CN119978072A - A succinic acid optical probe and its preparation method and application - Google Patents

A succinic acid optical probe and its preparation method and application Download PDF

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CN119978072A
CN119978072A CN202311508503.5A CN202311508503A CN119978072A CN 119978072 A CN119978072 A CN 119978072A CN 202311508503 A CN202311508503 A CN 202311508503A CN 119978072 A CN119978072 A CN 119978072A
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succinic acid
optical probe
sequence
succinate
polypeptide
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杨弋
赵玉政
李写
张秀泽
卢世洁
唐伟韬
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East China University of Science and Technology
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East China University of Science and Technology
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Abstract

本发明涉及一种琥珀酸光学探针及其制备方法和应用。一方面，本发明涉及一种光学探针，包含琥珀酸敏感多肽或其功能变体和光学活性多肽或其功能变体，其中光学活性多肽或其功能变体位于琥珀酸敏感多肽或其功能变体的序列内。本发明也涉及上述探针的制备方法及其在检测琥珀酸中的应用。The present invention relates to a succinic acid optical probe and a preparation method and application thereof. On the one hand, the present invention relates to an optical probe comprising a succinic acid sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is located within the sequence of the succinic acid sensitive polypeptide or a functional variant thereof. The present invention also relates to a preparation method of the above-mentioned probe and its application in detecting succinic acid.

Description

Succinic acid optical probe and preparation method and application thereof
Technical Field
The invention relates to the technical field of optical probes, in particular to a succinic acid optical probe and a preparation method and application thereof.
Background
Succinic acid is an important natural organic acid, and abnormal succinic acid metabolism is closely related to various diseases, including cardiovascular diseases, tumors, etc. Prag, H.A et al found that succinic acid and the signal axis of the succinic acid receptor SUCNR act as a metabolite sensing mechanism to regulate leptin dynamics in a biologically clock-related manner, thereby controlling the energy balance of the organism.
The existing detection methods of succinic acid comprise a colorimetric method, a liquid chromatography-mass spectrometry method, a high performance liquid chromatography and the like. The detection and analysis methods either need professional analysis instruments or complicated steps, cannot distinguish succinic acid from xanthine, are prone to human errors, are only suitable for in vitro detection, and cannot monitor the concentration change of succinic acid in living cells in real time. Therefore, development of a new detection method is needed to realize the real-time positioning, quantification and high-throughput detection of succinic acid in cells, outside cells, simply, conveniently and rapidly with high specificity.
Disclosure of Invention
The invention aims to provide a probe and a method for detecting succinic acid in real time in a cell and outside the cell in a high-throughput and quantitative manner. In order to achieve the above object, the present invention provides the following technical solutions:
the first aspect of the present invention provides a succinic acid binding protein variant which:
(1) Having the sequence shown in SEQ ID NO. 1 and having a mutation at 1,2 or 3,4, 5 or 6 positions S154, F155, Y160, N182, V183, S184, said mutation comprising a modification, substitution or deletion of an amino acid,
(2) Is a truncated variant of (1) having amino acids 65-320, or
(3) Is a sequence having at least 70% sequence identity to the sequence of (1) or (2) and having the mutation of (1) and retaining the ability to bind succinic acid.
In one or more embodiments, the mutation comprises a mutation at any position selected from the group consisting of S154, F155, Y160, N182, V183, S184.
In one or more embodiments, the mutation comprises a mutation at a site selected from any of (1) F155 and Y160, (2) Y160 and N182, (3) Y160, N182 and V183, (4) Y160, N182 and S184, (5) S154, Y160, N182 and S184;
In one or more embodiments, S154 is mutated to R. In one or more embodiments, F155 is mutated to T, P, Q or C. In one or more embodiments, Y160 is mutated to Y or W. In one or more embodiments, N182 is mutated to S. In one or more embodiments, V183 is mutated to Y or W. In one or more embodiments, S184 is mutated to L.
In one or more embodiments, the mutation comprises a mutation selected from any one of (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N S and S184L.
In another aspect, the invention provides a succinic acid optical probe comprising a succinic acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the succinic acid-sensitive polypeptide.
In one or more embodiments, the succinic acid-sensitive polypeptide comprises a succinic acid-binding protein or a functional variant thereof. In one or more embodiments, the succinic acid-sensitive polypeptide is derived from agrobacterium tumefaciens (Agrobacterium tumefaciens).
In one or more embodiments, the succinic acid-sensitive polypeptide has:
(1) The sequence shown in SEQ ID No. 1 or a truncated variant thereof having amino acids 65 to 320, or a sequence which has at least 70% sequence identity thereto and retains binding activity to succinic acid,
(2) The sequence of the variant succinic acid binding protein according to any one of the embodiments of the first aspect herein, or
(3) Sequences having at least 70% sequence identity to the sequence of (2) and having the mutation of (2) and retaining sensitivity to succinic acid.
In one or more embodiments, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one or more embodiments, the fluorescent protein is selected from the group consisting of yellow fluorescent protein (cpYFP as shown in SEQ ID NO: 2), orange fluorescent protein (cpmOrange as shown in SEQ ID NO: 3), red fluorescent protein (mKate as shown in SEQ ID NO:4 or 8, mcherry as shown in SEQ ID NO: 5), green fluorescent protein (cpGFP as shown in SEQ ID NO: 6), blue fluorescent protein (cpBFP as shown in SEQ ID NO: 7), apple red fluorescent protein (cpmApple as shown in SEQ ID NO: 9). Preferably, the optically active polypeptide is cpYFP. In one or more embodiments, the fluorescent protein has the sequence set forth in any one of SEQ ID NOs 2-9.
In one or more embodiments, the fluorescent protein functional variant has (1) the sequence shown in SEQ ID NO. 2 and has mutations at 1,2,3, 4,5 or 6 positions M9, S132, Y141, F201, N207, Y245, said mutations comprising modifications, substitutions or deletions of amino acids, the numbering of the amino acids corresponding to the sequence of the fluorescent protein, or (2) a sequence having at least 70% sequence identity to the sequence of (1). In one or more embodiments, the mutation of the fluorescent protein is selected from any one or more of M9T, S132R, Y141N, F201S, N207T and Y245F. In one or more embodiments, the mutations in the fluorescent protein are M9T, S132R, Y141N, F201S, N T and Y245F.
In one or more embodiments, the optical probe comprises a succinic acid binding protein variant as described in any of the embodiments herein and a fluorescent protein functional variant as described above, and comprises mutations selected from any of (1) F155T and Y160W of the succinic acid binding protein, (2) F155P and Y160W of the succinic acid binding protein, (3) F155Q and Y160W of the succinic acid binding protein, (4) F155C and Y160W of the succinic acid binding protein, (5) Y160W and N182S of the succinic acid binding protein, (6) Y160W, N S and V183Y of the succinic acid binding protein, (7) Y160W, N S and V183W of the succinic acid binding protein, (8) Y160W, N182S and S184L of the succinic acid binding protein, (9) S154R, Y160W, N S and S184L of the succinic acid binding protein, (10) Y160W, N S and S184L of the succinic acid binding protein, and M9 38328 56132S and Y207S 184L of the fluorescent protein, and Y160T 35S 245S 182S and Y245L of the fluorescent protein, (8) Y160W, N S182S and Y184L of the succinic acid binding protein, and Y35F 35 and Y245S 245L of the fluorescent protein.
In one or more embodiments, the optically active polypeptide is located between residues 108-112, 181-185, 196-201, and 148-156 of the succinic acid-sensitive polypeptide, numbering corresponding to the full length of the succinic acid-sensitive polypeptide. Preferably, the optically active polypeptide is located at one or more ：108/109,108/110,108/111,108/112,109/110,109/111,109/112,110/111,110/112,111/112,181/182,181/183,181/184,181/185,182/183,182/184,182/185,183/184,183/185,184/185,196/197,196/198,196/199,196/200,196/201,197/198,197/199,197/200,197/201,198/199,198/200,198/201,199/200,199/201,200/201,148/149,148/150,148/151,148/152,148/153,148/154,148/155,148/156,149/150,149/151,149/152,149/153,149/154,149/155,149/156,150/151,150/152,150/153,150/154,150/155,150/156,151/152,151/153,151/154,151/155,151/156,152/151,152/152,152/153,152/154,152/155,152/156,153/151,153/152,153/153,153/154,153/155,153/156,154/151,154/152,154/153,154/154,154/155,154/156,155/156. of the following sites of the succinic acid-sensitive polypeptide, more preferably, the optically active polypeptide is located at any one or more of the following sites of the succinic acid-sensitive polypeptide ：150/152,150/153,150/155,150/156,151/153,152/152,152/153、152/154,153/152,153/153,153/154,154/151,154/152,154/154,181/182.
In one or more embodiments, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the invention may be any amino acid sequence of any length. In one or more embodiments, the optically active polypeptide is flanked by linkers of no more than 5 amino acids, e.g., 0,1, 2,3, 4 amino acids. In one or more embodiments, the linker flanking the optically active polypeptide comprises amino acid Y. In one or more embodiments, the linker Y is located at the N-terminus and/or the C-terminus of the optically active polypeptide. In one or more embodiments, the optical probe is shown as a first portion B1-Y of the succinic acid-sensitive polypeptide and a second portion B2 of the succinic acid-sensitive polypeptide. In one or more embodiments, the optical probes of the present invention do not comprise a linker.
In one or more embodiments, the optical probes of the invention further comprise a localization sequence for localizing the probe to a specific organelle, e.g., a cell.
In one or more embodiments, the optically active polypeptide is cpYFP or a variant thereof, which is located at any one or more ：108/110、108/111、108/112、181/182、181/185、182/183、182/185、183/184、148/150、148/153、148/155、149/150、149/151、149/152、149/154、150/152、150/153、150/154、150/155、150/156、151/152、151/153、151/154、151/156、152/151、152/152、152/153、152/154、152/156、153/152、153/153、153/154、153/156、154/151、154/152、154/153、154/154、154/155、155/156,(, preferably 150/152,150/153,150/155,150/156,151/153,152/152,152/153、152/154,153/152,153/153,153/154,154/151,154/152,154/154,181/182),, of the succinic acid-sensitive polypeptide selected from the group consisting of (1) a truncated variant of the succinic acid-sensitive polypeptide shown in SEQ ID No. 1 or having amino acids 65-320, or (2) a succinic acid-binding protein variant according to any one of the embodiments of the first aspect herein. Preferably, the succinic acid binding protein variant has a mutation selected from any one of (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.
In one or more embodiments, the cpYFP variant has the sequence shown in SEQ ID NO. 2 and has mutations at 1,2 or 3,4, 5 or 6 positions M9, S132, Y141, F201, N207, Y245, including modifications, substitutions or deletions of amino acids.
In one or more embodiments, the mutation of the cpYFP variant is M9T, S132R, Y141N, F201S, N207T, Y245F.
In one or more embodiments, the optically active polypeptide is cpGFP, which is located at any one or more ：181/182、183/185、184/185、197/198、198/200、198/201、199/200、199/201、200/201、148/152、148/154、148/156、149/150、149/151、149/156、150/151、150/152、150/153、150/155、150/156、151/152、151/153、151/154、151/155、151/156、152/152、152/153、152/154、152/155、152/156、153/151、153/153、153/155、153/156、154/151、154/154、154/155、154/156、155/156,(, preferably 150/153,151/152,152/153,152/154, of the succinic acid-sensitive polypeptide (1) as shown in SEQ ID No. 1 or a truncated variant thereof having amino acids 65-320, or (2) is a succinic acid-binding protein variant according to any one of the embodiments of the first aspect herein. Preferably, the succinic acid binding protein variant has a mutation selected from any one of (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.
