Construction of force response bioluminescent protein and application of force response bioluminescent protein in ultrasonic regulation and control of cell luminescence

Abstract

The invention relates to the technical field of biomechanical luminescence diagnosis and treatment, in particular to a fusion protein and a reagent for ultrasonically regulating and controlling cell luminescence. A force response bioluminescent protein system composed of mechanical force sensitive ion channel (Msc) protein and jellyfish luminescent protein (AEQ) is constructed in cells, and transmembrane conduction of Ca < 2 + > under the action of ultrasonic mechanical force is achieved. And Ca < 2 + > is specifically combined with jellyfish luminescent protein, and the oxidized coelenterazine substrate emits light, so that the bioluminescence process regulated and controlled by ultrasound is achieved. The Ca < 2 + >-sensitive jellyfish luminescent protein is used as an ultrasonic mechanical force response bioluminescent carrier, and can generate instantaneous luminescence under ultrasonic control, so that the ultrasonic has the switch function characteristic of controlling the luminescence of the protein. Meanwhile, based on the non-invasiveness and excellent tissue penetrating capacity of ultrasound, accurate space-time positioning in the body can be achieved. Therefore, bioprotein luminescence triggered by ultrasound is not limited by treatment depth, and the technical bottleneck of in-vivo application of light therapy is broken through.

Description

Technical Field

[0001] The present invention relates to the technical field of bioluminescence diagnosis and treatment, and particularly to the construction of force-responsive bioluminescent proteins and the application of ultrasonic regulation of cell luminescence. Background Art

[0002] Photodynamic therapy (PDT) is the emerging fourth cancer treatment method following surgery, chemotherapy, and radiotherapy. Through the photodynamic reaction of photosensitizers under the excitation of a specific wavelength light source, reactive oxygen species (ROS) are generated to play an anti-tumor role. Different from traditional tumor treatment methods, PDT uses the selective enrichment of photosensitizers in tumor tissues and the selective activation of photosensitizers in target tissues by specific light sources, thereby reducing damage to normal tissues and avoiding systemic side effects. The PDT process requires the application of light to activate photosensitizers to exert anti-tumor efficacy. However, the maximum optical penetration depth of visible light in human tissues is about 3 mm, resulting in the inability of sensitizers inside tumors to be activated by light irradiation, which greatly weakens the synergistic treatment effect of PDT. The penetration ability of light through human tissues depends on the wavelength. Light with a longer wavelength has a stronger penetration ability than light with a shorter wavelength. Increasing the light irradiation wavelength is one of the methods to solve this problem.

[0003] Near-infrared light (NIR) has low absorption and strong scattering in biological tissues, thus having a large optical penetration depth and being known as the optical window of the human body. Using NIR to excite sensitizers can improve the depth of light therapy to a certain extent. However, NIR also has a penetration depth limitation (the illumination depth of 980 nm NIR is 1.5 cm), and it is still difficult to transfer photon energy to deeper lesion sites in the body. To break through the penetration depth limitation in traditional PDT treatment, many researchers have developed in vivo luminescence technology as a PDT treatment light source in recent years. Without the need for external light source excitation, bioluminescent proteins can directly activate the photodynamic process of photosensitizers by enzymatically oxidizing small molecule substrates to produce luminescence. In vivo bioluminescence as an induced light source for PDT can achieve good therapeutic effects and can solve the penetration depth limitation of the light source in traditional PDT. Bioluminescent proteins, as an innovative implantable tumor light source, have been widely used in the field of PDT research. For example, attenuated Salmonella expressing firefly luciferase is injected into tumors, and in situ luminescence can be produced after binding to the substrate luciferin, which can activate the photosensitizer chlorin e6 to exert photodynamic activity. However, although this strategy shows great potential, there is still a lack of effective regulatory mechanisms for the in vivo luminescence time and light intensity of the currently used bioluminescent proteins. This limitation not only affects the depth and breadth of PDT treatment but also restricts its flexibility and adaptability in different tumor environments and individual patients. Therefore, improving the controllability of bioluminescence has become an urgent task. By deeply studying the molecular mechanism of bioluminescence and developing technologies that can precisely control the luminescence time and light intensity, it is expected to bring revolutionary progress to PDT treatment. This will not only improve the targeting and effectiveness of treatment but also significantly reduce side effects, bringing safer and more effective treatment options for cancer patients. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to construct a force-responsive luminescent fusion protein and realize cell luminescence by ultrasonic regulation.

[0005] The fusion protein or protein combination provided by the present invention includes fragment A and fragment B;

[0006] Wherein:

[0007] Fragment A is a mechanosensitive ion channel protein;

[0008] Fragment B is a calcium-sensitive photoprotein.

[0009] The present invention constructs a force-responsive bioluminescent protein system composed of a mechanosensitive ion channel (Msc) protein and a calcium-sensitive photoprotein in cells; uses the mechanical force generated by ultrasound to activate the Msc ion channel and mediate Ca 2+ transmembrane conduction; Ca 2+Specifically bind to calcium ion-sensitive photoproteins, oxidize the coelenterazine substrate to produce luminescence, and achieve a bioluminescence process regulated by ultrasound.

