CN108728326B - Far-red light regulation gene expression loop control system, construction method and application - Google Patents
Far-red light regulation gene expression loop control system, construction method and application Download PDFInfo
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Abstract
The invention discloses a far-red light regulation gene expression loop control system, which comprises a photoreceptor for sensing a far-red light source, a processor for processing signals transmitted by the photoreceptor, and an effector for responding the signals transmitted by the processor. The invention also discloses application of the far-red light regulation gene expression loop control system in treating diseases, such as diabetes. The invention also discloses a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the far-red light regulatory gene expression loop control system. The invention can quickly regulate and control gene expression by far-red light, accurately control gene expression quantity, and has the characteristics of high regulation and control gene expression multiple, high space-time specificity, strong tissue penetration, no toxic or side effect and the like.
Description
Technical Field
The invention relates to the interdisciplinary fields of synthetic biology, optogenetics and the like, in particular to a far-red light regulation gene expression loop control system and a construction method and application thereof.
Background
Synthetic biology is a comprehensive discipline that takes engineering theory as guidance, connects genes into networks, designs and synthesizes various complex biological function modules, and allows cells to complete various tasks assumed by designers. Research in synthetic biology has progressed rapidly in recent years and has made significant progress in a number of areas. In particular, in the field of mammalian cell synthetic biology, a variety of controllable genetic circuits have been designed and constructed for the diagnosis and treatment of metabolic diseases, cancer, immune system diseases, and the like.
In the fields of synthetic biology and disease treatment, molecular switches for artificially and precisely regulating gene expression have become an indispensable means in disease treatment. There are many systems for inducing gene expression through artificial regulation in the world, and the gene expression system is induced and regulated through chemical inducers or physical methods.
In recent years, the induction and regulation of gene expression systems by chemical substances has also been greatly developed. For example vitamins [ Weber, W. et al., Metab Eng,2006,8(3):273-80], amino Acids [ Hartenbach, S. et al., Nucleic Acids Res,2007,35(20): e136], phloretin [ Gitzinger, M. et al., Proc Natl Acad Sci USA,2009,106(26):10638-43], and parabens [ Wang, H. et al., Nucleic Acids Res,2015,43(14): e91], among others. These chemical inducers present their own potential problems such as toxicity, pleiotropic properties, non-specificity, and poor tissue penetration. When the induction expression is carried out in vivo, the chemical inducer needs to be injected into the body, and the normal metabolic process of the human body can be influenced. In addition, it is difficult to achieve precise control of chemical substances in terms of time and space when the expression of genes is induced and controlled.
Compared with chemical substance regulation systems, regulation systems induced by physical methods are more and more concerned by people due to the advantages of convenience, no need of in vivo injection of drugs and the like, and comprise ultraviolet-induced regulation and control 'caging' technology [ Keyes W M. et al, Trends in biotechnology,2003,21(2):53-55], far infrared light controlled heat shock effect induced regulation and control gene expression systems [ KameiY. et al, Nature methods,2009,6(1):79-81], radio controlled temperature induced regulation and control gene expression systems [ Stanley S A. et al, Science,2012,336(6081): 604-. Although these systems for inducing gene expression by physical means can also induce gene expression, these systems are generally very toxic to cells, may cause irreversible damage to cells and even death of cells, and involve expensive and complicated equipment; in addition, these systems have great limitations in their application and can cause significant damage to the body.
Light is an ideal inducer of gene expression. It is ubiquitous in nature, readily available, spatio-temporal specific, and non-toxic. Therefore, the research of using light as an inducer to regulate gene expression and further treat various diseases has great application value. In our previous research work, synthetic biology was used to design and synthesize gene loops for blue light-regulated transgene expression, and melanoid blue light system was used to treat type II diabetes [ Ye H. et al, Science,2011,332(6037): 1565-. The subject group of professor Yange Yangyi university of eastern science and technology reports the Light On blue Light regulatory gene expression switch, and uses the LOV blue Light system to treat type I diabetes [ Wang X. et al, Nature methods,2012,9(3): 266-. Although the transgenic expression system regulated by blue light does not need to add small-molecule chemical substances into cells, the blue light has great limitation on regulating gene expression in vivo. The transdermal efficiency of blue light is low, the target genes are not easy to be deactivated through skin or abdominal cavity, and the clinical application of the light control system is greatly limited. A far-red light controlled transgenic expression control system is developed by professor Martin Fussenegger of the biological system engineering system of the university of Federal rational Engineers of Zurich, Switzerland [ Fussenegger M. et al, Nature communication,2014,5:5392 ], but the far-red light control system does not show good gene expression intensity and has low system expression times which are only ten times or so, so that the clinical application of the light control system is limited. The far-red light regulation gene expression loop control system is a better far-red light system version optimized on the basis of a far-red light system taught by Martin Fussenegger, optimizes the amount of a processor in the system, and also optimizes a promoter in an effector. The optimized novel far-red light regulation and control gene expression loop control system has the advantages of lower background (the expression quantity of SEAP is only 1-8U/L), higher expression multiple (about 50 times), more sensitivity to far-red light, and more benefit to deep development and clinical application of future light regulation and control systems.
Diabetes is one of the chronic epidemic diseases that currently threaten global human health. With the improvement of living standard and the change of life style of people, the proportion of diabetics increases year by year. Diabetes is a chronic lifelong disease affecting human health, which cannot be completely cured but can be controlled. After onset, the patients mainly have high blood sugar level, and with the progress of the disease, they will affect various vital organs and tissues of the body, causing various complications. However, diabetes cannot be treated radically so far and needs to be administered for life. At present, the treatment modes of diabetes mainly comprise insulin injection, medicine taking, diet control and the like. Diabetic patients need to take hypoglycemic drugs or inject insulin every day to maintain stable blood sugar. However, due to the limitations of these treatment methods, oral administration of hypoglycemic drugs or insulin injection cannot achieve controlled release of insulin, which is very likely to cause hypoglycemia. There is a need to find new treatment modes to improve the treatment effect, reduce the treatment risk and improve the convenience of treatment.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a novel far-red light regulation gene expression loop control system for the first time. In the invention, the photon energy of far-red light is much lower than that of blue light, and the toxic and side effect on cells is far less than that of the blue light. The penetrability of far-red light is far greater than that of blue light, and the far-red light can permeate 7-8 cm of skin and muscle tissues, so that the target cells transplanted in the abdominal cavity can be regulated and controlled without traces to express target genes, and even the target genes can be regulated and controlled by specific tissues and organs in vivo. Moreover, the far-red light control system can be directly activated by far-red light without adding any photosensitive pigment additionally. The invention provides a novel far-red light regulation and control gene expression loop control system by regulating the amount of a processor in the system and the type of a promoter in an effector, the background is lower (the expression amount of SEAP is only 1-8U/L), the toxicity to cells is lower, the expression multiple of the system is higher (about 50 times), the system is more sensitive to far-red light, and the system is more beneficial to deep development and clinical application of a future light regulation and control system. The far-red light regulation gene expression loop control system can design different target protein expressions, is used for treating various diseases such as diabetes and the like, has great potential application value, and can be widely popularized in clinical application.
The nucleotide sequences or amino acid sequences can be prepared by adopting an artificial synthesis method.
The invention provides an artificially designed and synthesized gene loop control system for regulating transgene expression based on far-red light. The invention provides a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector of a far-red light regulation gene expression loop control system. The invention also provides a kit for each component of the far-red light regulatory gene expression loop control system. The invention also provides a novel diabetes therapy based on far-red light regulation. The invention can quickly regulate and control gene expression and regulate and control gene expression quantity, and has the characteristics of high regulation and control expression multiple, high space-time specificity, strong tissue penetrating power, no toxic or side effect and the like. The expression multiple of the existing STING far-red light regulatory gene expression loop control system is only about 10 times, while the expression multiple of the far-red light regulatory gene expression loop control system provided by the invention is as high as 50 times, and the expression background is low (the expression amount of SEAP is only 1-8U/L), so that the potential application value is great.
The invention provides a far-red light regulation gene expression loop control system, which comprises: a photoreceptor that senses a far-red light source; a processor for processing the signals transmitted by the photoreceptors; an effector responsive to a signal delivered by the processor.
