WO2021164668A1 - Pyruvic acid optical probe, preparation method therefor, and application thereof - Google Patents

Pyruvic acid optical probe, preparation method therefor, and application thereof Download PDF

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WO2021164668A1
WO2021164668A1 PCT/CN2021/076317 CN2021076317W WO2021164668A1 WO 2021164668 A1 WO2021164668 A1 WO 2021164668A1 CN 2021076317 W CN2021076317 W CN 2021076317W WO 2021164668 A1 WO2021164668 A1 WO 2021164668A1
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pyruvate
optical probe
polypeptide
sequence
protein
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Chinese (zh)
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杨弋
赵玉政
李写
张则一
张秀泽
黄立
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华东理工大学
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds

Definitions

  • the invention relates to the technical field of optical probes, in particular to a pyruvate optical probe and a preparation method and application thereof.
  • Pyruvate is an organic acid with two functional groups, carbonyl and carboxyl, and is also the simplest ⁇ -keto acid, which is widely present in various animals and plants. Pyruvate is inseparable from energy metabolism. During glycolysis, one molecule of glucose or glycogen and two molecules of ATP are consumed to produce two molecules of NADH, four molecules of ATP and two molecules of pyruvate. In the cytoplasm, pyruvate can be reduced to supply energy, and can also be used as the main fuel for transport to the mitochondria to become the tricarboxylic acid cycle. Pyruvate enters the mitochondria under aerobic conditions.
  • the pyruvate dehydrogenase complex catalyzes oxidative decarboxylation to produce NADH, CO2 and acetyl-CoA.
  • Acetyl-CoA enters the tricarboxylic acid cycle and oxidative phosphorylation is completely oxidized to CO2 and H2O and released The energy of this process can produce a large amount of ATP. This is the aerobic oxidation process of sugar and the main way for the body to obtain ATP.
  • pyruvate can generate alanine through transamination, which is used as a raw material for protein synthesis. Pyruvate can also realize the mutual conversion of sugars, fats and amino acids in the body through the acetyl-CoA and tricarboxylic acid cycle. In summary, pyruvate plays an important pivotal role in the metabolic connection of the three major nutrients, and is a key molecule that is vital to many aspects of eukaryotic and human metabolism.
  • pyruvate can directly inhibit hydrogen peroxide through a non-enzymatic decanoation reaction, and has the effect of preventing free radical damage. It has been proven to protect the body against functional damage in cardiac reperfusion injury and acute renal failure. . Supplementing pyruvate can enhance the citric acid cycle. When the production of citric acid increases, it inhibits phosphofructokinase and enters the pentose phosphate bypass to produce reduced coenzyme II (NADPH), thereby indirectly increasing the antioxidant capacity of glutathione (GSH) System capabilities.
  • NADPH reduced coenzyme II
  • pyruvate detection methods for pyruvate include ultraviolet spectrophotometer, high performance liquid chromatography, dehydrogenase colorimetry and so on.
  • detection methods for pyruvate include ultraviolet spectrophotometer, high performance liquid chromatography, dehydrogenase colorimetry and so on.
  • time-consuming sample processing procedures are required, such as cell disruption, separation, extraction and purification, etc.; in situ, real-time, dynamic, and high-speed analysis in living cells and subcellular organelles cannot be performed. Detection of throughput and high temporal and spatial resolution.
  • the art still needs a method capable of real-time positioning, quantitative, and high-throughput detection of pyruvate inside and outside the cell.
  • the purpose of the present invention is to provide a probe and method for real-time positioning, high-throughput, and quantitative detection of pyruvate inside and outside the cell.
  • the present invention provides a pyruvate optical probe, comprising a pyruvate sensitive polypeptide or its functional variant and an optically active polypeptide or its functional variant, wherein the optically active polypeptide or its functional variant is located in the pyruvate sensitive polypeptide or its functional variant Within the sequence.
  • the pyruvate sensitive polypeptide or its functional variant is divided into a first part and a second part by the optically active polypeptide or its functional variant.
  • the present invention provides a pyruvate optical probe, comprising pyruvate sensitive polypeptide B and optically active polypeptide A, wherein the optically active polypeptide A is located in the sequence of pyruvate sensitive polypeptide B, and the pyruvate sensitive polypeptide B is divided into the first part B1 And the second part B2, forming a probe structure of formula B1-A-B2.
  • the pyruvate sensitive polypeptide includes a pyruvate binding protein or a pyruvate binding domain thereof. In one embodiment, the pyruvate sensitive polypeptide is derived from Escherichia coli. In one embodiment, the pyruvate sensitive polypeptide is a pyruvate binding protein or a functional fragment thereof. In one or more embodiments, the pyruvate binding protein is a PdhR protein. In one embodiment, the pyruvate-sensitive polypeptide has the sequence shown in SEQ ID NO: 1, or at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% of the sequence shown in SEQ ID NO: 1.
  • the functional fragment of the pyruvate-sensitive polypeptide has amino acids 96-254 of the sequence shown in SEQ ID NO:1, or at least 35%, at least 40%, at least 50%, at least 60%, or at least Sequences that are 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identical and retain pyruvate binding function.
  • the optically active polypeptide is a fluorescent protein or a functional variant thereof.
  • the fluorescent protein is selected from yellow fluorescent protein (cpYFP shown in SEQ ID NO: 2), orange fluorescent protein, red fluorescent protein, and green fluorescent protein (cpGFP shown in SEQ ID NO: 3) , Blue fluorescent protein (cpBFP shown in SEQ ID NO: 4), Apple red fluorescent protein (cpmApple shown in SEQ ID NO: 5).
  • the optically active polypeptide is cpYFP.
  • the fluorescent protein has a sequence shown in any one of SEQ ID NO: 2-5.
  • the optical probe further comprises one or more linkers flanking the optically active polypeptide.
  • the linker of the present invention can be any amino acid sequence of any length.
  • the optically active polypeptide flanking includes a linker of no more than 5 amino acids, such as a linker of 0, 1, 2, 3, or 4 amino acids.