In one or more embodiments, the optically active polypeptide is cpBFP, which is located at any one or more ：108/109、108/110、110/111、110/112、181/185、183/184、183/185、197/201、200/201、148/150、148/151、148/152、148/153、149/151、149/153、149/155、150/151、150/152、150/155、151/152、151/153、151/154、151/156、152/151、152/152、152/154、152/155、152/156、153/151、153/152、153/153、153/156、154/151、154/152、155/156,(, preferably 148/152, 148/153, 151/154, 152/155, 152/156, 153/151, 154/152, of a succinic acid-sensitive polypeptide (1) as shown in SEQ ID No.1 or a truncated variant thereof having amino acids 65-320, or (2) is a succinic acid-binding protein variant according to any one of the embodiments of the first aspect herein. Preferably, the succinic acid binding protein variant has a mutation selected from any one of (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.
In one or more embodiments, the optically active polypeptide is cpmApple, which is located at any one or more ：108/109、108/110、110/111、196/201、197/201、199/200、200/201、148/149、148/151、148/155、149/152、149/156、150/155、150/156、151/156、152/154、152/155、152/156、153/151、153/152、153/153、154/152、154/153、154/156,(, preferably 152/154, 153/153, of a succinic acid-sensitive polypeptide (1) as shown in SEQ ID No. 1 or a truncated variant thereof having amino acids 65-320, or (2) is a succinic acid-binding protein variant according to any one of the embodiments of the first aspect herein. Preferably, the succinic acid binding protein variant has a mutation selected from any one of (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L.
In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NOS 10-31 or variants thereof. In one or more embodiments, the optical probes provided herein comprise sequences that are 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NOs 10-31. Preferably, the optical probes provided by the present invention comprise a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NOs 10-31.
In another aspect, the invention also provides fusion polypeptides comprising an optical probe as described herein and other polypeptides. In some embodiments, the optical probes described herein further comprise additional polypeptides fused thereto. Other polypeptides described herein do not affect the properties of the optical probe. In some embodiments, the other polypeptide is located at the N-terminus and/or the C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize the optical probe to a different organelle or subcellular organelle, tags for purification, or tags for immunoblotting. The fusion polypeptides described herein may have a linker between the optical probe and the other polypeptides.
In another aspect, the invention provides a nucleic acid molecule comprising (a) a coding sequence for a polypeptide or probe as described in any of the embodiments herein, or (b) a complement of (a), or (c) a fragment of (a) or (b). The fragments are primers.
In one or more embodiments, the nucleic acid sequence comprises an amino acid sequence encoding any one of SEQ ID NOs 25 to 31. Preferably, the nucleic acid sequence comprises any one of the nucleotide sequences SEQ ID NOs 30 to 31 or variants thereof. More preferably, the nucleic acid sequence comprises a sequence having 99%, 95%, 90%, 80%, 70% or 50% identity to any of the nucleotide sequences SEQ ID NO. 30-31, or comprises a nucleotide sequence substantially similar or identical to any of the nucleotide sequences SEQ ID NO. 30-31.
The invention also relates to the complementary sequences of the above-mentioned nucleic acid sequences or variants thereof, which may comprise nucleic acid sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the optical probes or fusion proteins of the invention or the complementary sequences thereof.
The invention also provides nucleic acid constructs comprising the nucleic acid molecules described herein. The nucleic acid sequence encodes an optical probe or fusion polypeptide of the invention.
In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector.
In one or more embodiments, the nucleic acid molecule is operably linked to an expression control sequence.
In some embodiments, the expression vector is selected from the group consisting of a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.
The invention also provides a host cell comprising (1) an optical probe or fusion polypeptide according to any of the embodiments of the invention, (2) a nucleic acid molecule according to any of the embodiments of the invention, or (3) a nucleic acid construct according to any of the embodiments of the invention. The host cell is preferably E.coli.
In another aspect the invention also provides a succinic acid detection kit comprising an optical probe as described herein or a fusion polypeptide or polynucleotide or an optical probe prepared by a method as described herein.
In one or more embodiments, the kit further comprises one or more reagents selected from the group consisting of buffers, media, succinic acid standards.
The present invention provides methods of making an optical probe described herein comprising providing a host cell expressing an optical probe or fusion polypeptide described herein, culturing the host cell under conditions in which the optical probe or fusion polypeptide is expressed, and isolating the optical probe or fusion polypeptide.
In one or more embodiments, a method of making a succinic acid optical probe or fusion polypeptide described herein comprises the steps of 1) transferring an expression vector encoding a succinic acid optical probe described herein into a host cell, 2) culturing the host cell under conditions suitable for expression of the expression vector, 3) isolating the succinic acid optical probe.
The invention also provides a method of detecting succinic acid in a sample comprising contacting an optical probe or fusion polypeptide as described herein or an optical probe or fusion polypeptide prepared as described herein with a sample and detecting a change in an optically active polypeptide. The detection may be performed in vivo, in vitro, subcellular or in situ. Such as blood.
Also provided herein are methods of quantifying succinic acid in a sample comprising contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a sample, detecting a change in an optically active polypeptide, and quantifying succinic acid in the sample based on the change in the optically active polypeptide.
The invention also provides a method of screening for a compound (e.g., a drug) comprising contacting an optical probe or fusion polypeptide described herein or prepared as described herein with a candidate compound in a succinic acid-containing system, detecting a change in an optically active polypeptide, and screening the compound for a change in the optically active polypeptide. The method can screen compounds with high throughput.
In one or more embodiments, the host cell described herein is contacted with a candidate compound in a succinic acid-containing system, and an optical change in the optically active polypeptide is indicative of whether the candidate compound is capable of modulating cellular uptake of succinic acid.
In another aspect the invention also provides a method of intracellular and/or extracellular localization of succinic acid comprising contacting a succinic acid-containing system with the optical probe or the host cell and detecting an optical change in an optically active polypeptide.
In one or more embodiments, the system is a solution system, a cell system, a subcellular system.
In a further aspect the invention provides the use of a succinic acid optical probe or fusion polypeptide or host cell as described herein for detecting succinic acid, for screening compounds or for intracellular/extracellular localization of succinic acid in a sample. In one or more embodiments, the positioning is real-time positioning.
The succinic acid optical probe provided by the application has the beneficial effects that the succinic acid optical probe is easy to mature, large in fluorescence dynamic change and good in specificity, can be expressed in cells by a gene operation method, can be used for positioning inside and outside the cells in real time, detecting succinic acid in a high-flux and quantitative manner, and omits the time-consuming step of processing samples. Experimental results show that the highest response of the succinic acid optical probe provided by the application to succinic acid reaches about 4 times of that of a control, and the succinic acid optical probe can be used for positioning, qualitatively and quantitatively detecting cells in subcellular structures such as cytoplasm, mitochondria, cell nucleus, nuclear exclusion and the like, and can be used for high-flux compound screening and quantitative detection of succinic acid in blood.
Drawings
The invention is further described below with reference to the drawings and examples.
FIG. 1 is a SDS-PAGE diagram of an exemplary succinic acid optical probe as described in example 1;
FIG. 2 is a graph of titration of response of an exemplary optical probe for succinic acid to varying concentrations of succinic acid as described in example 6;
FIG. 3 is an illustration of the affinity of an exemplary succinic acid optical probe for succinic acid described in example 8;
FIG. 4 is a bar graph of the specific detection of 4 succinic acid analogs by an exemplary optical succinic acid probe described in example 8;
FIG. 5 is a photograph of subcellular organelle localization of an exemplary succinic acid optical probe in mammalian cells according to example 9;
FIG. 6 is a schematic representation of dynamic monitoring of the concentration of succinic acid in the cytosol in a mammalian cell using an exemplary optical probe for succinic acid as described in example 9;
FIG. 7 is a plot of high throughput compound screening at the living cell level for an exemplary succinic acid optical probe described in example 10;
FIG. 8 is a bar graph of the quantification of succinic acid in mouse and human blood using an exemplary optical probe for succinic acid described in example 11;
Detailed Description
When a value or range is given, the term "about" as used herein means that the value or range is within 20%, within 10% and within 5% of the given value or range.
The terms "comprising," including, "and equivalents thereof as used herein include the meaning of" containing "and" consisting of, for example, a composition "comprising" X may consist of X alone or may contain other materials, such as X+Y.
The term "succinic acid-sensitive polypeptide" or "succinic acid-responsive polypeptide" as used herein refers to a polypeptide that responds to succinic acid, including any response of a chemical, biological, electrical or physiological parameter of the polypeptide that is associated with the interaction of the sensitive polypeptide. Responses include small changes, e.g., changes in the orientation of amino acids or peptide fragments of a polypeptide, e.g., changes in the primary, secondary, or tertiary structure of a polypeptide, including, e.g., changes in protonation, electrochemical potential, and/or conformation. A "conformation" is a three-dimensional arrangement of the primary, secondary and tertiary structures of a molecule comprising pendant groups in the molecule, which changes when the three-dimensional structure of the molecule changes. Examples of conformational changes include a transition from an alpha-helix to a beta-sheet or from a beta-sheet to an alpha-helix. It will be appreciated that the detectable change need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is altered. The succinic acid-sensitive polypeptides described herein may also include functional variants thereof. Functional variants of a succinic acid-sensitive polypeptide include, but are not limited to, variants that can interact with succinic acid to effect the same or similar changes as the parent succinic acid-sensitive polypeptide.
The term "optical probe" as used herein refers to a succinic acid-sensitive polypeptide fused to an optically active polypeptide, into which an optically active polypeptide (e.g., a fluorescent protein) is operably inserted. The inventors have found that when an optically active polypeptide is fused to a succinic acid-sensitive polypeptide, such as a succinic acid binding protein, conformational changes that result from the binding of a succinic acid-sensitive polypeptide specifically to a physiological concentration of succinic acid result in a conformational change in the optically active polypeptide (e.g., a fluorescent protein), which in turn results in an alteration in the optical properties of the optically active polypeptide. The presence and/or level of succinic acid can be detected and analyzed by plotting a standard curve from the fluorescence of the fluorescent protein measured at different succinic acid concentrations. The succinic acid-sensitive polypeptides of the invention include, but are not limited to, succinic acid-binding protein DctBp or variants that have greater than 90% homology thereto. The exemplary succinic acid binding protein DctBp of the present invention is derived from Agrobacterium tumefaciens (Agrobacterium tumefaciens). Exemplary DctBp proteins are shown in SEQ ID NO. 1, and exemplary DctBp truncated variants are fragments of SEQ ID NO. 1 comprising amino acids 65-320. When describing the optical probes, succinic acid-sensitive polypeptides or succinic acid-binding proteins of the invention (e.g. when describing insertion sites or mutation sites), reference is made to the amino acid residue numbers all referring to SEQ ID NO:1.
A protein-based "optically active polypeptide" is a polypeptide that has the ability to emit fluorescence. Fluorescence is an optical property of an optically active polypeptide that can be used as a means to detect the responsiveness of an optical probe of the invention. As used herein, the term "fluorescent properties" refers to molar extinction coefficient, fluorescence quantum efficiency, shape of excitation spectrum or emission spectrum, excitation wavelength maximum and emission wavelength maximum, amplitude of excitation at two different wavelengths, emission amplitude ratio at two different wavelengths, excited state lifetime or fluorescence anisotropy at an appropriate excitation wavelength. The measurable difference in any of these properties between active and inactive states is sufficient for the utility of the fluorescent protein substrates of the invention in activity assays. The measurable difference can be determined by determining the amount of any quantitative fluorescent property, for example, the amount of fluorescence at a particular wavelength or the integration of fluorescence over the emission spectrum. Preferably, the protein substrate is selected to have fluorescent properties that are readily distinguishable in the unactivated and activated conformational state. Optically active polypeptides described herein can also include functional variants thereof. Functional variants of an optically active polypeptide include, but are not limited to, variants that can undergo a change in the same or similar fluorescent properties as the parent optically active polypeptide.
