CN107177498A

CN107177498A - A kind of application of the gene cyclic system of remote control and regulation in treatment diabetes

Info

Publication number: CN107177498A
Application number: CN201610136478.6A
Authority: CN
Inventors: 叶海峰; 邵佳伟; 于袁欢; 薛帅; 余贵玲; 杨雪平; 朱苏承; 白郁
Original assignee: Suzhou Sai Sai Biotechnology Co Ltd
Current assignee: Shanghai Zhennuo Biotechnology Co ltd
Priority date: 2016-03-10
Filing date: 2016-03-10
Publication date: 2017-09-19
Anticipated expiration: 2036-03-10
Also published as: CN107177498B

Abstract

Control system is expressed the invention discloses a kind of far-red light gene loop, including experiences the photoreceptor of far-red light light source；Handle the processor that the photoreceptor transmits signal；And processor described in response transmits the effector of signal.The invention also discloses a kind of gene loop remote control and regulation system, including far-red light gene loop expression control system；Control device for sending remote control commands；And far-red light light supply apparatus.Invention additionally discloses the application of far-red light gene loop expression control system and the gene loop remote control and regulation system in treatment diabetes.The invention also discloses the carrier for expression of eukaryon, engineering cell or engineering cell transplantation carrier that control system is expressed containing the far-red light gene loop.The present invention quickly controlling gene can be expressed, with very-long-range control, intelligent control gene expression amount, regulating and expressing multiple height, height Space-time speciality, strong tissue penetration, good insulating and the features such as have no toxic side effect.

Description

A kind of application of the gene cyclic system of remote control and regulation in treatment diabetes

Technical field

The present invention relates to the multi-crossed disciplines fields such as synthetic biology, light science of heredity, electronic engineering, and in particular to one kind bag The gene loop for including far-red light gene loop expression control system including far-red light gene loop expression control system is long-range Regulator control system, and its regulation and control methods and applications.

Background technology

Synthetic biology is, using engineering theory as guidance, to design and synthesize various complex biological functional modules, system very To artificial life, and one applied to the production of specified chemical thing, biomaterial manufacture, gene therapy, organizational project etc. is comprehensive Close subject.Synthetic biology is developed rapidly by last decade, is made substantial progress in multiple application fields.Especially exist Mammalian cell synthetic biology field, people design, construct a variety of controllable gene genetic circuits for diagnosing and controlling Treat metabolic disease, cancer, disease of immune system etc..

In synthetic biology and disease treatment field, the molecular switch of artificial accuracy controlling gene expression is in disease treatment Have become a kind of indispensable means.Have in the world at present and be by artificial regulatory inducible gene expression a lot System, can be largely classified into two major classes：(1) by chemical substance come induction regulating controlling gene expression system；(2) by physical method come Induction regulating controlling gene expression system.

Chemical inducer is mainly some small-molecule substances, for example tetracycline induction regulating controlling gene expression system [Gossen, M., Proc Natl Acad Sci USA, 1992,89 are waited:5547-5551], acetaldehyde induction regulating controlling gene expression system [, Nat such as Weber, W. Biotechnol., 2004Nov；22(11):1440-1444], benzoic acid and vanillic acid induction regulating controlling Gene expression system [, the Nucleic Acids such as Xie, M. Res.2014Aug；42(14):] etc..These systems are generally with change Material is inducer, and some systems obtain very excellent induction performance by optimization for many years.Thus past Between several years, chemical substance carrys out induction regulating controlling gene expression system and is widely used on the time regulating and controlling the effective expression of gene.But That the small molecule inducer that chemical substance to be related in induction regulating controlling gene expression system has the problem of some are potential, such as it is malicious Property, pleiotropism, non-specificity and tissue permeability difference etc..And it is difficult to accomplish that chemical substance, which comes induction regulating controlling gene expression system, The accuracy controlling on space-time.

Physical method includes come the regulator control system induced：Ultraviolet induction regulation and control " prisoner's cage (cage) " technology [Keyes, WM., Trends Biotechnol.2003Feb are waited；21(2):53-5.], far red light control heat shock effect induction regulating controlling base Because of the expression system [, Nat such as Kamei, Y. Methods.2009Jan；6(1):79-81.], radio control temperature sense lures Lead the controlling gene expression system [, Science.2012May such as Stanley, SA. 4；336(6081):604-8.] etc..These Toxicity by the system of physical method inducible gene expression generally to cell is very big, may cause irreversible to cell Injury even makes cell death, and some apparatus for being related to of these systems are costly and complex operation.

Just a kind of preferable gene expression inducer, because its generally existing in nature, is readily available, highly may be used Control, without toxicity, it is most important that, it can realize accuracy controlling over time and space.Therefore derivant is used light as It is biologists always in the target of pursuit to carry out controlling gene to express and then regulate and control the various metabolic activities of life entity. In our previous research work, attempt the method design using synthetic biology, synthesized blue light regulation and control transgene expression Gene loop, switch deactivation, regulating and expressing gene [Ye, H. etc., Science, 2011.332 (6037) are used as by the use of blue light: p.1565-8.].East China University of Science Yang Yi professor seminar report LightOn blue lights controlling gene expression switch [Wang, X. etc., Nat Methods, 2012.9 (3):p.266-9.].But blue light is used to regulate and control gene expression in vivo and has great limitation Property --- the transdermal efficiency of blue light is low, is difficult to deactivate target gene through skin or abdominal cavity, significantly limit light regulator control system Deep level development and clinical practice.The Wilfired Weber professors seminar of Freiburg, Germany university develops feux rouges regulation and control Transgene expression controlling switch [M ü ller K. etc., Nucleic Acids Res, 2013,41 (7):e77.Nat Protoc, 2014,9(3):622-32], but this feux rouges control system does not embody preferable Gene expression intensities, it is also not any Experimental data shows the feux rouges controlling switch effect that controlling gene is expressed in animal model mouse body.Importantly, this is red Photocontrol switch needs additionally to add a kind of phytochrome algocyan (Phycocyanobilin) ability activated gene expression.And , need to be artificial synthesized in laboratory and this pigment source is extremely inconvenient, it is impossible to obtained by commercially available, synthesis step is complicated, Synthetic product is unstable and yield is extremely low.These unfavorable factors all significantly limit further should for the feux rouges control system With.

Diabetes are one of most important chronic epidemic diseases of current threat global human health.Diabetes are a kind of Influence main based on blood glucose rise etc. after the chronic lifelong disease of human health, morbidity, and adding with the state of an illness Weight, it will involve each important organ of body and body tissue, triggers multiple complications, it is impossible to thoroughly cures, can only control.Closely Decades, global diabetic's number increases with surprising rapidity.One multinational joint study shows that 1980-2008 is global Diabetes number increases to 3.47 hundred million people by 1.53 hundred million people.14, the whole nation of diabetology branch of Chinese Medical Association tissue coverage provinces and cities Diabetes Epidemiological Investigation data display, China's maturity-onset diabetes number of patients is up to 92,400,000, the incidence of disease 9.7%.With The improvement of people's living standards, living-pattern preservation, fat and overweight ratio increases year by year, makes diabetes " reserve duty portion Team " is continually.Type 1 diabetes are a kind of chronic autoimmune diseases, are due to the beta Cell of islet of excreting insulin in pancreas By the targeted disruption of autoreactive T cell, insulin secretion is caused to reduce, so that body loses regulation blood glucose Ability, and then body metabolism is influenceed, patient usually requires to receive insulin therapy all the life.The distinctive mark of diabetes B It is abnormal sugar balance, this abnormal balance is also that as caused by insulin deficit, and insulin deficit comes from beta Cell of islet Functionally inactive or insulin resistance, that is, target organ, the tissue of insulin action drop to the reactivity of its biological effect Low or forfeiture, body needs to secrete the next compensatory this defect of more insulin.So frequently resulting in other serious has life The second dangerous complication, such as angiocardiopathy, kidney failure and PVR.But so far, diabetes there is no method root Control and need to be administered all the life.Diabetic needs daily oral hypoglycaemic medicine or insulin injection to maintain glucostasis, especially It is that insulin injection is unable to reach controllably uelralante, easily causes risk of hypoglycemia.

The content of the invention

To overcome the problem of prior art is present, control is expressed present invention firstly provides a kind of new far-red light gene loop System processed and a kind of express control system, control device and far-red light light supply apparatus including the far-red light gene loop Gene loop remote control and regulation system.In the present invention, the transdermal efficiency far of far-red light is better than blue light, can pass through skin, musculature 7-8 centimetres, it is possible to achieve long-range, seamless slash regulation and control are implanted in intraperitoneal target cell expression target gene, or even controllable Internal particular organization organ expression target gene.The photon energy of far-red light is more much lower than blue light photon energy, and cell is produced Toxic side effect to be far smaller than blue light.And, far-red light control system of the present invention need not additionally add any phytochrome just Can directly it be activated by far-red light.Present system and far-red light controlling switch have very big potential using value, can be widely popularized In clinical practice.

The present invention proposes a kind of engineer, synthesis and based on intelligent mobile phone platform very-long-range regulates and controls transgene expression Gene loop control system.The system controls far-red light light source by smart mobile phone by LAN WiFi or 2G/3G/4G network Gene loop control system two parts composition of intelligence control system and far-red light regulation and control transgene expression.The present invention provides intelligence The intelligence control system of mobile phone control far-red light light source, the eukaryotic expression comprising smart mobile phone regulation and control transgenic expression system are carried Body, in host cell smart mobile phone regulation and control transgene expression method and it is transplanted in Mice Body smart mobile phone regulation and control and is turned The method of gene expression.The invention provides the kit that above-mentioned smart mobile phone regulates and controls transgenic expression system each component.This hair It is bright to additionally provide a kind of novel diabetes therapy regulated and controled based on intelligent mobile phone platform very-long-range.The present invention can quickly regulate and control base Because of expression, with very-long-range control, intelligent control gene expression amount, regulating and expressing multiple height, height Space-time speciality, strong tissue Penetration power, good insulating and the features such as have no toxic side effect.

Far-red light gene loop proposed by the present invention expresses control system, and it includes：Experience the photoreception of far-red light light source Device；Handle the processor that the photoreceptor transmits signal；Processor described in response transmits the effector of signal.

Wherein, the photoreceptor includes photosensitive two guanylate cyclases Bphs, and it changes GTP under the conditions of far-red light For c-di-GMP.Wherein, the photosensitive two guanylate cyclases Bphs is one of most critical albumen, is used as core component.

Wherein, the photosensitive two guanylate cyclases Bphs of the photoreceptor is by the 1-511 amino acids of BphG albumen Merged with the 175-343 amino acids of Slr1143 albumen, and 587 arginine of fusion protein are sported into alanine (R587A) prepare；Wherein, the coding BphG GFPs can be synthesized by artificial chemistry, can be from class ball In red bacterium (Rhodobacter sphaeroides)；The Slr1143 albumen may come from cytoalgae (Synechocystis sp.), can also artificial chemistry synthesis, the gene of coding Slr1143 albumen of the present invention by Artificial chemistry is synthesized.

Wherein, the structure form of the photoreceptor includes：

A) the photosensitive two guanylate cyclases Bphs encoding genes (Bphs) of artificial synthesized bacterium；

B) the photosensitive two guanylate cyclases Bphs encoding genes of artificial synthesized bacterium are dropped by 2A sequences and c-di-GMP Solve enzyme YhjH encoding genes and be connected (Bphs-2A-YhjH)；

C) the photosensitive two guanylate cyclases Bphs encoding genes of artificial synthesized bacterium are closed by 2A sequences and phytochrome It is connected (Bphs-2A-Bpho) into enzyme Bpho encoding genes；

D) the photosensitive two guanylate cyclases Bphs encoding genes of artificial synthesized bacterium are closed by 2A sequences and phytochrome It is connected into enzyme Bpho encoding genes, then is connected by 2A sequences with c-di-GMP digestive enzyme YhjH encoding genes (Bphs-2A- Bpho-2A-YhjH)；

Wherein, the 2A sequences can be substituted by internal ribosome entry site sequence IRES；The phytochrome synthesis Enzyme Bpho has the function of synthesis phytochrome biliverdin (biliverdin)；The digestive enzyme YhjH of the c-di-GMP has will C-di-GMP is degraded to pGpG function.

Wherein, described Bphs, Bpho, YhjH amino acid sequence are selected from sequence 45,46,48.

Wherein, the promoter of the expression photoreceptor includes：a)SV40；b)CMV；c)hEF1α；d)mPGK；e)CAG.

Wherein, the processor includes：Believe as the polypeptide of DNA binding domain and c-di-GMP binding domain, as nuclear location Number NLS polypeptide, the polypeptide as link field and the polypeptide as transcriptional regulatory domain.

Wherein, the polypeptide as DNA binding domain and c-di-GMP binding domain, it is energy after being combined with c-di-GMP The albumen combined with specific DNA sequence dna, such as BldD albumen, the BldD albumen can come from streptomyces venezuelae BldD albumen in (Streptomyces venezuelae), can also be artificial synthesized, and its amino acid sequence is selected from sequence 49, The BldD albumen of the present invention is by artificial synthesized.

Wherein, the polypeptide as nuclear localization signal NLS, it can copy diversified forms, its amino acid sequence for 1-3 For selected from sequence 54.

Wherein, the polypeptide as linkage function domain (Linker), its length can be from a variety of shapes of 0-30 amino acid Formula, its amino acid sequence is selected from sequence 55.

Wherein, the polypeptide as transcriptional regulatory domain, it has the domain protein of transcriptional activation function, bag for all Include NF- Κ B p65 subunits transcriptional activation domain, Features of The Heat Shock Transcription Factor HSF1 transcription activating domains, Herpes simplex virus particles The transcriptional activation domain VP64 of albumen VP16 transcriptional activation domains and its 4 copy；Its amino acid sequence be selected from sequence 50, 51、52、53。

Wherein, the polypeptide as transcriptional regulatory domain is placed in the polypeptide of the DNA binding domain and c-di-GMP binding domain BldD N-terminal or C-terminal.

Wherein, it can be directly connected to or be connected by connecting peptide between the polypeptide in difference in functionality domain in the processor.

Wherein, the effector includes P_FRL-reporter；The effector includes promoter sequence and treats transcription factor Nucleotide sequence.

Wherein, the promoter sequence comprising the protein bound DNA sequence dnas of processor BldD and promotor gene express it is weak Promoter.

Wherein, the protein bound DNA sequence dnas of processor BldD, it is DNA binding domain and c-di-GMP binding domain The DNA sequence dna that polypeptid specificity is recognized and combined, can be from streptomyces venezuelae (S.venezuelae) bldM and The partial sequence of whiG promoter regions, can also artificial chemistry synthesis, BldD specific recognitions of the invention and the DNA combined Sequence bldM and whiG promoter partial sequence is synthesized by artificial chemistry.

Wherein, the nucleotide sequence of the bldM is selected from sequence 18；The nucleotide sequence of the whiG is selected from sequence 19.

Wherein, the partial sequence of the bldM and whiG promoter regions, it is copied for 1-10.

Wherein, the weak promoter of promotor gene expression includes all weak promoters, and it includes TATA box, big and small Cellular virus CMV minimal promoters and its mutant CMVmin 3G.

