CN117187285A

CN117187285A - Prokaryotic far-red light regulation transcription activation device, construction method thereof and application thereof in tumor treatment

Info

Publication number: CN117187285A
Application number: CN202210607219.2A
Authority: CN
Inventors: 叶海峰; 牛灵雪; 乔龙亮; 王智浩; 管宁子
Original assignee: East China Normal University
Current assignee: East China Normal University
Priority date: 2022-05-31
Filing date: 2022-05-31
Publication date: 2023-12-08

Abstract

The invention discloses a prokaryotic far-red light regulation and transcription activation device (NITE device)/system and a construction method thereof, and application of the device/system in tumor treatment. The far-red light regulated transcription activation device comprises a far-red light sensing element, a gene transcription activation element and a far-red light response element. The far-red light regulation and transcription activation device has high efficiency of activating transcription expression of reporter genes in prokaryotes, can accurately regulate and control gene transcription of prokaryotes, and has the characteristics of no toxicity, high efficiency, space-time specificity, adjustability and the like. By utilizing the characteristic that salmonella targets tumor tissues, the device is engineered and modified to attenuate salmonella enteritidis3934 delta XV, has good treatment effect in the treatment of tumors, and provides a more accurate tool for the development of bacterial therapy in the field of tumor medicine.

Description

Prokaryotic far-red light regulation transcription activation device, construction method thereof and application thereof in tumor treatment

Technical Field

The invention relates to the field of multidisciplinary intersection of microorganism synthesis biology, optogenetics, tumor therapy, tumor immunology and the like, in particular to a prokaryotic far-red light regulation and transcription activation device, a construction method thereof and application thereof in tumor therapy, which can efficiently induce the transcriptional expression activation of prokaryotic genes under the irradiation of far-red light.

Background

Tumor incidence increases year by year and gradually tends to be younger, and tumor treatment is a long-term business currently being struggled with worldwide humans. The tumor targeting treatment achieves the effect of killing tumor locally by guiding the drug to target the tumor area in an accurate orientation and minimizing the damage to surrounding normal tissues, and has important significance in tumor treatment.

Salmonella as a facultative anaerobic bacteria can target the hypoxic tumor microenvironment and enrich the surrounding tumor tissue. By utilizing the advantage of synthetic biology from bottom to top, salmonella is engineered to express drug protein for tumor treatment, so that a drug missile for treating tumors is constructed. Since salmonella is a high risk of infection in clinical use, it is a critical challenge for researchers to balance the excessive toxicity of bacteria with therapeutic efficacy. The three virulence genes (delta purI, delta rpoS and delta lpxM) are knocked out on the basis of Salmonella enteritidis 3934 delta XII to obtain an attenuated Salmonella enteritidis 3934 delta XV, so that the virulence of salmonella to organisms is greatly reduced, and a safe drug 'active carrier' is provided for bacterial therapy in tumor treatment.

By engineering bacteria for bacterial therapy of tumor treatment, the expression system of the bacterial therapy usually adopts constitutive promoters, anaerobic promoters or small-molecule regulated inducible promoters to express drug proteins for tumor treatment, and accurate control of drug expression release is difficult to realize. Because the light has good space-time specificity and no toxicity, a new development direction is provided for realizing accurate tumor treatment. Therefore, by combining optogenetics and engineering attenuated salmonella, accurate and controllable tumor treatment can be realized, and the method has important medical application value. At present, optogenetics has a wide range of applications in microorganisms, and general light control systems are control systems based on blue light activation or deactivation. Due to the defects of poor tissue penetrability, weak toxicity to cells and the like of blue light, development and clinical application of light-controlled bacterial therapy to tumor treatment are greatly limited.

Disclosure of Invention

In order to overcome the limitations in the prior art, the invention provides a prokaryotic far-red light regulation and transcription activation device (NITE, near-infrared light triggered genetic control system)/system with strong tissue penetration capability and no toxicity, which can efficiently activate the transcription expression of a reporter gene through far-red light. The device/system is engineered to realize the accurate controllability of the bacterial therapy in treating tumors by attenuated Salmonella enteritidis 3934 delta XV, and has important clinical application value for tumor treatment.

The invention provides a far-infrared regulation transcription activation device (NITE device)/system using prokaryote as chassis cells, which combines the advantages of salmonella of good tumor targeting, and can efficiently activate the expression (55 times) of reporter genes through 710nm far-infrared irradiation. In addition, the device (NITE) has the characteristics of space-time specificity, adjustability, chassis cell universality, strong tissue penetrating power combined with 710nm far-red light, no phototoxicity and the like, can provide a powerful tool for accurate tumor bacterial therapy, can effectively induce and secrete tumor drug proteins in attenuated Salmonella enteritidis 3934 delta XV, and has good treatment effect on tumors. The prokaryotic far-red light activated transcription activation device (NITE)/system has great potential application value and can be widely popularized in clinical application.

The term "space-time specificity" refers to the fact that when a target gene is induced to be expressed by a specific factor, the target gene is influenced by the action time and action space of the induction factor, and the dependence of the gene expression on the time and space of the induction factor is shown.

The nucleotide sequence or the amino acid sequence of the invention can be prepared by adopting an artificial synthesis method.

The invention provides an artificial design and synthesis prokaryotic far-red light regulation and transcription activation device (NITE). In the invention, the photon energy of far-red light is much lower than that of blue light, the toxic and side effect on cells is much smaller than that of blue light, and the far-red light has stronger tissue penetrability, so that the efficient and accurate regulation and control of microorganisms in animal bodies can be realized. Due to the limitations of prokaryotes on element size and composition during engineering, the use of large and complex far-red/red optogenetic tools for modular elements in prokaryotes is constrained and efficient fold induction is difficult to achieve. In the invention, the prokaryotic far-red light regulation transcription activation device breaks through the limitation of the prokaryotic on elements, and shows good transcription activation efficiency under the induction of far-red light.

The invention provides a prokaryotic far-red light regulation transcription activation device (NITE), which comprises: far red light sensing elements (far red light sensing proteins), gene transcription activating elements, and far red light response elements.

In the present invention, the far-red light sensing element includes bacterial photosensitive diguanylate cyclase PadC 4/PadC 10, heme oxygenase BphO and degradation enzyme YhjH of cyclodiguanylate c-di-GMP.

Wherein, the amino acid sequences of the bacterial photosensitive diguanylate cyclases PadC4 and PadC10 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2; the heme oxygenase BphO can generate BV pigment, and the amino acid sequence of the BV pigment is shown as SEQ ID NO. 3; the amino acid sequence of the c-di-GMP degrading enzyme YhjH is shown in SEQ ID No. 4.

In the invention, the gene transcription activation element comprises a transcription activation factor MrkH, and the amino acid sequence of the gene transcription activation element is shown as SEQ ID NO. 5.

In the present invention, the far-red light response element comprises an inducible promoter P _MrkA And a downstream reporter gene.

Wherein the inducible promoter P _MrkA The nucleotide sequence of the polypeptide is shown as SEQ ID NO. 6; the downstream reporter gene is the gene sequence of any meaningful protein, wherein the reporter gene comprises luciferase LuxCDABE, the nucleotide sequence of which is shown as SEQ ID NO.7, tumor therapeutic protein Azurin Azurin, the amino acid sequence of which is shown as SEQ ID NO.15, tumor therapeutic protein cytolysin A (ClyA), the amino acid sequence of which is shown as SEQ ID NO.16, tumor therapeutic protein nano-antibody anti-PD-L1 nanobody, the amino acid sequence of which is shown as SEQ ID NO.17, and tumor therapeutic protein nano-antibody anti-CTLA4 nanobody, the amino acid sequence of which is shown as SEQ ID NO. 18.

Where there are two or more proteins of interest, the simultaneous expression of both proteins on the same vector is facilitated by the addition of a Ribosome Binding Site (RBS) between the gene sequences of the two proteins.

Wherein the inducible promoter P _MrkA Can be specifically combined by transcription activator MrkH, and the nucleotide sequence is shown as SEQ ID NO. 6; constitutive promoter P _lac The nucleotide sequence is shown as SEQ ID NO.13, and BphO and PadC4/PadC10 expression is started; constitutive promoter P _tac The nucleotide sequence is shown as SEQ ID NO.14, and the expression of YhjH and MrkH is started.

The prokaryotic far-red light regulation and transcription activation device/system and the transcription activation mechanism thereof are shown in figure 1, and the specific text is as follows:

bacterial photosensitive diguanylate cyclase PadC4/PadC10 produced by catalysis of heme oxidase BphOWith the aid of biliverdin BV, red light is felt to activate the own activity, converting GTP into second messenger c-di-GMP. YhjH is a protease which can convert c-di-GMP into pGpGpG, and can degrade the c-di-GMP generated in the microorganism by expressing YhjH so as to reduce the background noise of the device in dark conditions. The transcriptional properties of the transcription factor MrkH activating gene are regulated by a second messenger c-di-GMP. MrkH consists of two domains: an N-terminal YcgR-N analog domain and a C-terminal pilZ domain. The PilZ domain is a classical c-di-GMP binding domain, while the function of the YcgR-N domain remains unclear. MrkH binds to the specific promoter P after high affinity for c-di-GMP _MrkA Transcriptional expression of downstream reporter genes was activated by recruiting RNA polymerase.

According to the invention, different bacterial photosensitive diguanylate cyclases PadC4 and PadC10 are selected, padC4 is selected according to the induction efficiency, and the amino acid sequence of the PadC4 is shown as SEQ ID NO. 1; the chassis cells of salmonella and the RBS sequences of c-di-GMP degrading enzyme YhjH are optimized, the nucleotide sequences of different RBSs are shown in SEQ ID No.8-12, and RBS4 is selected according to the induction efficiency. The NITE device has optimal transcription and expression efficiency of the reporter gene through the optimization.

The invention can efficiently induce Salmonella enteritidis 3934DeltaXII to express the reporter gene, can regulate and control the expression quantity of the reporter gene according to the illumination time and intensity, and has the characteristics of good induction expression multiple, high space-time specificity, strong adjustability, no toxicity and the like.

The prokaryotic far-red light regulation and transcription activation device provided by the invention has good far-red light induction expression efficiency in Salmonella enteritidis 3934 DeltaXII, and has good far-red light regulation and gene expression effects in any type of prokaryotic microorganism, such as Ecoil BL21, salmonella typhimurium VNP20009, ecoil TOP10, ecoil JM109 and the like.

Wherein the wavelength of far-red light in the NITE device is 660-730nm, and the illumination intensity is 0-2.5 mW/cm ² The method comprises the steps of carrying out a first treatment on the surface of the The illumination time is 0-7h; the illumination mode is continuous irradiation or selective irradiation of the produced mould on a specific space, and different positions are controlledGene transcription expression of the cells. Different illumination intensities and illumination times are generated by controlling the far-red light source, so that the induction of different degrees of the reporter gene is realized. The far-red light source is an LED bulb, an infrared therapeutic device, a laser lamp and the like with the wavelength of 710 nm.

The invention also provides a prokaryotic expression vector, which comprises the prokaryotic far-red light regulation transcription activation device.

The invention also provides an engineered bacterium comprising a prokaryotic far-red light regulated transcriptional activation device as described above.

The invention also provides a prokaryotic far-red light regulation and transcription activation system, which comprises the prokaryotic far-red light regulation and transcription activation device.

The invention also provides a kit containing the prokaryotic far-red light regulation and transcription activation device, and the kit comprises a prokaryotic expression vector containing the prokaryotic far-red light regulation and transcription activation device, engineering bacteria and corresponding instructions.

The invention also provides an application of the prokaryotic far-red light regulation transcription activation device, a prokaryotic expression vector, engineering bacteria and a kit in preparing products for treating tumors.

Wherein, when tumor therapeutic proteins, including Azurin Azurin and cytolysin A ClyA, nanobody anti-PD-L1 nanobody and nanobody anti-CTLA4 nanobody are used as reporter genes, the product can be used for bacterial therapy to treat tumors.

The prokaryotic far-red light regulation and transcription activation device disclosed by the invention is used for additionally knocking out three virulence genes delta purI, delta rpoS and delta lpxM on the basis of Salmonella enteritidis 3934 delta XII to obtain the attenuated Salmonella enteritidis 3934 delta XV. Wherein purI gene codes for phosphorus fiber triglycine glycine amidine synthetase (FGAM synthetase) which is a key gene in adenine synthesis process, rpoS gene codes for sigma factor of RNA polymerase, lpxM gene codes for lipid A (lipidA) which is a key structure in Lipopolysaccharide (LPS) and is necessary for lipid acylation to endotoxin.

The invention also provides an application of the prokaryotic far-red light regulation and transcription activation device in tumor treatment, wherein the DeltaXV-NITE is obtained by engineering Salmonella enteritidis 3934 DeltaXV.

Wherein, the N end of the drug protein for tumor treatment is fused and expressed with a secretion signal yopE, so that the drug protein which is induced and activated by far-red light can be secreted into a tumor microenvironment, and the secretion signal yopE has an amino acid sequence shown as SEQ ID NO. 19.

In order to detect the secretion effect of the drug protein for tumor treatment, the C end of the drug protein is fused with corresponding tag proteins, including 3 xFlag, HA and 6 xHis, and the amino acid sequences of the tag proteins are shown in SEQ ID NO. 20-22.

The invention also provides a construction method of the prokaryotic far-red light regulation transcription activation device, the far-red light sensing element, the gene transcription activation element and the far-red light response element are constructed on corresponding vectors by genetic engineering technology, bphO and PadC4 in the far-red light sensing element are constructed on pGN62 plasmid vectors, and the far-red light sensing element YhjH, the gene transcription activation element MrkH and the far-red light response element P _MrkA And the reporter gene is constructed on a pYC214 plasmid vector.

The invention also provides a construction method of the prokaryotic far-red light regulation transcription activation device, which comprises the following steps: (1) constructing a far-red light sensing element; (2) constructing a gene transcription activation element; (3) constructing far-red light effect elements.

In the step (1), the bacterial photosensitive diguanylate cyclase PadC4 is induced by far-red light with the assistance of biliverdin BV, and can convert guanosine triphosphate GTP into cyclic diguanylate phosphate c-di-GMP; heme oxygenase BphO can catalyze the production of biliverdin BV molecules; yhjH degrades the natural second messenger c-di-GMP in bacterial cells, achieving a reduction in background noise generated under dark conditions.

In the step (2), mrkH is a transcriptional activator, has no transcriptional activation activity in a natural state, has the activity of recruiting RNA polymerase after being combined with c-di-GMP in a high affinity mode, and specifically binds to a promoter P _MrkA And activating transcriptional expression of the downstream gene.

In step (3), P _MrkA An inducible promoter specifically identified and bound to MrkH, recruiting RNA polymerase by the transcription factor MrkH, activating expression of downstream genes; the reporter gene is the gene sequence of any protein of interest.

The invention also provides a prokaryotic far-red light regulation transcription activation device constructed by the method.