[0010] In the present invention, the mechanosensitive ion channel protein and the calcium ion-sensitive photoprotein can exist in the form of a fusion protein or in the form of a composition, and the present invention does not limit this.

[0011] In the examples, by constructing an expression vector containing nucleic acids encoding a calcium ion-sensitive photoprotein and a mechanosensitive ion channel protein, a fusion protein of the two is obtained. After its intracellular expression, the 2A peptide self-cleaves to form two independent proteins. However, this is only a feasible way, and other schemes for expressing the two proteins in the same cell are also within the protection scope of this application.

[0012] In the present invention, the calcium ion-sensitive photoprotein and the mechanosensitive ion channel protein can be transferred into cells by the same means or by different means, and the present invention does not limit this. For example, they can be transferred into cells by chemical transfection, electroporation, microinjection, virus vector-mediated method, gene gun-mediated method. Or, they can also be transferred into cells with the help of the CRISPR / Cas system. For example, both the calcium ion-sensitive photoprotein and the mechanosensitive ion channel protein can be transferred into cells by chemical transfection, or one can be transferred into cells by chemical transfection and the other by virus vector-mediated method.

[0013] Taking chemical transfection as an example, in order to make the cells luminesce to produce a photodynamic therapy effect, the calcium ion-sensitive photoprotein and the mechanosensitive ion channel protein need to be expressed in the cells, and they can be transferred into cells by one vector or by two vectors respectively, and the present invention does not limit this.

[0014] When the calcium ion-sensitive photoprotein and the mechanosensitive ion channel protein are transferred into cells by one vector, the nucleic acids encoding the calcium ion-sensitive photoprotein and the mechanosensitive ion channel protein can be expressed in two expression units or in one expression unit, and the present invention also does not limit this.

[0015] The structure of the fusion protein described in the present invention is:

[0016] [(Fragment A)a-(Fragment B)b]c;

[0017] Or [(Fragment B)b-(Fragment A)a]c;

[0018] Wherein: a, b, and c independently selected from non-zero integers.

[0019] In the fusion protein delivery structure of the present invention, a represents the number of repeats of fragment A, b represents the number of repeats of fragment B, and c represents the number of repeats of the unit composed of fragment A and fragment B in the fusion protein.

[0020] In the present invention, a, b, and c are independently selected from non-zero integers. a, b, and c may be equal or unequal, and the present invention does not make any limitation in this regard. As a feasible example, 1 ≤ a ≤ 10, 1 ≤ b ≤ 10, and 1 ≤ c ≤ 10.

[0021] Taking a = 1, b = 1, and c = 1 as an example, the structure of the fusion protein of the present invention is fragment A - fragment B or fragment B - fragment A.

[0022] Taking a = 1, b = 2, and c = 1 as an example, the structure of the fusion protein of the present invention is fragment A - fragment B - fragment B or fragment B - fragment B - fragment A.

[0023] Taking a = 2, b = 1, and c = 2 as an example, the structure of the fusion protein of the present invention can be expressed as: [(fragment A)2 - (fragment B)]2; or [(fragment B) - (fragment A)2] 2。 That is: its structure is fragment A - fragment A - fragment B - fragment A - fragment A - fragment B or fragment B - fragment A - fragment A - fragment B - fragment A - fragment A.

[0024] In the present invention, the calcium-sensitive photoprotein and the mechanosensitive ion channel protein are expressed as a fusion protein, and after being transferred into cells, they can form two independent proteins after self-cleavage by the 2A peptide, or they may not be separated. The present invention does not make any limitation in this regard.

[0025] In the present invention, a linker may or may not be included between fragment A and fragment B. In the embodiments of the present invention, in order to maintain the stability of the structures of the two proteins, the fusion described in the present invention further includes a linker fragment.

[0026] In the present invention, the linker is located between two adjacent fragments, such as between fragment A and fragment B, or between two fragment As, or between two fragment Bs. The present invention does not make any limitation in this regard.

[0027] Optionally, the length of the linker is 2 - 20 amino acids, or 5 - 15 amino acids, or 10 - 15 amino acids. The present invention also does not make any limitation in this regard.

[0028] In the present invention, the linker may or may not contain a self-cleavable or cleavable enzyme cleavage site. The present invention also does not make any limitation in this regard.

[0029] In a specific embodiment, the linker is selected from any one of P2A, T2A, E2A, and F2A.

[0030] The T2A peptide sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO. 4).

[0031] In the present invention, the end of the fusion protein may or may not be linked to a detection tag, and the present invention does not limit this. As a feasible case, the detection tag is located at the N-terminus or C-terminus of the fusion protein. It may be a Flag tag, HA tag, Myc tag, GST tag, MBP tag, GFP tag, RFP tag or 6×His tag.

[0032] In the present invention, the mechanosensitive ion channel protein is from Escherichia coli.

[0033] In some embodiments, the mechanosensitive ion channel protein has an amino acid sequence as shown in SEQ ID NO: 1, or a sequence in which at least one amino acid is substituted, deleted or added in the sequence shown in SEQ ID NO: 1, or has an amino acid sequence having more than 80% identity with SEQ ID NO: 1.

