CN116925985A - Intelligent controllable microbial drug factory and application thereof in skin wound healing - Google Patents

Intelligent controllable microbial drug factory and application thereof in skin wound healing Download PDF

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Publication number
CN116925985A
CN116925985A CN202210326231.6A CN202210326231A CN116925985A CN 116925985 A CN116925985 A CN 116925985A CN 202210326231 A CN202210326231 A CN 202210326231A CN 116925985 A CN116925985 A CN 116925985A
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glucose
expression
gene expression
control system
strong promoter
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叶海峰
管宁子
高纤云
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East China Normal University
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East China Normal University
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Priority to PCT/CN2023/081260 priority patent/WO2023185443A1/en
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Abstract

The invention discloses an intelligent controllable microbial drug factory and application thereof in skin wound healing, wherein the intelligent controllable microbial drug factory comprises a gene expression loop control system and chassis probiotics. The invention also discloses a glucose regulation gene expression loop control system and application thereof in regulating blood sugar, wherein the system comprises a transcription inhibitor HexR, a glucose inducible strong promoter and a sequence to be transcribed. The glucose regulation gene expression loop control system has the characteristics of micro-regulation and reversible expression dynamics. The invention also discloses a prokaryotic expression vector, an engineering cell and application thereof in medicines for treating diabetes, promoting skin wound healing and preventing and/or treating metabolic diseases. The invention relates to the fields of synthesis biology and cell therapy, combines microorganism synthesis biology with disease treatment, and has wide application prospect.

Description

Intelligent controllable microbial drug factory and application thereof in skin wound healing
Technical Field
The invention relates to the fields of synthetic biology and cell therapy, which combines microorganism synthesis biology with disease therapy, in particular to an intelligent controllable microorganism drug factory and application thereof in skin wound healing.
Background
The biosensor can directly use the whole microbial cells or cell fragments as sensitive materials, and uses enzymes and metabolic systems in the body to identify and detect various substances. The development of synthetic biology in recent years has greatly prompted the development of biosensor functions in which various physiological indicators or disease signals, including stimuli and external signals in its host environment, can be well detected. The probiotics can specifically target the intestinal tract to be used as an intestinal tract directional administration carrier, almost all requirements of the biological therapeutic agent are met, and the development of the biosensor by utilizing the probiotics is an important means for realizing accurate medicine.
At present, the treatment mode of diabetes mainly comprises insulin injection, medicine taking, diet control and the like, but the current medical level can not thoroughly cure the diabetes, and diabetics need to orally take hypoglycemic medicines or inject insulin every day to maintain stable blood sugar. With oral or injectable administration, the active ingredients of most clinical drugs currently in use are deactivated and only a small fraction is able to act on the intended target site. If the required dosage of the drug is increased rapidly due to insufficient or even too low efficiency of the drug action, the health of the patient is often impaired and even a number of side effects occur. And the insulin cannot be controllably released by injecting the insulin, so that the risk of hypoglycemia is very easily caused. The blood glucose concentration in the human body can be automatically monitored in real time, and the blood glucose concentration can be accurately and timely adjusted, so that the blood glucose concentration monitoring device is very important for treating diabetes. Therefore, development of new therapeutic methods to improve therapeutic effects, reduce therapeutic risks, and improve therapeutic convenience is needed.
On the other hand, impaired wound healing is an increasingly serious medical problem closely related to metabolic diseases and aging. Open wounds on the skin cause serious discomfort and provide access for bacterial invasion. In the inflammatory phase of wound healing, immune cells accumulate under the action of alarm signals, cytokines and chemokines released by injured or activated cells, thus playing a vital role in wound healing. Macrophages and neutrophils are the primary immune cell population at the wound site and they can also promote the healing process by secreting additional chemokines, growth factors, matrix digestive enzymes, and the like while preventing microbial invasion.
Chronic wounds are often associated with potential pathological processes that increase wound susceptibility or decrease healing capacity, such as inadequate arterial/venous function, diabetes, or are undergoing systemic steroid therapy. Taking diabetic foot ulcers as an example, as one of the common complications of diabetes, it can lead to long-term pain, reduced activity, and even possible need for amputation. Standard treatments for chronic wounds such as diabetic foot ulcers include surgery or chemical removal of necrotic tissue, repeated dressing changes, infection control using antibiotics, and the like, with unstable results and concomitant adverse side effects. In addition, different types of chronic wounds are also treated clinically by topical application of growth factors or in combination with different biological materials, but the effect is not significant. Among them, the proteolytic nature of the wound limits the availability of the drug and is a major obstacle to chronic wound drug therapy. Therefore, there is a need to develop new therapeutic methods for stable and safe treatment of chronic skin wounds that are difficult to heal, improving the therapeutic effect and the convenience of treatment.
Disclosure of Invention
The invention provides an intelligent controllable microbial drug factory, which comprises a chassis microorganism and a gene expression loop control system uploaded into the chassis microorganism. In a specific embodiment, the gene expression loop control system is located on any one of the chromosome or plasmid of the chassis microorganism.
The intelligent controllable microbial drug factory, preferably a glucose-regulated probiotic drug factory, is an engineering cell containing a glucose-regulated gene expression loop control system. Other modes of regulation are also possible, namely, engineered cells containing other modes of regulation of the gene expression loop control system. In particular embodiments, the engineered cell may be any probiotic, such as Escherichia coli Nissle 1917 (EcN), and the like.
Wherein the gene expression loop control system comprises, but is not limited to, a glucose regulation gene expression loop control system, and gene expression loop control systems of other regulation modes, such as a xylose-induced gene expression system, an arabinose-induced gene expression system, a uric acid-induced gene expression system, and the like.
In the invention, the glucose regulation gene expression loop control system comprises a transcription inhibitor HexR, a glucose-inducible strong promoter and a sequence to be transcribed. In a specific embodiment, the transcription repressor HexR can bind to the glucose-inducible strong promoter and thereby repress the expression of the downstream sequence to be transcribed; the glucose-inducible strong promoter is formed by combining a constitutive strong promoter and a DNA sequence specifically combined with HexR; the sequence to be transcribed comprises a single coding expression report protein such as LuxCDABE (amino acid sequence Genbank accession number: EF 173694) or a functional protein such as human acidic fibroblast growth factor rhaFGF135 amino acid sequence shown as SEQ ID NO.14, a chemokine CXCL12 amino acid sequence shown as SEQ ID NO.15, a human interleukin 4 factor (hIL 4) amino acid sequence shown as SEQ ID NO.75 and the like, or multiple proteins are expressed in series. In particular embodiments, the transcriptional repressor is operably linked to the strong glucose-inducible promoter in the absence of glucose, thereby repressing expression of the downstream sequence to be transcribed; in the presence of glucose, glucose is metabolized via the Entner-Doudoroff pathway to the metabolic intermediate 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is blocked by KDG, allowing the transcription repressor to dissociate from the glucose-inducible strong promoter, initiating expression of the sequence to be transcribed downstream.
In the present invention, the gene expression loop control system comprises a transcription inhibitor, an inducible strong promoter and a sequence to be transcribed. In particular embodiments, the transcription repressor, the inducible strong promoter and the sequence to be transcribed may be constructed on one or two plasmid vectors.
In the invention, the intelligently controllable microbial pharmaceutical factory can express a target gene in the presence of an inducer. In specific embodiments, the inducer is added exogenously or is produced in vivo.
Preferably, the chassis microorganisms comprise probiotics. Preferably, the probiotics include, but are not limited to, escherichia coli Nissle 1917, lactococcus lactis, lactobacillus plantarum, bacillus subtilis, and the like. In a specific embodiment, the intelligently controllable microbiological pharmaceutical factory is recombinant probiotics.
The intelligently controllable microbial pharmaceutical factory according to the invention includes, but is not limited to, can be in a liquid or freeze-dried, spray-dried.
The invention also provides a construction method of the intelligent controllable microbial drug factory, which comprises the following steps:
a) Combining a DNA operator sequence specifically recognized by the transcription repressing protein with a constitutive strong promoter to construct an inducible strong promoter;
b) Constructing a coding gene for expressing the drug protein to the downstream of an inducible promoter in a single or tandem mode, and constructing a report module; wherein the types and the quantity of the drug proteins can be correspondingly replaced and changed according to actual demands;
c) Functional modules are constructed that constitutively express the transcriptional repressor protein.
The invention provides a construction method of an intelligent controllable microbial drug factory, which is characterized in that plasmids containing functional modules and report modules constructed in the step b) and the step c) are transformed into probiotic competent cells, or the functional modules and the report modules constructed in the step b) and the step c) are integrated onto probiotic chromosomes through an RED recombinase system or a CRISPR system, so as to prepare inducible engineering cells, namely the intelligent controllable microbial drug factory.
The invention also provides an expression vector and an engineering cell, which contain the intelligent controllable microbial drug factory. In particular, a prokaryotic expression vector containing a glucose regulatory gene expression loop control system.
The invention also provides application of the intelligent controllable microbial drug factory in preparing drugs or products for preventing and/or treating diseases. Such drugs or products include, but are not limited to, drugs that promote healing of skin wounds, drugs that prevent and/or treat metabolic disorders, drugs that prevent and/or treat other disorders, and the like.
In particular embodiments, the skin wound includes, but is not limited to, chronic skin wounds, diabetic foot ulcers, and the like.
The invention also provides application of the intelligent controllable microorganism (preferably probiotics) pharmaceutical factory in promoting healing of diabetic skin wounds. The application of promoting the wound healing of the diabetic skin is realized by a method of dripping an intelligent controllable probiotic medicine factory on the wound in situ, namely, by a method of constructing the intelligent controllable probiotic medicine factory to induce and generate growth factors, cytokines and chemokines through blood sugar, namely, the treatment method is realized by utilizing the intelligent controllable probiotic medicine factory to accurately regulate and control the expression and secretion of the growth factors, the cytokines and the chemokines so as to promote the wound healing.
