-77所示的其中一种或几种。本发明还提出了一种引物对，其序列如编号1-30所示 引物对之一种或几种。如表1、表2所示。
本发明还提出一种智能可控的微生物药物工厂，其包括底盘微生物，以及上载至所述底盘微生物中的基因表达环路控制系统。在具体实施方案要中，所述基因表达环路控制系统位于所述底盘微生物的染色体或质粒中的任意一种。
本发明所述智能可控的微生物药物工厂，优选地为葡萄糖调控的益生菌药物工厂，为含有葡萄糖调控基因表达环路控制系统的工程化细胞。还可以是其他调控方式的微生物药物工厂，即含有其他调控方式的基因表达环路控制系统的工程化细胞。在具体实施方案中，所述工程化细胞可以为任意益生菌，如Escherichia coli Nissle 1917(EcN)等。
其中，所述基因表达环路控制系统包括但不限于葡萄糖调控基因表达环路控制系统、以及其他调控方式的基因表达环路控制系统，如木糖诱导的基因表达系统、阿拉伯糖诱导的基因表达系统、尿酸诱导的基因表达系统等。
本发明中，所述葡萄糖调控基因表达环路控制系统包括转录抑制子HexR，葡萄糖诱导型强启动子和待转录序列三部分。在一具体实施方案中，所述转录抑制子HexR可结合至所述葡萄糖诱导型强启动子上并进而阻遏下游待转录序列的表达；所述葡萄糖诱导型强启动子是由组成型强启动子与HexR特异结合的DNA序列组合而成；所述待转录序列包括单一编码表达报告蛋白如LuxCDABE(氨基酸序列Genbank登录号：EF173694)或功能型蛋白如SEQ ID NO.14所示的人酸性成纤维生长因子rhaFGF135氨基酸序列、如SEQ ID NO.15所示的趋化因子CXCL12氨基酸序列、如SEQ ID NO.75所示的人源白介素4因子(hIL4)氨基酸序列等，或串联表达多种蛋白。具体实施方案中，调控方式为，当葡萄糖不存在时，所述转录抑制子可结合至所述葡萄糖诱导型强启动子上，进而阻遏下游待转录序列的表达；当葡萄糖存在时，葡萄糖经Entner-Doudoroff途径代谢生成代谢中间物2-keto-3-deoxy-6-phosphogluconate(KDPG)，KDPG阻断其结合，使所述转录抑制子从所述葡萄糖诱导型强启动子上解离下来，启动下游待转录序列的表达。
本发明中，所述基因表达环路控制系统包括转录抑制子，诱导型强启动子和待转录序列。在具体实施方案中，所述转录抑制子、诱导型强启动子和待转录序列可构建在一个或两个质粒载体上。
本发明中，所述智能可控的微生物药物工厂可以在一种诱导物的存在下表达目的基因。在具体实施方案中，所述诱导物通过外源添加或由体内产生。
优选地，所述底盘微生菌包括益生菌。优选地，所述益生菌包括但不限大肠杆菌Nissle 1917、乳酸乳球菌、植物乳杆菌、枯草芽孢杆菌等。在具体实施方案中，所述智能可控的微生物药物工厂即重组益生菌。
本发明所述智能可控的微生物药物工厂包括但不限于可以处于液体中，或是经冷冻干燥、喷雾干燥的。
本发明还提出了一种所述智能可控的微生物药物工厂的构建方法，包括以下步骤：
a)将转录抑制蛋白特异性识别的DNA操纵子序列与组成型强启动子组合，构建诱导型强启动子；
b)将表达药物蛋白的编码基因以单一或串联的方式构建至诱导型启动子下游，构建报告模块；其中药物蛋白的种类和数量可根据实际需求进行相应的替换改变；
c)构建组成型表达转录抑制蛋白的功能模块。
本发明提出的智能可控的微生物药物工厂的构建方法，将包含所述步骤b)和步骤c)构建的功能模块与报告模块的质粒转化至益生菌感受态细胞中，或将所述步骤b)和步骤c)构建的功能模块与报告模块通过RED重组酶系统或CRISPR系统整合至益生菌染色体上，制备得到可诱导的工程化细胞，即制备得到所述智能可控的微生物药物工厂。
本发明还提出了表达载体、工程化细胞，其含有所述智能可控的微生物药物工厂。具体实施中，一种含有葡萄糖调控基因表达环路控制系统的原核表达载体。
本发明还提出了所述智能可控的微生物药物工厂在在制备预防和/或治疗疾病的药物或产品中的应用。所述药物或产品包括但不限于促进皮肤伤口愈合的药物、预防和/或治疗代谢性疾病的药物、预防和/或治疗其他疾病的药物等。
在具体实施方案中，所述皮肤伤口包括但不限于慢性皮肤伤口、糖尿病患皮肤伤口、糖尿病足溃疡等。
本发明还提出了智能可控的微生物(优选为益生菌)药物工厂在促进糖尿病皮肤伤口愈合中的应用。所述促进糖尿病皮肤伤口愈合的应用是通过伤口原位滴 涂智能可控的益生菌药物工厂的方法实现，即通过构建智能可控的益生菌药物工厂，使其通过血糖诱导产生生长因子、细胞因子和趋化因子的方法实现，即，所述治疗方法是利用智能可控的益生菌药物工厂精确调控生长因子、细胞因子和趋化因子的表达与分泌来促进伤口愈合。