[0034] In a specific embodiment, the amino acid sequence of the mechanosensitive ion channel protein is as shown in SEQ ID NO: 1 or 2.

[0035] Among them, the MSC protein shown in SEQ ID NO: 1 is from Escherichia coli, and the Msc-G22S mutant of Msc shown in SEQ ID NO: 2 has its 22nd glycine (Gly) replaced by serine (Ser). Compared with the wild-type Msc protein, the Msc-G22S mutant enables the Msc channel to be activated under lower mechanical pressure, thus showing higher sensitivity to ultrasonic stimulation.

[0036] In the present invention, the calcium-sensitive photoprotein is at least one of aequorin, obelin, pennalum, calmodulin, and clytia.

[0037] In some embodiments, the calcium-sensitive photoprotein is apoaequorin.

[0038] In a specific embodiment, the apoaequorin is from Aequorea victoria. ApoAEQ has 3 calcium-binding EF-hand structures and causes coelenterazine to oxidize and emit light in the presence of calcium.

[0039] More specifically, the calcium ion-sensitive photoprotein has an amino acid sequence as shown in SEQ ID NO:3, or a sequence in which at least one amino acid is substituted, deleted or added in the sequence shown in SEQ ID NO:3, or an amino acid sequence having more than 80% identity with SEQ ID NO:3.

[0040] The present invention also provides biological materials, including any one of the following:

[0041] I) Nucleic acids encoding the fusion protein as described above;

[0042] II) Expression units containing the nucleic acids described in I);

[0043] III) Plasmid vectors containing the nucleic acids described in I) or the expression units described in II);

[0044] IV) Hosts transformed or transfected with the vectors described in III), or hosts in which the nucleic acids described in I) or the expression units described in II) are integrated into the genome;

[0045] V) Expression products of the hosts described in IV).

[0046] In the present invention, the nucleic acid can be DNA or RNA, and it can be single-stranded or double-stranded. The present invention does not make any limitation in this regard.

[0047] In the expression unit of the present invention, it includes a promoter and a coding nucleic acid. Among them, the promoter is a eukaryotic promoter or a prokaryotic promoter, and the present invention does not make any limitation in this regard. In specific embodiments, the promoter is at least one of CMV promoter, SV40 promoter, EF1-α promoter, Ubiquitin promoter, β-actin promoter, CMV IE promoter, PGK promoter, T7 promoter, SP6 promoter, LTR promoter, HSP70 promoter, TK promoter, EFS promoter, CAG promoter, CBh promoter, ROSA26 promoter, U6 promoter, H1 promoter, EF1β promoter or GAPDH promoter.

[0048] On this basis, the expression unit further includes a terminator and / or an enhancer. For example, the enhancer is selected from at least one of SV40 enhancer, CMV enhancer, HSV-TK enhancer, Ig enhancer, β-globin enhancer, E1A enhancer, LTR enhancer, Polyoma enhancer, IgH enhancer, EF1α enhancer, CAG enhancer, Ubiquitin enhancer, Albumin enhancer, MHC enhancer, AFP enhancer, Fibrinogen enhancer, Collagen enhancer, Elastase enhancer, Insulin enhancer or Keratin enhancer. The terminator is selected from at least one of SV40 terminator, BGH (bovine growth hormone) terminator, T7 terminator, SP6 terminator, T3 terminator, PolyA terminator, HSP70 terminator, Ad2 (adenovirus type 2) terminator, SV40polyA terminator, TKpolyA terminator, EF1α terminator, CAG terminator, Ubiquitin terminator, PGK terminator, β-actin terminator, ColE1 terminator, rrnB terminator, Tφ terminator, λt0 terminator, TetO terminator.

[0049] The vector containing the nucleic acid or the expression unit according to the present invention is used for the storage, amplification of the nucleic acid or the expression unit, or the expression of the target protein. It is a plasmid vector or a viral vector. In some embodiments, the vector is a plasmid vector, which includes at least one of pUC19 plasmid vector, pBR322 plasmid vector, pcDNA3.1 plasmid vector, pET series vectors (such as pET-28a, etc.), pGEX series vectors (such as pGEX-4T-1, etc.), pMAL series vectors (such as pMAL-c2x, etc.), pEGFP-N1 vector, pDsRed-Express vector, pBacPAK8 baculovirus expression vector or pCMV-Sport6 vector.

[0050] The host according to the present invention is used for the preservation, amplification of the plasmid vector as described above, or the expression of the fusion protein. In the present invention, the host is a eukaryotic host or a prokaryotic host. The eukaryotic host includes but is not limited to yeast, insect cells, and renal epithelial cells. The prokaryotic host includes but is not limited to Escherichia coli.

[0051] Furthermore, the present invention also provides a method for preparing the fusion protein as described above, which includes: culturing the host as described above to obtain a culture product containing the fusion protein.

[0052] Even further, the present invention also provides the application of the fusion protein or protein combination in at least one of the following:

[0053] i), Regulating cell luminescence;

[0054] ii), Serving as an excitation light source for photodynamic therapy.

[0055] Furthermore, the present invention also provides a reagent for regulating cell luminescence, which includes at least one of the fusion protein or protein combination as described above, or the biomaterial as described above.