  • the linker flanking the optically active polypeptide comprises amino acid Y.
  • the linker Y is located at the N-terminus and/or C-terminus of the optically active polypeptide.
  • the optical probe is as follows: the first part of the pyruvate sensitive polypeptide B1-Y-optically active polypeptide A-the second part of the pyruvate sensitive polypeptide B2.
  • the optical probe of the present invention does not include a joint.
  • the optically active polypeptide of the present invention can be located at any position of the pyruvate sensitive polypeptide.
  • the optically active polypeptide is located in one or more positions of the pyruvate sensitive polypeptide selected from the group consisting of residues 117-121, 140-143, 160-164, 174-176, 191-195, and/or 210-214, numbering corresponds to the full length of the pyruvate binding protein.
  • the optically active polypeptide replaces one or more amino acids in one or more positions of the pyruvate-sensitive polypeptide selected from the following: residues 117-121, 140-143, 160-164, 174-176, 191-195 and/or 210-214.
  • the optically active polypeptide is inserted into one or more positions of the pyruvate sensitive polypeptide selected from the following: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 140/141, 140/142, 140/143, 141/142, 141/143, 142/143, 160/161, 160/162, 160/ 163, 160/164, 161/162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191/192, 191/193, 191/194, 191/195, 192/193, 192/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/211, 210/212, 210/213, 210/214, 211/ 212, 211/213, 211/214, 212/213, 212/214 and/or 213/214.
  • the optically active polypeptide is located at one or more positions of the pyruvate sensitive polypeptide selected from the following: 117/121, 141/143, 191/192, 191/193, 191/194, 191/195, 192/193 , 192/194, 192/195, 193/194, 193/195, 194/195 and 210/214.
  • the optical probe of the present invention may be a probe selected from one or more of the following sites: 117/120, 117/121, when cpYFP is located in the pyruvate binding protein or its functional fragment.
  • the optical probe of the present invention may be a probe selected from one or more of the following sites when cpYFP is located in a pyruvate binding protein or a functional fragment thereof: 117/121, 119/120, 119/ 121, 120/121, 140/143, 141/142, 141/143, 160/161, 160/164, 161/163, 191/192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/212, 210/214, 211/213 and 211/214.
  • the optical probe of the present invention may be a probe selected from one or more of the following positions when cpYFP is located in a pyruvate binding protein or a functional fragment thereof: 117/121, 141/143, 191/192, 191/ 193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195 and 210/214.
  • the functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
  • the optical probe of the present invention may be a probe selected from one or more of the following sites when cpGFP is located in the functional fragment of pyruvate binding protein: 117/120, 118/119, 119/ 120, 140/141, 140/142, 141/142, 141/143, 142/143, 160/161, 161/163, 161/164, 162/163, 174/175, 191/193, 210/212, 211/212, 211/214, 212/213, 212/214 and/or 213/214.
  • the optical probe of the present invention may be a probe selected from one or more of the following positions when cpGFP is located in the functional fragment of pyruvate binding protein: 118/119, 140/141, 160/161, 191/193, 210/212, 212/213, 212/214 and/or 213/214.
  • the functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
  • the optical probe of the present invention may be a probe selected from one or more of the following sites when cpBFP is located in a pyruvate binding protein or a functional fragment thereof: 117/118, 117/120, 119/ 120, 119/121, 140/141, 141/142, 160/162, 160/163, 161/163, 174/176, 191/192, 191/194, 192/193, 192/195, 193/194, 193/195, 194/195, 210/212, 211/213 or 212/213.
  • the optical probe of the present invention may be a probe selected from one or more of the following sites when cpBFP is located in a pyruvate binding protein or a functional fragment thereof: 141/142, 160/163, 192/ 193, 192/195, 193/194, 193/195, 194/195, 210/212, 211/213.
  • the functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
  • the optical probe of the present invention may be a probe selected from one or more of the following positions when cpmApple is located in a pyruvate binding protein or a functional fragment thereof: 117/119, 117/120, 118/ 119, 118/120, 119/121, 140/141, 140/142, 141/142, 160/161, 160/162, 160/164, 162/163, 174/176, 175/176, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 210/212, 211/213, 211/214, 212/214.
  • the optical probe of the present invention can be a probe selected from one or more of the following positions when cpmApple is located in a pyruvate binding protein or a functional fragment thereof: 117/120, 118/120, 160/ 162, 162/163, 191/195, 192/193, 192/194, 192/195, 210/212, 211/213.
  • the functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
  • the optical probe of the present invention has or consists of the sequence shown in SEQ ID NO: 6-18.
  • the present invention also provides pyruvate sensitive polypeptides with one or more mutations.
  • the mutation is located at 1, 2, 3, 4, 5, 6, or 7 positions in Q138, S190, R191, R192, E193, M194, and L195 of the pyruvate binding protein or its functional fragments .
  • the mutation is selected from 1, 2, 3, 4, 5, 6, or 7 of the following: Q138S, Q138Y, Q138C, Q138L, Q138P, Q138H, Q138R, Q138W, Q138I, Q138T, Q138N, Q138K, Q138F, Q138V, Q138A, Q138D, Q138E, Q138M, Q138A, S190E, R191S, R191Y, R191C, R191L, R191P, R191H, R191Q, R191W, R191I, R191T, R191N, R191K, R191F, R191F, R191A, R191D, R191E, R191M, R191A, R192D, E193S, E193Y, E193C, E193L, E193P, E193H, E193Q, E193W, E193R, E193I, E193T, E
  • the present invention also provides an optical probe comprising a pyruvate sensitive polypeptide with one or more mutations.
  • the optical probe is any optical probe inserted with an optically active polypeptide as described above, and the pyruvate-sensitive polypeptide in the optical probe is selected from Q138, S190, R191, R192 , E193, M194, and L195 have mutations at 1, 2, 3, 4, 5, 6, or 7 positions.