Wherein, the nucleotide sequence of the promoter sequence be selected from sequence 20,21,22,23,24,25,26,27,28,29, 30、31、32、33、34、35、36、37、38、39。

Wherein, the albumen for treating that transcribable nucleic acid sequence coding schedule reaches can be all significant albumen, including conduct The albumen of reporter gene and/or pharmaceutical protein or the nucleotide sequence of small peptide as treatment disease；Wherein, it is described to be used as report base The albumen of cause includes SEAP (SEAP), enhanced green fluorescence protein (EGFP, EYFP), luciferase (Luciferase) nucleotide sequence；Include insulin (Insulin), the high blood of pancreas as the pharmaceutical protein or small peptide for the treatment of disease The nucleotide sequence of sugared element sample peptide (GLP-1).

Wherein, all described significant albumen can express multiple eggs simultaneously by 2A sequences on an expression vector In vain, including SEAP-2A-Insulin, EGFP-2A-Insulin, EGFP-SEAP-2A-Insulin；Wherein, the 2A sequences can To be substituted by internal ribosome entry site sequence IRES.

In one embodiment, far-red light gene loop expression control system of the present invention includes：Experience far-red light Photoreceptor Bphs；Processing photoreceptor transmits the processor P65-VP64-BldD of signal；Reply process device transmits signal Effector P_FRL-reporter。

Far-red light gene loop of the present invention expresses control system, thirdly part can be loaded by three kinds of plasmids.

First, be loaded with the photoreceptor expression vector plasmid for experiencing far-red light, the photoreceptor comprising Bphs, Bpho, YhjH and 2A.

Wherein Bphs is merged by BphG 1-511 amino acids and Slr1143 175-343 amino acids, and And the 587th amino acids arginine of the fusion protein is sported into alanine (R587A) got；The gene of the coding BphG Hydrogenlike silicon ion (Rhodobacter sphaeroides) is may come from, can also be synthesized by artificial chemistry, institute of the present invention The coding BphG GFPs of use are synthesized by artificial chemistry；The coding Slr1143 GFPs may come from cytoalgae (Synechocystis sp.), can also be synthesized by artificial chemistry, Slr1143 GFPs fragment of the present invention Synthesized by artificial chemistry.Bphs 1-511 amino acids are PAS-GAF-PHY domains, can experience far-red light；512- 680 amino acids are DGC domains, GTP can be transformed into c-di-GMP after activation.The light receptor can be from other Pseudomonas The albumen with identical function domain, such as BphG [Ryu M H. etc., ACS synthetic biology, 2014,3 (11): 802-810.], BphG etc. can also be synthesized by artificial chemistry.

Bpho is the blood red oxidizing ferment being present in hydrogenlike silicon ion (Rhodobacter sphaeroides), and it is encoded Gene can also be synthesized by artificial chemistry, and it can synthesize phytochrome biliverdin (biliverdin), be Bphs synthesis c- Di-GMP provides phytochrome [Ryu M H. etc., ACS synthetic biology, 2014,3 (11):802-810.].

YhjH comes from Escherichia coli (E.coli.), and its gene can be synthesized by artificial chemistry, and it has c-di- GMP is degraded to pGpG function [Ryu M H. etc., ACS synthetic biology, 2014,3 (11):802-810.], its Digestive enzyme can also be other all albumen with the function that c-di-GMP is degraded to pGpG.

2A is the amino acid sequence that same promoter expresses two different albumen, can there is T2A, F2A,

[Doronina VA etc., Molecular the and cellular biology, 2008,28 (13) such as P2A:4227‐ 4239].2A sequences wherein used can be substituted by internal ribosome entry site sequence IRES.

When expressing Bphs, Bpho and YhjH in mammalian cell, in far-red light irradiation and phytochrome biliverdin In the presence of, Bphs can perceive far-red light and GTP is synthesized into c-di-GMP.When intensity of illumination or different light application time, it can be closed Into the c-di-GMP of different amounts, and intensity of illumination or light application time are within the specific limits with producing c-di-GMP amount in just It is related.When not needing c-di-GMP, it can be degraded by YhjH.

Second, being loaded with the processor plasmid that processing photoreceptor transmits signal.The processor by as DNA binding domain and The polypeptide BldD of c-di-GMP binding domain, as the polypeptide NLS for entering nuclear signal domain, is used as the polypeptide Linker and work of link field Polypeptide P65, VP64, VP16 and HSF1 for transcription activating domain etc. are constituted.Wherein BldD albumen may come from Venezuela's chain Mould (Streptomyces venezuelae) [Tschowri N. etc., Cell, 2014,158 (5):1136-1147.], also may be used To be synthesized by artificial chemistry, coding BldD GFPs of the invention are artificial chemical synthesis, and VP16 is herpes simplex virus Granule protein transcriptional activation domain, p65 are that NF- Κ B p65 subunits transcriptional activation domain, HSF1 are Features of The Heat Shock Transcription Factor Transcription activating domain [Konermann S. etc., Nature.2015Jan 29；517(7536):583-8.].

NLS can be 1-3 copies etc., and it is directly connected in BldD N-terminal, and P65, VP64, HSF1 etc. is used as transcription activating domain Then can respectively or combination of two or three's series connection are connected between BldD N-terminal or C-terminal, its each component then to be used as linkage function Polypeptide Linker (0-30 amino acid) be connected.Specific be built with can have following several forms：NLS-BldD- VP16、VP64-Linker-NLS-BldD、VP64-Linker-NLS-BldD-Linker-VP64、NLS-BldD-Linker- VP64、P65-Linker-VP64-Linker-NLS-BldD、P65-HSF1-Linker-NLS-BldD、NLS-BldD- Linker-P65-HSF1 and VP64-NLS-BldD-Linker-P65-HSF1 etc..

The processor is when c-di-GMP is not present, and it can not be with specific sequence identification, the combination in effector；When When c-di-GMP exists and forms the tetramer, two molecular processor can be combined with the c-di-GMP tetramer, form one six Dimeric complexes [Tschowri N. etc., Cell, 2014,158 (5):1136-1147.], and can be with the DNA sequence dna in effector Recognize and combine.

Third, being loaded with the plasmid of effector.The effector is by insulating signal, promoter sequence and treats transcribable nucleic acid sequence group Into.

Wherein, insulating signal can be simian virus 40 PolyA (SV40PolyA), bovine growth hormone gene PolyA (BGH PolyA) etc., with the function of blocking upstream promoter influence.Promoter sequence is by the BldD DNA sequence dnas combined and promotor gene The weak promoter sequence composition of expression.The DNA sequence dna that BldD is combined is from streptomyces venezuelae (S.venezuelae) The partial sequence of bldM and whiG promoter regions, 1 repeats to a variety of wild types or the mutant forms such as 10 repetitions, is characterized in Can be by upstream processor BldD institute's specific recognitions and the sequence combined, the partial sequence of the bldM and whiG promoter regions Can also artificial chemistry synthesis.

The weak promoter of promotor gene expression, it includes TATA box, cytomegalovirus CMV minimal promoters and mutant (CMV_min3G), when upstream processor is not present, it does not express or hardly expressed downstream nucleotide sequence to be transcribed.

It can be any significant albumen to treat transcribable nucleic acid sequence, and it can be the alkaline phosphorus of reporter gene secreting type Sour enzyme (SEAP), enhanced green fluorescence protein (EGFP, EYFP), luciferase (Luciferase) etc.；Can also be treatment The functional form albumen of disease, such as insulin (Insulin), glucagon-like peptide (GLP-1).Different required albumen It can also be connected with 2A, realize a promoter while expressing multiple albumen.Such as SEAP-2A-Insulin, EGFP-2A- Insulin, EGFP-2A-SEAP-2A-Insulin etc..

In the presence of c-di-GMP, its tetramer formed and two molecular processors are formed in six aggressiveness, recognition effect device Specific sequence and combination, recruit transcription factor start transcriptional expression downstream gene.When c-di-GMP is not present, processor It can not be attached on effector, then can not open the transcriptional expression of gene.

Another object of the present invention is to provide a kind of new gene loop remote control and regulation system.With development of Mobile Internet technology Fast development, mobile phone remote control triggers a change in various equipment applications, and market prospects are huge.Mobile phone very-long-range control System combination processed hardware resource and internet cloud end platform, can pass through LAN WiFi by hardware device and access Internet of Things cloud End, by mobile phone A PP softwares easily very-long-range control device, even across intercontinental control.The present invention is by the super remote of mobile phone Journey intelligence control system is combined with far-red light gene loop expression control system, is proposed super remote based on intelligent mobile phone platform The gene loop tele-control system of journey controlling gene expression.

Gene loop remote control and regulation system of the present invention includes：The far-red light gene loop expresses control system, for sending out Go out the control device and far-red light light supply apparatus of remote control commands；Wherein, the control device is by sending control instruction Far-red light light supply apparatus described in remote control and regulation, the far-red light sent by the far-red light light supply apparatus regulates and controls the far-red light base Because loop expresses control system, regulation and control transgene expression is realized.

Wherein, the control device sends remote control commands to regulate and control by LAN WiFi or 2G/3G/4G network State the different working condition of far-red light light supply apparatus.

Wherein, the different working condition includes the light that is turned on and off, can adjust on demand of the far-red light light source According to intensity, light application time or illuminating method.

Wherein, the intensity of illumination is 0-5mW/cm²；The irradiation time is 0-72h；The illuminating method includes pulse Formula irradiation, Continuous irradiation, direct irradiation or the projection card portrayed with hollow out spatially control the base of the cell of diverse location Because of the irradiation of expression.

Wherein, the control device includes smart mobile phone App and/or Intelligence Remote Controller.The intelligent remote control Device includes all controllers with remote control function, and it includes single channel output and/or the intelligent remote control of multiple-channel output Device.

In a particular embodiment, the present invention is based on intelligent mobile phone platform very-long-range gene loop remote control and regulation system, bag Include：Smart mobile phone controls intelligent controlling device, the far-red light light of far-red light light source by LAN WiFi or 2G/3G/4G network Source device and far-red light regulate and control the gene loop remote control and regulation system of transgene expression.Wherein, smart mobile phone passes through LAN WiFi or 2G/3G/4G networks control the intelligent controlling device of far-red light light source, including smart mobile phone App and/or intelligent remote control Device processed.

First, smart mobile phone App clients.Smart mobile phone controls far-red light by LAN WiFi or 2G/3G/4G network The intelligence control system of light source is to complete control to far-red light light source by APP clients input instruction on smart mobile phone, Instruction can be sent to Intelligence Remote Controller by it by LAN WiFi or mobile phone mobile data through cloud server.Tool There is input very-long-range to select which kind of far-red light light source and its working condition opened or closed, very-long-range regulation and control far-red light light source works strong The function of the instructions such as degree, working time, so as to realize to target gene controlling type induced expression.The App can install with it is any One smart mobile phone, it is adaptable to the operating system such as Android, iOS.

Second, the remote intelligent controller with multiple output function.Smart mobile phone passes through LAN WiFi or 2G/3G/ The intelligence control system of 4G networks control far-red light light source is to receive to be sent to high in the clouds by mobile phone by Intelligence Remote Controller to take Control of the signal and execution of business device to far-red light light source.Its control condition can be：A) very-long-range is controlled, intelligent remote control Device reception mobile phone processed, which is sent to the signal of cloud server, can realize very-long-range control, pass through LAN WiFi or 2G/3G/4G Network, no matter control object and control target different cities, country not even with intercontinental can realize very-long-range Control；B) equipment supports multi-channel control, can independent control, multi-output switch power source realization per road.Control effect is pushed away in real time Mobile phone A PP clients are delivered to, realize that control far-red light light source produces different intensities of illumination, so as to realize that smart mobile phone is remotely adjusted Control the variable expression of gene；C) equipment supports time switch, realizes time switch and a key switch function, realizes that control is remote Red-light source produces different light application times, so as to realize the variable expression of smart mobile phone remote control and regulation gene.

Wherein, the far-red light light supply apparatus is that can produce the light supply apparatus that wavelength is 600-900nm far-red lights.

Wherein, the far-red light light supply apparatus can produce the light source of 600-900nm wavelength far-red lights, can be 600- 900nm LED, infrared therapeutic device, laser lamp etc..In one embodiment, experiment in vitro uses 720nm in the present invention Far-red light LED, realize that the control of smart mobile phone very-long-range, different light application time controlling gene expression and different illumination intensity are adjusted Control gene expression etc..Experiment in vivo is then by controlling to transplant in feux rouges physiotherapy equipment or 720nmLED bodies, being powered reality through radio Existing smart mobile phone very-long-range control, different light application time controlling gene expression and the expression of different illumination intensity controlling gene etc.. Smart mobile phone controls the intelligence control system of far-red light light source to control far-red light light by LAN WiFi or 2G/3G/4G network Source has three kinds of modes to realize that it is：Mobile phone and far-red light light source access same LAN WiFi, and mobile phone is accessed by it LAN WiFi very-long-ranges control far-red light light source；Or mobile phone by 2G/3G/4G networks very-long-range control far-red light light source and In emergency circumstances manual key controls far-red light light source.

In a particular embodiment, the present invention based on the control such as intelligent mobile phone platform, remote control by LAN WiFi or The gene loop remote control and regulation system of 2G/3G/4G network very-long-ranges controlling gene expression, wherein, far-red light light supply apparatus is by referring to Make reception device and light source constitute, far-red light light source is controlled by mobile phone, remote control etc., produce varying strength, light application time, irradiation Far-red light gene loop described in the far-red light induced expression of mode expresses control system, shown in such as Fig. 2 (a).When dynamic in mammality Expression far-red light photoreceptor, processor, effector in thing cell carrier, when no far-red light is induced, in photoreceptor Bphs is not activated, no c-di-GMP synthesis, so that processor can not be made to be attached to the promoter that far-red light induction starts (P_FRL), then it can not drive downstream destination gene expression.When far-red light is induced, the Bphs in photoreceptor is activated, and synthesizes c- Di-GMP, makes processor be attached to the promoter (P that far-red light induction starts_FRL), recruit transcription factor and start transcription and translation, drive Dynamic downstream destination gene expression, shown in such as Fig. 2 (b).

The invention also provides carrier for expression of eukaryon, the engineering that control system is expressed containing the far-red light gene loop Change cell or engineering cell transplantation carrier.

The invention also provides prepare containing the far-red light gene loop express control system carrier for expression of eukaryon, It is engineered cell or the method for being engineered cell transplantation carrier.

It is thin that the carrier for expression of eukaryon includes the mammal containing far-red light gene loop expression control system Cellular expression carrier.The expression vector can be the independent carrier containing far-red light photoreceptor encoding gene or individually contain place The carrier or the independent carrier containing effector encoding gene of device encoding gene are managed, described effector contains far-red light response Promoter, but without needing transcribable nucleic acid sequence.Or or the expression that all contains containing two of which or three kinds carry Body.The building mode of foregoing all mammalian cell expression vectors refers to table 1.

The invention also provides the carrier for expression of eukaryon of control system is expressed containing the far-red light gene loop in system Application in standby treatment diabetes medicament.

Another object of the present invention is, it is proposed that express control system or the gene using the far-red light gene loop The loop remote control and regulation system method that controlling gene is expressed in host cell.It the described method comprises the following steps：A) will be described Far-red light gene loop expression control system is built in eucaryon plasmid expression vector；B) host cell is imported through transfection In；C) condition is irradiated come host cell described in induction regulating controlling by regulation and control or remote control and regulation far-red light, realizes that the effector is compiled Code gene expression.Wherein, the host cell includes mammalian cell.