Herein, "response fold" is the normalized fluorescence ratio. The more the response fold of the probe deviates from 1 (whether greater or lesser), the greater the fold change in response of the probe to the substrate or response relative to the control. For example, the present embodiment calculates the response times by detecting the change in the Ratio of the fluorescence intensity at 420nm excitation 528nm emission to the fluorescence intensity at 485nm excitation 528nm emission (Normalized Ratio 485/420), as follows:
Correction of the fluorescent signal values was performed by subtracting the detection signal values of cells not expressing the probe protein. Correction data were obtained by dividing the probe detection signal from the parallel experimental group with the control detection signal to eliminate pH sensitive interference.
F=Fsample＝FBLK
F represents the fluorescence intensity (Fluorescence intensity), F sample represents the total fluorescence intensity of the sample expressing the fluorescent probe, F BLK represents the background fluorescence intensity of the sample not expressing the fluorescent probe, and F cpYFP represents the fluorescence intensity of the sample as a pH control. F 485 represents the fluorescence intensity emitted at 528nm of excitation at 485nm of the fluorescent protein sample, and F 420 represents the fluorescence intensity emitted at 528nm of excitation at 420nm of the fluorescent protein sample. Ratio sensor represents the fluorescence intensity Ratio of the probe, and Ratio cpYFP represents the fluorescence intensity Ratio of the pH control fluorescent protein of the corresponding probe. Normalized Ratio 485/420 is the multiple of the change or response of the probe. A Normalized Ratio 485/420 that deviates from 1 (whether larger or smaller) indicates a larger fold change or response of the probe.
"Linker" or "junction region" refers to an amino acid or nucleotide sequence that connects two parts in a polypeptide, protein or nucleic acid of the invention. Illustratively, the amino acid number of the amino terminal of the linking region of the succinic acid-sensitive polypeptide and the optically active polypeptide in the present invention is selected to be 0 to 3, the amino acid number of the carboxy terminal is selected to be 0 to 2, and when the recombinant optical probe is linked to the functional protein as a basic unit, the amino acid or the carboxy terminal of the recombinant optical probe may be fused. The linker sequence may be a short peptide chain consisting of one or more flexible amino acids, such as Y.
As used herein, the terms "chromophore", "fluorophore" and "fluorescent protein" are synonymous and refer to proteins that fluoresce upon irradiation with excitation light. Fluorescent protein is used as basic detection means in the field of bioscience, such as green fluorescent protein GFP, cyclic rearranged blue fluorescent protein (cpBFP) derived from mutation of the protein, cyclic rearranged green fluorescent protein (cpGFP), cyclic rearranged yellow fluorescent protein (cpYFP) and the like, as well as red fluorescent protein RFP, which is commonly used in the field of technology, and cyclic rearranged proteins derived from the protein, such as cpmApple, cpmOrange, cpmKate and the like. Exemplary fluorescent proteins have the sequence shown in any one of SEQ ID NOS.2-9.
The succinic acid optical probes of the present invention include a succinic acid-sensitive polypeptide B, such as a succinic acid-binding protein or variant thereof, and an optically active polypeptide a, such as a fluorescent protein. The optical active polypeptide A is inserted into the succinic acid sensitive polypeptide B, the B is divided into a first part B1 and a second part B2 to form a probe structure of a formula B1-A-B2, and the interaction of the succinic acid sensitive polypeptide B and the succinic acid leads to the optical signal of the optical active polypeptide A to be strengthened.
In the optical probes of the invention, the optically active polypeptide can be located anywhere on the succinic acid-sensitive polypeptide. In one or more embodiments, the optically active polypeptide is located in the N-C direction in the region of the N-C direction of the succinic acid-sensitive polypeptide that is amino acid residues 108-112,181-185,196-201 and the 148-156 region. Illustratively, the optically active polypeptide is located at 108/109,108/110,108/111,108/112,109/110,109/111,109/112,110/111,110/112,111/112,181/182,181/183,181/184,181/185,182/183,182/184,182/185,183/184,183/185,184/185,196/197,196/198,196/199,196/200,196/201,197/198,197/199,197/200,197/201,198/199,198/200,198/201,199/200,199/201,200/201,148/149,148/150,148/151,148/152,148/153,148/154,148/155,148/156,149/150,149/151,149/152,149/153,149/154,149/155,149/156,150/151,150/152,150/153,150/154,150/155,150/156,151/152,151/153,151/154,151/155,151/156,152/151,152/152,152/153,152/154,152/155,152/156,153/151,153/152,153/153,153/154,153/155,153/156,154/151,154/152,154/153,154/154,154/155,154/156,155/156. of the amino acid sequence of the succinic acid binding protein.
Herein, in the site indicated by the "X/Y" form, the optically active polypeptide has a portion of the succinic acid-sensitive polypeptide at each of its two ends, wherein the N-terminus of the optically active polypeptide is the N-terminal starting amino acid (e.g., any one of amino acids 1 to 65) to the X-th amino acid of the succinic acid-sensitive polypeptide sequence, and the C-terminus of the optically active polypeptide is the Y-th amino acid to the C-terminal end amino acid (e.g., any one of amino acids Y to 320) of the succinic acid-sensitive polypeptide sequence. Wherein, if two numbers in the positions represented in the form of "X/Y" are consecutive integers, then it is indicated that the optically active polypeptide is located between the amino acids recited in that number, e.g., insertion position 152/153 indicates that the optically active polypeptide is located between amino acids 152 and 153 of the succinic acid-sensitive polypeptide; if two numbers in the position indicated in the form of "X/Y" are not consecutive integers and X is less than Y, then it is indicated that the optically active polypeptide replaces the amino acid between the amino acids indicated in the numbers, e.g., insertion position 150/153 indicates that the optically active polypeptide replaces amino acids 151-152 of the succinic acid-sensitive polypeptide, and if X in the position indicated in the form of "X/Y" is greater than or equal to Y, it is indicated that the portion of the succinic acid-sensitive polypeptide located at the N-terminus of the optically active polypeptide terminates at amino acid X of the succinic acid-sensitive polypeptide sequence, and the portion of the succinic acid-sensitive polypeptide located at the C-terminus of the optically active polypeptide begins at amino acid Y of the succinic acid-sensitive polypeptide sequence, e.g., insertion position 131/126 indicates that the optically active polypeptide is fused at the N-terminus to amino acids N-terminus of the succinic acid-sensitive polypeptide sequence (e.g., any amino acid 1-654) to amino acid 126-to amino acid C-terminus of the succinic acid-sensitive polypeptide sequence, and the structure of the optically active polypeptide is (e.g., amino acid 320) from amino acid 65-position(s) - (320) of the optically active polypeptide sequence).
In one or more embodiments, the optical probe comprises, in order from the N-terminus to the C-terminus, the 65-X residue of SEQ ID No. 1, the optically active polypeptide shown in any one of SEQ ID nos. 2-9 or a variant thereof, and the Y-320 residue of SEQ ID No. 1, wherein X and Y are selected from any one of the following groups:
(1) X is 108, Y is 109,
(2) X is 108, Y is 110,
(3) X is 108, Y is 111,
(4) X is 108, Y is 112,
(5) X is 109, Y is 110,
(6) X is 109, Y is 111,
(7) X is 109, Y is 112,
(8) X is 110, Y is 111,
(9) X is 110, Y is 112,
(10) X is 111, Y is 112,
(11) X is 181, Y is 182,
(12) X is 181, Y is 183,
(13) X is 181, Y is 184,
(14) X is 181, Y is 185,
(15) X is 182, Y is 183,
(16) X is 182, Y is 184,
(17) X is 182, Y is 185,
(18) X is 183, Y is 184,
(19) X is 183, Y is 185,
(20) X is 184, Y is 185,
(21) X is 196, Y is 197,
(22) X is 196, Y is 198,
(23) X is 196, Y is 199,
(24) X is 196, Y is 200,
(25) X is 196, Y is 201,
(26) X is 197, Y is 198,
(27) X is 197, Y is 199,
(28) X is 197, Y is 200,
(29) X is 197, Y is 201,
(30) X is 198, Y is 199,
(31) X is 198, Y is 200,
(32) X is 198, Y is 201,
(33) X is 199, Y is 200,
(34) X is 199, Y is 201,
(35) X is 200, Y is 201,
(36) X is 148, Y is 149,
(37) X is 148, Y is 150,
(38) X is 148, Y is 151,
(39) X is 148, Y is 152,
(40) X is 148, Y is 153,
(41) X is 148, Y is 154,
(42) X is 148, Y is 155,
(43) X is 148, Y is 156,
(44) X is 149, Y is 150,
(45) X is 149, Y is 151,
(46) X is 149, Y is 152,
(47) X is 149, Y is 153,
(48) X is 149, Y is 154,
(49) X is 149, Y is 155,
(50) X is 149, Y is 156,
(51) X is 150, Y is 151,
(52) X is 150, Y is 152,
(53) X is 150, Y is 153,
(54) X is 150, Y is 154,
(55) X is 150, Y is 155,
(56) X is 150, Y is 156,
(57) X is 151, Y is 152,
(58) X is 151, Y is 153,
(59) X is 151, Y is 154,
(60) X is 151, Y is 155,
(61) X is 151, Y is 156,
(62) X is 152, Y is 151,
(63) X is 152, Y is 152,
(64) X is 152, Y is 153,
(65) X is 152, Y is 154,
(66) X is 152, Y is 155,
(67) X is 152, Y is 156,
(68) X is 153, Y is 151,
(69) X is 153, Y is 152,
(70) X is 153, Y is 153,
(71) X is 153, Y is 154,
(72) X is 153, Y is 155,
(73) X is 153, Y is 156,
(74) X is 154, Y is 151,
(75) X is 154, Y is 152,
(76) X is 154, Y is 153,
(77) X is 154, Y is 154,
(78) X is 154, Y is 155,
(79) X is 154, Y is 156,
(80) X is 155 and Y is 156.
Preferably, the optically active polypeptide is located at 150/152,150/153,150/155,150/156,151/153,152/152,152/153、152/154,153/152,153/153,153/154,154/151,154/152,154/154,181/182 of the amino acid sequence of the succinic acid binding protein, as shown in SEQ ID NO 10-24.
The term "variant" or "mutant" as used herein in reference to a polypeptide or protein includes variants having the same function but different sequences of the polypeptide or protein. Variants of a polypeptide or protein may include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. Such variants include, but are not limited to, sequences obtained by deleting, inserting and/or substituting one or more (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5) amino acids in the sequence of the polypeptide or protein, and adding one or more (usually within 20, preferably within 10, more preferably within 5) amino acids at the carboxy-terminal and/or amino-terminal end thereof. Without wishing to be bound by theory, amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, i.e., function-conservative mutations. For example, in the art, substitution with amino acids having similar or similar properties typically does not alter the function of the polypeptide or protein. Amino acids of similar properties are often referred to in the art as families of amino acids with similar side chains, which are well defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, succinic acid), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, succinic acid, histidine). As another example, the addition of one or more amino acids at the amino-and/or carboxy-terminus typically does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for many commonly known non-genetically encoded amino acids are known in the art. Conservative substitutions of other non-coding amino acids may be determined based on a comparison of their physical properties with those of the genetically encoded amino acid. It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. As another example, to construct a fusion protein, facilitate expression of a recombinant protein, obtain an automatic secretion of a recombinant protein outside a host cell, or facilitate purification of a recombinant protein, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, a tag such as 6His or Flag, or factor Xa or a proteolytic enzyme site of thrombin or enterokinase, for example. Variants of a polypeptide or protein may include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants. These variants may also comprise a polypeptide or protein having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the polypeptide or protein. Exemplary DctBp protein truncations are fragments of SEQ ID NO. 1 comprising amino acids 65-320 which retain the binding function of the DctBp protein to succinic acid and do not affect the change in optical properties of the inserted optically active polypeptide in response to succinic acid binding.