The photoreceptor of the present invention comprises a promoter for expressing the photoreceptor; the coding gene sequence of the bacterial photosensitive diguanylate cyclase BphS is shown as SEQ ID NO. 1; and c-di-GMP degrading enzyme YhjH, and a gene sequence coding the same is Genebank accession number: ANK 04038.
In the invention, the photoreceptor can also comprise a phytochrome synthetase BphO, and the coding gene sequence of the phytochrome synthetase BphO is shown as SEQ ID NO. 2. The photosensitive diguanylate cyclase BphS converts GTP into c-di-GMP under the condition of far-red light, is one of the most key proteins and serves as a core component of a photoreceptor. The photoreceptor photosensitive diguanylate cyclase BphS is prepared by fusing the 1 st-511 th amino acid of BphG protein and the 175 st-343 st amino acid of Slr1143 protein and mutating the 587 th arginine of the fusion protein into alanine (R587A), wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1; wherein, the BphG protein can be from Rhodobacter sphaeroides (Rhodobacter sphaeroides) or be artificially synthesized, and the BphG protein adopted by the invention is artificially synthesized; the Slr1143 protein can be from Synechocystis sp or be artificially synthesized, and the Slr1143 protein adopted by the invention is artificially synthesized.
Wherein, the degrading enzyme YhjH of the c-di-GMP is derived from Escherichia coli (E.coli), can also be artificially synthesized, and the nucleotide sequence of the coding gene thereof is Genebank accession number: ANK 04038. The YhjH has the function of degrading c-di-GMP to pGpGpG [ Ryu M H. et al, ACS synthetic biology,2014,3(11): 802-. Wherein the phytochrome synthetase BphO is a red blood oxidase existing in Rhodobacter sphaeroides (Rhodobacter sphaeroides), and the BphO can also be artificially synthesized, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2. It has the function of synthesizing photosensitive pigment biliverdin, and provides photosensitive pigment for synthesizing c-di-GMP by BphS [ Ryu M H, et al, ACS synthetic biology,2014,3(11):802-810 ]. Wherein the amino acid sequences of BphS and BphO are respectively shown as SEQ ID NO.18 and SEQ ID NO.19, the amino acid sequence of YhjH is Genebank accession number: NP _ 417982.
Wherein, the promoter for expressing photoreceptors can be any promoter capable of expressing photoreceptors in mammalian cells, including but not limited to: a) the nucleotide sequence of the simian vacuolating virus promoter SV40 is shown as SEQ ID NO. 4; b) EF1 α (which may be human EF1 α (hEF1 α)) promoter, the nucleotide sequence Genebank accession number: AY 043301; c) PGK (which may be murine PGK (mpgk)) -promoter, the nucleotide sequence of which Genebank accession no: HZ 040569; d) cytomegalovirus early enhancer combined with chicken β -actin promoter (CAG), nucleotide sequence Genebank accession no: HQ 456319; e) cytomegalovirus promoter CMV, its nucleotide sequence Genebank accession number: KY 199427.
Wherein the processor comprises the promoter CMV (cytomegalovirus promoter), the nucleotide sequence of which Genebank accession no: KY 199427; and an immune signaling molecule, the promoter CMV driving expression of an immune signaling molecule that is capable of binding to c-di-GMP to form a binary complex and self-activate the immune signaling molecule, the immune signaling molecule comprising: a natural immune signaling molecule STING, whose nucleotide sequence Genebank accession number: NM-198282, wherein the STING source can be human or mouse, and it contains 2 functional regions, namely N-terminal functional region with 5-times transmembrane structure and globular carboxyl terminal (i.e. C-terminal) functional region CTD. When STING is expressed in mammalian cells, C-di-GMP binds to the functional region CTD of STING to form a binary complex and activates it, activated STING is activated by recruitment of TBK1 through the C-terminal domain, activated TBK1 phosphorylates IRF3, and IRF3 subsequently dimerizes into the nucleus. The invention can make the background of the far-red light regulatory gene expression loop control system lower (the expression quantity of SEAP is only 5-8U/L) and the toxicity to cells lower by adjusting the STING quantity of the processor in the system, and the mass ratio of the processor to the photoreceptors and effectors is (0.5-2): (0.5-40): (0.5-40); preferably, 1: (1-20): (1-20); further preferably, is 1:10: 10; can be adjusted according to different requirements.
Wherein the effector comprises a promoter PFRLAnd a target gene reporter, denoted as PFRL-reporter。
Wherein, the promoter PFRLComprising a DNA sequence that is recognized and bound by dimerized IFR3 and a weak promoter sequence that promotes gene expression. The dimerized IFR3 recognizes and binds to a DNA sequence that is specifically recognized and bound by an IFR3 polypeptide, which is a partial sequence of the hIFN-RE-ISRE promoter region. Wherein the hIFN-RE-ISRE consists of a nucleotide sequence of a synthetic human interferon response element hIFN-RE shown in SEQ ID NO.7 and an interferon stimulation response element ISRE shown in SEQ ID NO. 8. Partial sequence of the hIFN-RE-ISRE promoter region is 1-10 copies.
Wherein, the weak promoter for promoting gene expression can be any weak promoter, such as T with a nucleotide sequence shown as SEQ ID NO.5ATA box, cytomegalovirus minimal promoter CMVmin as shown in nucleotide sequence SEQ ID NO.6, mutant CMVmin 3G, and the like, which do not express or hardly express downstream target genes (nucleotide sequences to be transcribed) when an upstream processor does not exist. The invention optimizes the DNA sequence and the weak promoter which are identified and combined by IFR3 in an effector, and the DNA sequence and the weak promoter which are identified and combined by IFR3 can be selected from P with the nucleotide sequence shown as SEQ ID NO.9FRL1(5 × ISRE-h _ CMVmin), P as shown in SEQ ID NO.10FRL2(hIFN-RE-h _ CMVmin), P shown as SEQ ID NO.11FRL3((hIFN-RE) -3 × ISRE-h _ CMVmin), P as shown in SEQ ID NO.12FRL4((hIFN-RE) -3 × ISRE-h _ min), P as shown in SEQ ID NO.13FRL5((hIFN-RE) -3 × ISRE- (hIFN-RE) -3 × ISRE-h _ min), P as shown in SEQ ID NO.14FRL6((hIFN-RE) -3 × ISRE-h _ min-40bp), P as shown in SEQ ID NO.15FRL7(hIFN-RE) -h _ min) nucleotide sequence, P shown as SEQ ID NO.16FRL8((hIFN-RE) -3 × ISRE- (hIFN-RE) -h _ min), P as shown in SEQ ID NO.17FRL9(3 × ISRE- (hIFN-RE) -h _ min), can make the system expression fold higher. The invention also optimizes the interval sequence between the weak promoter for starting gene expression and the gene initiation codon ATG as 40bp, the gene expression multiple is higher (about 50 times), and the system background is lower (the SEAP expression quantity is only 5-8U/L), so that the system is more sensitive to far-red light and is more beneficial to the deep development and clinical application of a future light control system.
Wherein, the protein coded by the target gene reporter (nucleotide sequence to be transcribed) can be all meaningful proteins, including proteins used as reporter genes and/or drug proteins or small peptides used for treating diseases; wherein the protein serving as the reporter gene comprises secreted alkaline phosphatase (SEAP), Enhanced Green Fluorescent Protein (EGFP) and Luciferase (Luciferase); pharmaceutical proteins or small peptides as therapeutics for diseases include Insulin (Insulin), glucagon-like peptide (GLP-1). Wherein the nucleotide sequence encoding said SEAP is Genebank accession no: AX036887, nucleotide sequence Genebank accession number encoding said EGFP: KY002200, nucleotide sequence encoding said Luciferase Genebank accession no: KJ561464, nucleotide sequence Genebank accession number of the nucleotide sequence encoding said GLP-1-Fc: AY 311599. That is, the treatment of various diseases or the expression of target proteins can be achieved by adjusting the type of target gene.
Wherein, when two or more of the said all interesting proteins are present, they can be expressed simultaneously by linking self-cleaving peptide 2A to an expression vector, such as SEAP-2A-Insulin, EGFP-2A-Insulin, etc.; wherein the nucleotide sequence of the self-cutting peptide 2A is SEQ ID NO.3 (the amino acid sequence of the self-cutting peptide 2A is shown as SEQ ID NO. 20). Wherein the 2A sequence used may be replaced by an internal ribosome entry site sequence IRES.