  • the optical probe containing the mutant pyruvate sensitive polypeptide has no less response to pyruvate than its non-mutated counterpart.
  • the mutation is selected from 1, 2, 3, 4, 5, 6, or 7 of the following: Q138S, Q138Y, Q138C, Q138L, Q138P, Q138H, Q138R , Q138W, Q138I, Q138T, Q138N, Q138K, Q138F, Q138V, Q138A, Q138D, Q138E, Q138M, Q138A, S190E, R191S, R191Y, R191C, R191L, R191P, R191H, R191Q, R191R191N, R191T, R191K , R191F, R191V, R191A, R191D, R191E, R191M, R191A, R192D, E193S, E193Y, E193C, E193L, E193P, E193H, E193Q, E193W, E193R, E193I, E193T, E1919
  • the optical probe of the B1-A-B2 type of the present invention can be a pyruvate synthin functional fragment with cpYFP inserted at positions 141/143, 191/193, 192/194 or 192/195 and having Q138P.
  • the optical probe of the present invention is a mutant based on different insertion sites, wherein the combination of insertion site and mutation is selected from: 191/193-Q138P, 191/193-Q138L, 191 /193-R191Y, 191/193-R191F, 191/193-R191L, 191/193-R191P, 191/193-E193Q, 191/193-E193L, 192/194-M194D, 192/194-M194V, 192/194 -M194H, 192/194-M194W, 192/194-M194V-S190E/R191N/R192D, 192/194-M194V/S190D/R191Y/R192T, 141/143-S190P/R191H/R192P, 141/143-S190R/R191S /R192P, 141/143-S190L/R191V, 14
  • the optical probe provided by the present invention comprises any one of the amino acid sequence SEQ ID NO: 6-30 or a variant thereof.
  • the optical probe provided by the present invention contains 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90% of the amino acid sequence SEQ ID NO: 6-30. %, 95%, 99% sequence identity.
  • the optical probe provided by the present invention contains a sequence substantially similar or identical to any one of the amino acid sequence SEQ ID NO: 6-30.
  • the optical probe provided by the present invention comprises or consists of any one of SEQ ID NO: 19-30.
  • the present invention also provides fusion polypeptides, including the optical probes described herein and other polypeptides.
  • other polypeptides are located at the N-terminus and/or C-terminus of the optical probe.
  • other polypeptides include polypeptides that localize optical probes to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting.
  • the present invention also provides nucleic acid sequences encoding the polypeptides, probes or proteins described herein or their complementary sequences.
  • the nucleic acid sequence of the present invention is selected from (1) the coding sequence of any amino acid sequence shown in SEQ ID NO: 6-30 or its complementary sequence, and (2) and (1) have at least 99%, Sequences that are at least 95%, at least 90%, at least 80%, at least 70%, or at least 50% identical, fragments of (3) (1) or (2).
  • the fragment is a primer.
  • the nucleic acid sequence of the present invention comprises the nucleotide sequence SEQ ID NO: 31 or a variant thereof, and the amino acid sequence encoded by the variant has the function of detecting tryptophan.
  • the present invention provides a nucleic acid sequence comprising at least 99%, at least 95%, at least 90%, at least 80%, at least 70% or at least 50% identical to the nucleotide sequence SEQ ID NO: 31
  • the sequence of sex also relates to a complementary sequence of the above-mentioned nucleic acid sequence or a variant thereof, which may comprise a nucleic acid sequence or a complementary sequence thereof encoding a fragment, analog, derivative, soluble fragment and variant of the optical probe or fusion polypeptide of the present invention.
  • the present invention also provides a nucleic acid construct comprising the nucleic acid sequence described herein or its complementary sequence, which nucleic acid sequence encodes the optical probe or fusion polypeptide of the present invention.
  • the nucleic acid construct is a cloning vector, expression vector or recombinant vector.
  • the nucleic acid sequence is operably linked to an expression control sequence.
  • the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors and viral vectors.
  • the present invention also provides a cell comprising the nucleic acid sequence or nucleic acid construct of the present invention.
  • the cell expresses the optical probe or fusion polypeptide described herein.
  • the present invention also provides a detection kit comprising the pyruvate optical probe or fusion polypeptide described herein or the pyruvate optical probe or fusion polypeptide prepared by the method described herein.
  • the present invention provides a method for preparing the optical probe described herein, including: providing a cell expressing the optical probe or fusion polypeptide described herein, culturing the cell under the condition of expression of the cell, and isolating the optical probe or fusion polypeptide .
  • the method for preparing the tryptophan optical probe or fusion polypeptide described herein includes: 1) transferring the expression vector encoding the pyruvate optical probe described herein into a host cell; 2) The host cell is cultured under the condition of expression vector expression, and 3) the optical probe is isolated.
  • the present invention also provides a method for detecting pyruvate in a sample, including: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, and detecting changes in the optically active polypeptide.
  • the detection can be performed in vivo, in vitro, subcellular or in situ.
  • the sample is for example blood.
  • This article also provides a method for quantifying pyruvate in a sample, including: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, detecting changes in the optically active polypeptide, and The change in the optically active peptide quantifies the pyruvate in the sample.
  • the present invention also provides a method for screening a compound (such as a drug), comprising: contacting an optical probe or fusion polypeptide as described herein or an optical probe or fusion polypeptide prepared as described herein with a candidate compound to detect changes in the optically active polypeptide , And screening compounds based on changes in optically active polypeptides.
  • the method can screen compounds with high throughput.
  • the present invention also provides the application of the pyruvate optical probe or fusion polypeptide described herein or the pyruvate optical probe or fusion polypeptide prepared by the method described herein in the intracellular/extracellular localization of pyruvate.
  • the positioning is real-time positioning.
  • the pyruvate optical probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, good specificity, and can be expressed in cells by genetic manipulation methods, and can be located inside and outside cells in real time, with high throughput, Quantitative detection of pyruvate saves time-consuming sample processing steps.
