Fusion protein for regulating pancreatic beta cell function based on optogenetics technology and application of fusion protein

Abstract

The invention discloses a fusion protein for regulating and controlling functions of pancreatic beta cells based on an optogenetics technology and application of the fusion protein, and relates to the technical field of biology. The fusion protein Opto LOVTRAP disclosed by the invention has very high temporal-spatial resolution, and rapid and accurate control on a single cell can be realized by adjusting blue light; opto LOVTRAP has reversibility, can effectively inhibit a pancreatic beta cell Ca < 2 + > channel under a dark condition, and can quickly stop playing a role after being stimulated by blue light, so that the purpose of reversibly and accurately regulating insulin secretion is achieved. Opto LOVTRAP is targeted to regulate the concentration of Ca < 2 + > in cells, and is a final triggering factor for release of pancreas islet vesicles, so that the opto LOVTRAP is not influenced by the KATP function, and the purpose of breaking through the bottleneck of a traditional clinical treatment mode is achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fusion protein based on optogenetics to regulate the function of pancreatic β cells and its applications. Background Technology

[0002] Congenital hyperinsulinism (CHI) is primarily caused by mutations in genes regulating insulin secretion from pancreatic β-cells, leading to dysregulation of insulin secretion. A key characteristic is the excessive insulin secretion by β-cells even during hypoglycemia. CHI is a major cause of refractory hypoglycemia in infants and young children, often presenting with seizures and coma during the neonatal or early childhood period. This can lead to irreversible brain damage, and delayed treatment can even result in death. The risk of permanent brain damage in infants with CHI is as high as 25%-50% if persistent, refractory hypoglycemia is not diagnosed and treated promptly. The etiology of this disease is complex, with a wide range of clinical manifestations, resulting in a very high risk of neurodevelopmental disorders. Therefore, clarifying the pathogenesis of CHI and achieving early diagnosis and treatment of congenital hyperinsulinemia is crucial.

[0003] The current treatment options for CHI are divided into two categories: surgical treatment and drug treatment. Its intervention methods are limited, mainly presenting the following problems: (1) Adenosine triphosphate (ATP)-sensitive potassium ion channels (K... ATP Diazide is the only first-line drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of CHI (congenital heart disease), and its main target is K+. ATP It is a potassium ion channel opener. Therefore, diazoxide is effective against potassium ion channels. ATP Ineffective for children with impaired channel function, only effective for K ATP K pathway is either fully or dominantly inherited. ATP The mutation-induced CHI is effective. However, most persistent CHI flare-ups are mainly caused by K. ATP The effects of diazoxide on patients vary greatly due to mutations in key gene mutation sites. In addition, diazoxide is not specific to pancreatic tissue and is prone to various adverse reactions, such as fluid retention and pulmonary hypertension, which can endanger the life of children in severe cases. Therefore, its application is limited. (2) Traditional drug treatment is lacking. In addition to diazoxide, drugs such as octreotide are also used for the chronic treatment of children with CHI, but all of them have adverse reactions. Octreotide can cause abdominal discomfort, diarrhea, gallstones, cholecystitis, etc., and growth deceleration, bradycardia and long QT syndrome can be seen; the most serious can lead to necrotizing colitis. Nifedipine has poor clinical treatment effect on CHI, and as a Ca 2+Channel blockers have significant drawbacks, including off-target effects, lack of spatial control, and irreversibility. (3) Postoperative complications are severe. If drug treatment fails, the child usually has no choice but to undergo surgery, which will lead to a variety of postoperative complications. Focal CHI patients are cured by partial pancreatectomy or selective pancreatectomy, but it should be noted that most CHI patients who undergo partial pancreatectomy have an increased risk of developing insulin-resistant diabetes. Diffuse CHI often requires near-total pancreatectomy, and nearly two-thirds of patients may develop problems such as pancreatic exocrine insufficiency, resulting in poor postoperative outcomes and a high number of complications.

