Programmable protein rapid secretion and release system and its construction method and application

Abstract

The present invention discloses a program-controllable protein rapid secretion and release PASS system, including a protein rapid secretion and release system regulated by clinically approved drugs, a protein rapid secretion and release system regulated by blue light, and a protein rapid secretion and release system that recognizes protein antigen signals. The PASS system of the present invention has the ability to respond to a variety of inducers and achieve rapid release of a variety of user-defined proteins by directly regulating protein secretion rather than inducing protein transcriptional expression. The PASS system of the present invention can quickly and efficiently regulate the secretion and release of target proteins, and has the characteristics of orthogonality, non-toxicity, high efficiency, low background leakage, and rapid induction of protein rapid secretion and release, providing an artificially controllable protein drug rapid, transient protein rapid delivery technology platform for basic research, genetic engineering, and regenerative medicine, and has great potential application value for treating diseases caused by insufficient protein secretion or damage.

Description

Technical Field

[0001] The present invention relates to multidisciplinary cross-fields such as synthetic biology and optogenetics, and specifically to a method for constructing a mammalian cell clinically approved drug, protein antigen, and blue light-induced protein rapid release system, as well as its application in mammalian genetic engineering and disease treatment. Background Art

[0002] The expression and secretion of proteins are involved in regulating various life activities of mammals, including cell proliferation, differentiation, execution of correct physiological functions, and growth and normal development of mammalian organisms. Impaired protein expression and secretion processes are often accompanied by the occurrence of a variety of diseases. High-risk metabolic syndromes that seriously affect human health, such as diabetes, hyperlipidemia, and hyperuricemia, are all related to insufficient protein secretion or impaired secretion processes. Secretory proteins are usually delivered by injection as therapeutic drugs, and it is difficult to reach drug therapeutic window concentrations or provide suitable pharmacokinetics. Gene therapy and cell therapy are common methods for delivering therapeutic secretory proteins. Protein drugs can be expressed stably and for a long time in gene-transfected somatic cells or externally transplanted cells. However, safe and effective gene and cell therapy require precise regulation of protein drug expression on demand, and continuous overexpression of protein drugs is not conducive to safe and effective treatment. In order to achieve controllable gene therapy, some antibiotic-regulated transgenic expression systems based on gene transcription levels have been designed and developed. The most typical tetracycline and rapamycin-regulated gene expression switches are used to regulate the expression of various therapeutic proteins. However, a common feature of this type of regulatory system is that protein induced expression and attenuation occur on a time scale of days, and protein expression release cannot be regulated on a time scale of minutes or hours.

[0003] With the development of synthetic biology, more and more gene circuits designed and assembled based on synthetic biology ideas have been developed and used for precise and controllable gene therapy and cell therapy. In particular, artificial customized cells loaded with synthetic gene circuits can be customized according to actual needs, perceive user-defined input signals, identify and process the signals, and then output corresponding protein therapeutic drugs, thereby achieving precise treatment of diseases. However, the expression and release of existing artificial customized cell drugs are mostly based on the induced expression and regulation of proteins. There is a delay in the analysis, processing, and output of external signals, and the release rate of induced proteins is slow. Usually, after being stimulated by the induced signal, such customized cells will express and release a certain amount of protein after 4-6 hours, and reach the peak of induced expression in 24-48 hours. This controllable switch system based on transcriptional regulation level takes a long time to regulate protein expression and release, so it is slow to take effect in disease treatment. However, for many diseases such as asthma, allergic reactions, acute myocardial infarction, angina pectoris, cerebral hemorrhage, epilepsy, hypertension, etc., it is crucial to quickly release therapeutic drugs for timely treatment. Therefore, how to design and construct a precise and controllable protein drug rapid release control system, respond to external signals in a timely manner and quickly release therapeutic proteins within minutes to achieve rapid on-demand drug delivery is a major challenge facing the field of gene therapy and cell therapy.

[0004] Precise regulation of protein secretion is of great significance for disease treatment. Under normal physiological conditions, cells often respond, analyze and process external signals very quickly. A variety of bioactive substances such as proteases, hormones, killer proteins and neurotransmitters are usually stored in cells after transcription and translation. When stimulated by external stimuli, they are quickly secreted outside the cells and play specific biological functions. For example, increased blood sugar stimulates pancreatic cells to rapidly release insulin stored in the cells, achieving the effect of lowering blood sugar in a short period of time; atrial natriuretic peptide (ANP) stored in myocardial cells is rapidly released when atrial pressure increases, thereby maintaining blood pressure homeostasis; in addition, immune cells recognize and respond to tumor cells very quickly. When immune cells (NK cells or T cells) recognize tumors, perforins and granzymes stored in cytotoxic granules are rapidly released to induce tumor cell apoptosis.

[0005] Directly regulating protein secretion, rather than indirectly achieving rapid protein release through gene transcription and translation, is the key to solving the problem of delayed regulatory protein release and increasing the rate of protein release. Previously, researchers designed an insulin release device that dissociates protein aggregates through rapamycin analogs, achieving a rapid response of cells to rapamycin. Recently, researchers used the KDEL endoplasmic reticulum retention signal to retain the translated insulin in the endoplasmic reticulum cavity. Rapamycin needs to enter the endoplasmic reticulum cavity to induce the removal of the retention signal to achieve insulin release. However, its protein release rate is low, its induction response ability is weak, and its design is relatively complex, with poor versatility, scalability, and modular interchangeability. In addition, rapamycin has toxic side effects such as activating intracellular signaling pathways, and its safety cannot be guaranteed when used in controllable gene therapy or cell therapy. Combined with electrogenetic technology, the researchers designed and developed an insulin release system that responds to electrical stimulation, and achieved the effect of lowering blood sugar in type 1 diabetic mice. The system is based on electrical stimulation of Ca 2+ However, various signaling pathways in cells can lead to Ca 2+ Inflow, so the system is susceptible to interference from external signals and has poor insulation and orthogonality. In addition, the chassis cells of this system must be pancreatic islet cells, and can only achieve insulin secretion, and cannot control the secretion and release of other protein drugs. These shortcomings greatly limit the breadth and depth of its application. Therefore, it is of great significance to establish a protein rapid release system with good orthogonality, safety, and remote control, and to regulate the chassis cells to release different target protein drugs quickly and instantaneously on demand through the recognition of external signals from different sources (clinical drugs, light, etc.) for the precise treatment of diseases. Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the present invention proposes a mammalian cell controllable protein rapid secretion release system (also known as a program-controlled protein rapid secretion release system) (PASS), which realizes highly sensitive, precisely controllable rapid release of protein drugs. The present invention has orthogonality, non-toxicity, high efficiency, low background leakage, high module replaceability, and the characteristics of inducing rapid release of target proteins, which well solves the problems existing in the current transcription expression system. The present invention can respond to different inducers, such as the antiviral drug Danoprevir (Danoprevir, DNV) approved by the U.S. Food and Drug Administration, the antiviral drug Grazoprevir (Grazoprevir, GZV), tumor antigens and 450nm blue light, and the module is small, simple and easy to operate; at the same time, the present invention can regulate a variety of proteins, can quickly respond to the stimulation of inducers, and realize the rapid release rate of proteins at the minute level; in addition, the present invention can also simultaneously respond to the stimulation of two different kinds of inducers, release one or more therapeutic proteins, and realize the precise treatment of diseases. In summary, the present invention provides a new technology for the rapid and instantaneous delivery of controllable protein drugs for gene and cell therapy, which is of great significance in the research of mammalian genetic engineering and biomedical fields.

[0007] The present invention proposes for the first time a mammalian cell-controlled protein rapid secretion and release system (programmable protein rapid secretion and release system) PASS. The clinically approved drugs, tumor antigens and blue light described in the present invention are non-cytotoxic. The protein rapid secretion and release system (chemPASS) regulated by clinically approved drugs has high sensitivity and can achieve minute-level protein rapid release when low-concentration drugs are added; the protein rapid secretion and release system (antigenPASS) regulated by protein antigen signals can specifically recognize antigens on the surface of tumor cells, mediate the precise release of tumor-killing proteins, and induce apoptosis of tumor cells; the protein rapid secretion and release system (optoPASS) regulated by blue light can safely, remotely and tracelessly control the release of multiple therapeutic proteins, and achieve spatiotemporal-specific drug delivery.

[0008] The present invention optimizes the dimerization fusion protein in the chemPASS system, achieves the optimal protein rapid release rate, enables the system to have the greatest responsiveness to drugs, and provides technical support for the realization of clinical transformation; the present invention replaces multiple antigens in the antigenPASS system, enables the system to respond to the regulation of different antigens, mediates the release of multiple proteins and cytokines, and can accurately kill target cells; the present invention optimizes the optoPASS system, enables the system to have the greatest responsiveness to 450nm blue light, and can achieve rapid reversible release of proteins under blue light and darkness. The mammalian cell PASS system described in the present invention can regulate the release of multiple proteins, provides a new tool for genetic engineering, has great potential application value, and can be widely promoted for clinical use.

[0009] The optoPASS system of the present invention can rapidly release atrial natriuretic peptide, analgesic peptide, insulin and other protein therapeutic drugs retained in the endoplasmic reticulum through light-regulated engineered cell activation protein secretion pathways, achieving the purpose of rapidly lowering blood pressure, relieving pain or lowering blood sugar in minutes. The antigenPASS system can recognize tumor-specific antigen signals and rapidly secrete cytokines to kill tumor cells.

[0010] The nucleotide sequence or amino acid sequence described in the present invention can be prepared by artificial synthesis.

[0011] The present invention also provides a eukaryotic expression vector, host cell and / or engineered cell of a mammalian cell programmable protein rapid secretion and release system; wherein the host cell and / or engineered cell is transfected with the eukaryotic expression vector.

[0012] The present invention proposes a program-controllable protein rapid secretion and release system PASS, which includes a protein rapid secretion and release system chemPASS regulated by clinically approved drugs, a protein rapid secretion and release system antigenPASS regulated by protein antigen signals, and a protein rapid secretion and release system optoPASS regulated by blue light.

[0013] The mammalian cell PASS system proposed in the present invention includes: a target protein endoplasmic reticulum recycler, a danoprevir DNV receptor, a grazoprevir GZV receptor, a tumor antigen receptor and a blue light receptor.

[0014] In the present invention, the protein rapid secretion and release system chemPASS regulated by clinically approved drugs (including but not limited to danoprevir and grazoprevir) includes an endoplasmic reticulum recycler that responds to the target protein and a clinical drug receptor.

[0015] In the present invention, the target protein endoplasmic reticulum recycler comprises a target protein -COOH end and a Golgi furin recognition amino acid sequence (Fcs), a CD4 transmembrane domain (TM), a tobacco etch virus protease (TEVp) or tobacco vein spot virus protease (TVMVp) recognition site (TEVcs or TVMVcs) and an endoplasmic reticulum protein recovery signal fused and expressed. The amino acid sequence of the furin recognition site Fcs is shown in SEQ ID NO.1, the amino acid sequence of the CD4 transmembrane domain TM is shown in SEQ ID NO.2, the amino acid sequence of the TEV protease recognition site TEVcs is shown in SEQ ID NO.3, the amino acid sequence of the TVMV protease recognition site TVMVcs is shown in SEQ ID NO.4, and the endoplasmic reticulum protein recovery signal is shown in SEQ ID NO.5-10.

[0016] In the present invention, the clinical drug receptors include Danoprevir DNV receptor and Grazoprevir GZV receptor.

[0017] In the present invention, the danoprevir receptor includes the hepatitis C NS3 protease domain NS3a and its interacting protein DNCR2. The amino acid sequence of the protein NS3a is shown in SEQ ID NO.11, and the amino acid sequence of the interacting protein DNCR2 is shown in SEQ ID NO.12.