The invention also proposes a method for promoting skin wound healing using said intelligently controllable microbiological (probiotic) pharmaceutical factory. The method comprises the following steps:
a) Artificially constructing a prokaryotic expression vector containing an expression regulation system of glucose-induced growth factors, macrophage M2 polarized cytokines and chemokines or a functional module for chromosome integration;
b) Preparing an engineering cell containing glucose-induced growth factors, macrophage M2 polarized cytokines and a chemokine expression control system;
c) The engineering cells prepared in the step b) are transplanted to skin wounds of diabetic model mice in the form of in-situ wound dripping;
d) Glucose closed-loop induction engineering cells at the skin wound of the mouse express and secrete growth factors, macrophage M2 polarized cytokines and chemokines so as to achieve the effect of promoting wound healing.
Wherein the growth factors are rhaFGF135, FGF2, FGF7, TGF-alpha, TGF-beta, PDGF, EGF, VEGF, IGF-1, IGF-2, PDGF, HGF; the macrophage M2 polarization cytokine is IL4, IL-10, IL-13, CSF1, IL34; the chemokines are CXCL12, CXCL13, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11 and CXCL10.
The invention also provides a plasmid, which is one or more of plasmids pGN233, pGN65, pGN299, pGN300 and pGN314, and the details are shown in the table 1.
The invention also provides a nucleotide sequence which is a glucose inducible strong promoter P HexR4 、P HexR9 One of which has the sequence shown in SEQ ID NO.4 and SEQ ID NO. 9.
The invention also provides a primer, and the primer sequence is one or more of SEQ ID NO.16-60, 62-65, 67-74 and 76-77. The invention also provides a primer pair, and the sequence of the primer pair is one or more than one of the primer pairs shown in the numbers 1-30. As shown in tables 1 and 2.
The invention also provides a glucose regulation gene expression loop control system, which comprises a transcription inhibitor HexR, a glucose inducible strong promoter and a sequence to be transcribed. Wherein the transcription repressor HexR is homodimerized and binds to a specific DNA sequence selected from the sequences SEQ ID NO. 78-80. Wherein the glucose-inducible strong promoter is formed by combining a constitutive strong promoter with the specific DNA sequence; including but not limited to any one of the nucleotide sequences set forth in SEQ ID NO. 1-10.
Wherein the transcriptional repressor is capable of binding to the glucose inducible strong promoter and thereby repressing expression of the downstream sequence to be transcribed. The sequence to be transcribed comprises single encoding of an expressed reporter protein or a functional protein, or tandem expression of multiple proteins. The transcription repressor, the glucose-inducible strong promoter and the sequence to be transcribed may be constructed on one or two plasmid vectors.
The invention also provides a glucose-induced gene expression regulation method, which is regulated and controlled by the glucose-induced gene expression loop control system. In particular embodiments, the transcriptional repressor may bind to the glucose-inducible strong promoter in the absence of glucose, thereby repressing expression of the downstream sequence to be transcribed; in the presence of glucose, glucose is metabolized via the Entner-Doudoroff pathway to the metabolic intermediate 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is blocked by KDG, allowing the transcription repressor to dissociate from the glucose-inducible strong promoter, initiating expression of the sequence to be transcribed downstream. In a specific embodiment, the manner of controlling the induction of gene expression is shown in FIG. 1.
In the invention, the glucose regulation gene expression loop control system consists of three parts, namely a glucose precise regulation gene, namely the expression and secretion of the glucose precise regulation gene, a transcription inhibitor HexR, a glucose inducible strong promoter and a sequence to be transcribed.
Wherein, the transcription inhibitor HexR is a repressor HexR of HexR operon system derived from Pseudomonas putida (amino acid sequence Genbank accession number: AE 015451), and is continuously expressed by a strong promoter.
Preferably, the transcription repressor HexR can be expressed by a different kind of strong promoter including but not limited to P BBA23100 (nucleotide sequence Genbank accession number: MG 649435), P Lac (nucleotide sequence Genebank accession No. LC 652750), P Tac (its nucleotide sequence Genebank accession)Recording: MN 913428), and the like.
Wherein, the glucose inducible strong promoter is formed by inserting HexR operon binding sites with different copy numbers between the strong promoter and RBS sites; the HexR operator binding site is derived from the operator element HexO of the HexR operator system. The glucose-inducible strong promoter can drive the expression of downstream genes. The HexR operon system consists of the repressor protein HexR and the operon element HexO, which was originally isolated from Pseudomonas putida and is tightly regulated by the glucose metabolite 2-keyo-3-deoxy-6-phosphogluconate (KDPG).
Preferably, the glucose-inducible strong promoter may constitute different types of fusion-type strong promoters according to the species of the strong promoter and different copy numbers of the operon HexO, including: a) Glucose inducible strong promoter P as shown in SEQ ID NO.1 HexR1 Nucleotide sequence P HexR1 (P Tac -HexO); b) Glucose inducible strong promoter P as shown in SEQ ID NO.2 HexR2 Nucleotide sequence P HexR2 (P Tac -2 x HexO); c) Glucose inducible strong promoter P as shown in SEQ ID NO.3 HexR3 Nucleotide sequence P HexR3 (P Tac -3 x HexO); d) Glucose inducible strong promoter P as shown in SEQ ID NO.4 HexR4 Nucleotide sequence P HexR4 (P Tac -4 x HexO); e) Glucose inducible strong promoter P as shown in SEQ ID NO.5 HexR5 Nucleotide sequence P HexR5 (P Tac -5 x HexO); f) Glucose inducible strong promoter P as shown in SEQ ID NO.6 HexR6 Nucleotide sequence P HexR6 (P Lac -HexO); g) Glucose inducible strong promoter P as shown in SEQ ID NO.7 HexR7 Nucleotide sequence P HexR7 (P Lac -2 x HexO); h) Glucose inducible strong promoter P as shown in SEQ ID NO.8 HexR8 Nucleotide sequence P HexR8 (P Lac -3 x HexO); i) Glucose inducible strong promoter P as shown in SEQ ID NO.9 HexR9 Nucleotide sequence P HexR9 (P Lac -4 x HexO); j) Glucose inducible strong promoter P as shown in SEQ ID NO.10 HexR10 Nucleotide sequence P HexR10 (P Lac -5 XHexO) and glucose inducible promoter P consisting of any constitutive promoter and HexO in any copy number HexRn 。
Wherein the sequence to be transcribed can be a single coding expression reporter protein such as sfGFP (amino acid sequence Genbank accession number: AB 971579), luxCDABE (amino acid sequence Genbank accession number: EF 173694) or a glucagon-like peptide GLP-1 amino acid sequence shown in SEQ ID NO.11 of a functional protein, a glucagon-like peptide EK-GLP-1 amino acid sequence shown in SEQ ID NO.12, a glucagon-like peptide GLP-1-Fc amino acid sequence shown in SEQ ID NO.13, a murine islet duodenal homology box-1 (mPDX-1) amino acid sequence shown in SEQ ID NO.61, a human-derived islet homology box-1 (hP-1) amino acid sequence shown in SEQ ID NO.66, and the like.
The glucose regulation gene expression loop control system can accurately regulate the expression of one or multiple reporter proteins and functional proteins connected in series in a polycistron mode.
Wherein, the glucose regulation gene expression loop control system is loaded by an artificially designed and synthesized double-plasmid system, and the related sequences in the double-plasmid system are shown in Table 1 in detail.
The invention also provides an expression vector, an engineering cell, a sensor or a recombinant probiotics, which contains the glucose regulatory gene expression loop control system. In specific embodiments, the glucose regulatory gene expression loop control system is located on any one of the chromosomes or plasmids of the engineered cell, sensor or recombinant probiotic.
The invention also provides an application of the glucose regulation gene expression loop control system, namely an application of the expression vector, the engineering cell, the sensor or the recombinant probiotics in preparing medicines for treating and/or preventing diabetes and medicines or products for regulating and controlling blood sugar.
In particular embodiments, the medicament or product includes, but is not limited to, various dosage forms such as oral, spread, injection, and the like. Preferably, oral administration is used. In specific embodiments, the regulation of blood glucose is achieved by orally administering recombinant probiotics (i.e., intelligently controllable microbial pharmaceutical factories) containing the glucose-regulated gene expression loop control system, by glucose-induced gene expression regulation, expressing a hypoglycemic peptide. That is, by orally administering the recombinant probiotic (engineered intelligent controlled probiotic pharmaceutical factory) to achieve the effect of producing glucagon-like peptide by blood glucose induction, the expression of GLP-1 or/and PDX-1 can be precisely regulated, and the effect of treating diabetes can be achieved. Specifically, in the application and the treatment method, the blood sugar is regulated and controlled by regulating and releasing glucagon-like peptide and the glucose-lowering protein of the islet-duodenum homologous box-1, so that the blood sugar level is regulated and controlled.
The invention also provides a method for regulating and controlling blood sugar, namely, the glucose regulating and controlling gene expression loop control system is utilized, and/or the expression vector, the engineering cell, the sensor or the recombinant probiotics and the like are utilized for regulating and controlling the blood sugar. In a specific embodiment, the method realizes the effect of regulating blood sugar by regulating the expression of GLP-1 and PDX-1 hypoglycemic proteins. Specifically, the method for regulating and controlling blood sugar comprises the following steps:
a) Artificially constructing a prokaryotic expression vector or a functional module for chromosome integration; the prokaryotic expression vector or the functional module for chromosome integration contains a glucose-induced GLP-1/PDX-1 expression regulation system;
b) Transforming the expression vector constructed in the step a) or the functional module for chromosome integration into a microbial cell or integrating the expression vector into a microbial cell chromosome to prepare an engineering cell containing a glucose-induced GLP-1/PDX-1 expression regulation system;
c) Transplanting the engineered cells prepared in the step b) into a diabetic model mouse in a gastral mode;
d) Glucose in mice is closed-loop to induce the engineered cells to express and secrete GLP-1/PDX-1, which is absorbed into the blood to reduce blood glucose.
In particular embodiments, the method of regulating blood glucose may be by way of oral administration of probiotics.