本发明还提出一种利用所述智能可控的微生物(益生菌)药物工厂从而促进皮肤伤口愈合的方法。所述方法包括以下步骤：
a)人工构建含有葡萄糖诱导生长因子、巨噬细胞M2极化细胞因子和趋化因子的表达调控系统的原核表达载体或用于染色体整合的功能模块；
b)制备含有葡萄糖诱导生长因子、巨噬细胞M2极化细胞因子和趋化因子表达调控系统的工程化细胞；
c)所述步骤b)制备的工程化细胞以伤口原位滴涂的形式移植至糖尿病模型鼠皮肤伤口处；
d)小鼠皮肤伤口处葡萄糖闭环诱导工程化细胞表达并分泌生长因子、巨噬细胞M2极化细胞因子和趋化因子，以实现促进伤口愈合的效果。
其中，所述生长因子为rhaFGF135、FGF2、FGF7、TGF-α、TGF-β、PDGF、EGF、VEGF、IGF-1、IGF-2、PDGF、HGF；所述巨噬细胞M2极化细胞因子为IL4、IL-10、IL-13、CSF1、IL34；所述趋化因子为CXCL12、CXCL13、CXCL8、CCL2、CCL3、CCL4、CCL5、CCL11、CXCL10。
本发明还提出了一种质粒，所述质粒为质粒pGN233、pGN65、pGN299、pGN300、pGN314之一种或几种，详见表1。
本发明还提出了一种核苷酸序列，其为葡萄糖诱导型强启动子PHexR4、PHexR9之一种，其序列分别如SEQ ID NO.4、SEQ ID NO.9所示。
本发明有益效果包括，本发明提供了一种促进慢性皮肤伤口愈合的新方法，通过所述智能可控的益生菌药物工厂、葡萄糖诱导生长因子、细胞因子和趋化因子表达调控系统，采用伤口原位滴涂益生菌的方式便可达到加速伤口愈合的效果。本发明提供了一种安全、可靠、可精确调控释放治疗因子加速慢性皮肤伤口、糖尿病足溃疡等慢性皮肤伤口愈合的新策略。所述系统可调控生长因子、巨噬细胞M2极化细胞因子和趋化因子的表达与分泌。本发明创新开发了一种将生长因子、巨噬细胞M2极化细胞因子和趋化因子直接传递到受伤皮肤的优化技术，以益生菌用作载体，用质粒或染色体基因组编码生长因子、巨噬细胞M2极化细胞 因子和趋化因子。
本发明有益效果包括，提供一种智能可控的益生菌药物工厂，包括基因表达环路控制系统和底盘益生菌。本发明所述葡萄糖调控基因表达环路控制系统具有微调性、可逆性的表达动力学特征。本发明还提供所述智能可控的益生菌药物工厂或含有葡萄糖调控基因表达环路控制系统的原核表达载体或含有葡萄糖调控基因表达环路控制系统的工程化细胞在治疗糖尿病、促进慢性皮肤伤口愈合、促进糖尿病足溃疡等慢性皮肤伤口愈合、预防和/或治疗代谢疾病的药物中的应用。
附图说明
图1是本发明葡萄糖调控基因表达环路控制系统及调控方法的原理示意图。
图2是本发明葡萄糖调控基因表达环路控制系统的优化研究，即利用不同启动子启动表达HexR的表达载体分别和葡萄糖诱导型强启动子PHexR1进行组合的实验结果。
图3是本发明葡萄糖调控基因表达环路控制系统的优化研究，即利用PTac启动表达HexR的表达载体分别和5种不同的响应元件进行组合的实验结果。
图4是本发明葡萄糖调控基因表达环路控制系统的优化研究，即利用RBS调控HexR的表达载体分别和葡萄糖诱导型强启动子PHexR4进行组合的实验结果。
图5是本发明葡萄糖调控基因表达环路控制系统具有微调性的表达动力学特征的实验结果。
图6是本发明葡萄糖调控基因表达环路控制系统具有可逆性的表达动力学特征的实验结果。
图7是在本发明葡萄糖调控基因表达环路控制系统中，不同的葡萄糖诱导时间对其基因表达影响的实验结果。
图8是本发明葡萄糖调控基因表达环路控制系统在野生型小鼠和1型糖尿病小鼠体内调控报告基因luxCDABE表达的实验结果。
图9是本发明调控胰高血糖素样肽表达分泌的益生菌药物工厂治疗1型糖尿病小鼠的实验结果。
图10是本发明调控胰高血糖素样肽表达分泌的益生菌药物工厂长期控制1型糖尿病小鼠血糖的实验结果。
图11是本发明葡萄糖调控的益生菌药物工厂在1型糖尿病的治疗过程中糖耐受的实验结果。
图12是本发明葡萄糖调控基因表达环路控制系统在糖尿病小鼠皮肤伤口处调控报告基因luxCDABE表达的实验结果。
图13是本发明益生菌药物工厂受葡萄糖调控诱导人酸性成纤维生长因子rhaFGF135表达分泌的实验结果。
图14是本发明益生菌药物工厂受葡萄糖调控诱导趋化因子CXCL12表达分泌的实验结果。
图15是本发明糖尿病小鼠伤口处激活益生菌药物工厂表达和分泌人酸性成纤维生长因子rhaFGF135和趋化因子CXCL12的实验结果。
图16是本发明葡萄糖调控的益生菌药物工厂促进糖尿病小鼠皮肤伤口愈合的实验结果。