[0018] In the present invention, the grazoprevir receptor includes the hepatitis C virus NS3 protease domain NS3a and its interacting protein GNCR1. The amino acid sequence of the protein NS3a is shown in SEQ ID NO.11, and the amino acid sequence of the interacting protein GNCR1 is shown in SEQ ID NO.13.

[0019] In the present invention, the antigenPASS includes an endoplasmic reticulum recycler and a tumor antigen receptor.

[0020] In the present invention, the construction of the endoplasmic reticulum recycler is the same as that of chemPASS.

[0021] In the present invention, the tumor antigen receptor comprises a single-chain variable region fragment of a tumor antigen monoclonal antibody, a core domain of a Notch receptor and a TEV protease; the amino acid sequence of the fusion protein expressed by fusion of the single-chain variable region of the tumor antigen monoclonal antibody with the core domain of a Notch receptor and a TEV protease is shown in SEQ ID NO.14.

[0022] In the present invention, the blue light-regulated protein rapid release system optoPASS comprises a target protein endoplasmic reticulum recycler and a blue light receptor.

[0023] In the present invention, the construction of the endoplasmic reticulum recycler is the same as that of chemPASS.

[0024] In the present invention, the photoreceptor comprises a blue light sensing protein pMag and an interacting protein nMag thereof, wherein the amino acid sequence of the pMag is shown in SEQ ID NO.15, and the amino acid sequence of the interacting protein nMag thereof is shown in SEQ ID NO.16.

[0025] The mechanism of action of the mammalian cell chemPASS system described in the present invention is as follows: the target protein is retained in the endoplasmic reticulum through the fusion-expressed endoplasmic reticulum recycling signal, and the endogenous furin recognition site Fcs, transmembrane domain TM and TEV protease recognition site TEVcs located in the Golgi apparatus are inserted between the two, so that it can rely on clinical drug receptors to regulate TEV protease activity to achieve controllable rapid release of proteins. The clinical drug receptor NS3a / DNCR2 or NS3a / GNCR1 is fused and expressed with the split -NH2 end and -COOH end of the TEV protease, respectively. In the presence of the inducer danoprevir or grazoprevir, NS3a specifically binds to DNCR2 or GNCR1 to form a complete structure of the split TEV protease, restore its hydrolase activity, and thus hydrolyze and remove the endoplasmic reticulum recycling signal fused to the target protein. After the recycling signal is removed, the target protein is recognized by furin when it passes through the Golgi apparatus through vesicle transport, and the transmembrane domain is removed, thereby achieving rapid release of the target protein.

[0026] The mechanism of action of the mammalian cell antigenPASS system described in the present invention is as follows: the target protein is retained in the endoplasmic reticulum through the endoplasmic reticulum recycling signal fused to its -COOH end, and the endogenous furin recognition site Fcs, transmembrane domain TM and TEV protease recognition site TEVcs located in the Golgi apparatus are inserted between the two, so that it can rely on antigen to regulate TEV protease to achieve controllable rapid release of protein. The single-chain variable region -COOH end of the antigen is fused with the Notch receptor core region to express transmembrane, and the TEV protease is anchored in the cell by fusion expression with the -COOH end of the above protein, and after specific recognition by the extracellular antigen antibody, the membrane-anchored protease is released, thereby removing the endoplasmic reticulum recycling signal fused to the target protein, and after the recycling signal is removed, the target protein is recognized by furin when it passes through the Golgi apparatus through vesicle transport, and the transmembrane domain is removed, thereby achieving rapid release of the target protein.

[0027] The mechanism of action of the mammalian cell optoPASS system described in the present invention is: the target protein is retained in the endoplasmic reticulum through the endoplasmic reticulum recycling signal fused to its -COOH end, and the endogenous furin recognition site Fcs, transmembrane domain TM and TVMV protease recognition site TVMVcs located in the Golgi apparatus are inserted between the two, so that it can rely on the blue light receptor to regulate the TVMV protease activity to achieve controllable rapid release of proteins. The blue light receptor pMag / nMag is fused and expressed with the split -NH2 end and -COOH end of the TVMV protease, respectively. Once the receptor is irradiated with 450nm blue light, pMag and nMag can quickly combine to form a heterodimer, so that the split TVMV protease forms a complete structure, restores its hydrolase activity, and thus hydrolyzes and removes the endoplasmic reticulum recycling signal fused to the target protein. After the recycling signal is removed, the target protein is recognized by furin when it passes through the Golgi apparatus through vesicle transport, and the transmembrane domain is removed, thereby achieving rapid release of the target protein.

[0028] The PASS system provided by the present invention can be delivered to chassis cells or host cells by cell engineering or genetic engineering technology, thereby achieving controlled and rapid release of the target protein. The mammalian PASS system provided by the present invention will not damage cell activity, and the chassis cells can be any type of mammalian cells, such as hMSC-TERT, HEK-293, Hana3A, iPSC, etc.

[0029] The present invention also provides a method for constructing a mammalian cell PASS system, comprising the following steps:

[0030] (1) Construction of the endoplasmic reticulum recycling unit for target protein

[0031] A fusion protein of the target protein, furin recognition site Fcs, CD4 transmembrane domain TM, protease recognition site and endoplasmic reticulum recycling signal is constructed as the target protein endoplasmic reticulum recycling device of the system, so that the target protein is retained in the endoplasmic reticulum.

[0032] The protease recognition site includes but is not limited to TEVp and TVMVp recognition sites, and the amino acid sequence is such as SEQ ID NO.3-4.

[0033] The endoplasmic reticulum recycling signals include but are not limited to KKRN, KKML, KKVV, KKFL, KKTL, and KKKI, among which KKRN has the best protein recovery effect. The amino acid sequences of the endoplasmic reticulum recycling signals are as shown in SEQ ID NO.5-10.

[0034] (2) Construction of Danoprevir receptor

[0035] Fusion proteins of NS3a and split TEV protease and fusion proteins of DNCR2 and split TEV protease were constructed as the system's danoprevir receptors.

[0036] Among them, the fusion expression of NS3a and the split TEV protease includes NS3a-nTEVp, NS3a-cTEVp, nTEVp-NS3a, cTEVp-NS3a, and the fusion expression of DNCR2 and the split TEV protease includes DNCR2-nTEVp, DNCR2-cTEVp, nTEVp-DNCR2, and cTEVp-DNCR2; among them, the combination of nTEVp-NS3a and DNCR2-cTEVp is the optimal combination.

[0037] (3) Construction of grazoprevir receptors

[0038] Fusion proteins of NS3a and split TEV protease, and fusion proteins of GNCR1 and split TEV protease were constructed as the system's grazoprevir receptors.

[0039] Among them, the fusion expression of NS3a and the split TEV protease includes NS3a-nTEVp, NS3a-cTEVp, nTEVp-NS3a, cTEVp-NS3a, and the fusion expression of GNCR1 and the split TEV protease includes GNCR1-nTEVp, GNCR1-cTEVp, nTEVp-GNCR1, and cTEVp-GNCR1; among them, the combination of nTEVp-NS3a and GNCR1-cTEVp is the optimal combination.

[0040] (4) Construction of a protein rapid release system responsive to tumor antigens

[0041] A fusion protein of the single-chain variable region of tumor antigen monoclonal antibody, Notch receptor core structure region and TEV protease was constructed as a protein rapid release system for tumor antigen response.

[0042] Wherein, the amino acid sequences of the single-chain variable region of the constructed CD19 tumor antigen or EGFP antigen monoclonal antibody are shown in SEQ ID NO.14 and SEQ ID NO.19 respectively.

[0043] (5) Construction of blue light receptors

[0044] The fusion protein of pMag and split TVMV protease, the fusion protein of nMag and split TVMV protease, the nuclear localization / nuclear export signals of the above two fusion proteins and the connecting peptide between the above two fusion proteins were constructed as the blue light receptor of the system.

[0045] The fusion expression of pMag and split TVMV protease includes pMag-nTVMVp, pMag-cTVMVp, nTVMVp-pMag, cTVMVp-pMag, and the fusion expression of nMag and split TVMV protease includes nMag-nTVMVp, nMag-cTVMVp, nTVMVp-nMag, and cTVMVp-nMag. The amino acid sequences of cTVMVp and nTVMVp proteins are shown in SEQ ID NO.20-21. The combination of nMag-cTVMVp and nTVMVp-pMag is the optimal combination.

[0046] The nuclear localization / nuclear export signals of the two fusion proteins are NLS / NES, and their amino acid sequences are shown in SEQ ID NO.22-23.

[0047] Wherein, the connecting peptide between the two fusion proteins is a peptide chain of 0-15 amino acids, and preferably its amino acid sequence is shown in SEQ ID NO.24-25.

[0048] The chemPASS system of the present invention can respond to micromole-level small molecule concentration stimulation to induce rapid release of proteins; the chemPASS system can induce release of proteins at the minute level.

[0049] The antigenPASS system described in the present invention can accurately identify a variety of tumor antigens, induce the release of cytokines and killer proteins, and quickly kill tumor cells.

[0050] The optoPASS system described in the present invention can respond to blue light stimulation at the minute level to induce the release of protein; the optoPASS system can respond to blue light stimulation with an intensity of microwatt level to induce the release of protein; the optoPASS system can induce the release of protein at the minute level; the optoPASS system can reversibly regulate the release of protein under blue light and darkness.

[0051] The PASS system of the present invention is suitable for the release of various proteins, has simple modules and high replaceability, and can be used to treat various diseases. The system can regulate the rapid release of proteins in different mammalian cell lines.

[0052] The present invention also provides a eukaryotic expression vector, which is the eukaryotic expression vector of the PASS system as described above.

[0053] The present invention also provides a host cell and / or engineered cell transplantation vector, wherein the host cell and / or engineered cell transplantation vector is transfected with the above-mentioned PASS system.

[0054] The invention also provides a method for preparing a eukaryotic expression vector, an engineered cell or an engineered cell transplantation vector containing the mammalian cell PASS system.

[0055] The eukaryotic expression vector includes a mammalian cell expression vector containing the mammalian cell PASS system. The expression vector can be a vector containing a target protein endoplasmic reticulum recycling device encoding gene alone or a vector containing an inducer receptor encoding gene alone. The construction methods of all the aforementioned mammalian cell expression vectors are detailed in Table 1.

[0056] The invention also proposes the application of the program-controllable protein rapid secretion and release system in mammalian genetic engineering and disease treatment.

[0057] The present invention also proposes the use of a eukaryotic expression vector containing a chemPASS system regulated by danoprevir in mammalian cells, or a host cell and / or an engineered cell transplantation vector in the treatment of diseases.

[0058] The present invention also proposes the use of a eukaryotic expression vector containing a mammalian cell optoPASS system, or a host cell and / or an engineered cell transplantation vector in disease treatment.

[0059] The beneficial effects of the present invention are: the mammalian cell program-controlled rapid protein secretion and release system of the present invention can respond to the stimulation of multiple inducers, and the protein rapid secretion system has the characteristics of good orthogonality and safety. The present invention provides an artificially controllable rapid and transient protein rapid delivery technology platform for basic research, genetic engineering and regenerative medicine, which can achieve precise control of the rapid and transient release of intracellular protein drugs, and has great potential application value for the treatment of diseases caused by insufficient or damaged protein secretion. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of the principle of the PASS system in mammalian cells.

[0061] Figure 2 The screening results of the endoplasmic reticulum recycling signal of the target protein endoplasmic reticulum recycling device in the PASS system of mammalian cells.

[0062] Figure 3 Target protein salvage toxicity assay in the PASS system in mammalian cells.