[0004] Under physiological conditions, K ATP The channels remain open in resting pancreatic β cells, allowing potassium ions to flow out and maintaining the β cells in a hyperpolarized state. When plasma glucose levels rise, glucose taken up by β cells undergoes glycolysis, which, along with many biochemical processes within the mitochondria, increases the intracellular ATP / ADP ratio, leading to a rise in ATP and potassium levels. ATP Kir6.2 binding on the channel promotes K ATP Channel closure triggers pancreatic β-cell membrane depolarization, initiating voltage-gated Ca2+. 2+ Channels open, intracellular Ca 2+ Increased concentration leads to increased intracellular Ca 2+ The exocytosis of insulin-dependent secretory granules promotes insulin release; abnormalities in any of these processes can lead to CHI (congenital hypoxic-ischemic attack). Intracellular calcium... 2+ Increased insulin concentration is the ultimate trigger for insulin vesicle release; therefore, inhibiting voltage-gated Ca2+ is crucial. 2+ Channels can be considered one of the effective ways to treat CHI.

[0005] Optogenetics, combining optics and genetics, enables the precise regulation of specific biological functions in cells, tissues, and organisms. This technology achieves unprecedented levels of spatial and temporal control over cellular physiology, offering advantages such as reversibility, millisecond-level temporal accuracy, high specificity, and high spatiotemporal resolution. By employing protein engineering and synthetic biology methods, it introduces gene-encoded light-sensitive proteins to control numerous biological processes and intervene in in vivo diseases, showing great promise for the development of precision medicine. Based on these technological characteristics, optogenetics holds the potential to overcome the off-target effects, multiple effects, cytotoxicity, and signal crosstalk associated with traditional pharmacological methods. In clinical treatment, optogenetics already has practical applications, fully demonstrating its feasibility and safety. With further research in optogenetics, it will become possible to develop personalized treatments and clinical translation strategies for various diseases based on optogenetic technology.

[0006] Given the above issues, current limited treatment options are insufficient to effectively control the disease and are prone to causing a series of adverse events. Therefore, there is an urgent need to advance key research into new therapies to specifically regulate pancreatic β-cell function and effectively improve the prognosis of affected children. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a fusion protein for regulating pancreatic β-cell function based on optogenetics and its applications. Based on optogenetics, a voltage-gated Ca2+ protein for light-controlled pancreatic β-cells is developed. 2+ This channel can precisely regulate pancreatic β-cell function, reversibly modulate insulin release, and provide a new approach for the treatment of CHI.

[0008] This study employed the LOVTRAP (LOV2 Trap and Release of Protein) system, a reversible photo-induced protein dissociation optogenetic method. Specifically, LOVTRAP uses gene editing technology to anchor LOV2 to the cell membrane, while ZDK2 and Rem... 1-266 The protein (amino acids 1-266 of the Rem protein) is linked together. In the dark, LOV2 and ZDK2 bind to enable the release of Rem proteins free in the cytoplasm during the dark state. 1-266 The protein anchors to the cell membrane, inhibits calcium ion channels, regulates pancreatic β-cell function, and suppresses pathological excessive insulin release. Under conditions of elevated blood glucose (such as after eating) and light exposure, LOV2 undergoes a conformational change, causing ZDK2 attached to LOV2 to dissociate into the cytoplasm, thereby achieving calcium ion release. 2+ The opening of the channels promotes insulin release, achieving precise targeted regulation of pancreatic β-cell function. In summary, this invention utilizes the ultra-high spatial resolution and precise regulatory capabilities of optogenetics to achieve precise and reversible regulation of pancreatic β-cell function. This invention can more directly achieve physiological regulation of abnormal pancreatic β-cell function.

[0009] The specific technical solution of the present invention is as follows:

[0010] This invention provides a fusion protein for regulating pancreatic β-cell function based on optogenetics. The fusion protein comprises a first fusion protein and a second fusion protein. The first fusion protein includes, from the N-terminus to the C-terminus, a membrane localization sequence (NLYN) for anchoring the LOV2 domain of the phytosensitive protein to the pancreatic β-cell membrane, miRFPnano3 fluorescent protein, and the LOV2 domain of the phytosensitive protein. The second fusion protein includes, from the C-terminus to the N-terminus, ZDK2 for binding the LOV2 domain of the phytosensitive protein, a calcium channel inhibitor, and mTagBFP2 fluorescent protein.

[0011] The calcium channel inhibitor protein is used to inhibit the opening of calcium channels;

[0012] In the dark, the LOV2 domain of the light-sensitive protein in the first fusion protein binds to the ZDK2 binding protein in the second fusion protein, and the calcium channel inhibitor in the second fusion protein inhibits the opening of calcium channels.