The optical probes of the invention may comprise succinic acid-sensitive polypeptides having mutations. A variant of succinic acid binding protein having a mutation at a site selected from the group consisting of S154, F155, Y160, N182, V183, S184 of SEQ ID NO1 or a truncated variant thereof exhibits a binding activity different from succinic acid. The amino acid mutation includes modification, substitution or deletion of amino acids. In preferred embodiments, the mutation of the succinic acid binding protein variant comprises a mutation at a site selected from any one of (1) F155 and Y160, (2) Y160 and N182, (3) Y160, N182 and V183, (4) Y160, N182 and S184, (5) S154, Y160, N182 and S184.
Wherein, as an example in the examples, in SEQ ID NO. 1 or a truncated variant thereof, S154 is mutated to R. In one or more embodiments, F155 is mutated to T, P, Q or C. In one or more embodiments, Y160 is mutated to Y or W. In one or more embodiments, N182 is mutated to S. In one or more embodiments, V183 is mutated to Y or W. In one or more embodiments, S184 is mutated to L.
In one or more embodiments, the mutation comprises a mutation selected from any one of (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N S and S184L. The present invention provides succinic acid binding protein variants having these mutations and optical probes comprising such succinic acid binding protein variants as succinic acid-sensitive polypeptides.
The optical probes of the invention may comprise optically active polypeptides having mutations. In some embodiments, the mutated optically active polypeptide has the sequence shown in SEQ ID NO. 2 and has mutations at 1, 2 or 3, 4, 5 or 6 positions M9, S132, Y141, F201, N207, Y245, said mutations comprising modifications, substitutions or deletions of amino acids, in one or more embodiments said mutations comprising mutations at any position selected from the group consisting of M9, S132, Y141, F201, N207, Y245. Specifically, the mutations are M9T, S132R, Y141N, F201S, N T and Y245F;
In one or more embodiments, the optical probe comprises any one of the amino acid sequences SEQ ID NOs 10-31 or a variant thereof. In one or more embodiments, the optical probes provided herein comprise sequences that are 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity to any one of the amino acid sequences SEQ ID NOs 10-31. In a preferred embodiment, the optical probes provided herein comprise a sequence substantially similar or identical to any one of the amino acid sequences SEQ ID NOs 10-31. Preferably, the optical probe has the sequence shown in SEQ ID NOS.25-31, and more preferably, the optical probe has the sequence shown in SEQ ID NOS.29-31.
In some embodiments, the succinic acid-sensitive polypeptide in the optical probe is as shown in amino acids 65-320 of SEQ ID NO:1, the optically active polypeptide is as shown in SEQ ID NO:2, the optically active polypeptide is at position 150/153 of the succinic acid-sensitive polypeptide, and the succinic acid-sensitive polypeptide has a mutation selected from any of (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N S and S184L, (9) S154R, Y160 35182S and S184L, or the optical probe has a mutation selected from any of (10) F160W, N S and S184L of the succinic acid-sensitive polypeptide, and M9 38328 383550325S and Y160W, (6) Y160W, N S and V183W, (8) Y160 56182S and S182S 35 and F245S 245L of the optically active polypeptide, and F35S 35 and F35.
The amino acid sequences of the optical probes shown in the (1) - (4) are shown as SEQ ID NO. 25, the amino acid sequence of the optical probe shown in the (5) is shown as SEQ ID NO. 26, the amino acid sequence of the optical probe shown in the (6) - (7) is shown as SEQ ID NO. 27, the amino acid sequence of the optical probe shown in the (8) is shown as SEQ ID NO. 28, the amino acid sequence of the optical probe shown in the (9) is shown as SEQ ID NO. 29, the amino acid sequence of the optical probe shown in the (10) is shown as SEQ ID NO. 30, and the amino acid sequence of the optical probe shown in the (11) is shown as SEQ ID NO. 31.
In two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percent identity" refers to two or more sequences or subsequences that are the same or wherein a percentage of amino acid residues or nucleotides are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) as compared and aligned for maximum correspondence over a comparison window or designated region, using methods known in the art, such as sequence comparison algorithms, by manual alignment and visual inspection. For example, preferred algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al (1977) Nucleic Acids Res.25:3389 and Altschul et al (1990) J.mol.biol.215:403, respectively.
It is well known to those skilled in the art that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed polypeptide or protein, without affecting the activity of the polypeptide or protein of interest. As another example, to construct a fusion protein, facilitate expression of a recombinant protein, obtain an automatic secretion of a recombinant protein outside a host cell, or facilitate purification of a recombinant protein, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, glutathione S-transferase (GST), maltose E binding protein, a tag such as 6His or Flag, or factor Xa or a proteolytic enzyme site of thrombin or enterokinase, for example.
The terms "functional variant," "derivative," and "analog" as used herein refer to a protein that retains substantially the same biological function or activity as the original polypeptide or protein (e.g., dctBp protein or fluorescent protein). The functional variant, derivative or analogue of a polypeptide or protein of the invention (e.g. DctBp protein or fluorescent protein) may be (i) a protein having one or more, preferably conservative or non-conservative amino acid residues substituted, which may or may not be encoded by the genetic code, or (ii) a protein having a substituent in one or more amino acid residues, or (iii) a protein formed by fusion of a mature protein with another compound (e.g. a compound that prolongs the half-life of the protein, such as polyethylene glycol), or (iv) a protein formed by fusion of an additional amino acid sequence to the protein sequence (e.g. a secretion sequence or a sequence used to purify the protein or a pro-protein sequence, or fusion protein with the formation of an antigen IgG fragment). Such functional variants, derivatives and analogs are within the scope of those skilled in the art, as determined by the teachings herein.
The difference between the analog and the original polypeptide or protein may be a difference in amino acid sequence, a difference in modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants may be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, as well as by site-directed mutagenesis or other known molecular biological techniques.
The analogs also include analogs having residues other than the natural L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It will be appreciated that the succinic acid-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives and analogues listed above. Modified (typically without altering the primary structure) forms include chemically derivatized forms of the protein, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the protein or during further processing steps. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Proteins modified to increase their proteolytic resistance or to optimize their solubility properties are also included.
Fusion polypeptides of the invention include optical probes and other polypeptides described herein. In some embodiments, the optical probes described herein further comprise additional polypeptides fused thereto. Other polypeptides described herein do not affect the properties of the optical probe. Other polypeptides may be located at the N-terminus and/or C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize the optical probe to a different organelle or subcellular organelle, tags for purification, or tags for immunoblotting. The fusion polypeptides described herein may have a linker between the optical probe and the other polypeptides.
Subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, golgi apparatus, lysosomes, peroxisomes, and the like. In some embodiments, the tag for purification or for immunoblotting comprises 6 histidine (6 xhis), glutathione-s-transferase (GST), flag.
The invention also provides a preparation method of the succinic acid optical probe, which comprises the following steps of 1) incorporating a nucleic acid sequence encoding the succinic acid optical probe into an expression vector, 2) transferring the expression vector into a host cell, 2) culturing the host cell under the condition suitable for the expression of the expression vector, and 3) separating the succinic acid optical probe.
The term "nucleic acid" or "nucleotide" as used herein may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The term "variant" as used herein when referring to a nucleic acid may be a naturally occurring allelic variant or a non-naturally occurring variant. Such nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution pattern of a nucleic acid, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the encoded protein. The nucleic acids of the invention may comprise a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the nucleic acid sequence. The invention also relates to nucleic acid fragments which hybridize to the sequences described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more in length. The nucleic acid fragments may be used in nucleic acid amplification techniques (e.g., PCR).
The full-length sequence of the optical probe or fusion protein of the present invention or a fragment thereof can be generally obtained by PCR amplification, artificial synthesis or recombinant methods. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, and the relevant sequences can be obtained by amplification using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the nucleotide sequence is larger than 2500bp, the PCR amplification is preferably carried out for 2-6 times, and then the amplified fragments are spliced together according to the correct sequence. The PCR amplification procedure and system are not particularly limited, and conventional PCR amplification procedures and systems in the art can be adopted. The sequences of interest can also be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into cells, and isolating and purifying the relevant polypeptide or protein from the proliferated host cells by conventional methods. Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In the present invention, when the nucleotide sequence of the optical probe is less than 2500bp, the optical probe can be synthesized by adopting an artificial synthesis method. The artificial synthesis method is a conventional DNA artificial synthesis method in the field, and has no other special requirements. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or functional variants, derivatives or analogues thereof) entirely by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art. Mutations can be introduced into the protein sequences of the present invention by mutation PCR or chemical synthesis, etc.
After the nucleotide sequence for coding the optical probe is obtained, the nucleotide sequence for coding the optical probe is incorporated into an expression vector to obtain a recombinant expression vector. The terms "expression vector" and "recombinant vector" are used interchangeably herein to refer to a prokaryotic or eukaryotic vector well known in the art, such as a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other vectors, which are capable of replication and stable expression in a host, an important feature of such recombinant vectors being that they typically contain expression control sequences. The term "expression control sequence" as used herein refers to an element operably linked to a gene of interest that regulates the transcription, translation and expression of the gene of interest, and may be an origin of replication, a promoter, a marker gene or a translational control element, including an enhancer, an operator, a terminator, a ribosome binding site, etc., the choice of expression control sequence being dependent upon the host cell used. Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In recombinant expression vectors, "operably linked" refers to the attachment of a nucleotide sequence of interest to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Methods for constructing expression vectors comprising the fusion protein coding sequences of the invention and appropriate transcriptional/translational control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are the lac or trp promoter of E.coli, the lambda phage PL promoter, eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses and some other known promoters which control gene expression in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one or more embodiments, the expression vector may be a commercially available pCDF vector, without other special requirements. Illustratively, the nucleotide sequence encoding the optical probe and the expression vector are double digested with HindIII and XhoI, respectively, and then the digested products of the two are ligated to obtain a recombinant expression vector. The specific steps and parameters of the digestion and the connection are not particularly limited, and the steps and parameters conventional in the art are adopted.
After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide comprising the fusion protein. Such transfer may be carried out by conventional techniques known to those skilled in the art, such as transformation or transfection. The host cell of the invention is a cell capable of receiving and accommodating recombinant DNA molecules, is a site for amplifying recombinant genes, and ideal recipient cells should satisfy both conditions of easy acquisition and proliferation. "host cells" according to the invention may include prokaryotic and eukaryotic cells, including in particular bacterial cells, yeast cells, insect cells and mammalian cells. Specific examples are bacterial cells of E.coli, streptomyces, salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS, HEK293, HEK293 cells, or Bowes melanoma cells, among others, including but not limited to those host cells described above. The host cell is preferably a variety of cells that facilitate expression or fermentative production of the gene product, such cells being well known and commonly used in the art. An exemplary host cell for use in embodiments of the invention is the E.coli BL21-DE3 strain. It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.
Methods of transferring to host cells described herein are conventional in the art and include calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical-mediated transfer, or electroporation. When the host is a prokaryote such as E.coli, the method is preferably a CaCl 2 method or MgCl 2 method treatment, using procedures well known in the art. When the host cell is eukaryotic, DNA transfection methods such as calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be used.