图17是本发明伤口原位滴涂益生菌药物工厂后小鼠的血液指标。
图18是本发明伤口原位滴涂益生菌药物工厂后重组益生菌在小鼠各脏器及血液中的残留。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明。这些实施例仅用于举例说明发明，而不对本发明的范围构成任何限制。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。以下实施例中所用的试剂、仪器等，以及未注明具体条件的实验方法，按照常规或商品供货商所建议的条件进行。
分子克隆
分子克隆技术构建本发明所有表达质粒，步骤为业内常识。
所有用于PCR的引物均由金唯智生物科技有限公司合成。本发明实施例中构建的表达质粒都经过序列测定，序列测定由金唯智生物科技有限公司完成。本发明实施例中所用的Phanta Max Super-Fidelity DNA聚合酶购自南京诺唯赞生物科技有限公司。核酸内切酶、T4DNA连接酶均购自TaKaRa公司。同源重组酶购自和元生物技术(上海)股份有限公司。PhantaMax Super-Fidelity DNA聚合酶购买时附带有相应的聚合酶缓冲液和dNTP。核酸内切酶、T4DNA连接酶、 同源重组酶购买时附带有相应的缓冲液。酵母提取物(Yeast Extract)、胰蛋白胨(Trypton)、琼脂粉、M9培养基、葡萄糖、氨苄青霉素(Amp)、卡那霉素(Kan)购自上海生工生物工程技术有限公司。DNA Marker DL5000、DNA Marker DL2000(宝生物工程有限公司)；核酸染料EB(广东国奥生物技术公司)；质粒小抽提取试剂盒(天根生化科技(北京)有限公司)；DNA胶回收试剂盒、PCR产物纯化试剂盒均购自康为世纪生物科技有限公司；实施例中提及的无水乙醇、NaCl等其余试剂均为国产分析纯产品。DNA片段的胶回收、纯化回收，其步骤根据DNA胶回收试剂盒、PCR产物纯化试剂盒(康为世纪生物科技有限公司)的操作说明书；质粒提取步骤根据质粒小抽(天根生化科技(北京)有限公司)提取试剂盒说明书。
细菌培养与转化
本发明实施例中用以下细菌底盘细胞和电转化为例说明葡萄糖调控基因表达环路控制系统在原核细胞及动物体内的工作情况，但不限制本发明保护范围。
细菌培养：大肠杆菌EcN培养于LB培养基中，培养基中加入100μg/ml的氨苄青霉素和50μg/ml的卡那霉素溶液；细菌培养于37℃、210rpm的摇床中。
大肠杆菌EcN感受态细胞的制备：所有用于感受态细胞制备的溶液和耗材均经过高温高压灭菌处理。将大肠杆菌EcN菌种划线于不含抗生素的平板上，37℃倒置培养12～16h；挑取一个单菌落于2mL不含抗生素的LB摇菌管中，37℃、210rpm振荡培养过夜。吸取1mL菌液转入100mL新鲜LB培养基中，37℃、210rpm摇床振荡培养至OD600在0.4～0.6之间。将培养液转入离心管中，冰上放置15min，然后于4℃、3500rpm离心10min，弃上清，依次用50mL和25mL预冷的ddH2O重悬，4℃、3500rpm离心10min，用25mL预冷的10％甘油重悬，4℃、3500rpm离心10min。弃上清，用预冷的10％甘油重悬，分装(100μl/管)，-80℃保存。
转化：EcN的转化使用优化后的电转化法。简单的说，即将预冷的质粒(300-500ng)和感受态(100μl)混合物加进电转杯底部，电转参数设置为2mm，2500V，电击一次。电击结束后，将培养基900μl加进电转杯，混匀后吸出置于灭菌的EP管中，37℃、210rpm孵育1h，将菌体涂布抗性平板(100μg/ml的Amp和50μg/ml的Kan)，37℃培养16-20h。
报告基因绿色荧光蛋白(sfGFP)的检测
绿色荧光蛋白sfGFP在蓝光下具有肉眼可见的荧光，且其荧光强度可用酶标仪Synergy H1测定。吸取100μl细菌悬液于黑色96孔酶标板中，放在酶标仪中，在波长为480nm的激发光作用下，检测菌悬液在520nm的发射光读数，即为绿色荧光强度。同时吸取100μl细菌悬液于透明96孔酶标板中，使用酶标仪检测细菌培养液在600nm处的吸光值，即为菌体细胞密度。用荧光强度/菌体细胞密度来表征报告基因的表达效率。
分泌蛋白提取
在2ml细菌培养液上清中分别加入8ml甲醇、2ml氯仿和8ml水，混匀后12000g离心10min，移除上层液体，加入8ml甲醇,混匀后12000g离心15min，弃尽上清，风干后加入30μl PBS缓冲液重悬沉淀。
蛋白免疫印记(Western Bloting)