The invention also provides a novel method for treating diabetes mellitus, and the effect of reducing blood sugar can be achieved by adopting an oral probiotic mode through the intelligent controllable probiotic drug factory and the glucose-induced GLP-1 or/and PDX-1 expression control system. The invention provides a novel strategy for treating diabetes by safely, reliably and accurately regulating and releasing glucagon-like peptide or/and islet duodenal homologous box-1. The invention provides a novel method and a novel strategy for treating diabetes. The system can regulate and control the expression of glucagon-like peptide GLP-1 and islet duodenal homologous box-1 PDX-1. The expression of the glucagon-like peptide GLP-1 comprises EK-GLP-1, GLP-1-Fc and the like. The expression of the islet duodenal homologous box-1 PDX-1 comprises mPDX-1, hPDX-1 and the like. The glucose regulation gene expression loop control system can rapidly regulate gene expression through glucose metabolites, and has the characteristics of precisely controlling gene expression quantity, regulating gene expression multiple and the like.
The glucose regulation gene expression loop control system has the expression dynamics characteristics of micromanipulation and reversibility. The fine tuning means that the downstream gene expression is precisely regulated and controlled by glucose and shows a dose-dependent relationship; the reversibility means that the whole process of regulating the gene expression by glucose is reversible, and the opening or closing of the gene expression can be realized by controlling the existence or non-existence of glucose. And the expression dynamics characteristics with micro-regulation and reversibility are also embodied in the glucose-induced gene expression regulation and control method, the blood sugar regulation and control method, the expression vector, the engineering cell, the sensor or the recombinant probiotics and the like.
The invention also provides a plasmid, which is one or more selected from plasmids 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 and pXG. The plasmids are detailed in Table 1.
The invention also provides a nucleotide sequence which is a glucose inducible strong promoter P HexR1 、P HexR2 、 P HexR3 、P HexR4 、P HexR5 、P HexR6 、P HexR7 、P HexR8 、P HexR9 、P HexR10 One of which has a nucleotide sequence shown in one of SEQ ID NO. 1-10, respectively.
The beneficial effects of the invention include that the invention provides a novel method for promoting the healing of chronic skin wounds, and the effect of accelerating the healing of wounds can be achieved by adopting a wound in-situ Tu Yisheng bacteria dripping mode through the intelligent controllable probiotic drug factory, the glucose-induced growth factor, the cytokine and the chemokine expression regulation system. The invention provides a novel strategy for accelerating the healing of chronic skin wounds such as chronic skin wounds, diabetic foot ulcers and the like by safely, reliably and accurately regulating and releasing therapeutic factors. The system can regulate the expression and secretion of growth factors, macrophage M2 polarized cytokines and chemokines. The invention innovatively develops an optimized technology for directly transmitting growth factors, macrophage M2 polarized cytokines and chemokines to injured skin, wherein probiotics are used as vectors, and plasmids or chromosome genomes are used for encoding the growth factors, the macrophage M2 polarized cytokines and the chemokines.
The beneficial effects of the invention include providing an intelligent controllable probiotic drug factory, comprising a gene expression loop control system and chassis probiotics. The glucose regulation gene expression loop control system has the expression dynamics characteristics of micromanipulation and reversibility. The invention also provides application of the intelligent controllable probiotic pharmaceutical factory or prokaryotic expression vector containing a glucose regulation gene expression loop control system or engineering cells containing the glucose regulation gene expression loop control system in medicines for treating diabetes, promoting chronic skin wound healing such as diabetic foot ulcers and the like, and preventing and/or treating metabolic diseases.
Drawings
FIG. 1 is a schematic diagram of a glucose regulatory gene expression loop control system and a regulatory method according to the present invention.
FIG. 2 is a graph showing the optimization of the control system of the glucose regulatory gene expression loop of the present invention, i.e., the use of different promoter-start tablesExpression vectors reaching HexR and glucose inducible strong promoter P respectively HexR1 Combined experimental results were performed.
FIG. 3 is a schematic diagram of the optimization study of the glucose regulatory gene expression loop control system of the present invention, i.e., using P Tac Experimental results for the initiation of expression vectors expressing HexR in combination with 5 different response elements, respectively.
FIG. 4 is an optimization study of the expression loop control system of the glucose regulatory gene of the present invention, namely, using RBS-regulated HexR expression vectors and glucose inducible strong promoter P, respectively HexR4 Combined experimental results were performed.
FIG. 5 is an experimental result of the expression dynamics characteristic of the glucose regulatory gene expression loop control system of the present invention with fine-tuning.
FIG. 6 is an experimental result of the expression dynamics characteristic of the glucose regulatory gene expression loop control system of the present invention with reversibility.
FIG. 7 is a graph showing the experimental results of the influence of different glucose induction times on the gene expression in the glucose regulatory gene expression loop control system of the present invention.
FIG. 8 is an experimental result of the glucose regulatory gene expression loop control system of the present invention for regulating the expression of reporter gene luxCDABE in wild type mice and type 1 diabetic mice.
FIG. 9 is the experimental results of a probiotic pharmaceutical factory for treating type 1 diabetic mice, which regulates expression and secretion of glucagon-like peptide of the present invention.
FIG. 10 is the experimental results of the long-term control of blood glucose in type 1 diabetic mice by a probiotic pharmaceutical factory of the present invention that regulates the expression and secretion of glucagon-like peptide.
FIG. 11 is an experimental result of glucose tolerance in a glucose-regulated probiotic pharmaceutical factory of the present invention during the treatment of type 1 diabetes.
FIG. 12 is an experimental result of the glucose regulatory gene expression loop control system of the present invention regulating the expression of reporter gene luxCDABE at skin wounds of diabetic mice.
FIG. 13 shows the experimental results of the expression and secretion of human acidic fibroblast growth factor rhaFGF135 induced by glucose control in the probiotic pharmaceutical factory according to the present invention.
FIG. 14 is an experimental result of the expression and secretion of chemokine CXCL12 induced by glucose control in a probiotic pharmaceutical factory according to the invention.
FIG. 15 is a graph showing the experimental results of the factory expression and secretion of human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12 by a probiotic drug for wound site activation in diabetic mice in accordance with the present invention.
FIG. 16 is a graph showing the experimental results of the glucose-regulated probiotic drug factory of the present invention for promoting skin wound healing in diabetic mice.
FIG. 17 is a graph showing blood index of mice after the in situ wound drip Tu Yisheng bacterial drug factory of the present invention.
Figure 18 shows the residue of recombinant probiotics in the individual organs and blood of mice after in situ wound drip Tu Yisheng bacterial drug factory according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings. These examples are only for illustrating the invention and do not limit the scope of the invention in any way. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited. The reagents, instruments, etc. used in the following examples were carried out according to the conditions suggested by the conventional or commercial suppliers, without specifying the specific conditions.
Molecular cloning
The molecular cloning technique constructs all expression plasmids of the invention, and the steps are common knowledge in the industry.
All primers used for PCR were synthesized by Jin Weizhi Biotechnology Co. The expression plasmids constructed in the examples of the present invention were all subjected to sequence determination, which was performed by Jin Weizhi biotechnology, inc. The Phanta Max Super-Fidelity DNA polymerase used in the examples of the present invention was purchased from Nanjinopran Biotechnology Co., ltd. Endonucleases, T4 DNA ligase were purchased from TaKaRa. Homologous recombinases were purchased from Shanghai Biotechnology (Shanghai) Inc. The Phanta Max Super-Fidelity DNA polymerase was purchased with the corresponding polymerase buffer and dNTPs. Endonucleases, T4 DNA ligase and homologous recombinases were purchased with corresponding buffers. Yeast Extract (Yeast Extract), tryptone (Trypton), agar powder, M9 medium, glucose, ampicillin (Amp), kanamycin (Kan) were purchased from Shanghai Biotechnology Co., ltd. DNA Marker DL5000, DNA Marker DL2000 (Takara Bio Inc.); nucleic acid dye EB (austic biotechnology company, guangdong); plasmid small extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.); DNA gel recovery kit and PCR product purification kit are all purchased from century biotechnology Co., ltd; the rest reagents such as absolute ethyl alcohol, naCl and the like mentioned in the examples are all domestic analytically pure products. The gel recovery, purification and recovery of the DNA fragment are carried out according to the operation instruction of a DNA gel recovery kit and a PCR product purification kit (Kangji Biotechnology Co., ltd.); plasmid extraction procedure the kit instructions were extracted according to plasmid xiaozhu (Tiangen Biochemical technology (Beijing) Co., ltd.).
Bacterial culture and transformation
The following bacterial chassis cells and electrotransformation are used as examples to illustrate the operation of the glucose regulatory gene expression loop control system in prokaryotic cells and animals, but the scope of the invention is not limited thereto.
Bacterial culture: coli EcN was cultured in LB medium, to which 100. Mu.g/ml ampicillin and 50. Mu.g/ml kanamycin solution were added; bacteria were cultured in a shaker at 37℃and 210 rpm.
Preparation of E.coli EcN competent cells: all solutions and consumables used for competent cell preparation were subjected to high temperature autoclaving. Streaking the escherichia coli EcN strain on a flat plate without antibiotics, and culturing for 12-16 hours at 37 ℃ in an inverted way; a single colony was picked up and cultured overnight at 37℃in 2mL LB shake tubes without antibiotics at 210 rpm. 1mL of bacterial liquid is absorbed and transferred into 100mL of fresh LB culture medium, and shake culture is carried out at 37 ℃ and 210rpm until OD 600 Between 0.4 and 0.6. Transferring the culture solution into a centrifuge tube, standing on ice for 15min, and isolating at 4deg.C and 3500rpmHeart for 10min, discard supernatant, sequentially use 50 mL and 25mL pre-chilled ddH 2 O was resuspended, centrifuged at 3500rpm for 10min at 4℃and 25mL of pre-chilled 10% glycerol was resuspended, and centrifuged at 3500rpm for 10min at 4 ℃. The supernatant was discarded, resuspended in pre-chilled 10% glycerol, and sub-packaged (100. Mu.l/tube), stored at-80 ℃.