In a particular embodiment, the present invention expresses control system or the gene ring using the far-red light gene loop The expression of great distance journey regulator control system controlling gene in mammalian cell, comprises the following steps：

A) intelligent mobile phone platform is expressed by LAN WiFi or 2G/3G/4G network very-long-range controlling gene Far-red light gene loop expression control system is built in mammalian cell expression vector；

B) plasmid is imported in mammalian cell；

C) condition is irradiated by the certain far-red light of LAN WiFi or 2G/3G/4G network overshoot control by smart mobile phone to induce Regulate and control the mammalian cell, make the effector P in the mammalian cell_FRL- reporter encoding genes (such as SEAP, EGFP, Luciferase, Insulin, GLP-1 etc.) expression.

D) respectively in the expression of tri- time point testing goal genes of 24h, 48h, 72h

Plasmid construction method reference material method and table 1 in the present invention.The side that plasmid is imported in mammalian cell Method includes：Calcium phosphate transfection, PEI transfections, liposome transfection electroporation transfection, virus infection etc..

Wherein, certain far-red light is regulated and controled by LAN WiFi or 2G/3G/4G network very-long-range by smart mobile phone and irradiates bar Part, includes selection and exposure intensity, the selection of time and illuminating method of light source.Wherein light source includes LED, far-red light physiotherapy Instrument etc.；Exposure intensity, it is 0-5mW/cm²Deng；Irradiation time, it is 0-72h etc., can be Continuous irradiation or discontinuous irradiation Deng；Illuminating method, including direct irradiation and the projection card portrayed with hollow out spatially control the base of the cell of diverse location Because of expression.

The invention also provides remotely adjusted using far-red light gene loop expression control system or the gene loop The method that control system carries out transgenic regulation expression in transplantation carrier.

Methods described includes step：A) the eucaryon plasmid table that control system is expressed containing the far-red light gene loop is prepared Up to carrier；B) the engineering cell that control system is expressed containing the far-red light gene loop is prepared；C) prepare containing described remote Feux rouges gene loop expresses the engineering cell transplantation carrier of control system；D) by remote control and regulation far-red light light source to engineering Cell transplantation carrier carries out induced expression, makes the effector P in the transplantation carrier_FRL- reporter encoding genes (such as SEAP, EGFP, Luciferase, Insulin, GLP-1 etc.) expression.E) respectively in tri- time point testing goal bases of 24h, 48h, 72h The expression of cause.

In a particular embodiment, the present invention expresses control system or the gene ring using the far-red light gene loop The expression of great distance journey regulator control system controlling gene in engineering cell transplantation carrier, comprises the following steps：

A) the eucaryon plasmid expression vector that control system is expressed containing the far-red light gene loop is prepared；

B) the engineering cell that control system is expressed containing the far-red light gene loop is prepared；

C) the engineering cell transplantation carrier that control system is expressed containing the far-red light gene loop is prepared；

D) induced expression is carried out to the transplantation carrier containing engineering cell by remote control and regulation far-red light light source, made described Effector P in transplantation carrier_FRL- reporter encoding genes (such as SEAP, EGFP, Luciferase, Insulin, GLP-1 Deng) expression；

E) respectively in the expression of tri- time point testing goal genes of 24h, 48h, 72h.

The method of described structure carrier for expression of eukaryon refers to table 1；The method of the engineering cell turns including calcium phosphate Dye, PEI transfections, liposome transfection electroporation transfection or virus infection；The preparation method bag of the engineering cell transplantation carrier Include：Microcapsules are prepared, sodium alginate blob of viscose skin is prepared, prepares hollow-fibre membrane grafts.

Wherein, regulate and control certain far-red light by LAN WiFi or 2G/3G/4G network very-long-range by smart mobile phone to irradiate Condition, includes selection and exposure intensity, the selection of time and illuminating method of light source.Wherein light source includes LED, far-red light reason Treat instrument etc.；Exposure intensity, it is 0-5mW/cm²Deng；Irradiation time, it is 0-72h etc., and irradiation time is more than 0h, Ke Yiwei Continuous irradiation or discontinuous irradiation etc..

The present invention is also provided expresses control system or the gene loop remote control and regulation system by the far-red light gene loop The method united in transplantation carrier implantation Mice Body, and far-red light gene loop expression control system or the gene loop The method that remote control and regulation system carries out transgenic regulation expression in Mice Body, including：

A) the eucaryon plasmid expression vector that control system is expressed containing the far-red light gene loop is prepared；B) prepare and contain The far-red light gene loop expresses the engineering cell of control system；C) prepare and express control containing the far-red light gene loop The transplantation carrier of system processed or the gene loop remote control and regulation system；D) control will be expressed containing the far-red light gene loop In the transplantation carrier implantation Mice Body of system or the gene loop remote control and regulation system；E) remote control and regulation far-red light light source is passed through Induced expression is carried out to the transplantation carrier containing engineering cell, makes the effector P in the transplantation carrier_FRL- reporter is compiled Code gene (such as SEAP, EGFP, Luciferase, Insulin, GLP-1) expression；F) respectively in 24h, 48h, 72h tri- Between put testing goal gene expression.

In a particular embodiment, the engineering cell transplantation of control system will be expressed containing above-mentioned far-red light gene loop Carrier, which is transplanted in Mice Body, carries out far-red light induced expression, and step is as follows：

C) prepare and express control system or the gene loop remote control and regulation system containing the far-red light gene loop Transplantation carrier；

D) shifting of control system or the gene loop remote control and regulation system will be expressed containing the far-red light gene loop Plant in carrier implantation Mice Body；

E) induced expression is carried out to the transplantation carrier containing engineering cell by remote control and regulation far-red light light source, made described Effector P in transplantation carrier_FRL- reporter encoding genes (such as SEAP, EGFP, Luciferase, Insulin, GLP-1 Deng) expression；

F) respectively in the expression of tri- time point testing goal genes of 24h, 48h, 72h.

The method for wherein preparing transplantation carrier prepares microcapsules, prepares sodium alginate blob of viscose skin, prepares hollow-fibre membrane Grafts；Implantation method can be intraperitoneal transplantation or subcutaneous transplantation etc.；

The method that controlling gene is expressed in Mice Body includes the selection of light source and the control of light source.Light source includes LED Lamp, physiotherapy equipment, laser lamp.Illumination method include light application time, intensity of illumination, illumination frequency it is selected.

The present invention also proposes to express control system or the gene loop remote control and regulation using the far-red light gene loop Application of the system in Remedies for diabetes is prepared.The diabetes include type i diabetes and/or type ii diabetes.This hair Bright system provide it is a kind of it is safe and reliable, can accuracy controlling uelralante and glucagon-like peptide treatment glycosuria on space-time The new strategy of disease.The invention provides the new method for the treatment of diabetes, new strategy.The system adjustable control insulin and/or pancreas Glucagon-like peptide GLP-1 expression.The expression of the insulin, which is built, includes SEAP-2A-Insulin, EGFP-2A- Insulin、EGFP-2A-SEAP-2A-Insulin.The expression of the glucagon-like peptide GLP-1 is including GLP-1-Fc etc..

The invention also provides a kind of kit, it contains the far-red light gene loop expression control system or containing State gene loop remote control and regulation system.The invention also provides a kind of kit, it, which is equipped with, contains the far-red light gene loop Express the carrier for expression of eukaryon of control system or/and transfected the host cell containing the carrier for expression of eukaryon and said accordingly Bright book.

The kit, including the intelligent mobile phone platform very-long-range regulation and control far-red light gene loop expression control system are each Component plasmid kit, the lactation containing the intelligent mobile phone platform very-long-range regulation and control far-red light gene loop expression control system Class zooblast kit.Including being adjusted equipped with the intelligent mobile phone platform by LAN WiFi or 2G/3G/4G network very-long-range Control the far-red light gene loop expression control system each component plasmid kit and pass through LAN containing intelligent mobile phone platform WiFi or 2G/3G/4G networks very-long-range regulates and controls the mammalian cell examination of the far-red light gene loop expression control system Agent box and corresponding specification.

Brief description of the drawings

Fig. 1 is that remote control far-red light gene loop of the present invention expresses principle of the control system in mammalian cell Diagram is intended to.

In Fig. 2：Fig. 2 (a) is that control device of the present invention is controlled far by LAN WiFi or 2G/3G/4G network very-long-range Red-light source schematic device；Fig. 2 (b) is the far-red light gene loop expression control that far-red light of the present invention regulates and controls transgene expression Schematic diagram of the system in mammalian cell.

Fig. 3 is the gene ring that smart mobile phone of the present invention controls far-red light light source by LAN WiFi or 2G/3G/4G network Express the APP schematic diagrames of control system in road.

Fig. 4 is that smart mobile phone of the present invention controls the intelligence of far-red light light source to control by LAN WiFi or 2G/3G/4G network The schematic diagram and pictorial diagram of the intelligent controller of system processed.

Fig. 5 is that smart mobile phone of the present invention controls the intelligence of far-red light light source to control by LAN WiFi or 2G/3G/4G network The schematic diagram and pictorial diagram of the multi-output switch power source of system processed.

Fig. 6 is that smart mobile phone of the present invention controls the intelligence of far-red light light source to control by LAN WiFi or 2G/3G/4G network The system wiring figure of the very-long-range intelligent controller with multiple output function of system processed.

Fig. 7 is that smart mobile phone of the present invention controls the intelligence of far-red light light source to control by LAN WiFi or 2G/3G/4G network The pictorial diagram of the very-long-range intelligent controller with multiple output function of system processed.

Fig. 8 is that smart mobile phone of the present invention controls the intelligence of far-red light light source to control by LAN WiFi or 2G/3G/4G network 24 far infrared LED circuit design drawings of system processed.

Fig. 9 is that smart mobile phone of the present invention controls the intelligence of far-red light light source to control by LAN WiFi or 2G/3G/4G network 24 far infrared LED circuit layouts of system processed.

Figure 10 is the intelligence that smart mobile phone of the present invention controls far-red light light source by LAN WiFi or 2G/3G/4G network 24 far infrared LED circuit pictorial diagrams of control system.

Figure 11 is the intelligence that smart mobile phone of the present invention controls far-red light light source by LAN WiFi or 2G/3G/4G network The total system schematic diagram of control system.

Figure 12 is the experiment that feux rouges of the present invention induction photoreceptor pWS189 produces c-di-GMP in mammalian cell Result figure.

Figure 13 expresses the experimental result picture of control system building block for proof far-red light gene loop of the present invention.

Figure 14 is the experimental result picture that far-red light gene loop of the present invention expresses the photoreceptor that control system difference is built.

Figure 15 is the experiment for the photoreceptor that far-red light gene loop of the present invention expresses the expression of control system different promoters Result figure.

Figure 16 is the experimental result picture that far-red light gene loop of the present invention expresses the processor that control system difference is built.

Figure 17, Figure 18 express different in the effector that control system difference is built for far-red light gene loop of the present invention The experimental result picture of the different repeat numbers of processor recognition site and recognition site.

Figure 19 is expressed in the effector that control system difference is built for far-red light gene loop of the present invention is containing difference The experimental result picture of insulating signal is added before the processor recognition site of repeat number.

Figure 20 expresses different types of in the effector that control system difference is built for far-red light gene loop of the present invention The experimental result picture of weak promoter.

Figure 21 is that far-red light gene loop of the present invention expresses what control system was expressed in different mammalian cells Experimental result picture.

Figure 22 is that smart mobile phone of the present invention sets different illumination by LAN WiFi or 2G/3G/4G network very-long-range The experimental result picture of time-controllable much different expression quantity of feux rouges gene loop expression control system.

Figure 23 is that smart mobile phone of the present invention sets different illumination by LAN WiFi or 2G/3G/4G network very-long-range The experimental result picture of the different expression quantity of intensity modulation far-red light gene loop expression control system.

Figure 24,25,26 and 27 prove smart mobile phone for the present invention exemplified by expressing SEAP, Luciferase, EGFP, GLP1 All can be expressed by regulating and controlling far-red light gene loop expression control system by LAN WiFi or 2G/3G/4G network very-long-range The experimental result of significant albumen.

Figure 28 and accompanying drawing 29 are that smart mobile phone of the present invention regulates and controls remote by LAN WiFi or 2G/3G/4G network very-long-range Feux rouges gene loop expression control system can express the experimental result picture of all two or more significant albumen simultaneously.

Figure 30 passes through LAN WiFi or 2G/3G/4G network overshoot control far-red light for present invention preparation containing smart mobile phone The experimental result picture of the microcapsules transplantation carrier of gene loop expression control systems engineering (CSE) cell.

Figure 31 regulates and controls far-red light containing smart mobile phone for the present invention by LAN WiFi or 2G/3G/4G network very-long-range Gene loop expresses control systems engineering (CSE) cell experimental result picture by far-red light regulating and expressing in microcapsules.

Figure 32 is prepared for the present invention regulates and controls remote containing smart mobile phone by LAN WiFi or 2G/3G/4G network very-long-range The experimental result picture of the hollow-fibre membrane grafts transplantation carrier of feux rouges gene loop expression control systems engineering (CSE) cell.

Figure 33 regulates and controls far-red light containing smart mobile phone for the present invention by LAN WiFi or 2G/3G/4G network very-long-range Gene loop expresses control systems engineering (CSE) cell experimental result by far-red light regulating and expressing in hollow-fibre membrane grafts Figure.

Figure 34, Figure 35 are that smart mobile phone of the present invention regulates and controls far-red light by LAN WiFi or 2G/3G/4G network very-long-range Gene loop expresses control system in experimental result picture of the Mice Body by the situation of far-red light regulating and expressing.

Figure 36 is that smart mobile phone of the present invention regulates and controls far-red light gene by LAN WiFi or 2G/3G/4G network very-long-range Loop expresses the fasting blood-glucose that control system accuracy controlling insulin expression in a type glycosuria model mouse treats IDDM Value.

Figure 37 is that smart mobile phone of the present invention regulates and controls far-red light gene by LAN WiFi or 2G/3G/4G network very-long-range Loop expresses the sugared tolerance test that control system accuracy controlling insulin expression in a type glycosuria model mouse treats IDDM As a result.

Figure 38 is that smart mobile phone of the present invention regulates and controls far-red light gene by LAN WiFi or 2G/3G/4G network very-long-range Loop expresses the fasting blood-glucose of control system accuracy controlling GLP-1 expression treatment type II diabetes in a type glycosuria model mouse Value.

Figure 39 is that smart mobile phone of the present invention regulates and controls far-red light gene by LAN WiFi or 2G/3G/4G network very-long-range Loop expresses the sugared tolerance test of control system accuracy controlling GLP-1 expression treatment type II diabetes in a type glycosuria model mouse As a result.

Figure 40 is that smart mobile phone of the present invention regulates and controls far-red light gene by LAN WiFi or 2G/3G/4G network very-long-range Loop expresses the insulin resistant of control system accuracy controlling GLP-1 expression treatment type II diabetes in a type glycosuria model mouse Experimental result.

Figure 41 is that smart mobile phone of the present invention regulates and controls far-red light gene by LAN WiFi or 2G/3G/4G network very-long-range Loop expresses control system high blood of pancreas expressed by accuracy controlling GLP-1 expression treatment type II diabetes in a type glycosuria model mouse The amount of sugared element.