[0013] Under light conditions, the LOV2 domain of the photosensitive protein in the first fusion protein and the ZDK2 binding protein in the second fusion protein dissociate, and the calcium channel inhibitor in the second fusion protein releases, causing the calcium channel to open.

[0014] The working principle of this invention: Under dark conditions, the optogenetic tool LOVTRAP causes LOV2 and ZDK2 to form a heterodimer, thereby enabling the targeting of Rem in the cytoplasm. 1-266 The spatiotemporal control of proteins inhibits voltage-gated calcium ion channels that are abnormally excited. First, LOV2 is anchored to the cell membrane. Under dark conditions, LOV2 binds to ZDK2, which can then inhibit free calcium ions in the cytoplasm. 2+ Channel inhibitors pull to voltage-gated Ca 2+ Channels that inhibit Ca 2+ Internal flow. Under illumination, the two can dissociate, thereby restoring Ca. 2+ The channel is open, enabling precise and reversible adjustment.

[0015] In some embodiments of the present invention, the membrane positioning sequence is as follows:

[0016] MGCIKSKRKDNLNDDE.

[0017] The calcium channel inhibitory protein is Rem 1-266 The protein, specifically the first 266 amino acids of the Rem protein, wherein the amino acid sequence of the first 266 amino acids of the Rem protein is as shown in the amino acid sequence of positions 252-517 of SEQ ID No. 2.

[0018] The LOV2 domain of the photosensitive protein can be selected as the AsLOV2 (Arabidopsis sativa LOV2) or AtLOV2 (Arabidopsis thaliana LOV2) domain. In the embodiments of the present invention, the AsLOV2 domain is used as an example.

[0019] Specifically, the fluorescent protein is either a red fluorescent protein or a blue fluorescent protein. The red fluorescent protein can be mCherry or miRFPnano3; the blue fluorescent protein can be mTagBFP2.

[0020] The LOV2 domain of the photosensitive protein was labeled with the near-infrared fluorescent protein miRFPnano3 to ensure its expression on the cell membrane.

[0021] Preferably, the amino acid sequence of the first fusion protein is shown in SEQ ID No. 1, and the amino acid sequence of the second fusion protein is shown in SEQ ID No. 2.

[0022] Specifically, firstly, this invention constructs a voltage-gated Ca 2+ Opto LOVTRAP (Optical Loop Transistor). K ATP CHI caused by mutations is the most common and severe type, accounting for 40%-50% of cases. However, current first-line drug treatments only target K+. ATP Effective in children with remaining functional cells. Intracellular Ca 2+ Elevated levels are the ultimate trigger for insulin vesicle release, and are K+. ATP Downstream sites. Therefore, based on optogenetics, Opto LOVTRAP was used to achieve transient and reversible regulation of voltage-gated Ca2+ in pancreatic β-cells. 2+ Channel, regardless of K ATP Children with both functional and non-functional disorders can reversibly regulate insulin release.

[0023] In addition, traditional Ca 2+ Channel blockers lack pancreatic tissue specificity, easily leading to various adverse reactions, and their clinical efficacy in treating CHI remains uncertain. Optogenetics, combining optics and genetics, can precisely regulate specific biological functions of cells, tissues, and organisms. This technology achieves unprecedented levels of spatial and temporal control over cellular physiology, offering advantages such as reversibility and high spatiotemporal resolution. Single-cell-level regulation of Ca2+ can be achieved through Opto LOVTRAP. 2+ Channels, enabling precise regulation of specific tissues or cells, show great promise for the development of precision medicine. This invention is expected to overcome the problems of off-target effects, multiple effects, cytotoxicity, and signal crosstalk caused by traditional pharmacological methods.

[0024] The present invention also provides the gene for the fusion protein.

[0025] In some embodiments of the present invention, the gene sequence encoding the first fusion protein in the gene is shown in SEQ ID No. 3, and the gene sequence encoding the second fusion protein is shown in SEQ ID No. 4.

[0026] The present invention also provides the use of the fusion protein in the preparation of a medicament for treating diseases caused by insulin secretion disorders of pancreatic β cells.

[0027] Preferably, the disease caused by the disorder of insulin secretion from pancreatic β cells is congenital hyperinsulinemia.