Conversion: the EcN conversion uses an optimized electroconversion process. Briefly, a mixture of pre-chilled plasmid (300-500 ng) and competent (100. Mu.l) was added to the bottom of the electrocuvette, and the electrotransformation parameters were set to 2mm,2500V, and shocked once. After the electric shock was completed, 900. Mu.l of the culture medium was added to an electric rotating cup, the mixture was aspirated and placed in a sterilized EP tube, incubated at 37℃and 210rpm for 1 hour, and the cells were plated on a resistance plate (100. Mu.g/ml Amp and 50. Mu.g/ml Kan) and incubated at 37℃for 16 to 20 hours.
Detection of reporter Green fluorescent protein (sfGFP)
The green fluorescent protein sfGFP has macroscopic fluorescence under blue light, and its fluorescence intensity can be measured with a microplate reader Synergy H1. 100 μl of bacterial suspension is sucked into a black 96-well ELISA plate, and is placed into an ELISA apparatus, and under the action of excitation light with the wavelength of 480nm, the reading of the emitted light of the bacterial suspension at 520nm is detected, namely the green fluorescence intensity. And simultaneously sucking 100 mu l of bacterial suspension into a transparent 96-well ELISA plate, and detecting the absorbance value of the bacterial culture solution at 600nm by using an ELISA instrument to obtain the bacterial cell density. The expression efficiency of the reporter gene was characterized by fluorescence intensity/cell density.
Secreted protein extraction
8ml of methanol, 2ml of chloroform and 8ml of water are respectively added into 2ml of bacterial culture supernatant, after uniform mixing, 12000g of the supernatant is centrifuged for 10min, 8ml of methanol is added, after uniform mixing, 12000g of the supernatant is centrifuged for 15min, the supernatant is discarded, and 30 mu l of PBS buffer solution is added for re-suspension precipitation after air drying.
Protein immunoblotting (Western blotting)
Protein samples were added to 10. Mu.l of 4 Xprotein loading buffer and subjected to SDS-PAGE. Transfer membrane (250 mA,1-1.5 h) was performed using wet electroblotting, blocking with TBST+5% nonfat dry milk for 1.5h, adding primary antibody after 3 TBST washes, incubating overnight at 4deg.C, adding secondary antibody after 3 TBST washes, incubating at room temperature for 2h, and performing development detection using a gel imaging system.
Example 1 construction of a glucose regulatory Gene expression Loop control System
The embodiment comprises a construction method of a plasmid vector related to a glucose regulatory gene expression loop control system. The detailed design scheme and steps are shown in table 1.
TABLE 1 plasmid construction Table
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Primer pairs (numbers 1-30) as shown in Table 2:
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example 2 optimization of the glucose regulatory Gene expression Loop control System in EcN, i.e., expression vectors that utilize different promoters to promote expression of HexR, and glucose-inducible strong promoter P, respectively HexR1 And (5) performing combination optimization.
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.
And in the second step, conversion. The transformation systems of this example can be divided into 4 groups, including pGN11, pGN11 and pGN69, pGN11 and pGN89, pGN11 and pGN90. Each of the above groups of plasmids was electrotransformed EcN electrotransformed competent.
And thirdly, culturing. Positive monoclonal transfer liquid LB medium was selected and cultured at 37℃and 210rpm for 12 hours.
Fourth, the expression level of the reporter gene sfGFP is detected. 100. Mu.L of bacterial suspension was pipetted into a black 96-well microplate and a transparent 96-well microplate, and the fluorescence intensity and the cell density of the cells were measured using an microplate reader. The expression efficiency of the reporter gene was characterized by fluorescence intensity/cell density.
The experimental results (see FIG. 2) show that the expression of the reporter gene sfgfp can be inhibited after exogenously expressing the HexR repressor protein, wherein the Tac promoter is most strongly repressed the expression of the reporter gene after the HexR (pGN 89) is expressed.
Example 3 optimization study of the glucose regulatory Gene expression Loop control System in EcN, i.e., using P Tac Expression vectors that initiate expression of HexR were optimized in combination with 5 different response elements, respectively.
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.
And in the second step, conversion. The transformation systems of this example can be divided into 5 groups, including pGN89 and pGN11, pGN89 and pGN227, pGN89 and pGN12, pGN89 and pGN228, pGN89 and pGN229. Each of the above groups of plasmids was electrotransformed EcN electrotransformed competent.
Third, culture (the specific procedure is the same as in example 2).
Fourth, induction. Centrifuging at 5000rpm for 5min, blowing with equal amount of M9 culture medium, and packaging into 48-well plate according to culture volume of 500 μl. Different experimental groups were induced by adding sterile glucose solutions of the corresponding concentrations, and were cultured at 37℃with shaking at 150rpm for 12h.
Fifth, the expression level of sfGFP as a reporter gene was examined (the procedure is the same as in example 2 of the present invention).
The experimental results (see fig. 3) show that different optimized combinations of the glucose regulatory gene expression loop control system can activate the expression of the reporter gene sfGFP, but the induction effects generated by the systems are different, wherein the combined induction times of pGN89 and pGN228 are optimal.
Example 4 optimization study of the glucose regulatory Gene expression Loop control System in EcN, i.e., expression vector for controlling HexR Using RBS and glucose inducible strong promoter P, respectively HexR4 And (5) performing combination optimization.
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.
And in the second step, conversion. The transformation systems of this example can be divided into 4 groups, including pGN89 and pGN228, pGN231 and pGN228, pGN232 and pGN228, pGN233 and pGN228. Each of the above groups of plasmids was electrotransformed EcN electrotransformed competent.
In the third step, the culture was carried out (the specific procedure is the same as in example 2 of the present invention).
Fourth, induction (specific procedure is the same as in example 3).
Fifth, the expression level of sfGFP as a reporter gene was examined (the procedure is the same as in example 2 of the present invention).
The experimental results (see fig. 4) show that different optimized combinations of the glucose regulatory gene expression loop control system can activate the expression of the reporter gene sfGFP, but the induction effects generated by the systems are different, wherein the combined induction times of pGN233 and pGN228 are optimal.
Example 5 study of expression dynamics of glucose regulatory Gene expression Loop control System with micro-regulation.
Firstly, inoculating and culturing the engineering bacteria with the optimal induction times obtained in the embodiment 4, centrifuging at 5000 rpm for 5min, blowing and beating the engineering bacteria uniformly with an equal amount of M9 culture medium by using a gun head, and sub-packaging the engineering bacteria into 48 pore plates according to a culture volume of 500 mu L. Different experimental groups were induced by adding sterile glucose solutions of different final concentrations, and were cultured at 37℃with shaking at 150rpm for 12h.
In the second step, the expression level of sfGFP as a reporter gene was examined (the specific procedure is the same as in example 2 of the present invention).
The experimental result (see figure 5) shows that the engineering bacteria of the glucose regulation gene expression loop control system are accurately regulated by glucose, namely, the gene expression is induced in a dose-dependent manner, so that the engineering bacteria have the expression dynamics characteristics of the micro-regulation.
Example 6 expression kinetics characterization of the glucose regulatory Gene expression Loop control System with reversibility.
Firstly, inoculating and culturing the engineering bacteria with the optimal induction times obtained in the embodiment 4, centrifuging at 5000 rpm for 5min, blowing and beating the engineering bacteria uniformly with an equal amount of M9 culture medium by using a gun head, and sub-packaging the engineering bacteria into 48 pore plates according to a culture volume of 500 mu L.
Second, the "ON-OFF-ON" experimental group was induced by adding a sterile glucose solution with a final concentration of 20mM at 0 h; 2h, centrifugally washing the original culture medium, adding an M9 culture medium without glucose, and carrying out resuspension culture; at 7h, the medium was washed off by centrifugation and resuspended in M9 medium containing a final concentration of 20mM glucose. The "OFF-ON-OFF" experimental group was induced by adding no glucose for 0 h; 2h, adding a sterile glucose solution with a final concentration of 20mM for induction; and 7h, centrifuging to wash the original culture medium, and adding an M9 culture medium without glucose for resuspension culture. Both experimental groups were aspirated at 1h intervals to detect the expression level of the reporter gene sfGFP (the specific procedure is the same as in example 2 of the present invention).
The experimental result (see figure 6) shows that the on-off of the system gene expression can be realized by controlling the existence of glucose, which indicates that the glucose regulation gene expression loop control system has good reversibility.
Example 7 study of the control of the gene expression by different induction times of glucose in a glucose regulatory gene expression loop control system.
Firstly, inoculating and culturing the engineering bacteria with the optimal induction times obtained in the embodiment 4, centrifuging at 5000 rpm for 5min, blowing and beating the engineering bacteria uniformly with an equal amount of M9 culture medium by using a gun head, and sub-packaging the engineering bacteria into 48 pore plates according to a culture volume of 500 mu L. The different experimental groups were incubated with sterile glucose solution at a final concentration of 20mM at 37℃and shaking at 150rpm for different times (0 h, 2h, 4h, 6h, 8h, 10h, 12 h).
In the second step, the expression level of sfGFP as a reporter gene was examined (the specific procedure is the same as in example 2 of the present invention).
The experimental result (see fig. 7) shows that the gene expression amount of the system is regulated and controlled by the glucose induction time, namely, the glucose regulation gene expression loop control system has good controllability.
Example 8 model mice for type 1 diabetes were constructed using the Streptozotocin (STZ) modeling method.
In the first step, fasting. Prior to dosing, 40C 57BL/6J mice weighing around 25g were selected for fasting for up to 16 hours.
In the second step, administration is performed. STZ was dissolved in a citric acid buffer (0.1 mol/L, pH 4.5), and then the mice were intraperitoneally injected at a dose of 40-50mg/kg, and the injection was continued for 5 days. Because STZ is easy to degrade, the whole process needs to ensure that the medicine is in a low-temperature light-shielding state, and the injection process needs to be rapid.
Third, the blood glucose level is measured. On day 9, blood glucose levels were measured 4 hours after starvation of the mice, and mice with blood glucose levels above 16.7mM were considered successful in molding.
Example 9 expression of the glucose regulatory Gene expression Loop control System expression studies of the reporter gene luxCDABE in type 1 diabetic mice.