Embodiment

With embodiment, the present invention is further elaborated below.These embodiments are only used for illustrating invention, without right The scope of the present invention constitutes any limitation.Implementation process, condition, reagent, experimental method of the present invention etc., are specially referred to except following Content outside, be the universal knowledege and generally acknowledged general knowledge of this area, content is not particularly limited in the present invention.In following examples Reagent, instrument used etc., and unreceipted actual conditions experimental method, according to the bar proposed by routine or goods supplier Part is carried out.

Materials and methods

Smart mobile phone controls the intelligent control system of far-red light light source by LAN WiFi or 2G/3G/4G network very-long-range The making of system

Intelligent controller

Make intelligent controller used and be purchased from smart home operating room, its parameter and function are as follows：

1. supporting mobile phone remote control, WiFi, 2G/3G/4G control models are supported；

2. maximum allowable 10A high currents, support multi-channel control, can independent control, control effect real time propelling movement to hand per road Machine App clients；

3. supporting time switch, scene mode, time switch and a key switch function are realized；

4. support Android, iPhone and flat board；Software support Custom Attributes；

5. with a software support multiple equipment, multiple switch；

6. support data backup and data recovery；Support scanning Quick Response Code to import equipment, can also share with other staff and set It is standby, easy to operate victory.

Multi-output switch power source

Multi-output switch power source used is purchased from cloud particle side's electronics workbench, and its parameter and function are as follows：

1. input voltage:5~23V, highest 23V, it is proposed that used in 20V, input is anti-, and reversed (voltage of input must be than output High more than the 1V of voltage)

2. adjustable output voltage scope 0V~16.5V continuously adjustabes, automatically saves last time setting voltage.

3. peak point current 3A, it is proposed that used in 2A.Precision 1%, minimum display 0.01, unit is A, more than 2A heating compared with Greatly, please voluntarily try every possible means to solve heat dissipation problem.

4. high conversion efficiency, up to 95% (efficiency is relevant with input, output voltage, electric current, pressure difference)

5. load regulation S (I)≤0.8%, voltage regulation factor S (u)≤0.8%

6. size 62mm × 44mm × 18mm

7. weight 45g

Far infrared LED

Used far infrared LED lamp bead is purchased from hundred photoelectricity, and far infrared LED lamp circuit plate is designed by microelectronic engineering teacher Bai Yu is completed, and processing is completed by Shenzhen Jie Duobang Science and Technology Ltd.s.Other materials is common home made materials.

1. input voltage：1.5V~4.8V, highest 6V is used；

2. the different voltage in 6 tunnels can arbitrarily switch；

3. with independent switch, it is provided with the potentiometer (under normal circumstances it is not recommended that using) of integrally-regulated brightness；

4. diode can be with independent disassembling；

Molecular cloning

The structure reagents of all expression plasmids of the present invention, specific constructive system and step are as follows.

All primers for PCR are synthesized by Jin Wei intelligence bio tech ltd.Built in the embodiment of the present invention Expression plasmid all passes through sequencing, and sequencing is completed by Jin Wei intelligence bio tech ltd.Institute in the embodiment of the present invention Phanta Max Super-Fidelity archaeal dna polymerases are purchased from Nanjing Vazyme Biotechnology Co., Ltd..In nucleic acid Enzyme cutting, T4 DNA ligases are purchased from TaKaRa companies.Homologous recombination enzyme is purchased from and the limited public affairs of first biotechnology (Shanghai) share Department.Phanta Max Super-Fidelity archaeal dna polymerases are accompanied with corresponding polymerase buffer and dNTP when buying.Core Corresponding buffer solution is accompanied with when sour restriction endonuclease, T4 DNA ligases, the purchase of homologous recombination enzyme.Yeast extract (Yeast Extract), the blue or green enzyme of tryptone (Trypton), agar powder, ammonia benzyl plain (Amp) is limited purchased from Shanghai life work biotechnology Company.DNA Marker DL5000, DNA Marker DL2000 (precious bioengineering Co., Ltd)；Nucleic acid dye EB (Guangdong Biotech company of National Olympic)；Plasmid is small to take out extracts kit (TIANGEN Biotech (Beijing) Co., Ltd.)；DNA glue reclaims are tried It is century bio tech ltd that agent box, PCR primer purification kit, which are purchased from health,；The absolute ethyl alcohol that is referred in embodiment, Remaining reagent such as NaCl is domestic analysis net product.

(1) PCR (PCR)

Amplification step (bp represents the nucleotides quantity of amplified fragments).

(2) endonuclease endonuclease reaction

(1. x is volume when representing plasmid quality as 1 μ g to the system of pair plasmid vector progress double digestion；N, which is represented, makes system Reach the sterilizing ultra-pure water μ L amounts added required for cumulative volume)

(2. x is volume when representing plasmid quality as 1 μ g to the system of pair PCR primer fragment progress double digestion；N, which is represented, to be made System reaches the sterilizing ultra-pure water μ L amounts added required for cumulative volume)

3. PCR primer fragment after double digestion is connected into the system that the cyclization of the plasmid vector after double digestion fills plasmid：

Note:The mass ratio of PCR primer fragment and carrier double digestion product is substantially 2:1-6:Between 1.

(3) homologous recombination coupled reaction

According to first biotechnology (Shanghai) limited company it is seamless clone (assembling) kit specification：PCR primer 15bp and the 15bp nucleic acid sequence homologous of linearized vector both sides nucleotide sequence, amplification are added during fragment amplification in both sides Obtained PCR primer both sides 15bp nucleotide sequences and linearized vector sequence both sides nucleotide sequences homologous, in homologous recombination enzyme In the presence of, PCR primer fragment is connected circlewise with linearized vector homologous recombination.

Note:N values are depending on the size, concentration of PCR primer fragment.

(4) preparation of competent escherichia coli cell

All solution prepared for competent cell and consumptive material are handled by autoclave sterilization.

Be inverted 1. e.colistraindh5α strain is lined on the flat board without antibiotic, at 37 DEG C culture 12~ 16h；

One single bacterium of picking falls within LBs of the 2mL without any antibiotic and shaken in tube, and 37 DEG C of 180rpm shaken cultivations are stayed overnight；

Culture is enlarged (by 1 2. drawing 1mL bacterium solutions and being transferred in 250mL triangular flask LB culture mediums:100 ratios expand training Support), 180rpm 2~3h of shaking table shaken cultivation to OD600 are between 0.4~0.5 at 37 DEG C；

3. nutrient solution is transferred in centrifuge tube, 10min is placed on ice, then centrifuges 10min (centrifugations in 3000rpm at 4 DEG C Machine needs precooling in advance), careful supernatant discarding；

4. add the 0.1M CaCl of precooling₂ 4mL(0.1M CaCl₂Precooling should be shifted to an earlier date), mixed after abundant precooling with vortex The quick suspension thalline of device, then two pipes are merged 1 and managed by ice bath 10min again；

5. 4 DEG C, 4000rpm centrifugations 6min；

6. abandoning supernatant, the ice-cold 0.1M CaCl of 4mL are added₂With the pre- cold sterilization pure glycerins of 0.1mL, suspend precipitation；

7. above-mentioned suspension is sub-packed in PCR pipe (PCR pipe is preferably placed in precooling on ice in advance), liquid nitrogen with 100 μ L/ pipes In save backup；

(5) connection product is converted into Escherichia coli

1. the competent cell prepared is thawed and (thawed on ice), after the connection product mixing for adding proper volume, ice Bathe 30min.The volume for being usually added into connection product is less than the 1/10 of competent cell volume；

2. heat shock 90s in 42 DEG C of water-baths, then places 5min on ice rapidly；

3. bacterium solution is added in 800 μ L LB fluid nutrient mediums (antibiotic-free), mix after 37 DEG C of shaken cultivation 40- 60min；

4. bacterium solution is gone in 1.5mL centrifuge tube, 4000rpm centrifugation 5min abandon part supernatant, stayed on 100 μ L or so Clearly, then by cell cell suspension is dispelled into；

5. above-mentioned suspension is coated on the LB solid mediums containing Amp, incubated overnight in 37 DEG C of incubators is inverted in；

The glue reclaim of remaining experimental implementation, such as DNA fragmentation, purifying are reclaimed, its step according to DNA glue reclaims kit, The operational manual of PCR primer purification kit (health is century bio tech ltd)；Plasmid extraction step is according to plasmid It is small to take out (TIANGEN Biotech (Beijing) Co., Ltd.) extracts kit specification.

The selection and making of light source

LED (L720-__AU, epitex, Japan) to 720nm wavelength and infrared therapy in the embodiment of the present invention Illustrate gene loop remote control and regulation system of the present invention exemplified by device (CQ-61, Chongqing in China space rocket Electron Technology Co., Ltd) Regulation and control method, but do not limit invention protection domain of the present invention.

Experiment LED used purchases Japanese epitex companies；Infrared therapeutic device is purchased from Chongqing in China space rocket electronics Technology Co., Ltd..Remaining accessory is domestic conventional consumptive material.

The LED of 720nm wavelength, to provide low power far-red light transmitter.4 × 6LED plates, its connected mode is：According to The arrangement of Tissue Culture Plate (24 hole), makes with parallel way to be connected between center of each LED correspondences per hole, each LED. Needed to adjust light application time according to experiment, intensity of illumination is tested.

Infrared therapeutic device, to provide powerful far-red light transmitter.Needed to adjust light application time, illumination according to experiment Intensity is tested.

Cell culture and transfection

With the gene for illustrating far-red light regulation and control transgene expression exemplified by following cell line and PEI transfections in the embodiment of the present invention Working condition of the loop remote control and regulation system in cell and animal body, but invention protection domain of the present invention is not limited.

The centrifuge tube of 10cm Tissue Culture Dish, Tissue Culture Plate (24 hole), 15mL and 50mL for cell culture is purchased From Thermo Fisher Scientific companies of the U.S. (Labserv)；Improved Eagle culture mediums, the tire ox blood used Clearly, penicillin and Streptomycin Solution are purchased from Gibico companies of the U.S.；Transfection PEI used is purchased from Polysciences companies；Carefully Born of the same parents' incubator is purchased from Thermo Fisher Scientific companies of the U.S..Remaining consumptive material is common domestic consumptive material.

Cell culture.Human embryonic kidney cell (HEK-293T, ATCC:CRL-11268), continuous expression RTP1, RTP2, REEP1 and G_αoλφHEK-293 (Hana3A), telomerase immortalized human mesenchymal stem cells (hMSC-TERT, ATCC: CRL-3220), human embryonic kidney epithelial cells (HEK-293A), human cervical carcinoma cell (HeLa, ATCC:CCL-2), mouse neuroma is female Cell (Neuro-2A, ATCC:CCL-131) it is incubated in improved Eagle culture mediums, 10% (v/v) is added in culture medium Hyclone and 1% (v/v) penicillin and Streptomycin Solution.All types of cells are all incubated at 37 DEG C, containing 5% 2 In the incubator for aoxidizing concentration of carbon.

Transfection.All cell line transfections use operating procedure (the Wieland M, Methods 56 (3) of the PEI after optimization: 351).Briefly, it is that 6x10 is planted per hole in cultivating system is the orifice plates of 500 μ L 24⁴Individual cell, after culture 16h, By the DNA of best proportion according to 3:1(PEI:DNA mass ratio) and PEI mixed dissolutions static 6h in 50 μ L culture medium.

The detection of reporter gene SEAP (SEAP)

Homoarginine, magnesium chloride, diethanol amine, HCl for configuring examining report Gene response buffer solution are purchased to life Work bioengineering (Shanghai) limited company；Chromogenic substrate (pNPP:P-Nitrophenylphosphate) purchase is brilliant to Shanghai Pure biochemical technology limited company (Aladdin).

(1) reagent is configured：

2x buffer:

20mM homoarginine is noted：It is to suppress endogenic alkaline phosphatase activities that it, which is acted on,

1mM magnesium chlorides

2% diethanol amine

PH to 9.8 is adjusted with HCl

Substrate solution：

120mM chromogenic substrates (pNPP:p-Nitrophenylphosphate)

·In 2x assay buffer

(2) experimental procedure：

1. cell culture supernatant liquid is drawn, 200uL (notes into centrifuge tube：Typically to exceed 150uL, because follow-up add A volume part can be lost in heat)

2. 65 DEG C of water-bath 30min (notes：Heating mainly allows endogenic alkaline phosphatase to inactivate, and SEAP high temperature resistants, It will not inactivate at this temperature).

3. drawing 80uL (experimental situation voluntarily dilutes) to 96 orifice plates, 2x preheated in advance is rapidly joined The μ l of the buffer 100 and μ l of substrate solution 20.

4. ELIASA 405nm is surveyed 10 times, per minor tick 1min (experimental situation can separately set condition).

(3) calculating of enzyme activity

The enzyme activity of alkaline phosphatase is defined:37 DEG C, during pH 9.8, with substrate 4-NPP in 1min (PNPP-Na₂) alkaline phosphatase for generating 1mol/L p-nitrophenols is reacted, it is defined as 1 unit of activity (1U).P-nitrophenyl Phenol has glassy yellow in itself, in wavelength 405nm, the corresponding different light absorption value of p-nitrophenol of various concentrations.Computational methods are： It is enzyme activity, unit U/L that different time points, which survey OD values and make slope of a curve * 256.8, during sample and substrate reactions.

The detection of reporter gene luciferase (Luciferase)

The dual luciferase reporter gene detection kit used is purchased from biotool companies of the U.S..Concrete operation step is detailed See product dual luciferase reporter gene detection kit (Cat:B17001,Lot71005,Bitool,Houston,TX,USA) Specification.

The preparation method of sodium alginate-PLL- sodium alginate micro gel capsules

Prepare the sodium alginate used in microcapsules, microcapsules granulation instrument and be purchased from Buchi companies of Switzerland, poly-D-lysine purchase From Sigma Co., USA, trisodium citrate (Na₃- Cirate), sodium chloride, calcium chloride, MOPS (Morpholinopropanesulfonic acid) is purchased from Shanghai Sheng Gong biotechnologies Co., Ltd.Used in experiment Preparation of reagents and operating procedure refer to microcapsules granulation instrument (Inotech Encapsulator Research Unit IE- 50R) operation manual.

Major parameter in preparation process is：The jet diameters of assembling are 200 μm, the frequency of ultrasonic vibration cutout is 1300Hz, the scattered electrostatic used by 1100V, injection speed be 20mL/min, the microcapsules per second formed be 500, it is every Microcapsule size is 300-350 μm；The cell number of each encapsulation is 200-250.

The preparation method of hollow-fibre membrane grafts

Experiment hollow-fibre membrane grafts used (Implant Membrane) purchase U.S. Spectrum Laboratories companies.The method of the preparation of hollow-fibre membrane grafts is referred toImplant Membrane are produced Product specification.

The detection method of insulin (Insulin)

Experiment detection kit for insulin (Mouse Insulin ELISA kit) used is public purchased from Sweden Mercodia Department, specific assay method refers to product description.

The detection method of hyperglycemic factor (GLP-1)

Experiment hyperglycemic factor detection kit used (Millipore Corporation, Billerica, MA01821USA, Cat.no.EGLP-35K, Lot.no.2639195) it is purchased from Millipore companies of the U.S., specific assay method Refer to product description.