[0028] Specifically, in application, a plasmid containing the fusion protein is transfected into the recipient tissue or cell, and the recipient tissue or cell is stimulated by light.

[0029] Preferably, the light stimulation conditions are: blue light stimulation, wavelength of 480nm, wavelength of 5ms, and frequency of 20Hz.

[0030] In summary, this invention utilizes the precise control capabilities of optogenetics with ultra-high spatial resolution to achieve precise regulation of calcium in pancreatic β-cells. 2+ Precise regulation of the channels provides theoretical support and technological innovation for the clinical treatment of infantile CHI, and is of great value for developing new technologies and methods for the clinical treatment of pancreatic secretory dysfunction-related disorders.

[0031] The beneficial effects of this invention are:

[0032] 1. Opto LOVTRAP has a high spatiotemporal resolution, enabling rapid and precise manipulation of individual cells by adjusting blue light;

[0033] 2. Opto LOVTRAP is reversible and can effectively inhibit pancreatic β-cell calcium deficiency under dark conditions. 2+ The blue light stimulation of the channel can quickly stop its effect, thus meeting the insulin secretion needs when blood sugar rises, such as after eating.

[0034] 3. Opto LOVTRAP targets and regulates intracellular calcium ion concentration, acting as the ultimate trigger for pancreatic vesicle release; therefore, it is not affected by potassium. ATP The functional impact allows for breakthroughs in overcoming the limitations of traditional clinical drug use. Attached Figure Description

[0035] Figure 1 The diagram shows the structure of Opto LOVTRAP (A) and the working principle of Opto LOVTRAP (B) of this invention.

[0036] Figure 2 This is a predicted structure diagram of the Opto LOVTRAP protein.

[0037] Figure 3 This diagram illustrates the construction of a CHI cell model. In the diagram, A represents a schematic diagram of the mutation sites in the in vitro CHI cell model constructed using gene editing technology; B represents a confocal fluorescence microscopy representation of intracellular calcium ion concentration in pancreatic β cells (INS-1 cells) with the ABCC8 gene mutation and control cells; C represents the quantitative plot of B. The scale bar is 10 μm.

[0038] Figure 4 To inhibit intracellular pathological hypercalcemia in the CHI pancreatic β-cell model under dark conditions, Opto LOVTRAP was used. 2+ Concentration. Scale bar: 5 μm.

[0039] Figure 5 To demonstrate that Opto LOVTRAP can reversibly regulate pancreatic β-cell function in a CHI pancreatic β-cell model. Scale bar: 2 μm.

[0040] Figure 6 To quantitatively analyze the role of Opto LOVTRAP in regulating insulin secretion in CHI pancreatic β-cells using ELISA. In a CHI cell model, a stable transgenic cell line overexpressing the Opto LOVTRAP light-controlled mechanism was constructed as the experimental group, and an INS-1 stable transgenic cell line overexpressing a portion of the LOV2 light-controlled mechanism (preventing calcium channel protein adhesion) was constructed as the control group. Both groups were further divided into dark and light groups. The control group consisted of a Control-Dark group and a Control-Light group, while the experimental group consisted of an OptoLOVTRAP-Dark group and an Opto LOVTRAP-Light group. Detailed Implementation

[0041] Example 1: Design of Opto LOVTRAP, a light control element

[0042] Rem 1-266 The protein (amino acid sequence shown in SEQ ID No. 1) belongs to the Ras-associated GTPase subfamily and can inhibit voltage-gated Ca2+. 2+ The channel exists primarily in the cytoplasm, and it can only exert its inhibitory effect on Ca when anchored to the cell membrane. 2+ The role of channels. Therefore, this invention will construct optogenetic tools to facilitate Rem 1-266 In the dark, it targets and locates itself on the cell membrane, effectively inhibiting Ca2+. 2+ The channel, under light conditions, returns to its free state in the cytoplasm, opening up Ca2+ channels. 2+ Channel. This enables precise reversible suppression of voltage-gated Ca. 2+ aisle.