[0063] Figure 4These are the results of combined screening of NS3a / GNCR1 and NS3a / DNCR2 fused with split TEV protease in the mammalian cell chemPASS system, where A is the schematic diagram of GZV-induced NS3a / GNCR1 protein dimerization; B is a schematic diagram of the plasmid construction for the fusion expression of NS3a / GNCR1 and split TEV protease; C is the optimal combination screening of NS3a / GNCR1 and split TEV protease; D is the schematic diagram of DNV-induced NS3a / DNCR2 protein dimerization; E is a schematic diagram of the plasmid construction for the fusion expression of NS3a / DNCR2 and split TEV protease; F is the optimal combination screening of NS3a / DNCR2 and split TEV protease.

[0064] Figure 5 is the rate of rapid protein release induced by the chemPASS system in mammalian cells, where A is the rate of rapid protein release induced by the chemPASS system in mammalian cells, DNV The system induces rapid protein release; B is chemPASS GZV The rate at which the system induces rapid release of proteins.

[0065] Figure 6 is the response of the mammalian cell chemPASS system to different concentrations of drug small molecules, where A is the chemPASS DNV The system's response to different concentrations of DNV drug small molecules; B is chemPASS GZV The system's response to stimulation with different concentrations of GZV drug small molecules.

[0066] Figure 7 Insulin secretion mediated by the DNV-regulated chemPASS system, an antiviral drug clinically approved for mammalian cells, is used to control blood glucose homeostasis in a type 1 diabetic mouse model, where A is chemPASS DNV Schematic diagram of system-mediated insulin secretion for blood glucose homeostasis control in type 1 diabetic mouse model; B is chemPASS DNV Schematic diagram of the experimental process of system-mediated insulin secretion for blood glucose control in type 1 diabetic mice; C is chemPASS DNV System monitoring of blood glucose homeostasis in type 1 diabetic mouse model; D is chemPASS DNV Detection of systemically mediated insulin secretion in vivo in a mouse model of type 1 diabetes.

[0067] Figure 8Protein release mediated by antigenPASS in mammalian cells, wherein A is a schematic diagram of protein release mediated by antigenPASS in mammalian cells; B is the release of Nanoluc mediated by antigenPASS in response to protein EGFP antigen; C is the release of Nanoluc mediated by antigenPASS in response to tumor antigen CD19; D is the release of granzyme B mediated by antigenPASS in response to tumor antigen CD19; E is the release of perforin mediated by antigenPASS in response to tumor antigen CD19.

[0068] Fig. 9 Apoptosis of target cells mediated by antigenPASS in mammalian cells, wherein A is a flow cytometric analysis of target cell apoptosis induced by antigenPASS-mediated granzyme B release detected by Annexin V-FITC / PI staining; B is a flow cytometric analysis of target cell apoptosis induced by antigenPASS-mediated perforin release detected by Annexin V-FITC / PI staining.

[0069] Fig.10 These are the screening results for the fusion expression of pMag / nMag and split TVMV protease in the optoPASS system of mammalian cells, where A is the schematic diagram of the optoPASS system; B is the schematic diagram of the plasmid construction for the fusion expression of pMag / nMag and split TVMV protease; and C is the screening for the optimal combination of pMag / nMag and split TVMV protease.

[0070] Fig.11 The figure shows the effect of nuclear localization signal on the rapid release rate of induced protein in the optoPASS system of mammalian cells, where A is a schematic diagram of the construction of optimized plasmid for nuclear localization signal of the optoPASS system; B is the screening of plasmid combination for nuclear localization signal optimization of the optoPASS system.

[0071] Fig.12 The figure shows the effect of the connecting peptide of nMag-cTVMVp and nTVMVp-pMag fusion protein on the rapid release rate of induced protein in the optoPASS system of mammalian cells, wherein A is the schematic diagram of plasmid construction for optimizing the connecting peptide of nMag-cTVMVp and nTVMVp-pMag fusion protein in the optoPASS system; B is the combined screening of connecting peptides of nMag-cTVMVp and nTVMVp-pMag fusion protein.

[0072] Fig.13The figure shows the effect of the localization of the nTVMVp-pMag fusion protein in the optoPASS system of mammalian cells on the rate of rapid protein release induced by the system. A is the schematic diagram of the optimal version of optoPASS inducing protein release; B is the comparison of the localization of the nTVMVp-pMag fusion protein in the optoPASS system.

[0073] Fig.14 The response of the optoPASS system of mammalian cells to blue light stimulation of different intensities.

[0074] Fig.15 The response of the optoPASS system of mammalian cells to blue light stimulation of different durations.

[0075] Fig.16 The rate at which the optoPASS system induces rapid protein release in mammalian cells.

[0076] Fig.17 To investigate the reversibility of the optoPASS system in mammalian cells.

[0077] Fig.18 The figures show the working conditions of the optoPASS system in mammalian cells in different cell lines, where A shows the optoPASS system mediating the release of protein SEAP in HEK293T cells; B shows the optoPASS system mediating the release of protein SEAP in MSC cells; C shows the optoPASS system mediating the release of protein SEAP in Hana3A cells; and D shows the optoPASS system mediating the release of protein SEAP in iPSC cells.

[0078] Fig.19 The optoPASS system in mammalian cells induces the rapid release of atrial natriuretic peptide ANP for the control of blood pressure in a hypertensive mouse model, wherein A is the schematic diagram of the optoPASS system-mediated release of atrial natriuretic peptide ANP for the control of blood pressure in a hypertensive mouse model; B is the schematic diagram of the experimental process of the optoPASS system-mediated release of atrial natriuretic peptide ANP for the control of blood pressure in a hypertensive mouse model; C is the optoPASS system-mediated release of atrial natriuretic peptide ANP after light exposure. ANP The system monitors the blood pressure homeostasis control of the hypertensive mouse model; D is the optoPASS after 15 minutes of light exposure ANP System rapid blood pressure lowering effect monitoring; E is optoPASS after 15min and 90min of light exposure ANP Detection of ANP secretion in vivo in a systemically mediated hypertensive mouse model.

[0079] Fig. 20The optoPASS system in mammalian cells induces rapid release of insulin for the treatment of type 1 diabetic mouse models, wherein A is a schematic diagram of the optoPASS system-mediated release of insulin for the control of blood glucose homeostasis in type 1 diabetic mouse models; B is a schematic diagram of the experimental process of the optoPASS system-mediated release of insulin for the control of blood glucose homeostasis in type 1 diabetic mouse models; C is a schematic diagram of the optoPASS system-mediated release of insulin after illumination insulin The system monitors blood glucose homeostasis in a type 1 diabetic mouse model; D is optoPASS after 15 minutes of light exposure insulin System rapid blood sugar lowering effect monitoring; E is optoPASS after 15min and 90min of light exposure insulin System-mediated in vivo insulin secretion detection in type 1 diabetes mouse model.

[0080] Fig.21 The optoPASS system in mammalian cells induces the rapid release of the analgesic peptide ssHwTx for the treatment of a chronic inflammatory pain mouse model, wherein A is a schematic diagram of the optoPASS system-mediated release of the analgesic peptide ssHwTx for analgesia in a chronic inflammatory pain mouse model; B is a schematic diagram of the experimental process of the optoPASS system-mediated release of the analgesic peptide ssHwTx for analgesia in a chronic inflammatory pain mouse model; C is a schematic diagram of the optoPASS system-mediated release of the analgesic peptide ssHwTx for analgesia in a chronic inflammatory pain mouse model after illumination ssHwTx Monitoring of the analgesic effect of the optoPASS system on a mouse model of chronic inflammatory pain; D is 15 minutes after light exposure ssHwTx Monitoring of the rapid analgesic effect of the system; E is optoPASS after 15min and 90min of light exposure ssHwTx Detection of ssHwTx secretion in vivo in a systemically mediated chronic inflammatory pain mouse model. DETAILED DESCRIPTION

[0081] The present invention is further described in detail in conjunction with the following specific examples and drawings. These examples are only used to illustrate the invention and do not constitute any limitation on the scope of the present invention. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no special restrictions. The reagents, instruments, etc. used in the following examples, as well as the experimental methods without specifying specific conditions, are carried out according to the conditions recommended by conventional or commodity suppliers.

[0082] Materials and Methods

[0083] The construction reagents, specific construction systems and steps of all expression plasmids of the present invention are as follows.

[0084] All primers used for PCR were synthesized by Shanghai Ruimian Biotechnology Co., Ltd. The expression plasmids constructed in the examples of the present invention were sequenced, and the sequence determination was completed by Shanghai Sunny Biotechnology Co., Ltd. Phanta Max Super-Fidelity DNA polymerase used in the examples of the present invention was purchased from Nanjing Novozyme Biotechnology Co., Ltd. Endonucleases and T4 DNA ligases were purchased from TaKaRa. Homologous recombinases were purchased from Heyuan Biotechnology (Shanghai) Co., Ltd. Phanta Max Super-Fidelity DNA polymerases were purchased with corresponding polymerase buffers and dNTPs. Endonucleases, T4 DNA ligases, and homologous recombinases were purchased with corresponding buffers. Yeast extract (Yeast Extract), tryptone (Trypton), agar powder, and ampicillin (Amp) were purchased from Shanghai Shenggong Biotechnology Co., Ltd. DNA Marker DL5000 and DNA Marker DL2000 (Bao Bioengineering Co., Ltd.); nucleic acid dye EB (Guangdong Guoao Biotechnology Co., Ltd.); plasmid mini-extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.); DNA gel recovery kit and PCR product purification kit were all purchased from Kangwei Century Biotechnology Co., Ltd.; the other reagents mentioned in the examples, such as anhydrous ethanol and NaCl, were all domestic analytical grade products.

[0085] 1. Polymerase chain reaction (PCR)

[0086]

[0087]

[0088] (II) Endonuclease digestion reaction

[0089] 1. System for double enzyme digestion of plasmid vector (x represents the volume when the plasmid mass is 1 μg; n represents the amount of sterile ultrapure water μL required to make the system reach the total volume)

[0090]

[0091] 2. Double enzyme digestion system for PCR product fragments (x represents the volume when the plasmid mass is 1 μg; n represents the amount of sterile ultrapure water μL required to make the system reach the total volume)

[0092]

[0093] 3. Connect the PCR product fragment after double enzyme digestion with the plasmid vector after double enzyme digestion to form a circular plasmid system:

[0094]

[0095]

[0096] Note: The mass ratio of PCR product fragment to vector double enzyme digestion product is roughly between 2:1-6:1.

[0097] (III) Homologous recombination ligation reaction

[0098] According to the instructions of the seamless cloning (assembly) kit of Heyuan Biotechnology (Shanghai) Co., Ltd.: When amplifying the PCR product fragment, 15bp nucleotide sequences homologous to the 15bp nucleic acid sequences on both sides of the linearized vector are added on both sides. The 15bp nucleic acid sequences on both sides of the amplified PCR product are homologous to the nucleotide sequences on both sides of the linearized vector sequence. Under the action of the homologous recombinase, the PCR product fragment and the linearized vector are homologously recombined and connected into a circle.

[0099]

[0100] Note: The n value depends on the size and concentration of the PCR product fragment.

[0101] (IV) Preparation of E. coli competent cells

[0102] All solutions and consumables used for competent cell preparation were sterilized by high temperature and high pressure.