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.
And in the second step, conversion. pGN65 and pGN233 were electrotransformed EcN electrotransformed competent.
In the third step, the culture was carried out (the specific procedure is the same as in example 2 of the present invention).
Fourth, cells are collected. Centrifugation at 5000rpm for 5min, washing 3 times with sterile PBS followed by resuspension (10 9 CFU/100μl)。
Fifth, the stomach is irrigated. Bacterial suspensions were respectively gavaged into wild type and type 1 diabetic mice (4 h fasted) with 100 μl (10 9 CFU engineering bacteria).
Sixth, the expression level of luxCDABE in mice is detected. After 6h of gastric lavage, the bioluminescence signal was detected by a small animal biopsy imager.
The experimental results (see FIG. 8) show that the glucose regulatory gene expression loop control system is capable of activating gene expression in type 1 diabetic mice, but not in wild type mice.
Example 10 engineering bacteria for glucose regulation of glucagon-like peptide expression and secretion were constructed and assayed.
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.
In the second step, the transformation (specific procedure is the same as in example 2 of the present invention). The transformation systems of 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 of the above groups of plasmids was electrotransformed EcN electrotransformed competent.
Third, the expression and secretion of glucagon-like peptides is identified. And inoculating the amplified engineering bacteria, and adding 20mM glucose for induction. After 12h, the content of intracellular and extracellular glucagon-like peptide was detected. Amplifying and preserving engineering bacteria with good secretion inducing effect.
Example 11 study of probiotics regulating glucagon-like peptide expression secretion in mice treated for type 2 diabetes (db).
In this example, a type 2 diabetic mouse is taken as an example, and the closed-loop treatment function of the probiotic glucose sensor on diabetes is demonstrated, but the protection scope of the invention is not limited. The method comprises the following specific steps:
in the first step, cells are collected. Amplifying and culturing the engineering bacteria with good glucagon-like peptide secretion induction effect obtained by screening in the example 10 and the engineering bacteria for regulating and controlling the expression of the reporter gene luxCDABE obtained by screening in the example 9, centrifuging at 5000rpm for 5min, washing 3 times by using sterile PBS, and re-suspending (10 9 CFU/100 μl)。
And secondly, stomach irrigation. The PBS and the two engineering bacteria suspensions amplified in the step one were respectively fed to the bodies of type 2 diabetic mice (fasted for 4 hours), 100. Mu.l (10 9 CFU engineering bacteria).
Third, the blood sugar of the mice is detected. After gastric lavage for 24h, mice were tested for blood glucose using a blood glucose test strip (mice fasted for 4 h). Wild-type mice were also tested for blood glucose as a control.
The experimental results (see fig. 9) show that oral administration of probiotics that regulate glucagon-like peptide expression and secretion can reduce blood glucose levels in type 2 diabetic mice.
Example 12 study of the long-term efficacy of probiotics regulating glucagon-like peptide expression and secretion in treating type 2 diabetic mice.
In the first step, cells were collected (the specific procedure is the same as in example 11 of the present invention).
And secondly, stomach irrigation. The procedure of example 11 of the present invention was followed for each 24h for 15 consecutive days.
In the third step, blood glucose in mice was measured (the procedure is the same as in example 11 of the present invention).
The experimental results (see fig. 10) show that probiotics that regulate glucagon-like peptide expression and secretion orally can maintain blood glucose homeostasis in type 2 diabetic mice for a long period of time.
Example 13 glucose-regulated probiotic pharmaceutical factory sugar tolerance study during the treatment of type 2 diabetes.
This example was developed after treatment of type 2 diabetes model mice in accordance with example 12 of the present invention, and the specific experimental procedure for glucose tolerance was as follows:
in the first step, the model mice were fasted for 16 hours.
In the second step, 125mg/ml glucose solution was prepared.
In the third step, 0 point blood glucose was measured in mice and intraperitoneal injection was performed at a glucose dose of 1.25 g/kg. Then, blood glucose values of the mice at 30, 60, 90, 120min were measured sequentially.
The experimental results (see fig. 11) show that compared with the control group, the hyperglycemia of the treatment group is well improved and controlled, namely, the probiotics for regulating and controlling the expression and secretion of glucagon-like peptide have remarkable effect on the treatment of type 2 diabetes.
Example 14 skin wound modeling of diabetic mice.
First, anesthesia. 40 model mice with type 1 diabetes were selected and anesthetized with the gas anesthetic isoflurane using inhalation anesthesia.
And secondly, dehairing. The hair on the back of the mice is removed by using small animal electric hair clippers and depilatory cream, and the mice are wiped clean by using alcohol cotton.
Third step, full-layer leatherSkin loss. Two identical circular areas (about 30mm in area) were marked on the dehairing sites on both sides of the dorsal vertebra of the mice 2 ) The skin and subcutaneous tissue are cut down the mark to the fascia layer, and the two resected portions are separated by the intact skin. The mice were grouped for dorsal wounds using a random grouping method.
Example 15 expression studies of the reporter luxCDABE in glucose regulated probiotic drug factories at skin wounds of type 1 diabetic mice.
In the first step, conversion is performed. pGN65 and pGN233 were electrotransformed EcN electrotransformed competent.
And secondly, culturing. Positive monoclonal transfer liquid LB medium was selected and cultured at 37℃and 210rpm for 12 hours.
Third, cells were collected. Centrifugation at 5000rpm for 5min, washing 3 times with sterile PBS followed by resuspension (10 9 CFU/10μl)。
Fourth, the skin is dripped in situ. Bacterial suspensions were applied drop wise to skin wounds in type 1 diabetic mice, 10 μl (10 9 CFU engineering bacteria).
Fifth, the expression level of luxCDABE at the skin wound of the mice is detected. After the engineering bacteria are dripped, the bioluminescence signal is detected by a small animal living body imager.
The experimental results (see FIG. 12) show that the glucose regulatory gene expression loop control system can continuously activate gene expression at skin wounds of diabetic mice, and the activation can be maintained for about 24 hours.
Example 16 a probiotic pharmaceutical factory with glucose regulated expression and secretion of human acidic fibroblast growth factor rhaFGF135 was constructed and tested.
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.
And in the second step, conversion. pGN299 and pGN233 were electrotransformed EcN electrotransformed competent.
Third, the expression and secretion of rhaFGF135 was identified. And inoculating the amplified engineering bacteria, and adding 20mM glucose for induction. After 12 hours, the supernatant was centrifuged, and the protein in the supernatant was extracted, and the content of rhaFGF135 was detected by western blotting. Amplifying and preserving engineering bacteria with good secretion inducing effect.
The experimental results (see fig. 13) show that glucose can regulate rhaFGF135 expression and secretion.
Example 17 a probiotic pharmaceutical factory for glucose regulating chemokine CXCL12 expression and secretion was constructed and tested.
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.
And in the second step, conversion. pXG22 and pGN233 were electrotransformed EcN to electrotransformation competence.
Third, the expression and secretion of CXCL12 is identified. And inoculating the amplified engineering bacteria, and adding 20mM glucose for induction. After 12 hours, the supernatant was centrifuged, and the protein in the supernatant was extracted, and the CXCL12 content was measured by western blotting. Amplifying and preserving engineering bacteria with good secretion inducing effect.
The experimental results (see fig. 14) show that glucose can regulate CXCL12 expression and secretion.
Example 18, validation of wound site activation of diabetic mice probiotic drug factory expression and secretion of human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12.
In the first step, bacteria are cultivated. The probiotics and wild EcN which are used for regulating and controlling the expression and secretion of human acid fibroblast growth factor rhaFGF135 and chemotactic factor CXCL12 by glucose are inoculated into a liquid LB culture medium, and the culture is carried out for 12 hours at 37 ℃ and 210 rpm.
In the second step, cells were collected (the specific procedure is the same as in example 4 of the present invention).
In the third step, the skin is applied by in situ drip (the specific procedure is the same as in example 4 of the present invention).
Fourth, skin tissue is sampled. After 6 hours, the mice were sacrificed and the skin at the wound of the mice was sheared.
Fifth, tissue is sectioned. Skin tissue was fixed in 4% paraformaldehyde for 24 hours, rinsed overnight with running water, and the skin was sequentially soaked in 30%, 50%, 75%, 85%, 95%, 100% ethanol, 50% ethanol and 50% xylene mixed solution for 1 hour each, and sequentially permeabilized in 100% xylene and fresh 100% xylene for 5 minutes each. The wax was then soaked in 50% xylene and 50% paraffin for 1 hour, soaked in 100% paraffin overnight, and transferred the next day to a new 100% paraffin soak for 1 hour. After embedding using an embedding machine, freezing overnight at-20 ℃. Tissue sections were performed using a paraffin microtome.
Sixth, immunofluorescence staining. Sequentially placing the slices into xylene I15 min-xylene II 15 min-absolute ethanol I5 min-absolute ethanol II 5min-85% ethanol 5min-75% ethanol 5 min-distilled water for washing. The tissue sections were placed in a repair box filled with EDTA antigen retrieval buffer (pH 8.0) and subjected to antigen retrieval in a microwave oven. Middle fire for 8min to boiling, stopping fire for 8min, and turning to middle and low fire for 7min. After natural cooling, the slide was washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each. The sections were slightly spun off, then circled around the tissue with a histochemical pen (to prevent antibody from running off), spun off PBS, BSA added dropwise, and blocked for 30min. The blocking solution is gently thrown away, PBS is dripped on the slice, the slice is horizontally placed in a wet box for incubation at 4 ℃ for overnight. The slide was washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each. And (3) dripping secondary antibody to cover tissues in the rings after the slices are slightly dried, and incubating for 50min at room temperature in a dark place. The slide was washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each. And (3) dripping DAPI dye solution into the ring after the slices are slightly dried, and incubating for 10min at room temperature in a dark place. The slide was washed 3 times with shaking in PBS (pH 7.4) on a decolorizing shaker for 5min each. Adding an autofluorescence quenching agent into the ring for 5min, and washing with running water for 10min. And (5) after the slices are slightly dried, sealing the slices by using an anti-fluorescence quenching sealing tablet. The sections were observed under a fluorescence microscope and images were collected (DAPI UV excitation wavelength 330-380nm, emission wavelength 420nm, blue emission; CY3 excitation wavelength 510-560, emission wavelength 590nm, red emission).