The present invention also provides a method for regulating gene expression in a prokaryote using a prokaryote far-red light-regulated transcriptional activation device as described above, the method comprising the steps of:

a) Constructing the prokaryotic far-red light regulation transcription activation device in a prokaryotic plasmid expression vector;

b) Transforming said prokaryotic cell with an expression vector;

c) Activating expression of the reporter gene in the prokaryotic cell by far-red light irradiation, so that the prokaryotic far-red light regulation transcription activation device activates gene expression in the host cell.

The invention also provides an application method of the prokaryotic far-red light regulation gene transcription activation device in tumor treatment, which comprises the following steps:

a) Constructing a report gene which is a prokaryotic far-red light regulation transcription activation device effector of a drug protein for treating tumors;

b) Preparation of an NITE device an engineered Salmonella enteritidis 3934 DeltaXV (DeltaXV-NITE) that activates expression of a tumor drug protein;

c) Tumor in situ injection of DeltaXV-NITE;

d) The 710nm far-red light induces the expression and secretion of tumor drug protein, thus realizing the treatment of tumor.

The tumor comprises a mouse colon cancer cell CT26 and a mouse B lymphoma cell A20;

the invention has the beneficial effects that: the invention constructs a far-red light-regulated transcriptional activation device/system in a first prokaryote, can regulate and control the transcriptional activation device through far-red light induction, realizes accurate regulation and control on the gene expression of the prokaryote, and has the characteristics of high induction gene expression multiple, high space-time specificity, strong tissue penetrating power, no toxic or side effect and the like. By combining with the advantage of bacterial therapy in treating tumors, the device can induce secretion of tumor therapeutic proteins in attenuated Salmonella enteritidis 3934 delta XV by engineering transformation of Salmonella enteritidis 3934 delta XV, successfully inhibits the growth of tumors, has better tumor therapeutic effect, has important clinical application value for tumor therapy, and has important significance in application in microbial engineering and regenerative medicine.

Drawings

FIG. 1 is a schematic diagram of a prokaryotic far-red light regulated transcriptional activation device NITE/system and transcriptional activation thereof;

FIG. 2 is a graph showing the results of screening a prokaryotic far-red light regulated transcriptional activator NITE for a bacterial photosensitive diguanylate cyclase;

FIG. 3 is a graph showing the result of screening the optimum illumination red wavelength by using a prokaryotic far-red light regulation transcription activation device NITE;

FIG. 4 is a graph showing the results of activation efficiency of the prokaryotic far-red light regulated transcriptional activator NITE at wild type Salmonella enteritidis 3934 (WT) and Salmonella enteritidis 3934 ΔXII (ΔXII) knocked out to produce the c-di-GMP gene;

FIG. 5 is a graph showing the results of the prokaryote far-red light regulated transcriptional activator NITE optimizing RBS of the c-di-GMP degrading enzyme YhjH;

FIG. 6 is a graph showing the results of the light intensity dependence of the expression of the NITE activation reporter gene of the prokaryotic far-red light regulation transcription activation device;

FIG. 7 is a graph showing the results of the light time dependence of the expression of the NITE activation reporter gene of the prokaryotic far-red light regulation transcription activation device;

FIG. 8 is a graph showing the results of the tunability of the expression of a prokaryotic far-red light regulation transcription activation device NITE activation reporter gene;

FIG. 9 is a graph showing the results of a far-red light phototoxicity test for prokaryote growth;

FIG. 10 is a graph showing the results of prokaryotic far-red light regulated transcriptional activation devices NITE capable of activating gene expression in a variety of prokaryotic cells;

FIG. 11 is a graph showing the results of prokaryotic far-red light regulated transcriptional activator NITE activation reporter gene expression with spatial specificity;

FIG. 12 is a graph showing the results of cell level verification of prokaryotic far-red light regulated transcriptional activator NITE secretion expression Azurin and ClyA killer tumor cells;

FIG. 13 is a graph showing the results of prokaryotic far-red light regulated transcriptional activation device NITE secretory expression of Azurin and ClyA with light time dependence;

FIG. 14 is a flow chart of a prokaryotic far-red light regulated transcription activation device NITE for treating CT26 tumor;

FIG. 15 is a graph showing the results of monitoring tumor volume of a prokaryotic far-red light regulated transcription activation device NITE for treating mouse CT26 tumor;

FIG. 16 is a graph showing the results of the use of a prokaryotic far-red light regulated transcriptional activator NITE for treating CT26 tumor in mice;

FIG. 17 is a flow chart of a prokaryotic far-red light regulated transcription activation device NITE for treating A20 tumor;

FIG. 18 is a graph showing the results of tumor volume monitoring of a prokaryotic far-red light regulated transcription activation device NITE for treating mouse A20 tumor;

FIG. 19 is a graph showing the results of prokaryotic far-red light regulated transcriptional activator NITE for weight monitoring in mice treated for CT26 tumors;

FIG. 20 is a graph showing the results of the use of a prokaryotic far-red light regulated transcriptional activator NITE for treating CT26 tumor in mice;

FIG. 21 is a flow chart of a prokaryotic far-red light regulated transcriptional activator device NITE for treating bilateral A20 tumors;

FIG. 22 is a graph showing the results of monitoring tumor volume on the treatment side of a double-sided A20 tumor in mice treated with a prokaryotic far-red light regulated transcription activation device NITE;

FIG. 23 is a graph showing the results of untreated side tumor volume monitoring of a prokaryotic far-red light regulated transcriptional activator apparatus NITE for treating bilateral A20 tumors in mice.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and drawings. These examples are only for illustrating the invention and do not limit the scope of the invention in any way. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited. The reagents, instruments, etc. used in the following examples were carried out according to the conditions suggested by the conventional or commercial suppliers, without specifying the specific conditions.

Materials and methods

Plasmid construction and related reagent configuration

The molecular cloning technique constructs all expression plasmids of the invention, and the steps are common knowledge in the industry.

All primers used for PCR were synthesized by Shanghai Rui, inc. The expression plasmids constructed in the embodiment of the invention are all sequenced, and sequencing is completed by Shanghai Paeno Biotechnology Co. The PhantaMaxSuper-FidelityDNA polymerase and homologous recombinase used in the examples of the present invention were purchased from Nanjinopran Biotechnology Co., ltd. Endonucleases were purchased from New England Biolabs; t4DNA ligase, DNA Marker DL15000, DNA Marker DL5000, DNA Marker DL2000 were purchased from Takara doctor materials technology (Beijing) Inc. Yeast extract, tryptone, agar powder, ampicillin (Amp), and agarose commercially available from Shanghai Corp. Nucleic acid dye GoldView was purchased from the company of Saint Biotechnology, inc. (Shanghai); the plasmid small extraction kit is purchased from Tiangen Biochemical technology (Beijing) Co., ltd; DNA gel recovery kit and PCR product purification kit are all purchased from century biotechnology Co., ltd; the rest reagents such as absolute ethyl alcohol, naCl and the like mentioned in the examples are all domestic analytically pure products.

The PCR system and program were set up according to the instructions provided by Phanta Max Super-Fidelity DNA polymerase (Bosun doctor technologies (Beijing)) incorporated; seamless cloning was performed according to the instructions provided by homologous recombinase (Nanjinouzan Biotechnology Co., ltd.) and T4 enzyme ligation was performed according to the instructions provided by T4DNA ligase (Bosun doctor technologies (Beijing Co., ltd.); the digestion of the DNA vector or fragment is performed according to the instructions provided by endonuclease (New England Biolabs); the gel recovery, purification and recovery of the DNA fragment are carried out according to the operation instruction of a DNA gel recovery kit and a PCR product purification kit (Kangji Biotechnology Co., ltd.); plasmid extraction procedure the kit instructions were extracted according to plasmid xiaozhu (Tiangen Biochemical technology (Beijing) Co., ltd.).

Configuration of related reagents

Preparation of DH5 alpha competence

The preparation of the transformation competence comprises the following specific steps of

1) Culturing DH5 alpha streak non-anti-solid LB plate overnight to reach the aim of activation;

2) Selecting a monoclonal and inoculating the monoclonal into 5mL of liquid LB culture medium, and culturing at 37 ℃ and 210rpm overnight;

3) 2mL of the seed solution was inoculated into 200mL of antibiotic-free liquid LB, and cultured at 37℃and 210rpm to OD ₆₀₀ ＝0.3-0.5；

4) Cooling the seed solution on ice for 20min, centrifuging at 4500rpm for 7min at 4deg.C, and discarding supernatant;

5) Adding 0.1M CaCl with the same volume as the bacterial liquid ₂ Gently stirring and mixing with a pipetting gun, centrifuging at 4500rpm for 7min at 4deg.C, and discarding supernatant;

6) Adding 0.1M CaCl 1/2 volume of the total bacterial liquid ₂ Gently stirring and mixing with a pipetting gun, centrifuging at 4500rpm for 7min at 4deg.C, and discarding supernatant;

7) Adding 0.1M CaCl 1/2 volume of the total bacterial liquid ₂ Mixing with 10% glycerol by gently blowing with a pipette,centrifuging at 4500rpm for 7min at 4deg.C, and discarding supernatant;

8) 10ml of 0.1M CaCl was used ₂ Re-suspending 10% glycerin, sub-packaging 100 μl/tube, and storing at-80deg.C;

the competence is tested in a sterile environment in the manufacturing process, and the ultracentrifuge and reagent consumable materials related to the competence need to be precooled at 4 ℃.

Conversion process

Chemical transformation (transformation competence)

1) 100ng of plasmid DNA was added to the competence (the volume of plasmid DNA added was not more than 1/10 of the total volume of competence), and left on ice for 30min;

2) Heat shock at 42 ℃ for 90s, and immediately standing on ice for 2min;

3) Adding 600 mu L of antibiotic-free liquid LB culture medium, and culturing for 1h in a shaking table at 37 ℃ and 210 rpm;

4) Centrifuging at 6000rpm for 2min, discarding a proper amount of supernatant, and culturing with a heavy suspension coating plate LB solid plate in a 37 ℃ incubator overnight;

Salmonella culture, transformation and related reagent configuration

The prokaryotic far-red light regulation and transcription activation device takes Salmonella enteritidis 3934 and a gene knockout strain thereof as chassis cells, and the culture conditions of other prokaryotic cells E.coli TOP10, E.coli BL21, E.coli JM109 and Salmonella typhimurium VNP20009 are the same as those of Salmonella enteritidis 3934. The culture medium of all strains was LB medium, and the culture conditions were 37℃and 210rpm, without specific explanation. Bacterial growth Density light absorbance at 600nm was measured using an Eppendorf spectrophotometer (0D ₆₀₀ ). 12mL of shaking tube, 48 pore plate, 96 pore plate, solid LB plate, and 1.5mL and 2mL of EP tube for prokaryotic culture are all common domestic consumables.

Salmonella transformation plasmids involved in the electrotransformation process, competent preparation and transformation modes were as follows:

the preparation of the electrotransformation method competence comprises the following specific steps:

1) Culturing the bacterial streak on an antibiotic-free solid LB plate overnight to achieve the aim of activation;

4) Cooling the bacterial liquid on ice for 20min, centrifuging at 4500rpm for 7min at 4 ℃, and discarding the supernatant;

5) Adding ddH with the same volume as the bacterial liquid ₂ O, lightly blowing and mixing by a pipetting gun, centrifuging at 4 ℃ and 4500rpm for 7min, and discarding the supernatant;

6) Adding ddH 1/2 volume of the sum bacterial liquid ₂ O, lightly blowing and mixing by a pipetting gun, centrifuging at 4 ℃ and 4500rpm for 7min, and discarding the supernatant;

7) Adding 10% glycerol 1/2 volume of the bacterial liquid, gently stirring with a pipetting gun, centrifuging at 4deg.C and 4500rpm for 7min, and discarding supernatant;

8) Resuspension with 10ml 10% glycerol, packaging with 100 μl/tube, and storing at-80deg.C;

The electrotransformation method comprises the following specific steps:

2) The tuning parameters of the electrotometer are 2.5kV,25 mu F,400 omega and electric excitation.

4) After centrifugation at 6000rpm for 2min, a proper amount of supernatant was discarded, and the culture was performed overnight in a 37℃incubator with a heavy suspension plate LB solid plate.

Reporter gene LuxCDABE and OD ₆₀₀ The detection of (2) comprises the following specific steps:

200 mu L of bacterial culture solution is sucked into a 96-well plate, and then a light-emitting value is detected by using a Synergy H1 hybridization multimode enzyme-labeling instrument (BioTek Instruments) to read the expression quantity of LuxCDABE and measure the absorbance OD ₆₀₀ Detecting the bacterial growth density, and finallyLuminescence value/OD in LuxCDABE ₆₀₀ As a final measurement.

Concentration of S.enteritidis 3934. DELTA. XV secreted protein (active):

1) Centrifuging the bacterial culture solution at 8000rpm for 5min, and collecting the supernatant;

2) The supernatant was filtered using a 0.22 μm filter;

3) Placing the supernatant in an inner groove of a 10KD ultrafiltration column, and centrifuging at 6000rpm at 4 ℃ for 1h;

4) PBS was resuspended and centrifuged at 6000rpm at 4℃for 30min;

5) -80 ℃/liquid nitrogen preservation of the concentrated protein.

Extraction of the enteritidis 3934 Δxv secreted protein (inactive):

the organic reagents used in the extraction and purification of the secretion expression protein of S.enteritidis 3934 delta XV are all of analytical grade, such as chloroform and methanol. The method adopted is methanol and chloroform protein precipitation. The specific operation steps are as follows:

1) Taking 3mL of bacterial culture solution, centrifuging at 8000rpm for 5min, and sucking 2mL of supernatant;

2) Adding 8mL of methanol, and vortex shaking;

3) Adding 2mL of chloroform, and vortex shaking;

4) Add 6mL of ddH ₂ O, vortex oscillation;

5) Centrifuging at 14000 Xg for 5min, wherein the centrifuge tube presents 3 layers, protein exists in the middle layer, and carefully removing the uppermost water layer by using a pipette;

6) Adding 8mL of methanol, and vortex shaking;

7) Centrifuging at 14000 Xg for 5min, discarding supernatant, drying, and eluting protein with 30 μLPBS;

8) mu.L of 5X SDS Loading Buffer was added and incubated at 100℃for 5-10min.

Western blotting and related reagent configuration

Glycine, tris, SDS, PBS, tween, protein Marker and the like used in the experimental process are all purchased from Shanghai Biotechnology Inc., 5× SDS Loading Buffer, protein gel kit, primary antibody/secondary antibody diluent are purchased from Shanghai Yase biological medicine technology Inc., and primary antibody/secondary antibody is purchased from Shanghai Biotechnology (Shanghai) Inc.