In the present invention, the type of the target gene in the system can be adjusted according to the type of the disease to be treated to prepare corresponding products, such as genes encoding insulin and/or glucagon-like peptides as the target gene of the effector for treating diabetes.
The mechanism of action of the invention is that when c-di-GMP is generated under the condition of illumination, the c-di-GMP is combined with STING to mediate STING-TBK1-IFR3 signal path activation, the dimerized IFR3 enters into nucleus, and the specific sequence in an effector is recognized and combined to start transcription and expression of downstream genes. When the illumination stops and the c-di-GMP can not be generated, the synthesized c-di-GMP is degraded, and the STING can not be activated, so that the transcription expression of the gene can not be started, and the closing of the gene switch is realized.
Three components of the far-red light regulation gene expression loop control system provided by the invention can be constructed in a eukaryotic expression vector by a genetic engineering technology, so that the transcription and expression of a regulation target gene are realized. The far-red light regulation gene expression loop control system provided by the invention can regulate the expression of a target gene in a eukaryotic host cell in time and space by using far-red light irradiation which hardly damages cells or organisms, and the host cell can be any type of mammalian cells, such as hMSC-TERT, Hana 3A, HEK-293A, HEK-293T and the like.
Wherein the illumination intensity of the far-red light is 0-5mW/cm2(ii) a The irradiation time is 0-72 h; the irradiation method comprises irradiating pulseImpact irradiation, continuous irradiation, direct irradiation or irradiation of spatially controlling the gene expression levels of cells at different positions using a projection card with a hollow depiction. Different expression amounts of the regulatory gene are realized by controlling different illumination time generated by the far-red light source. The far-red light source can generate 600-900nm wavelength far-red light, and can be 600-900nm LED, infrared therapeutic device, laser lamp, etc.
The invention also provides a construction method of the far-red light regulatory gene expression loop control system. (1) The invention optimizes the relationship between STING and the mass of the photoreceptors and the processor under the expression of the promoter CMV, and finds that the effect is optimal when the ratio of STING to the mass of the photoreceptors and the processor is 1:10: 10. (3) The invention optimizes effectors that include promoter PFRLAnd a target gene reporter, denoted as PFRL-reporter. Wherein, the promoter PFRLComprising a DNA sequence that is recognized and bound by dimerized IFR3 and a weak promoter sequence that promotes gene expression. The weak promoter for promoting gene expression can be any weak promoter, such as TATA box with a nucleotide sequence shown as SEQ ID NO.5 and cytomegalovirus minimal promoter CMV with a nucleotide sequence shown as SEQ ID NO.6minAnd a mutant CMVmin 3G thereof. (4) The invention optimizes the DNA sequence and the promoter which are identified and combined by IFR3 in a plurality of versions of effectors, and the DNA sequence and the promoter which are identified and combined by IFR3 can be selected from P with the nucleotide sequence shown as SEQ ID NO.9FRL1(5×ISRE-h_CMVmin)P as shown in SEQ ID NO.10FRL2(hIFN-RE-h_CMVmin)P as shown in SEQ ID NO.11FRL3((hIFN-RE)-3×ISRE-h_CMVmin)P as shown in SEQ ID NO.12FRL4((hIFN-RE)-3×ISRE-h_min)P as shown in SEQ ID NO.13FRL5((hIFN-RE)-3×ISRE-(hIFN-RE)-3×ISRE-h_min)P as shown in SEQ ID NO.14FRL6((hIFN-RE)-3×ISRE-h_min-40bp)P as shown in SEQ ID NO.15FRL7((hIFN-RE)-h_min)Nucleotide sequence, P shown as SEQ ID NO.16FRL8((hIFN-RE)-3×ISRE-(hIFN-RE)-h_min)P as shown in SEQ ID NO.17FRL9(3×ISRE-(hIFN-RE)-h_min)The system can be made to express two foldThe number is higher. (5) The invention also optimizes the interval sequence between the weak promoter and the gene initiation codon ATG as the gene expression multiple is higher (about 50 times) when the length of the interval sequence is 40bp, and the system background is lower (the expression quantity of SEAP is only 5-8U/L), so that the system is more sensitive to far-red light, and is more favorable for deep development and clinical application of a future light control system.
The invention also provides a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the far-red light regulatory gene expression loop control system; wherein, the engineering cell transplantation carrier comprises a hollow fiber membrane transplantation tube, a sodium alginate gel block and the like.
The invention also provides a kit which contains the far-red light regulatory gene expression loop control system. The invention also provides a kit which is provided with a eukaryotic expression vector containing the far-red light regulatory gene expression loop control system and/or a host cell transfected with the eukaryotic expression vector and/or an engineered cell transplantation vector and a corresponding instruction.
In the invention, the kit comprises a plasmid kit for regulating and controlling each component of the far-red light regulation and control gene expression loop control system, a mammalian cell kit containing the far-red light regulation and control gene expression loop control system and a corresponding instruction.
The invention also provides a method for preparing the eukaryotic expression vector, the engineered cell or the engineered cell transplantation vector containing the far-red light regulatory gene expression loop control system. The eukaryotic expression vector comprises a mammalian cell expression vector containing the far-red light regulatory gene expression loop control system. The expression vector can be a vector containing a far-red light photoreceptor coding gene alone or a vector containing a processor coding gene alone or a vector containing an effector coding gene alone, wherein the effector contains a far-red light response promoter but does not contain a nucleic acid sequence to be transcribed. Or the expression vector comprises two or three of a vector of the far-red light photoreceptor encoding gene, a vector of the processor encoding gene and a vector of the effector encoding gene. The construction of all the aforementioned mammalian cell expression vectors is detailed in Table 1.
The invention also provides application of the eukaryotic expression vector containing the far-red light regulatory gene expression loop control system in preparation of a medicament/product for treating diabetes, wherein the diabetes comprises type I diabetes and/or type II diabetes.
The invention also provides a method for regulating and controlling gene expression in host cells by using the far-red light regulation and control gene expression loop control system. The method comprises the following steps:
a) constructing the far-red light regulatory gene expression loop control system in a eukaryotic plasmid expression vector;
b) is introduced into a host cell by transfection
Wherein the mass ratio of the processor to the photoreceptors to the effectors is 0.1:10: 10. c) And inducing and regulating the host cell by regulating and controlling the red light irradiation condition to realize the expression of the effector coding gene. Wherein the host cell is derived from a mammalian cell.
The plasmid construction method of the present invention is referred to the materials method and table 1. The method of introducing a plasmid into a mammalian cell comprises: calcium phosphate transfection, PEI transfection, lipofection, electroporation transfection, viral infection, and the like.
The invention also provides a method for regulating and controlling transgene expression in a transplanting vector by using the far-red light regulation and control gene expression loop control system, which comprises the following steps:
a) preparing a eukaryotic plasmid expression vector containing the far-red light regulatory gene expression loop control system;
b) preparing an engineered cell containing the far-red light regulatory gene expression loop control system;
c) preparing an engineered cell transplantation carrier containing the far-red light regulatory gene expression loop control system;
d) inducing and expressing a transplantation carrier containing engineered cells by regulating a far-red light source to ensure that an effector P in the transplantation carrierFRL-expression of reporter encoding genes (e.g. SEAP, EGFP, Luciferase, Insulin, GLP-1-Fc, etc.); e) are respectively atAnd detecting the expression condition of the target gene at three time points of 24h, 48h and 72 h.
The invention also provides a method for implanting the far-red light regulatory gene expression loop control system transplanting carrier into a mouse body, and a method for performing transgenic regulatory expression on the far-red light regulatory gene expression loop control system in the mouse body, which comprises the following steps:
a) preparing a transplanting carrier containing the far-red light regulatory gene expression loop control system;
b) implanting a transplantation carrier containing the far-red light regulatory gene expression loop control system into a mouse body;
c) inducing and expressing a transplantation carrier containing engineered cells by regulating a far-red light source to ensure that an effector P in the transplantation carrierFRL-expression of reporter encoding genes (e.g. SEAP, EGFP, Luciferase, Insulin, GLP-1-Fc, etc.); d) and detecting the expression condition of the target gene at three time points of 24h, 48h and 72h respectively.