将蛋白样品中加入10μl 4×蛋白上样缓冲液，进行SDS-PAGE电泳。使用湿式电印迹法进行转膜(250mA，1-1.5h)，使用TBST+5％的脱脂奶粉封闭1.5h，TBST清洗3次后加入一抗，于4℃孵育过夜，TBST清洗3次后加入二抗，于室温孵育2h，使用凝胶成像系统进行显影检测。
实施例1，葡萄糖调控基因表达环路控制系统的构建
本实施例中包含了葡萄糖调控基因表达环路控制系统所涉及质粒载体的构建方法。详细设计方案及步骤见附表1。
表1质粒构建表



引物对(编号1-30)，如表2所示：

实施例2，葡萄糖调控基因表达环路控制系统在EcN中的优化研究，即利用不同启动子启动表达HexR的表达载体分别和葡萄糖诱导型强启动子PHexR1进行组合优化。
第一步，质粒构建。本实施例中的质粒构建详见附表1。
第二步，转化。本实施例的转化体系可分为4组，包括pGN11，pGN11和pGN69，pGN11和pGN89，pGN11和pGN90。将上述每组质粒电转化EcN电转感受态。
第三步，培养。挑取阳性单克隆转接液体LB培养基，37℃、210rpm培养12h。
第四步，检测报告基因sfGFP的表达量。吸取100μL细菌悬液于黑色96孔酶标板中和透明96孔酶标板中，使用酶标仪检测荧光强度和菌体细胞密度。用荧光强度/菌体细胞密度来表征报告基因的表达效率。
实验结果(见图2)显示外源表达HexR阻遏蛋白后，均可抑制报告基因sfgfp的表达，其中Tac启动子启动表达HexR(pGN89)后报告基因的表达阻遏最强。
实施例3，葡萄糖调控基因表达环路控制系统在EcN中的优化研究，即利用PTac启动表达HexR的表达载体分别和5种不同的响应元件进行组合优化。
第一步，质粒构建。本实施例中的质粒构建详见附表1。
第二步，转化。本实施例的转化体系可分为5组，包括pGN89和pGN11,pGN89和pGN227，pGN89和pGN12，pGN89和pGN228，pGN89和pGN229。将上述每组质粒电转化EcN电转感受态。
第三步，培养(具体步骤同实施例2)。
第四步，诱导。5000rpm离心5min，以等量M9培养基用枪头吹打均匀，按500μL的培养体积分装至48孔板。不同实验组添加相应浓度的无菌葡萄糖溶液诱导，37℃、150rpm振荡培养12h。
第五步，检测报告基因sfGFP的表达量(具体步骤同本发明实施例2)。
实验结果(见图3)显示葡萄糖调控基因表达环路控制系统的不同优化组合均能激活报告基因sfGFP的表达，但系统所产生的诱导效果各不相同，其中，pGN89和pGN228的组合诱导倍数最佳。
实施例4，葡萄糖调控基因表达环路控制系统在EcN中的优化研究，即利用RBS调控HexR的表达载体分别和葡萄糖诱导型强启动子PHexR4进行组合优化。
第一步，质粒构建。本实例中的质粒构建详见附表1。
第二步，转化。本实施例的转化体系可分为4组，包括pGN89和pGN228，pGN231和pGN228，pGN232和pGN228，pGN233和pGN228。将上述每组质粒电转化EcN电转感受态。
第三步，培养(具体步骤同本发明实施例2)。
第四步，诱导(具体步骤同本发明实施例3)。
第五步，检测报告基因sfGFP的表达量(具体步骤同本发明实施例2)。
实验结果(见图4)显示葡萄糖调控基因表达环路控制系统的不同优化组合均能激活报告基因sfGFP的表达，但系统所产生的诱导效果各不相同，其中，pGN233和pGN228的组合诱导倍数最佳。
实施例5，葡萄糖调控基因表达环路控制系统具有微调性的表达动力学特征研究。
第一步，将本发明实施例4获得的诱导倍数最佳的工程菌接种、培养，5000rpm离心5min，以等量M9培养基用枪头吹打均匀，按500μL的培养体积分装至48孔板。不同实验组添加不同终浓度的无菌葡萄糖溶液诱导，37℃、150rpm振荡培养12h。
第二步，检测报告基因sfGFP的表达量(具体步骤同本发明实施例2)。
实验结果(见图5)显示葡萄糖调控基因表达环路控制系统的工程菌受葡萄糖的精确调控，即剂量依赖性地诱导基因表达，进而表明其具有微调性的表达动力学特征。
实施例6，葡萄糖调控基因表达环路控制系统具有可逆性的表达动力学特征研究。
第一步，将本发明实施例4获得的诱导倍数最佳的工程菌接种、培养，5000rpm离心5min，以等量M9培养基用枪头吹打均匀，按500μL的培养体积分装至48孔板。