Configuration of related reagents

The Western blotting specifically comprises the following steps:

1) Preparing a gel, namely 12.5% of a separating gel;

2) Electrophoresis, 80V 30min,120V 60min;

3) Transferring film, 0.22-0.25A,60min;

4) Sealing, incubating with skimmed milk powder for 1-2h, and cleaning with PBST for 3 times each for 10min;

5) Incubating the primary antibody at 4 ℃ overnight, and washing with PBST for 3 times for 10min each time;

6) Incubating the secondary antibody for 2-3h at 4 ℃ and cleaning the PBST for 3 times for 10min each time;

7) And (5) scanning film imaging by the instrument.

Cell culture

In the examples of the present invention, the following tumor cell lines are used as examples to illustrate that the prokaryotic far-red light regulated transcriptional activator has an inhibitory effect on tumor growth by the therapeutic protein secreted by Salmonella enteritidis 3934 DeltaXV expression, but does not limit the scope of the present invention.

The principle schematic diagram of the prokaryotic far-red light regulation and transcription activation device NITE/system and the transcription activation thereof are shown in figure 1, and the specific mechanism is as follows:

bacterial photosensitive IIGuanylate cyclase PadC4/PadC10 senses red activating self-activity with the aid of biliverdin BV produced by catalysis of heme oxidase BphO to convert GTP to a second messenger c-di-GMP. YhjH is a protease which can convert c-di-GMP into pGpGpG, and can degrade the c-di-GMP generated in the microorganism by expressing YhjH so as to reduce the background noise of the device in dark conditions. The transcriptional properties of the transcription factor MrkH activating gene are regulated by a second messenger c-di-GMP. MrkH consists of two domains: an N-terminal YcgR-N analog domain and a C-terminal pilZ domain. The PilZ domain is a classical c-di-GMP binding domain, while the function of the YcgR-N domain remains unclear. MrkH binds to the specific promoter P after high affinity for c-di-GMP _MrkA Transcriptional expression of downstream reporter genes was activated by recruiting RNA polymerase.

10cm cell culture dishes, 15mL and 50mL centrifuge tubes for cell culture were all purchased from U.S. Thermo Fisher Scientific (Labserv); modified Dulbecco's Modified Eagle's Medium (DMEM), RPMI-1640 medium, fetal bovine serum, penicillin and streptomycin solutions were used and were purchased from Gibico corporation, U.S.A.; cell incubator is purchased from us Thermo Fisher Scientific company; the rest of the consumables are common domestic consumables.

The tumor cells related in the invention comprise mouse colon cancer cells CT26-WT (product number C11875500 BT), and mouse B lymphoma cells A20 (donated by Guangzhou medical university) purchased from a cell bank of the Chinese sciences. Wherein CT26-WT and A20 cells are cultured in PRMI-1640 cell culture medium, and 10% (v/v) fetal bovine serum and 1% (v/v) penicillin and streptomycin solution are added into the culture medium; the cells were cultured at 37℃with 5% CO ₂ Is provided.

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Example 1

In this example, luxCDABE was used as a reporter gene to verify the efficiency of expression of different bacterial photosensitive diguanylate cyclase activating genes in a far-red light regulated transcription activation (NITE) device, but the scope of protection of the present invention is not limited. The method comprises the following specific steps:

In the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1;

and in the second step, conversion. pQL88 (P) _lac -PadC10-BphO-T _T7 )/pQL93(P _lac -PadC4-BphO- T _T7 )、pQL128(P _tac -YhjH-MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) Two plasmid combinations (100 ng each) were transformed into Salmonella enteritidis 3934 by electrotransformation (see methods materials for specific steps);

third, plating cultures and activates the monoclonal. The engineered Salmonella enteritidis 3934 coated plates contain LB solid plates with corresponding resistance, are placed in a dark condition at 37 ℃ for inversion overnight culture, and are selected and placed in a liquid LB culture medium for culture for 12 hours in the dark condition at 210rpm at 37 ℃ to serve as seed liquid;

fourth, inoculating. Seed solution was inoculated into 48-well plates (500. Mu.L per well) at an inoculation ratio of 1%, and was divided into a dark group and an light group, and cultured at 37℃under dark conditions at 150 rpm;

and fifthly, illuminating. Culturing for 3h, OD ₆₀₀ =0.3-0.5, placing the light group at 660nm wavelength and light intensity of 2mW/cm ² The LED of the (2) is illuminated for 7 hours, and the dark group is always cultured in dark condition;

sixth, the reporter gene is detected (see methods materials for specific steps).

The result shows that the prokaryotic far-red light regulation transcription activation device NITE has higher activation efficiency when red light sensitive bacteria photosensitive diguanylate cyclase PadC4 is selected. Experimental data are detailed in figure 2 of the accompanying description, all data are presented as n=3 independent replicates.

Example 2

In the embodiment, luxCDABE is used as a reporter gene to verify the activation efficiency of transcription and expression of different red light wavelength activated genes in a prokaryotic far-red light regulation transcription and activation (NITE) device, but the protection scope of the invention is not limited. The method comprises the following specific steps:

in the first step, plasmid construction. The plasmid construction in this example is detailed in Table 1.

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 )、pQL128(P _tac -RBS1-YhjH- MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) (100 ng each plasmid) was transformed into Salmonella enteritidis 3934 by electrotransformation (see methods materials for specific procedures);

third, plating cultures and activates the monoclonal. The engineered Salmonella enteritidis 3934 plates contained LB solid plates with corresponding resistances, were incubated overnight at 37℃in the dark, and the monoclonal was selected and incubated in liquid LB medium at 210rpm in the dark for 12h as seed solution;

fourth, inoculating. Seed solution was inoculated into 48 well plates (500. Mu.L per well) at an inoculation ratio of 1%, 4 plates (numbered 1, 2, 3, 4) were inoculated in total, and cultured at 37℃under dark conditions at 150 rpm;

and fifthly, illuminating. Culturing for 3h, OD ₆₀₀ The dark group of No. 1, no. 2 under the LED with the wavelength of 660nm, no. 3 under the LED with the wavelength of 710nm, no. 4 under the LED with the wavelength of 730nm, is always cultured in dark condition, and the light is illuminated for 7h;

The result shows that the prokaryotic far-red light regulated transcription activation device NITE has higher activation efficiency when irradiated by far-red light with the wavelength of 710 nm. Experimental data are detailed in figure 3 of the description, all data are presented as n=3 independent replicates.

Example 3

In this example, luxCDABE was used as a reporter gene to verify the effect of a prokaryotic far-red light regulated transcriptional activation (NITE) device on activating transcriptional expression of genes in Salmonella enteritidis 3934 (WT) and Salmonella enteritidis 3934 ΔXII (ΔXII) after knocking out genes that produce c-di-GMP, but without limiting the scope of protection of the present invention. The method comprises the following specific steps:

Second oneAnd (3) transforming. pQL93 (P) _lac -PadC4-BphO-T _T7 )、pQL128(P _tac -RBS1-YhjH- MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) (100 ng each of each plasmid) was transformed into wild type Salmonella enteritidis 3934 (WT) and knockout Salmonella enteritidis 3934 ΔXII (ΔXII) by electrotransformation (see methods materials for specific steps);

third, plating cultures and activates the monoclonal. The engineered Salmonella enteritidis 3934 (WT/DeltaXII) plates contained LB solid plates of the corresponding resistances, were incubated overnight at 37℃in the dark, and the monoclonal was selected and incubated in liquid LB medium at 37℃in the dark at 210rpm for 12h as seed solution;

and fifthly, illuminating. Culturing for 3h, OD ₆₀₀ =0.3-0.5, placing the light group at 710nm wavelength with light intensity of 2mW/cm ² The LED of the (2) is illuminated for 7 hours, and the dark group is always cultured in dark condition;

The result shows that the prokaryotic far-red light regulated transcription activation device NITE has lower background noise and higher activation efficiency in the DeltaXII. Experimental data are detailed in figure 4 of the accompanying description, all data are presented as n=3 independent replicates.

Example 4

In this example, luxCDABE is used as a reporter gene to verify the effect of the transcription and expression of different RBS activating genes of c-di-GMP degrading enzyme YhjH in a prokaryotic far-red light regulated transcription activation (NITE) device, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 ) Respectively with pQL (P) _tac -RBS1- YhjH-MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 )、pNX3(P _tac -RBS2-YhjH-MrkH-T _T7 ；P _MrkA - LuxCDABE-T _T7 )、pNX4(P _tac -RBS3-YhjH-MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 )、 pNX5(P _tac -RBS4-YhjH-MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 )、pNX6(P _tac -RBS5-YhjH- MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) (wherein 100ng of each plasmid was transformed) into Salmonella enteritidis 3934 ΔXII (see methods materials for specific procedures);

Third, plating cultures and activates the monoclonal. The engineered Salmonella enteritidis 3934 DeltaXII coated plate contains LB solid plates with corresponding resistance, and is cultured overnight at 37 ℃ in dark condition, and the monoclonal is selected and cultured in liquid LB culture medium at 37 ℃ in 210rpm in dark condition for 12 hours to be used as seed liquid;

The results show that the prokaryotic far-red light regulated transcriptional activator NITE, c-di-GMP degrading enzyme YhjH has higher activation efficiency when RBS4 is used. Experimental data are detailed in figure 5 of the accompanying description, all data are presented as n=3 independent replicates.

Example 5

In this example, luxCDABE is used as a reporter gene to verify the effect of illumination intensity in a prokaryotic far-red light regulated transcription activation (NITE) device on activation of gene transcription expression, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 )、pNX5(P _tac -RBS4-YhjH- MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) (wherein 100ng of each plasmid was transformed) into Salmonella enteritidis 3934 ΔXII (see methods materials for specific procedures);

fourth, inoculating. The activated seed solution was inoculated into 48 well plates (500. Mu.L per well) at an inoculation ratio of 1%, 9 plates (numbered 1, 2, 3, 4, 5, 6, 7, 8, 9) were inoculated in total, and cultured at 37℃under dark conditions at 150 rpm;

and fifthly, illuminating. Culturing for 3h, OD ₆₀₀ The dark group of No. 1, no. 1 was cultured in the dark, and the light intensities of No. 2 to No. 9 were 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5 mW/cm ² Illuminating for 7 hours under the 710nm LED;

The result shows that the expression of the prokaryotic far-red light regulated transcription activation device NITE activation reporter gene has good light intensity dependence. Experimental data are detailed in figure 6 of the accompanying description, all data are presented as n=3 independent replicates.

Example 6

fourth, inoculating. Seed solution was inoculated into 48 well plates (500. Mu.L per well) at an inoculation ratio of 1%, 9 plates (numbered 1, 2, 3, 4, 5, 6, 7, 8, 9) were inoculated in total, and cultured at 37℃under dark conditions at 150 rpm;

and fifthly, illuminating. Culturing for 3h, OD ₆₀₀ The dark group of No. 1, no. 2-9, no. 0.3-0.5, was cultured under dark conditions, and the light intensity was 2mW/cm at 710nm ² The LEDs of the culture medium are respectively illuminated for 0.5, 1, 2, 3, 4, 5, 6 and 7 hours, and the culture medium is placed under dark condition for continuous culture after illumination for corresponding time;

The result shows that the expression of the prokaryotic far-red light regulated transcription activation device NITE activation reporter gene has good illumination time dependence. Experimental data are detailed in figure 7 of the accompanying description, all data are presented as n=3 independent replicates.

Example 7

In the embodiment, luxCDABE is used as a reporter gene to verify the adjustability of the transcription expression of the activation gene of a prokaryotic far-red light regulation transcription activation (NITE) device, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 )、pNX5(P _tac -RBS4-YhjH- MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) (wherein 100ng of each plasmid was transformed) into Salmonella enteritidis 3934. DELTA.XII (specific)Step (2) a method material);

Fourth, inoculating. The activated seed solution was inoculated into a 48-well plate (500. Mu.L per well) at an inoculation ratio of 1%, 3 plates (numbered 1, 2, 3) were inoculated together, and cultured at 37℃under dark conditions at 150 rpm;

and fifthly, illuminating. Culturing for 3h, OD ₆₀₀ The dark group of No. 1 is always placed in dark condition for culture, no. 2 is placed in 710nm, light is applied for 2 hours, then placed in dark condition for culture, no. 2 is placed in 710nm, light is applied for 7 hours;

The result shows that the expression of the NITE activated reporter gene of the prokaryotic far-red light regulated transcriptional activation device is activated by irradiation of 710nm far-red light, and the expression capacity of the transcriptional activated reporter gene is lost when the 710nm far-red light is removed, so that the transcriptional activation device has good adjustability. Experimental data are detailed in figure 8 of the accompanying description, all data are presented as n=3 independent replicates.

Example 8

The embodiment uses OD ₆₀₀ As a detection basis, the phototoxicity of far-red light to the growth of prokaryotes is verified, but the protection scope of the invention is not limited. The method comprises the following specific steps:

in the first step, the monoclonal is activated. The Salmonella enteritidis 3934. DELTA.XII monoclonal was streaked on a non-resistant LB plate and cultured with 5mL of non-resistant liquid LB medium at 37℃for 12 hours at 210rpm as a seed solution.

And step two, inoculating. Inoculating the activated seed solution into a 48-well plate (500 mu L of each well) at an inoculation ratio of 1%, and culturing at 37 ℃ under the dark condition in an illumination group and a dark group;

and thirdly, illuminating. Culturing to OD ₆₀₀ After =0.3-0.5, the dark group was always cultured under dark conditions, and the light group was culturedPlacing at 710nm with illumination intensity of 2mW/cm ² Is illuminated under the LED;

fourth step, detecting OD ₆₀₀ . OD measurement at 1h intervals ₆₀₀ (specific steps see method materials);

the result shows that far-red light has no influence on the growth of prokaryotes and has the characteristics of no toxic or side effect. Experimental data are detailed in figure 9 of the accompanying description, all data are presented as n=3 independent replicates.

Example 9

In this embodiment, luxCDABE is used as a reporter gene to verify the activation effect of a prokaryotic far-red light regulated transcription activation (NITE) device in activating gene transcription to express different bacterial cells, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 )、pNX5(P _tac -RBS4-YhjH- MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) Co-transformation (where 100ng of each plasmid was transformed) into different bacteria, such as Salmonella enteritidis 3934ΔXII, E.coli Top10, E.coli BL21, E.coli JM109, salmonella typhimurium VNP20009 (see methods materials for specific steps);

Third, plating cultures and activates the monoclonal. The engineered bacteria coated plate contains LB solid plates with corresponding resistance, and is placed in a dark condition at 37 ℃ for overnight culture, and monoclonal is selected and placed in a liquid LB culture medium for culturing for 12 hours in the dark condition at 210rpm at 37 ℃ to be used as seed liquid;

fourth, inoculating. Inoculating the activated seed solution into a 48-well plate (500 mu L of each well) at an inoculation ratio of 1%, and culturing at 37 ℃ under dark conditions and 150rpm in a light group;

and fifthly, illuminating. Culturing to OD ₆₀₀ After=0.3-0.5, the dark group was always incubated in dark condition, the light group was incubated at 710nm, and the light intensity was 2mW/cm ² Is illuminated for 6 hours under the LED;

The result shows that the prokaryotic far-red light regulated transcription activation device NITE can activate transcription of the reporter gene in different prokaryotic cells, and has chassis cell universality. Experimental data are detailed in figure 10 of the accompanying description, all data are presented as n=3 independent replicates.