The experimental results (see fig. 15) show that the probiotic drug factory, which can activate glucose regulation at the wound of the diabetic mice, expresses and secretes human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12.
Example 19 study of probiotic drug factories regulating expression and secretion of human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12 to promote skin wound healing in diabetic mice.
In this example, the skin wound of a type 1 diabetic mouse is taken as an example, and the promotion of the skin wound healing of the mouse by the glucose-regulated probiotic drug factory is demonstrated, but the protection scope of the invention is not limited. The method comprises the following specific steps:
in the first step, bacteria were cultured (the specific procedure is the same as in example 8 of the present invention).
In the second step, cells are collected. Recombinant probiotics obtained by screening in examples 5-7 and having good secretion of human acidic fibroblast growth factor rhaFGF135 and chemokine CXCL12 and wild-type EcN were amplified and cultured, centrifuged at 5000rpm for 5min, washed 3 times with sterile PBS (pH 6.35) and resuspended (10) 9 CFU/10 μl)。
Thirdly, bacteria are dripped in situ. PBS, human acid fibroblast growth factor rhaFGF135, wild-type EcN, probiotics for regulating expression and secretion of human acid fibroblast growth factor rhaFGF135 and probiotic suspension for regulating expression and secretion of chemokine CXCL12 are respectively dripped on skin wounds of diabetic mice in situ, wherein each mouse has a concentration of 10 mu l (10 9 CFU engineering bacteria).
Fourth, the size of skin wound of the mice was examined. The skin wounds of the mice were photographed every 24 hours and the wound areas were counted using Image J software.
The experimental results (see fig. 16) show that in situ drip application of probiotics that regulate rhaFGF135 and CXCL12 expression and secretion can significantly promote healing of skin wounds of diabetic mice.
Example 20 safety verification of probiotic drug factories to promote skin wound healing.
In the first step, bacteria were cultured (the specific procedure is the same as in example 7 of the present invention).
In the second step, cells were collected (the specific procedure is the same as in example 4 of the present invention).
Thirdly, the bacteria are applied in situ (the specific procedure is the same as in example 4 of the present invention).
Fourth, sampling. After 24 hours of bacteria coating, blood was taken from the mice, the mice were then sacrificed, and the heart, liver, spleen, lung, kidney were taken, respectively, and ground after adding PBS buffer.
And fifthly, safety verification. Performing routine blood analysis on the blood of the mice; the tissue grinding fluid is sucked up, the LB plate is coated, and after stationary culture is carried out for 12 hours at 37 ℃, colony counting is carried out.
The experimental results (see fig. 17 and 18) show that the in-situ drip Tu Yisheng bacteria does not affect the blood index of the mice, the bacteria does not infect the organs of the mice, and no residues exist in the blood after 24 hours.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that would occur to one skilled in the art are included in the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is defined by the appended claims.
SEQUENCE LISTING
<110> university of east China
<120> Intelligent controllable microbiological pharmaceutical factory and its use in skin wound healing
<160> 80
<170> PatentIn version 3.3
<210> 1
<211> 64
<212> DNA
<213> glucose inducible strong promoter PHexR1
<400> 1
ttgacatccc acctcacgta tgctataatg tgtgcagtct gacgcggcgt tgttggaatt 60
acaa 64
<210> 2
<211> 79
<212> DNA
<213> glucose inducible strong promoter PHexR2
<400> 2
ttgacatccc acctcacgta tgctataatg tgtgcagtct gacgcggcgt tgttggaatt 60
acaattgttg gaattacaa 79
<210> 3
<211> 94
<212> DNA
<213> glucose inducible strong promoter PHexR3
<400> 3
ttgacatccc acctcacgta tgctataatg tgtgcagtct gacgcggcgt tgttggaatt 60
acaattgttg gaattacaat tgttggaatt acaa 94
<210> 4
<211> 109
<212> DNA
<213> glucose inducible strong promoter PHexR4
<400> 4
ttgacatccc acctcacgta tgctataatg tgtgcagtct gacgcggcgt tgttggaatt 60
acaattgttg gaattacaat tgttggaatt acaattgttg gaattacaa 109
<210> 5
<211> 124
<212> DNA
<213> glucose inducible strong promoter PHexR5
<400> 5
ttgacatccc acctcacgta tgctataatg tgtgcagtct gacgcggcgt tgttggaatt 60
acaattgttg gaattacaat tgttggaatt acaattgttg gaattacaat tgttggaatt 120
acaa 124
<210> 6
<211> 91
<212> DNA
<213> glucose inducible strong promoter PHexR6
<400> 6
tttacacttt atgcttccgg ctcgtatgtt gttgttggaa ttacaattgt tggaattaca 60
attgttggaa ttacaattgt tggaattaca a 91
<210> 7
<211> 81
<212> DNA
<213> glucose inducible strong promoter PHexR7
<400> 7
tttacacttt atgcttccgg ctcgtatgtt ggtgtgcagt ctgacgcggc gttgttggaa 60
ttacaattgt tggaattaca a 81
<210> 8
<211> 96
<212> DNA
<213> glucose inducible strong promoter PHexR8
<400> 8
tttacacttt atgcttccgg ctcgtatgtt ggtgtgcagt ctgacgcggc gttgttggaa 60
ttacaattgt tggaattaca attgttggaa ttacaa 96
<210> 9
<211> 111
<212> DNA
<213> glucose inducible strong promoter PHexR9
<400> 9
tttacacttt atgcttccgg ctcgtatgtt ggtgtgcagt ctgacgcggc gttgttggaa 60
ttacaattgt tggaattaca attgttggaa ttacaattgt tggaattaca a 111
<210> 10
<211> 126
<212> DNA
<213> glucose inducible strong promoter PHexR10
<400> 10
tttacacttt atgcttccgg ctcgtatgtt ggtgtgcagt ctgacgcggc gttgttggaa 60
ttacaattgt tggaattaca attgttggaa ttacaattgt tggaattaca attgttggaa 120
ttacaa 126
<210> 11
<211> 31
<212> PRT
<213> glucagon-like peptide (GLP-1)
<400> 11
His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asn Gly Arg Gly
20 25 30
<210> 12
<211> 36
<212> PRT
<213> glucagon-like peptide EK-GLP-1
<400> 12
Asp Asp Asp Asp Lys His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser
1 5 10 15
Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val
20 25 30
Asn Gly Arg Gly
35
<210> 13
<211> 270
<212> PRT
<213> glucagon-like peptide GLP-1-Fc
<400> 13
His Ser Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly
1 5 10 15
Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asn Gly Arg Gly Gly
20 25 30
Gly Gly Gly Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr
35 40 45
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
50 55 60
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
65 70 75 80
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
85 90 95
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
100 105 110
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
115 120 125
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
130 135 140
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
145 150 155 160
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
165 170 175
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
180 185 190
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
195 200 205
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
210 215 220
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
225 230 235 240
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
245 250 255
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
260 265 270
<210> 14
<211> 136
<212> PRT
<213> human acid fibroblast factor (rhaFGF 135)
<400> 14
Met Ala Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly
1 5 10 15
His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp
20 25 30
Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly
35 40 45
Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp
50 55 60
Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu
65 70 75 80
Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys
85 90 95
Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser
100 105 110
Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe
115 120 125
Leu Pro Leu Pro Val Ser Ser Asp
130 135
<210> 15
<211> 69
<212> PRT
<213> chemokine (CXCL 12)
<400> 15
Met Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys Arg Phe Phe Glu
1 5 10 15
Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr
20 25 30
Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg
35 40 45
Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu
50 55 60
Lys Ala Leu Asn Lys
65
<210> 16
<211> 35
<212> DNA
<213> artificial sequence
<400> 16
ttgttggaat tacaacccct ctagaaataa ttttg 35
<210> 17
<211> 35
<212> DNA
<213> artificial sequence
<400> 17
ttgtaattcc aacaacgccg cgtcagactg cacac 35
<210> 18
<211> 48
<212> DNA
<213> artificial sequence
<400> 18
ggagaaatac tagatggcag atcttgtgcg aaacctcctg gaacagat 48
<210> 19
<211> 46
<212> DNA
<213> artificial sequence
<400> 19
tttctttacc agactcgagg gtacctcagt tgaggtcgtc gtcctc 46
<210> 20
<211> 50
<212> DNA
<213> artificial sequence
<400> 20
ttgtaattcc aacaattgta attccaacaa cgccgcgtca gactgcacac 50
<210> 21
<211> 50