Wherein the transplantation carrier comprises a sodium alginate gel block skin, a hollow fiber membrane transplantation tube and the like; the method of implantation may be subcutaneous implantation.
The invention also provides application of the far-red light regulation and control gene expression loop control system in preparing a medicament for treating diabetes, wherein the diabetes comprises type I diabetes and/or type II diabetes. The invention provides a novel strategy for treating diabetes, which is safe and reliable and can accurately regulate and control the release of insulin and glucagon-like peptide in time and space. The present invention provides new methods and strategies for treating diabetes. The system can modulate the expression of insulin and/or glucagon-like peptide, GLP-1. The expression construction of the Insulin comprises SEAP-2A-Insulin, EGFP-2A-Insulin and EGFP-2A-SEAP-2A-Insulin. The expression of the glucagon-like peptide GLP-1 comprises GLP-1-Fc and the like.
Drawings
FIG. 1 is a schematic diagram showing the principle of the far-red light regulatory gene expression loop control system of the present invention in mammalian cells.
FIG. 2 is a diagram showing the experimental results of photoreceptors expressed by different promoters of the far-red light regulatory gene expression loop control system of the present invention.
FIG. 3 is a diagram showing the experimental results of processors with different quantities in the far-red light regulatory gene expression loop control system of the present invention.
FIG. 4 is an experimental result diagram of effectors differently constructed by the far-red light regulatory gene expression loop control system of the present invention.
FIG. 5 is a diagram showing the results of experiments on the expression of far-red light regulatory gene expression loop control system in different mammalian cells.
FIG. 6 is a graph showing the experimental results of different expression levels of the far-red light regulatory gene expression loop control system according to the present invention under different illumination times.
FIG. 7 shows the effect of different illumination intensities on the control of target protein expression level by far-red light regulatory gene expression loop.
FIG. 8 shows the effect of different illumination times on the expression level of the activity of the target protein FLuc expressed by the far-red light regulatory gene expression loop control system.
FIG. 9 shows the effect of different illumination times on the GLP-1 expression level of the far-red light regulatory gene expression system expressed target protein.
FIG. 10 is a diagram showing the results of green fluorescence experiments in which the far-red light-regulated gene expression loop control system of the present invention can simultaneously express two or more proteins of interest.
FIG. 11 is a diagram showing the results of an insulin experiment in which the far-red light-regulating gene expression loop control system of the present invention can simultaneously express two or more proteins of interest.
FIG. 12 is a diagram showing the experimental results of preparing a hollow fiber membrane graft vessel graft carrier containing far-red light regulatory gene expression loop control system engineered cells according to the present invention.
FIG. 13 is a graph showing the far-red light toxicity test results of the present invention.
FIG. 14 shows the background measurement results of the far-red light regulatory gene expression loop control system of the present invention.
FIG. 15 is a diagram showing the experimental results of the far-red light regulated expression of the far-red light regulated gene expression loop control system of the present invention in a mouse.
FIG. 16 shows that the far-red light regulated gene expression loop control system of the present invention can precisely regulate the fasting blood glucose level of insulin expression in type I diabetes mellitus model mice for treating type I diabetes mellitus.
FIG. 17 shows the results of glucose tolerance experiments in the present invention of far-red light regulated gene expression loop control system for accurately regulating insulin expression in type I diabetic mice to treat type I diabetes.
FIG. 18 shows the fasting blood glucose level of the far-red light regulatory gene expression loop control system for accurately regulating GLP-1-Fc expression in type II diabetic mice for treating type II diabetes.
FIG. 19 shows the results of glucose tolerance experiments in the present invention of a far-red light regulated gene expression loop control system for the precise regulation of GLP-1-Fc expression in type II diabetic mice for the treatment of type II diabetes.
FIG. 20 shows the results of an insulin resistance experiment in which the far-red light regulatory gene expression loop control system of the present invention precisely regulates GLP-1-Fc expression in type II diabetic rats to treat type II diabetes.
FIG. 21 shows that the far-red light regulatory gene expression loop control system of the present invention precisely regulates the amount of glucagon expressed by GLP-1-Fc expression therapy of type II diabetes in type II diabetes model mice.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited. The implementation process, conditions, reagents, experimental methods and the like of the present invention are general knowledge and common general knowledge in the field except for the contents specifically mentioned below, and the present invention is not particularly limited.
The material and the method are as follows:
all primers used for PCR were synthesized by Kingzhi Biotech, Inc. The expression plasmids in the embodiment of the invention are all carried out according to the conventional molecular cloning process, and the constructed expression plasmids are subjected to sequence determination which is performed by goldZhi Biotechnology Limited. The DNA polymerase, endonuclease, and T4DNA ligase used in the examples of the present invention were purchased from Nanjing Novozam Biotech, Inc. The dual-luciferase reporter gene detection kit used was purchased from biotool, usa. Hollow fiber membrane graft tube for experiment (
Implant Membrane) purchased from Spectrum Laboratories, Inc., USA. The Insulin detection kit (Mouse Insulin ELISAkit) used for the experiments was purchased from Mercodia, Sweden. Glucagon detection kits for the experiments (Millipore Corporation, Billerica, MA 01821 USA, Cat. No. EGLP-35K, Lot. No.2639195) were purchased from Millipore Corporation, USA.
Example 1 construction of Gene Loop regulatory System elements for far-Red light regulation of transgene expression
The embodiment of the invention includes a method for constructing a representative element in a gene loop remote control system for regulating transgene expression by far-red light, but the invention is not limited to the protection scope of the invention. The detailed design scheme and procedure are shown in table 1.
Example 2 photoreceptor expressed by different promoters of the far-red light-regulated Gene expression Loop control System for far-red light-regulated transgene expression
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-5 groups. Within 16 to 24h of inoculation of cells, 0.1. mu.g of pWS50 (expressed from the promoter SV 40) in group 1 of the dark and light groups, 0.1. mu.g of pWS189 (expressed from the promoter CMV) in group 2, 0.1. mu.g of pWS51 (expressed from the promoter hEF 1. alpha.) in group 3, 0.1. mu.g of pWS55 (expressed from the promoter mPGK) in group 4, 0.1. mu.g of pWS59 (expressed from the promoter CAG) in group 5, and 0.01. mu.g of processor pSTING, 0.1. mu.g of effector pWS67, PEI transfection reagent were mixed with serum-free DMEM and added to 24-well culture plates after standing at room temperature for 15 min. Fourthly, the solution is irradiated, after 14 to 18 hours of solution changing, the solution is irradiatedPlacing at wavelength of 720nm and illumination intensity of 1mW/cm2Under the LED for 4 h. And fifthly, detecting the reporter gene.
As shown in FIG. 2, in the gene loop control system for far-red light to control transgene expression, photoreceptors expressed by different promoters can work normally in mammalian cells under the induction of far-red light. However, photoreceptors expressed by different promoters under the induction of the same far-red light cause different response intensities of effectors, and the photoreceptor expressed by the CMV promoter has the highest induction multiple according to experimental results.
Example 3 Gene Loop control System with far-Red light for regulating transgene expression varying amounts of processor
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-8 groups. Within 16 to 24h of cell inoculation, 0ng of pSTING in group 1, 5ng of pSTING in group 2, 10ng of pSTING in group 3, 20ng of pSTING in group 4, 40ng of pSTING in group 5, 60ng of pSTING in group 6, 80ng of pSTING in group 7, 100ng of pSTING in group 8, 100ng of pSTING, and 100ng of the far-red receptor pWS189, 100. mu.g of the effector pWS67, PEI transfection reagent were mixed with serum-free DMEM and were added to 24-well plates evenly after standing for 15min at room temperature. And step four, illuminating (the concrete steps are the same as those of the example 2). And fifthly, detecting the reporter gene.
The results are shown in FIG. 3, in the gene loop control system for far-red light to regulate transgene expression, different amounts of processors under the same far-red light induction cause different effector response intensities. The expression fold was highest at 10ng processor, 100ng photoreceptor and 100ng effector and background was low (SEAP expression was only 5-8U/L), the expression fold was highest at 40ng processor, 100ng photoreceptor and 100ng effector but not at 10ng processor due to high background; the optimal amount of processors can be selected according to experimental needs.