第二步，“ON-OFF-ON”实验组在0h加入终浓度20mM的无菌葡萄糖溶液诱导；第2h，离心洗去原有的培养基，加入不含葡萄糖的M9培养基重悬培养；第7h，离心洗去培养基，加入含终浓度20mM葡萄糖的M9培养基重悬培养。而“OFF-ON-OFF”实验组则在0h加入不加葡萄糖诱导；第2h，加入终浓度20mM的无菌葡萄糖溶液诱导；第7h，离心洗去原有的培养基，加入不含葡萄糖的M9培养基重悬培养。两实验组均每隔1h吸取菌液检测报告基因sfGFP的表达量(具体步骤同本发明实施例2)。
实验结果(见图6)表明通过控制葡萄糖的存在与否可以实现其系统基因表达的开启或关闭，说明葡萄糖调控基因表达环路控制系统具有良好的可逆性。
实施例7，在葡萄糖调控基因表达环路控制系统中，葡萄糖的不同诱导时间控制其基因表达的研究。
第一步，将本发明实施例4获得的诱导倍数最佳的工程菌接种、培养，5000rpm离心5min，以等量M9培养基用枪头吹打均匀，按500μL的培养体积分装至48孔板。不同实验组添加终浓度20mM的无菌葡萄糖溶液，37℃、150rpm振荡培养不同时间(0h、2h、4h、6h、8h、10h、12h)。
第二步，检测报告基因sfGFP的表达量(具体步骤同本发明实施例2)。
实验结果(见图7)显示系统的基因表达量受到葡萄糖诱导时间的调控，即说明葡萄糖调控基因表达环路控制系统具有良好的可控性。
实施例8，利用链脲霉素(STZ)造模法构建1型糖尿病模型鼠。
第一步，禁食。给药前，选择40只体重为25g左右，雄性的C57BL/6J小鼠进行长达16h的禁食。
第二步，给药。将STZ溶解于柠檬酸缓冲液(0.1mol/L，pH 4.5)中，然后以40-50mg/kg的给药剂量进行小鼠腹腔注射，并连续注射5天。由于STZ易降解，因此整个过程需要保证药品处于低温避光的状态，且注射过程需快速。
第三步，测定血糖值。第9天，在小鼠饥饿4小时后检测血糖水平，血糖值高于16.7mM的小鼠可视为造模成功。
实施例9，葡萄糖调控基因表达环路控制系统在1型糖尿病小鼠体内调控报告基因luxCDABE的表达研究。
第一步，质粒构建。本实施例中的质粒构建详见附表1。
第二步，转化。将pGN65和pGN233电转化EcN电转感受态。
第三步，培养(具体步骤同本发明实施例2)。
第四步，细胞收集。5000rpm离心5min，用无菌的PBS洗涤3次后重悬(109CFU/100μl)。
第五步，灌胃。将细菌悬浮液分别灌胃至野生型小鼠和1型糖尿病小鼠体内(禁食4h)，每只小鼠100μl(109CFU工程菌)。
第六步，检测小鼠体内luxCDABE的表达量。灌胃6h后，通过小动物活体成像仪检测生物发光信号。
实验结果(见图8)显示葡萄糖调控基因表达环路控制系统在1型糖尿病小鼠体内能激活基因表达，而在野生型小鼠体内不能激活基因表达。
实施例10，构建并测定葡萄糖调控胰高血糖素样肽表达和分泌的工程菌。
第一步，质粒构建。本实施例中的质粒构建详见附表1。
第二步，转化(具体步骤同本发明实施例2)。本实施例的转化体系可分为8组，包括pGN237和pGN233,pGN238和pGN233，pGN239和pGN233，pGN241和pGN233，pGN242和pGN233，pGN243和pGN233，pGN288和pGN233，pGN308和pGN233。将上述每组质粒电转化EcN电转感受态。
第三步，鉴定胰高血糖素样肽的表达和分泌。将扩增好的工程菌接种后加入20mM葡萄糖诱导。12h后检测胞内外胰高血糖素样肽的含量。将诱导分泌效果佳的工程菌进行扩增、保存。
实施例11，调控胰高血糖素样肽表达分泌的益生菌治疗2型糖尿病(dbdb)小鼠的研究。
本实施例以2型糖尿病小鼠为例，举例证明益生菌葡萄糖传感器对于糖尿病的闭环治疗功能，但不对本发明的保护范围有所限制。具体步骤如下：
第一步，细胞收集。将实施例10筛选获得的诱导胰高血糖素样肽分泌效果 佳的工程菌和本发明实施例9中筛选获得的调控报告基因luxCDABE表达的工程菌扩增培养，5000rpm离心5min，用无菌的PBS洗涤3次后重悬(109CFU/100μl)。
第二步，灌胃。分别将PBS和步骤一扩增后的两种工程菌悬浮液灌胃至2型糖尿病小鼠体内(禁食4h)，每只小鼠100μl(109CFU工程菌)。
第三步，检测小鼠血糖。灌胃24h后，使用血糖试纸条检测小鼠血糖(小鼠禁食4h)。同时检测野生型小鼠血糖作为对照。
实验结果(见图9)显示口服调控胰高血糖素样肽表达与分泌的益生菌可降低2型糖尿病鼠的血糖水平。
实施例12，调控胰高血糖素样肽表达分泌的益生菌治疗2型糖尿病小鼠的长期性的效果研究。
第一步，细胞收集(具体步骤同本发明实施例11)。
第二步，灌胃。连续15天每24h按照本发明实施例11的步骤进行灌胃。
第三步，检测小鼠血糖(具体步骤同本发明实施例11)。