Embodiment 1, smart mobile phone controls the intelligence of far-red light light source by LAN WiFi or 2G/3G/4G network very-long-range The making of energy control system

The intelligent controller made in the present invention with smart home operating room and its APP, 6 multiple-channel outputs that match Exemplified by Switching Power Supply and 24 far infrared LEDs are as far-red light light source, illustrate that smart mobile phone passes through LAN WiFi or 2G/ The preparation method of the intelligence control system of 3G/4G networks very-long-range control far-red light light source, but present invention invention protection is not limited Scope.

The first step, the realization of APP clients.Experiment material and side (are specifically shown in by the intelligent controller manufacturer bought Method) provide the APP matched, it is specifically set and its application method refers to producer's operation instructions.APP schematic diagrames are referred to Figure of description 3.

Second step, the realization of remote intelligent controller.(concrete function and parameter refer to reality to intelligent controller in the present invention Proved recipe method and material) smart home operating room is purchased from, by it smart mobile phone can be used to utilize LAN WiFi or 2G/3G/ 4G Internet resources realize that very-long-range controls far-red light light source.Intelligent controller schematic diagram and pictorial diagram refer to Figure of description 4.

3rd step, multi-output switch power source is realized.Multi-output switch power source (concrete function and parameter in the present invention Refer to experimental method and material) cloud particle side is purchased from, respectively by No. 6 independent switch plant-grid connection intelligence of cloud particle side's electronics workbench Controller, passes through cut-offfing come the setting to the different brightness of same far-red light light source, so as to realize for mobile phone A PP 6 road power supplys of control The design of multi-output switch power source.Multi-output switch power source schematic diagram and pictorial diagram refer to Figure of description 5.

4th step, the realization of the very-long-range intelligent controller with multiple output function.In the present invention, with multiple-channel output The preparation method of the very-long-range intelligent controller of function is：By the output port of 6 independent Switching Power Supplies respectively with intelligent control The wherein 6 road input ports of device processed are connected, then by the 6 corresponding road output ports of intelligent controller with far infrared LED's Input port is connected, and is used as far infrared LED powered by direct current port.Wherein the power supply of whole system is fitted by AC/DC power supplys Orchestration is completed, and parameter is 3A fuse in the negative lead series connection of AC/DC power supply adaptors, and protection multichannel is defeated Go out the very-long-range intelligent controller of the function normal work in the range of safe current, system wiring figure refers to bright book accompanying drawing 6.Work as intelligence When installing the APP software supporting with intelligent controller on energy mobile phone, smart mobile phone and intelligent controller configuration access local jointly , can be by operating the control of smart mobile phone APP softwares to switch the switch electricity of 6 tunnel independences when netting WiFi or 2G/3G/4G Internet resources The opening in source and closing, and the time of the opening and closing of the Switching Power Supply of 6 tunnel independences can be set, so that very-long-range is controlled The different working condition of far-red light light source, such as different intensities of illumination, different light application times etc..With multiple output function The pictorial diagram of very-long-range intelligent controller refers to accompanying drawing 7.

5th step, the realization of far infrared LED.By 24 far infrared LED respectively and resistant series, afterwards again and alliance Electricity.6 road feeder ears of the PCB design of the far-red light light source, wherein 6 road feeder ears are often located to add voltage clamping Diode, is effectively protected to LED circuit, and adds a master control self-lock switch in whole power supply, so as to realize manually Control cut-offfing for lamp plate circuit.24 far infrared LED circuit design drawings refer to accompanying drawing 8.In PCB circuit layouts, to ensure light According to distributing homogeneity, 24 LEDs are equidistantly uniformly placed, according to the Column Layout of 4 row 6, line space column pitch is 2cm. 24 far infrared LED circuit layouts refer to accompanying drawing 9；24 far infrared LED circuit pictorial diagrams refer to accompanying drawing 10.

In summary, APP clients, remote intelligent controller, multi-output switch power source, far infrared LED are integrated After connection, the intelligence that above-mentioned smart mobile phone controls far-red light light source by LAN WiFi or 2G/3G/4G network very-long-range is formed The making of control system.Total system schematic diagram refers to accompanying drawing 11.

Embodiment 2, the structure of the gene loop remote control and regulation system element of far-red light regulation and control transgene expression

Have in the gene loop remote control and regulation system that far-red light regulation and control transgene expression is contained in the present embodiment and represent The construction method of property element, but it is not restricted to the scope of the present invention.Detailed design plan and step are shown in Table 1.

Embodiment 3, checking far-red light induction photoreceptor pWS189 produces c-di-GMP in mammalian cell

The first step, plasmid construction.Plasmid construction in this example refers to table 1.

Second step, inoculating cell.By the good HEK-293T cells of growth conditions kind after 0.25% pancreatin digestion In 7 piece of 24 orifice plate, per hole kind 6 × 10⁴Individual cell, and add DMEM culture mediums of the 500 μ L containing 10%FBS.

3rd step, transfection.In inoculating cell 16 to 24h, by 0.1 μ g photoreceptor pWS189, PEI transfection reagents and nothing The DMEM of serum is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 well culture plates.Preparation cumulative volume wherein per hole is 50 μ L, plasmid is 1 with PEI mass ratioes:3.DMEM culture mediums of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination.After changing liquid 14-18 hours, 7 groups (numbering 1,2,3,4,5,6,7 respectively) are divided into, except No. 1 Outer whole is placed in wavelength for 720nm, and intensity of illumination is 1mW/cm²LED under (specific connected mode is with reference to experiment material and side Method) irradiation 1h.

5th step, collects cell.After illumination one hour respectively 0,5,10,15,30,60min (respectively with numbering 2,3,4, 5th, 6,7 correspondence；24 orifice plates of numbering 1 are under dark condition all the time) harvesting.Culture medium in 24 orifice plates is removed, plus Enter 200 μ L PBS, hole-specifically piping and druming makes cell detachment repeatedly successively with pipettor.It is transferred into a new 2mL centrifuge tube In, 1000 × g centrifugations 5min collects cell.

6th step, cell lysis.200 μ L lysis buffer is added into the cell of above-mentioned collection, in -80 DEG C of placements 15min, rapid to take out 37 DEG C of placement 5min, 3 times repeatedly, middle concussion is mixed.5000 × g centrifuges 5min, takes supernatant Detection.

7th step, the measure of c-di-GMP amounts.With c-di-GMPELISA kit measurement each groups c-di-GMP expression Amount.Experimental data refers to Figure of description 12.

Embodiment 4, the composition of the gene loop remote control and regulation system of far-red light regulation and control transgene expression

This example is using SEAP as reporter gene, and citing proves that far-red light regulates and controls the gene loop remote control and regulation of transgene expression The building block of system, but protection scope of the present invention has not been limited.Comprise the following steps that：

Second step, inoculating cell.By the good HEK-293T cells of growth conditions kind after 0.25% pancreatin digestion In 2 piece of 24 orifice plate, per hole kind 6 × 10⁴Individual cell, and add DMEM culture mediums of the 500 μ L containing 10%FBS.

3rd step, transfection.2 piece of 24 orifice plate is divided into dark group and light group, every group is divided into 1-8 group.In inoculation Cell 16 is in 24h, wherein by 0.3 μ g pcDNA3.1 (+) in the 1st group of dark group and light group, by 0.1 in the 2nd group It is in μ g pWS189 and 0.2 μ g pcDNA3.1 (+), the 3rd group that 0.1 μ g pGY32 and 0.2 μ g pcDNA3.1 (+), the 4th is small By 0.1 μ g pXY34 and 0.2 μ g pcDNA3.1 (+) in group, by 0.1 μ g pWS189,0.1 μ g pGY32 and 0.1 in the 5th group By 0.1 μ g pWS189,0.1 μ g pXY34 and 0.1 μ g pcDNA3.1 (+), the 7th group in μ g pcDNA3.1 (+), the 6th group It is middle by 0.1 μ g pGY32,0.1 μ g pXY34 and 0.1 μ g pcDNA3.1 (+) and the 8th group by 0.1 μ g pWS189,0.1 μ g pGY32 and 0.1 μ g pXY34 are mixed with the DMEM of PEI transfection reagents and serum-free respectively, are stored at room temperature after 15min uniform It is added drop-wise in 24 well culture plates.Preparation cumulative volume wherein per hole is 50 μ L, and plasmid is 1 with PEI mass ratioes:3.Changed after transfection 6h Enter DMEM culture mediums of the 500 μ L containing 10%FBS to be cultivated.

4th step, illumination.Change after liquid 14-18h, light group is placed in wavelength for 720nm, intensity of illumination is 1mW/cm²'s (specific connected mode reference material and method) illumination 4h under LED, and dark group is then placed at dark and cultivated always.

5th step, examining report gene.The cell culture supernatant of dark group and light group is taken to survey after culture 48h respectively Determine SEAP expression quantity (specific method reference material and method).

As a result show, only when far-red light gene loop expresses the photoreceptor of control system, processor and effector Exist simultaneously in host cell and when far-red light is induced, the system could normal work.If lacking wherein any one The individual part or system can not work under dark condition.Experimental data refers to Figure of description 13.

Embodiment 5, the photoreceptor of the gene loop control system difference structure of far-red light regulation and control transgene expression

In the present embodiment, the far-red light gene loop for proving far-red light regulation and control transgene expression for citing expresses control system Difference build photoreceptors in mammalian cell by far-red light regulating and expressing situation, but not to the present invention protection model It is with and is limited.Comprise the following steps that：

Second step, inoculating cell (specific steps be the same as Example 3).

3rd step, transfection.In inoculating cell 16 to 24h, 2 piece of 24 orifice plate is divided into dark group and light group, every group equal It is divided into 1-4 group.In inoculating cell 16 to 24h, wherein by 0.1 μ g pWS46 in the 1st group of dark group and light group, By 0.1 μ g pWS48 in 2nd group, by 0.1 μ g pWS1 in the 3rd group, in the 4th group by 0.1 μ g pWS50 respectively with 0.1 μ G pGY32,0.1 μ g pXY34, PEI transfection reagents and serum-free DMEM are mixed, and are stored at room temperature after 15min and are uniformly added drop-wise to 24 In well culture plate.Preparation cumulative volume wherein per hole is 50 μ L, and plasmid is 1 with PEI mass ratioes:3.500 μ L are changed to after transfection 6h DMEM culture mediums containing 10%FBS are cultivated.

4th step, illumination (specific steps be the same as Example 3).

5th step, examining report gene (specific steps be the same as Example 3).

As a result show, the light of the far-red light gene loop expression control system difference structure of far-red light regulation and control transgene expression Receptor, far-red light induction under can in mammalian cell normal work.But under the induction of identical far-red light The photoreceptor of different structures makes the response intensity of effector different.Experimental data refers to Figure of description 14.

Embodiment 6, the far-red light gene loop expression control system different promoters table of far-red light regulation and control transgene expression The photoreceptor reached

In the present embodiment, the far-red light gene loop for proving far-red light regulation and control transgene expression for citing expresses control system Photoreceptor expressed with different promoter, its in mammalian cell by far-red light regulating and expressing situation, but not The scope of the present invention has been limited.Comprise the following steps that：

Second step, inoculating cell (specific steps be the same as Example 3).

3rd step, transfection.In inoculating cell 16 to 24h, 2 piece of 24 orifice plate is divided into dark group and light group, every group equal It is divided into 1-5 group.In inoculating cell 16 to 24h, wherein by 0.1 μ g in the 1st group of dark group and light group By 0.1 μ g pWS50 in pWS189, the 2nd group, by 0.1 μ g pWS51 in the 3rd group, in the 4th group by 0.1 μ g pWS55, In 5th group by 0.1 μ g pWS59 processor pGY32 respectively with 0.1 μ g, 0.1 μ g effector pXY34, PEI transfection reagents with The DMEM of serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 well culture plates.Wherein often the preparation cumulative volume in hole is 50 μ L, plasmid is 1 with PEI mass ratioes:3.DMEM culture mediums of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination (specific steps be the same as Example 3).

5th step, examining report gene (specific steps be the same as Example 3).

As a result show, in the gene loop control system of far-red light regulation and control transgene expression, expressed with different promoters Photoreceptor, far-red light induction under can in mammalian cell normal work.But in the induction of identical far-red light The photoreceptor of lower different promoters expression makes the response intensity of effector different.Experimental data refers to Figure of description 15。

Embodiment 7, the processor of the gene loop control system difference structure of far-red light regulation and control transgene expression

In the present embodiment, the different of gene loop control system for proving far-red light regulation and control transgene expression for citing build Processor in mammalian cell by far-red light regulating and expressing situation, but do not limit the scope of the present invention.Specifically Step is as follows：

Second step, inoculating cell (specific steps be the same as Example 3).

3rd step, transfection.In inoculating cell 16 to 24h, 2 piece of 24 orifice plate is divided into dark group and light group, every group equal It is divided into 1-8 group.In inoculating cell 16 to 24h, wherein by 0.1 μ g in the 1st group of dark group and light group By 0.1 μ g pXY34 in pWS200, the 2nd group, by 0.1 μ g pXY35 in the 3rd group, in the 4th group by 0.1 μ g pXY36, In 5th group by 0.1 μ g pGY28, the 6th group by 0.1 μ g pGY32, the 7th group by 0.1 μ g pGY33, the 8th group Middle far-red light receptor pWS189,0.1 μ g effector pXY24, PEI transfection reagents by 0.1 μ g pGY34 respectively with 0.01 μ g Mixed with the DMEM of serum-free, be stored at room temperature after 15min and be uniformly added drop-wise in 24 well culture plates.Preparation cumulative volume wherein per hole For 50 μ L, plasmid is 1 with PEI mass ratioes:3.DMEM culture mediums of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination (specific steps be the same as Example 3).

5th step, examining report gene (specific steps be the same as Example 3).

As a result show, in the gene loop control system of far-red light regulation and control transgene expression, different processors are remote red Under photoinduction can in mammalian cell normal work.But the place of structure different under the induction of identical far-red light Reason device makes the response intensity of effector different, can be needed to select different processors according to experiment.Experimental data is referred to Bright book accompanying drawing 16.

Embodiment 8, the effector of the gene loop control system difference structure of far-red light regulation and control transgene expression

In the present embodiment, for the different effects built for the gene loop control system for proving far-red light regulation and control transgene expression Answer situation of the device by far-red light regulating and expressing in mammalian cell.With different processor recognition sites, recognition site Different repeat numbers, whether containing exemplified by insulating signal and different types of weak promoter, illustrate different effect device in lactation Situation about being regulated and controled in class zooblast by far-red light, but the scope of the present invention has not been limited.

The different repeat numbers of different processor recognition sites and recognition site.Specifically comprise the following steps that：

Second step, inoculating cell (specific steps be the same as Example 3).