Example 4 Effector with different constructs of Gene Loop control System for far-Red light Regulation of transgene expression
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. Within 16 to 24h of seeding the cells, 2 24-well plates were divided into dark and light groups, each divided into 1-9 groups. Within 16 to 24h of seeding cells, 0.1. mu.g of pWS32 in group 1, 0.1. mu.g of pWS33 in group 2, 0.1. mu.g of pWS35 in group 3, 0.1. mu.g of pWS54 in group 4, 0.1. mu.g of pWS58 in group 5, 0.1. mu.g of pWS67 in group 6, 0.1. mu.g of pYW25 in group 7, 0.1. mu.g of pYW28 in group 8, 0.1. mu.g of pYW29 in group 9, far-receptor mix with 0.1. mu.g of each, red light transfection reagent and serum-free DMEM were mixed, and dropped into 24-well plates after standing for 15min at room temperature. And step four, illuminating (the concrete steps are the same as those of the example 2). And fifthly, detecting the reporter gene.
The results are shown in FIG. 4, in the gene loop control system for far-red light to regulate transgene expression, pWS32 (P)FRL1(5×ISRE-h_CMVmin))、pWS33(PFRL2(hIFN-RE-h_CMVmin))、pWS35(PFRL3((hIFN-RE)-3×ISRE-h_CMVmin))、pWS54(PFRL4((hIFN-RE)-3×ISRE-h_min))、pWS58(PFRL5((hIFN-RE)-3×ISRE-(hIFN-RE)-3×ISRE-h_min))、pWS67(PFRL6((hIFN-RE)-3×ISRE-h_min-40bp))、pYW25(PFRL7((hIFN-RE)-h_min))、pYW28(PFRL8((hIFN-RE)-3×ISRE-(hIFN-RE)-h_min))、pYW29(PFRL9(3×ISRE-(hIFN-RE)-h_min)) Different processor recognition sites and different numbers of repeats of the recognition sites and different kinds of effectors of weak promoters can work normally in mammalian cells under the induction of far-red light. However, under the same far-red light induction, the response intensity of effectors containing different processor recognition sites and different repetition numbers of the recognition sites is different, and according to the experimental results, the expression multiple of the pWS67 as an effector is the highest, while the expression level of the pWS35 as an effector is the highest, and different effectors can be selected according to the experimental needs.
Example 5 Gene Loop control System for far-Red light-regulated transgene expression in different mammalian cells
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. Within 16 to 24 hours of cell inoculation, 0.1. mu.g of pWS189, 0.01. mu.g of pSTING, 0.1. mu.g of pWS67 and PEI transfection reagent were mixed with serum-free DMEM, and the mixture was allowed to stand at room temperature for 15min and then added dropwise to a 24-well culture plate. And step four, illuminating (the concrete steps are the same as those of the example 2). And fifthly, detecting the reporter gene.
As shown in FIG. 5, the far-red light-regulated transgenic gene loop control system of the present invention can be expressed in different mammalian cells (e.g., hMSC-TERT, Hana 3A, HEK-293A, HEK-293T) under the induction of far-red light. Therefore, the far-red light transgenic gene loop control system can be expressed in various mammalian cell types and can be suitable for various mammalian cells.
Example 6 control of different expression levels of Gene Loop control System for regulating transgene expression by far-Red light with different illumination time
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. Within 16 to 24 hours of cell inoculation, 0.1. mu.g of photoreceptor pWS189, 0.01. mu.g of pSTING, 0.1. mu.g of pWS67, PEI transfection reagent and serum-free DMEM were mixed well, left to stand at room temperature for 15min and then added dropwise to 24-well culture plates. And fourthly, controlling different illumination time to illuminate. After the liquid is changed for 14-18h, the liquid is divided into 13 groups, and the groups are placed at a wavelength of 720nm and an illumination intensity of 1mW/cm2Under the LED of (1). The different illumination times were 0, 0.01, 0.1, 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48, 72h, respectively (the group with illumination 0h was always cultured in the dark). And fifthly, detecting the reporter gene. After 72 hours of culture, the supernatant of the cell culture medium of each group was collected to determine the expression level of SEAP.
The experimental result is shown in fig. 6, the gene loop control system capable of inducing far-red light to regulate and control transgene can regulate and control different expression quantities of target genes by controlling different illumination time, and the longer the illumination induction time is, the higher the expression quantity is, and illumination time-dependent expression is presented within 0-72 h.
Example 7 Gene Loop control System for regulating far-Red light-regulated transgene expression by controlling different illumination intensities
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. Third, transfection (the same procedure as in example 6). And fourthly, controlling different illumination intensities. After the liquid is changed for 14-18h, the liquid is divided into 11 groups, and the groups are respectively placed at the wavelength of 720nm and the illumination intensity of 0, 25, 50, 75, 100, 250, 500, 750, 1000, 1500 and 2000 mu W/cm2Under the LED of (1). The light time was 4 hours (the group in which the light intensity was 0 was always cultured in the dark). And fifthly, detecting the reporter gene. After 72 hours of culture, the supernatant of the cell culture medium of each group was collected to determine the expression level of SEAP.
The experimental result is shown in fig. 7, the gene loop control system which can induce the far-red light to regulate the transgene expression can regulate the different expression levels of the target gene by controlling different illumination intensities, and the stronger the illumination intensity is, the higher the expression level is, and illumination intensity dependent expression is presented within 0-72 h.
Example 8 Gene Loop control System for regulating transgenes
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. 0.1. mu.g of pWS189, 0.01. mu.g of pSTING, 0.1. mu.g of pGY45 (expressing Luciferase) within 16 to 24h of seeding cells; 0.1. mu.g of pWS189, 0.01. mu.g of pSTING, 0.1. mu.g of pWS152 (expressing GLP-1-FC) and the PEI transfection reagent were mixed with serum-free DMEM, and the mixture was allowed to stand at room temperature for 15min and then added dropwise to a 24-well culture plate. Fourthly, illuminating (the specific steps are the same as the example 2), and measuring the expression quantity of Luciferase (Luciferase) after 4 hours of illumination; the expression level of GLP-1 under different illumination time is determined immediately after illumination for 0, 0.1, 0.25, 0.5, 1 and 2 hours respectively. The dark group was cultured in the dark. And fifthly, detecting the reporter gene. Measuring the expression quantity (the activity of the protein of interest FLuc) of Luciferase (Luciferase) by using an ELISA kit at 24h and 48h (figure 8); the expression level of GLP-1 was measured at 48h with ELISA kit under different illumination time (FIG. 9).
As a result, as shown in FIGS. 8 and 9, the gene loop control system for regulating transgene expression of the present invention can induce expression of various proteins, such as Luciferase, GLP-1-Fc, etc., well, and there is no particular limitation on the types of proteins. The transgenic gene loop control system of the invention is therefore suitable for the expression of all proteins of interest.
Meanwhile, as can be seen from fig. 10 and 11, the gene loop control system for regulating the expression of the transgene can accurately regulate the expression of EGFP and insulin, and the expression level of EGFP and insulin is in positive correlation with the illumination time.
Example 9 Gene Loop control System for regulating transgenes can simultaneously express two or more proteins of all interest
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. Within 16 to 24 hours of cell inoculation, 0.1. mu.g of pWS189, 0.01. mu.g of pSTING, 0.1. mu.g of pWS174, and PEI transfection reagent were mixed with serum-free DMEM, and the mixture was allowed to stand at room temperature for 15min and then added dropwise to a 24-well culture plate. And fourthly, illuminating. After the liquid is changed for 14-18h, the illumination group is placed at the wavelength of 720nm and the set illumination intensity is 1mW/cm2The measurement was carried out in a batch manner immediately after 0, 0.1, 0.25, 0.5, 1, and 2 hours of the LED illumination. And fifthly, detecting the reporter gene.
As a result, as shown in FIGS. 10 and 11, the gene loop control system for regulating transgene expression of the present invention can express two different proteins (linked by 2A) at the same time, and the two different proteins expressed at the same time are time-dependent. Thus, the transgenic gene loop control system of the invention can be used to express two or more proteins of interest simultaneously.