Example 10

In the embodiment, luxCDABE is taken as a reporter gene, and the fact that the transcription and expression of the prokaryotic far-red light regulation and transcription activation (NITE) device activation gene are spatially regulated and controlled by far-red light is verified, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 )、pNX5(P _tac -RBS4-YhjH- MrkH-T _T7 ；P _MrkA -LuxCDABE-T _T7 ) Co-transformation (where 100ng of each plasmid was transformed) into Salmonella enteritidis 3934ΔXII (see methods materials for specific procedures);

third, plating cultures and activates the monoclonal. The engineered Salmonella enteritidis 3934 DeltaXII coated plate contains LB solid plates with corresponding resistance, and is placed in a dark condition at 37 ℃ for overnight culture, and a monoclonal is selected and placed in a liquid LB culture medium, and is cultured for 12 hours under the dark condition at 210rpm at 37 ℃ to be used as seed liquid;

fourth, coating the plate. Uniformly coating the activated seeds in a solid LB plate, and culturing in an inverted mode under a dark condition;

and fifthly, illuminating. The plate was placed at 710nm with an illumination intensity of 2mW/cm ² Is 1 h under LED;

sixth, detecting the reporter gene. The expression of luciferase LuxCDABE in the plates was photographed using Clinx imaging equipment (Chemiscope 4300Pro, clinx, shanghai, china);

the result shows that the expression of the prokaryotic far-red light regulated transcription activation device NITE activation reporter gene has good space specificity. Experimental data are detailed in figure 11 of the accompanying specification, all data are presented as n=3 independent replicates.

Example 11

In the embodiment, azurin or ClyA is used as a reporter gene, and the effect of the prokaryotic far-red light regulation and transcription activation (NITE) device on killing tumor cells by secreting tumor therapeutic proteins Azurin and ClyA is verified by expressing in Salmonella Enteritidis DeltaXV, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 ) Respectively with pQL263 (P) _tac -RBS4- YhjH-MrkH-T _T7 ；P _MrkA -Azurin-T _T7 )/pQL274(P _tac -RBS4-YhjH-MrkH-T _T7 ；P _MrkA - ClyA-T _T7 ) (wherein 100ng of each plasmid was transformed) into Salmonella enteritidis 3934. DELTA. XV (see methods materials for specific procedures);

third, plating cultures and activates the monoclonal. The engineered Salmonella enteritidis 3934 DeltaXV coated plates contained LB solid plates with corresponding resistances, were incubated overnight at 37℃in the dark, and the monoclonal was selected and incubated in liquid LB medium at 37℃under 210rpm in the dark for 12h as seed solution;

and fifthly, illuminating. Culturing for 3h, OD ₆₀₀ The dark group is always cultured under dark condition, the light group is placed at 710nm, and the light intensity is 2mW/cm ² Is illuminated for 6 hours under the LED;

sixthly, concentrating the protein in the supernatant to ensure the activity of the protein (specific steps are shown in the method materials);

seventh, cells are seeded. CT26 cells are digested by pancreatin, inoculated into a 96-well plate and cultured for 16 hours;

eighth, incubating the concentrated protein with CT26 cells for 8 hours;

and ninth, detecting. Detecting death of tumor cells using a CCK-8 detection kit;

the result shows that the prokaryotic far-red light regulation transcription activation device has killing effect on CT26 colon cancer cells by secreted Azurin and ClyA under the induction of red light. Experimental data are detailed in figure 12 of the accompanying specification, all data are presented as n=3 independent replicates.

Example 12

In the embodiment, the Azurin and ClyA are used as reporter genes, and the prokaryotic far-red light regulation and transcription activation (NITE) device is verified to express and secrete tumor therapeutic proteins Azurin and ClyA in Salmonella enteritidis 3934 delta XV according to the irradiation time, but the protection scope of the invention is not limited. The method comprises the following specific steps:

And in the second step, conversion. Will pQL93 (P _lac -PadC4-BphO-T _T7 )、pNX178(P _tac -RBS4- YhjH-MrkH-T _T7 ；P _MrkA -Azurin-ClyA-T _T7 ) (wherein 100ng of each plasmid was transformed) Salmonella enteritidis 3934ΔXV (see methods materials for specific procedures);

sixth, extracting protein from the supernatant (specific steps see method materials);

seventh, western blotting (specific steps see methods materials);

eighth, detecting the reporter gene.

The result shows that the prokaryotic far-red light regulation transcription activation device has illumination intensity dependence on secretion of Azurin and ClyA under the induction of red light. Experimental data are detailed in figure 13 of the accompanying description, all data are presented as n=3 independent replicates.

Example 13

In this example, by constructing a tumor model of a CT26 colon cancer mouse, it was verified that the prokaryotic far-red light regulated transcription activation (NITE) device expressed and secreted tumor therapeutic proteins Azurin and ClyA in salmonella enteritidis 3934 Δxv had therapeutic effects on tumors, but did not limit the scope of protection of the present invention.

The method comprises the following specific steps:

in the first step, tumor is carried. 6 week female BALB/c mice, 10 subcutaneous injections in the back ⁶ CT26 cells. After about 9 days, the tumor volume grew to 100mm ³ -200 mm ³ 。

In the second step, plasmid construction. The plasmid construction in this example is detailed in Table 1.

And thirdly, converting. pQL93 (P) _lac -PadC4-BphO-T _T7 )、pNX178(P _tac -RBS4-YhjH- MrkH-T _T7 ；P _MrkA -Azurin-ClyA-T _T7 ) (wherein 100ng of each plasmid was transformed) into Salmonella enteritidis 3934. DELTA. XV (see methods materials for specific procedures);

fourth, plating and activating the monoclonal. Engineered Salmonella enteritidis 3934 DeltaXV (. DELTA.XV-NITE) _A+C ) The coated plate contains LB solid plates with corresponding resistance, and is placed in a dark condition at 37 ℃ for overnight culture, and a monoclonal is selected and placed in 5mL of liquid LB culture medium, and is cultured for 12 hours in the dark condition at 210rpm at 37 ℃ to be used as seed liquid;

fifth step, intratumoral injection engineering DeltaXV-NITE _A+C . Centrifuging the seed solution at 6000rpm for 5min, re-suspending with appropriate amount of sterile PBS, and adjusting final concentration to 10 ⁵ CFUs/. Mu.L, tumor was injected in situ by 50. Mu.L (5X 10) ⁶ CFUs), once every 3 days, three times in total;

and sixthly, illuminating. Grouping illuminationThe mice were placed at a wavelength of 710nm with an illumination intensity of 20 mW/cm, respectively ² Illuminating for 2 hours per day under the LED of (2), and raising other groups in a normal environment;

sixth, tumor volume and survival of mice were monitored. The length and width of the tumor were measured with a vernier caliper, and the volume calculation formula was (V _Tumor(s) =long×wide×wide/2).

The result shows that the prokaryotic far-red light regulated transcription activation device has a good tumor volume inhibition effect on treating CT26 colon cancer, and the survival rate is improved. Experimental data are detailed in figures 14-16 of the description, all of which are presented as n=6 independent replicates.

Example 14

In this example, by constructing a tumor model of a mice with a20B lymphoma, it was verified that the prokaryotic far-red light regulated transcription activation (NITE) device expressed and secreted nanobodies anti-PD-L1 nanobody and anti-CTLA4 nanobody in salmonella enteritidis 3934 Δv had therapeutic effects on tumors, but did not limit the scope of protection of the present invention. The method comprises the following specific steps:

in the first step, tumor is carried. Female BALB/c mice 6 weeks dorsally injected with 2.5X10 ⁶ A20 cells. After about 9 days, the tumor volume grew to 100mm ³ -200 mm ³ 。

In the second step, plasmid construction. The construction of the plasmids in this example is detailed in Table 1.

And thirdly, converting. pQL93 (P) _lac -PadC4-BphO-T _T7 )、pNX179(P _tac -RBS4-YhjH- MrkH-T _T7 ；P _MrkA -anti-PD-L1 nanobody-anti-CTLA4 nanobody-T _T7 ) (wherein 100ng of each plasmid was transformed) into Salmonella enteritidis 3934. DELTA. XV (see methods materials for specific procedures);

fourth, plating and activating the monoclonal. Engineered Salmonella enteritidis 3934 DeltaXV (. DELTA.XV-NITE) _P+C ) The coated plate contains LB solid plates with corresponding resistance, and is placed in a dark condition at 37 ℃ for overnight culture, and a monoclonal is selected and placed in 5mL of liquid LB culture medium, and is cultured for 12 hours in the dark condition at 210rpm at 37 ℃ to be used as seed liquid;

fifth stepIntratumoral injection engineering DeltaXV-NITE _P+C . Centrifuging the seed solution at 6000rpm for 5min, re-suspending with appropriate amount of sterile PBS, and adjusting final concentration to 10 ⁵ CFUs/. Mu.L, tumor was injected in situ by 50. Mu.L (5X 10) ⁶ CFUs), once every 3 days, three times in total;

and sixthly, illuminating. Placing mice in an illumination group at a wavelength of 710nm and illumination intensities of 20 mW/cm respectively ² Illuminating for 2 hours per day under the LED of (2), and raising other groups in a normal environment;

sixth, tumor volume, body weight and survival of mice were monitored. The length and width of the tumor were measured with a vernier caliper, and the volume calculation formula was (V _Tumor(s) =long×wide×wide/2).

The result shows that the prokaryotic far-red light regulated transcriptional activation device has a good tumor volume inhibition effect on treating A20B lymphoma, the weight of mice is not obviously changed, and the survival rate is improved. Experimental data are detailed in figures 17-20 of the description, all of which are presented as n=6 independent replicates.

Example 15

In the embodiment, by constructing a bilateral A20B lymphoma mouse tumor model, the prokaryotic far-red light regulation and transcription activation device is verified to express and secrete nano-antibody anti-PD-L1 nanobody and nano-antibody anti-CTLA4 nanobody in Salmonella enteritidis 3934 delta XV to generate a long-distance inhibition effect on tumor treatment, but the protection scope of the invention is not limited. The method comprises the following specific steps:

In the first step, tumor is carried. Female BALB/c mice of 6 weeks, injected 2.5X10 respectively on both sides ⁶ A20 cells. After about 12 days, the tumor volume grew to 100mm ³ -200mm ³ 。

And thirdly, converting. pQL93 (P) _lac -PadC4-BphO-T _T7 )、pNX179(P _tac -RBS4-YhjH- MrkH-T _T7 ；P _MrkA -anti-PD-L1 nanobody-anti-CTLA4 nanobody-T _T7 ) (wherein 100ng of each plasmid was transformed) into Salmonella enteritidis 3934. DELTA. XV (specific procedure)See method materials);

fifth step, intratumoral injection engineering DeltaXV-NITE _P+C . Centrifuging the seed solution at 6000rpm for 5min, re-suspending with appropriate amount of sterile PBS, and adjusting final concentration to 10 ⁵ CFUs/. Mu.L, 50. Mu.L (5X 10) ⁶ CFUs) on left tumor, once every 3 days, three total times;

sixth, the volumes of the tumors on both sides were monitored separately. The length and width of the tumor were measured with a vernier caliper, and the volume calculation formula was (V _Tumor(s) =long×wide×wide/2).

The result shows that the prokaryotic far-red light regulation transcription activation device has an inhibition effect on both tumors when used for treating double-sided A20B lymphomas. Experimental data are detailed in figures 21-23 of the description, all of which are presented as n=6 independent replicates.

TABLE 1 plasmid construction Table

SEQ ID NO.1: amino acid sequence of bacterial photosensitive diguanylate cyclase PadC4

MAADLGSDDISKLIAACDQEPIHIPNAIQPFGAMLIVEKDTQQIVYASANSAEY FSVADNTIHELSDIKQANINSLLPEHLISGLASAIRENEPIWVETDRLSFLGWRH ENYYIIEVERYHVQTSNWFEIQFQRAFQKLRNCKTHNDLINTLTRLIQEISGYD RVMIYQFDPEWNGRVIAESVRQLFTSMLNHHFPASDIPAQARAMYSINPIRIIPD VNAEPQPLHMIHKPQNTEAVNLSSGVLRAVSPLHMQYLRNFGVSASTSIGIFN EDELWGIVACHHTKPRAIGRRIRRLLVRTVEFAAERLWLIHSRNVERYMVTVQ AAREQLSTTADDKHSSHEIVIEHAADWCKLFRCDGIGYLRGEELTTYGETPDQ TTINKLVEWLEENGKKSLFWHSHMLKEDAPGLLPDGSRFAGLLAIPLKSDAD LFSYLLLFRVAQNEVRTWAGKPEKLSVETSTGTMLGPRKSFEAWQDEVSGKS QPWRTAQLYAARDIARDLLIVADSMQISQLNQRLERLATRDHLTGLWNRYRM EGAIEQEVAAAERYGRPCALVMFDIDHFKRFNDTWGHDAGDEVLVRIATTVS TQMRDTDLAGRWGGEEFLVLAANTDLEGAARLAERLRAAIAALEIRDYGHV TASFGVAVYREGDQSRDIVKRADLALYAAKEGGRNRVEVSAD

SEQ ID NO.2: amino acid sequence of bacterial photosensitive diguanylate cyclase PadC10

MDEPELVAALEACEREPIHIPNAIQPFGAMLIVEKDTQQIVYASANSAEYFSVA DNTIHELSDIKQANINSLLPEHLISGLASAIRENEPIWVETDRLSFLGWRHENY YIIEVERYHVQTSNWFEIQFQRAFQKLRNCKTHNDLINTLTRLIQEISGYDRVM IYQFDPEWNGRVIAESVRQLFTSMLNHHFPASDIPAQARAMYSINPIRIIPDVNA EPQPLHMIHKPQNTEAVNLSSGVLRAVSPLHMQYLRNFGVSASTSIGIFNEDEL WGIVACHHTKPRAIGRRIRRLLVRTVEFAAERLWLIHSRNVERYMVTVQAARE QLSTTADDKHSSHEIVIEHAADWCKLFRCDGIGYLRGEELTTYGETPDQTTIN KLVEWLEENGKKSLFWHSHMLKEDAPGLLPDGSRFAGLLAIPLKSDADLFSY LLLFRVAQAETRIWAGNPEKTIDRQSGRLSPRESFASWKETVSGKSQPWRTAQ LYAARDIARDLLIVADSMQLNLLNDQLADANENLEKLASFDDLTGIFNRRRME DRLESEVKEAQRYKKQFGILLFDLDKFKSVNDTYGHNIGDQILQNTCAAVSET LRDTDKFGRWGGEEFLIIAPQTGMPELMQLGERVRAAVEKMQHKDLPAVTISI GVAEFQNDTRWDHMIDRADKAMYRAKENGRNQVCSQ