实验结果(见图10)显示口服调控胰高血糖素样肽表达与分泌的益生菌可长期维持2型糖尿病鼠的血糖稳态。
实施例13，葡萄糖调控的益生菌药物工厂在2型糖尿病的治疗过程中糖耐受研究。
本实施例是在2型糖尿病模型鼠进行本发明实施例12的治疗后展开的，其糖耐受的具体实验方法如下：
第一步，模型鼠进行16小时的禁食。
第二步，配制125mg/ml的葡萄糖溶液。
第三步，测量小鼠的0点血糖，并按照1.25g/kg的葡萄糖剂量进行腹腔注射。然后，依次测量小鼠在30，60，90，120min的血糖值。
实验结果(见图11)显示相比于对照组，治疗组的高血糖症得到了良好的改善和控制，即说明调控胰高血糖素样肽表达分泌的益生菌在2型糖尿病治疗上有显著的效果。
实施例14，糖尿病小鼠皮肤伤口造模。
第一步，麻醉。选择40只1型糖尿病模型鼠，使用吸入式麻醉法以气体麻 醉剂异氟烷麻醉。
第二步，脱毛。用小动物电推剪和脱毛膏脱去小鼠背部毛发，并用酒精棉擦拭干净。
第三步，全层皮肤损失。于小鼠背部脊椎两侧脱毛处标记两个相同的圆形区域(面积约30mm2)，沿标记剪去皮肤及皮下组织深至筋膜层，两个切除部分由完整的皮肤分开。采用随机分组法对小鼠背部伤口进行分组。
实施例15，葡萄糖调控的益生菌药物工厂在1型糖尿病小鼠皮肤伤口处调控报告基因luxCDABE的表达研究。
第一步，转化。将pGN65和pGN233电转化EcN电转感受态。
第二步，培养。挑取阳性单克隆转接液体LB培养基，37℃、210rpm培养12h。
第三步，细胞收集。5000rpm离心5min，用无菌的PBS洗涤3次后重悬(109CFU/10μl)。
第四步，皮肤原位滴涂。将细菌悬浮液分别滴涂至1型糖尿病小鼠皮肤伤口处，每只小鼠10μl(109CFU工程菌)。
第五步，检测小鼠皮肤伤口处luxCDABE的表达量。工程菌滴涂后，通过小动物活体成像仪检测生物发光信号。
实验结果(见图12)显示葡萄糖调控基因表达环路控制系统在糖尿病小鼠皮肤伤口处能持续激活基因表达，激活约可维持24小时。
实施例16，构建并测定葡萄糖调控人酸性成纤维生长因子rhaFGF135表达和分泌的益生菌药物工厂。
第一步，质粒构建。本实施例中的质粒构建详见附表1。
第二步，转化。将pGN299和pGN233电转化EcN电转感受态。
第三步，鉴定rhaFGF135的表达和分泌。将扩增好的工程菌接种后加入20mM葡萄糖诱导。12h后离心取上清，提取上清中的蛋白，通过western bloting检测rhaFGF135的含量。将诱导分泌效果佳的工程菌进行扩增、保存。
实验结果(见图13)显示葡萄糖可调控rhaFGF135的表达和分泌。
实施例17，构建并测定葡萄糖调控趋化因子CXCL12表达和分泌的益生菌药物工厂。
第一步，质粒构建。本实施例中的质粒构建详见附表1。
第二步，转化。将pXG22和pGN233电转化EcN电转感受态。
第三步，鉴定CXCL12的表达和分泌。将扩增好的工程菌接种后加入20mM葡萄糖诱导。12h后离心取上清，提取上清中的蛋白，通过western bloting检测CXCL12的含量。将诱导分泌效果佳的工程菌进行扩增、保存。
实验结果(见图14)显示葡萄糖可调控CXCL12的表达和分泌。
实施例18，验证糖尿病小鼠伤口处激活益生菌药物工厂表达和分泌人酸性成纤维生长因子rhaFGF135和趋化因子CXCL12。
第一步，细菌培养。将葡萄糖调控人酸性成纤维生长因子rhaFGF135和趋化因子CXCL12表达和分泌的益生菌及野生型EcN接种液体LB培养基，37℃、210rpm培养12h。
第二步，细胞收集(具体步骤同本发明实施例4)。
第三步，皮肤原位滴涂(具体步骤同本发明实施例4)。
第四步，皮肤组织取样。6小时后，处死小鼠，剪取小鼠伤口处皮肤。
第五步，组织切片。将皮肤组织置于4％多聚甲醛中固定24小时，流水过夜冲洗，将皮肤依次经过30％、50％、75％、85％、95％、100％的乙醇、50％乙醇和50％的二甲苯混合液中各浸泡1小时，再依次经过100％二甲苯和新的100％二甲苯中各通透5分钟。随后于50％二甲苯和50％的石蜡中浸蜡1小时，于100％石蜡中浸泡过夜，第二天转移至新的100％石蜡中浸泡1小时。使用包埋机进行包埋后于-20℃冷冻过夜。使用石蜡切片机进行组织切片。