Meanwhile, as can be seen from fig. 11, the gene loop control system for regulating the transgene expression can accurately regulate the insulin expression in vitro, and the expression level thereof is positively correlated with the illumination time.
Example 10: preparation of hollow fiber membrane transplantation vessel transplantation carrier containing gene loop control system engineered cells for regulating transgene expression
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. Step two, cell inoculation step three, transfection: within 16 to 24h of cell seeding, 0.1. mu.g of pWS189, 0.01. mu.g of pSTING, 0.1. mu.g of pWS67, PEI transfection reagent and serum-free DMEM were mixed well, left for 15min at room temperature and added dropwise to 24-well cell culture plates. After 6h of transfection, 10mL of DMEM medium containing 10% FBS was added for culture. And fourthly, preparing the hollow fiber membrane transplanting tube of the gene loop control system engineering cells for regulating and controlling the transgene expression. After the liquid is changed for 14-18h, pancreatin digestion and cell collection by centrifugation are carried out. The hollow fiber membrane graft tube was produced according to the production method.
The experimental results are detailed in fig. 12, the prepared hollow fiber membrane graft tube, nutrients required for cell growth and small molecules of target proteins secreted by the engineered cells can freely pass through the membrane system. But cells and other macromolecular proteins cannot pass through the membrane system. Therefore, the cells wrapped by the hollow fiber membrane transplantation tube can be transplanted into a mouse body to grow normally.
Example 11 far-red light toxicity test on cells
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded.
And thirdly, transfection. Within 16 to 24h of cell inoculation, 0.1. mu.g of pSEAP2control and PEI transfection reagent was mixed well with serum-free DMEM, and was allowed to stand at room temperature for 15min and then was dropped into 24-well culture plates. After transfection for 6 hours, 500. mu.L of DMEM medium containing 10% FBS was added and cultured. And fourthly, illuminating. After the liquid is changed for 14-18h, the liquid is divided into 10 groups, and the groups are respectively placed at the wavelength of 720nm and the set illumination intensity of 1mW/cm2The measurement was carried out uniformly immediately after 0, 0.1, 0.5, 1, 2, 6, 12, 24, 48, and 72 hours of the illumination under the LED. And fifthly, detecting the reporter gene.
The experimental results are shown in FIG. 13, and the expression level of SEAP after 72h of illumination is basically not different from that of dark, which indicates that far-red light has no toxicity to cells.
Example 12 background assay of far-Red light regulatory Gene expression Loop control System of the present invention
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, cells are seeded. And thirdly, transfection. Within 16 to 24h of seeding the cells, 0.1. mu.g of pWS189, 0.01. mu.g of pSTING, 0.1. mu.g of pWS 67; 0.1 mu g of pWS189, 0.1 mu g of pGY32 and 0.1 mu g of pXY34 are respectively mixed uniformly in a PEI transfection reagent and serum-free DMEM, and the mixture is placed for 15min at room temperature and then uniformly dripped into a 24-well culture plate. And fourthly, after 6 hours of transfection, 500 mu L of DMEM medium containing 10% FBS is added, wrapped by tinfoil paper and placed in an incubator for culture. And fifthly, detecting the reporter gene. After 48 hours of culture, the supernatant of the cell culture medium of each group was collected to determine the expression level of SEAP.
The experimental result is shown in fig. 14, and the expression levels of two groups of SEAPs after 72h of culture can show that the background of the far-red light regulatory gene expression loop control system is low, the expression level of the SEAP is only 5-8U/L, and under the same conditions, the application number of the SEAP filed in 2016 is 201610136462.5, the background of the far-red light gene loop expression control system in the title of 'a far-red light gene loop expression control system and a eukaryotic expression vector thereof' is 16-19U/L.
Example 13 cases where Gene Loop control System regulating transgene expression is regulated by far-Red light in mice
In the first step, a hollow fiber membrane graft tube was prepared (see example 10 for details). And secondly, implanting the hollow fiber membrane transplanting tube into the back of the mouse. And thirdly, illuminating. Control infrared therapeutic equipment (10 mW/cm)2) The cells were irradiated for 2h, 8h, 26h and 32h after transplantation. And fourthly, detecting the reporter gene. The amount of reporter gene was measured at 24h and 48h orbital hemospasia, respectively, post-transplant.
The results of the experiment are shown in FIG. 15, which shows that the gene loop control system of the present invention is also under far-red light control in mice and has time dependence.
Example 14 Gene Loop control System for regulating transgene expression in type I diabetic mice for the treatment of type I diabetes mellitus by accurately regulating insulin expression
Firstly, constructing a mouse model of type I diabetes. We used multiple low dose streptozotocin (Streptozocin, STZ, from Sigma S0130, 18883-6, 6-4) dosing induction model. 25C 57BL/6J mice (from Chinese academy of sciences), 8 weeks old, male, were injected intraperitoneally (fasted for 12-16h before injection) for 5 consecutive days with sodium citrate buffer dissolved with STZ (at a dose of 40-50 mg/kg). In the second step, the gene loop for regulating the expression of the transgene controls the preparation of the engineered cell of the system, as described in example 10. And thirdly, preparing a hollow fiber membrane transplanting tube of the gene loop control system engineering cells for regulating and controlling the transgene expression, which is specifically referred to example 10. And fourthly, implanting a transplantation tube of the gene loop control system engineered cells for regulating and controlling the transgene expression into the back of the mouse. Step five, light irradiation, the third step of example 13 was specifically referred to. Sixthly, determining the fasting blood glucose value of the type I diabetic mice. After 8h of transplantation, the mice were fasted (fed with water) for 16h, and then blood was taken from the tail, and fasting blood glucose values were measured. Experimental data indicate that the expressed insulin has good blood sugar lowering effect (FIG. 16). Seventh step, sugar tolerance experiment. 24h after transplantation, sugar tolerance experiments were performed. Experiments showed that diabetic mice had a very good improvement in glucose tolerance (fig. 17).
Example 15 Gene Loop control System regulating transgene expression for treatment of type II diabetes by precise Regulation of GLP-1 expression in type II diabetes model mice
In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1. In the second step, type II diabetes model mice db/db mice 20 (from Chinese academy of sciences), 8 weeks old, female, divided into four groups. The groups are respectively a non-transplanted non-illumination group, a non-transplanted illumination group, a transplanted non-illumination group and a transplanted illumination group. And thirdly, preparing far-red light engineering cells, and particularly referring to example 10. And fourthly, preparing a transplanting tube of the gene loop control system engineered cells for regulating and controlling the expression of the transgenes, which is specifically referred to the embodiment 10. And fifthly, transplanting a transplantation tube of the gene loop control system engineered cells for regulating and controlling the transgene expression to the back of the mouse, specifically referring to the second step of the example 14. Sixth step, illumination, third step with specific reference to example 14. Seventhly, determining the fasting blood glucose value of the type II diabetic mice. After 8h of transplantation, the mice were fasted (fed with water) for 16h, and then blood was taken from the tail, and fasting blood glucose values were measured. The experimental results are shown in fig. 18, and the data show that the expressed glucagon has a good blood glucose reduction effect. Eighth, a sugar tolerance test is performed. 24h after transplantation, sugar tolerance experiments were performed. Reference is made in particular to example 18. The results of the experiment are shown in fig. 19, and the glucose tolerance of the diabetic mice is well improved. And the ninth step, carrying out insulin resistance experiment. 24h after transplantation, insulin resistance experiments were performed. The results of the experiment are shown in fig. 20, and the insulin resistance of the diabetic mice is improved well. And step ten, measuring the expression of GLP-1 in the type II diabetic mouse by a gene loop control system for regulating and controlling the transgene expression. Blood was collected at the inner canthus 48h after transplantation, and the GLP-1(activie) content in serum was measured using GLP-1(7-36) activie ELISA kit. The experimental results are shown in fig. 21, and the gene loop control system for regulating the expression of the transgene can accurately regulate the expression of glucagon in vivo.
TABLE 1 plasmid construction Table
The primers, cleavage sites and the seamless cloned fragments are underlined.