[0056] Using the reagent for regulating cell luminescence according to the present invention, with a Ca 2+ -sensitive luminescent protein as a bioluminescent carrier responsive to ultrasonic mechanical force, instantaneous luminescence can be generated under ultrasonic control, endowing ultrasound with the switch function characteristic of controlling protein luminescence.

[0057] In the present invention, the reagent for regulating cell luminescence is used for cell luminescence of prokaryotes or eukaryotes, and the present invention does not limit this. For example, it can be used for cell detection and / or imaging. The cells include but are not limited to bacteria, fungi, plant cells, insect cells or human cells. For example, when applied to the human body, it can achieve imaging of human tissues or specific cells. Or when applied to bacteria, fungi, etc., it can achieve detection of pathogenic bacteria. When it is necessary to image or detect specific pathogenic bacteria or cells, in order to achieve a better targeting effect, a targeting component can be added to the fusion protein. When imaging human cells or tissues, the cells or tissues can be tumor cells or tissues, or can also be normal cells and / or tissues, and the present invention does not limit this.

[0058] In the embodiments of the present invention, the reagent for regulating cell luminescence further includes coelenterazine or its derivatives and / or Ca 2 + . Further, the reagent for regulating cell luminescence may further include a nucleic acid transfection reagent, such as a liposome transfection reagent.

[0059] In the present invention, the derivatives of coelenterazine in the reagent for regulating cell luminescence include but are not limited to coelenterazine h, coelenterazine 400a, coelenterazine cp, coelenterazine f, coelenterazine n, coelenterazine e or dimethyl coelenterazine.

[0060] In the present invention, the Ca 2+ in the reagent for regulating cell luminescence can be endogenous Ca 2+ from the cell, or can also be exogenously added Ca 2+ . As a feasible case, the source of the Ca 2+ can be calcium chloride, calcium nitrate, calcium hypochlorite and / or calcium gluconate.

[0061] Furthermore, the present invention also provides a method for regulating cell luminescence, which includes contacting cells overexpressing mechanosensitive ion channel proteins and calcium-sensitive photoproteins with coelenterazine and Ca 2+ to produce fluorescence in the cells under ultrasonic conditions.

[0062] Specifically, the method for regulating cells in the present invention includes transferring the plasmid vector as described above into cells, and then contacting the cells with coelenterazine and Ca 2+ to cause the cells to emit light under ultrasonic conditions.

[0063] More specifically, the method includes:

[0064] Using a transfection reagent to introduce the recombinant plasmid into cells, and culturing the cells under appropriate culture conditions (37 °C, 5% CO2) for 48 hours to allow sufficient time for the target gene to be expressed;

[0065] Trypsinize the cells and adjust the cell density to 3×10 5 cells / mL, take 1 mL and add it to a polystyrene cell culture tube for ultrasonic irradiation;

[0066] Use free-field water bath ultrasound, ultrasonic conditions: 0.5 - 10 MHz, 1 - 20 W / cm 2 , 1 - 30 min;

[0067] Immediately after ultrasound, add a coelenterazine solution with a final concentration of 1 mM, and bioluminescence signals can be detected.

[0068] The present invention utilizes the self-cleavage property of 2A peptides to ensure the simultaneous expression of two proteins and reduce the mutual influence between them. Embed the target gene sequence into the multiple cloning site of the mammalian expression vector pcDNA3.1+ plasmid, and add 6×His as a detection tag to construct the pcDNA3.1-apoAEQ-2A-Msc-His recombinant vector ( Figure 1 ). Use a transfection reagent to introduce the recombinant plasmid into cells, and verify the functionality of the ultrasound-responsive bioluminescent protein through a three-step process of "gene introduction - protein expression - functional evaluation" to achieve the intracellular bioluminescence process regulated by ultrasound.

[0069] In the examples of the present invention, the ultrasound is free-field low-intensity non-focused ultrasound, and the intensity of the ultrasound is 1 W / cm 2 - 20 W / cm 2 , and the ultrasound time is 1 - 30 min.

[0070] For example, the intensity of the ultrasound is 1 W / cm 2 , 2 W / cm 2 , 3 W / cm 2 , 4 W / cm 2, 5 W / cm 2 , 6 W / cm 2 , 7 W / cm 2 , 8 W / cm 2 , 9 W / cm 2 , 10 W / cm 2 , 11 W / cm 2 , 12 W / cm 2 , 13 W / cm 2 , 14 W / cm 2 , 15 W / cm 2 , 16 W / cm 2 , 17 W / cm 2 , 18 W / cm 2 , 19 W / cm 2 or 20 W / cm 2 .

[0071] For example, the time of the ultrasound is 1 min, 3 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 20 min, 25 min or 30 min.

[0072] In some specific embodiments, the intensity of the ultrasound is 5 W / cm 2 , and the ultrasound time is 10 min.

[0073] Furthermore, the present invention also provides an excitation light source for photodynamic therapy, which comprises at least one of the fusion proteins as described above or the biomaterials as described above.

[0074] In the excitation light source of the present invention, coelenterazine or its derivatives, photosensitizers and / or Ca 2+ .