[0103] 1. Streak the E. coli DH5α strain on a plate without antibiotics and incubate it upside down at 37°C for 12-16 hours; pick a single colony and place it in a 2 mL LB shake tube without any antibiotics and culture it at 37°C with shaking at 180 rpm overnight;

[0104] 2. Transfer 1 mL of bacterial solution into a 250 mL Erlenmeyer flask with LB medium for expansion culture (expand culture at a ratio of 1:100), and culture at 37°C at 180 rpm for 2 to 3 hours until OD600 is between 0.4 and 0.5;

[0105] 3. Transfer the culture medium into a centrifuge tube, place on ice for 10 minutes, then centrifuge at 3000 rpm for 10 minutes at 4°C (the centrifuge needs to be pre-cooled in advance), and carefully discard the supernatant;

[0106] 4. Add pre-cooled 0.1M CaCl 2 4 mL (0.1 M CaCl 2 The tubes should be pre-cooled in advance). After sufficient pre-cooling, use a vortex mixer to quickly suspend the cells, then place them in an ice bath for 10 minutes, combine the two tubes into one tube; centrifuge at 5.4°C, 4000 rpm for 6 minutes;

[0107] 5. Discard the supernatant and add 4 mL of ice-cold 0.1 M CaCl2 and 0.1 mL of pre-cooled sterilized pure glycerol to suspend the precipitate;

[0108] 6. Dispense the above suspension into PCR tubes at 100 μL / tube and store in liquid nitrogen for later use;

[0109] (V) Transformation of ligation products into E. coli

[0110] 1. Thaw the prepared competent cells (thaw on ice), add an appropriate volume of ligation product, mix well, and place on ice for 30 minutes. Usually, the volume of ligation product added is less than 1 / 10 of the volume of competent cells;

[0111] 2. Heat shock in a 42℃ water bath for 90s, then quickly place on ice for 5min;

[0112] 3. Add the bacterial solution to 800 μL LB liquid medium (without antibiotics), mix well and culture at 37°C with shaking for 40-60 minutes;

[0113] 4. Transfer the bacterial solution to a 1.5 mL centrifuge tube, centrifuge at 4000 rpm for 5 min, discard part of the supernatant, keep about 100 μL of supernatant, and then blow the cells into a cell suspension;

[0114] 5. The above suspension was spread on LB solid culture medium containing ampicillin and inverted in a 37°C incubator for overnight culture; the remaining experimental operations, such as gel recovery and purification recovery of DNA fragments, were based on the operating instructions of the DNA gel recovery kit and PCR product purification kit (Kangwei Century Biotechnology Co., Ltd.); the plasmid extraction steps were based on the plasmid mini-extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.).

[0115] Selection and production of light source

[0116] In the embodiment of the present invention, a 450nm wavelength LED is used as an example to illustrate the control method of the remote control system of the present invention, but it does not limit the scope of protection of the present invention. The LEDs used in the experiment were purchased from Japan's Epitex Company. The remaining accessories are all domestic conventional consumables. The 450nm wavelength LED provides a low-power blue light emitter. The connection method of the 4×6 LED board is: according to the arrangement of the cell culture plate (24-well plate), each LED corresponds to the center position of each well, and each LED is connected in parallel. Adjust the illumination time and illumination intensity according to the experimental needs to conduct the experiment.

[0117] Cell culture and transfection

[0118] In the examples of the present invention, the following cell lines and PEI transfection are used as examples to illustrate the working conditions of the mammalian cell controllable protein rapid secretion and release system in cells, but the protection scope of the present invention is not limited. The 10 cm cell culture dish, cell culture plate (24 wells), 15 mL and 50 mL centrifuge tubes used for cell culture were purchased from Thermo Fisher Scientific (Labserv), USA; the modified Eagle medium, fetal bovine serum, penicillin and streptomycin solution used were purchased from Gibico, USA; the PEI used for transfection was purchased from Polysciences; the cell culture box was purchased from Thermo Fisher Scientific, USA; the remaining consumables were ordinary domestic consumables. Cell culture: The cells involved in the patent include human embryonic kidney cells (HEK-293T, ATCC: CRL-11268), stably integrated with a copy of the E1 gene (ThermoFisher, R70507), human mesenchymal cells with telomeres (hMSC-TERT), and HEK-293-derived Hana3A cells cultured in modified Eagle medium (DMEM) supplemented with 10% (v / v) fetal bovine serum and 1% (v / v) penicillin and streptomycin solution; mouse iPSCs cells cultured in modified Eagle medium (GMEM) supplemented with 15% (v / v) fetal bovine serum, 1% (v / v) non-essential amino acids, 1×10 -3 M GlutaMAX, 0.1×10 -3 Mβ-mercaptoethanol, 100U / mL mouse recombinant leukemia inhibitory factor; human acute T-cell leukemia cell line (Jurkat T, ATCC-TIB-152) was cultured in 1640 medium, 10% (v / v) fetal bovine serum and 1% (v / v) penicillin and streptomycin solution were added to the medium, and all cells were cultured in an incubator at 37°C with a 5% carbon dioxide concentration. Transfection: There are three methods for transfection of all cell lines. The first transfection method uses optimized PEI. Simply put, 6×10 cells were inoculated in a 10mL 10cm cell culture dish. 4 After culturing for 18 h, the optimal ratio of DNA was mixed with PEI at a mass ratio of 3:1 (PEI:DNA) and dissolved in the culture medium for 6 h. (Polyethyleneimine, molecular weight 40,000, stock solution 1 mg / mL in ddH 2 O; Polysciences; Cat. no. 24765) cells were counted using Countess II automated cell counter. TM3000: a. One day before transfection, seed 50,000 cells per well into a 24-well plate and culture for 16 hours to reach a confluence of about 70% during transfection. b. Take a clean centrifuge tube and add DMEM to dilute Lipo TM c. Take a clean centrifuge tube and add DMEM and the plasmid required for transfection. TM = Add P300 in a ratio of 1:2 (w:w) TM d. Mix the diluted Lipo TM 3000 solution added P300 TM The solution was blown up and down to mix well, and then allowed to stand for 15 minutes before being added to the 24-well plate. The third transfection method was to use lentivirus infection: a. Cell subculture and transfection: HEK293T cells were amplified and plated on 15 cm culture dishes, with a cell inoculation volume of 6 × 10 6 , transfection was performed 16 hours later. The plasmid packaging system includes packaging plasmid psPAX2, pMD2G and vector plasmid, the ratio of the three is 2:1:2, the total amount of plasmid per dish is 30μg, the liquid is changed 6 hours after transfection, and the plasmid used in virus production is the endotoxin-free plasmid; b. Lentivirus collection: 48 hours after the liquid is changed, collect the supernatant containing the virus into a 50mL centrifuge tube, centrifuge at 800×g for 10 minutes to remove cell debris, and filter with a 0.45μm filter after centrifugation; c. Lentivirus concentration: 15000×g, centrifuge at 4℃ for 2.5h. Remove the supernatant, resuspend the precipitate with PBS, aliquot and store at -80℃. d. Lentivirus infection: 1×10 6 Jurkat T cells were plated in 6-well plates, 100 μL of concentrated virus was added, and fresh medium was replaced 24 h after infection.

[0119] Detection of reporter gene secretory alkaline phosphatase (SEAP): Homoarginine, magnesium chloride, diethanolamine, and HCl used to prepare the reaction buffer for detecting the reporter gene were purchased from Sangon Biotech (Shanghai) Co., Ltd.; the chromogenic substrate (p-nitrophenol phosphate) was purchased from Shanghai Jingchun Biochemical Technology Co., Ltd. (Aladdin).

[0120] (1) Reagent configuration: 2x buffer:

[0121] 20mM Homoarginine Note: Its function is to inhibit endogenous alkaline phosphatase activity

[0122] 1 mM magnesium chloride

[0123] 2% diethanolamine

[0124] Adjust pH to 9.8 with HCl

[0125] Substrate solution:

[0126] 120 mM chromogenic substrate (p-nitrophenol phosphate)

[0127] In 2x assay buffer

[0128] (2) Experimental steps:

[0129] 1. Pipette 200 μL of the cell culture supernatant into a centrifuge tube (Note: generally more than 150 μL is required because subsequent heating will consume part of the volume).

[0130] 2.65℃ water bath for 30min (Note: Heating is mainly to inactivate endogenous alkaline phosphatase, but SEAP is resistant to high temperature and will not be inactivated at this temperature).

[0131] 3. Pipette 80 μL (dilute according to the experimental situation) into a 96-well plate, and quickly add 100 μL of preheated 2x buffer and 20 μL of substrate solution.

[0132] 4. Measure 10 times at 405nm using an ELISA reader, with an interval of 1 minute between each measurement (other conditions can be set depending on the experimental situation).

[0133] (3) Calculation of enzyme activity

[0134] The enzyme activity of alkaline phosphatase (SEAP) is defined as: at 37°C and pH 9.8, the alkaline phosphatase that reacts with the substrate disodium p-nitrophenyl phosphate (PNPP-Na2) within 1 minute to generate 1 mol / L p-nitrophenol is defined as 1 activity unit (1U). p-Nitrophenol itself has a bright yellow color. At a wavelength of 405nm, different concentrations of p-nitrophenol correspond to different absorbance values. The calculation method is: the slope of the curve of the OD values ​​measured at different time points during the reaction of the sample and the substrate * 256.8 is the enzyme activity, in units of U / L.

[0135] Reporter luciferase Luciferase detection

[0136] 8 μL of cell supernatant was taken and incubated with 8 μL of buffer containing detection substrate in a 384 white plate for 5 min, and the fluorescence intensity was detected using a Synergy H1 hybrid multi-mode microplate reader.

[0137] AntigenPASS-mediated tumor cell apoptosis detection

[0138] Annexin V-FITC / Propidium Iodide (PI) double staining apoptosis detection kit was used to evaluate antigenPASS-mediated target cell apoptosis. Briefly, Jurkat T cells were infected with lentivirus expressing antiCD19-synNotch-TEVp receptor and ER-loaded granzyme B (or perforin). Jurkat T cells (2×10 5 ) were co-cultured with CD19+ or CD19- HEK293T cells at a ratio of 1:1 in 48-well plates for 24 h. After 24 h, the cells were collected and washed three times with pre-cooled 1× PBS, gently suspended in 100 μL Annexin V-FITC binding buffer, and then 5 μL Annexin V-FITC and 5 μL PI staining solution were added, gently mixed, and then incubated at room temperature in the dark for 10 minutes, followed by the addition of 400 μL Annexin V-FITC binding buffer and analyzed using Becton Dickinson LSR Fortessa flow cytometry.

[0139] Cell microencapsulation

[0140] Alginate-polylysine-alginate microcapsules were prepared using the Enotech Encapsulator. Each microcapsule has a diameter of about 400 μm and contains about 200 cells / microcapsule. The capsule membrane of the microcapsule can intercept molecules larger than 110,000 Kd, so that immunoglobulin molecules and immune-active cells cannot penetrate the capsule membrane and enter the capsule to destroy the mammalian cells therein, thus having a good immune protection effect. The production process is as follows: mixing cells with sodium alginate solution; dripping into spheres; polylysine coating microspheres; sodium alginate coating microspheres; depolymerizing sodium alginate in the spheres, forming microcapsules, etc.

[0141] Construction of L-NAME-induced hypertension model mice

[0142] After one week of adaptive feeding, L-NAME was dissolved in the drinking water of the experimental mice at a dose of 1 mg / mL, and the experimental mice were allowed to drink water freely for one month. During the modeling period of the hypertensive model mice, fresh drinking water was replaced every week. After one month of continuous drinking water, the water was replaced with normal drinking water to allow the mice to excrete L-NAME in their bodies, and the systolic blood pressure of the mice was measured using the tail pressure method.

[0143] Type 1 diabetes model mouse model

[0144] After one week of adaptive feeding, STZ was injected intraperitoneally (dissolved in 0.1 mmol / L citric acid buffer at pH 4 before use, 65 mg / kg, once a day for 9 consecutive days). The fasting blood glucose of rats was measured by a blood glucose tester (fasting for 12 hours before the test). A blood glucose level greater than 16.7 mmol / L was defined as a type 1 diabetes model.