SEQ ID NO.3: amino acid sequence of heme oxygenase BphO

MPLSRDLREKTGMLHNRAETLLGLPSGIMGWADYVDWLRHFLALYDPIERRI VAFGGWSGLASFDPDPGHSRRLIQDLHALGIDTDRIPRAPAEYCPPLTNFARAL GARYVLEGSALGGRVILHHLKKRIGDEIGNATAFFGGPSHGTATHWRAFQAAL DRFGAAHPDKRADVLAGAAATFTALLEWFTPFVAARRV

SEQ ID NO.4: amino acid sequence of c-di-GMP degrading enzyme YhjH

MIRQVIQRISNPEASIESLQERRFWLQCERAYTWQPIYQTCGRLMAVELLTVVT HPLNPSQRLPPDRYFTEITVSHRMEVVKEQIDLLAQKADFFIEHGLLASVNIDG PTLIALRQQPKILRQIERLPWLRFELVEHIRLPKDSTFASMCEFGPLWLDDFGTG MANFSALSEVRYDYIKIARELFVMLRQSPEGRTLFSQLLHLMNRYCRGVIVEG VETPEEWRDVQNSPAFAAQGWFLSRPAPIETLNTAVLAL

SEQ ID NO.5: amino acid sequence of transcription factor MrkH

MTEGTIKTSKYEIIAIFREELRKRTEIEIFFNNTSIITQLTRVDFAEFHIQTHRKIPS GHKIRFLLHSDSGKIEFNAALTKHDNSGVDKGIRYAFSLPECLQVVQRRRDPR FRLRHEHDFYCRGRHKNGENYLFDIKDISDGGCALMTKTPNLKFLSHNALLK NAVLMLAEYGEITIDLVVKNVIVITLDNANEESESYYQISCQFKFRHLDDQRRI EKILLDLILEAKRKKRI

SEQ ID NO.6: inducible promoter P _MrkA Nucleotide sequence of (2)

GCTGCGCTGTAAACAACCACCCTCGCGTTTTCATCTATCAATGGCTGTTTAT TAATAGTCGATGGTTATCTGTTATATAACTTAATGAAACGTGAACAAATGTAT ATTTGTCGGCGAATAAATAGCATTCTTTGACGCCGATAGCACCAG

SEQ ID NO.7: nucleotide sequence of LuxCDABE gene cluster

ATGGCTAATATGACTAAAAAAATTTCATTCATTATTAACGGCCAGGTTGAAA TTTTTCCCGAAAGTGATGATTTAGTGCAATCCATTAATTTTGGTGATAATAGT GTTTACCTGCCAATATTGAATAATTCTCATGTAAAAAACATTATTGATTATAAT GAAAATAATAAATTACGGTTGCATAATATTGTCAATTTTCTCTATACGGTAGG GCAAAGATGGAAAAATGAAGAATATTCAAGACGCAGGACATACATTCGTGA TTTAAAAAAATATATGGGATATTCAGAAGCAATGGCCAAGTTAGAGGCCAA CTGGATATCTATGATTTTATGTTCTAAAGGTGGCCTTTATGATGTTGTAGAAA ATGAACTTGGTTCTCGCCATATCATGGATGAATGGCTACCTCAGGATGAAAG TTATATTAAGGCTTTTCCGAAAGGTAAGTCTATACATCTGTTGGCAGGTAAT GTTCCATTATCTGTGATCATGTCTATATTACGCGCAATTTTAACCAAGAATCA GTGTATTATAAAAACATCGTCAACCGATCCCTTTACCGCTAATGCATTAGCG TTAAGCTTTATCGATGTAGACCCTAATCATCCGATAACGCGCTCTTTGTCTGT TGTATATTGGCCACACCAAGGTGATACATCACTCGCAAAAGAAATTATGCA ACATATGGATGTTATTGTCGCTTGGGGAGGGGAAGATGCGATTAATTGGGCT GTAGAACATGCACCACCCTATGCTGACGTGATTAAATTTGGCTCTAAAAAG AGTTTTTGCATTATTGATAATCCAGTTGATTTAACGTCAGCAGCTACCGGTG CGGCTCATGATATTTGTTTTTACGATCAGCGCGCTTGTTTTTCTGCCCAAAA CATATATTACATGGGAAATCAGTATGAGGAATTTAAGTTAGCGTTGATAGAA AAACTTAATCTATATGCGCATATATTACCAAACGCCAAAAAAGATTTTGATG AAAAGGCGGCCTATTCTTTAGTCCAAAAAGAGAGCTTATTTGCTGGATTAA AAGTAGAGGTGGATGTTCATCAACGTTGGATGATTATTGAGTCAAATGCGG GTGTGGAATTTAATCAACCACTTGGCAGATGTGTGTATCTTCATCACGTCGA TAATATTGAGCAAGTATTGCCTTATGTTCAAAAAAATAAGACACAAACCATA TCTATTTTTCCTTGGGAATCCGCATTTAAGTATCGAGATGCGTTGGCATTAAG AGGTGCGGAAAGGATTGTAGAAGCAGGAATGAATAATATATTTCGAGTTGG TGGATCTCATGACGGAATGAGGCCGTTACAACGATTAGTGACATATATTTCT CATGAGAGGCCATCTCATTATACTGCTAAGGATGTTGCGGTTGAAATAGAAC AGACTCGATTCCTGGAAGAAGATAAGTTCCTTGTATTTGTCCCGTAATAGGT AAAAAGTATGGAAAATAAATCCAAATATAAAACCATCGACCATGTTCTTTGT GTTGAAGGAAATAAAAAAATTCATGTTTGGGAAACGCTGCCAGAAGAAAC CAGCCCAAAGAGAAAGAATCCCATTATTATTGCGTCGGGTTTTGCCCGAAG GATGGATCATTTTGCTGGTTTAGCGGAATATTTATCGCGGAATGGGTTTCATG TGATTCGCTATGATTCACTTCACCACGTTGGGTTGAGTTCAGGGACAATTGA TGAATTTACAATGTCTATAGGAAAACAGAGCCTATTAGCCGTGGTTGATTGG TTAAATACACGAAAAATAAATAACCGTGGTATTTTGGCTTCAAGCTTATCTG CACGGATAGTTTATGCAAGTCTATCTGAAATTAATGTTTCATTTTTAATCACC GCAGTCGGTGTTGTTAACTTAAGATATACGCTTGAAAGAGCTTTAGGATTTG ATTATCTCAGTTTACCCATTAATGAATTGCCGAATAATTTGGATTTTGAAGGC CATAAATTGGGTGCTGAAGTCTTTGCGAGAGATTGCCTTGATTTTGGCTGG GAAGATTTAACTTCTACAATCAATAGCATGATGTATCTTGATATACCGTTTAT TGCTTTTACTGCAAATAACGACAATTGGGTAAAGCAAGATGAAGTTATCAC ATTGTTATCAAATATTCGTAGTAATCGATGCAAGATACATTCTTTGTTAGGAA GTTCGCATGACTTGTGTGTTTTCTTAGTGGTCCTGCGCAATTTTTATCAATCG GTTACGAAGGCTGCTATCGCGATGGATAATGATCGTCTGGATATTGATGTTG ATATTATTGAACCATCATTCGAACATCTAACTATTGCGACAGTCAATGAACG TCGAATGAAAATTGAGATTGAAAATCAAGCGATTTCGCTGTCTTAAAACCT ATTGGGATAGATATTACCCTATAGATTTCAAGATGGATCGCGACGGCAAGGG AGCGAATCCCGGGAGCATAGCAAACTATGTGACCGGGGTGAGTGAGTGCA GCCAACAAAGAAGCAACTTGAAAGATAACGGGTATAGTTAATTCTATCACT CAAATATAAGGGCTCTCTATGAAATTTGGAAACTTTTTGCTTACATACCAAC CCCCCCAATTTTCTCAAACAGAAGTAATGAAACGTTTGGTTAAATTAGGTC GTATTTCTGAGGAGTGTGGTTTTGATACTGTATGGTTACTGGAGCATCATTT CACGGAGTTTGGTTTGCTTGGTAACCCTTATGTCGCTGCTGCATATTTACTT GGTGCAACCAAAAAATTGAATGTAGGGACTGCGGCTATTGTTCTTCCCACC GCTCATCCAGTGCGCCAACTTGAAGATGTGAATTTATTGGATCAAATGTCAA AAGGACGATTTCGGTTTGGTATTTGTCGGGGGCTTTACAATAAAGACTTTCG CGTATTTGGCACGGATATGAATAACAGTCGCGCTTTAACGGAGTGCTGGTAC GGGTTGATAAAAAATGGCATGACAGAGGGATATATGGAAGCTGATAATGAA CATATCAAGTTCCATAAGGTAAAAGTAAACCCGACAGCATATAGTAAAGGT GGAGCCCCTGTTTATGTGGTTGCTGAATCAGCCTCGACAACTGAATGGGCC GCTCAATTTGGTTTACCGATGATATTAAGTTGGATTATAAATACTAACGAAAA GAAAGCACAGCTTGAGCTTTATAACGAGGTGGCTCAAGAATATGGGCACGA TATTCATAATATCGACCATTGCTTATCATATATAACATCTGTAAATTATGACTC AAATAAAGCGAAAGAGATTTGTCGGAAATTTCTAGGGCATTGGTATGATTCT TATGTGAATGCCACGACCATTTTTGATGATTCAGACAAAACAAGAGGTTAT GATTTCAATAAAGGGCAGTGGCGTGACTTTGTATTAAAGGGACATAGAGAT ACTAATCGCCGCATTGATTACAGTTACGAAATCAATCCCGTGGGAACCCCG CAGGAATGCATTGACATAATTCAAAAAGACATTGATGCCACGGGAATATCA AATATCTGTTGTGGGTTTGAAGCGAATGGAACAGTAGACGAAATTATTGCTT CCATGAAGCTCTTCCAGTCTGATGTCATGCCGTTTCTTAAAGAAAAACAAC GTTCGCTATTATAGTAGCTAAGGAAAAAGAAATGAAATTTGGATTGTTCTTC CTTAACTTCATCAATTCAACAACTGTTCAAGAACAAAGTATAGTTCGCATGC AGGAAATAACGGAGTATGTTGATAAGTTGAATTTTGAACAGATTTTGGTGTA TGAAAATCATTTTTCAGGTAATGGTGTTGTCGGTGCTCCTCTGACTGTTTCT GGTTTTTTGCTCGGTTTAACAGAAAAAATTAAAATTGGCTCATTGAATCACA TCATTACAACTCATCATCCTGTCCGAATAGCGGAGGAGGCTTGCTTACTGGA TCAATTAAGCGAAGGGAGATTTATTTTAGGGTTTAGTGATTGTGAAAAAAA AGATGAAATGCGTCTTTTTAATCGCCCTGTTGAATATCAACAGCAACTATTT GAAGAGTGTTATGAAATCATTAACGATGCTTTAACAACAGGCTATTGTAATC CCGATAATGATTTTTATAGTTTCCCTAAAATATCGGTAAACCCCCACGCTTAT ACCCAAGGCGGGCCTCGGAGATATATTACAGCAACCAGTCATCATATTGTTG AATGGGCGGCTAAAAAAGGCATTCCTCTCATCTTTAAGTGGGATGACTCCA ATGATGTTAGATATGAATATGCTGAAAGGTATAAAGCCGTTGCTGATAAATAT GGCATTGACTTATCAGCGATAGATCATCAGTTAATGGTATTGGTTAACTATAA CGAAGATAGTCACAAAGCTAAACAAGAGACGCGTGCATTTATCCGTGATTA TGTTCTTGAAATGTATCCTAATGAAAATCTCGAAAATAAACTTGAAGAGATA ATCACAGAAAACGCTGTCGGAGATTATACGGAATGTATAGCTGCGGCTAAG CTGGCAATTGAAAAGTGCGGTGCAAAAAGGGTATTATTATCCTTTGAACCA ATGAATGACTTGATGCACCAAAAAAATGTAATCAATATTGTTGATGATAATAT TAAAAAGTACCACATGTAGTAAAAGAATATGGCAGCAACGCTGCCATATTC TCTAAATTATTTGGAGGGGTAAAACAGGTATGACTTCATATGTTGATAAACA AGAGATCATAGCAAGCTCAGAAATTGATGATTTGATTTTTTCCAGCGATCCA TTAGCTTGGTCTTACGATGAACAGGAAAAAATCAGAAACAAATTTGTTCTT GATGCATTTCGTAATCACTATAAACATTGTCAAGAATACCGTCACTACTGTC AGGTACACAAAGTAGACGACAATATTACGGAAATTGATGACATACCTGTATT CCCAACATCAGTTTTTAAGTTTACTCGCTTATTAACTTCTCAGGAGAACGAG ATTGAAAGTTGGTTTACCAGCAGCGGCACGAGTGGTTTAAAAAGTCAGGT GGCGCGTGACAGACTAAGTATTGAGAGACTCTTAGGCTCTGTGAGTTATGG CATGAAATATGTTGGTAGTTGGTTTGATCATCAAATAGAGTTGGTCAACTTA GGGCCAGATAGATTTAATGCTCATAACATTTGGTTTAAATATGTTATTAGTTT GGTAGAATTATTATATCCCACGACATTTACCGTAATGGAAGAACGAATAGAT TTTGTTAAGACATTGAATAGCCTTGAGCGAATAAAAAATCAAGGGAAAGAT ATTTGTCTTATCGGCTCACCATACTTTATTTATTTGCTCTGCCAGTATATGAAA GATAAAAACATCTCATTTTATGGGGATAAAAACCTTTATATCATAACGGGGG GCGGCTGGAAAAGTTATGAAAAAGAGTCCCTAAAACGCGATGATTTCAATC ATCTTTTATTCGACACGTTCAACCTCAATAATATTAGTCAAATCCGCGATATA TTTAATCAAGTTGAACTCAACACTTGTTTCTTTGAGGATGAAATGCAACGT AAACGTGTTCCGCCGTGGGTATATGCGCGAGCACTTGATCCTGAAACATTG AAACCTGTACCTGATGGAATGCCGGGTTTGATGAGTTATATGGATGCGTCAT CAACGAGTTATCCGGCATTTATTGTTACCGATGATGTCGGGATAATGAGCAG AGAATATGGTCAATATCCTGGTGTACTTGTTGAGATTTTACGTCGCGTCAAT ACGAGGGCACAGAAAGGGTGTGCTTTAAGCTTAAACCAAGCATTTAATAGT TGA