[0043] A schematic diagram of the Opto LOVTRAP of the present invention is shown below. Figure 1As shown in Figure A. This embodiment uses a fusion of the following proteins as the first fusion protein: a membrane localization sequence (MGCIKSKRKDNLNDDE, used in this example) to anchor the LOV2 domain of the light-sensitive protein to the pancreatic β-cell membrane; miRFPnano3; and the blue light-sensing protein AsLOV2. The internal ribosome entry site IRES (which is transcribed into an mRNA along with the preceding and following sequences during transcription, and can directly recruit ribosomes to translate downstream sequences into proteins during translation, thus enabling independent translation of multiple genes on the same mRNA) is also included. Other fusion proteins include the blue fluorescent protein mTagBFP2 and the calcium channel inhibitor Rem... 1-266 The protein and ZDK2, which binds to the blue light-sensing protein AsLOV2, are used as the second fusion protein. The two fusion proteins yield the fusion protein Opto LOVTRAP. The amino acid sequence of the first fusion protein is shown in SEQ ID No. 1, the amino acid sequence of the second fusion protein is shown in SEQ ID No. 2, the gene sequence encoding the first fusion protein is shown in SEQ ID No. 3, and the gene sequence encoding the second fusion protein is shown in SEQ ID No. 4. Figure 1 The sequences of the coding gene for the first fusion protein, the internal ribosome entry site IRES, and the coding gene for the second fusion protein in A are shown in SEQ ID No. 5.

[0044] The working principle diagram of Opto LOVTRAP is as follows: Figure 1 As shown in Figure B, in the dark, LOV2 and ZDK2 combine to achieve the release of Rem in the cytoplasm under darkness. 1-266 The protein anchors to the cell membrane, inhibiting abnormally excited Ca2+. 2+ Channel. Under blue light, ZDK2 attached to LOV2 dissociates into the cytoplasm, thereby enabling Ca2+ channeling. 2+ Channel opening. Light-controlled Rem 1-266 The translocation of proteins from the cell membrane to the cytoplasm enables the control of Rem... 1-266 Spatiotemporal control of proteins, thereby remotely regulating Ca 2+ Channel. Its protein structure was predicted using AlphaFold 3.0 as follows: Figure 2 As shown.

[0045] Example 2: Establishment of an in vitro model of congenital hyperinsulinemia

[0046] An in vitro CHI cell model was established. Based on clinical case reports of CHI patients, gene mutation sites in the ABCC8 gene were detected. Nucleotide and protein alignment was performed using BLAST (basic local alignment search tools) to identify genomic sequences homologous to locus 1506 of the human SUR1 gene (encoded by the ABCC8 gene). The GAA to AAA mutation from the human SUR1-E1506K carrier was introduced into 8.6 kb of mouse and rat genomic DNA in exons 30-39 of the ABCC8 gene. Mouse pancreatic β-cell lines were selected, and the mutated K+ was integrated using lentiviral technology. ATP The key gene (ABCC8, mouse: Gene ID: 20927; rat: Gene ID: 25559) was injected into the genome of target cells to construct an in vitro CHI cell model. VIRUS-Free™ vectors were constructed for the experimental group (pPB[Exp]-EF1A>EGFP-CAG>rAbcc8[NM_013039.3]*(E1507K)-PGK>puro) and the control group (pPB[Exp]-EF1A>EGFP-CAG>[ORF-stuffer]-PGK>puro). Large-scale extraction of transfection-grade low-endotoxin nucleic acid was performed.

[0047] Choose Neon TM Transfection instrument (Cat#MPK5000) was used to collect target cells (mouse pancreatic β cell line MIN6 and rat pancreatic β cell line INS-1) in good logarithmic growth phase. The cells were digested, centrifuged to remove the supernatant, resuspended in PBS, counted, and 1×10⁻⁶ cells were collected. 6 Transfer the cells to EP tubes and centrifuge to obtain cell pellet. Resuspend the cells in electroporation buffer, add plasmid and mix thoroughly. Input the optimal electroporation parameters and electroporate the mixture. Electroporate the control group under the same parameters. Transfer the electroporated cells to their respective culture dishes and incubate them in an incubator. Observe the cell status after 24 hours and determine the electroporation efficiency based on the EGFP control group. After 48 hours of cell culture, screen the electroporated target cells with puromycin antibiotic. After the screening cycle, remove the drug-containing medium and replace it with fresh medium. Culture for 24-48 hours to restore cell status. Continue to culture stable cell lines with puromycin antibiotic for expansion. Identify the transfection efficiency of the mutant ABCC8 gene by RT-qPCR and select appropriate cell batches. Since elevated calcium ion concentration is the ultimate trigger for insulin release, intracellular calcium ion concentration was labeled using a calcium ion probe (jRCaMP1b, red; addgene#100851). Intracellular calcium ion concentration was observed using fluorescence confocal microscopy, and insulin secretion was detected by ELISA to verify whether the CHI model was successfully constructed.