  • the experimental results show that the highest response of the pyruvate optical probe provided in this application to pyruvate is more than 10 times that of the control, and it can be used in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosome, and Golgi apparatus. It can locate, qualitatively and quantitatively detect cells, and can perform high-throughput compound screening and quantitative detection of pyruvate in blood.
  • Fig. 1 is an SDS-PAGE chart of the exemplary pyruvate optical probe described in Example 1;
  • FIG. 2 is a graph showing the response change of the exemplary pyruvate optical probe containing cpYFP and pyruvate binding protein to pyruvate described in Example 2;
  • FIG. 3 is a graph showing the response change of the exemplary pyruvate optical probe containing cpGFP and pyruvate binding protein to pyruvate described in Example 3;
  • FIG. 4 is a graph showing the response of the exemplary pyruvate optical probe containing cpBFP and pyruvate binding protein to pyruvate described in Example 4;
  • Example 5 is a graph showing the response change of the exemplary pyruvate optical probe containing cpmApple and pyruvate binding protein to pyruvate described in Example 5;
  • FIG. 6 shows the response of an exemplary pyruvate optical probe with mutations to pyruvate based on the insertion of cpYFP at positions 191/193, 192/194 or 192/195 of the pyruvate binding protein described in Example 6;
  • FIG. 7A-B are titration curves of the exemplary pyruvate optical probe described in Example 7 to different concentrations of pyruvate;
  • Example 8A-C are graphs of the fluorescence spectrum properties of the exemplary pyruvate optical probe described in Example 8.
  • Example 9 is a histogram of the specific detection of the exemplary pyruvate optical probe described in Example 8.
  • FIG. 10 is a photograph of the subcellular organelle location of the exemplary pyruvate optical probe described in Example 9 in mammalian cells;
  • FIG. 11 is a schematic diagram of the response of the exemplary pyruvate optical probe described in Example 10 to exogenous pyruvate in mammalian cells;
  • Fig. 12 is a point diagram of the exemplary pyruvate optical probe described in Example 11 for high-throughput compound screening at the level of living cells;
  • Example 13 is a bar graph of the exemplary pyruvate optical probe described in Example 12 for quantifying pyruvate in the blood of mice and humans.
  • pyruvate sensitive polypeptide or “pyruvate responsive polypeptide” refers to a polypeptide that responds to pyruvate, and the response includes any of the chemical, biological, electrical or physiological parameters of the polypeptide related to the interaction of the sensitive polypeptide. response.
  • the response includes small changes, for example, changes in the orientation of the amino acids or peptide fragments of the polypeptide and, for example, changes in the primary, secondary, or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential, and/or conformation.
  • Conformation refers to the three-dimensional arrangement of the primary, secondary and tertiary structure of the molecule containing side groups; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include conversion from ⁇ -helix to ⁇ -sheet or from ⁇ -sheet to ⁇ -helix. It is understandable that as long as the fluorescence of the fluorescent protein portion is changed, the detectable change need not be a conformational change.
  • the pyruvate sensitive polypeptides described herein may also include functional variants thereof. Functional variants of the pyruvate-sensitive polypeptide include, but are not limited to, variants that can interact with pyruvate to undergo the same or similar changes as the parent pyruvate-sensitive polypeptide.
  • the pyruvate sensitive polypeptides of the present invention include but are not limited to pyruvate binding protein PdhR or variants with more than 90% homology.
  • the exemplary pyruvate binding protein PdhR of the present invention is derived from Escherichia coli.
  • PdhR is a bacterial transcription factor composed of a pyruvate binding/regulatory domain and a DNA binding domain.
  • An exemplary PdhR protein is shown in SEQ ID NO:1.
  • the pyruvate sensitive polypeptide comprises the pyruvate binding domain of the PdhR protein, that is, amino acids 96-254, but does not include the DNA binding domain.
  • optical probe refers to a pyruvate sensitive polypeptide fused to an optically active polypeptide.
  • the inventors found that the conformational changes produced by pyruvate-sensitive polypeptides such as pyruvate-binding proteins that specifically bind to physiological concentrations of pyruvate will cause conformational changes of optically active polypeptides (such as fluorescent proteins), which in turn leads to optically active polypeptides.
  • optically active polypeptides such as fluorescent proteins
  • the optically active polypeptide (for example, fluorescent protein) is operably inserted into the pyruvate-sensitive polypeptide.
  • Protein-based "optically active polypeptides" are polypeptides that have the ability to emit fluorescence. Fluorescence is an optical property of optically active polypeptides, which can be used as a means to detect the responsiveness of the optical probe of the present invention.
  • the protein substrate is selected to have fluorescent properties that are easily distinguishable in the unactivated and activated conformational states.
  • the optically active polypeptides described herein may also include functional variants thereof. Functional variants of an optically active polypeptide include, but are not limited to, variants that can have the same or similar changes in fluorescence properties as the parent optically active polypeptide.
  • Linker refers to an amino acid or nucleotide sequence that connects two parts in the polypeptide, protein or nucleic acid of the present invention.
  • the number of amino acids at the amino terminus of the connecting region of the pyruvate sensitive polypeptide and the optically active polypeptide is selected from 0 to 3
  • the number of amino acids at the carboxy terminus is selected from 0 to 2; when the recombinant optical probe is used as the basic unit When connected to a functional protein, it can be fused to the amino acid or carboxyl end of the recombinant optical probe.
  • the linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.
  • fluorescent protein refers to a protein that fluoresces under excitation light.
  • green fluorescent protein GFP and circularly rearranged blue fluorescent protein (cpBFP) derived from the mutation of the protein circularly rearranged green fluorescent protein (cpGFP), circularly rearranged yellow fluorescent protein (cpYFP), etc.
  • red fluorescent protein RFP commonly used in the art
  • circular rearranged proteins derived from this protein such as cpmApple, cpmOrange, cpmKate, etc.
  • the fluorescent protein and its sequence that can be used in the present invention are known in the art. Exemplarily, cpYFP is shown in SEQ ID NO: 2; cpGFP is shown in SEQ ID NO: 3; cpBFP is shown in SEQ ID NO: 4; cpmApple is shown in SEQ ID NO: 5.