[0075] In the present invention, the derivatives of coelenterazine in the excitation light source include but are not limited to coelenterazine h, coelenterazine 400a, coelenterazine cp, coelenterazine f, coelenterazine n, coelenterazine e or dimethyl coelenterazine.

[0076] In the present invention, the photosensitizers in the excitation light source are selected from at least one of hematoporphyrin derivatives, verteporfin, hematoporphyrin monomethyl ether, dihydrochlorin c4, dihydrochlorin p6 or pyropheophorbide a. In a specific embodiment, in the drug of the present invention, the photosensitizer is dihydrochlorin e6.

[0077] In the present invention, the Ca in the excitation light source2+ Can be endogenous Ca from cells 2+ , or exogenously added Ca 2+ As a feasibility case, the Ca 2+ The source of calcium can be calcium chloride, calcium nitrate, calcium hypochlorite and / or calcium gluconate.

[0078] In the excitation light source of the present invention, the molar ratio of coelenterazine to photosensitizer is (1-5):(2-10). In a specific embodiment, the molar ratio of coelenterazine to photosensitizer is 2:5.

[0079] Furthermore, the present invention also provides a method for using an excitation light source for photodynamic therapy, which comprises reacting cells overexpressing a mechanical force-sensitive ion channel protein and a calcium ion-sensitive photoprotein with a photosensitizer, coelenterazine and Ca 2+ Contact with ultrasound causes cell death.

[0080] In the present invention, the excitation light source of the photodynamic therapy as described above can be applied to tumors and / or other diseases, and the other diseases include but are not limited to acne, condyloma acuminatum, psoriasis, port wine stains, etc. The tumors include but are not limited to at least one of skin cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck tumors (including oral cancer, nasopharyngeal cancer, laryngeal cancer, etc.), brain glioma, melanoma, thyroid cancer, anal canal cancer, penile cancer, and vulvar cancer.

[0081] In the present invention, the subject of photodynamic therapy includes but is not limited to humans and / or mammals, for example, the mammals include but are not limited to mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, horses, cows, sheep, macaques or tree shrews.

[0082] Treatments described herein include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or total).

[0083] In the photodynamic therapy method of the present invention, Ca 2+ Sensitive aequorin, as a bioluminescent carrier that responds to ultrasonic mechanical force, can be controlled by ultrasound to produce instantaneous luminescence, giving ultrasound the switch function of controlling protein luminescence. At the same time, based on the non-invasiveness and excellent tissue penetration ability of ultrasound, precise temporal and spatial positioning in the body can be achieved. Therefore, the use of ultrasound to trigger bioprotein luminescence is not limited by the depth of treatment, breaking through the technical bottleneck of light therapy in the body.

[0084] In the present invention, in the embodiments of the present invention, the ultrasound is free-field low-intensity non-focused ultrasound, and the intensity of the ultrasound is 0.1 W / cm 2 ~5 W / cm 2 , and the ultrasound time is 1 to 30 min.

[0085] For example, the intensity of the ultrasound is 0.1 W / cm 2 、0.2 W / cm 2 、0.3 W / cm 2 、0.4 W / cm 2 、0.5 W / cm 2 、0.6 W / cm 2 、0.7 W / cm 2 、0.8 W / cm 2 、0.9 W / cm 2 、1 W / cm 2 、2 W / cm 2 、3 W / cm 2 、4 W / cm 2 or 5 W / cm 2 .

[0086] For example, the ultrasound time is 1 min, 3 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 20 min, 25 min or 30 min.

[0087] In some specific embodiments, the intensity of the ultrasound is 5 W / cm 2 , and the ultrasound time is 10 min.

[0088] In the present invention, a force-responsive bioluminescent protein system composed of a mechanosensitive ion channel (Msc) protein and aequorin (AEQ) is constructed in cells to realize the transmembrane conduction of Ca 2+ under the action of ultrasonic mechanical force. Ca 2+ specifically binds to aequorin, oxidizes the coelenterazine substrate to produce luminescence, achieving a bioluminescence process regulated by ultrasound. Different from the traditional in-vivo luminescence technology of biological proteins, using Ca 2+ -sensitive aequorin as a bioluminescent carrier responsive to ultrasonic mechanical force, instantaneous luminescence can be generated under the control of ultrasound, endowing ultrasound with the switch function characteristics of controlling protein luminescence. At the same time, based on the non-invasive nature and excellent tissue penetration ability of ultrasound, precise spatio-temporal localization in vivo can be achieved. Therefore, triggering bioluminescence of biological proteins by ultrasound is not limited by the treatment depth, breaking through the technical bottleneck of light therapy in in-vivo applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1It is the map of the pcDNA3.1-apoAEQ-2A-Msc-His recombinant plasmid;

[0090] Figure 2 It is the expression of the target protein detected by Western blot;

[0091] Figure 3 It is the expression level of the target gene mRNA detected by real-time quantitative PCR;

[0092] Figure 4 It is the schematic diagram of the free-field ultrasound device;

[0093] Figure 5 It is to detect the intracellular Ca 2+ level in different cells after ultrasound using the fluo-4 probe;

[0094] Figure 6 It is the luminescence imaging of the cell suspension after ultrasound;

[0095] Figure 7 It is the cell viability under different treatment conditions;

[0096] Figure 8 It is the schematic diagram of the principle of the solution of the present invention. Detailed implementation manners

[0097] The present invention provides the construction of a force-responsive bioluminescent protein and the application of ultrasound-regulated cell luminescence. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all such substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0098] Unless otherwise defined in the present invention, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0099] In addition, unless otherwise specified in this article, terms in the singular form in this article shall include the plural form, and terms in the plural form shall include the singular form. More specifically, as used in this specification and the appended claims, unless otherwise clearly indicated, the singular forms "a" and "the" include plural referents.