第六步，免疫荧光染色。依次将切片放入二甲苯Ⅰ15min-二甲苯Ⅱ15min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-85％酒精5min-75％酒精5min-蒸馏水洗。组织切片置于盛满EDTA抗原修复缓冲液(pH 8.0)的修复盒中于微波炉内进行抗原修复。中火8min至沸，停火8min，再转中低火7min。自然冷却后将玻片置于PBS(pH 7.4)中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后用组化笔在组织周围画圈(防止抗体流走)，甩干PBS，滴加BSA，封闭30min。轻轻甩掉封闭液，在切片上滴加PBS按一定比例配好的一抗，切片平放于湿盒内4℃孵育过夜。玻片置于PBS(pH 7.4)中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后在圈内滴加二抗覆盖组织，避光室温孵育50min。玻片置 于PBS(pH 7.4)中在脱色摇床上晃动洗涤3次，每次5min。切片稍甩干后在圈内滴加DAPI染液，避光室温孵育10min。玻片置于PBS(pH 7.4)中在脱色摇床上晃动洗涤3次，每次5min。在圈内加入自发荧光淬灭剂5min，流水冲洗10min。切片稍甩干后用抗荧光淬灭封片剂封片。切片于荧光显微镜下观察并采集图像(DAPI紫外激发波长330-380nm，发射波长420nm，发蓝光；CY3激发波长510-560，发射波长590nm，发红光)。
实验结果(见图15)显示糖尿病小鼠伤口处可激活葡萄糖调控的益生菌药物工厂表达和分泌人酸性成纤维生长因子rhaFGF135和趋化因子CXCL12。
实施例19，调控人酸性成纤维生长因子rhaFGF135和趋化因子CXCL12表达分泌的益生菌药物工厂促进糖尿病小鼠皮肤伤口愈合的研究。
本实施例以1型糖尿病小鼠皮肤伤口为例，举例证明葡萄糖调控的益生菌药物工厂对于小鼠皮肤伤口愈合的促进，但不对本发明的保护范围有所限制。具体步骤如下：
第一步，细菌培养(具体步骤同本发明实施例8)。
第二步，细胞收集。将实例5-7筛选获得的诱导人酸性成纤维生长因子rhaFGF135和趋化因子CXCL12分泌效果佳的重组益生菌和野生型的EcN扩增培养，5000rpm离心5min，用无菌的PBS(pH 6.35)洗涤3次后重悬(109CFU/10μl)。
第三步，原位滴涂细菌。分别将PBS、人酸性成纤维生长因子rhaFGF135、野生型EcN、调控人酸性成纤维生长因子rhaFGF135表达分泌的益生菌、调控趋化因子CXCL12表达分泌的益生菌悬浮液原位滴涂至糖尿病小鼠皮肤伤口处，每只小鼠每24小时10μl(109CFU工程菌)。
第四步，检测小鼠皮肤伤口大小。每24h对小鼠皮肤伤口进行拍照，使用Image J软件统计伤口面积。
实验结果(见图16)显示原位滴涂调控rhaFGF135和CXCL12表达与分泌的益生菌可显著促进糖尿病小鼠皮肤伤口的愈合。
实施例20，益生菌药物工厂促进皮肤伤口愈合的安全性验证。
第一步，细菌培养(具体步骤同本发明实施例7)。
第二步，细胞收集(具体步骤同本发明实施例4)。
第三步，原位滴涂细菌(具体步骤同本发明实施例4)。
第四步，取样。涂菌24小时后取小鼠血液，随后处死小鼠，分别取心、肝、脾、肺、肾，加入PBS缓冲液后进行研磨。
第五步，安全验证。对小鼠血液进行血常规分析；吸取组织研磨液涂布LB平板，37℃静置培养12小时后进行菌落计数。
实验结果(见图17、图18)显示原位滴涂益生菌不影响小鼠的血液指标，且细菌不会侵染小鼠的脏器，24小时后血液中也没有残留。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。

Claims (21)

  1. 一种葡萄糖调控基因表达环路控制系统，其特征在于，其包括转录抑制子HexR，葡萄糖诱导型强启动子和待转录序列。
  2. 如权利要求1所述的葡萄糖调控基因表达环路控制系统，其特征在于，所述转录抑制子HexR可同源二聚化并与特异的DNA序列相结合，所述特异的DNA序列选自序列SEQ ID NO.78～80。
  3. 如权利要求2所述的葡萄糖调控基因表达环路控制系统，其特征在于，所述葡萄糖诱导型强启动子是由组成型强启动子与所述特异的DNA序列组合而成；所述葡萄糖诱导型强启动子包括如SEQ ID NO.1-10之任一种核苷酸序列。
  4. 如权利要求1所述的葡萄糖调控基因表达环路控制系统，其特征在于，所述转录抑制子为可结合至所述葡萄糖诱导型强启动子上并进而阻遏下游待转录序列的表达。
  5. 如权利要求1所述的葡萄糖调控基因表达环路控制系统，其特征在于，所述待转录序列包括单一编码表达报告蛋白或功能型蛋白，或串联表达多种蛋白。
  6. 如权利要求1所述的葡萄糖调控基因表达环路控制系统，其特征在于，所述转录抑制子、葡萄糖诱导型强启动子和待转录序列可构建在一个或两个质粒载体上。