  • the pyruvate optical probe of the present invention includes amino acids 96-254 of the pyruvate binding domain of a pyruvate sensitive polypeptide (B), such as pyruvate binding protein or its variants, and an optically active polypeptide (A), such as fluorescent protein .
  • the optically active polypeptide (A) is inserted into the 96-254 amino acids of the pyruvate binding domain of the pyruvate sensitive polypeptide (B), and B is divided into two parts, B1 and B2, to form a probe structure of formula B1-A-B2;
  • the interaction between the pyruvate sensitive polypeptide B and pyruvate causes the optical signal of the optically active polypeptide (A) to become stronger.
  • the optically active polypeptide can be located at any position of the pyruvate sensitive polypeptide.
  • the optically active polypeptide is located at any position of the pyruvate sensitive polypeptide in the N-C direction in the N-C direction.
  • the optically active polypeptide is located in the flexible region of the pyruvate-sensitive polypeptide.
  • the flexible region refers to some specific structures such as loop domains that exist in the high-level structure of the protein. These domains are compared to other high-level structures of the protein. It has higher mobility and flexibility, and the region can dynamically change its spatial structure and conformation after the protein and ligand are combined.
  • the flexible region described in the present invention mainly refers to the region where the insertion site in the pyruvate binding protein is located, such as the region of amino acid residues 117-121, 140-143, 160-164, 174-176, 191-195 and 210-214 .
  • the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120 ,118/121,119/120,119/121,120/121,140/141,140/142,140/143,141/142,141/143,142/143,160/161,160/162,160 /163, 160/164, 161/162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191/192, 191/193 , 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/211, 210/212, 210/213, 210/214, 211 /212, 211/213, 211/214, 212/213, 212/214 or 213/214.
  • the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein or functional fragment thereof: 117/121, 141/143, 191/192, 191/193, 191 /194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195 or 210/214.
  • 117/121, 141/143, 191/192, 191/193, 191 /194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195 or 210/214 if the two numbers in the position expressed in the form of "X/Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids described in the number.
  • the insertion site 117/118 indicates that the optically active polypeptide is located between amino acids 117 and 118 of the pyruvate sensitive polypeptide or its functional fragment.
  • the optically active polypeptide replaces the amino acids between the amino acids indicated by the numbers.
  • the insertion site 191/195 indicates that the optically active polypeptide replaces amino acids 192-194 of the pyruvate sensitive polypeptide or a functional fragment thereof.
  • the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein or functional fragment thereof: 117/118, 117/119, 117/120, 117/121, 118 /119, 118/120, 118/121, 119/120, 119/121, 120/121, 140/141, 140/142, 140/143, 141/142, 141/143, 142/143, 160/161 , 160/162, 160/163, 160/164, 161/162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191 /192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/211, 210/212, 210/213 , 210/214, 211/212, 211/213, 211/214, 212/213, 212/214 or 213/214,
  • the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein functional fragment containing amino acids 96-254: 117/121, 141/143, 191 /192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195 or 210/214, such as SEQ ID NO: 6 -18 shown.
  • variants include variants that have the same function of the polypeptide or protein but have different sequences.
  • variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants. These variants include but are not limited to: deletion, insertion and/or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1- 10, preferably 1-5) amino acids, and one or more (usually within 20, preferably within 10, more preferably within 5) at the carboxyl and/or amino terminus ) Sequence obtained from amino acids.
  • variants may also comprise sequence identity with the polypeptide or protein of 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% polypeptide or protein.
  • amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, that is, functional conservative mutations.
  • amino acids with similar or similar properties when amino acids with similar or similar properties are substituted, the functions of the polypeptide or protein are usually not changed.
  • amino acids with similar properties often refer to amino acid families with similar side chains, which have been clearly defined in the art.
  • amino acids with basic side chains such as lysine, arginine, histidine
  • amino acids with acidic side chains such as aspartic acid, glutamic acid
  • uncharged polar Side chain amino acids e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • amino acids with non-polar side chains e.g., alanine, valine, Leucine, isoleucine, arginine, phenylalanine, methionine, tryptophan
  • amino acids with ⁇ -branched side chains e.g.
  • amino acids with aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine.
  • amino acids with aromatic side chains such as tyrosine, phenylalanine, tryptophan, histidine.
  • adding one or several amino acids to the amino terminus and/or carboxy terminus usually does not change 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-encoded amino acids can be determined based on the comparison of their physical properties with those of genetically encoded amino acids.
  • identity refers to the comparison window or designated area, using methods known in the art such as sequence comparison algorithms, through manual alignment and visual inspection
  • sequence comparison algorithms through manual alignment and visual inspection
  • two or more sequences or subsequences are the same or a certain percentage of amino acid residues or nucleotides in the specified region are the same (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , At least 99% or 100% the same).
  • the preferred algorithms suitable for determining the percentage of sequence identity and the percentage of sequence similarity are BLAST and BLAST 2.0 algorithms, respectively, see Altschul et al. (1977) Nucleic Acids Res. 25: 3389 and Altschul et al. (1990) J. Mol. Biol. 215: 403.
  • suitable linker peptides including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, such as 6His or Flag tag, or factor Xa or proteolytic enzyme site of thrombin or enterokinase.
  • GST glutathione S-transferase
  • maltose E binding protein protein A, such as 6His or Flag tag, or factor Xa or proteolytic enzyme site of thrombin or enterokinase.
  • the optical probe of the present invention may comprise a pyruvate sensitive polypeptide with mutation.
  • Such mutations are, for example, Q138, S190, R191, R192, E193, M194, and/or L195 allele mutations.