After the expression vector is transferred into a host cell, the host cell transferred into the expression vector is amplified, expressed and cultured, and the succinic acid optical probe is obtained by separation. The host cell amplification expression culture can be carried out by adopting a conventional method. The medium used in the culture may be various conventional media depending on the kind of host cell used. The culture is carried out under conditions suitable for the growth of the host cell.
In the present invention, the optical probe is expressed in a cell, on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated or purified by various isolation methods using their physical, chemical and other properties. The method for separating the succinic acid fluorescent protein is not particularly limited, and a method for separating fusion proteins conventional in the art can be adopted. Such methods are well known to those skilled in the art and include, but are not limited to, conventional renaturation treatment, salting-out methods, centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. In one or more embodiments, the separation of the optical probe is performed using His-tag affinity chromatography.
The invention also provides application of the succinic acid optical probe in succinic acid real-time positioning, quantitative detection and high-flux compound screening. In one aspect, the succinic acid optical probe is preferably connected with signal peptides at different parts of a cell, is transferred into the cell, performs real-time localization of succinic acid by detecting the intensity of fluorescent signals in the cell, and performs quantitative detection of corresponding succinic acid by using a succinic acid standard dripping curve. The succinic acid standard dripping curve is drawn according to fluorescent signals of succinic acid optical probes under the condition of different concentrations of succinic acid. The succinic acid optical probe is directly transferred into cells, and a time-consuming sample treatment process is not needed in the succinic acid real-time positioning and quantitative detection process, so that the succinic acid optical probe is more accurate. When the succinic acid optical probe is used for high-flux compound screening, different compounds are added into a cell culture solution, and the change of the succinic acid content is measured, so that the compounds with influence on the change of the succinic acid content are screened. The application of the succinic acid optical probe in succinic acid real-time positioning and quantitative detection and high-flux compound screening is non-diagnosis and treatment purposes, and does not relate to diagnosis and treatment of diseases.
Concentrations, amounts, percentages, and other numerical values may be expressed herein in terms of ranges. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include the numerical values explicitly recited as the limits of the range, as well as to include all the individual numerical values or sub-ranges encompassed within that range.
Examples
The succinic acid optical probes provided by the present invention are described in detail with reference to examples below, but they should not be construed as limiting the scope of the present invention.
I. experimental materials and reagents
In the examples, the conventional cloning method, cell culture and imaging methods of the genetically engineered molecular biology are mainly used, and these methods are well known to those skilled in the art, for example, jianluo Skems et al, J. Sambrook, D.W. Lassel, huang Peitang et al, ind. Molecular cloning laboratory Manual (third edition, 8 months 2002, published by Sci.11, beijing), fei Leixie ni et al, ind. Basic technical Specification (fifth edition), zhang Jingbo, xu Cunshuan et al, J.S. Bonefferson, M.dar et al, ind. Cell biological laboratory Manual, zhang Jingbo et al.
The pCDF-cpYFP-based, pCDF-succinic acid binding protein plasmid used in the examples was constructed by the protein laboratory of the university of Dongpo, and the pCDF plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai JieRui Bioengineering Co.Ltd. The expression plasmids constructed in the examples were all subjected to sequence determination, which was performed by Huada gene company and Jie Li Cexu company. Taq DNA polymerase used in each example was purchased from Dongsheng, pfu DNA polymerase was purchased from Tiangen Biochemical technology (Beijing) Co., ltd, PRIMESTAR DNA polymerase was purchased from TaKaRa Co., ltd, and the corresponding polymerase buffer and dNTPs were added when all three polymerases were purchased. BamHI, bglII, hindIII, ndeI, xhoI, ecoRI, speI, etc., a T4 ligase, a T4 phosphorylase (T4 PNK) are purchased from Fermentas, inc., and corresponding buffers are added thereto. Transfection reagent Lip2000 Kit was purchased from Invitrogen company. Succinic acid and other compounds were purchased from Sigma. Unless otherwise specified, chemical reagents such as inorganic salts were purchased from Sigma-Aldrich corporation. HEPES salts, ampicillin (Amp) and puromycin were purchased from Ameresco. 96-well assay blackboard, 384 Kong Yingguang assay blackboard, purchased from Grenier company.
The DNA purification kit used in the examples was purchased from BBI (Canada), and the ordinary plasmid minipump kit was purchased from Tiangen Biochemical technology (Beijing) Co. Clone strain Mach1 was purchased from Invitrogen. The nickel column affinity chromatography column and desalting column packing were all from GE HEALTHCARE company.
The main instruments used in the examples include Biotek Synergy 2 multifunctional enzyme-labeled instrument (Bio-Tek Co., ltd., U.S.A.), X-15R high-speed refrigerated centrifuge (Beckman Co., ltd., U.S.A.), microfuge22R bench-type high-speed refrigerated centrifuge (Beckman Co., ltd., U.S.A.), PCR amplification instrument (Biometa, germany), ultrasonoscope (Ningbo Zhi Co., ltd.), nucleic acid electrophoresis instrument (Shencan Bo Co., fluorescent spectrophotometer (Varian Co., ltd.), CO 2 constant temperature cell incubator (SANYO), inverted fluorescent microscope (Nikon Co., ltd.).
II molecular biology method and cell experiment method
II.1 Polymerase Chain Reaction (PCR):
1. Amplification of the fragment of interest PCR:
The method is mainly used for gene fragment amplification and colony PCR identification of positive clones. The PCR amplification reaction system comprises 0.5-1 mu L of template sequence, 0.5 mu L of forward primer (25 mu M), 0.5 mu L of reverse primer (25 mu M), 5 mu L of 10 Xpfu buffer solution, 0.5 mu L of pfu DNA polymerase, 1 mu L of dNTP (10 mM), 41.5-42 mu L of sterilized ultrapure water (ddH 2 O) and 50 mu L of total volume. The PCR amplification procedure was as follows, denaturation at 95℃for 2-10 min, 30 cycles (94-96℃for 30-45 seconds, 50-65℃for 30-45 seconds, 72℃for a certain time (600 bp/min)), and extension at 72℃for 10 min.
2. Long fragment (> 2500 bp) amplification PCR:
The long fragment amplification used in the present invention is mainly an inverse PCR amplification vector, a technique for obtaining site-directed mutagenesis in the following examples. Reverse PCR primers were designed at the mutation sites, wherein the 5' end of one primer contained the mutated nucleotide sequence. The amplified product contains the corresponding mutation site. The long fragment amplification PCR reaction was performed with 1. Mu.L of the template sequence (10 pg-1 ng), 0.5. Mu.L of the forward primer (25. Mu.M), 0.5. Mu.L of the reverse primer (25. Mu.M), 10. Mu.L of 5X PRIMERSTAR buffer, 0.5. Mu.L of PRIMERSTAR DNA polymerase, 4. Mu.L of dNTPs (2.5 mM), 33.5. Mu.L of sterilized ultrapure water (ddH 2 O), and a total volume of 50. Mu.L. The PCR amplification procedure was either 95℃for 5 minutes, 30 cycles (98℃for 10 seconds, 50-68℃for 5-15 seconds, 72℃for a certain time (1000 bp/min)), 72℃for 10 minutes, or 95℃for 5 minutes, 30 cycles (98℃for 10 seconds, 68℃for a certain time (1000 bp/min)), 72℃for 10 minutes.
II.2 endonuclease cleavage reaction:
The plasmid vector was digested simultaneously with 20. Mu.L (about 1.5. Mu.g) of the plasmid vector, 5. Mu.L of 10 Xbuffer, 1-2. Mu.L of restriction enzyme 1 and 1-2. Mu.L of restriction enzyme 2, and the total volume was made up to 50. Mu.L with sterilized ultrapure water. The reaction conditions were 37℃for 1-7 hours.
II.3 5' -terminal phosphorylation of DNA fragments
The plasmid or genome extracted from the microorganism contains phosphate groups at the terminal, but the PCR product does not, so that the 5' -terminal base of the PCR product needs to be subjected to phosphate group addition reaction, and only DNA molecules containing phosphate groups at the terminal can undergo ligation reaction. The phosphorylation reaction system was as follows, the DNA sequence of the PCR product fragment was 5-8. Mu.L, 10 XT 4 ligase buffer 1. Mu.L, T4 polynucleotide kinase (T4 PNK) 1. Mu.L, sterilized ultrapure water 0-3. Mu.L, and total volume 10. Mu.L. The reaction conditions were 37℃and after 30 minutes to 2 hours the reaction was inactivated at 72℃for 20 minutes.
II.4 ligation of the fragment of interest and the vector
The ligation methods between different fragments and vectors are different, and three ligation methods are used in the present invention
1. Blunt end ligation of blunt end short fragments and linearized vectors
The principle of the method is that a blunt end product obtained by PCR carries out phosphorylation reaction on the 5' end of a DNA fragment under the action of T4 PNK, and then is connected with a linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system was 4. Mu.L of T4 PNK treated DNA fragment, 4. Mu.L of linearized vector fragment, PEG4000 1. Mu.L, 10 XT 4 ligase buffer 1. Mu.L, T4 DNA ligase 1. Mu.L, and a total of 10. Mu.L. The reaction conditions were 22 ℃ for 30 minutes.
2. Ligation of DNA fragments containing cohesive ends and vector fragments containing cohesive ends
DNA fragments cleaved by restriction enzymes will typically produce protruding cohesive ends and thus can be ligated to cohesive end vector fragments containing sequence complementarity to form recombinant plasmids. The ligation reaction system was 1-7. Mu.L of the digested PCR fragment DNA, 0.5-7. Mu.L of the digested plasmid, 1. Mu.L of 10 XT 4 ligase buffer solution, 1. Mu.L of T4DNA ligase, and 10. Mu.L of sterilized ultrapure water were added to the total volume. The reaction conditions are 16 ℃ and 4-8 hours.
3. Ligation of the product of 5' -phosphorylated DNA fragments by self-cyclization after introduction of site-directed mutagenesis by inverse PCR
The DNA fragment with phosphorylated 5' end is connected with the 3' end and the 5' end of the linearization vector through self cyclization connection reaction to obtain the recombinant plasmid. The self-circularization ligation reaction system was 10. Mu.L of the phosphorylation reaction system, 0.5. Mu.L of T4 ligase (5U/. Mu.L), and a total volume of 10.5. Mu.L. The reaction conditions are 16 ℃ and 4-16 hours.
II.5 preparation and transformation of competent cells
Preparation of competent cells:
1. single colonies (e.g., mach 1) were picked and inoculated into 5mL LB medium, and shaken overnight at 37 ℃.
2. 0.5-1ML of the overnight cultured bacterial liquid is transferred to 50mL of LB culture medium, and cultured for 3 to 5 hours at 37 ℃ and 220rpm until OD 600 reaches 0.5.
3. The cells were pre-chilled in an ice bath for 2 hours.
Centrifuge at 4000rpm for 10 min at 4.4 ℃.
5. The supernatant was discarded, and the cells were resuspended in 5mL of pre-chilled buffer, and after homogenization, the resuspension buffer was added to a final volume of 50mL.
6. Ice bath for 45 minutes.
The bacteria were resuspended by centrifugation at 4000rpm at 7.4℃for 10 minutes with 5mL of ice-chilled storage buffer.
8. Mu.L of bacterial liquid was placed in each EP tube and frozen at-80℃or with liquid nitrogen.
Resuspension buffer CaCl 2(100mM)、MgCl2 (70 mM), naAc (40 mM)
Storage buffer 0.5mL DMSO, 1.9mL 80% glycerol, 1mL 10 XCaCl 2(1M)、1mL10×MgCl2(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH2 O
Transformation of competent cells:
1. 100. Mu.L of competent cells were thawed on an ice bath.