[0145] Establishment of mouse model of chronic inflammatory pain

[0146] After one week of adaptive feeding, 20 μL of freshly emulsified Freund's adjuvant (1:1 dissolved in sterile PBS solution) was injected into the surface of the left hind foot of the experimental mouse using a 30G Hamilton syringe. After 24 hours, the modeling condition of the chronic inflammatory pain model mouse was judged according to the measurement value of MNT (mechanical pain threshold).

[0147] Example 1 Screening of the ER recycling signal of the target protein ER recycling device in the PASS system of mammalian cells

[0148] This example uses SEAP as the target protein to verify the screening of the endoplasmic reticulum recycling signal of the target protein in the PASS system of mammalian cells, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0149] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0150] The second step is to seed cells. HEK-293T cells that are growing well are digested with 0.25% trypsin and plated at 6×10 cells per well. 4 The cells were seeded in a 24-well plate at a seeding density of 10 cells / well and placed in a 37°C incubator for 16-18 h.

[0151] Step 3: Transfection. The inoculated cells were divided into 7 groups, and pcDNA3.1(+) and target proteins pZQ215, pZQ216, pZQ217, pZQ218, pZQ219, and pZQ220 with different endoplasmic reticulum recycling signals were added to each group at a ratio of 1:1 (w:w), mixed with PEI transfection reagent and serum-free DMEM medium, and allowed to stand at room temperature for 15 minutes before being added to a 24-well plate. The total volume of each well transfected was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture was added for culture.

[0152] Step 4: Detect the secretion of the target protein. 24 hours after transfection, aspirate 200 μL of cell supernatant into a 96-well plate and measure the enzyme activity of SEAP. (For details, see Materials and Methods)

[0153] The results showed that when the target protein was fused with the endoplasmic reticulum recycling signal KKRN, the endoplasmic reticulum recycling efficiency was the most significant. Figure 2 , All data are presented as n = 3 independent replicate experiments.

[0154] Example 2 Toxicity detection of target protein recoverer in mammalian cell PASS system

[0155] This example uses EGFP as the target protein to verify the effect of the target protein collector on cell activity in the PASS system of mammalian cells, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0156] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0157] The second step is to seed cells. HEK-293T cells that are growing well are digested with 0.25% trypsin and plated at 1.5 × 10 cells per well. 4 The cells were seeded in a 96-well plate at a seeding density of 10 cells / well and placed in a 37°C incubator for 16-18 h.

[0158] The third step is transfection. The inoculated cells were divided into 7 groups. Each group was added with pcDNA3.1(+) and different masses of the target protein pZQ273 plasmid with KKRN endoplasmic reticulum recycling signal at a total transfection amount of 75ng, mixed with PEI transfection reagent and serum-free DMEM medium, and placed at room temperature for 15 minutes before adding to a 96-well plate. The total volume of each well transfected was 12.5μL, and the mass ratio of plasmid to PEI was 1:3. After 6 hours of transfection, 125μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture was added for culture.

[0159] Step 4: Detect cell activity: 24 hours after transfection, add 15 μL of CCK8 solution to each well, incubate in the cell culture incubator for 1 hour, and then detect the absorbance of each well at 450 nm.

[0160] The results showed that the PASS system target protein collector had no effect on cell activity, indicating that the PASS system is not cytotoxic. Figure 3 , all data are presented as n = 6 independent replicate experiments.

[0161] Example 3 Combination screening of fusion expression of NS3a / DNCR2 and NS3a / GNCR1 with split TEVp protease in mammalian cell chemPASS system

[0162] This example uses SEAP as the target protein to verify the combined screening of fusion expression of NS3a / DNCR2 and NS3a / GNCR1 with the split TEV protease in the mammalian cell chemPASS system, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0163] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0164] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0165] The third step is transfection. The inoculated cells were divided into a drug-added group and a non-drug-added group, and each group was divided into 8 subgroups. The system induced by danoprevir was added with pZQ328 and pKL72 and pKL77, pKL72 and pKL79, pKL73 and pKL76, pKL73 and pKL78, pKL74 and pKL77, pKL74 and pKL79, pKL75 and pKL76, pKL75 and pKL78 in a ratio of 1:2:2 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM medium. The mixture was incubated at room temperature. The system induced by grazoprevir was added with pZQ328 and pKL72 and pKL81, pKL72 and pKL83, pKL74 and pKL81, pKL74 and pKL83, pKL73 and pKL80, pKL73 and pKL82, pKL75 and pKL80, pKL75 and pKL82 in a ratio of 1:2:2 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM medium, and then allowed to stand at room temperature for 15 minutes and added to the 24-well plate. The total volume of each well transfected was 50 μL, the mass ratio of plasmid to PEI was 1:3, and after transfection for 6 hours, 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture was replaced for culture.

[0166] Step 4: Adding drugs. 24 hours after transfection, the chemPASS system induced by danoprevir (chemPASS DNV ) were added with 0.5 μL of DNV solution to make the final drug concentration in the 24-well plate 10 μM; the chemPASS system (chemPASS GZV ) were added with 0.5 μL of GZV solution to make the final drug concentration in the 24-well plate reach 10 μM.

[0167] Step 5: Detect the amount of target protein released. Take the supernatant of the cell fluid 24 hours after drug addition to detect the enzyme activity of SEAP to determine the amount of protein released. (For specific methods, see Materials and Methods)

[0168] The results showed that mammalian cell chemPASS DNV The combination of nTEVp-NS3a and DNCR2-cTEVp in the system induces the best multiple of protein rapid release; mammalian cell chemPASS GZV The combination of nTEVp-NS3a and GNCR1-cTEVp in the system induces the best multiple of protein rapid release. Figure 4 , All data are presented as n = 3 independent replicate experiments.

[0169] Example 4 Response of mammalian cell chemPASS system to drug stimulation at different concentrations

[0170] This example uses SEAP as the target protein to verify the response of the mammalian cell chemPASS system to drug stimulation of different concentrations, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0171] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0172] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0173] Step 3: Transfection. Mammalian Cell chemPASS DNV In the system, the inoculated cells were divided into 6 groups, and pZQ328, pKL72, and pKL79 were added to each group in a ratio of 1:2:2 (w / w / w) and mixed with PEI transfection reagent and serum-free DMEM, and then allowed to stand at room temperature for 15 minutes and added to a 24-well plate; mammalian cell chemPASS GZV In the system, the inoculated cells were divided into 6 groups, and pZQ328, pKL72, and pKL83 were added to each group in a ratio of 1:2:2 (w / w / w), mixed with PEI transfection reagent and serum-free DMEM, and placed at room temperature for 15 minutes before being added to a 24-well plate. The total volume of each well for transfection was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture was added for culture.

[0174] Step 4: Add drug. 24 hours after transfection, chemPASS DNV 1 μL of DNV solution of different concentrations was added to the system to make the final concentrations 0-10 μM; chemPASS GZV 1 μL of GZV solution of different concentrations was added to the system to make the final concentrations of 0-10 μM.

[0175] Step 5: Detection: 4 hours after drug addition, the cell supernatant was collected to determine the amount of SEAP released (the specific steps are the same as those in Example 3 of the present invention).

[0176] The results showed that mammalian cell chemPASS DNV The ability of the system to induce rapid protein release increased with the increase of DNV concentration and was close to saturation at a DNV concentration of 10 μM; mammalian cell chemPASS GZV The ability of the system to induce rapid protein release increases with the increase of GZV concentration and is close to saturation when the DNV concentration is 10μM. Figure 5 , All data are presented as n = 3 independent replicate experiments.

[0177] Example 5: Rapid release of proteins induced by the chemPASS system in mammalian cells

[0178] This example uses SEAP as the target protein to verify the rate of protein release induced by the chemPASS system in mammalian cells, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0179] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0180] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0181] Step 3: Transfection. Mammalian cell chemPASS DNV In the system, the inoculated cells were divided into a drug-added group and a non-drug-added group, each group was divided into 10 groups, and pZQ328, pKL72, and pKL79 were added to each group in a ratio of 1:2:2 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM, and then allowed to stand at room temperature for 15 minutes and added to a 24-well plate; mammalian cell chemPASS GZV In the system, the inoculated cells were divided into a drug-added group and a non-drug-added group, each group was divided into 10 groups, and pZQ328, pKL72, and pKL83 were added to each group at a ratio of 1:2:2 (w:w:w), mixed with PEI transfection reagent and serum-free DMEM, and placed at room temperature for 15 minutes before being added to a 24-well plate. The total volume of each well transfected was 50 μL, the mass ratio of plasmid to PEI was 1:3, and 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture was replaced after 6 hours of transfection for culture.

[0182] Step 4: Add drug. 24 hours after transfection, chemPASS DNV 0.5 μL of DNV solution (10 mM) was added to the system; chemPASS GZV0.5 μL of GZV solution (10 mM) was added to the system.

[0183] Step 5: Detection: The cell supernatant of the drug-added group and the non-drug-added group was collected at different times (0-210 min) after drug addition to determine the secretion of SEAP (for specific methods, refer to Materials and Methods).

[0184] The results showed that the mammalian cell chemPASS system could significantly induce protein release 30 minutes after drug addition, and the amount of induction increased with time. Figure 6 , All data are presented as n = 3 independent replicate experiments.

[0185] Example 6 Mammalian Cell ChemPASS DNV Systemically induced rapid release of insulin for the treatment of type 1 diabetes mouse models

[0186] This example uses type 1 diabetic mice as an example to demonstrate the use of mammalian cell chemPASS DNV The system is used for the treatment of type 1 diabetes, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0187] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0188] The second step is to inoculate cells (the specific steps are the same as those in Example 1 of the present invention).

[0189] Step 3: Transfection. Within 16 to 24 hours after inoculating the cells, mix 2μg pKL125, 6μg pKL72, 6μg pKL79, PEI transfection reagent and serum-free DMEM, let stand at room temperature for 15 minutes, and then evenly add to a 10cm cell culture dish. The total volume of each dish is 2mL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, replace with 10mL DMEM medium containing 10% FBS for culture.

[0190] Step 4: Preparation of cell microcapsules (see Materials and Methods for details)

[0191] Step 5, in vivo experimental process and experimental results determination. The prepared microcapsules were transplanted to the back of type 1 diabetes model mice. 30 hours after transplantation, the mice were divided into drug administration, PBS group, wild type group and oral DNV group. All experimental mice were fasted for 4 hours before drug administration, and the blood glucose of all mice was monitored by blood sampling from the tail vein at different times after drug administration (0-120 minutes). Blood samples were taken from the inner canthus of the eye at 15 minutes and 90 minutes after illumination to measure the release of insulin in the blood of diabetic model mice in the drug administration group and the non-drug administration group.

[0192] The results showed that mammalian cell chemPASS DNV The system can also quickly induce insulin release in mice, and the blood sugar of diabetic mice in the treatment group basically dropped to normal levels after 120 minutes of light exposure. In this example, the hypoglycemic protein insulin was used as the target protein, and the insulin rapid release system regulated by danoprevir was transplanted into the type 1 diabetes mouse model in the form of microcapsules. The application potential of the protein rapid release system regulated by danoprevir in disease treatment was verified in the type 1 diabetes mouse model. For detailed experimental data, please see the attached Figure 7 .

[0193] Example 7 Response of mammalian cell antigenPASS system to different antigen stimulations

[0194] This example uses nanoLuciferase as the target protein to verify the response of the mammalian cell antigenPASS system to different antigenic stimuli, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0195] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0196] The second step is virus infection. 6 Jurkat T cells were inoculated into 12-well plates, and antiCD19-synNotch-TEVp or antiEGFP-synNotch-TEVp and nanoLuci-TM-KKRN were added, with a multiplicity of infection of MOI = 3, and 6-8 μg / ml Polybrene was added to increase the infection efficiency. After 24 hours of infection, the virus was removed by centrifugation and the cells were placed in an incubator for further culture.