<212> DNA
<213> artificial sequence
<400> 21
ttgttggaat tacaattgtt ggaattacaa cccctctaga aataattttg 50
<210> 22
<211> 80
<212> DNA
<213> artificial sequence
<400> 22
ttgtaattcc aacaattgta attccaacaa ttgtaattcc aacaattgta attccaacaa 60
cgccgcgtca gactgcacac 80
<210> 23
<211> 65
<212> DNA
<213> artificial sequence
<400> 23
ttgtaattcc aacaattgta attccaacaa ttgttggaat tacaacccct ctagaaataa 60
ttttg 65
<210> 24
<211> 54
<212> DNA
<213> artificial sequence
<400> 24
tttacacttt atgcttccgg ctcgtatgtt ggtgtgcagt ctgacgcggc gttg 54
<210> 25
<211> 50
<212> DNA
<213> artificial sequence
<400> 25
cggaagcata aagtgtaaac ttaagcatat ggtgcactct cagtacaatc 50
<210> 26
<211> 48
<212> DNA
<213> artificial sequence
<400> 26
tttgtttaac tttaagaagg agatataccg tgcgaaacct cctggaac 48
<210> 27
<211> 57
<212> DNA
<213> artificial sequence
<400> 27
tctccttctt aaagttaaac aaaattattt ctagacatta tacgagccga tgattaa 57
<210> 28
<211> 45
<212> DNA
<213> artificial sequence
<400> 28
gactcgtctt ataaggaaag ttaacggtgc gaaacctcct ggaac 45
<210> 29
<211> 53
<212> DNA
<213> artificial sequence
<400> 29
gttaactttc cttataagac gagtctattc attatacgag ccgatgatta att 53
<210> 30
<211> 45
<212> DNA
<213> artificial sequence
<400> 30
acaacaaagc aaaaataggg ggttcggtgc gaaacctcct ggaac 45
<210> 31
<211> 51
<212> DNA
<213> artificial sequence
<400> 31
aaccccctat ttttgctttg ttgtgctcat tatacgagcc gatgattaat t 51
<210> 32
<211> 60
<212> DNA
<213> artificial sequence
<400> 32
ttaagaagga gatataccat gggcagcagc atgactaaaa aaatttcatt cattattaac 60
<210> 33
<211> 50
<212> DNA
<213> artificial sequence
<400> 33
agcttgtcga cggagctcga attctcaact attaaatgct tggtttaagc 50
<210> 34
<211> 68
<212> DNA
<213> artificial sequence
<400> 34
tgctctggct ctgctgccgc tgctgtttac cccggtaacc aaggcacatt ccgaaggcac 60
ctttacct 68
<210> 35
<211> 52
<212> DNA
<213> artificial sequence
<400> 35
agaaggagat ataccatgat gaaacagtcc accattgctc tggctctgct gc 52
<210> 36
<211> 40
<212> DNA
<213> artificial sequence
<400> 36
tgctcgagtg cggccgctca cttgtcatcg tcatccttgt 40
<210> 37
<211> 56
<212> DNA
<213> artificial sequence
<400> 37
tgcactggct ggtttcgcga ccgtggcaca ggctcattcc gaaggcacct ttacct 56
<210> 38
<211> 65
<212> DNA
<213> artificial sequence
<400> 38
agaaggagat ataccatgat gaaaaaaacc gctatcgcaa tcgccgttgc actggctggt 60
ttcgc 65
<210> 39
<211> 61
<212> DNA
<213> artificial sequence
<400> 39
atagtactaa cgaccccgat tgcgattagc tcttttgcgc atagcgaagg caccttcacc 60
t 61
<210> 40
<211> 59
<212> DNA
<213> artificial sequence
<400> 40
agaaggagat ataccatgag ggctaaatta ttgggaatag tactaacgac cccgattgc 59
<210> 41
<211> 57
<212> DNA
<213> artificial sequence
<400> 41
tagcggtggc tgtggcggcg ggtgttatga gcgcacaggc acatagcgag ggcacct 57
<210> 42
<211> 60
<212> DNA
<213> artificial sequence
<400> 42
agaaggagat ataccatgat gataacattg aggaaattac ccctagcggt ggctgtggcg 60
<210> 43
<211> 56
<212> DNA
<213> artificial sequence
<400> 43
tgcgggcctg ttgttgctgg ctgcgcagcc ggcaatggcg catagcgagg gcacct 56
<210> 44
<211> 58
<212> DNA
<213> artificial sequence
<400> 44
agaaggagat ataccatgaa atatttacta cccacagcag ctgcgggcct gttgttgc 58
<210> 45
<211> 56
<212> DNA
<213> artificial sequence
<400> 45
aagctttata tctacaagtt tgccgctgcc gacccatagc gaaggtacgt ttacct 56
<210> 46
<211> 51
<212> DNA
<213> artificial sequence
<400> 46
agaaggagat ataccatgaa aatttcaagc tttatatcta caagtttgcc g 51
<210> 47
<211> 42
<212> DNA
<213> artificial sequence
<400> 47
ccgaccgacg acgacgacaa gcatagcgaa ggtacgttta cc 42
<210> 48
<211> 39
<212> DNA
<213> artificial sequence
<400> 48
cgctatgctt gtcgtcgtcg tcggtcggca gcggcaaac 39
<210> 49
<211> 42
<212> DNA
<213> artificial sequence
<400> 49
ccgaccgacg acgacgacaa gcatagcgaa ggtacgttta cc 42
<210> 50
<211> 36
<212> DNA
<213> artificial sequence
<400> 50
tggtggtgct cgagtttacc cggagacagg gagagg 36
<210> 51
<211> 25
<212> DNA
<213> artificial sequence
<400> 51
taaactcgag caccaccacc accac 25
<210> 52
<211> 39
<212> DNA
<213> artificial sequence
<400> 52
cgctatgctt gtcgtcgtcg tcggtcggca gcggcaaac 39
<210> 53
<211> 64
<212> DNA
<213> artificial sequence
<400> 53
agcagcttta ttagtaccag tctgccgctg ccgaccgcta actataaaaa accgaaactg 60
ctgt 64
<210> 54
<211> 52
<212> DNA
<213> artificial sequence
<400> 54
ttaagaagga gatataccat gaagatcagc agctttatta gtaccagtct gc 52
<210> 55
<211> 65
<212> DNA
<213> artificial sequence
<400> 55
gtcgacggag ctcgaattct taagcgtaat ctggaacatc gtatgggtag tccgacgaca 60
ccggc 65
<210> 56
<211> 76
<212> DNA
<213> artificial sequence
<400> 56
atagcgattg ctgtagcgct ggcgggtttt gctaccgttg ctcaggcgaa accggtgagt 60
ctgagttatc gttgcc 76
<210> 57
<211> 58
<212> DNA
<213> artificial sequence
<400> 57
taactttaag aaggagatat accatgaaaa agacagcaat agcgattgct gtagcgct 58
<210> 58
<211> 79
<212> DNA
<213> artificial sequence
<400> 58
gtcgacggag ctcgaattct taagcgtaat ctggaacatc gtatgggtat ttattcagtg 60
ctttttccag atattcctg 79
<210> 59
<211> 74
<212> DNA
<213> artificial sequence
<400> 59
attacgctta agaattcaat agactcgtct tataaggaaa gttaacgatg aaaaagacag 60
caatagcgat tgct 74
<210> 60
<211> 47
<212> DNA
<213> artificial sequence
<400> 60
tggtggtggt ggtgctcgag tttattcagt gctttttcca gatattc 47
<210> 61
<211> 284
<212> PRT
<213> murine islet-duodenal homology box-1 (mPDX-1)
<400> 61
Met Asn Ser Glu Glu Gln Tyr Tyr Ala Ala Thr Gln Leu Tyr Lys Asp
1 5 10 15
Pro Cys Ala Phe Gln Arg Gly Pro Val Pro Glu Phe Ser Ala Asn Pro
20 25 30
Pro Ala Cys Leu Tyr Met Gly Arg Gln Pro Pro Pro Pro Pro Pro Pro
35 40 45
Gln Phe Thr Ser Ser Leu Gly Ser Leu Glu Gln Gly Ser Pro Pro Asp
50 55 60
Ile Ser Pro Tyr Glu Val Pro Pro Leu Ala Ser Asp Asp Pro Ala Gly
65 70 75 80
Ala His Leu His His His Leu Pro Ala Gln Leu Gly Leu Ala His Pro
85 90 95
Pro Pro Gly Pro Phe Pro Asn Gly Thr Glu Pro Gly Gly Leu Glu Glu
100 105 110
Pro Asn Arg Val Gln Leu Pro Phe Pro Trp Met Lys Ser Thr Lys Ala
115 120 125
His Ala Trp Lys Gly Gln Trp Ala Gly Gly Ala Tyr Thr Ala Glu Pro
130 135 140
Glu Glu Asn Lys Arg Thr Arg Thr Ala Tyr Thr Arg Ala Gln Leu Leu
145 150 155 160
Glu Leu Glu Lys Glu Phe Leu Phe Asn Lys Tyr Ile Ser Arg Pro Arg
165 170 175
Arg Val Glu Leu Ala Val Met Leu Asn Leu Thr Glu Arg His Ile Lys
180 185 190
Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Glu Glu Asp Lys
195 200 205
Lys Arg Ser Ser Gly Thr Pro Ser Gly Gly Gly Gly Gly Glu Glu Pro
210 215 220
Glu Gln Asp Cys Ala Val Thr Ser Gly Glu Glu Leu Leu Ala Val Pro
225 230 235 240
Pro Leu Pro Pro Pro Gly Gly Ala Val Pro Pro Gly Val Pro Ala Ala
245 250 255
Val Arg Glu Gly Leu Leu Pro Ser Gly Leu Ser Val Ser Pro Gln Pro
260 265 270
Ser Ser Ile Ala Pro Leu Arg Pro Gln Glu Pro Arg
275 280
<210> 62
<211> 39
<212> DNA
<213> artificial sequence
<400> 62
caccaccacc accaccacat gaacagtgag gagcagtac 39
<210> 63
<211> 61
<212> DNA
<213> artificial sequence
<400> 63
ttaacgacga cgctggcggc gttttttacg gccgtaaccg ccaccccggg gttcctgcgg 60
t 61
<210> 64
<211> 38
<212> DNA
<213> artificial sequence
<400> 64
gccagcgtcg tcgttaatga gatccggctg ctaacaaa 38
<210> 65
<211> 34
<212> DNA
<213> artificial sequence
<400> 65
tggtggtggt ggtggtgggt cggcagcggc aaac 34
<210> 66
<211> 283
<212> PRT
<213> human islet duodenal homologous Box-1 (hPDX-1)
<400> 66
Met Asn Gly Glu Glu Gln Tyr Tyr Ala Ala Thr Gln Leu Tyr Lys Asp
1 5 10 15
Pro Cys Ala Phe Gln Arg Gly Pro Ala Pro Glu Phe Ser Ala Ser Pro
20 25 30
Pro Ala Cys Leu Tyr Met Gly Arg Gln Pro Pro Pro Pro Pro Pro His
35 40 45
Pro Phe Pro Gly Ala Leu Gly Ala Leu Glu Gln Gly Ser Pro Pro Asp
50 55 60
Ile Ser Pro Tyr Glu Val Pro Pro Leu Ala Asp Asp Pro Ala Val Ala
65 70 75 80
His Leu His His His Leu Pro Ala Gln Leu Ala Leu Pro His Pro Pro
85 90 95
Ala Gly Pro Phe Pro Glu Gly Ala Glu Pro Gly Val Leu Glu Glu Pro
100 105 110
Asn Arg Val Gln Leu Pro Phe Pro Trp Met Lys Ser Thr Lys Ala His
115 120 125
Ala Trp Lys Gly Gln Trp Ala Gly Gly Ala Tyr Ala Ala Glu Pro Glu
130 135 140
Glu Asn Lys Arg Thr Arg Thr Ala Tyr Thr Arg Ala Gln Leu Leu Glu
145 150 155 160
Leu Glu Lys Glu Phe Leu Phe Asn Lys Tyr Ile Ser Arg Pro Arg Arg
165 170 175
Val Glu Leu Ala Val Met Leu Asn Leu Thr Glu Arg His Ile Lys Ile
180 185 190
Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys Glu Glu Asp Lys Lys
195 200 205
Arg Gly Gly Gly Thr Ala Val Gly Gly Gly Gly Val Ala Glu Pro Glu
210 215 220
Gln Asp Cys Ala Val Thr Ser Gly Glu Glu Leu Leu Ala Leu Pro Pro
225 230 235 240
Pro Pro Pro Pro Gly Gly Ala Val Pro Pro Ala Ala Pro Val Ala Ala
245 250 255
Arg Glu Gly Arg Leu Pro Pro Gly Leu Ser Ala Ser Pro Gln Pro Ser
260 265 270
Ser Val Ala Pro Arg Arg Pro Gln Glu Pro Arg
275 280
<210> 67
<211> 37
<212> DNA
<213> artificial sequence
<400> 67
caccaccacc accaccacat gaacggcgag gagcagt 37
<210> 68
<211> 62
<212> DNA
<213> artificial sequence
<400> 68
ttaacgacga cgctggcggc gttttttacg gccgtaaccg ccacctcgtg gttcctgcgg 60
cc 62
<210> 69
<211> 38
<212> DNA
<213> artificial sequence
<400> 69
gccagcgtcg tcgttaatga gatccggctg ctaacaaa 38
<210> 70
<211> 34
<212> DNA
<213> artificial sequence
<400> 70