3rd step, transfection.In inoculating cell 16 to 24h, 2 piece of 24 orifice plate is divided into dark group and light group, every group equal It is divided into 1-10 group.In inoculating cell 16 to 24h, wherein by 0.1 μ g in the 1st group of dark group and light group By 0.1 μ g pXY20 in pXY19, the 2nd group, by 0.1 μ g pXY21 in the 3rd group, by 0.1 μ g pXY22, in the 4th group In 5 groups by 0.1 μ g pXY23, the 6th group by 0.1 μ g pXY16, the 7th group by 0.1 μ g pXY17, the 8th group By will be remote red with 0.1 μ g respectively by 0.1 μ g pXY32 in 0.1 μ g pXY31, the 10th group in 0.1 μ g pXY18, the 9th group Photoreceptor pWS189,0.1 μ g processor pGY32, PEI transfection reagents and serum-free DMEM are mixed, and are stored at room temperature after 15min Uniformly it is added drop-wise in 24 well culture plates.Preparation cumulative volume wherein per hole is 50 μ L, and plasmid is 1 with PEI mass ratioes:3.Transfect 6h DMEM culture mediums of the 500 μ L containing 10%FBS is changed to afterwards to be cultivated.

4th step, illumination (specific steps be the same as Example 3).

5th step, examining report gene (specific steps be the same as Example 3).

As a result show, in the gene loop control system of far-red light regulation and control transgene expression, different processor identification positions The effector of point and different repeat number recognition site under far-red light induction can in mammalian cell normal work Make.But the effector of different processor recognition site and different repeat number recognition sites is anti-under the induction of identical far-red light Answer intensity different, can be needed to select different processors according to experiment.Experimental data refers to Figure of description 17,18.

Insulating signal is added before the processor recognition site containing different repeat numbers.Comprise the following steps that：

Second step, inoculating cell (specific steps be the same as Example 3).

3rd step, transfection.In inoculating cell 16 to 24h, 2 piece of 24 orifice plate is divided into dark group and light group, every group equal It is divided into 1-5 group.In inoculating cell 16 to 24h, wherein by 0.1 μ g pXY33 in the 1st group of dark group and light group, By 0.1 μ g pXY28 in 2nd group, by 0.1 μ g pXY34 in the 3rd group, by 0.1 μ g pXY39, the 5th group in the 4th group It is middle by 0.1 μ g pXY40 far-red light receptor pWS189 respectively with 0.1 μ g, 0.1 μ g processor pGY32, PEI transfection reagents with The DMEM of serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 well culture plates.Wherein often the preparation cumulative volume in hole is 50 μ L, plasmid is 1 with PEI mass ratioes:3.DMEM culture mediums of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination (specific steps be the same as Example 3).

5th step, examining report gene (specific steps be the same as Example 3).

As a result show, in the gene loop control system of far-red light regulation and control transgene expression, containing different repeat numbers The effector that insulating signal is added before processor recognition site can be in mammalian cell just under far-red light induction Often work.But the effect of insulating signal is added before the processor recognition site containing different repeat numbers under the induction of identical far-red light Answer the response intensity of device different, can be needed to select different processors according to experiment.Experimental data refers to Figure of description 19。

Different types of weak promoter.With promoter h_CMV_min3GExemplified by explanation can use different weak promoters.Tool Body step is as follows：

Second step, inoculating cell (specific steps be the same as Example 3).

3rd step, transfection.In inoculating cell 16 to 24h, 2 piece of 24 orifice plate is divided into dark group and light group, every group equal It is divided into 1-5 group.In inoculating cell 16 to 24h, wherein by 0.1 μ g pGY36 in the 1st group of dark group and light group, By 0.1g pGY37 in 2nd group, by 0.1 μ g pGY38 in the 3rd group, by 0.1 μ g pGY39, the 5th group in the 4th group By 0.1 μ g pGY40 far-red light receptor pWS189 respectively with 0.1 μ g, 0.1 μ g processor pGY32, PEI transfection reagents with The DMEM of serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 well culture plates.Wherein often the preparation cumulative volume in hole is 50 μ L, plasmid is 1 with PEI mass ratioes:3.DMEM culture mediums of the 500 μ L containing 10%FBS is changed to after transfection 6h to be cultivated.

4th step, illumination (specific steps be the same as Example 3).

5th step, examining report gene (specific steps be the same as Example 3).

As a result show, in the gene loop control system of far-red light regulation and control transgene expression, contain not in effector With weak promoter far-red light induction under can in mammalian cell normal work.But the weak promoter and other The intensity of weak promoter response is different.Experimental data refers to Figure of description 17,20.

Embodiment 9, far-red light regulation and control transgenosis gene loop control system in different mammalian cells table Reach

In the present embodiment, prove the gene loop control system of far-red light regulation and control transgene expression in the different food in one's mouths for citing By the situation of far-red light regulating and expressing in newborn class cell, but the scope of the present invention is not limited.Comprise the following steps that：

Second step, inoculating cell.By the good Neuro-2A cells of growth conditions, HeLa cells, hMSC-TERT cells, Hana3A cells, HEK-293A cells and HEK-293T cells are planted (every kind of thin in 2 pieces respectively after being digested with 0.25% pancreatin Born of the same parents plant 4 holes on every block of plate) in different 24 orifice plates, per hole kind 6 × 10⁴Individual cell, and 500 μ L are added containing 10%FBS's DMEM culture mediums.

3rd step, transfection.In inoculating cell 16 to 24h, by 0.1 μ g pWS189,0.1 μ g pGY32,0.1 μ g The DMEM of pXY34, PEI transfection reagent and serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 well culture plates.Its In preparation cumulative volume per hole be 50 μ L, plasmid and PEI mass ratioes are 1:3.500 μ L are changed to containing 10%FBS's after transfection 6h DMEM culture mediums are cultivated.

4th step, illumination (specific steps be the same as Example 3).

5th step, examining report gene (specific steps be the same as Example 3).

As a result show, the gene loop control system of the far-red light regulation and control transgene expression in the present invention can be different By far-red light induced expression in mammalian cell.Therefore the gene loop control of the far-red light regulation and control transgenosis in the present invention System is applied to a variety of mammalian cells.Experimental data refers to Figure of description 21.Embodiment 10, smart mobile phone passes through office Domain net WiFi or 2G/3G/4G networks very-long-range controls the gene of different light application time regulation and control far-red lights regulation and control transgene expressions The different expression quantity of loop control system

In the present embodiment, when smart mobile phone controls different illumination by LAN WiFi or 2G/3G/4G network very-long-range Between can be that pulsed illumination can also be continuous illumination, discontinuous light, light application time is changeable.This example proves for citing Smart mobile phone controls different light application times (continuous) to be adjusted with effector by LAN WiFi or 2G/3G/4G network very-long-range The relation of expression quantity is controlled, but protection scope of the present invention has not been defined.Comprise the following steps that：

Second step, inoculating cell.By the good HEK-293T cells of growth conditions kind after 0.25% pancreatin digestion In 12 piece of 24 orifice plate, per hole kind 6 × 10⁴Individual cell, and add DMEM culture mediums of the 500 μ L containing 10%FBS.

3rd step, transfection.In inoculating cell 16 to 24h, by 0.1 μ g photoreceptors pWS189,0.1 μ g pGY32,0.1 The DMEM of μ g pXY34, PEI transfection reagents and serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 well culture plates. Preparation cumulative volume wherein per hole is 50 μ L, and plasmid is 1 with PEI mass ratioes:3.500 μ L are changed to containing 10%FBS's after transfection 6h DMEM culture mediums are cultivated.

4th step, smart mobile phone controls different light application times to enter by LAN WiFi or 2G/3G/4G network very-long-range Row illumination.Change after liquid 14-18h, be divided into 12 groups, be placed in wavelength for 720nm, intensity of illumination is 1mW/cm²LED under (tool Body connected mode reference material and method).It is respectively 0 that its smart mobile phone, which controls different light application times, 0.1,0.25,0.5,1, 2nd, 4,6,12,24,48,72h (being placed in always at dark for wherein light group 0h is cultivated).

5th step, examining report gene.The cell culture supernatant of each group is taken to determine SEAP table after culture 72h respectively Up to amount.

Test result indicates that, the different illumination that smart mobile phone is controlled by LAN WiFi or 2G/3G/4G network very-long-range The gene loop control system that time can induce far-red light to regulate and control transgenosis regulates and controls the variable expression of target gene, and illumination Induction time is longer, and its expression quantity is higher, and light application time dependent expression is presented.Experimental data refers to Figure of description 22.

Embodiment 11, smart mobile phone controls different intensities of illumination by LAN WiFi or 2G/3G/4G network very-long-range Regulate and control the different expression quantity of the gene loop control system of far-red light regulation and control transgene expression

In the present embodiment, prove that smart mobile phone passes through LAN WiFi or 2G/3G/4G network very-long-range for citing The relation of the different intensity of illumination of control and effector regulating and expressing amount, but protection scope of the present invention has not been defined.Tool Body step is as follows：

3rd step, is transfected (specific steps be the same as Example 8).

4th step, smart mobile phone controls different intensities of illumination to enter by LAN WiFi or 2G/3G/4G network very-long-range Row illumination.Change after liquid 14-18h, be divided into 7 groups, be respectively placed in wavelength for 720nm, intensity of illumination is respectively 0,50,75, 100、250、500、750、1000、1500、2000μW/cm²LED under (specific connected mode reference material and method).Its light It is 4h according to the time (wherein intensity of illumination is placed at dark culture always for 0).

Test result indicates that, the different illumination that smart mobile phone is controlled by LAN WiFi or 2G/3G/4G network very-long-range The gene loop control system that intensity can induce far-red light to regulate and control transgene expression regulates and controls the variable expression of target gene, and Intensity of illumination is stronger, and its expression quantity is higher, and intensity of illumination dependent expression is presented.Experimental data refers to Figure of description 23.

Embodiment 12, smart mobile phone regulates and controls the gene ring of transgenosis by LAN WiFi or 2G/3G/4G network very-long-range Path control system can express all significant albumen

Smart mobile phone regulates and controls the gene loop control of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range System processed can be expressed in all significant albumen, the present embodiment, to express reporter gene SEAP (SEAP), illustrate that far-red light regulation and control turn exemplified by enhanced green fluorescence protein (EGFP) and glucagon-like peptide (GLP-1) The gene loop control system of gene expression can with regulating and expressing all significant albumen.But not to protection scope of the present invention Define.Comprise the following steps that：

Second step, inoculating cell.By the good HEK-293T cells of growth conditions with after 0.25% pancreatin digestion points Other kind is in 24 different orifice plates, per hole kind 6 × 10⁴Individual cell, and add DMEM culture mediums of the 500 μ L containing 10%FBS.

3rd step, transfection.In inoculating cell 16 to 24h, by 0.1 μ g pWS189,0.1 μ g pGY32,0.1 μ g pXY34；0.1μg pWS189、0.1μg pGY32、0.1μg pGY44；0.1μg pWS189、0.1μg pGY32、0.1μg PWS212 is mixed with the DMEM of PEI transfection reagents and serum-free respectively, is stored at room temperature after 15min and is uniformly added drop-wise to 24 well culture plates In.Preparation cumulative volume wherein per hole is 50 μ L, and plasmid is 1 with PEI mass ratioes:3.500 μ L are changed to after transfection 6h containing 10%FBS DMEM culture mediums cultivated.

4th step, smart mobile phone sets intensity of illumination by LAN WiFi or 2G/3G/4G network very-long-range and carries out light According to.Change after liquid 14-18h, light group is placed in wavelength for 720nm, the intensity of illumination set is 1mW/cm²LED under (specifically connect Connect mode reference material and method) illumination 4h, and dark group is then placed at dark and cultivated always.

5th step, examining report gene.Respectively in 24h, 48h, 72h and measure SEAP amount；Respectively 0h, 6h, 12h, 24h, 48h shoot fluorescence photo；In 24,48,72h luciferase is detected with dual luciferase reporter gene detection kit Amount；48h with ELISA kit determine GLP-1 under different light application times expression quantity (specific method with reference to experiment material with Method).

As a result show, the smart mobile phone in the present invention turns base by the regulation and control of LAN WiFi or 2G/3G/4G network very-long-range Because the gene loop control system of expression can be very good the different albumen of induced expression.Therefore the smart mobile phone in the present invention leads to The gene loop control system for crossing LAN WiFi or 2G/3G/4G network very-long-range regulation and control transgenosis is cut with suitable for expression one The albumen of meaning.Experimental data refers to Figure of description 24,25,26,27.

Embodiment 13, smart mobile phone regulates and controls the gene ring of transgenosis by LAN WiFi or 2G/3G/4G network very-long-range Path control system can express all two or more significant albumen simultaneously

Smart mobile phone regulates and controls the gene loop control of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range System processed can be expressed in two different albumen (being connected with 2A), the present embodiment simultaneously, to express enhanced green fluorescence egg Illustrate that smart mobile phone is super remote by LAN WiFi or 2G/3G/4G network exemplified by -2A- insulin (EGFP-2A-Insulin) in vain The gene loop control system of journey regulation and control transgene expression can express two different albumen (being connected with 2A) simultaneously.But it is right Protection scope of the present invention has been defined.Comprise the following steps that：

3rd step, transfection.In inoculating cell 16 to 24h, by 0.1 μ g pWS189,0.1 μ g pGY32,0.1 μ g The DMEM of pWS213, PEI transfection reagent and serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 well culture plates.Its In preparation cumulative volume per hole be 50 μ L, plasmid and PEI mass ratioes are 1:3.500 μ L are changed to containing 10%FBS's after transfection 6h DMEM culture mediums are cultivated.

4th step, illumination (specific steps be the same as Example 11).

5th step, examining report gene.The fluorescent protein map of different light application times is shot in 48h；Use ELISA kit Determine expression quantity of the Insulin under different light application times (specific method is with reference to experiment material and method).

As a result show, the smart mobile phone in the present invention turns base by the regulation and control of LAN WiFi or 2G/3G/4G network very-long-range Because the gene loop control system of expression can be very good while expressing two different albumen (being connected with 2A).Therefore it is of the invention In smart mobile phone by LAN WiFi or 2G/3G/4G network very-long-range regulate and control transgenosis gene loop control system can For expressing multiple albumen simultaneously.Experimental data refers to Figure of description 28,29.

Embodiment 14：Prepare and pass through LAN WiFi or 2G/3G/4G network very-long-range regulation and control transgenosis containing smart mobile phone The gene loop control system of expression is engineered the microcapsules transplantation carrier of cell

In the present embodiment, turned with preparing containing smart mobile phone by the regulation and control of LAN WiFi or 2G/3G/4G network very-long-range Illustrate the preparation side of transplantation carrier exemplified by the microcapsules transplantation carrier of the gene loop control system engineering cell of gene expression Method, but protection scope of the present invention has not been defined.Comprise the following steps that：

Second step, inoculating cell.By the good HEK-293T cells of growth conditions kind after 0.25% pancreatin digestion In 10 cm cell culture dishes, per ware kind 4 × 10⁶Individual cell, and add DMEM culture mediums of the 10mL containing 10%FBS.

3rd step, in inoculating cell 16 to 24h, 4 μ g pWS189,4 μ g pGY32,4 μ g pXY34, PEI transfections are tried The DMEM of agent and serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 10 cm cell culture dishes.Wherein matching somebody with somebody per ware Cumulative volume processed is 2mL, and plasmid is 1 with PEI mass ratioes:3.DMEM culture mediums of the change 10mL containing 10%FBS after 6h is transfected to carry out Culture.

It is prepared by the 4th step, microcapsules.Change after liquid 14-18h, cell is collected by centrifugation in pancreatin digestion.Prepared using microcapsules Instrument prepares microcapsules (specific equipment and parameter refer to experimental method and material).