[0048] like Figure 3 As shown, by comparing K ATP The ABCC8 base sequence of patients with type CHI was used to design the ABCC8 gene mutation site E1506K (GAA→AAA) in mice and rats. Experiments showed that the intracellular calcium ion concentration of mutated ABCC8 E1506K insulin β cells (INS-1 ABCC8 mutE1506K) was significantly higher than that of control cells (INS-1Control).

[0049] Example 3: Verification at the cellular level of the reversible regulation of voltage-gated Ca2+ by Opto LOVTRAP 2+ aisle

[0050] Qualitative and quantitative analysis of the ability of Opto LOVTRAP to regulate insulin secretion in a CHI cell model.

[0051] The CHI in vitro cell model (INS-1ABCC8) constructed in Example 2 was used. E1506K The cell lines were cultured and plated in perforated dishes. After 24 hours, transfection was performed using liposome transfection technology (using Lipo3000 transfection reagent). The dosage was: 1.5 μg plasmid (fusion protein and calcium ion probe from Example 1) and 3 μl P3000 per well. TM Reagent, 2 μl Lipo3000) was used to transfect OptoLOVTRAP plasmid and red light responsive calcium ion probe jRCaMP1b (red; addgene#100851).

[0052] Twenty-four hours after transfection, the location of the calcium ion probes in cells (LOV2 labeled with miRFPnano3 excited at 640 nm and ZDK2 labeled with mTagBFP2 excited at 405 nm) and the red-light-responsive calcium ion probe jRCaMP1b (561 nm) were observed using fluorescence confocal microscopy. First, the calcium ion probes and the location of Opto LOVTRAP in cells without blue light stimulation were excited using lasers at 561 nm, 640 nm, and 405 nm, and the changes in intracellular calcium ion concentration were observed in the dark. Then, Opto LOVTRAP was stimulated with a 488 nm laser at 1% laser power for 20 s / frame, and the 488 nm laser was turned off after 200 s. Single cells were selected using the point stimulation function of fluorescence confocal microscopy for blue light stimulation to verify the inhibition voltage-gated calcium ion concentration of Opto LOVTRAP. 2+ Channels that regulate intracellular Ca2+ 2+ The ability to measure concentration was compared with changes in intracellular calcium ion concentration under dark conditions.

[0053] The results are as follows Figure 4 As shown, experimental results indicate that Opto LOVTRAP anchors to the cell membrane in the absence of blue light stimulation, inhibiting voltage-gated Ca2+. 2+ This inhibits the flow of calcium ions through the channel, thereby suppressing intracellular calcium ion concentration. Upon stimulation with blue light, ZDK2 fuses with the calcium channel inhibitor Rem... 1-266 The protein rapidly diffuses into the cytoplasm, thereby opening the voltage-gated Ca2+. 2+ This pathway increases intracellular calcium ion concentration and promotes calcium ion concentration-dependent insulin release.

[0054] Similarly, such as Figure 5 As shown, the reversibility of Opto LOVTRAP was verified. After the blue light stimulation ended and the cells were returned to darkness for 20 minutes, the changes in intracellular calcium ion concentration were observed again using a fluorescence confocal microscope, confirming that Opto LOVTRAP had an effect on intracellular calcium... 2+ Reversible regulation of concentration. Results showed that it re-anchored to the cell membrane after the removal of blue light stimulation, demonstrating good reversibility. Opto LOVTRAP exhibits high temporal and spatial resolution, possesses local regulatory capabilities, and demonstrates a reversible regulatory effect on the pathological hyperinsulinemia in CHI in vitro cell model.