tggtggtggt ggtggtgggt cggcagcggc aaac 34
<210> 71
<211> 70
<212> DNA
<213> artificial sequence
<400> 71
atccggctgc taacaaagga aataattttg tttaacttta agaaggagat ataccatgaa 60
aatttcaagc 70
<210> 72
<211> 39
<212> DNA
<213> artificial sequence
<400> 72
caactcagct tcctttcggg ttaacgacga cgctggcgg 39
<210> 73
<211> 22
<212> DNA
<213> artificial sequence
<400> 73
aaggaagctg agttggctgc tg 22
<210> 74
<211> 24
<212> DNA
<213> artificial sequence
<400> 74
tcctttgtta gcagccggat ctca 24
<210> 75
<211> 129
<212> PRT
<213> human interleukin 4 factor (hIL 4)
<400> 75
His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser
1 5 10 15
Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile
20 25 30
Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala
35 40 45
Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg
50 55 60
Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile
65 70 75 80
Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu
85 90 95
Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe
100 105 110
Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser
115 120 125
Ser
<210> 76
<211> 49
<212> DNA
<213> artificial sequence
<400> 76
ggaagatctt ccaaagagga gaaatactag atgaaaatct cctctttca 49
<210> 77
<211> 33
<212> DNA
<213> artificial sequence
<400> 77
cggggtaccc cgccggatct cagtggtggt ggt 33
<210> 78
<211> 15
<212> DNA
<213> artificial sequence
<400> 78
ttgtggtttt tacta 15
<210> 79
<211> 15
<212> DNA
<213> artificial sequence
<400> 79
ttgtaattcc aacaa 15
<210> 80
<211> 16
<212> DNA
<213> artificial sequence
<400> 80
ttgtttaaat atacaa 16

Claims (23)

1. An intelligent controllable microbiological pharmaceutical factory characterized in that it comprises a chassis microorganism and a gene expression loop control system uploaded into said chassis microorganism.
2. The intelligently controllable microbiological pharmaceutical factory of claim 1 wherein the chassis microorganisms comprise probiotics.
3. The intelligently controllable microbial pharmaceutical factory according to claim 2, wherein the probiotics comprise escherichia coli Nissle 1917, lactococcus lactis, lactobacillus plantarum, bacillus subtilis.
4. The intelligently controllable microbial pharmaceutical factory according to claim 1, wherein the gene expression loop control system is located on any one of the chromosomes or plasmids of the chassis microorganisms.
5. The intelligently controllable microbial pharmaceutical plant of claim 1, wherein the gene expression loop control system comprises a glucose regulated gene expression loop control system, a xylose-induced gene expression system, an arabinose-induced gene expression system, a uric acid-induced gene expression system.
6. The intelligent controllable microbiological pharmaceutical factory of claim 5 wherein said plant can express a gene of interest in the presence of an inducer.
7. The intelligently controllable microbial pharmaceutical factory according to claim 6, wherein the inducer is added exogenously or generated in vivo.
8. The intelligently controllable microbial pharmaceutical plant of claim 1, wherein the gene expression loop control system comprises a transcription repressor, an inducible strong promoter, and a sequence to be transcribed.
9. The intelligently controllable microbial pharmaceutical factory according to claim 8, wherein the transcription repressor, the inducible strong promoter and the sequence to be transcribed can be constructed on one or two plasmid vectors.
10. The intelligently controllable microbial pharmaceutical plant of claim 5, wherein the glucose regulatory gene expression loop control system comprises a transcription repressor HexR, a glucose inducible strong promoter, and a sequence to be transcribed.
11. The intelligently controllable microbial pharmaceutical plant according to claim 10, wherein the transcription inhibitor HexR can bind to the glucose-inducible strong promoter and thereby repress the expression of downstream sequences to be transcribed; the glucose-inducible strong promoter is formed by combining a constitutive strong promoter and a DNA sequence specifically combined with HexR; the sequence to be transcribed comprises single encoding of an expressed reporter protein or a functional protein, or tandem expression of multiple proteins.
12. The intelligently controllable microbial pharmaceutical factory according to claim 11, wherein said transcriptional repressor is capable of binding to said glucose inducible strong promoter in the absence of glucose, thereby repressing the expression of a downstream sequence to be transcribed; in the presence of glucose, glucose is metabolized via the Entner-Doudoroff pathway to the metabolic intermediate 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is blocked by KDG, allowing the transcription repressor to dissociate from the glucose-inducible strong promoter, initiating expression of the sequence to be transcribed downstream.
13. The intelligently controllable microbiological pharmaceutical factory according to claims 1-12, wherein said intelligently controllable microbiological pharmaceutical factory is in a liquid or is freeze-dried, spray-dried.
14. The method for constructing an intelligent controllable microbiological pharmaceutical factory according to claim 1, comprising the steps of:
a) Combining a DNA operator sequence specifically recognized by the transcription repressing protein with a constitutive strong promoter to construct an inducible strong promoter;
b) Constructing a coding gene for expressing the drug protein to the downstream of an inducible promoter in a single or tandem mode, and constructing a report module;
c) Constructing a functional module for constitutive expression of the transcription repressor protein;
the method converts the plasmid containing the functional module constructed in the step b) and the reporting module constructed in the step c) into probiotic competent cells, or integrates the functional module constructed in the step b) and the reporting module constructed in the step c) onto probiotic chromosomes through an RED recombinase system or a CRISPR system, so as to prepare the inducible engineering cells, namely the intelligent controllable microbial drug factory.
15. An expression vector, an engineered cell, comprising a intelligently controllable microbial pharmaceutical factory according to any one of claims 1-13.
16. Use of a intelligently controllable microbiological pharmaceutical factory according to any one of claims 1 to 13 for the preparation of a medicament.
17. The use according to claim 16, wherein the medicament comprises a medicament for promoting healing of skin wounds, a medicament for preventing and/or treating metabolic disorders.
18. The use of claim 17, wherein the skin wound comprises a chronic skin wound, a diabetic foot ulcer.
19. A method of promoting skin wound healing using a intelligently controllable microbiological pharmaceutical factory according to any one of claims 1 to 13, the method comprising the steps of:
a) Artificially constructing a prokaryotic expression vector containing an expression regulation system of glucose-induced growth factors, macrophage M2 polarized cytokines and chemokines or a functional module for chromosome integration;
b) Preparing an engineering cell containing glucose-induced growth factors, macrophage M2 polarized cytokines and a chemokine expression control system;
c) The engineering cells prepared in the step b) are transplanted to skin wounds of diabetic model mice in the form of in-situ wound dripping;
d) Glucose closed-loop induction engineering cells at the skin wound of the mouse express and secrete growth factors, macrophage M2 polarized cytokines and chemokines so as to achieve the effect of promoting wound healing.
20. The method of claim 13, wherein the growth factor is rhaFGF135, FGF2, FGF7, TGF- α, TGF- β, PDGF, EGF, VEGF, IGF-1, IGF-2, PDGF, HGF; the macrophage M2 polarization cytokine is IL4, IL-10, IL-13, CSF1, IL34; the chemokines are CXCL12, CXCL13, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL11 and CXCL10.
21. A plasmid, characterized in that the plasmid is one or more of plasmids pGN233, pGN65, pGN299, pGN300 and pGN 314.
22. A nucleotide sequence which is a glucose-inducible strong promoter P HexR4 、P HexR9 One of them has the nucleotide sequence shown in SEQ ID NO.4 and SEQ ID NO. 9.
23. A primer/primer pair, which is characterized in that the primer sequence is one or more of SEQ ID NO.16-60, 62-65, 67-74 and 76-77; the sequences of the primer pairs are one or more of the primer pairs shown in the numbers 1-30.
CN202210326231.6A 2022-03-30 2022-03-30 Intelligent controllable microbial drug factory and application thereof in skin wound healing Pending CN116925985A (en)

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