2. Add the appropriate volume of ligation product, gently blow mix, ice bath for 30 minutes. The ligation product is typically added in a volume of less than 1/10 of the competent cell volume.
3. The bacterial liquid is placed in a 42 ℃ water bath for heat shock for 90 seconds, and is quickly transferred to an ice bath for 5 minutes.
4. Mu.L of LB was added and incubated at 200rpm on a 37℃constant temperature shaker for 1 hour.
5. The bacterial liquid was centrifuged at 4000rpm for 3 minutes, 200. Mu.L of supernatant was left to blow the bacterial cells evenly, and the cells were spread evenly on the surface of an agar plate containing the appropriate antibiotics, and the plate was inverted overnight in a 37℃incubator.
II.6 expression, purification and fluorescence detection of proteins
1. Expression vectors (e.g., pCDF-based succinic optical probe expression vectors) were transformed into BL21 (DE 3) cells, cultured upside down overnight, cloned into 250ml Erlenmeyer flasks were picked from plates, placed in a 37℃shaker, cultured at 220rpm to OD=0.4-0.8, 1/1000 (v/v) of IPTG (1M) was added, and expression was induced at 18℃for 24-36 hours.
2. After the induction expression was completed, the cells were collected by centrifugation at 4000rpm for 30 minutes, and the cell pellet was resuspended in 50mM phosphate buffer and sonicated until the cells were clarified. Centrifugation was performed at 9600rpm at 4℃for 20 minutes.
3. The supernatant was purified by self-contained nickel column affinity chromatography to obtain protein, and the protein after nickel column affinity chromatography was further passed through self-contained desalting column to obtain protein dissolved in 100mM HEPES buffer (pH 7.4).
4. After SDS-PAGE identification of the purified proteins, the probes were diluted with assay buffer (100mM HEPES,100mM NaCl,pH 7.4) to a final concentration of 0.2-5. Mu.M protein solution. Succinic acid was formulated with assay buffer (100mM HEPES,100mM NaCl,pH 7.4) as stock solution with a final concentration of 50 mM.
5. 100 Μl of 1 μM protein solution was incubated at 37deg.C for 10 min, succinic acid was added for titration, and the fluorescence intensities of 528nm emission after 420nm excitation and 528nm emission after 485nm excitation were measured. The fluorescence excitation and emission measurement of the sample are completed by a multifunctional fluorescence enzyme-labeling instrument.
6. 100 Μl of 1 μM protein solution was incubated at 37deg.C for 10min, succinic acid was added, and the absorption spectrum and fluorescence spectrum of the protein were determined. The measurement of the absorption spectrum and fluorescence spectrum of the sample is performed by a spectrophotometer and a fluorescence spectrophotometer.
II.7 transfection and fluorescence detection of mammalian cells
1. The pcdna3.1+ based succinic acid optical probe plasmid was transfected into HEK293 by the transfection reagent Lipofectamine2000 (Invitrogen) and incubated in a cell incubator at 37 ℃,5% CO 2. And (4) performing fluorescence detection after the exogenous gene is fully expressed for 24-36 hours.
2. After the induction of expression is completed, the adherent HEK293 cells are washed three times by PBS and placed in HBSS solution for detection by a fluorescence microscope and an enzyme-labeled instrument respectively.
EXAMPLE 1 succinic acid binding protein plasmid
The DctBp (65-320) gene in the E.coli gene was amplified by PCR, and after gel electrophoresis, the PCR product was recovered and digested with BamHI and XhoI, and the pCDF vector was subjected to the corresponding double digestion. After ligation with T4 DNA ligase, DH 5. Alpha. Was transformed with the product, and the transformed DH 5. Alpha. Was plated on LB plates (streptomycin 100. Mu.g/mL) and incubated overnight at 37 ℃. The growing DH5 alpha transformants were subjected to plasmid extraction and PCR identification. The positive plasmid is sequenced correctly and then the subsequent plasmid construction is carried out.
Example 2 expression and detection of cpYFP optical probes at different insertion sites
In this example, the following sites were selected for insertion cpYFP based on pCDF-DctBp to give the corresponding pCDF-DctBp-cpYFP plasmid ：108/109,108/110,108/111,108/112,109/110,109/111,109/112,110/111,110/112,111/112,181/182,181/183,181/184,181/185,182/183,182/184,182/185,183/184,183/185,184/185,196/197,196/198,196/199,196/200,196/201,197/198,197/199,197/200,197/201,198/199,198/200,198/201,199/200,199/201,200/201,148/149,148/150,148/151,148/152,148/153,148/154,148/155,148/156,149/150,149/151,149/152,149/153,149/154,149/155,149/156,150/151,150/152,150/153,150/154,150/155,150/156,151/152,151/153,151/154,151/155,151/156,152/151,152/152,152/153,152/154,152/155,152/156,153/151,153/152,153/153,153/154,153/155,153/156,154/151,154/152,154/153,154/154,154/155,154/156,155/156.
And amplifying by using a PCR technology to obtain a cpYFP DNA fragment and a pCDF-DctBp linearization vector, wherein the 5 'and 3' extreme ends of the DNA fragment respectively have completely identical sequences (15 bp-20 bp) corresponding to the two extreme ends of cpYFP. The linearized pCDF-DctBp and cpYFP fragments were subjected to homologous recombination under the action of Hieff Clone Enzyme. The product was transformed into DH 5. Alpha. And the transformed DH 5. Alpha. Was plated on LB plates (streptomycin 100 ug/mL) and incubated overnight at 37 ℃. Positive clones identified by PCR were sequenced after drawing the plasmid. Sequencing was accomplished by Jie Li Cexu.
After sequencing correctly, the recombinant plasmid was transformed into BL21 (DE 3) to induce expression, and the protein was purified and sized around 57kDa by SDS-PAGE. The size of the recombinant DNA is consistent with the size of the DctBp-cpYFP fusion protein containing the His-tag purification tag expressed by pCDF-DctBp-cpYFP. The results are shown in FIG. 1.
The disrupted supernatant of E.coli expressing DctBp-cpYFP fusion protein was used for succinic acid response screening, and the detection signal of the fusion fluorescent protein containing succinic acid was divided by the detection signal of the fusion fluorescent protein without succinic acid. The results are shown in Table 1, and the detection results show that:
An optical probe having a response to succinic acid of more than 1.1 times or less than 0.9 times ：108/110、108/111、108/112、181/182、181/185、182/183、182/185、183/184、148/150、148/153、148/155、149/150、149/151、149/152、149/154、150/152、150/153、150/154、150/155、150/156、151/152、151/153、151/154、151/156、152/151、152/152、152/153、152/154、152/156、153/152、153/153、153/154、153/156、154/151、154/152、154/153、154/154、154/155、155/156;
There are 15 optical probes that respond more than 2-fold to succinic acid, at position 150/152,150/153,150/155,150/156,151/153,152/152,152/153、152/154,153/152,153/153,153/154,154/151,154/152,154/154,181/182.
TABLE 1
Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change
108/109 1.09 182/185 1.80 199/200 1.02 149/155 0.95 152/154 3.98
108/110 1.10 183/184 1.40 199/201 1.01 149/156 0.95 152/155 1.08
108/111 1.18 183/185 0.99 200/201 1.05 150/151 1.03 152/156 1.10
108/112 1.19 184/185 0.98 148/149 0.95 150/152 2.59 153/151 1.01
109/110 1.03 196/197 1.01 148/150 1.15 150/153 3.32 153/152 5.92
109/111 1.01 196/198 0.99 148/151 0.87 150/154 1.59 153/153 3.65
109/112 1.03 196/199 0.97 148/152 0.96 150/155 0.34 153/154 3.19
110/111 1.05 196/200 0.96 148/153 1.30 150/156 0.20 153/155 1.03
110/112 1.05 196/201 1.09 148/154 0.97 151/152 1.28 153/156 1.18
111/112 1.01 197/198 0.97 148/155 0.88 151/153 4.50 154/151 6.49
181/182 0.45 197/199 1.03 148/156 1.08 151/154 0.74 154/152 6.03
181/183 0.97 197/200 1.02 149/150 0.81 151/155 0.93 154/153 0.70
181/184 0.94 197/201 0.99 149/151 0.56 151/156 0.84 154/154 6.63
181/185 0.81 198/199 1.03 149/152 0.82 152/151 1.28 154/155 1.26
182/183 0.71 198/200 1.05 149/153 0.97 152/152 2.89 154/156 0.91
182/184 0.98 198/201 1.05 149/154 1.14 152/153 8.89 155/156 1.32
cpYFP 1.00
Example 3 expression and detection of cpGFP optical probes at different insertion sites
The green fluorescent protein succinate fluorescent probe was constructed by substituting cpYFP with cpGFP according to the procedure in example 2. As shown in table 3, the detection results showed that:
An optical probe having a response to succinic acid of more than 1.1 times or less than 0.9 times ：181/182、183/185、184/185、197/198、198/200、198/201、199/200、199/201、200/201、148/152、148/154、148/156、149/150、149/151、149/156、150/151、150/152、150/153、150/155、150/156、151/152、151/153、151/154、151/155、151/156、152/152、152/153、152/154、152/155、152/156、153/151、153/153、153/155、153/156、154/151、154/154、154/155、154/156、155/156;
An optical probe that responds more than 1.5 times to succinic acid has a position 150/153,151/152,152/153,152/154.
TABLE 2
Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change
108/109 0.97 182/185 0.97 199/200 1.42 149/155 0.97 152/154 2.21
108/110 0.99 183/184 0.94 199/201 1.29 149/156 1.20 152/155 1.45
108/111 1.00 183/185 1.18 200/201 1.10 150/151 1.24 152/156 1.26
108/112 1.01 184/185 1.14 148/149 0.89 150/152 1.41 153/151 1.39
109/110 1.03 196/197 1.00 148/150 1.04 150/153 1.89 153/152 1.00
109/111 1.01 196/198 1.02 148/151 1.07 150/154 1.09 153/153 1.41
109/112 1.00 196/199 1.00 148/152 1.28 150/155 1.39 153/154 1.04
110/111 0.97 196/200 0.97 148/153 1.04 150/156 1.25 153/155 1.44
110/112 0.99 196/201 0.99 148/154 1.20 151/152 2.01 153/156 1.37
111/112 1.00 197/198 0.89 148/155 1.07 151/153 1.30 154/151 1.29
181/182 1.25 197/199 0.98 148/156 1.25 151/154 1.32 154/152 1.04
181/183 1.09 197/200 1.00 149/150 0.89 151/155 1.39 154/153 0.99
181/184 1.00 197/201 1.04 149/151 1.19 151/156 1.41 154/154 1.89
181/185 0.96 198/199 1.02 149/152 1.08 152/151 1.07 154/155 0.85
182/183 1.08 198/200 1.20 149/153 1.05 152/152 1.24 154/156 1.49
182/184 0.93 198/201 1.31 149/154 0.98 152/153 2.08 155/156 1.28
cpGFP 1.00
Example 4 expression and detection of cpBFP optical probes at different insertion sites
The blue fluorescent protein succinate fluorescent probe was constructed by substituting cpYFP with cpBFP as in example 2. As shown in table 3, the detection results showed that:
an optical probe having a response to succinic acid of more than 1.1 times or less than 0.9 times ：108/109、108/110、110/111、110/112、181/185、183/184、183/185、197/201、200/201、148/150、148/151、148/152、148/153、149/151、149/153、149/155、150/151、150/152、150/155、151/152、151/153、151/154、151/156、152/151、152/152、152/154、152/155、152/156、153/151、153/152、153/153、153/156、154/151、154/152、155/156;
Optical probes that respond more than 1.3 times to succinic acid have sites at 148/152, 148/153, 151/154, 152/155, 152/156, 153/151, 154/152.