SEQ ID NO.8: RBS1 nucleotide sequence of c-di-GMP degrading enzyme YhjH

AATAATTTTGTTTAACTTTAAGAAGGAGATATACC

SEQ ID NO.9: RBS2 nucleotide sequence of c-di-GMP degrading enzyme YhjH

AAGGAGATATACATAAA

SEQ ID NO.10: RBS3 nucleotide sequence of c-di-GMP degrading enzyme YhjH

TCTAGAGAAAGAGGAGAAATACTAG

SEQ ID NO.11: RBS4 nucleotide sequence of c-di-GMP degrading enzyme YhjH

GCCTACGTAACACAAATAAGGAAGGGATT

SEQ ID NO.12: RBS5 nucleotide sequence of c-di-GMP degrading enzyme YhjH

CAGCCGCTAGGCCGAGATTCAGGAAAGTAAA

SEQ ID NO.13: nucleotide sequence of Ptac promoter

TGACAATTAATCATCGGCTCGTATAATGT

SEQ ID NO.14: nucleotide sequence of Plac promoter

TTTACACTTTATGCTTCCGGCTCGTATGTTG

SEQ ID NO.15: amino acid sequence of tumor therapeutic protein Azurin

MLRKLAAVSLLSLLSAPLLAAECSVDIQGNDQMQFNTNAITVDKSCKQFTVN

LSHPGNLPKNVMGHNWVLSTAADMQGVVTDGMASGLDKDYLKPDDSRVIA

HTKLIGSGEKDSVTFDVSKLKEGEQYMFFCTFPGHSALMKGTLTLK

SEQ ID NO.16: amino acid sequence of tumor therapeutic protein ClyA

MTEIVADKTVEVVKNAIETADGALDLYNKYLDQVIPWQTFDETIKELSRFKQE YSQAASVLVGDIKTLLMDSQDKYFEATQTVYEWCGVATQLLAAYILLFDEYN EKKASAQKDILIKVLDDGITKLNEAQKSLLVSSQSFNNASGKLLALDSQLTND FSEKSSYFQSQVDKIRREAYAGAAAGVVAGPFGLIISYSIAAAVVEGKLIPELKN KLKSVQNFFTTLSNTVKQANKDIDAAKLKLTTEIAAIGEIKTETETTRFYVDYD DLMLSLLKEAAKKMINTCNEYQKRHGKKTLFEVPEV

SEQ ID NO.17: amino acid sequence of nano antibody anti-PD-L1 nanobody

MKKLLPTAAAGLLLLAAQPAQAMMAQVQLVETGGGLVQPGGSLRLSCTASG FTFSMHAMTWYRQAPGKQRELVAVITSHGDRANYTDSVRGRFTISRDNTKNM VYLQMNSLKPEDTAVYYCNVPRYDSWGQGTQVTVSSGGLPETGG

SEQ ID NO.18: amino acid sequence of nano antibody anti-CTLA4 nanobody

MQVQLQESGGGSVQAGGSLRLSCTASGFGVDGTDMGWYRQAPGNECELVSS ISSIGIGYYSESVKGRFTISRDNAKNTVYLQMNSLRPDDTAVYYCGRRWIGYR CGNWGRGTQVTVSS

SEQ ID NO.19: amino acid sequence of tumor therapeutic protein secretion signal peptide yopE

MKISSFISTSLPLPT

SEQ ID NO.20: amino acid sequence of 3×flag tag

DYKDHDGDYKDHDIDYKDDDDK

SEQ ID NO.21: amino acid sequence of HA tag

YPYDVPDYA

SEQ ID NO.22: amino acid sequence of His tag

HHHHHH.

SEQUENCE LISTING

<110> university of east China

<120> a prokaryotic far-red light regulation transcription activation device, its construction method and application in tumor treatment