<110> Suzhou Xinsai Biotechnology Ltd
<120> far-red light regulation gene expression loop control system, construction method and application
<160> 20
<210> 1
<211> 2064
<212> DNA
<213> Artificial sequence
<400> 1
ATGGCTAGAGGCTGCCTCATGACCATTAGCGGCGGCACATTCGATCCTAGCATCTGTGAGATGGAGCCCATCGCTACCCCTGGAGCTATTCAACCTCACGGCGCCCTGATGACCGCCAGAGCTGACAGCGGAAGGGTCGCCCATGCCTCCGTCAATCTGGGCGAAATCCTGGGACTGCCTGCTGCTTCCGTGCTCGGAGCCCCCATCGGAGAGGTGATTGGCAGGGTGAACGAGATCCTGCTCAGAGAGGCCAGAAGATCCGGAAGCGAGACCCCCGAGACAATCGGCTCCTTTAGAAGGTCCGACGGCCAGCTGCTCCATCTCCATGCCTTCCAAAGCGGCGATTATATGTGCCTCGACATCGAGCCCGTGAGAGACGAAGATGGCAGACTGCCCCCCGGCGCTAGACAGTCCGTGATTGAGACCTTCTCCAGCGCCATGACACAGGTGGAGCTGTGCGAACTCGCCGTCCACGGCCTCCAGCTCGTCCTCGGCTACGATAGAGTGATGGCCTACAGATTCGGCGCTGATGGACACGGCGAGGTGATCGCTGAGAGGAGAAGGCAGGATCTGGAACCCTACCTGGGACTGCATTATCCCGCCAGCGACATCCCCCAGATCGCCAGAGCCCTGTACCTGAGGCAGAGGGTCGGCGCCATCGCCGATGCTTGCTACAGGCCTGTCCCTCTGCTGGGACATCCCGAACTGGACGACGGAAAGCCTCTCGACCTGACACACAGCAGCCTGAGGAGCGTCAGCCCTGTGCACCTGGACTATATGCAGAACATGAATACCGCTGCCTCCCTGACCATCGGCCTGGCCGATGGAGACAGGCTGTGGGGAATGCTGGTGTGTCACAATACCACCCCCAGGATTGCCGGACCCGAATGGAGAGCTGCTGCTGGCATGATCGGCCAGGTGGTCTCCCTGCTGCTGAGCAGGCTGGGCGAGGTCGAGAATGCTGCCGAGACACTCGCCAGACAGAGCACCCTGTCCACCCTCGTGGAGAGGCTCAGCACCGGAGACACACTCGCCGCCGCTTTTGTGGCTGCTGACCAGCTCATCCTGGATCTCGTCGGAGCTTCCGCCGCCGTGGTCAGACTGGCCGGACAGGAGCTGCACTTCGGCAGGACACCTCCCGTCGACGCTATGCAGAAAGTGCTGGACAGCCTGGGAAGACCCTCCCCCCTGGAGGTGCTGAGCCTGGACGATGTGACCCTGAGACATCCTGAGCTGCCCGAGCTGCTGGCTGCCGGGAGCGGAATTCTGCTGCTGCCCCTCACCTCCGGAGACGGAGACCTGATCGCCTGGTTCAGGCCTGAGCACGTGCAGACCATCACCTGGGGAGGCAACCCCGCCGAACATGGAACCTGGAATCCTGCTACCCAAAGGATGAGACCCAGGGCCTCCTTCGACGCCTGGAAAGAGACCGTGACCGGCAGAAGCCTGCCCTGGACCAGCGCCGAGAGAAATTGCGCCAGGGAACTGGGCGAGGCTATCGCCGCTGAAATGGCCCAGAGAACCAGAGCCGAAGAACTCGAAAGGGTGGCTATGGTCGACAGCCTCACAAGGCTCTGGAATAGGCTGGGCATTGAGACACTGCTGAAGAGAGAGTGGGAGTATGCCACCAGAAAGAATTCCCCCATCAGCATCGTGATGATCGACTTTGACAACTTCAAGCAGATTAACGATCAGCACGGACACCTGGTCGGCGACGAGGTGCTGCAGGGAAGCGCTAGGCTCATCATCAGCGTGCTGGCCTCCTATGACATTCTGGGAAGATGGGGAGGCGACGAGTTTATGCTGATTCTGCCCGGAAGCGGAAGGGAGCAGACAGCCGTCCTGCTCGAGAGAATCCAGGCCACCATTGCTCAGAACCCCGTCCCTACCTCCGCCGGACCTATGGCTATCAGCCTGTCCATGGGCGGCGTGAGCGTGTTCACCAATCAGGGAGAGGCCCTGCAGTACTGGGTGGAGCAAGCTGACAACCAACTGATGAAAGTGAAGAGGCTCGGCAAAGGCAACTTCCAGCTCGCCGAGTACCACCACCACCATCACCACTAG
<210> 2
<211> 594
<212> DNA
<213> Artificial sequence
<400> 2
ATGCCCCTGAGCAGGGACCTGAGGGAGAAGACAGGAATGCTGCACAATAGAGCCGAGACCCTGCTGGGCCTGCCTAGCGGAATTATGGGCTGGGCCGACTACGTGGACTGGCTGAGGCACTTCCTGGCCCTGTACGACCCCATCGAGAGAAGGATTGTCGCCTTCGGCGGCTGGAGCGGACTGGCCTCCTTTGATCCTGATCCCGGCCATTCCAGAAGACTGATCCAGGACCTGCACGCCCTCGGCATTGACACAGACAGAATCCCCAGGGCTCCCGCCGAGTACTGTCCCCCTCTGACCAATTTTGCTAGAGCCCTGGGAGCCAGGTATGTCCTGGAGGGAAGCGCCCTCGGAGGCAGGGTCATTCTGCACCATCTGAAGAAGAGGATCGGCGACGAAATTGGCAACGCCACCGCCTTTTTTGGCGGACCTTCCCACGGCACCGCCACCCATTGGAGGGCCTTCCAGGCTGCCCTGGACAGGTTTGGCGCTGCCCATCCCGATAAAAGGGCCGATGTGCTGGCCGGAGCTGCTGCTACCTTCACAGCTCTGCTCGAGTGGTTTACCCCCTTTGTGGCCGCCAGAAGGGTGTAG
<210> 3
<211> 72
<212> DNA
<213> Artificial sequence
<400> 3
GGGAGCGGCGCCACAAACTTTTCCCTCCTGAAGCAGGCTGGAGATGTGGAGGAGAATCCCGGACCTAGCGGA
<210> 4
<211> 209
<212> DNA
<213> Artificial sequence
<400> 4
GATCTGCGATCTGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATCGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAA
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence
<400> 5
TAGAGGGTATATAATGGAAGCTCG
<210> 6
<211> 149
<212> DNA
<213> Artificial sequence
<400> 6
TCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG
<210> 7
<211> 31
<212> DNA
<213> Artificial sequence
<400> 7
CATGACAAAGGGAAACTGAAAGGGAAACTGA
<210> 8
<211> 15
<212> DNA
<213> Artificial sequence
<400> 8
TTTCACTTTCCCTAG
<210> 9
<211> 235
<212> DNA
<213> Artificial sequence
<400> 9
TTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTTCGAATCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG
<210> 10
<211> 206
<212> DNA
<213> Artificial sequence
<400> 10
CATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTGCGATCTAAGTAAGCTTCGAATCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG
<210> 11
<211> 255
<212> DNA
<213> Artificial sequence
<400> 11
CATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTTCGAATCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCG
<210> 12
<211> 140
<212> DNA
<213> Artificial sequence
<400> 12
CATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTTAGAGGGTATATAATGGAAGCTCG
<210> 13
<211> 256
<212> DNA
<213> Artificial sequence
<400> 13
CATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTCATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTTAGAGGGTATATAATGGAAGCTCG
<210> 14
<211> 184
<212> DNA
<213> Artificial sequence
<400> 14
CATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTTGGGCTAGCCCGGGCTCGAGAGAGGGTATATAATGGAAGCTCGAATTCCAGAAGCTTATACTCAGTGCCCTGACTATATACTCAGTGCCCTGACTATG
<210> 15
<211> 73
<212> DNA
<213> Artificial sequence
<400> 15
ATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTAGACTCTAGAGGGTATATAATGGAAGCTCGAATTCCAG
<210> 16
<211> 213
<212> DNA
<213> Artificial sequence
<400> 16
CATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTTGGGCTAGCATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTAGACTCTAGAGGGTATATAATGGAAGCTCGAATTCCAG
<210> 17
<211> 177
<212> DNA
<213> Artificial sequence
<400> 17
TAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCTAGTTTCACTTTCCCAAGCTTGGGCTAGCATGACAAAGGGAAACTGAAAGGGAAACTGAGATCTAGACTCTAGAGGGTATATAATGGAAGCTCGAATTCCAG
<210> 18
<211> 687
<212> DNA
<213> Artificial sequence
<400> 18
MARGCLMTISGGTFDPSICEMEPIATPGAIQPHGALMTARADSGRVAHASVNLGEILGLPAASVLGAPIGEVIGRVNEILLREARRSGSETPETIGSFRRSDGQLLHLHAFQSGDYMCLDIEPVRDEDGRLPPGARQSVIETFSSAMTQVELCELAVHGLQLVLGYDRVMAYRFGADGHGEVIAERRRQDLEPYLGLHYPASDIPQIARALYLRQRVGAIADACYRPVPLLGHPELDDGKPLDLTHSSLRSVSPVHLDYMQNMNTAASLTIGLADGDRLWGMLVCHNTTPRIAGPEWRAAAGMIGQVVSLLLSRLGEVENAAETLARQSTLSTLVERLSTGDTLAAAFVAADQLILDLVGASAAVVRLAGQELHFGRTPPVDAMQKVLDSLGRPSPLEVLSLDDVTLRHPELPELLAAGSGILLLPLTSGDGDLIAWFRPEHVQTITWGGNPAEHGTWNPATQRMRPRASFDAWKETVTGRSLPWTSAERNCARELGEAIAAEMAQRTRAEELERVAMVDSLTRLWNRLGIETLLKREWEYATRKNSPISIVMIDFDNFKQINDQHGHLVGDEVLQGSARLIISVLASYDILGRWGGDEFMLILPGSGREQTAVLLERIQATIAQNPVPTSAGPMAISLSMGGVSVFTNQGEALQYWVEQANDQLMKVKRLGKGNFQLAEYHHHHHH