TABLE 3 Table 3
Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change
108/109 1.30 182/185 1.02 199/200 1.07 149/155 1.29 152/154 1.20
108/110 1.23 183/184 1.14 199/201 0.93 149/156 1.02 152/155 1.39
108/111 1.01 183/185 1.29 200/201 0.85 150/151 1.30 152/156 1.44
108/112 1.04 184/185 1.03 148/149 1.03 150/152 1.29 153/151 2.04
109/110 0.98 196/197 1.05 148/150 1.20 150/153 0.96 153/152 1.49
109/111 0.99 196/198 0.99 148/151 1.19 150/154 0.96 153/153 1.24
109/112 0.97 196/199 0.96 148/152 1.50 150/155 0.81 153/154 1.00
110/111 1.21 196/200 1.04 148/153 1.42 150/156 1.02 153/155 1.04
110/112 1.19 196/201 1.07 148/154 1.02 151/152 1.30 153/156 1.18
111/112 1.03 197/198 0.96 148/155 0.98 151/153 1.59 154/151 1.30
181/182 1.00 197/199 1.04 148/156 1.05 151/154 1.83 154/152 1.50
181/183 1.05 197/200 1.08 149/150 1.06 151/155 0.92 154/153 0.92
181/184 1.08 197/201 1.10 149/151 0.89 151/156 0.83 154/154 1.00
181/185 0.89 198/199 1.04 149/152 0.92 152/151 0.81 154/155 0.93
182/183 0.94 198/200 1.06 149/153 0.81 152/152 0.82 154/156 1.03
182/184 1.02 198/201 0.97 149/154 1.04 152/153 0.93 155/156 1.19
cpBFP 1.00
Example 5 expression and detection of cpmApple optical probes at different insertion sites
The fluorescent probe for succinic acid red fluorescent protein was constructed by substituting cpYFP with cpmApple according to the method in example 2. As shown in table 4, the detection results showed that:
An optical probe having a response to succinic acid of more than 1.1 times or less than 0.9 times ：108/109、108/110、110/111、196/201、197/201、199/200、200/201、148/149、148/151、148/155、149/152、149/156、150/155、150/156、151/156、152/154、152/155、152/156、153/151、153/152、153/153、154/152、154/153、154/156;
Optical probes that respond more than 1.2 times to succinic acid have positions 152/154, 153/153.
TABLE 4 Table 4
Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change Insertion site Multiple of change
108/109 0.89 182/185 0.95 199/200 1.19 149/155 0.93 152/154 1.21
108/110 0.83 183/184 1.03 199/201 1.01 149/156 0.82 152/155 1.17
108/111 0.98 183/185 1.08 200/201 1.17 150/151 1.04 152/156 1.16
108/112 0.93 184/185 1.02 148/149 1.11 150/152 1.02 153/151 1.20
109/110 1.02 196/197 0.97 148/150 1.02 150/153 1.00 153/152 1.10
109/111 1.06 196/198 0.93 148/151 0.86 150/154 0.92 153/153 1.29
109/112 1.08 196/199 1.01 148/152 0.92 150/155 0.85 153/154 0.92
110/111 1.10 196/200 1.04 148/153 0.94 150/156 0.82 153/155 0.95
110/112 0.92 196/201 1.13 148/154 0.97 151/152 1.04 153/156 1.02
111/112 1.02 197/198 1.02 148/155 0.89 151/153 1.01 154/151 1.04
181/182 1.04 197/199 1.07 148/156 1.03 151/154 1.02 154/152 0.85
181/183 1.08 197/200 1.04 149/150 1.02 151/155 1.00 154/153 0.82
181/184 1.00 197/201 0.82 149/151 1.07 151/156 1.12 154/154 0.94
181/185 0.97 198/199 0.92 149/152 0.89 152/151 1.04 154/155 0.97
182/183 0.93 198/200 0.95 149/153 0.92 152/152 1.03 154/156 1.11
182/184 0.91 198/201 1.02 149/154 1.04 152/153 0.98 155/156 1.03
cpmApple 1.00
Example 6 Properties of the optical Probe
For the optical probes having a response to succinic acid of more than 2 times obtained in example 2, namely, 15 optical probes inserted at ：150/152,150/153,150/155,150/156,151/153,152/152,152/153、152/154,153/152,153/153,153/154,154/151,154/152,154/154,181/182 sites, succinic acid detection was performed in a concentration gradient (0 to 100 mM), and the change in the ratio of the fluorescence intensity at the 528nm emission at 420nm and the fluorescence intensity at the 528nm emission at 485nm was detected. The K d (binding constant) of 8 succinic acid optical probes with insertion sites of 150/152, 153/153, 153/154, 154/151, 154/152, 154/154, 181/182 was too large to fit and unsuitable for detection. In addition, K d (binding constant) of the 7 succinic acid optical probes with insertion sites 150/153,150/155,150/156,151/153,152/152,152/153, 152/154 were 0.7mM,1.9mM,3.1mM,0.8mM,9.3mM,1.9mM,1.5mM, respectively. The results are shown in FIG. 2.
EXAMPLE 7 expression and detection of mutated cpYFP optical probes
The optical probe mutant is constructed on the basis of DctBp-150/153-cpYFP. The plasmid pCDF-DctBp-150/153-cpYFP was linearized by PCR, the primer contained the base sequence of the desired mutation site, the resulting PCR product was subjected to homologous recombination to give the S154, F155, Y160, N182, V183, S184 of succinic acid-sensitive polypeptide and the mutation plasmid of the 12 sites M9, S132, Y141, F201, N207, Y245 of optically active polypeptide, and sequencing was completed by Jie Li Cexu company.
The successfully constructed mutant plasmid is transformed into BL21 (DE 3) to induce expression, the broken supernatant of the escherichia coli expressing the probe protein is used for response screening of succinic acid and other nonspecific substrates, and the detection signal of the fusion fluorescent protein containing succinic acid or other nonspecific substrates is divided by the detection signal of the fusion fluorescent protein without succinic acid. The results are shown in Table 6, and the optical probes having a response to succinic acid of more than 2-fold and better specificity are shown below.
TABLE 6
Example 8 Properties of optical Probe mutants
The succinic acid optical probes in Table 6 described in example 7 were subjected to succinic acid detection with a concentration gradient (0 to 100 mM). After 10 minutes of probe treatment, the change in the ratio of the fluorescence intensity at 528nm emission from 420nm excitation to the fluorescence intensity at 528nm emission from 485nm excitation was detected. The results of the probe titration are shown in FIG. 3, and the results show that different mutants have different affinities for succinic acid.
The succinic acid probe in Table 6 was specifically detected, and the results of the specific detection with succinic acid structural analogues, fumaric acid, malic acid, alpha-ketoglutaric acid, citric acid, aspartic acid, pyruvic acid, oxaloacetic acid and malonic acid showed good specificity, as shown in FIG. 4.
Example 9 subcellular organelle localization of optical probes and the Performance of optical probes within subcellular organelles
In this example, different localization signal peptides were used to fuse with the optical probes DctBp-S154R/Y160W/N182S/S184L-cpYFP-M9T/S132R/Y141N/F201S/N207/Y245F to localize the optical probes to different organelles. HEK293 cells were transfected with optical probe plasmids fused with different localization signal peptides for 36 hours, rinsed with PBS, placed in HBSS solution and fluorescence detected under FITC channel using an inverted fluorescence microscope. The results are shown in FIG. 5. The succinic acid optical probe can be localized to the plasma, mitochondria, nucleus, nuclear exclusion, outer membrane, endoplasmic reticulum by fusion with different specific localization signal peptides. Fluorescence is shown in different subcellular structures, and the distribution and intensity of fluorescence are different.
HEK293 cells were transfected with cytoplasmic expression optical probe plasmid for 36 hours, rinsed with PBS, placed in HBSS solution, and the change in the ratio of fluorescence intensity at 420nm excitation 528nm emission to fluorescence intensity at 485nm excitation 528nm emission was detected over a 30min period. The results are shown in FIG. 6. 10mM succinic acid was added and detection was continued for 30 minutes. The 485/420 of the succinic acid added sample gradually increased up to 4 times the initial value, while the 485/420 of the control group without succinic acid added remained essentially unchanged.
Example 10 high throughput Compound screening in living cells based on optical probes
In this example, we used HEK293 cells expressing DctBp-S154R/Y160W/N182S/S184L-cpYFP-M9T/S132R/Y141N/F201S/N207/Y245F for high throughput compound screening.
Transfected HEK293 cells were rinsed with PBS, placed in HBSS solution (without succinic acid) for 1 hour, and then treated with 10 μm compound for 1 hour. Succinic acid was added dropwise to each sample. The change in the ratio of the fluorescence intensity at the 528nm emission of 420nm excitation to the fluorescence intensity at the 528nm emission of 485nm excitation was recorded using a microplate reader. Samples not treated with any compound were normalized as controls. The results are shown in FIG. 7. Of the 2000 compounds used, the vast majority of compounds had minimal effect on succinic acid entry into the cells. There are 7 compounds that increase the uptake of succinic acid by cells and 4 compounds that significantly decrease the uptake of succinic acid by cells.
Example 11 quantitative detection of succinic acid in blood by optical Probe
In this embodiment, purified DctBp-S154R/Y160W/N182S/S184L-cpYFP-M9T/S132R/Y141N/F201S/N207/Y245F succinic acid in mouse and human blood supernatants were used for the analysis.
After mixing DctBp-S154R/Y160W/N182S/S184L-cpYFP-M9T/S132R/Y141N/F201S/N207/Y245F with the diluted blood supernatant for 10 minutes, the ratio of the fluorescence intensity at 528nm excitation at 420nm to the fluorescence intensity at 528nm excitation at 485nm excitation was detected using a microplate reader. As a result, as shown in FIG. 8, the succinic acid content in the blood of the mice was about 32. Mu.M, and the succinic acid content in the blood of the human was about 16. Mu.M.
The embodiment shows that the succinic acid optical probe provided by the invention has the advantages of relatively small molecular weight, easiness in maturation, large dynamic change of fluorescence, good specificity, capability of expressing in cells by a gene operation method, capability of positioning and quantitatively detecting succinic acid inside and outside the cells in real time, and capability of performing high-flux compound screening.
Other embodiments
This specification describes a number of embodiments. It will be appreciated that various modifications may be made by those skilled in the art from a reading of this specification without departing from the spirit and scope of the invention, and are intended to be included within the scope of the appended claims.