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 678

<212> PRT

<213> bacterial photosensitive diguanylate cyclase PadC4

<400> 1

Met Ala Ala Asp Leu Gly Ser Asp Asp Ile Ser Lys Leu Ile Ala Ala

1 5 10 15

Cys Asp Gln Glu Pro Ile His Ile Pro Asn Ala Ile Gln Pro Phe Gly

20 25 30

Ala Met Leu Ile Val Glu Lys Asp Thr Gln Gln Ile Val Tyr Ala Ser

35 40 45

Ala Asn Ser Ala Glu Tyr Phe Ser Val Ala Asp Asn Thr Ile His Glu

50 55 60

Leu Ser Asp Ile Lys Gln Ala Asn Ile Asn Ser Leu Leu Pro Glu His

65 70 75 80

Leu Ile Ser Gly Leu Ala Ser Ala Ile Arg Glu Asn Glu Pro Ile Trp

85 90 95

Val Glu Thr Asp Arg Leu Ser Phe Leu Gly Trp Arg His Glu Asn Tyr

100 105 110

Tyr Ile Ile Glu Val Glu Arg Tyr His Val Gln Thr Ser Asn Trp Phe

115 120 125

Glu Ile Gln Phe Gln Arg Ala Phe Gln Lys Leu Arg Asn Cys Lys Thr

130 135 140

His Asn Asp Leu Ile Asn Thr Leu Thr Arg Leu Ile Gln Glu Ile Ser

145 150 155 160

Gly Tyr Asp Arg Val Met Ile Tyr Gln Phe Asp Pro Glu Trp Asn Gly

165 170 175

Arg Val Ile Ala Glu Ser Val Arg Gln Leu Phe Thr Ser Met Leu Asn

180 185 190

His His Phe Pro Ala Ser Asp Ile Pro Ala Gln Ala Arg Ala Met Tyr

195 200 205

Ser Ile Asn Pro Ile Arg Ile Ile Pro Asp Val Asn Ala Glu Pro Gln

210 215 220

Pro Leu His Met Ile His Lys Pro Gln Asn Thr Glu Ala Val Asn Leu

225 230 235 240

Ser Ser Gly Val Leu Arg Ala Val Ser Pro Leu His Met Gln Tyr Leu

245 250 255

Arg Asn Phe Gly Val Ser Ala Ser Thr Ser Ile Gly Ile Phe Asn Glu

260 265 270

Asp Glu Leu Trp Gly Ile Val Ala Cys His His Thr Lys Pro Arg Ala

275 280 285

Ile Gly Arg Arg Ile Arg Arg Leu Leu Val Arg Thr Val Glu Phe Ala

290 295 300

Ala Glu Arg Leu Trp Leu Ile His Ser Arg Asn Val Glu Arg Tyr Met

305 310 315 320

Val Thr Val Gln Ala Ala Arg Glu Gln Leu Ser Thr Thr Ala Asp Asp

325 330 335

Lys His Ser Ser His Glu Ile Val Ile Glu His Ala Ala Asp Trp Cys

340 345 350

Lys Leu Phe Arg Cys Asp Gly Ile Gly Tyr Leu Arg Gly Glu Glu Leu

355 360 365

Thr Thr Tyr Gly Glu Thr Pro Asp Gln Thr Thr Ile Asn Lys Leu Val

370 375 380

Glu Trp Leu Glu Glu Asn Gly Lys Lys Ser Leu Phe Trp His Ser His

385 390 395 400

Met Leu Lys Glu Asp Ala Pro Gly Leu Leu Pro Asp Gly Ser Arg Phe

405 410 415

Ala Gly Leu Leu Ala Ile Pro Leu Lys Ser Asp Ala Asp Leu Phe Ser

420 425 430

Tyr Leu Leu Leu Phe Arg Val Ala Gln Asn Glu Val Arg Thr Trp Ala

435 440 445

Gly Lys Pro Glu Lys Leu Ser Val Glu Thr Ser Thr Gly Thr Met Leu

450 455 460

Gly Pro Arg Lys Ser Phe Glu Ala Trp Gln Asp Glu Val Ser Gly Lys

465 470 475 480

Ser Gln Pro Trp Arg Thr Ala Gln Leu Tyr Ala Ala Arg Asp Ile Ala

485 490 495

Arg Asp Leu Leu Ile Val Ala Asp Ser Met Gln Ile Ser Gln Leu Asn

500 505 510

Gln Arg Leu Glu Arg Leu Ala Thr Arg Asp His Leu Thr Gly Leu Trp

515 520 525

Asn Arg Tyr Arg Met Glu Gly Ala Ile Glu Gln Glu Val Ala Ala Ala

530 535 540

Glu Arg Tyr Gly Arg Pro Cys Ala Leu Val Met Phe Asp Ile Asp His

545 550 555 560

Phe Lys Arg Phe Asn Asp Thr Trp Gly His Asp Ala Gly Asp Glu Val

565 570 575

Leu Val Arg Ile Ala Thr Thr Val Ser Thr Gln Met Arg Asp Thr Asp

580 585 590

Leu Ala Gly Arg Trp Gly Gly Glu Glu Phe Leu Val Leu Ala Ala Asn

595 600 605

Thr Asp Leu Glu Gly Ala Ala Arg Leu Ala Glu Arg Leu Arg Ala Ala

610 615 620

Ile Ala Ala Leu Glu Ile Arg Asp Tyr Gly His Val Thr Ala Ser Phe

625 630 635 640

Gly Val Ala Val Tyr Arg Glu Gly Asp Gln Ser Arg Asp Ile Val Lys

645 650 655

Arg Ala Asp Leu Ala Leu Tyr Ala Ala Lys Glu Gly Gly Arg Asn Arg

660 665 670

Val Glu Val Ser Ala Asp

675

<210> 2

<211> 677

<212> PRT

<213> bacterial photosensitive diguanylate cyclase PadC10

<400> 2

Met Asp Glu Pro Glu Leu Val Ala Ala Leu Glu Ala Cys Glu Arg Glu

1 5 10 15

Pro Ile His Ile Pro Asn Ala Ile Gln Pro Phe Gly Ala Met Leu Ile

20 25 30

Val Glu Lys Asp Thr Gln Gln Ile Val Tyr Ala Ser Ala Asn Ser Ala

35 40 45

Glu Tyr Phe Ser Val Ala Asp Asn Thr Ile His Glu Leu Ser Asp Ile

50 55 60

Lys Gln Ala Asn Ile Asn Ser Leu Leu Pro Glu His Leu Ile Ser Gly

65 70 75 80

Leu Ala Ser Ala Ile Arg Glu Asn Glu Pro Ile Trp Val Glu Thr Asp

85 90 95

Arg Leu Ser Phe Leu Gly Trp Arg His Glu Asn Tyr Tyr Ile Ile Glu

100 105 110

Val Glu Arg Tyr His Val Gln Thr Ser Asn Trp Phe Glu Ile Gln Phe

115 120 125

Gln Arg Ala Phe Gln Lys Leu Arg Asn Cys Lys Thr His Asn Asp Leu

130 135 140

Ile Asn Thr Leu Thr Arg Leu Ile Gln Glu Ile Ser Gly Tyr Asp Arg

145 150 155 160

Val Met Ile Tyr Gln Phe Asp Pro Glu Trp Asn Gly Arg Val Ile Ala

165 170 175

Glu Ser Val Arg Gln Leu Phe Thr Ser Met Leu Asn His His Phe Pro

180 185 190

Ala Ser Asp Ile Pro Ala Gln Ala Arg Ala Met Tyr Ser Ile Asn Pro

195 200 205

Ile Arg Ile Ile Pro Asp Val Asn Ala Glu Pro Gln Pro Leu His Met

210 215 220

Ile His Lys Pro Gln Asn Thr Glu Ala Val Asn Leu Ser Ser Gly Val

225 230 235 240

Leu Arg Ala Val Ser Pro Leu His Met Gln Tyr Leu Arg Asn Phe Gly

245 250 255

Val Ser Ala Ser Thr Ser Ile Gly Ile Phe Asn Glu Asp Glu Leu Trp

260 265 270

Gly Ile Val Ala Cys His His Thr Lys Pro Arg Ala Ile Gly Arg Arg

275 280 285

Ile Arg Arg Leu Leu Val Arg Thr Val Glu Phe Ala Ala Glu Arg Leu

290 295 300

Trp Leu Ile His Ser Arg Asn Val Glu Arg Tyr Met Val Thr Val Gln

305 310 315 320

Ala Ala Arg Glu Gln Leu Ser Thr Thr Ala Asp Asp Lys His Ser Ser

325 330 335

His Glu Ile Val Ile Glu His Ala Ala Asp Trp Cys Lys Leu Phe Arg

340 345 350

Cys Asp Gly Ile Gly Tyr Leu Arg Gly Glu Glu Leu Thr Thr Tyr Gly

355 360 365

Glu Thr Pro Asp Gln Thr Thr Ile Asn Lys Leu Val Glu Trp Leu Glu

370 375 380

Glu Asn Gly Lys Lys Ser Leu Phe Trp His Ser His Met Leu Lys Glu

385 390 395 400

Asp Ala Pro Gly Leu Leu Pro Asp Gly Ser Arg Phe Ala Gly Leu Leu

405 410 415

Ala Ile Pro Leu Lys Ser Asp Ala Asp Leu Phe Ser Tyr Leu Leu Leu

420 425 430

Phe Arg Val Ala Gln Ala Glu Thr Arg Ile Trp Ala Gly Asn Pro Glu

435 440 445

Lys Thr Ile Asp Arg Gln Ser Gly Arg Leu Ser Pro Arg Glu Ser Phe

450 455 460

Ala Ser Trp Lys Glu Thr Val Ser Gly Lys Ser Gln Pro Trp Arg Thr

465 470 475 480

Ala Gln Leu Tyr Ala Ala Arg Asp Ile Ala Arg Asp Leu Leu Ile Val

485 490 495

Ala Asp Ser Met Gln Leu Asn Leu Leu Asn Asp Gln Leu Ala Asp Ala

500 505 510

Asn Glu Asn Leu Glu Lys Leu Ala Ser Phe Asp Asp Leu Thr Gly Ile

515 520 525

Phe Asn Arg Arg Arg Met Glu Asp Arg Leu Glu Ser Glu Val Lys Glu

530 535 540

Ala Gln Arg Tyr Lys Lys Gln Phe Gly Ile Leu Leu Phe Asp Leu Asp

545 550 555 560

Lys Phe Lys Ser Val Asn Asp Thr Tyr Gly His Asn Ile Gly Asp Gln

565 570 575

Ile Leu Gln Asn Thr Cys Ala Ala Val Ser Glu Thr Leu Arg Asp Thr

580 585 590

Asp Lys Phe Gly Arg Trp Gly Gly Glu Glu Phe Leu Ile Ile Ala Pro

595 600 605

Gln Thr Gly Met Pro Glu Leu Met Gln Leu Gly Glu Arg Val Arg Ala

610 615 620

Ala Val Glu Lys Met Gln His Lys Asp Leu Pro Ala Val Thr Ile Ser

625 630 635 640

Ile Gly Val Ala Glu Phe Gln Asn Asp Thr Arg Trp Asp His Met Ile

645 650 655

Asp Arg Ala Asp Lys Ala Met Tyr Arg Ala Lys Glu Asn Gly Arg Asn

660 665 670

Gln Val Cys Ser Gln

675

<210> 3

<211> 197

<212> PRT

<213> heme oxygenase BphO

<400> 3

Met Pro Leu Ser Arg Asp Leu Arg Glu Lys Thr Gly Met Leu His Asn

1 5 10 15

Arg Ala Glu Thr Leu Leu Gly Leu Pro Ser Gly Ile Met Gly Trp Ala

20 25 30

Asp Tyr Val Asp Trp Leu Arg His Phe Leu Ala Leu Tyr Asp Pro Ile

35 40 45

Glu Arg Arg Ile Val Ala Phe Gly Gly Trp Ser Gly Leu Ala Ser Phe

50 55 60

Asp Pro Asp Pro Gly His Ser Arg Arg Leu Ile Gln Asp Leu His Ala

65 70 75 80

Leu Gly Ile Asp Thr Asp Arg Ile Pro Arg Ala Pro Ala Glu Tyr Cys

85 90 95

Pro Pro Leu Thr Asn Phe Ala Arg Ala Leu Gly Ala Arg Tyr Val Leu

100 105 110

Glu Gly Ser Ala Leu Gly Gly Arg Val Ile Leu His His Leu Lys Lys

115 120 125

Arg Ile Gly Asp Glu Ile Gly Asn Ala Thr Ala Phe Phe Gly Gly Pro

130 135 140

Ser His Gly Thr Ala Thr His Trp Arg Ala Phe Gln Ala Ala Leu Asp

145 150 155 160

Arg Phe Gly Ala Ala His Pro Asp Lys Arg Ala Asp Val Leu Ala Gly

165 170 175

Ala Ala Ala Thr Phe Thr Ala Leu Leu Glu Trp Phe Thr Pro Phe Val

180 185 190

Ala Ala Arg Arg Val

195

<210> 4

<211> 255

<212> PRT

<213> c-di-GMP degrading enzyme YhjH

<400> 4

Met Ile Arg Gln Val Ile Gln Arg Ile Ser Asn Pro Glu Ala Ser Ile

1 5 10 15

Glu Ser Leu Gln Glu Arg Arg Phe Trp Leu Gln Cys Glu Arg Ala Tyr

20 25 30

Thr Trp Gln Pro Ile Tyr Gln Thr Cys Gly Arg Leu Met Ala Val Glu

35 40 45

Leu Leu Thr Val Val Thr His Pro Leu Asn Pro Ser Gln Arg Leu Pro

50 55 60

Pro Asp Arg Tyr Phe Thr Glu Ile Thr Val Ser His Arg Met Glu Val

65 70 75 80

Val Lys Glu Gln Ile Asp Leu Leu Ala Gln Lys Ala Asp Phe Phe Ile

85 90 95

Glu His Gly Leu Leu Ala Ser Val Asn Ile Asp Gly Pro Thr Leu Ile

100 105 110

Ala Leu Arg Gln Gln Pro Lys Ile Leu Arg Gln Ile Glu Arg Leu Pro

115 120 125

Trp Leu Arg Phe Glu Leu Val Glu His Ile Arg Leu Pro Lys Asp Ser

130 135 140

Thr Phe Ala Ser Met Cys Glu Phe Gly Pro Leu Trp Leu Asp Asp Phe

145 150 155 160

Gly Thr Gly Met Ala Asn Phe Ser Ala Leu Ser Glu Val Arg Tyr Asp

165 170 175

Tyr Ile Lys Ile Ala Arg Glu Leu Phe Val Met Leu Arg Gln Ser Pro

180 185 190

Glu Gly Arg Thr Leu Phe Ser Gln Leu Leu His Leu Met Asn Arg Tyr

195 200 205

Cys Arg Gly Val Ile Val Glu Gly Val Glu Thr Pro Glu Glu Trp Arg

210 215 220

Asp Val Gln Asn Ser Pro Ala Phe Ala Ala Gln Gly Trp Phe Leu Ser

225 230 235 240

Arg Pro Ala Pro Ile Glu Thr Leu Asn Thr Ala Val Leu Ala Leu

245 250 255

<210> 5

<211> 234

<212> PRT

<213> transcription factor MrkH

<400> 5

Met Thr Glu Gly Thr Ile Lys Thr Ser Lys Tyr Glu Ile Ile Ala Ile

1 5 10 15

Phe Arg Glu Glu Leu Arg Lys Arg Thr Glu Ile Glu Ile Phe Phe Asn

20 25 30

Asn Thr Ser Ile Ile Thr Gln Leu Thr Arg Val Asp Phe Ala Glu Phe

35 40 45

His Ile Gln Thr His Arg Lys Ile Pro Ser Gly His Lys Ile Arg Phe

50 55 60

Leu Leu His Ser Asp Ser Gly Lys Ile Glu Phe Asn Ala Ala Leu Thr

65 70 75 80

Lys His Asp Asn Ser Gly Val Asp Lys Gly Ile Arg Tyr Ala Phe Ser

85 90 95

Leu Pro Glu Cys Leu Gln Val Val Gln Arg Arg Arg Asp Pro Arg Phe

100 105 110

Arg Leu Arg His Glu His Asp Phe Tyr Cys Arg Gly Arg His Lys Asn

115 120 125

Gly Glu Asn Tyr Leu Phe Asp Ile Lys Asp Ile Ser Asp Gly Gly Cys

130 135 140

Ala Leu Met Thr Lys Thr Pro Asn Leu Lys Phe Leu Ser His Asn Ala

145 150 155 160

Leu Leu Lys Asn Ala Val Leu Met Leu Ala Glu Tyr Gly Glu Ile Thr

165 170 175

Ile Asp Leu Val Val Lys Asn Val Ile Val Ile Thr Leu Asp Asn Ala

180 185 190

Asn Glu Glu Ser Glu Ser Tyr Tyr Gln Ile Ser Cys Gln Phe Lys Phe

195 200 205

Arg His Leu Asp Asp Gln Arg Arg Ile Glu Lys Ile Leu Leu Asp Leu

210 215 220

Ile Leu Glu Ala Lys Arg Lys Lys Arg Ile

225 230

<210> 6

<211> 150

<212> DNA

<213> inducible promoter PMrkA

<400> 6

gctgcgctgt aaacaaccac cctcgcgttt tcatctatca atggctgttt attaatagtc 60

gatggttatc tgttatataa cttaatgaaa cgtgaacaaa tgtatatttg tcggcgaata 120

aatagcattc tttgacgccg atagcaccag 150

<210> 7

<211> 5810

<212> DNA

<213> LuxCDABE Gene Cluster

<400> 7

atggctaata tgactaaaaa aatttcattc attattaacg gccaggttga aatttttccc 60

gaaagtgatg atttagtgca atccattaat tttggtgata atagtgttta cctgccaata 120

ttgaataatt ctcatgtaaa aaacattatt gattataatg aaaataataa attacggttg 180

cataatattg tcaattttct ctatacggta gggcaaagat ggaaaaatga agaatattca 240

agacgcagga catacattcg tgatttaaaa aaatatatgg gatattcaga agcaatggcc 300

aagttagagg ccaactggat atctatgatt ttatgttcta aaggtggcct ttatgatgtt 360

gtagaaaatg aacttggttc tcgccatatc atggatgaat ggctacctca ggatgaaagt 420

tatattaagg cttttccgaa aggtaagtct atacatctgt tggcaggtaa tgttccatta 480

tctgtgatca tgtctatatt acgcgcaatt ttaaccaaga atcagtgtat tataaaaaca 540

tcgtcaaccg atccctttac cgctaatgca ttagcgttaa gctttatcga tgtagaccct 600

aatcatccga taacgcgctc tttgtctgtt gtatattggc cacaccaagg tgatacatca 660

ctcgcaaaag aaattatgca acatatggat gttattgtcg cttggggagg ggaagatgcg 720

attaattggg ctgtagaaca tgcaccaccc tatgctgacg tgattaaatt tggctctaaa 780

aagagttttt gcattattga taatccagtt gatttaacgt cagcagctac cggtgcggct 840

catgatattt gtttttacga tcagcgcgct tgtttttctg cccaaaacat atattacatg 900

ggaaatcagt atgaggaatt taagttagcg ttgatagaaa aacttaatct atatgcgcat 960

atattaccaa acgccaaaaa agattttgat gaaaaggcgg cctattcttt agtccaaaaa 1020

gagagcttat ttgctggatt aaaagtagag gtggatgttc atcaacgttg gatgattatt 1080

gagtcaaatg cgggtgtgga atttaatcaa ccacttggca gatgtgtgta tcttcatcac 1140

gtcgataata ttgagcaagt attgccttat gttcaaaaaa ataagacaca aaccatatct 1200

atttttcctt gggaatccgc atttaagtat cgagatgcgt tggcattaag aggtgcggaa 1260

aggattgtag aagcaggaat gaataatata tttcgagttg gtggatctca tgacggaatg 1320

aggccgttac aacgattagt gacatatatt tctcatgaga ggccatctca ttatactgct 1380

aaggatgttg cggttgaaat agaacagact cgattcctgg aagaagataa gttccttgta 1440

tttgtcccgt aataggtaaa aagtatggaa aataaatcca aatataaaac catcgaccat 1500

gttctttgtg ttgaaggaaa taaaaaaatt catgtttggg aaacgctgcc agaagaaacc 1560

agcccaaaga gaaagaatcc cattattatt gcgtcgggtt ttgcccgaag gatggatcat 1620

tttgctggtt tagcggaata tttatcgcgg aatgggtttc atgtgattcg ctatgattca 1680

cttcaccacg ttgggttgag ttcagggaca attgatgaat ttacaatgtc tataggaaaa 1740

cagagcctat tagccgtggt tgattggtta aatacacgaa aaataaataa ccgtggtatt 1800

ttggcttcaa gcttatctgc acggatagtt tatgcaagtc tatctgaaat taatgtttca 1860

tttttaatca ccgcagtcgg tgttgttaac ttaagatata cgcttgaaag agctttagga 1920

tttgattatc tcagtttacc cattaatgaa ttgccgaata atttggattt tgaaggccat 1980

aaattgggtg ctgaagtctt tgcgagagat tgccttgatt ttggctggga agatttaact 2040

tctacaatca atagcatgat gtatcttgat ataccgttta ttgcttttac tgcaaataac 2100

gacaattggg taaagcaaga tgaagttatc acattgttat caaatattcg tagtaatcga 2160

tgcaagatac attctttgtt aggaagttcg catgacttgt gtgttttctt agtggtcctg 2220

cgcaattttt atcaatcggt tacgaaggct gctatcgcga tggataatga tcgtctggat 2280

attgatgttg atattattga accatcattc gaacatctaa ctattgcgac agtcaatgaa 2340

cgtcgaatga aaattgagat tgaaaatcaa gcgatttcgc tgtcttaaaa cctattggga 2400

tagatattac cctatagatt tcaagatgga tcgcgacggc aagggagcga atcccgggag 2460

catagcaaac tatgtgaccg gggtgagtga gtgcagccaa caaagaagca acttgaaaga 2520

taacgggtat agttaattct atcactcaaa tataagggct ctctatgaaa tttggaaact 2580

ttttgcttac ataccaaccc ccccaatttt ctcaaacaga agtaatgaaa cgtttggtta 2640

aattaggtcg tatttctgag gagtgtggtt ttgatactgt atggttactg gagcatcatt 2700

tcacggagtt tggtttgctt ggtaaccctt atgtcgctgc tgcatattta cttggtgcaa 2760

ccaaaaaatt gaatgtaggg actgcggcta ttgttcttcc caccgctcat ccagtgcgcc 2820

aacttgaaga tgtgaattta ttggatcaaa tgtcaaaagg acgatttcgg tttggtattt 2880

gtcgggggct ttacaataaa gactttcgcg tatttggcac ggatatgaat aacagtcgcg 2940

ctttaacgga gtgctggtac gggttgataa aaaatggcat gacagaggga tatatggaag 3000

ctgataatga acatatcaag ttccataagg taaaagtaaa cccgacagca tatagtaaag 3060

gtggagcccc tgtttatgtg gttgctgaat cagcctcgac aactgaatgg gccgctcaat 3120

ttggtttacc gatgatatta agttggatta taaatactaa cgaaaagaaa gcacagcttg 3180

agctttataa cgaggtggct caagaatatg ggcacgatat tcataatatc gaccattgct 3240

tatcatatat aacatctgta aattatgact caaataaagc gaaagagatt tgtcggaaat 3300

ttctagggca ttggtatgat tcttatgtga atgccacgac catttttgat gattcagaca 3360

aaacaagagg ttatgatttc aataaagggc agtggcgtga ctttgtatta aagggacata 3420