[0100] In this text, the terms "comprising", "including", and "having" are used interchangeably and are intended to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that when using "comprising", "including", and "having" to describe in this text, the solution of "consisting of..." is also provided.

[0101] The term "and / or" in this text describes the association relationship of associated objects and indicates that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0102] The term "at least one" in this text means one or more, and "a plurality" means two or more. "At least one of the following (items)" or its similar expressions refer to any combination of these items, including any combination of single items (items) or plural items (items).

[0103] The term "identity" in this text is used to refer to the sequence matching situation between two polypeptides or two nucleic acids. When a certain position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a certain position in each of two DNA molecules is occupied by adenine, or a certain position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared × 100.

[0104] The test materials used in this invention are all ordinary commercially available products and can all be purchased in the market.

[0105] The sequences involved in this invention are as follows:

[0106] Wild-type Msc protein, SEQ ID NO.1:

[0107] MSIIKEFREFAMRGNVVDLAVGVIIGAAFGKIVSSLVADIIMPPLGLLIG GIDFKQFAVTLRDAQGDIPAVVMHYGVFIQNVFDFLIVAFAIFMAIKLINKLN RKKEEPAAAPAPTKEEVLLTEIRDLLKEQNNRS.

[0108] Mutant Msc-G22S of Msc, SEQ ID NO.2:

[0109] MSIIKEFREFAMRGNVVDLAVSVIIGAAFGKIVSSLVADIIMPPLGLLIG GIDFKQFAVTLRDAQGDIPAVVMHYGVFIQNVFDFLIVAFAIFMAIKLINKLN RKKEEPAAAPAPTKEEVLLTEIRDLLKEQNNRS。

[0110] Nucleic acid encoding the mutant Msc-G22S of Msc, SEQ ID NO.9:

[0111] ATGCGGTGACGTCGAGGAGAATCCTGGCCCAATGAGCATTATTAAAGAATTTCGCGAATTTGCGATGCGCGGGAACGTGGTGGATTTGGCGGTGAGTGTCATTATCGGTGCGGCATTCGGGAAGATTGTCTCTTCACTGGTTGCCGATATCATCATGCCTCCTCTGGGCTTATTAATTGGCGGGATCGATTTTAAACAGTTTGCTGTCACGCTACGCGATGCGCAGGGGGATATCCCTGCTGTTGTGATGCATTACGGTGTCTTCATTCAAAACGTCTTTGATTTTCTGATTGTGGCCTTTGCCATCTTTATGGCGATTAAGCTAATCAACAAACTGAATCGGAAAAAAGAAGAACCAGCAGCCGCACCTGCACCAACTAAAGAAGAAGTATTACTGACAGAAATTCGTGATTTGCTGAAAGAGCAGAATAACCGCTCT

[0112] Apoaequorin (apoAEQ), SEQ ID NO:3:

[0113] MTSKQYSVKLTSDFDNPRWIGRHKHMFNFLDVNHNGKISLDEMVYKASDIVINNLGATPEQAKRHKDAVEAFFGGAGMKYGVETDWPAYIEGWKKLATDELEKYAKNEPTLIRIWGDALFDIVDKDQNGAITLDEWKAYTKAAGIIQSSEDCEETFRVCDIDESGQLDVDEMTRQHLGFWYTMDPACEKLYGGAVP。

[0114] Nucleic acid encoding apoaequorin, SEQ ID NO.10:

[0115] ATGACAAGCAAACAATACTCAGTCAAGCTTACATCAGACTTCGACAACCCAAGATGGATTGGACGACACAAGCATATGTTCAATTTCCTTGATGTCAACCACAATGGAAAAATCTCTCTTGACGAGATGGTCTACAAGGCATCTGATATTGTCATCAATAACCTTGGAGCAACACCTGAGCAAGCCAAACGACACAAAGATGCTGTAGAAGCCTTCTTCGGAGGAGCTGGAATGAAATATGGTGTGGAAACTGATTGGCCTGCATATATTGAAGGATGGAAAAAATTGGCTACTGATGAATTGGAGAAATACGCCAAAAACGAACCAACGCTCATCCGTATATGGGGTGATGCTTTGTTTGATATCGTTGACAAAGATCAAAATGGAGCCATTACACTGGATGAATGGAAAGCATACACCAAAGCTGCTGGTATCATCCAATCATCAGAAGATTGCGAGGAAACATTCAGAGTGTGCGATATTGATGAAAGTGGACAACTCGATGTTGATGAGATGACAAGACAACATTTAGGATTTTGGTACACCATGGATCCTGCTTGCGAAAAGCTCTACGGTGGAGCTGTCCCC

[0116] It should be understood that in various embodiments of the present application, the magnitude of the serial numbers of the above processes does not mean the sequence of execution. Some or all of the steps may be executed in parallel or sequentially. The execution sequence of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application. The present invention will be further described below in conjunction with embodiments:

[0117] Example 1 Intracellular Expression of Ultrasound-Responsive Bioluminescent Protein

[0118] Design a target gene sequence encoding a mechanosensitive ion channel (Msc) and apoaequorin (apoAEQ), screen plasmid vectors, add additional elements to optimize the sequence, and ensure precise control of the efficient expression of the exogenous gene coding sequence in tumor cells.