[0055] Example 4: Opto LOVTRAP for the treatment of congenital hyperinsulinemia

[0056] A characteristic of CHI is the ability to secrete excessive insulin even in the presence of hypoglycemia. The CHI in vitro cell model (INS-1ABCC8) constructed in Example 2... E1506K Based on the stable cell line, a stable CHI cell model overexpressing the Opto LOVTRAP light control tool (nucleotide sequence shown in SEQ ID No. 5) was constructed using the aforementioned lentiviral infection method as the experimental group, and an INS-1 stable cell line overexpressing a portion of the LOV2 light control tool (preventing calcium channel inhibitors from membrane deposition; nucleotide sequence shown in SEQ ID No. 4) served as the control group. INS-1 cells from both the control and experimental groups were transferred to 12-well plates. The control group was further subdivided into two subgroups: a Control-Dark group and a Control-Light group; the experimental group was divided into an OptoLOVTRAP-Dark group and an Opto LOVTRAP-Light group.

[0057] After 24 hours of plating, the cells were first starved for 30 minutes using 0.1% FBS / KRBH. Then, the two light-treated groups were stimulated with blue light at a specific frequency using LED plates. The light frequency was 1 second on, followed by 10 seconds of darkness. After 30 minutes of stimulation, the cell supernatant from each group was collected and centrifuged at 3000×g for 10 minutes. Changes in insulin secretion in the cell supernatant were then detected using ELISA.

[0058] The results are as follows Figure 6 As shown in the figure, the experiment demonstrates that Opto LOVTRAP can regulate insulin secretion in the CHI cell model. Under dark conditions, the Opto LOVTRAP experimental group significantly inhibited hyperinsulinemia. After blue light stimulation, insulin secretion in the Opto LOVTRAP experimental group increased and was not significantly different from the control group, indicating the regulatory effect of Opto LOVTRAP on insulin secretion in the CHI cell model.

Claims

1. A fusion protein for regulating pancreatic β-cell function based on optogenetics, characterized in that, The fusion protein includes a first fusion protein and a second fusion protein. The first fusion protein includes a membrane localization sequence for anchoring the LOV2 domain of the photosensitive protein to the pancreatic β-cell membrane and the LOV2 domain of the photosensitive protein, which are connected sequentially from the N-terminus to the C-terminus. The second fusion protein includes a binding protein ZDK2 for binding the LOV2 domain of the photosensitive protein, a calcium channel inhibitor protein, and a fluorescent protein, which are connected sequentially from the C-terminus to the N-terminus. The calcium channel inhibitor protein is used to inhibit the opening of calcium channels. In the dark, the LOV2 domain of the light-sensitive protein in the first fusion protein binds to the ZDK2 binding protein in the second fusion protein, and the calcium channel inhibitor in the second fusion protein inhibits the opening of calcium channels. Under light conditions, the LOV2 domain of the photosensitive protein in the first fusion protein and the ZDK2 binding protein in the second fusion protein dissociate, and the calcium channel inhibitor in the second fusion protein releases, causing the calcium channel to open.

2. The fusion protein as described in claim 1, characterized in that, The membrane localization sequence is MGCIKSKRKDNLNDDE.

3. The fusion protein as described in claim 1, characterized in that, The calcium channel inhibitor protein is amino acids 1-266 of the Rem protein. The amino acid sequence of the Rem protein from position 1 to 266 is the amino acid sequence from position 252 to 517 as shown in SEQ ID No.

2.

4. The fusion protein as described in claim 1, characterized in that, The fluorescent protein is either a red fluorescent protein or a blue fluorescent protein.

5. The fusion protein as described in claim 1, characterized in that, The amino acid sequence of the first fusion protein is shown in SEQ ID No. 1, and the amino acid sequence of the second fusion protein is shown in SEQ ID No.

2.

6. A gene encoding the fusion protein as described in any one of claims 1 to 5.

7. The gene as described in claim 6, characterized in that, The gene sequence encoding the first fusion protein in the gene is shown in SEQ ID No. 3, and the gene sequence encoding the second fusion protein is shown in SEQ ID No.

4.

8. The use of the fusion protein according to any one of claims 1 to 5 in the preparation of a medicament for treating diseases caused by insulin secretion disorders of pancreatic β cells.

9. The application as described in claim 8, characterized in that, The disease caused by the disorder of insulin secretion from pancreatic β cells is congenital hyperinsulinemia.

10. The application as described in claim 9, characterized in that, In application, a plasmid or adenovirus containing the fusion protein is transfected into the recipient tissue or cell, and the recipient tissue or cell is stimulated by light.

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