  • the mutation is selected from one or more of the following: Q138S, Q138Y, Q138C, Q138L, Q138P, Q138H, Q138R, Q138W, Q138I, Q138T, Q138N, Q138K, Q138F, Q138V, Q138A, Q138D, Q138E, Q138M, Q138A, S190E, R191S, R191Y, R191C, R191L, R191P, R191H, R191Q, R191W, R191I, R191T, R191N, R191K, R191F, R191V, R191A, R191D, R191E, R191M, R191A, S190E,
  • the mutation is selected from one or more of the following: Q138P, Q138L, S190E, R191Y, R191N, R191F, R191L, R191P, R192D, E193Q, E193L, M194D, M194V, M194H, M194W, M194V and S190E and R191N and R192D (M194V/S190E/R191N/R192D), M194V and S190D and R191Y and R192T (M194V/S190D/R191Y/R192T), S190P and R191H and R192P (S190P/R191H/R192P), S190R and R191S and R192 (S190R/R191S and R192P), S190L and R191V (S190L/R191V), S190T and R191Q and R192E (S190T/R191Q/R192E (S190T/
  • the B1-A-B2 optical probe of the present invention may be PdhR (96-254) selected from 117/121, 141/143, 191/192, 191/193, 191/194, 191 /195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/214 are inserted into one or more positions of cpYFP and have one or more selected from the following Mutation probes: Q138P, Q138L, R191Y, R191F, R191L, R191P, E193Q, E193L, M194D, M194V, M194H, M194W, M194V and S190E and R191N and R192D (M194V/S190E/R191N/R192D), M194V and S190D and R191Y and R192T (M194V/S190D/R191Y/R192T), S190P and R191H and
  • the optical probe is a probe with Q138P, Q138L, R191Y, R191F, R191L, R191P, E193Q or E193L mutation inserted into the 191/193 position of PdhR(96-254), or PdhR(96 -254) has a cpYFP inserted at position 192/194 and has a M194D, M194V, M194H or M194W mutation probe.
  • the combination of the insertion site of the optically active polypeptide in the pyruvate-sensitive polypeptide and the mutation of the pyruvate-sensitive polypeptide is selected from one or more of the following: 191/193-Q138P, 191/193-Q138L, 191/ 193-R191Y, 191/193-R191F, 191/193-R191L, 191/193-R191P, 191/193-E193Q, 191/193-E193L, 192/194-M194D, 192/194-M194V, 192/194- M194H, 192/194-M194W192/194-M194V/S190E/R191N/R192D, 192/194-M194V/S190D/R191Y/R192T, 141/143-S190P/R191H/R192P, 141/143-S190R/R191S/R192P, 141/143-S190
  • a functional variant, derivative or analogue of the polypeptide or protein (e.g., PdhR protein or fluorescent protein) of the present invention may be (i) having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) A protein that is substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins with substitution groups in one or more amino acid residues, or (iii) mature proteins A protein formed by fusion with another compound (such as a compound that prolongs the half-life of a protein, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the protein sequence to form a protein (such as a secretory sequence or used to purify the protein).
  • another compound such as a compound that prolongs the half-life of a protein, such as polyethylene glycol
  • an additional amino acid sequence fused to the protein sequence to form a protein (such as a secretory sequence or used to purify the protein).
  • sequence of the protein or the proprotein sequence, or the fusion protein formed with the antigen IgG fragment belongs to the scope well known to those skilled in the art.
  • the difference between the analog and the original polypeptide or protein may be a difference in the amino acid sequence, a difference in the modified form that does not affect the sequence, or both.
  • These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.
  • the fusion polypeptide of the present invention includes the optical probe described herein and other polypeptides.
  • the optical probe described herein further includes other polypeptides fused to it.
  • the other polypeptides described herein do not affect the properties of the optical probe.
  • Other polypeptides can be located at the N-terminus and/or C-terminus of the optical probe.
  • other polypeptides include polypeptides that localize optical probes to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting.
  • the optical probe in the fusion polypeptide described herein may have a linker between the other polypeptide.
  • the subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, peroxisomes, and the like.
  • the tag used for purification or the tag used for immunoblotting includes 6 histidine (6*His), glutathione sulfur transferase (GST), and Flag.
  • the expression vector of the present invention comprises the nucleic acid sequence of the present invention or its complementary sequence operably linked to an expression control sequence, and the nucleic acid sequence encodes the optical probe or fusion polypeptide of the present invention.
  • the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors and viral vectors.
  • the prokaryotic expression vector is preferably obtained by operably linking the plasmid pCDFDuet-1 with the nucleic acid sequence described herein.
  • the expression control sequence includes an origin of replication, promoter, enhancer, operator, terminator, ribosome binding site.
  • the present invention also provides a method for preparing the above-mentioned pyruvate optical probe, including the following steps: 1) incorporating the nucleic acid sequence encoding the pyruvate optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 2 ) Culturing the host cell under conditions suitable for the expression of the expression vector, 3) Isolating the pyruvate optical probe.
  • nucleic acid or “nucleotide” or “polynucleotide” or “nucleic acid sequence” used in the present invention may be in the form of DNA or RNA.
  • the form of DNA includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • variant as used herein may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants.
  • the nucleic acid of the present invention may comprise a sequence identity with the nucleic acid sequence of at least about 50%, at least about 60%, 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% of the nucleotide sequence.
  • the present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences.
  • the nucleic acid sequence is shown in SEQ ID NO: 31, which represents the coding sequence of the probe with cpYFP inserted at position 192/194 of the pyruvate synthin functional fragment and with the M194V mutation.
  • nucleic acid fragment has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).
  • the full-length sequence or fragments of the optical probe or fusion protein of the present invention can usually be obtained by PCR amplification method, artificial synthesis method or recombination method.
  • primers can be designed according to the nucleotide sequence disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as a template, and the relevant amplification can be obtained. sequence.
  • the nucleotide sequence is greater than 2500 bp, it is preferable to perform 2-6 PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the present invention does not specifically limit the PCR amplification program and system, and the conventional PCR amplification program and system in this field can be used.
  • the recombination method can also be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then separating and purifying it from the proliferated host cell by conventional methods to obtain the relevant polypeptide or protein.
  • artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short.