The microcapsules prepared have APA

(Alginate-Polylysine-Alginate-membrane) nutrition needed for trilamellar membrane system, cell growth Small molecule destination protein secreted by material and the cell of engineering can pass freely through the membranous system.But cell and other High molecular weight protein can not then pass through the membranous system.Therefore it can be transplanted in Mice Body and normally given birth to by the cell of encapsulation It is long.Experimental data refers to Figure of description 30.

Embodiment 15, transgene expression is regulated and controled containing smart mobile phone by LAN WiFi or 2G/3G/4G network very-long-range Gene loop control system be engineered cell in microcapsules by far-red light regulating and expressing

The present embodiment is cultivated according to the operating method of embodiment 14, obtained microcapsules in 6 orifice plates, while with infrared Line therapeutic equipment, intensity of illumination is 5mW/cm², light application time be 2h induced expressions contain smart mobile phone by LAN WiFi or 2G/3G/4G networks very-long-range regulation and control transgene expression gene loop control system engineering cell in microcapsules, exist respectively 24h, 48h, 72h examining report gene.Turned containing smart mobile phone by the regulation and control of LAN WiFi or 2G/3G/4G network very-long-range The gene loop control system engineering cell of gene expression is can be very good in microcapsules by far-red light regulating and expressing.Experiment Data refer to Figure of description 31.

Embodiment 16：Prepare and pass through LAN WiFi or 2G/3G/4G network very-long-range regulation and control transgenosis containing smart mobile phone The gene loop control system of expression is engineered the hollow-fibre membrane grafts transplantation carrier of cell

In the present embodiment, turned with preparing containing smart mobile phone by the regulation and control of LAN WiFi or 2G/3G/4G network very-long-range Explanation transplanting is carried exemplified by the hollow-fibre membrane grafts transplantation carrier of the gene loop control system engineering cell of gene expression The preparation method of body, but protection scope of the present invention has not been defined.Comprise the following steps that：

Second step, inoculating cell.By the good HEK-293T cells of growth conditions kind after 0.25% pancreatin digestion In 24 porocyte culture plates, per hole kind 6 × 10⁴Individual cell, and add DMEM culture mediums of the 500 μ L containing 10%FBS.

3rd step, in inoculating cell 16 to 24h, by 0.1 μ g pWS189,0.1 μ g pGY32,0.1 μ g pXY34, PEI The DMEM of transfection reagent and serum-free is mixed, and is stored at room temperature after 15min and is uniformly added drop-wise in 24 porocyte culture plates.Wherein per hole Preparation cumulative volume be 50 μ L, plasmid and PEI mass ratioes are 1:3.Transfect and DMEM culture mediums of the 10mL containing 10%FBS is changed to after 6h Cultivated.

It is prepared by the 4th step, hollow-fibre membrane grafts.Change after liquid 14-18h, cell is collected by centrifugation in pancreatin digestion.According to system Make method and make hollow-fibre membrane grafts (specific operation process refers to experimental method and material).

The hollow-fibre membrane grafts prepared, nutriment needed for cell growth and secreted by the cell of engineering Small molecule destination protein can pass freely through the membranous system.But cell and other high molecular weight proteins can not then pass through the membrane system System.Therefore the cell wrapped up by hollow-fibre membrane grafts can be transplanted to normal growth in Mice Body.Experimental data is referred to Bright book accompanying drawing 32.

Embodiment 17, transgene expression is regulated and controled containing smart mobile phone by LAN WiFi or 2G/3G/4G network very-long-range Gene loop control system be engineered cell in hollow-fibre membrane grafts by far-red light regulating and expressing

The present embodiment is cultivated according to the operating method of embodiment 16, obtained hollow-fibre membrane grafts in 6 orifice plates, Infrared therapeutic device is controlled to carry out illumination, illumination by LAN WiFi or 2G/3G/4G network very-long-range with smart mobile phone simultaneously Intensity is 5mW/cm², light application time is that 2h induced expressions contain the gene loop control system that far-red light regulates and controls transgene expression The hollow-fibre membrane grafts of cell are engineered, respectively in 24h, 48h, 72h examining report gene.Turn base containing far-red light regulation and control Because the gene loop control system engineering cell of expression can be very good to be regulated and controled by far-red light in hollow-fibre membrane grafts Expression.Experimental data refers to Figure of description 33.

Embodiment 18, smart mobile phone regulates and controls the base of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range Because loop control system is in situation of the Mice Body by far-red light regulating and expressing

Smart mobile phone regulates and controls the gene loop control of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range System processed is had a variety of in Mice Body by the mode of far-red light regulating and expressing, and the present embodiment passes through local to transplant containing smart mobile phone The gene loop control system for netting WiFi or 2G/3G/4G networks very-long-range regulation and control transgene expression is engineered the hollow fibre of cell Exemplified by dimension film grafts, it was demonstrated that the gene loop control system of far-red light regulation and control transgene expression is regulated and controled in Mice Body by far-red light The situation of expression.But protection scope of the present invention is not defined.The mode that transplantation carrier is transplanted in Mice Body has a variety of, The present embodiment illustrates that smart mobile phone is regulated and controled by LAN WiFi or 2G/3G/4G network very-long-range so that dorsal sc is transplanted as an example The gene loop control system of transgene expression is transplanted to the method in Mice Body.Comprise the following steps that：

The first step, prepares hollow-fibre membrane grafts (concrete mode is with reference to embodiment 16).

Hollow-fibre membrane grafts are implanted into mouse back (specific method is with reference to experiment material and method) by second step.

3rd step, illumination.Infrared ray is controlled to control by LAN WiFi or 2G/3G/4G network very-long-range with smart mobile phone Treat device (10mW/cm²) distinguish 2h, 8h, 26h, 32h illumination 2h after the transfer.

4th step, examining report gene.24h and 48h eye sockets take the amount of blood examining report gene (specific after the transfer respectively Method refers to experiment material and method).Detailed Experimental data are shown in accompanying drawing 34,35.

Embodiment 19, smart mobile phone regulates and controls the base of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range Because of the external accuracy controlling insulin expression of loop control system

Second step, inoculating cell.By the good HEK-293T cells of growth conditions kind after 0.25% pancreatin digestion In 6 piece of 24 orifice plate, per hole 6 × 104 cells of kind, and DMEM culture mediums of the 500 μ L containing 10%FBS is added.

3rd step, transfection.In inoculating cell 16 to 24h, by 0.1 μ g photoreceptors pWS189,0.1 μ g processor PGY32,0.1 μ g effector pWS213, PEI transfection reagents and serum-free DMEM are mixed, and are stored at room temperature after 15min and are uniformly added dropwise Into 24 well culture plates.Preparation cumulative volume wherein per hole is 50 μ L, and plasmid is 1 with PEI mass ratioes:3.Transfection is changed after 6 hours Enter DMEM culture mediums of the 500 μ L containing 10%FBS to be cultivated.

4th step, illumination.After changing liquid 14-18 hours, six blocks of plates are divided into 6 groups, wavelength are placed in for 720nm, intensity of illumination For under 1mW/cm2 LED, smart mobile phone respectively by LAN WiFi or 2G/3G/4G network very-long-range set light application time as 0,0.1,0.25,0.5,1,2h (illumination 0 hour remain stored in dark).

5th step, detects the expression of insulin.Culture shoots the expression of the green fluorescent protein of each group after 48 hours, And the expression quantity of each group insulin is determined with insulin ELISA kit.

Experiment shows that smart mobile phone regulates and controls the base of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range Because loop control system can accuracy controlling insulin expression in vitro, and its expression quantity and intensity of illumination are into positive correlation.Experiment Data refer to Figure of description 28,29.

Embodiment 20, smart mobile phone turns the gene of gene expression by LAN WiFi or 2G/3G/4G network very-long-range Loop control system accuracy controlling insulin expression in I type glycosuria model mouse treats IDDM

The first step, the structure of IDDM mouse model.We use multiple low dosage streptozotocin (Streptozocin, STZ, purchased from Sigma companies S0130,18883-6,6-4) dose regimen guidance model.C57BL/6J mouse 25 (coming from the Chinese Academy of Sciences), 8 week old, male, continuous 5 days abdominal cavities (fasting 12-16h before injection) injection is dissolved with STZ (agent Measure as 40-50mg/kg) sodium citrate buffer solution.STZ is dissolved in fresh citrate buffer solution during injection (0.1mol/L, PH 4.5), prepare on ice, note lucifuge.Complete injection after dissolving in 30min as far as possible.

Second step smart mobile phone turns the gene ring of gene expression by LAN WiFi or 2G/3G/4G network very-long-range The preparation process and the 3rd step of path control system engineering cell contain smart mobile phone and pass through LAN WiFi or 2G/3G/4G Network very-long-range turn gene expression gene loop control system engineering cell grafts preparation process referring in particular to Embodiment 16.

4th step contains the base that smart mobile phone turns gene expression by LAN WiFi or 2G/3G/4G network very-long-range Because the grafts of loop control system engineering cell are to mouse back and the 5th step illumination, referring in particular to embodiment 18.

6th step, determines a patients with type Ⅰ DM mouse fasting blood sugar.It is after transplanting 8 hours, mouse fasting (water supply) 16 is small Shi Hou, blood is taken through afterbody, determines fasting blood sugar.Experimental data shows that the insulin of expression has good blood sugar decreasing effect. Experimental data refers to Figure of description 36.

7th step, carries out sugared tolerance test.Transplant after 24h, carry out sugared tolerance test.First, fresh glucose is configured The aqueous solution (125mg/ml) 10mL, the body weight (1.25g/kg) further according to every mouse calculates the volume injected of D/W (body weight (g) of abdominal cavity note volume injected V=10 × mouse, unit：μL).Then measuring every mouse, (starvation 16 is small before measurement When) 0 blood glucose, above-mentioned glucose solution is injected intraperitoneally.Finally, the blood glucose value of every mouse 30,60,90,120min is determined. Experiment shows that the sugar tolerance of diabetic mice has good improvement.Experimental data refers to Figure of description 37.

Embodiment 21, smart mobile phone turns the gene of gene expression by LAN WiFi or 2G/3G/4G network very-long-range The expression of the external accuracy controlling hyperglycemic factor (GLP-1) of loop control system

Second step, inoculating cell.By the good HEK-293T cells of growth conditions kind after 0.25% pancreatin digestion In 6 pieces of 24 different orifice plates, per hole kind 6*10⁴Individual cell, and add DMEM culture mediums of the 500 μ L containing 10%FBS.

3rd step, transfection.In inoculating cell 16 to 24h, by 0.1 μ g photoreceptors pWS189,0.1 μ g processor PGY32,0.1 μ g effector pWS212, PEI transfection reagents and serum-free DMEM are mixed, and are stored at room temperature after 15min and are uniformly added dropwise Into 24 well culture plates.Preparation cumulative volume wherein per hole is 50 μ L, and plasmid is 1 with PEI mass ratioes:3.Transfection is changed after 6 hours Enter DMEM culture mediums of the 500 μ L containing 10%FBS to be cultivated.

5th step, determines smart mobile phone and regulates and controls transgene expression by LAN WiFi or 2G/3G/4G network very-long-range Gene loop control system vivoexpression GLP1-Fc amount.Culture uses Active GLP-1ELISA kit measurements after 48 hours Each group GLP1-Fc expression quantity.

Experiment shows that smart mobile phone regulates and controls the base of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range Because loop control system can accuracy controlling hyperglycemic factor in vitro expression, and its expression quantity and intensity of illumination are into positive Close.Experimental data refers to Figure of description 27.

Embodiment 22, smart mobile phone regulates and controls the base of transgene expression by LAN WiFi or 2G/3G/4G network very-long-range Because of loop control system in a type glycosuria model mouse accuracy controlling GLP-1 expression treatment type II diabetes

Second step, type II diabetes model mouse db/db mouse 20 (come from the Chinese Academy of Sciences), 8 week old, and female is divided into Four groups.Do not transplant respectively not light group, do not transplant light group, transplant not light group, transplanting light group.

The preparation process and the 4th step of 3rd step far-red light engineering cell contain smart mobile phone and pass through LAN WiFi Or the preparation of the grafts of the gene loop control system engineering cell of 2G/3G/4G networks very-long-range regulation and control transgene expression Process is referring in particular to embodiment 16.

4th step contains smart mobile phone and regulates and controls transgene expression by LAN WiFi or 2G/3G/4G network very-long-range The grafts of gene loop control system engineering cell are to mouse back and the 5th step illumination, referring in particular to embodiment 18.

6th step, determines a patients with type Ⅰ DM mouse fasting blood sugar.It is after transplanting 8 hours, mouse fasting (water supply) 16 is small Shi Hou, blood is taken through afterbody, determines fasting blood sugar.Experimental data shows that the hyperglycemic factor of expression has good effect of reducing blood sugar Really.Experimental data refers to Figure of description 38.

7th step, carries out sugared tolerance test.Transplant after 24h, carry out sugared tolerance test.First, fresh glucose is configured The aqueous solution (125mg/ml) 10mL, the body weight (1.25g/kg) further according to every mouse calculates the volume injected of D/W (body weight (g) of abdominal cavity note volume injected V=10 × mouse, unit：μL).Then measuring every mouse, (starvation 16 is small before measurement When) 0 blood glucose, above-mentioned glucose solution is injected intraperitoneally.Finally, the blood glucose value of every mouse 30,60,90,120min is determined. Experiment shows that the sugar tolerance of diabetic mice has good improvement.Experimental data refers to Figure of description 39.

8th step, carries out insulin resistant experiment.Transplant after 24h, carry out insulin resistant experiment.First, configure fresh Insulin solutions (0.1U/ml), further according to the body weight (1.1U/kg) of every mouse, the volume injected for calculating insulin (is noted in abdominal cavity The body weight (g) of volume injected V=1.1 × mouse, unit：μL).Then 0 blood glucose of every mouse is determined (hungry 4 before measurement Hour), above-mentioned insulin solutions are injected intraperitoneally.Finally, the blood glucose value of every mouse 30,60,90,120min is determined.Test table Bright, the insulin resistant of diabetic mice has good improvement.Experimental data refers to Figure of description 40.

9th step, determines smart mobile phone and regulates and controls transgene expression by LAN WiFi or 2G/3G/4G network very-long-range Gene loop control system GLP-1 in type II diabetes mouse expression.48h takes blood by eyeball endocanthion after transplanting, uses GLP-1 (activie) content in GLP-1 (7-36) activieELISA kits, detection serum.Experiment shows, smart mobile phone The gene loop control system for regulating and controlling transgene expression by LAN WiFi or 2G/3G/4G network very-long-range can be in vivo The expression of accuracy controlling hyperglycemic factor.Experimental data refers to Figure of description 41.

The plasmid construction table of table 1.

Primer:Restriction enzyme site and seamless cloned sequence are marked with underscore.