Claims (11)

1.一种琥珀酸结合蛋白变体，其：1. A succinate binding protein variant, which: (1)具有SEQ ID NO:1所示的序列并且在以下的1个、2个或3个、4个、5个或6个位点具有突变：S154，F155，Y160，N182，V183，S184，所述突变包括氨基酸的修饰、取代或缺失，(1) having the sequence shown in SEQ ID NO: 1 and having a mutation at one, two, three, four, five or six of the following positions: S154, F155, Y160, N182, V183, S184, wherein the mutation comprises a modification, substitution or deletion of an amino acid, (2)是(1)的具有第65-320位氨基酸的截短变体，或(2) is a truncated variant of (1) having amino acids 65-320, or (3)是与(1)或(2)的序列具有至少70％序列相同性并具有(1)所述突变并保留对琥珀酸结合能力的序列，(3) a sequence having at least 70% sequence identity with the sequence of (1) or (2) and having the mutation described in (1) and retaining the ability to bind to succinate, 优选地，所述突变的位点选自以下任一组中的1个、2个、3个、4个、5个或6个：S154，F155，Y160，N182，V183，S184；Preferably, the mutated sites are selected from 1, 2, 3, 4, 5 or 6 of any one of the following groups: S154, F155, Y160, N182, V183, S184; 更优选地，所述突变包括选自以下任一组的位点处的突变：(1)F155和Y160，(2)Y160和N182，(3)Y160、N182和V183，(4)Y160、N182和S184，(5)S154、Y160、N182和S184；More preferably, the mutation includes a mutation at a site selected from any one of the following groups: (1) F155 and Y160, (2) Y160 and N182, (3) Y160, N182 and V183, (4) Y160, N182 and S184, (5) S154, Y160, N182 and S184; 更优选地，所述S154突变为R；F155突变为T、P、Q或C；Y160突变为Y或W；N182突变为S；V183突变为Y或W；S184突变为L；More preferably, the S154 mutates to R; F155 mutates to T, P, Q or C; Y160 mutates to Y or W; N182 mutates to S; V183 mutates to Y or W; S184 mutates to L; 更优选地，所述突变包含选自以下任一组的突变：(1)F155T和Y160W，(2)F155P和Y160W，(3)F155Q和Y160W，(4)F155C和Y160W，(5)Y160W和N182S，(6)Y160W、N182S和V183Y，(7)Y160W、N182S和V183W，(8)Y160W、N182S和S184L，(9)S154R、Y160W、N182S和S184L。More preferably, the mutation comprises a mutation selected from any one of the following groups: (1) F155T and Y160W, (2) F155P and Y160W, (3) F155Q and Y160W, (4) F155C and Y160W, (5) Y160W and N182S, (6) Y160W, N182S and V183Y, (7) Y160W, N182S and V183W, (8) Y160W, N182S and S184L, (9) S154R, Y160W, N182S and S184L. 2.一种光学探针，包含琥珀酸敏感多肽和光学活性多肽，其中所述光学活性多肽位于琥珀酸敏感多肽的残基108-112、181-185、196-201或148-156之间，所述琥珀酸敏感多肽是琥珀酸结合蛋白或其功能变体，所述光学活性多肽是荧光蛋白或其功能变体，2. An optical probe comprising a succinate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located between residues 108-112, 181-185, 196-201 or 148-156 of the succinate-sensitive polypeptide, the succinate-sensitive polypeptide is a succinate-binding protein or a functional variant thereof, and the optically active polypeptide is a fluorescent protein or a functional variant thereof, 优选地，所述琥珀酸敏感多肽具有：Preferably, the succinate-sensitive polypeptide has: (1)SEQ ID NO:1所示的序列或其具有第65-320位氨基酸的截短变体，或与他们有至少70％序列相同性并保留对琥珀酸结合活性的序列，(1) the sequence shown in SEQ ID NO: 1 or a truncated variant thereof having amino acids 65 to 320, or a sequence having at least 70% sequence identity therewith and retaining succinate binding activity, (2)权利要求1所述的琥珀酸结合蛋白变体的序列，或(2) the sequence of the succinate binding protein variant according to claim 1, or (3)与(2)所述序列具有至少70％序列相同性并具有(2)所述突变并保留对琥珀酸敏感性的序列，(3) a sequence having at least 70% sequence identity with the sequence described in (2) and having the mutation described in (2) and retaining sensitivity to succinic acid, 优选地，所述光学活性多肽具有：Preferably, the optically active polypeptide has: (a)SEQ ID NO:2-9中任一所示的序列，(a) a sequence shown in any one of SEQ ID NOs: 2-9, (b)SEQ ID NO:2所示的序列并且在选自以下的1个、2个或3个、4个、5个或6个位点具有突变：M9，S132，Y141，F201，N207，Y245，所述突变包括氨基酸的修饰、取代或缺失；优选地，所述突变选自以下任意一个或多个：M9T、S132R、Y141N、F201S、N207T和Y245F，或(b) the sequence shown in SEQ ID NO: 2 and having a mutation at one, two or three, four, five or six positions selected from the group consisting of: M9, S132, Y141, F201, N207, Y245, wherein the mutation comprises a modification, substitution or deletion of an amino acid; preferably, the mutation is selected from any one or more of the following: M9T, S132R, Y141N, F201S, N207T and Y245F, or (c)与(a)或(b)有至少70％序列相同性并保留荧光蛋白功能的变体序列。(c) A variant sequence having at least 70% sequence identity with (a) or (b) and retaining fluorescent protein function. 3.如权利要求2所述的光学探针，其特征在于，所述光学探针包含(2)所述的琥珀酸结合蛋白变体和(b)所述的光学活性多肽，并且包含选自以下任一组的突变：(1)琥珀酸结合蛋白的F155T和Y160W，(2)琥珀酸结合蛋白的F155P和Y160W，(3)琥珀酸结合蛋白的F155Q和Y160W，(4)琥珀酸结合蛋白的F155C和Y160W，(5)琥珀酸结合蛋白的Y160W和N182S，(6)琥珀酸结合蛋白的Y160W、N182S和V183Y，(7)琥珀酸结合蛋白的Y160W、N182S和V183W，(8)琥珀酸结合蛋白的Y160W、N182S和S184L，(9)琥珀酸结合蛋白的S154R、Y160W、N182S和S184L，(10)琥珀酸结合蛋白的Y160W、N182S和S184L，和荧光蛋白的M9T、S132R、Y141N、F201S、N207T和Y245F，(11)琥珀酸结合蛋白的S154R、Y160W、N182S和S184L，和荧光蛋白的M9T、S132R、Y141N、F201S、N207T和Y245F。3. The optical probe according to claim 2, characterized in that the optical probe comprises the succinate binding protein variant described in (2) and the optically active polypeptide described in (b), and comprises mutations selected from any one of the following groups: (1) F155T and Y160W of succinate binding protein, (2) F155P and Y160W of succinate binding protein, (3) F155Q and Y160W of succinate binding protein, (4) F155C and Y160W of succinate binding protein, (5) Y160W and N182S of succinate binding protein, (6) Y160W, N182S and V183Y of succinate binding protein, (7) Y160W, N182S and V183W of (8) Y160W, N182S and S184L of succinate binding protein, (9) S154R, Y160W, N182S and S184L of succinate binding protein, (10) Y160W, N182S and S184L of succinate binding protein, and M9T, S132R, Y141N, F201S, N207T and Y245F of fluorescent protein, (11) S154R, Y160W, N182S and S184L of succinate binding protein, and M9T, S132R, Y141N, F201S, N207T and Y245F of fluorescent protein. 4.如权利要求2或3所述的光学探针，其特征在于，光学活性多肽位于琥珀酸敏感多肽的选自下述位点中的一个或多个：108/109，108/110，108/111，108/112，109/110，109/111，109/112，110/111，110/112，111/112，181/182，181/183，181/184，181/185，182/183，182/184，182/185，183/184，183/185，184/185，196/197，196/198，196/199，196/200，196/201，197/198，197/199，197/200，197/201，198/199，198/200，198/201，199/200，199/201，200/201，148/149，148/150，148/151，148/152，148/153，148/154，148/155，148/156，149/150，149/151，149/152，149/153，149/154，149/155，149/156，150/151，150/152，150/153，150/154，150/155，150/156，151/152，151/153，151/154，151/155，151/156，152/151，152/152，152/153，152/154，152/155，152/156，153/151，153/152，153/153，153/154，153/155，153/156，154/151，154/152，154/153，154/154，154/155，154/156，155/156。4. The optical probe according to claim 2 or 3, wherein the optically active polypeptide is located at one or more of the following sites of the succinate sensitive polypeptide: 108/109, 108/110, 108/111, 108/112, 109/110, 109/111, 109/112, 110/111, 110/112, 111/112, 181/182, 181/183, 181/184, 181/185, 182/183 , 182/184, 182/185, 183/184, 183/185, 184/185, 196/197, 196/198, 196/199, 196/200, 196/201, 197/198, 197/199, 197/200, 197/201, 198/199, 198/200, 198/201, 199/200, 199/201, 200/201, 148/149, 148/1 50, 148/151, 148/152, 148/153, 148/154, 148/155, 148/156, 149/150, 149/151, 149/152, 149/153, 149/154, 149/155, 149/156, 150/151, 150/152, 150/153, 150/154, 150/155, 150/156, 151/152, 151/153, 15 1/154, 151/155, 151/156, 152/151, 152/152, 152/153, 152/154, 152/155, 152/156, 153/151, 153/152, 153/153, 153/154, 153/155, 153/156, 154/151, 154/152, 154/153, 154/154, 154/155, 154/156, 155/156. 5.如权利要求4所述的光学探针，其特征在于，所述光学探针具有SEQ ID NO:10-31中任一所示的序列。5 . The optical probe according to claim 4 , wherein the optical probe has a sequence shown in any one of SEQ ID NOs: 10-31. 6.一种核酸分子，其包含：(a)权利要求2-5中任一项所述的光学探针的编码序列，或(b)(a)的互补序列。6. A nucleic acid molecule comprising: (a) a coding sequence of the optical probe according to any one of claims 2 to 5, or (b) a complementary sequence of (a). 7.核酸构建物，包括权利要求6所述的核酸分子，7. A nucleic acid construct comprising the nucleic acid molecule according to claim 6, 优选地，所述核酸构建物是克隆载体、表达载体或重组载体。Preferably, the nucleic acid construct is a cloning vector, an expression vector or a recombinant vector. 8.一种宿主细胞，所述宿主细胞：8. A host cell, wherein: (1)表达权利要求2-5中任一项所述的光学探针；(1) An optical probe as described in any one of claims 2 to 5; (2)包含权利要求6所述的核酸分子；和/或(2) comprising the nucleic acid molecule according to claim 6; and/or (3)包含权利要求7所述的核酸构建物。(3) comprising the nucleic acid construct according to claim 7. 9.一种检测试剂盒，其包含：9. A detection kit comprising: (1)权利要求2-5中任一项所述的光学探针，(1) The optical probe according to any one of claims 2 to 5, (2)权利要求6所述的核酸分子，(2) The nucleic acid molecule according to claim 6, (3)权利要求7所述的核酸构建物，(3) The nucleic acid construct according to claim 7, (4)权利要求8所述的宿主细胞，(4) The host cell according to claim 8, 所述检测试剂盒任选还包含利用光学探针检测琥珀酸所需的其他试剂，The detection kit optionally further comprises other reagents required for detecting succinic acid using an optical probe. 优选地，所述检测试剂盒还包含选自以下的一种或多种试剂：缓冲液、培养基、琥珀酸标准品。Preferably, the detection kit further comprises one or more reagents selected from the following: a buffer, a culture medium, and a succinic acid standard. 10.制备权利要求2-5中任一项所述光学探针的方法，包括：培养权利要求8所述的宿主细胞，和由培养物分离所述光学探针。10. A method for preparing the optical probe according to any one of claims 2 to 5, comprising: culturing the host cell according to claim 8, and isolating the optical probe from the culture. 11.权利要求2-5中任一项所述的光学探针、权利要求6所述的核酸分子、权利要求7所述的核酸构建物和/或权利要求8所述的宿主细胞在检测样品中的琥珀酸、筛选化合物或琥珀酸细胞内和/或外定位中的应用。11. Use of the optical probe according to any one of claims 2 to 5, the nucleic acid molecule according to claim 6, the nucleic acid construct according to claim 7 and/or the host cell according to claim 8 in detecting succinic acid in a sample, screening compounds or the intracellular and/or extracellular localization of succinic acid.
CN202311508503.5A 2023-11-13 2023-11-13 A succinic acid optical probe and its preparation method and application Pending CN119978072A (en)

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