  • the nucleotide sequence of the optical probe is less than 2500 bp, it can be synthesized by artificial synthesis.
  • the artificial synthesis method is a conventional DNA synthesis method in the field, and there are no other special requirements. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the DNA sequence of the polypeptide of the present invention can also be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.
  • the mutation can be introduced into the protein sequence of the present invention by mutation PCR or chemical synthesis.
  • the present invention also provides a detection kit comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe or fusion polypeptide prepared by the method described herein.
  • the kit also optionally contains other reagents required for the detection of pyruvate using an optical probe. Such other reagents are conventionally known in the art.
  • the present invention also relates to nucleic acid constructs, which contain the polynucleotides described herein, and one or more regulatory sequences operably linked to these sequences.
  • the polynucleotide of the present invention can be manipulated in a variety of ways to ensure the expression of the polypeptide or protein.
  • the nucleic acid construct can be manipulated according to the different or requirements of the expression vector before inserting the nucleic acid construct into the vector. Techniques for using recombinant DNA methods to alter polynucleotide sequences are known in the art.
  • the nucleic acid construct is a vector.
  • the vector can be a cloning vector, an expression vector, or a gene knock-in vector, such as a homologous recombination vector.
  • the polynucleotide of the present invention can be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Cloning vectors can be used to provide coding sequences for the proteins or polypeptides of the invention.
  • the expression vector can be provided to the cell in the form of a bacterial vector or a viral vector.
  • the expression of the polynucleotide of the present invention is usually achieved by operably linking the polynucleotide of the present invention to a promoter, and incorporating the construct into an expression vector.
  • the vector may be suitable for replication and integration of eukaryotic cells.
  • a typical expression vector contains expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.
  • Gene knock-in vectors are used to knock-in or integrate the expression cassettes described herein into the host genome.
  • expression control sequence refers to an element that can be operatively linked to the target gene that regulates the transcription, translation, and expression of a target gene. It can be an origin of replication, a promoter, a marker gene, or a translation control element, including enhancers and operons. , Terminator, ribosome binding site, etc. The choice of expression control sequence depends on the host cell used.
  • "operably linked" means that the target nucleotide sequence and the regulatory sequence are linked in a manner that allows the expression of the nucleotide sequence.
  • promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti The LTR of the transcription virus and some other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector can be a commercially available pRSETb vector without other special requirements.
  • the nucleotide sequence encoding the optical probe and the expression vector are double digested with BamHI and EcoRI respectively, and then the digested products of the two are connected to obtain a recombinant expression vector.
  • the specific steps and parameters of restriction digestion and ligation are not particularly limited, and conventional steps and parameters in this field can be used.
  • the vector is transformed into a host cell to produce a protein or peptide including a fusion protein.
  • a host cell refers to a cell capable of receiving and accommodating recombinant DNA molecules, and is a place for recombinant gene amplification.
  • the ideal recipient cell should meet the two conditions of easy acquisition and proliferation.
  • the "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells.
  • the host cell is preferably a variety of cells that are conducive to gene product expression or fermentation production, and such cells are well-known and commonly used in the art. Those of ordinary skill in the art know how to select appropriate vectors, promoters, enhancers and host cells.
  • the method of transferring to host cells according to the present invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical mediation Guided transfer or electroporation.
  • the method is preferably the CaCl 2 method or the MgCl 2 method, and the steps used are well-known in the art.
  • the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the host cell transferred into the expression vector is subjected to amplification and expression culture, and the pyruvate optical probe is isolated.
  • Conventional methods may be used for the amplification and expression culture of the host cell.
  • the medium used in the culture may be various conventional mediums. The culture is carried out under conditions suitable for the growth of the host cell.
  • the optical probe is expressed in the cell, on the cell membrane, or secreted out of the cell.
  • the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other characteristics.
  • the present invention does not specifically limit the method for separating the pyruvate fluorescent protein, as long as the conventional method for separating the fusion protein in the art can be used.
  • the invention also provides the application of the pyruvate optical probe in the real-time positioning, quantitative detection and high-throughput compound screening of pyruvate.
  • the pyruvate optical probe is preferably connected to signal peptides in different parts of the cell and transferred to the cell.
  • the real-time localization of pyruvate is performed by detecting the strength of the fluorescent signal in the cell; Add the curve for the quantitative detection of the corresponding pyruvate.
  • the standard dropping curve of pyruvate according to the present invention is drawn according to the fluorescence signal of the pyruvate optical probe under different concentrations of pyruvate.
  • the pyruvate optical probe of the present invention is directly transferred into cells, and in the process of real-time positioning and quantitative detection of pyruvate, the time-consuming sample processing process is not required, which is more accurate.
  • the pyruvate optical probe of the present invention performs high-throughput compound screening, different compounds are added to the cell culture solution to measure the change in the content of pyruvate, thereby screening compounds that have an effect on the change in the content of pyruvate.
  • the application of the pyruvate optical probe described in the present invention in the real-time positioning, quantitative detection and high-throughput compound screening of pyruvate is for non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.
  • the pRSETb-pyruvate binding protein particles used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pRSETb plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. The expression plasmids constructed in the examples have all undergone sequence determination, which was completed by Huada Gene Company and Jie Li Sequencing Company.
  • the Taq DNA polymerase used in each example was purchased from Dongsheng Bio, pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., primeSTAR DNA polymerase was purchased from TaKaRa Company, and the three polymerases were purchased with a corresponding gift Polymerase buffer and dNTP. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and the corresponding buffer was included with the purchase.
  • the transfection reagent Lip2000Kit was purchased from Invitrogen.
  • Amino acids such as pyruvate were purchased from Sigma. Unless otherwise stated, chemical reagents such as inorganic salts are purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp) and puromycin were purchased from Ameresco. 96-well detection blackboard and 384-well fluorescence detection blackboard were purchased from Grenier Company.
  • the DNA purification kit used in the examples was purchased from BBI Company (Canada), and the common plasmid mini-purge kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • the cloned strain Mach1 was purchased from Invitrogen. Both the nickel column affinity chromatography column and the desalting column packing materials are from GE Healthcare.