<210> 19
<211> 197
<212> DNA
<213> Artificial sequence
<400> 19
MPLSRDLREKTGMLHNRAETLLGLPSGIMGWADYVDWLRHFLALYDPIERRIVAFGGWSGLASFDPDPGHSRRLIQDLHALGIDTDRIPRAPAEYCPPLTNFARALGARYVLEGSALGGRVILHHLKKRIGDEIGNATAFFGGPSHGTATHWRAFQAALDRFGAAHPDKRADVLAGAAATFTALLEWFTPFVAARRV
<210> 20
<211> 24
<212> DNA
<213> Artificial sequence
<400> 20
GSGATNFSLLKQAGDVEENPGPSG
Claims (8)
1. A far-red light regulation gene expression loop control system, which is characterized by comprising: a photoreceptor for sensing a far-red light source, a processor for processing signals transmitted by said photoreceptor, and an effector for responding to signals transmitted by said processor;
wherein the photoreceptor comprises a promoter; the coding gene sequence of the photosensitive diguanylate cyclase BphS is shown as SEQ ID NO. 1; and c-di-GMP degrading enzyme YhjH, and a gene sequence coding the same is Genebank accession number: ANK 04038;
the processor comprises a promoter; and an immune signaling molecule STING, encoding the gene sequence Genebank accession number: NM-198282;
the ratio between the immune signaling molecule STING and the mass of the photoreceptors and the processor is 1:10: 10;
the effector comprises a promoter PFRLAnd a target gene reporter, denoted as PFRL-reporter, promoter P in said effectorFRLAny one selected from SEQ ID NOs.9 to 17;
the mass ratio of the processor to the photoreceptors and the effectors is (0.5-2): (0.5-40): (0.5-40);
promoter P in the EffectorFRLA DNA sequence and a weak promoter that are recognized and bound by trichosanthes kirilowii IFR 3; wherein the DNA sequence and weak promoter sequence recognized and bound by IFR3 are selected from the group consisting of: a) p shown in SEQ ID NO.9FRL1(5×ISRE-h_CMVmin)B) P shown in SEQ ID NO.10FRL2(hIFN-RE-h_CMVmin)C) P shown in SEQ ID NO.11FRL3((hIFN-RE)-3×ISRE-h_CMVmin)D) P shown in SEQ ID NO.12FRL4((hIFN-RE)-3×ISRE-h_min)E) P shown in SEQ ID NO.13FRL5((hIFN-RE)-3×ISRE-(hIFN-RE)-3×ISRE-h_min)F) P shown in SEQ ID NO.14FRL6((hIFN-RE)-3×ISRE-h_min-40bp)G) P represented by SEQ ID NO.15FRL7((hIFN-RE)-h_min)Nucleotide sequence, h) P shown as SEQ ID NO.16FRL8((hIFN-RE)-3×ISRE-(hIFN-RE)-h_min)I) P shown in SEQ ID NO.17FRL9(3×ISRE-(hIFN-RE)-h_min)And the spacing sequence between the weak promoter and the gene initiation codon ATG is 40 bp.
2. The far-red light regulatory gene expression loop control system of claim 1, wherein the photoreceptor promoter is selected from the group consisting of: a) SV40, the nucleotide sequence of which is shown as SEQ ID NO. 4; b) EF1 α promoter, the nucleotide sequence Genebank accession number: AY 043301; c) PGK promoter, its nucleotide sequence Genebank accession number: HZ 040569; d) cytomegalovirus early enhancer and chicken beta-actin promoter combination promoter CAG, the nucleotide sequence of which Genebank accession no: HQ 456319; e) cytomegalovirus promoter CMV, its nucleotide sequence Genebank accession number: KY 199427.
3. The far-red light regulatory gene expression loop control system of claim 1, wherein when two or more of the genes of interest are present, they are simultaneously expressed by linking them to an expression vector through self-cleaving peptide 2A.
4. Eukaryotic expression vectors, engineered cells or engineered cell transplantation vectors, kits comprising the far-red light-regulated gene expression loop control system of any one of claims 1 to 3.
5. Use of a far-red light-regulating gene expression loop control system according to any one of claims 1 to 3 for the preparation of a product for treating a disease, wherein when a gene of interest of an effector in the far-red light-regulating gene expression loop control system expresses insulin and/or glucagon-like peptide, the product is used for treating diabetes.
6. A method for constructing a gene loop control system as claimed in claim 1, wherein a photoreceptor which senses a far-red light source, a processor which processes a signal transmitted from said photoreceptor, and an effector which responds to a signal transmitted from said processor are constructed on a carrier by a genetic engineering technique.
7. A method of regulating gene expression in a host cell for purposes other than disease treatment, the method comprising the steps of:
a) constructing the far-red light regulatory gene expression loop control system of any one of claims 1-3 in a eukaryotic plasmid expression vector;
b) transfected into said host cell;
c) and inducing and regulating the host cell by regulating and controlling far-red light irradiation conditions to realize the expression of the target gene in the effector.
8. A method of regulating gene expression in a mouse for non-disease treatment purposes, the method comprising the steps of:
a) preparing a eukaryotic plasmid expression vector containing the far-red light-regulating gene expression loop control system of any one of claims 1 to 3;
b) preparing an engineered cell containing the far-red light regulatory gene expression loop control system;
c) preparing an engineered cell transplantation carrier containing the far-red light regulatory gene expression loop control system, and transplanting the engineered cell transplantation carrier into a mouse body;
d) the transplantation carrier containing the engineered cells is induced and expressed by regulating and controlling a far-red light source.
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Non-Patent Citations (3)
| Title |
|---|
| A red far red light responsive bi stable toggle switch to control gene expression in mammalian cells;Konrad Muller等;《Nucleic Acids Research》;20130125;第41卷(第7期);第e77,1-11页 * |
| Mind-controlled transgene expression by a wireless-powered optogenetic designer cell implant;Marc folcher等;《Nature communications》;20141111(第5期);摘要，第2页第1栏最后1-2段，第4页第2栏第1段，图1、2、5及其注释 * |
| Near-infrared Light Responsive Synthetic c di-GMP Module for Optogenetic Applications;第11期;《Synthetic photobiology》;20140128;第3卷(第11期);图1以及第808页第1栏第1段 * |
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