[0197] Step 3: Expression of tumor antigens. HEK293T cells were plated according to Example 1, 300 ng of pRC64 or pZQ546 was mixed with PEI transfection reagent and serum-free DMEM, and then evenly added to a 24-well plate after standing at room temperature for 15 min. After transfection for 6 h, 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture was added to continue culturing.

[0198] Step 4: Engineered Jurkat T cells are co-incubated with target cells. 5 Engineered Jurkat T cells were co-cultured with 2 × 10 5 CD19 / EGFP-negative or CD19 / EGFP-positive HEK293T cells were co-incubated in a 48-well plate for 24 hours, and the expression level of nanoLuci in the supernatant was collected. (For specific methods, see Materials and Methods)

[0199] The results showed that when engineered Jurkat T cells were stimulated by specific antigens, they were able to release the target protein. Figure 8 , All data are presented as n = 3 independent replicate experiments.

[0200] Example 8: Mammalian cell antigenPASS system induces the release of perforin and granzyme B and mediates target cell killing

[0201] This example uses perforin and granzyme B as target proteins to verify the cell killing mediated by the antigenPASS system of mammalian cells, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0202] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0203] Step 2: Virus infection. (Specific steps are the same as those in Example 1 of the present invention)

[0204] Step 3: Expression of tumor antigens. (Specific steps are the same as those in Example 1 of the present invention)

[0205] Step 4: Co-incubation of engineered Jurkat T cells with CD19-positive cells. Engineered Jurkat T cells were co-incubated with CD19-negative or -positive HEK293T cells at a ratio of 1:1. After 24 hours of incubation, cells were collected and flow cytometry was used to calculate the cell killing efficiency.

[0206] The results showed that when engineered Jurkat T cells were co-incubated with CD19-positive cells, they could release perforin and granzyme B, and the released cytokines could kill target cells. Fig. 9 , All data are presented as n = 3 independent replicate experiments.

[0207] Example 9 Screening of pMag / nMag and split TVMV protease fusion expression in the mammalian cell optoPASS system

[0208] This example uses SEAP as the target protein to verify the screening of pMag / nMag and split TVMV protease fusion expression in the mammalian cell optoPASS system, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0209] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0210] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0211] The third step is transfection. The inoculated cells were divided into a light group and a dark group, and each group was divided into 8 subgroups. pZQ428 and pYZ408 and pYZ409, pYZ408 and pZQ363, pYZ409 and pZQ364, pZQ363 and pZQ364, pZQ365 and pZQ367, pZQ366 and pZQ367, pZQ365 and pZQ368, pZQ366 and pZQ368 were added to each group at a ratio of 1:2:2 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM, and then allowed to stand at room temperature for 15 minutes and added to a 24-well plate. The total transfection volume of each well was 50 μL, the mass ratio of plasmid to PEI was 1:3, and 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture was added for culture 6 hours after transfection.

[0212] Step 4: Illumination. 32 hours after transfection, place the 24-well plate under a light source with a wavelength of 450 nm and an intensity of 0.5 mW / cm 2 The LED was irradiated for 24 h in a 1 min on / 2 min off cycle.

[0213] Step 5: Detection. After the illumination, the cell supernatant of the illumination group and the dark group was taken to measure the release of SEAP. (For specific methods, please refer to Materials and Methods)

[0214] The results showed that the combination of nMag-cTVMVp and nTVMVp-pMag in the blue light-regulated protein rapid release system of mammalian cells had the best multiple of protein rapid release. Fig.10 , All data are presented as n = 3 independent replicate experiments.

[0215] Example 10 Effect of nuclear localization signal on the rapid release rate of induced protein in the optoPASS system of mammalian cells

[0216] This example uses SEAP as the target protein to verify the effect of nuclear localization signals on the rapid release rate of induced proteins in the optoPASS system of mammalian cells, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0217] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0218] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0219] Step 3, transfection. The inoculated cells were divided into a light group and a dark group, each group was divided into 9 groups, and pZQ428 and pZQ365 and pZQ367, pYQ69 and pZQ367, pYQ68 and pZQ367, pYQ71 and pYQ365, pYQ69 and pYQ71, pYQ68 and pYQ71, pYQ70 and pZQ365, pYQ69 and pYQ70, pYQ68 and pYQ71 were added to each group at a ratio of 1:2:2 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM, and then allowed to stand at room temperature for 15 minutes and added to a 24-well plate. The total transfection volume of each well was 50 μL, the mass ratio of plasmid to PEI was 1:3, and 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture was added for culture 6 hours after transfection.

[0220] The fourth step is illumination (the specific steps are the same as those in Example 9 of the present invention).

[0221] The fifth step is detection (the specific steps are the same as those in Example 9 of the present invention).

[0222] The results showed that when the nMag-cTVMVp fusion protein in the mammalian cell optoPASS system had a nuclear localization signal and the nTVMVp-pMag fusion protein had a nuclear export signal, the system induced the highest multiple of protein rapid release. Fig.11 All data are presented as n = 3 independent replicate experiments.

[0223] Example 11 Effect of the connecting peptide of nMag-cTVMVp and nTVMVp-pMag fusion proteins in the mammalian cell optoPASS system on the rate of rapid protein release

[0224] This example uses SEAP as the target protein to verify the effect of the connecting peptide of nMag-cTVMVp and nTVMVp-pMag fusion protein in the mammalian cell optoPASS system on the rapid release rate of the induced protein, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0225] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0226] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0227] The third step is transfection. The inoculated cells were divided into a light group and a dark group, and each group was divided into 4 groups. pZQ428 and pYQ68 and pYQ71, pYQ68 and pZQ396, pYQ71 and pYQ393, pZQ393 and pZQ396 were added to each group at a ratio of 1:2:2 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM, and allowed to stand at room temperature for 15 minutes before adding to a 24-well plate. Among them, the total volume of each well transfection was 50 μL, the mass ratio of plasmid to PEI was 1:3, and 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture was replaced with culture after 6 hours of transfection.

[0228] The fourth step is illumination (the specific steps are the same as those in Example 9 of the present invention).

[0229] The fifth step is detection (the specific steps are the same as those in Example 9 of the present invention).

[0230] The results showed that in the mammalian cell optoPASS system, when the connecting peptide between the nMag-cTVMVp and nTVMVp-pMag fusion proteins was EAAAK, the system induced the highest multiple of protein rapid release. Fig.12 All data are presented as n = 3 independent replicate experiments.

[0231] Example 12 Effect of the localization of nTVMVp-pMag fusion protein in the mammalian cell optoPASS system on the rate of rapid protein release induced by the system

[0232] This example uses SEAP as the target protein to verify the effect of the localization of the nTVMVp-pMag fusion protein in the mammalian cell optoPASS system on the rapid release rate of the system-induced protein, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0233] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0234] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0235] The third step is transfection. The inoculated cells were divided into a light group and a dark group, and each group was divided into two small groups. pZQ428 and pZQ396 and pKL20, pZQ439 and pKL20 were added to each group in a ratio of 2:3:7 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM. The mixture was allowed to stand at room temperature for 15 minutes and added to a 24-well plate. The total volume of each well transfected was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture was added for culture.

[0236] The fourth step is illumination (the specific steps are the same as those in Example 9 of the present invention).

[0237] The fifth step is detection (the specific steps are the same as those in Example 9 of the present invention).

[0238] The results showed that in the mammalian cell blue light-regulated protein rapid release system, when the nTVMVp-pMag fusion protein was localized on the endoplasmic reticulum membrane, the system induced protein rapid release with the highest multiple. Fig.13 All data are presented as n = 3 independent replicate experiments.

[0239] Example 13 Response of mammalian cell optoPASS system to blue light stimulation of different intensities

[0240] This example uses SEAP as the target protein to verify the response of the optoPASS system of mammalian cells to blue light stimulation of different intensities, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0241] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0242] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0243] The third step is transfection. The inoculated cells were divided into a light group and a dark group, and each group was divided into 7 groups. pZQ428, pZQ439, and pKL20 were added to each group in a ratio of 2:3:7 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM, and then allowed to stand at room temperature for 15 minutes and added to a 24-well plate. Among them, the total volume of each well transfection was 50 μL, the mass ratio of plasmid to PEI was 1:3, and 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin and streptomycin mixture was replaced with culture medium 6 hours after transfection.

[0244] Step 4: Illumination. 32 hours after transfection, the cells were exposed to light at a wavelength of 450 nm and different intensities (0-2 mW / cm2 ) (refer to the experimental materials and methods for the specific connection method) for 1 min and then immediately placed in the dark after light treatment.

[0245] Step 5: Detection. Take the cell supernatant at different times (15-120 min) after the end of illumination to measure the amount of SEAP released. (For specific methods, refer to Materials and Methods)

[0246] The results showed that when the mammalian cell optoPASS system was irradiated with blue light for 1 minute, the rate of protein rapid release increased with the increase of light intensity. When the stimulation lasted for about 1 minute, the light intensity was 0.5 mW / cm 2 The ability of the system to induce rapid protein release no longer increases with increasing light intensity. Fig.14 , All data are presented as n = 3 independent replicate experiments.

[0247] Example 14 Response of mammalian cell blue light regulated protein rapid release system to blue light stimulation at different times

[0248] This example uses SEAP as the target protein to verify the response of the optoPASS system of mammalian cells to blue light stimulation at different times, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0249] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0250] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0251] Step 3: Transfection. Divide the inoculated cells into 7 groups, add pZQ428, pZQ439, and pKL20 to each group in a ratio of 2:3:7 (w:w:w), mix with PEI transfection reagent and serum-free DMEM, let stand at room temperature for 15 minutes, and add to a 24-well plate. The total volume of each well transfected is 50 μL, the mass ratio of plasmid to PEI is 1:3, and 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture is added after transfection for 6 hours for culture.

[0252] Step 4: Illumination. 32 hours after transfection, one group of cells was placed in the dark, and the rest of the cells were placed under a light illumination of 450 nm and 0.5 mW / cm 2 The samples were irradiated with LED (refer to experimental materials and methods for specific connection methods) for different lengths of time (1-30 min) and placed in the dark immediately after light treatment.

[0253] Step 5: Detection. The specific steps are the same as those in Example 9 of the present invention.

[0254] The results showed that the mammalian cell optoPASS system was activated by blue light at 0.5 mW / cm 2 Under the condition of , the ability to induce rapid protein release increases with the increase of light exposure time. Fig.15 , All data are presented as n = 3 independent replicate experiments.

[0255] Example 15: Rapid release of proteins induced by the optoPASS system in mammalian cells

[0256] This example uses SEAP as the target protein to verify the speed of rapid protein release induced by the optoPASS system in mammalian cells, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0257] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0258] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0259] The third step is transfection. The inoculated cells were divided into a light group and a dark group. pZQ428, pZQ439, and pKL20 were added to each group at a ratio of 2:3:7 (w:w:w) and mixed with PEI transfection reagent and serum-free DMEM. The mixture was allowed to stand at room temperature for 15 minutes and added to a 24-well plate. The total volume of each well for transfection was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS and 1% (volume / volume) penicillin and streptomycin mixture was added for culture.

[0260] Step 4: Illumination. 32 hours after transfection, the dark group cells were placed in the dark, and the rest of the cells were placed under a light with a wavelength of 450nm and an intensity of 0.5mW / cm 2 The samples were irradiated with LED (refer to experimental materials and methods for specific connection method) for 5 min and then placed in darkness immediately after light treatment.