[0119] Specifically, a 2A peptide sequence was used to link the Msc mutant Msc-G22S gene and the apoAEQ gene, and 6×His was added as a detection tag. The target gene sequence was inserted into the multiple cloning site of the mammalian expression vector pcDNA3.1+ plasmid, with the restriction enzyme sites EcoRI and Xhol, to construct the pcDNA3.1-apoAEQ-2A-Msc-His recombinant vector( Figure 1 ).

[0120] Cell transfection: The recombinant plasmid was introduced into human breast cancer cells (MCF 7) using the lipo3000 liposome transfection reagent. The specific steps were as follows: After MCF 7 cells were cultured and adhered, lipo3000 liposomes and the recombinant plasmid were mixed in proportion and added to the culture medium. This step needed to be carried out under serum-free conditions to ensure transfection efficiency. After transfection, the cells were placed under appropriate culture conditions (37°C, 5% CO2), and serum was added to promote cell growth.

[0121] Immunoblotting assay (Western blot) detection: Ensure that the cells were cultured for 48 hours to allow sufficient time for the target gene to express. Trypsin or a cell scraper was used to collect the transfected cells. The collected cells were suspended in an appropriate lysis buffer and lysed on ice to release the intracellular proteins. The lysate was centrifuged to remove cell debris, and the supernatant, which contained the target protein, was collected. The extracted cell proteins were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane using a constant current (300 mA, 20 min). The non-specific binding sites on the membrane were blocked with 5% skim milk powder. Anti-histag tag antibody was added and incubated overnight at 4°C. The membrane was washed with TBST to remove the unbound antibody. Secondary antibody was added and incubated at room temperature for 2 hours; the membrane was washed with TBST again, and the signal was detected using ECL, and the image was captured using an imaging system( Figure 2 ).

[0122] Real-time quantitative PCR detection: Similar to the above protein extraction, first, the cells transfected and cultured for 48 hours were collected, and RNA was extracted using an animal cell total RNA extraction kit; the extracted RNA was reverse transcribed to synthesize the corresponding cDNA. Specific primers for the target gene were designed, and the nucleotide sequences of the apoAEQ primers were as shown in SEQ ID NO.5 and 6:

[0123] SEQ ID NO.5 (forward primer):

[0124] 5’-TGGATTGGACGACACAAGCA-3’

[0125] SEQ ID NO.6 (reverse primer):

[0126] 5’-GTGTCGTTTGGCTTGCTCAG-3’

[0127] The nucleotide sequences of the Msc primers are shown in SEQ ID NO.7 and 8:

[0128] SEQ ID NO.7 (forward primer):

[0129] 5’-GTCTCTTCACTGGTTGCCGA-3’

[0130] SEQ ID NO.8 (reverse primer):

[0131] 5’-TGCATCACAACAGCAGGGAT-3’

[0132] Prepare the PCR system, including cDNA, upstream and downstream primers of the target gene or reference gene, and SYBR Green qPCR Mix. Perform qPCR according to the steps of pre-denaturation, denaturation, annealing, extension, and signal acquisition. Use a real-time quantitative PCR instrument to collect the fluorescence signal data of each cycle. Compared with the wild-type (WT) tumor cells without transfected plasmid, the target genes Msc and apoAEQ can be highly expressed in tumor cells ( Figure 3 ).

[0133] Example 2 Functional verification of ultrasound-responsive bioluminescent protein in cells

[0134] (1) Ultrasound-opened Msc mediates calcium influx: The DMEM medium used for cell culture contains approximately 1.8 mM Ca 2+ , which can directly simulate the Ca 2+ level under physiological conditions. Use a calcium fluorescence probe (Fluo-4) to detect the change in intracellular Ca 2+ concentration after the cells are irradiated with ultrasound. The specific steps are as follows: Collect the cells transfected in Example 1 48 h later and wild-type (WT) cells after trypsin digestion; preferably, adjust the cell density to 3×10 5 cells / mL, take 1 mL for ultrasound treatment; preferably, use free-field water bath ultrasound ( Figure 4 ), the ultrasound intensity is 5 W / cm 2 , and the ultrasound time is 10 min. Centrifuge to discard the supernatant, add the diluted Fluo-4 probe and incubate for 30 min, and then use a flow cytometer to detect the intracellular fluorescence intensity. After ultrasound treatment, the change in intracellular Ca 2+ concentration is as shown in Figure 5 .