gagatactaa tcgccgcatt gattacagtt acgaaatcaa tcccgtggga accccgcagg 3480

aatgcattga cataattcaa aaagacattg atgccacggg aatatcaaat atctgttgtg 3540

ggtttgaagc gaatggaaca gtagacgaaa ttattgcttc catgaagctc ttccagtctg 3600

atgtcatgcc gtttcttaaa gaaaaacaac gttcgctatt atagtagcta aggaaaaaga 3660

aatgaaattt ggattgttct tccttaactt catcaattca acaactgttc aagaacaaag 3720

tatagttcgc atgcaggaaa taacggagta tgttgataag ttgaattttg aacagatttt 3780

ggtgtatgaa aatcattttt caggtaatgg tgttgtcggt gctcctctga ctgtttctgg 3840

ttttttgctc ggtttaacag aaaaaattaa aattggctca ttgaatcaca tcattacaac 3900

tcatcatcct gtccgaatag cggaggaggc ttgcttactg gatcaattaa gcgaagggag 3960

atttatttta gggtttagtg attgtgaaaa aaaagatgaa atgcgtcttt ttaatcgccc 4020

tgttgaatat caacagcaac tatttgaaga gtgttatgaa atcattaacg atgctttaac 4080

aacaggctat tgtaatcccg ataatgattt ttatagtttc cctaaaatat cggtaaaccc 4140

ccacgcttat acccaaggcg ggcctcggag atatattaca gcaaccagtc atcatattgt 4200

tgaatgggcg gctaaaaaag gcattcctct catctttaag tgggatgact ccaatgatgt 4260

tagatatgaa tatgctgaaa ggtataaagc cgttgctgat aaatatggca ttgacttatc 4320

agcgatagat catcagttaa tggtattggt taactataac gaagatagtc acaaagctaa 4380

acaagagacg cgtgcattta tccgtgatta tgttcttgaa atgtatccta atgaaaatct 4440

cgaaaataaa cttgaagaga taatcacaga aaacgctgtc ggagattata cggaatgtat 4500

agctgcggct aagctggcaa ttgaaaagtg cggtgcaaaa agggtattat tatcctttga 4560

accaatgaat gacttgatgc accaaaaaaa tgtaatcaat attgttgatg ataatattaa 4620

aaagtaccac atgtagtaaa agaatatggc agcaacgctg ccatattctc taaattattt 4680

ggaggggtaa aacaggtatg acttcatatg ttgataaaca agagatcata gcaagctcag 4740

aaattgatga tttgattttt tccagcgatc cattagcttg gtcttacgat gaacaggaaa 4800

aaatcagaaa caaatttgtt cttgatgcat ttcgtaatca ctataaacat tgtcaagaat 4860

accgtcacta ctgtcaggta cacaaagtag acgacaatat tacggaaatt gatgacatac 4920

ctgtattccc aacatcagtt tttaagttta ctcgcttatt aacttctcag gagaacgaga 4980

ttgaaagttg gtttaccagc agcggcacga gtggtttaaa aagtcaggtg gcgcgtgaca 5040

gactaagtat tgagagactc ttaggctctg tgagttatgg catgaaatat gttggtagtt 5100

ggtttgatca tcaaatagag ttggtcaact tagggccaga tagatttaat gctcataaca 5160

tttggtttaa atatgttatt agtttggtag aattattata tcccacgaca tttaccgtaa 5220

tggaagaacg aatagatttt gttaagacat tgaatagcct tgagcgaata aaaaatcaag 5280

ggaaagatat ttgtcttatc ggctcaccat actttattta tttgctctgc cagtatatga 5340

aagataaaaa catctcattt tatggggata aaaaccttta tatcataacg gggggcggct 5400

ggaaaagtta tgaaaaagag tccctaaaac gcgatgattt caatcatctt ttattcgaca 5460

cgttcaacct caataatatt agtcaaatcc gcgatatatt taatcaagtt gaactcaaca 5520

cttgtttctt tgaggatgaa atgcaacgta aacgtgttcc gccgtgggta tatgcgcgag 5580

cacttgatcc tgaaacattg aaacctgtac ctgatggaat gccgggtttg atgagttata 5640

tggatgcgtc atcaacgagt tatccggcat ttattgttac cgatgatgtc gggataatga 5700

gcagagaata tggtcaatat cctggtgtac ttgttgagat tttacgtcgc gtcaatacga 5760

gggcacagaa agggtgtgct ttaagcttaa accaagcatt taatagttga 5810

<210> 8

<211> 35

<212> DNA

<213> RBS1 of c-di-GMP degrading enzyme YhjH

<400> 8

aataattttg tttaacttta agaaggagat atacc 35

<210> 9

<211> 17

<212> DNA

<213> RBS2 of c-di-GMP degrading enzyme YhjH

<400> 9

aaggagatat acataaa 17

<210> 10

<211> 25

<212> DNA

<213> RBS3 of c-di-GMP degrading enzyme YhjH

<400> 10

tctagagaaa gaggagaaat actag 25

<210> 11

<211> 29

<212> DNA

<213> RBS4 of c-di-GMP degrading enzyme YhjH

<400> 11

gcctacgtaa cacaaataag gaagggatt 29

<210> 12

<211> 31

<212> DNA

<213> RBS5 of c-di-GMP degrading enzyme YhjH

<400> 12

cagccgctag gccgagattc aggaaagtaa a 31

<210> 13

<211> 29

<212> DNA

<213> Ptac promoter

<400> 13

tgacaattaa tcatcggctc gtataatgt 29

<210> 14

<211> 31

<212> DNA

<213> Plac promoter

<400> 14

tttacacttt atgcttccgg ctcgtatgtt g 31

<210> 15

<211> 148

<212> PRT

<213> tumor therapeutic protein Azurin

<400> 15

Met Leu Arg Lys Leu Ala Ala Val Ser Leu Leu Ser Leu Leu Ser Ala

1 5 10 15

Pro Leu Leu Ala Ala Glu Cys Ser Val Asp Ile Gln Gly Asn Asp Gln

20 25 30

Met Gln Phe Asn Thr Asn Ala Ile Thr Val Asp Lys Ser Cys Lys Gln

35 40 45

Phe Thr Val Asn Leu Ser His Pro Gly Asn Leu Pro Lys Asn Val Met

50 55 60

Gly His Asn Trp Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val

65 70 75 80

Thr Asp Gly Met Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp

85 90 95

Asp Ser Arg Val Ile Ala His Thr Lys Leu Ile Gly Ser Gly Glu Lys

100 105 110

Asp Ser Val Thr Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln Tyr

115 120 125

Met Phe Phe Cys Thr Phe Pro Gly His Ser Ala Leu Met Lys Gly Thr

130 135 140

Leu Thr Leu Lys

145

<210> 16

<211> 303

<212> PRT

<213> tumor therapeutic protein ClyA

<400> 16

Met Thr Glu Ile Val Ala Asp Lys Thr Val Glu Val Val Lys Asn Ala

1 5 10 15

Ile Glu Thr Ala Asp Gly Ala Leu Asp Leu Tyr Asn Lys Tyr Leu Asp

20 25 30

Gln Val Ile Pro Trp Gln Thr Phe Asp Glu Thr Ile Lys Glu Leu Ser

35 40 45

Arg Phe Lys Gln Glu Tyr Ser Gln Ala Ala Ser Val Leu Val Gly Asp

50 55 60

Ile Lys Thr Leu Leu Met Asp Ser Gln Asp Lys Tyr Phe Glu Ala Thr

65 70 75 80

Gln Thr Val Tyr Glu Trp Cys Gly Val Ala Thr Gln Leu Leu Ala Ala

85 90 95

Tyr Ile Leu Leu Phe Asp Glu Tyr Asn Glu Lys Lys Ala Ser Ala Gln

100 105 110

Lys Asp Ile Leu Ile Lys Val Leu Asp Asp Gly Ile Thr Lys Leu Asn

115 120 125

Glu Ala Gln Lys Ser Leu Leu Val Ser Ser Gln Ser Phe Asn Asn Ala

130 135 140

Ser Gly Lys Leu Leu Ala Leu Asp Ser Gln Leu Thr Asn Asp Phe Ser

145 150 155 160

Glu Lys Ser Ser Tyr Phe Gln Ser Gln Val Asp Lys Ile Arg Arg Glu

165 170 175

Ala Tyr Ala Gly Ala Ala Ala Gly Val Val Ala Gly Pro Phe Gly Leu

180 185 190

Ile Ile Ser Tyr Ser Ile Ala Ala Ala Val Val Glu Gly Lys Leu Ile

195 200 205

Pro Glu Leu Lys Asn Lys Leu Lys Ser Val Gln Asn Phe Phe Thr Thr

210 215 220

Leu Ser Asn Thr Val Lys Gln Ala Asn Lys Asp Ile Asp Ala Ala Lys

225 230 235 240

Leu Lys Leu Thr Thr Glu Ile Ala Ala Ile Gly Glu Ile Lys Thr Glu

245 250 255

Thr Glu Thr Thr Arg Phe Tyr Val Asp Tyr Asp Asp Leu Met Leu Ser

260 265 270

Leu Leu Lys Glu Ala Ala Lys Lys Met Ile Asn Thr Cys Asn Glu Tyr

275 280 285

Gln Lys Arg His Gly Lys Lys Thr Leu Phe Glu Val Pro Glu Val

290 295 300

<210> 17

<211> 147

<212> PRT

<213> nanobody anti-PD-L1 nanobody

<400> 17

Met Lys Lys Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Gln Ala Met Met Ala Gln Val Gln Leu Val Glu Thr

20 25 30

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr

35 40 45

Ala Ser Gly Phe Thr Phe Ser Met His Ala Met Thr Trp Tyr Arg Gln

50 55 60

Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Val Ile Thr Ser His Gly

65 70 75 80

Asp Arg Ala Asn Tyr Thr Asp Ser Val Arg Gly Arg Phe Thr Ile Ser

85 90 95

Arg Asp Asn Thr Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Lys

100 105 110

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Pro Arg Tyr Asp Ser

115 120 125

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Leu Pro Glu

130 135 140

Thr Gly Gly

145

<210> 18

<211> 118

<212> PRT

<213> nanobody anti-CTLA4 nanobody

<400> 18

Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly

1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Gly Val Asp Gly

20 25 30

Thr Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Glu Cys Glu Leu

35 40 45

Val Ser Ser Ile Ser Ser Ile Gly Ile Gly Tyr Tyr Ser Glu Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Gly Arg Arg Trp Ile Gly Tyr Arg Cys Gly Asn Trp Gly Arg Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 19

<211> 15

<212> PRT

<213> tumor therapeutic protein secretion Signal peptide yopE

<400> 19

Met Lys Ile Ser Ser Phe Ile Ser Thr Ser Leu Pro Leu Pro Thr

1 5 10 15

<210> 20

<211> 22

<212> PRT

<213> 3×flag tag

<400> 20

Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr

1 5 10 15

Lys Asp Asp Asp Asp Lys

20

<210> 21

<211> 9

<212> PRT

<213> HA tag

<400> 21

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

1 5

<210> 22

<211> 6

<212> PRT

<213> His tag

<400> 22

His His His His His His

1 5

Claims

1. A prokaryotic far-red light regulated transcriptional activation device, the device comprising: a far-red light sensing element, a gene transcription activating element and a far-red light response element.

2. The prokaryotic far-red light-regulated transcriptional activation device according to claim 1, wherein the far-red light-sensing element comprises bacterial photosensitive diguanylate cyclase PadC4, heme oxygenase BphO, and a degradation enzyme YhjH of cyclodiguanylate c-di-GMP.

3. The prokaryotic far-red light-regulated transcriptional activation device according to claim 2, wherein the amino acid sequence of PadC4 is shown in SEQ ID No. 1; the amino acid sequence of BphO is shown as SEQ ID NO. 3; the amino acid sequence of YhjH is shown as SEQ ID NO. 4.

4. The prokaryotic far-red light regulation transcription activation device according to claim 1, wherein the gene transcription activation element comprises a transcription activation factor MrkH, and the amino acid sequence of the gene transcription activation element is shown as SEQ ID No. 5.

5. The prokaryotic far-red light regulated transcriptional activation device according to claim 1, wherein the far-red light response element comprises an inducible promoter P _MrkA And a downstream reporter gene, wherein the inducible promoter P _MrkA The nucleotide sequence of the polypeptide is shown as SEQ ID NO. 6; the downstream reporter gene is the gene sequence of any protein of interest.

6. The prokaryotic far-red light regulation and transcription activation device according to claim 5, wherein the reporter gene comprises luciferase LuxCDABE, the nucleotide sequence of which is shown in SEQ ID No.7, tumor therapeutic protein Azurin, the amino acid sequence of which is shown in SEQ ID No.15, tumor therapeutic protein cytolysin a, the amino acid sequence of which is shown in SEQ ID No.16, tumor therapeutic protein nanobody anti-PD-L1 nanobody, the amino acid sequence of which is shown in SEQ ID No.17, and tumor therapeutic protein anti-CTLA4 nanobody, the amino acid sequence of which is shown in SEQ ID No. 18.

7. The prokaryotic far-red light regulation and transcription activation device according to claim 6, wherein the secretion signal yopE fused at the N-terminus of the tumor therapeutic protein HAs an amino acid sequence shown in SEQ ID No.19, and the tag proteins 3 xflag, HA and 6 xHis fused at the C-terminus of the tumor therapeutic protein have amino acid sequences shown in SEQ ID nos. 20-22, respectively.

8. The prokaryotic far-red light-regulated transcriptional activator of claim 6, wherein when said transcription factor MrkH induces expression of two of said tumor therapeutic proteins, said two tumor therapeutic proteins are expressed on the same vector, split by a ribosome binding site, RBS.

9. A prokaryotic vector, an engineered prokaryote, a kit, or a system comprising a prokaryote far-red light-regulated transcriptional activation device of any one of claims 1-8.

10. Use of a prokaryotic far-red light regulated transcriptional activation device according to any one of claims 1-8, a prokaryotic vector according to claim 9, an engineered prokaryote, a kit or a system for the preparation of a product for the treatment of tumors.

11. The use according to claim 10, wherein the product is useful for bacterial therapy in the treatment of tumors by using tumor-treating proteins, including Azurin and cytolysin a ClyA, nanobodies anti-PD-L1 nanobody and nanobodies anti-CTLA4 nanobody as reporter genes.

12. The method for constructing a prokaryotic far-red light regulated transcriptional activator according to claim 1, wherein a far-red light sensing element, a gene transcriptional activator and a far-red light effector are constructed on corresponding vectors by genetic engineering technology, wherein BphO and PadC4 in the far-red light sensing element are constructed on pGN62 plasmid vectors, and the c-di-GMP degrading enzyme YhjH, the gene transcriptional activator MrkH and the far-red light effector P _MrkA And the reporter gene is constructed on a pYC214 plasmid vector.

13. The construction method of the prokaryotic far-red light regulation transcription activation device is characterized by comprising the following steps:

(1) Constructing a far-red light sensing element, wherein the far-red light sensing element comprises a bacterial photosensitive diguanylate cyclase PadC4, heme oxygenase BphO and a degradation enzyme YhjH of cyclodiguanylate c-di-GMP;

(2) Constructing a gene transcription activation element comprising a transcription activator MrkH;

(3) Constructing a far-red light response element comprising an inducible promoter P _MrkA And a downstream reporter gene.

14. The method of claim 13, wherein in step (1), the bacterial photosensitive diguanylate cyclase converts guanosine triphosphate GTP into c-di-GMP with cyclodiguanylate phosphate under the induction of far-red light with the aid of biliverdin BV; the heme oxygenase BphO may produce BV pigment molecules that activate bacterial photosensitive diguanylate cyclase; the YhjH degrades native c-di-GMP within bacterial cells to reduce background noise generated under dark conditions.

15. The method of claim 13, wherein in step (2), mrkH is a transcriptional activator that binds and activates a specific promoter P by binding to c-di-GMP with high affinity _MrkA Transcriptional expression of downstream genes.

16. The method of claim 13, wherein in step (3), the P _MrkA An inducible promoter specifically bound to MrkH, recruiting RNA polymerase by the transcription factor MrkH, activating expression of downstream genes; the reporter gene is the gene sequence of any protein of interest.

17. The method of claim 13, wherein the PadC4, yhjH, and BphO are defined as in claim 2; and/or, the MrkH is defined as set forth in claim 4; and/or, the inducible promoter P _MrkA And the reporter gene are defined as in claims 5-7.

18. A prokaryotic far-red light regulated transcriptional activator device constructed by the method of any one of claims 13-17.

19. A method for regulating gene expression in a prokaryote using a prokaryote far-red light-regulated transcriptional activation device according to any one of claims 1-8, said method comprising the steps of:

a) Constructing the prokaryotic far-red light-regulated transcriptional activation device of any one of claims 1-8 in a prokaryotic plasmid expression vector;

b) Transforming said prokaryotic cell with an expression vector;

20. A method of using a prokaryotic far-red light regulated gene transcription activation device in tumor treatment, the method comprising:

b) Preparing an engineering Salmonella enteritidis 3934DeltaXV (DeltaXV-NITE) of a prokaryotic far-red light regulation transcription activation device for activating the expression of tumor drug proteins;

c) Tumor in situ injection of DeltaXV-NITE;