[0261] Step 5: Detection: After different time periods (5-120 min) after the illumination, the supernatant of the cell fluid was taken to determine the amount of SEAP released (for specific methods, refer to Materials and Methods).

[0262] The results showed that the mammalian cell optoPASS system was activated by blue light at 0.5 mW / cm 2 After 5 minutes of illumination, the protein release was obviously induced, and the induction amount increased with the extension of time. Fig.16 , All data are presented as n = 3 independent replicate experiments.

[0263] Example 16 Study on the reversibility of the optoPASS system in mammalian cells

[0264] This example uses SEAP as the target protein to verify the reversibility of the optoPASS system in mammalian cells, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0265] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0266] Step 2: Inoculate cells. (Specific steps are the same as those in Example 1 of the present invention)

[0267] The third step is transfection. Divide 4 24-well plates into OFF group and ON group, select 3 wells in each group as a group, and divide them into 15 groups in total. Add pZQ428, pZQ439, and pKL120 to each group in a ratio of 2:3:7 (w / w / w), mix with PEI transfection reagent and serum-free DMEM, let stand at room temperature for 15 minutes, and then evenly add to the 24-well culture plate. The total preparation volume of each well is 50μL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, replace with 500μL of DMEM medium containing 10% FBS, 1% (volume / volume) penicillin, streptomycin mixture and 5μM PCB for culture.

[0268] Step 4: Illumination. 32 hours after transfection, the 24-well plates of the OFF group were placed in the dark to avoid light, and the 24-well plates of the ON group were placed in a light with a wavelength of 450 nm and an intensity of 0.5 mW / cm 2 The cells were irradiated with LED (refer to experimental materials and methods for specific connection methods) for 1 min and placed in the dark immediately after the light treatment. At the same time, the corresponding 24-well plate was changed to a light condition (450 nm / darkness) and fresh culture medium every 2 h.

[0269] Step 5: Detection. After each illumination, take the samples from the 24-well plates in the OFF group and the 24-well plates in the ON group.

[0270] A corresponding group of cell culture supernatants were collected and the expression level of SEAP was determined (for specific methods, see Materials and Methods).

[0271] The results show that the mammalian cell optoPASS system has good reversibility and controllability. Fig.17 , All data are presented as n = 3 independent replicate experiments.

[0272] Example 17: Working status of the mammalian cell optoPASS system in different cell lines

[0273] This example uses SEAP and green fluorescent protein as target proteins to verify the working conditions of the mammalian cell optoPASS system in different cell lines, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0274] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0275] Step 2: Cell inoculation. HEK-293T cells, Hana3A cells, hMSC-TERT cells, and iPSC cells that are in good growth condition were digested with 0.25% trypsin and inoculated into two different 24-well plates (each cell type was inoculated into 4 wells on each plate), with 6×10 cells per well. 4 cells and add 500 μL of DMEM medium containing 10% FBS.

[0276] The third step is transfection. The inoculated cells were divided into a light group and a dark group, each group was divided into 8 groups. 16-18 hours after the cells were inoculated, pKL428, pZQ439, and pKL120 were added to the HEK-293T cell group, Hana3A cell group, and hMSC-TERT cell group at a ratio of 2:3:7 (w / w / w) and mixed with PEI transfection reagent and serum-free DMEM. After standing at room temperature for 15 minutes, the mixture was evenly added to a 24-well culture plate. The total preparation volume of each well was 50 μL, and the mass ratio of plasmid to PEI was 1:3. After 6 hours of transfection, 500 μL of DMEM medium containing 10% FBS, 1% (volume / volume) of penicillin and streptomycin mixture and 5 μM PCB was replaced for culture. Add pKL32, pZQ439, and pKL120 to the Mouse iPSC cell group at a ratio of 2:3:7 (w / w / w) and mix well with Lip3000TM transfection reagent, and follow the instructions in Materials and Methods.

[0277] The fourth step is illumination. The dark group cells were placed in darkness, and the light group cells were placed in the light group at a wavelength of 450nm and an intensity of 0.5mW / cm 2 The samples were illuminated under an LED light (refer to the experimental materials and methods for the specific connection method) for 5 min and then immediately placed in darkness after illumination treatment.

[0278] Step 5: Detection. For the HEK-293T cell group, Hana3A cell group, and hMSC-TERT cell group, the cell supernatant was taken at different times (0-4h) after the end of illumination to determine the release of SEAP. For the mouse iPSC cell group, green fluorescent protein was photographed using an inverted fluorescence microscope at different times (0-4h) after the end of illumination, and the fluorescence intensity of green fluorescent protein was statistically analyzed using imageJ software.

[0279] The results show that the mammalian cell optoPASS system can work in a variety of different cell lines and has a broad spectrum. Fig.18 , attached Fig.18 The data in (AC) are presented in the form of n = 3 independent replicate experiments; Fig.18 The data in (D) are presented in the form of n=5 independent replicate experiments. Example 18 The rapid release of atrial natriuretic peptide (ANP) induced by the optoPASS system in mammalian cells is used to treat a hypertensive mouse model

[0280] This example uses hypertensive diabetic mice as an example to illustrate the treatment of hypertension by the mammalian cell optoPASS system, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0281] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0282] Step 2: Inoculate cells. HEK-293T cells that are growing well were digested with 0.25% trypsin and then inoculated in 10 cm cell culture dishes, with 4×10 cells per dish. 6 cells and add 10 mL of DMEM medium containing 10% FBS.

[0283] Step 3: Transfection. Within 16 to 24 hours after inoculating the cells, mix 2μg pKL84, 4μg pZQ439, 8μg pKL120, PEI transfection reagent and serum-free DMEM, let stand at room temperature for 15 minutes, and then evenly drop into a 10cm cell culture dish. The total volume of each dish is 2mL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, replace with 10mL DMEM medium containing 10% FBS for culture.

[0284] Step 4: Preparation of cell microcapsules (see Materials and Methods for details)

[0285] Step 5: In vivo experimental process and experimental results. The prepared microcapsules were transplanted into the backs of the hypertensive model mice, and the transplanted mice were placed in the dark. 30 hours after the transplantation, the mice were divided into a dark group and a light group. The dark group was placed in the dark, and the light group was placed in a light with a wavelength of 450nm and an intensity of 10mW / cm 2 The mice were illuminated under a blue light. The blood pressure of the mice was monitored by tail pressure method at different times (0-120min) during illumination. The blood pressure of the non-transplanted group and wild-type mice was also monitored. Blood samples were taken from the inner canthus of the eyeball 15min and 90min after illumination to measure the release of ANP in the blood of the hypertensive model mice in the dark group and the light group.

[0286] The results showed that the mammalian cell optoPASS system can also quickly induce protein release in mice, and the blood pressure of the hypertensive mice in the treatment group was basically reduced to normal levels after 2 hours of light exposure. In this example, the antihypertensive protein ANP was used as the target protein, and the blue light-regulated ANP rapid release system was transplanted into the hypertensive mouse model in the form of microcapsules. The application potential of the optoPASS system in disease treatment was verified in the hypertensive mouse model. For detailed experimental data, please see the attached Fig.19 .

[0287] Example 19: Rapid release of insulin induced by the optoPASS system in mammalian cells for the treatment of type 1 diabetes mouse model

[0288] This example uses hypertensive diabetic mice as an example to illustrate the treatment of hypertension by the mammalian cell optoPASS system, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0289] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0290] The second step is to inoculate cells (the specific steps are the same as those in Example 18 of the present invention).

[0291] Step 3: Transfection. Within 16 to 24 hours after inoculating the cells, mix 2μg pZQ475, 4μg pZQ439, 8μg pKL120, PEI transfection reagent and serum-free DMEM, let stand at room temperature for 15 minutes, and then evenly add to a 10cm cell culture dish. The total volume of each dish is 2mL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, replace with 10mL DMEM medium containing 10% FBS for culture.

[0292] Step 4: Preparation of cell microcapsules (see Materials and Methods for details)

[0293] Step 5: In vivo experimental process and experimental results. The prepared microcapsules were transplanted into the backs of type 1 diabetes model mice, and the transplanted mice were placed in darkness. 30 hours after transplantation, the mice were divided into a dark group and a light group. The dark group was placed in darkness, and the light group was placed under a wavelength of 450nm and a light intensity of 10mW / cm 2 All experimental mice were fasted for 4 hours before illumination. Blood glucose was monitored by taking blood from the tail vein at different times (0-120 minutes) during illumination. Blood pressure of the non-transplanted group and wild-type mice was also monitored. Blood samples were taken from the inner canthus of the eyeball 15 minutes and 90 minutes after illumination to measure the release of insulin in the blood of diabetic model mice in the dark group and the light group.

[0294] The results showed that the mammalian cell optoPASS system can also rapidly induce insulin release in mice, and the blood sugar of diabetic mice in the treatment group basically dropped to normal levels after 90 minutes of light exposure. In this example, the hypoglycemic protein insulin was used as the target protein, and the blue light-regulated insulin rapid release system was transplanted into the type 1 diabetic mouse model in the form of microcapsules to verify the application potential of the optoPASS system in disease treatment in the type 1 diabetic mouse model. For detailed experimental data, please see the attached Fig. 20 .

[0295] Example 20: Mammalian cell optoPASS system induces rapid release of analgesic peptide ssHwTx for the treatment of chronic inflammatory pain mouse model

[0296] This example uses chronic inflammatory pain mice as an example to illustrate the treatment of pain mouse models by the mammalian cell optoPASS system, but does not limit the scope of protection of the present invention. The specific steps are as follows:

[0297] The first step is plasmid construction. The plasmid construction in this example is shown in Table 1.

[0298] The second step is to inoculate cells (the specific steps are the same as those in Example 18 of the present invention).

[0299] Step 3: Transfection. Within 16 to 24 hours after inoculating the cells, mix 2μg pKL46, 4μg pZQ439, 8μg pKL120, PEI transfection reagent and serum-free DMEM, let stand at room temperature for 15 minutes, and then evenly drop into a 10cm cell culture dish. The total volume of each dish is 2mL, and the mass ratio of plasmid to PEI is 1:3. After 6 hours of transfection, replace with 10mL DMEM medium containing 10% FBS for culture.

[0300] Step 4: Preparation of cell microcapsules (see Materials and Methods for details)

[0301] Step 5: In vivo experimental process and experimental results. The prepared microcapsules were transplanted into the backs of pain model mice, and the transplanted mice were placed in darkness. 30 hours after transplantation, the mice were divided into a dark group and a light group. The dark group was placed in darkness, and the light group was placed under a wavelength of 450nm and a light intensity of 10mW / cm 2 The mice were illuminated under a blue light. During the illumination, the blood pressure of the mice was monitored in a top-down manner at different times (0-120 min) to determine the pain threshold of the mice (see Materials and Methods for specific measurement methods). The non-transplanted group and wild-type mice were also monitored. Blood samples were taken from the inner canthus of the eyeball 15 min and 90 min after illumination to measure the release of the analgesic peptide ssHwTx in the blood of the diabetic model mice in the dark group and the light group.

[0302] The results showed that the mammalian cell optoPASS system can also quickly induce the release of the analgesic peptide ssHwTx in mice, and the pain threshold of the pain mice in the treatment group was close to the normal level after 120 minutes of light exposure. In this example, the analgesic peptide ssHwTx was used as the target protein, and the blue light-regulated ssHwTx rapid release system was transplanted into the type 1 diabetes mouse model in the form of microcapsules. The application potential of the optoPASS system in disease treatment was verified in the chronic inflammatory pain mouse model. For detailed experimental data, please see the attached Fig.21 .