Abbreviation：

P_hCMV, human cytomegalovirus's promoter；P_SV40, simian virus 40 promoter；P_hCMVmin3G, cytomegalovirus CMV minimal promoter mutant；P_CAG, artificial constructed cytomegalovirus early stage enhancer combines with chicken β-actin promoter Promoter；EGFP, enhanced green fluorescence protein；SEAP, the outer secreting alkaline phosphorus phytase of the mankind；GLP-1, glucagon-like peptide 1；VP64, the herpesviral transcriptional activation domain tetramer；BldD, far-red light processor；2A, carbon teminal contains a D (V/I) The independent oligopeptides of EXNPGP domains；PA, polyadenosine acid signal；Bphs, photosensitive two guanylate of artificial synthesized bacterium Enzyme；YhjH, c-di-GMP digestive enzyme；Bpho, pigment synthesis enzyme；PCR, PCR.

In sequence table：

Sequence 1：Photosensitive two guanylate cyclase (Bphs) nucleotide sequence of artificial synthesized bacterium

Sequence 2：Bright pigment synthesis enzyme (Bpho) nucleotide sequence

Sequence 3：Self cleavage 2A peptide nucleotide sequences

Sequence 4：C-di-GMP digestive enzymes (YhjH) nucleotide sequence

Sequence 5：Simian virus 40 promoter (SV40) nucleotide sequence

Sequence 6：EF1 α (hEF1 α) the promoter nucleotide sequence in people source

Sequence 7：PGK (mPGK) the promoter nucleotide sequence in mouse source

Sequence 8：Artificial constructed cytomegalovirus early stage enhancer starts sub-portfolio promoter (CAG) with avian beta-actin Nucleotide sequence

Sequence 9：Cytomegalovirus promoter CMV nucleotide sequences

Sequence 10：Far-red light processor (BldD) nucleotide sequence

Sequence 11：Herpes simplex virus particles protein transcription activation structure domain (VP16) nucleotide sequence

Sequence 12：Herpes simplex virus particles protein transcription activation structure domain the core sequence tetramer (VP64) nucleotide sequence

Sequence 13：NF- Κ B p65 subunit transcriptional activation domain nucleotide sequences

Sequence 14：Features of The Heat Shock Transcription Factor transcription activating domain (HSF1) nucleotide sequence

Sequence 15：TATA Box nucleotide sequence

Sequence 16：Cytomegalovirus minimal promoter CMV_minNucleotide sequence

Sequence 17：Cytomegalovirus minimal promoter CMV_minMutant CMV_min3GNucleotide sequence

Sequence 18：BldD binding sites (bldM) nucleotide sequence

Sequence 19：BldD binding sites (whiG) nucleotide sequence

Sequence 20：Far-red light effector promoter P_FRL0(1×bldM-h-CMV_min) nucleotide sequence

Sequence 21：Far-red light effector promoter P_FRL1(2×bldM-h_-CMV_min) nucleotide sequence

Sequence 22：Far-red light effector promoter P_FRL2(3×bldM-h-CMV_min) nucleotide sequence

Sequence 23：Far-red light effector promoter P_FRL3(4×bldM-h-CMV_min) nucleotide sequence

Sequence 24：Far-red light effector promoter P_FRL4(5×bldM-h-CMV_min) nucleotide sequence

Sequence 25：Far-red light effector promoter P_FRL5(1×whiG-h-CMV_min) nucleotide sequence

Sequence 26：Far-red light effector promoter P_FRL6(2×whiG-h-CMV_min) nucleotide sequence

Sequence 27：Far-red light effector promoter P_FRL7(3×whiG-h-CMV_min) nucleotide sequence

Sequence 28：Far-red light effector promoter P_FRL8(4×whiG-h_-CMV_min) nucleotide sequence

Sequence 29：Far-red light effector promoter P_FRL9(5×whiG-h-CMV_min) nucleotide sequence

Sequence 30：Far-red light effector promoter P_FRL10(SV40PolyA-1×whiG-h-CMV_min) nucleotide sequence

Sequence 31：Far-red light effector promoter P_FRL11(SV40PolyA-2×whiG-h-CMV_min) nucleotide sequence

Sequence 32：Far-red light effector promoter P_FRL12(SV40PolyA-3×whiG-h-CMV_min) nucleotide sequence

Sequence 33：Far-red light effector promoter P_FRL13(SV40PolyA-4×whiG-h-CMV_min) nucleotide sequence

Sequence 34：Far-red light effector promoter P_FRL14(SV40PolyA-5×whiG-h-CMV_min) nucleotide sequence

Sequence 35：Far-red light effector promoter P_FRL15(1×whiG-h-CMV_min3G) nucleotide sequence

Sequence 36：Far-red light effector promoter P_FRL16(2×whiG-h-CMV_min3G) nucleotide sequence

Sequence 37：Far-red light effector promoter P_FRL17(3×whiG-h-CMV_min3G) nucleotide sequence

Sequence 38：Far-red light effector promoter P_FRL18(4×whiG-h-CMV_min3G) nucleotide sequence

Sequence 39：Far-red light effector promoter P_FRL19(4×whiG-h_-CMV_min3G) nucleotide sequence

Sequence 40：The nucleotide sequence for treating transcription sequence SEAP (SEAP) of far-red light effector expression

Sequence 41：The nucleotide sequence for treating transcription sequence green fluorescent protein (EGFP) of far-red light effector expression

Sequence 42：The nucleotide sequence for treating transcription sequence luciferase (Luciferase) of far-red light effector expression

Sequence 43：The nucleotides for treating transcription sequence glucagon-like-peptide-1 (GLP-1-Fc) of far-red light effector expression Sequence

Sequence 44：The core of the nucleotide sequence (EGFP-2A-Insulin) for treating transcription sequence of far-red light effector expression Nucleotide sequence

Sequence 45：Photosensitive two guanylate cyclase (Bphs) amino acid sequence of artificial synthesized bacterium

Sequence 46：Bright pigment synthesis enzyme (Bpho) amino acid sequence

Sequence 47：Self cleavage 2A peptide nucleotide sequences

Sequence 48：C-di-GMP digestive enzymes (YhjH) amino acid sequence

Sequence 49：Far-red light processor (BldD) amino acid sequence

Sequence 50：Herpes simplex virus particles protein transcription activation structure domain (VP16) amino acid sequence

Sequence 51：Herpes simplex virus particles protein transcription activation structure domain the core sequence tetramer (VP64) nucleotide sequence

Sequence 52：NF- Κ B p65 subunit transcriptional activation domain amino acid sequences

Sequence 53：Features of The Heat Shock Transcription Factor transcription activating domain (HSF1) amino acid sequence

Sequence 54：The amino acid sequence of nuclear localization signal (NLS)

Sequence 55：The amino acid sequence of linkage function peptide (Linker)

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Claims

1. application of the gene loop remote control and regulation system in Remedies for diabetes is prepared；The diabetes include I type glycosurias Disease and/or type ii diabetes；

The gene loop remote control and regulation system includes：

Far-red light gene loop expresses control system；

Control device for sending remote control commands；And

Far-red light light supply apparatus；

Wherein, the far-red light gene loop expression control system includes：

Experience the photoreceptor of far-red light light source；

Handle the processor that the photoreceptor transmits signal；And

Processor described in response transmits the effector of signal；

The control device passes through the far-red light light source by sending far-red light light supply apparatus described in control instruction remote control and regulation The far-red light that device is sent regulates and controls the far-red light gene loop expression control system, realizes regulation and control transgene expression.

2. application as claimed in claim 1, it is characterised in that described in the far-red light gene loop expression control system Photoreceptor includes photosensitive two guanylate cyclases Bphs, and GTP is changed into c-di-GMP by it under the conditions of far-red light.

3. application as claimed in claim 2, it is characterised in that described in the far-red light gene loop expression control system The photosensitive two guanylate cyclases Bphs of photoreceptor be by BphG albumen 1-511 amino acids and Slr1143 albumen 175-343 amino acids are merged, and 587 arginine of fusion protein are sported into alanine R587A prepared.

4. application as claimed in claim 2, it is characterised in that described in the far-red light gene loop expression control system The structure form of photoreceptor includes：

A) the photosensitive two guanylate cyclases Bphs encoding genes Bphs of artificial synthesized bacterium；

B) the photosensitive two guanylate cyclases Bphs encoding genes of artificial synthesized bacterium pass through 2A sequences and c-di-GMP digestive enzymes YhjH encoding genes are connected Bphs-2A-YhjH；

C) the photosensitive two guanylate cyclases Bphs encoding genes of artificial synthesized bacterium pass through 2A sequences and phytochrome synzyme Bpho encoding genes are connected Bphs-2A-Bpho；

D) the photosensitive two guanylate cyclases Bphs encoding genes of artificial synthesized bacterium pass through 2A sequences and phytochrome synzyme Bpho encoding genes are connected, then are connected by 2A sequences with c-di-GMP digestive enzyme YhjH encoding genes Bphs-2A-Bpho-2A- YhjH；

Wherein, the 2A sequences can be substituted by internal ribosome entry site sequence IRES；

The phytochrome synzyme Bpho has the function of synthesis phytochrome biliverdin；

The digestive enzyme YhjH of the c-di-GMP has the function that c-di-GMP is degraded to pGpG.

5. application as claimed in claim 4, it is characterised in that described in the far-red light gene loop expression control system Bphs, Bpho, YhjH amino acid sequence are selected from sequence 45,46,48.

6. application as claimed in claim 1, it is characterised in that described in the far-red light gene loop expression control system The promoter of expression photoreceptor includes：a)SV40；b)CMV；c)hEF1α；d)mPGK；e)CAG.

7. application as claimed in claim 1, it is characterised in that described in the far-red light gene loop expression control system Processor includes：As the polypeptide of DNA binding domain and c-di-GMP binding domain, the polypeptide as nuclear localization signal NLS, as even Connect the polypeptide and the polypeptide as transcriptional regulatory domain in domain.

8. application as claimed in claim 7, it is characterised in that described in the far-red light gene loop expression control system As the polypeptide of DNA binding domain and c-di-GMP binding domain, it, can be with specific DNA sequence dna knot for after being combined with c-di-GMP The albumen of conjunction, including BldD albumen, the amino acid sequence of the BldD albumen are selected from sequence 49.

9. application as claimed in claim 7, it is characterised in that described in the far-red light gene loop expression control system As nuclear localization signal NLS polypeptide, it can copy diversified forms for 1-3, and its amino acid sequence is selected from sequence 54.

10. application as claimed in claim 7, it is characterised in that described in the far-red light gene loop expression control system As the polypeptide in linkage function domain, its length can be selected from sequence 55 from 0-30 amino acid diversified forms, its amino acid sequence.

11. application as claimed in claim 7, it is characterised in that described in the far-red light gene loop expression control system As the polypeptide of transcriptional regulatory domain, it has the domain protein of transcriptional activation function, including NF- Κ B p65 subunits for all Transcriptional activation domain, Features of The Heat Shock Transcription Factor HSF1 transcription activating domains, Herpes simplex virus particles albumen VP16 transcriptional activations The transcriptional activation domain VP64 of domain and its 4 copy；Its amino acid sequence is selected from sequence 50,51,52,53.

12. application as claimed in claim 7, it is characterised in that described in the far-red light gene loop expression control system The polypeptide BldD of the DNA binding domain and c-di-GMP binding domain N-terminal or C-terminal are placed in as the polypeptide of transcriptional regulatory domain.

13. application as claimed in claim 7, it is characterised in that described in the far-red light gene loop expression control system It can be directly connected to or be connected by connecting peptide between the polypeptide in difference in functionality domain in processor.

14. application as claimed in claim 1, it is characterised in that described in the far-red light gene loop expression control system Effector includes P_FRL-reporter；The effector includes promoter sequence and treats the nucleotide sequence of transcription factor.

15. application as claimed in claim 14, it is characterised in that in the far-red light gene loop expression control system, institute State the weak promoter that promoter sequence is expressed comprising the protein bound DNA sequence dnas of processor BldD and promotor gene.

16. application as claimed in claim 15, it is characterised in that in the far-red light gene loop expression control system, institute The protein bound DNA sequence dnas of processor BldD are stated, it is recognized simultaneously for the polypeptid specificity of DNA binding domain and c-di-GMP binding domain With reference to DNA sequence dna, be the partial sequence of bldM and whiG promoter regions.

17. application as claimed in claim 16, it is characterised in that in the far-red light gene loop expression control system, institute The nucleotide sequence for stating bldM is selected from sequence 18；The nucleotide sequence of the whiG is selected from sequence 19.

18. application as claimed in claim 17, it is characterised in that in the far-red light gene loop expression control system, institute The partial sequence of bldM and whiG promoter regions is stated, it is copied for 1-10.

19. application as claimed in claim 16, it is characterised in that in the far-red light gene loop expression control system, institute Stating the weak promoter of promotor gene expression includes all weak promoters, and it includes TATA box, cytomegalovirus CMV minimums and opened Mover and its mutant CMVmin 3G.

20. application as claimed in claim 15, it is characterised in that in the far-red light gene loop expression control system, institute State promoter sequence nucleotide sequence be selected from sequence 20,21,22,23,24,25,26,27,28,29,30,31,32,33,34, 35、36、37、38、39。

21. application as claimed in claim 15, it is characterised in that in the far-red light gene loop expression control system, institute State and treat that the albumen that transcribable nucleic acid sequence coding schedule reaches can be all significant albumen, including be used as the albumen of reporter gene And/or pharmaceutical protein or the nucleotide sequence of small peptide as treatment disease；Wherein, the albumen as reporter gene includes dividing Secrete the nucleotide sequence of type alkaline phosphatase, enhanced green fluorescence protein, luciferase；As treatment disease pharmaceutical protein or Small peptide includes insulin, the nucleotide sequence of glucagon-like peptide.

22. application as claimed in claim 21, it is characterised in that in the far-red light gene loop expression control system, institute State all significant albumen can by 2A sequences on an expression vector while express multiple albumen, including SEAP-2A- Insulin、EGFP-2A-Insulin、EGFP-SEAP-2A-Insulin；Wherein, the 2A sequences can be by internal ribosome Entry site sequence IRES is substituted.

23. application as claimed in claim 1, it is characterised in that the control device passes through LAN WiFi or 2G/3G/4G Network sends remote control commands to regulate and control the different working condition of the far-red light light supply apparatus.

24. application as claimed in claim 23, it is characterised in that the different working condition includes the far-red light light source Intensity of illumination, light application time or the illuminating method that be turned on and off, can be adjusted on demand.

25. application as claimed in claim 24, it is characterised in that the intensity of illumination is 0-5mW/cm²；The irradiation time For 0-72h；The illuminating method includes pulsed exposure, Continuous irradiation, direct irradiation or the projection card portrayed with hollow out and existed The spatially irradiation of the gene expression dose of the cell of control diverse location.

26. application as claimed in claim 1, it is characterised in that the control device includes smart mobile phone App and/or intelligence Remote controllers.

27. application as claimed in claim 1, it is characterised in that the Intelligence Remote Controller includes all with long-range control The controller of function processed, it includes the Intelligence Remote Controller of single channel output and/or multiple-channel output.

28. application as claimed in claim 1, it is characterised in that the far-red light light supply apparatus is that to produce wavelength be 600- The light supply apparatus of 900nm far-red lights.

29. application as claimed in claim 1, it is characterised in that the system regulation insulin and/or glucagon-like peptide GLP-1 expression.

30. application as claimed in claim 23, it is characterised in that the expression of the insulin, which is built, includes SEAP-2A- Insulin、EGFP-2A-Insulin、EGFP-2A-SEAP-2A-Insulin；The expression of the glucagon-like peptide GLP-1 Including GLP-1-Fc.