[0135] (2) Ultrasound triggers intracellular bioluminescence: Add the luminescent substrate coelenterazine to the above-mentioned cells after ultrasound, and then immediately use a luminescence imager to detect the luminescence signal of the cell suspension (Figure 6 )。

[0136] Example 3: Ultrasound-Regulated Intracellular Bioluminescence for Photodynamic Therapy

[0137] Add a photosensitizer (chlorin e6) to the cells transfected in Example 1 48 h after transfection at a concentration of 50 mM; after co-incubating for 4 h, aspirate the culture medium, digest the cells with trypsin and adjust the cell density to 3×10 5 cells / mL, take 1 mL for sonication; use free-field water bath sonication with a sonication intensity of 2 W / cm 2 , and the sonication time is 10 min. Immediately after sonication, add 20 mM coelenterazine and add 10,000 cells per well to a 96-well plate; incubate for 24 h and detect the cell viability using the CCK8 method. The sonication-triggered bioluminescence can directly activate the photosensitizer inside the cells, thereby generating reactive oxygen species and killing tumor cells, resulting in a decrease in cell viability ( Figure 7 )。

[0138] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A fusion protein or protein combination, comprising fragment A and fragment B; Wherein: Fragment A is a mechanically sensitive ion channel protein; Fragment B is a calcium-sensitive photoprotein.

2. The fusion protein or protein combination according to claim 1, wherein Its structure is: [(Fragment A)a-(Fragment B)b]c; Or [(Fragment B)b-(Fragment A)a]c; Wherein: a, b, and c independently are selected from non-zero integers.

3. The fusion protein or protein combination according to claim 1 or 2, characterized in that The mechanically sensitive ion channel protein is from Escherichia coli. The calcium-sensitive photoprotein is at least one of aequorin, obelin, clytin, camgaroo luciferase, and mitrocomin.

4. The fusion protein or protein combination according to claim 3, characterized in that, The mechanically sensitive ion channel protein has the amino acid sequence shown in SEQ ID NO:1, or a sequence in which at least one amino acid is substituted, deleted, or added in the sequence shown in SEQ ID NO:1, or has an amino acid sequence having more than 80% identity with SEQ ID NO:

1.

5. The fusion protein or protein combination according to claim 4, wherein The amino acid sequence of the mechanically sensitive ion channel protein is shown in SEQ ID NO:1 or 2.

6. The fusion protein or protein combination according to claim 1 or 2, characterized in that, The calcium-sensitive photoprotein is apoaequorin.

7. The fusion protein or protein combination according to claim 6, characterized in that, The apoaequorin is from Aequorea victoria.

8. The fusion protein or protein combination according to claim 6, characterized in that, The calcium-sensitive photoprotein has the amino acid sequence shown in SEQ ID NO:3, or a sequence in which at least one amino acid is substituted, deleted, or added in the sequence shown in SEQ ID NO:3, or has an amino acid sequence having more than 80% identity with SEQ ID NO:

3.

9. The fusion protein or protein combination according to claim 1 or 2, characterized in that, A linker fragment is further included between fragment A and fragment B; the linker is selected from P2A, T2A, E2A, F2A.

10. Biological material: including any one of the following: I), a nucleic acid encoding the fusion protein according to any one of claims 1 to 9; II), an expression unit containing the nucleic acid described in I); III), a plasmid vector containing the nucleic acid described in I) or the expression unit described in II); IV), a host transformed or transfected with the vector described in III), or a gene integrated with the nucleic acid described in I) or the expression unit described in II); V), the expression product of the host described in IV).

11. A method for preparing the fusion protein according to any one of claims 1 to 9, characterized in that, Including: Culturing the host described in claim 10 to obtain a culture product containing the fusion protein.

12. The application of the fusion protein or protein combination according to any one of claims 1 to 9 in at least one of the following: i), regulating cell luminescence; ii), serving as an excitation light source for photodynamic therapy.

13. A reagent for regulating cell luminescence, which includes at least one of the fusion protein or protein combination according to any one of claims 1 to 9, or the biological material according to claim 10.

14. The reagent according to claim 13, wherein It also includes coelenterazine or its derivatives and / or Ca 2+ .

15. A method for regulating cell luminescence, which includes causing cells overexpressing mechanosensitive ion channel proteins and calcium-sensitive photoproteins to contact coelenterazine and Ca 2+ and generating fluorescence in the cells under ultrasonic conditions.

16. The method according to claim 15, characterized in that The intensity of the ultrasound is 5 W / cm 2 , and the ultrasound time is 10 min.

17. An excitation light source for photodynamic therapy, which includes at least one of the fusion protein or protein combination according to any one of claims 1 to 9, or the biological material according to claim 10.

18. The excitation light source according to claim 17, wherein, It also includes coelenterazine or its derivatives, photosensitizers, and / or Ca 2+ .

19. The excitation light source according to claim 18, characterized in that, The photosensitizer is chlorin e6; the molar ratio of coelenterazine or its derivative to the photosensitizer is 2:

5.

20. Method of using an excitation light source for photodynamic therapy, which includes contacting cells overexpressing mechanosensitive ion channel proteins and calcium-sensitive photoproteins with a photosensitizer, coelenterazine, and Ca 2+ and causing cell death under ultrasonic conditions.

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