[0303] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

[0304] Table 1. Plasmid construction table

[0305]

[0306]

[0307]

[0308] SEQ ID NO.1: Fcs amino acid sequence RKKR

[0309] SEQ ID NO.2:CD4 amino acid sequence

[0310] ESNIKVLPTWSTPVQPMALIVLGGVAGLLLFIGLGIFFCVRCRHRRR

[0311] SEQ ID NO.3: TEVcs amino acid sequence

[0312] ENLYFQS

[0313] SEQ ID NO.4: TVMVcs amino acid sequence

[0314] ETVRFQS

[0315] SEQ ID NO.5: Endoplasmic reticulum recycling signal

[0316] KKR

[0317] SEQ ID NO.6: Endoplasmic reticulum recycling signal

[0318] KKML

[0319] SEQ ID NO.7: Endoplasmic reticulum recycling signal

[0320] KVV

[0321] SEQ ID NO.8: Endoplasmic reticulum recycling signal

[0322] KKFL

[0323] SEQ ID NO.9: Endoplasmic reticulum recycling signal

[0324] KKT

[0325] SEQ ID NO.10: Endoplasmic reticulum recycling signal

[0326] KKTI

[0327] SEQ ID NO.11: NS3a amino acid sequence

[0328] MKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSINGVLWT

[0329] VYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSR

[0330] GSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPVESLETTMRSP

[0331] SEQ ID NO.12: DNCR2 amino acid sequence

[0332] SSDEEEARELIERAKEAAERAQEAAERTGDPRVRELARELKRLAQEAAEEVKRDPSSSDVNEALKLIVE

[0333] AIEAAVDALEAAERTGDPEVRELARELVRLAVEAAEEVQRNPSSSDVNEALHSIVYAIEAAIFALEAAE

[0334] RTGDPEVRELARELVRLAVEAAEEVQRNPSSRNVEHALMRIVLAIYLAEENLREAEESGDPEKREKARE

[0335] RVREAVERAEEVQRDPSGWLNH

[0336] SEQ ID NO.13: Amino acid sequence of GNCR1

[0337] DIEKLCKKAEEEAKEAQEKADELRQRHPDSQAAEDAEDLANLAVAAVLTACLLAQEHPNADIAKLCIKA

[0338] ASEAAEAASKAAELAQRHPDSQAARDAIKLASQAARAVILAIMLAAENPNADIAKLCIKAASEAAEAAS

[0339] KAAELAQRHPDSQAARDAIKLASQAAEAVERAIWLAAENPNADIAKKCIKAASEAAEEASKAAEEAQRH

[0340] PDSQKARDEIKEASQKAEEVKERCKS

[0341] SEQ ID NO.14: Amino acid sequence of antiCD19-synNotch

[0342] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGT

[0343] DYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQ

[0344] SLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSL

[0345] QTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSILDYSFTGGAGRDIPPPQIEEACELPECQVDAGN

[0346] KVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCN

[0347] PLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSH

[0348] VLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVY

[0349] LEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVL

[0350] LFFVGCGVLLSRKRRR

[0351] SEQ ID NO.15: Amino acid sequence of pMag

[0352] MHTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVL

[0353] GRNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETG

[0354] EYRYSMGFQCETE

[0355] SEQ ID NO.16: Amino acid sequence of nMag

[0356] HTLYAPGGYDIMGYLDQIGNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGYSNAEVLG

[0357] RNCRFLQSPDGMVKPKSTRKYVDSNTINTIRKAIDRNAEVQVEVVNFKKNGQRFVNFLTIIPVRDETGE

[0358] YRYSMGFQCETE

[0359] SEQ ID NO.17: Amino acid sequence of cTEVp

[0360] KSMSSMVSDTSCTFPSSDGTFWKHWIQTKDGQCGNPLVSTRDGFIVGIHSASNFTNTNNYFASVPKNFM

[0361] ELLTNQEAQQWVSGWRLNADSVLWGGHKVFMVKPEEPFQPVKEATQLMN

[0362] SEQ ID NO.18: nTEVp amino acid sequence

[0363] GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNT

[0364] TTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQT

[0365] SEQ ID NO.19: LaG17 amino acid sequence

[0366] MADVQLVESGGGLVQAGGSLRLSCAASGRTISMAAMSWFRQAPGKEREFVAGISRSAGSAVHADSVKGR

[0367] FTISRDNTKNTLYLQMNSLKAEDTAVYYCAVRTSGFFGSIPRTGTAFDYWGQGTQVTVS

[0368] SEQ ID NO.20: cTVMVp amino acid sequence

[0369] KSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGNILGIHSLTHTTNGSYYFVEFPEKFV

[0370] ATYLDAADGWCKNWKFNADKISWGSFTLVEDAPEDDFMAKKTVAAIMD

[0371] SEQ ID NO.21: nTVMVp amino acid sequence

[0372] SKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYIIANQHLFRRNNGELTIKTMHGEFKVKNS

[0373] TQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQPTIKDRVCMVSTNFQQ

[0374] SEQ ID NO.22: Nuclear localization signal NLS amino acid sequence

[0375] PKKKRKV

[0376] SEQ ID NO.23: Nuclear export signal NES amino acid sequence

[0377] LPPLERLTL

[0378] SEQ ID NO.24: amino acid sequence of connecting peptide L1

[0379] GGGGSGGGGSGGGGSGGGGS

[0380] SEQ ID NO.25: amino acid sequence of connecting peptide L2

[0381] EAAAK

Claims

1. A programmable protein rapid secretion and release system, characterized in that: include: Clinically approved drug-regulated protein rapid secretion and release system, protein antigen signal-regulated protein rapid secretion and release system, and blue light-regulated protein rapid secretion and release system; The clinically approved drug-regulated protein rapid secretion and release system comprises: a target protein endoplasmic reticulum recycler and a clinical drug receptor; The target protein endoplasmic reticulum recycler comprises: the target protein -COOH end is fused with the Golgi furin recognition site Fcs, the CD4 transmembrane domain TM, the tobacco etch virus protease TEVp recognition site TEVcs, the tobacco vein spot virus protease TVMVp recognition site TVMVcs and the endoplasmic reticulum protein recycling signal; The amino acid sequence of the furin recognition site Fcs is shown in SEQ ID NO.1, the amino acid sequence of the CD4 transmembrane domain TM is shown in SEQ ID NO.2, the amino acid sequence of the tobacco etch virus protease TEVp recognition site TEVcs is shown in SEQ ID NO.3, the amino acid sequence of the tobacco vein spot virus protease recognition site TVMVcs is shown in SEQ ID NO.4, and the endoplasmic reticulum protein recovery signal is shown in SEQ ID NO.5-10; The clinical drug receptors include: danoprevir receptor and grazoprevir receptor; the danoprevir receptor includes: hepatitis C virus NS3 protease domain NS3a and its interacting protein DNCR2; the grazoprevir receptor includes: hepatitis C virus NS3 protease domain NS3a and its interacting protein GNCR1; The amino acid sequence of the hepatitis C virus NS3 protease domain NS3a is shown in SEQ ID NO.11, the amino acid sequence of the protein DNCR2 is shown in SEQ ID NO.12, and the amino acid sequence of the protein GNCR1 is shown in SEQ ID NO.13; The fusion protein of NS3a and split TEV protease and the fusion protein of DNCR2 and split TEV protease were used as the danoprevir receptors of the system; The fusion protein of NS3a and split TEV protease, and the fusion protein of GNCR1 and split TEV protease were used as the system's grazoprevir receptors; The protein antigen signal-regulated protein rapid secretion and release system comprises: a target protein endoplasmic reticulum recycler and a tumor antigen receptor; the tumor antigen receptor comprises: a single-chain variable region of a tumor antigen monoclonal antibody, a core structural region of a Notch receptor, and a TEV protease; The amino acid sequence of the single-chain variable region of the tumor antigen monoclonal antibody fused with the core domain of the Notch receptor and the TEV protease is shown in SEQ NO.14; The blue light-regulated protein rapid release system comprises: a target protein endoplasmic reticulum recycler and a blue light receptor; The blue light receptor comprises: a blue light response protein pMag and an interacting protein nMag; The amino acid sequence of the blue light responsive protein pMag is shown in SEQ ID NO.15, and the amino acid sequence of its interacting protein nMag is shown in SEQ ID NO.16; The fusion protein of pMag and split TVMV protease, the fusion protein of nMag and split TVMV protease, the nuclear localization / nuclear export signals of the above two fusion proteins and the connecting peptide between the above two fusion proteins are used as the blue light receptors of the system.

2. A method for constructing a programmable protein rapid secretion and release system, characterized in that: The following steps are involved: (1) Construction of the endoplasmic reticulum recycling device for target protein Construct a fusion protein of the target protein with the furin recognition site, the CD4 transmembrane domain, the TEV and TVMV protease recognition sites, and the endoplasmic reticulum recycling signal; (2) Construction of Danoprevir receptor Constructing a fusion protein of NS3a / DNCR2 and split TEV protease; (3) Construction of grazoprevir receptor Construction of fusion protein of NS3a / GNCR1 and split TEV protease; (4) Construction of tumor antigen receptors Constructing a fusion protein of the single-chain variable region of CD19 tumor antigen or EGFP antigen monoclonal antibody, the Notch receptor core structural region, and TEV protease; (5) Construction of blue light receptors The fusion protein of pMag and split TVMV protease, the fusion protein of nMag and split TVMV protease, the nuclear localization / nuclear export signals of the above two fusion proteins and the connecting peptide between the above two fusion proteins were constructed as the blue light receptor of the system.

3. The construction method according to claim 2, characterized in that: In step (2), the fusion protein of NS3a and the split TEV protease includes: NS3a-nTEVp, NS3a-cTEVp, nTEVp-NS3a, cTEVp-NS3a; the fusion protein of DNCR2 and the split TEV protease includes: DNCR2-nTEVp, DNCR2-cTEVp, nTEVp-DNCR2, cTEVp-DNCR2, and the amino acid sequences of the split TEV proteases are shown in SEQ ID NO.17-18 respectively.

4. The construction method according to claim 2, characterized in that: In step (3), the fusion protein of NS3a and the split TEV protease includes: NS3a-nTEVp, NS3a-cTEVp, nTEVp-NS3a, cTEVp-NS3a; the fusion protein of GNCR1 and the split TEV protease includes: GNCR1-nTEVp, GNCR1-cTEVp, nTEVp-GNCR1, cTEVp-GNCR1.

5. The construction method according to claim 2, characterized in that: In step (4), the single-chain variable region of the CD19 tumor antigen or EGFP antigen monoclonal antibody is constructed, and its amino acid sequence is shown in SEQ ID NO.14 and SEQ ID NO.19 respectively.

6. The construction method according to claim 2, characterized in that: In step (5), the fusion protein of pMag and the split TVMV protease includes: pMag-nTVMVp, pMag-cTVMVp, nTVMVp-pMag, cTVMVp-pMag; the fusion protein of nMag and the split TVMV protease includes: nMag-nTVMVp, nMag-cTVMVp, nTVMVp-nMag, cTVMVp-nMag, and the amino acid sequences of cTVMVp and nTVMVp are shown in SEQ ID NO.20 and SEQ ID NO.21, respectively.

7. A eukaryotic expression vector, characterized in that: The eukaryotic expression vector is the eukaryotic expression vector of the program-controlled rapid protein secretion and release system as claimed in claim 1.

8. A host cell and / or engineered cell transplantation vector, characterized in that: The host cells and / or engineered cell transplantation vectors are transfected with the programmable protein rapid secretion and release system as claimed in claim 1.

9. Use of the programmable protein rapid secretion and release system as claimed in claim 1, or the eukaryotic expression vector as claimed in claim 7, or the host cell and / or engineered cell transplantation vector as claimed in claim 8 in mammalian genetic engineering.

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