  7. 一种葡萄糖诱导基因表达调控方法，其特征在于，所述方法是由如权利要求1所述的葡萄糖调控基因表达环路控制系统进行调控。
  8. 如权利要求7所述的葡萄糖诱导基因表达调控方法，其特征在于，
    当葡萄糖不存在时，所述转录抑制子可结合至所述葡萄糖诱导型强启动子上，进而阻遏下游待转录序列的表达；
    当葡萄糖存在时，葡萄糖经Entner-Doudoroff途径代谢生成代谢中间物2-keto-3-deoxy-6-phosphogluconate(KDPG)，KDPG阻断其结合，使所述转录抑制子从所述葡萄糖诱导型强启动子上解离下来，启动下游待转录序列的表达。
  9. 一种表达载体、工程化细胞、传感器或重组益生菌，其特征在于，其含有如权利要求1所述的葡萄糖调控基因表达环路控制系统。
  10. 如权利要求9所述的表达载体、工程化细胞、传感器或重组益生菌，其特征在于，所述葡萄糖调控基因表达环路控制系统位于所述工程化细胞、传感器或重组益生菌的染色体或质粒中的任意一种。
  11. 如权利要求1所述的葡萄糖调控基因表达环路控制系统，如权利要求9所述的表达载体、工程化细胞、传感器或重组益生菌在制备治疗和/或预防糖尿病的药物、制备调控血糖的药物中的应用。
  12. 如权利要求11所述的应用，其特征在于，所述应用包括口服药物。
  13. 如权利要求11所述的应用，其特征在于，所述应用包括通过调控释放胰高血糖素样肽、胰岛十二指肠同源盒-1降糖蛋白来调控血糖。
  14. 一种调控血糖的方法，其特征在于，所述方法利用如权利要求1所述的葡萄糖调控基因表达环路控制系统，和/或，利用如权利要求9所述的表达载体、工程化细胞、传感器或重组益生菌来调控血糖。
  15. 如权利要求14所述的方法，其特征在于，所述方法调控GLP-1、PDX-1降糖蛋白的表达。
  16. 如权利要求15所述的方法，其特征在于，所述方法包括：
    a)人工构建原核表达载体或用于染色体整合的功能模块；所述原核表达载体或用于染色体整合的功能模块含有葡萄糖诱导GLP-1/PDX-1表达调控系统；
    b)将步骤a)构建的表达载体或用于染色体整合的功能模块转化至微生物细胞中或整合至微生物细胞染色体上，制备含有葡萄糖诱导GLP-1/PDX-1表达调控系统的工程化细胞；
    c)将步骤b)制备的所述工程化细胞以灌胃的形式移植至糖尿病模型鼠体内；
    d)小鼠体内葡萄糖闭环诱导所述工程化细胞表达并分泌GLP-1/PDX-1，吸收至血液中以降血糖。
  17. 如权利要求14所述的方法，其特征在于，所述方法包括采用口服益生菌的方式。
  18. 如权利要求1所述的的葡萄糖调控基因表达环路控制系统，如权利要求7所述的葡萄糖诱导基因表达调控方法，如权利要求9所述的表达载体、工程化细胞、传感器或重组益生菌，如权利要求14所述的调控血糖的方法，其特征在于，所述葡萄糖调控基因表达环路控制系统具有微调性、可逆性的表达动力学特征；所述微调性是指下游的基因表达受葡萄糖的精准调控，并呈现出剂量依赖性的关系；所述可逆性是指葡萄糖调控基因表达的整个过程是可逆的，可以通过控制葡萄糖的存在与否实现基因表达的开启或关闭。
  19. 一种质粒，其特征在于，所述质粒为选自质粒pGN11、pGN69、pGN89、pGN90、 pGN227、pGN12、pGN228、pGN229、pGN220、pGN221、pGN222、pGN223、pGN224、pGN231、pGN232、pGN233、pGN65、pGN237、pGN238、pGN239、pGN241、pGN242、pGN243、pGN288、pGN308、pGN306、pGN307、pXG58之一种或几种。
  20. 一种核苷酸序列，其特征在于，其为葡萄糖诱导型强启动子PHexR1、PHexR2、PHexR3、PHexR4、PHexR5、PHexR6、PHexR7、PHexR8、PHexR9、PHexR10之一种，其核苷酸序列分别如SEQ ID NO.1～10所示。
  21. 一种引物/引物对，其特征在于，所述引物序列如SEQ ID NO.16-60、62-65、67-74、76-77所示的其中一种或几种；所述引物对的序列如编号1-30所示引物对之一种或几种。
PCT/CN2023/081232 2022-03-30 2023-03-14 葡萄糖调控基因表达环路控制系统及其调控血糖的应用 WO2023185442A1 (zh)

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