  • the main instruments used in the examples include: Biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), and Microfuge 22R desktop high-speed refrigerated centrifuge (Beckman, USA) , PCR amplification instrument (Germany Biometra company), ultrasonic disruptor (Ningbo Xinzhi company), nucleic acid electrophoresis system (Sheneng Group), fluorescence spectrophotometer (U.S. Varian company), CO 2 constant temperature cell incubator (SANYO) , Inverted fluorescence microscope (Nikon, Japan).
  • Target fragment amplification PCR 1.
  • the PCR amplification reaction system is as follows: template sequence 0.5-1 ⁇ L, forward primer (25 ⁇ M) 0.5 ⁇ L, reverse primer (25 ⁇ M) 0.5 ⁇ L, 10 ⁇ pfu buffer 5 ⁇ L, pfu DNA polymerase 0.5 ⁇ L, dNTP( 10mM) 1 ⁇ L, sterile ultrapure water (ddH2O) 41.5-42 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification procedure is as follows: denaturation at 95°C for 2-10 minutes, 30 cycles (94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, 72°C for a certain time (600bp/min)), Extend at 72°C for 10 minutes.
  • the long fragment amplification used in the present invention is mainly an inverse PCR amplification vector, which is a technique for obtaining site-directed mutation in the following examples. Design the reverse PCR primers at the mutation site, and one of the primers contains the mutation nucleotide sequence at the 5'end. The amplified product contains the corresponding mutation site.
  • the PCR reaction system for long-range amplification is as follows: template sequence (10pg-1ng) 1 ⁇ L, forward primer (25 ⁇ M) 0.5 ⁇ L, reverse primer (25 ⁇ M) 0.5 ⁇ L, 5 ⁇ PrimerSTAR buffer 10 ⁇ L, PrimerSTAR DNA polymerase 0.5 ⁇ L, dNTP (2.5mM) 4 ⁇ L, sterilized ultrapure water (ddH2O) 33.5 ⁇ L, total volume 50 ⁇ L.
  • the PCR amplification procedure is as follows: denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 50-68°C for 5-15 seconds, 72°C for a certain time (1000bp/min)), 72°C for 10 minutes; Or denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 68°C for a certain time (1000bp/min)), 72°C for 10 minutes extension.
  • the system for double digestion of plasmid vector is as follows: plasmid vector 20 ⁇ L (about 1.5 ⁇ g), 10 ⁇ buffer 5 ⁇ L, restriction endonuclease 11-2 ⁇ L, restriction endonuclease 21-2 ⁇ L, sterilized ultrapure water Make up to a total volume of 50 ⁇ L.
  • the reaction conditions were 37°C, 1-7 hours.
  • Plasmids extracted from microorganisms or genome ends contain phosphate groups, but PCR products do not, so it is necessary to perform phosphate group addition reaction on the 5'end bases of PCR products. Only DNA molecules with phosphate groups at the end can occur. Connection reaction.
  • the phosphorylation reaction system is as follows: PCR product fragment DNA sequence 5-8 ⁇ L, 10 ⁇ T4 ligase buffer 1 ⁇ L, T4 polynucleotide kinase (T4 PNK) 1 ⁇ L, sterilized ultrapure water 0-3 ⁇ L, total volume 10 ⁇ L. The reaction conditions were 37°C, and inactivation at 72°C for 20 minutes after 30 minutes to 2 hours.
  • connection methods between different fragments and vectors are different. Three connection methods are used in the present invention.
  • the principle of this method is that the blunt-end product obtained by PCR undergoes phosphorylation reaction on the 5'end of the DNA fragment under the action of T4 PNK, and is ligated with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid.
  • the homologous recombination ligation system is as follows: T4 PNK treated DNA fragment 4 ⁇ L, linearized vector fragment 4 ⁇ L, PEG4000 1 ⁇ L, 10 ⁇ T4 ligase buffer 1 ⁇ L, T4 DNA ligase 1 ⁇ L, total 10 ⁇ L.
  • the reaction conditions were 22°C, 30 minutes.
  • DNA fragments cleaved by restriction endonucleases usually produce protruding sticky ends, so they can be ligated with vector fragments containing complementary sticky ends to form recombinant plasmids.
  • the ligation reaction system is as follows: 1-7 ⁇ L of digested PCR product fragment DNA, 0.5-7 ⁇ L of digested plasmid, 1 ⁇ L of 10 ⁇ T4 ligase buffer, 1 ⁇ L of T4 DNA ligase, sterilized ultrapure water to make up to the total volume 10 ⁇ L.
  • the reaction conditions were 16°C, 4-8 hours.
  • the mass ratio of PCR product fragments and vector double enzyme digestion products is roughly between 2:1-6:1.
  • the 5'end phosphorylated DNA fragment is ligated with the 3'end and 5'end of the linearized vector through self-circularization ligation reaction to obtain a recombinant plasmid.
  • the self-cyclization ligation reaction system is as follows: phosphorylation reaction system 10 ⁇ L, T4 ligase (5U/ ⁇ L) 0.5 ⁇ L, total volume 10.5 ⁇ L. The reaction conditions were 16°C, 4-16 hours.
  • the resuspension buffer CaCl 2 (100 mM), MgCl 2 (70 mM), NaAc (40 mM).
  • Storage buffer 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10 ⁇ CaCl 2 (1M), 1 mL 10 ⁇ MgCl 2 (700 mM), 1 mL 10 ⁇ NaAc (400 mM), 4.6 mL ddH 2 O.
  • the bacteria are harvested by centrifugation at 4000 rpm for 30 minutes, and 50 mM phosphate buffer is added to resuspend the bacterial pellet and ultrasonically broken until the bacterial cells are clear. Centrifuge at 9600 rpm for 20 minutes at 4°C.
  • the centrifugal supernatant is purified by a self-packed nickel column affinity chromatography column to obtain protein, and the protein after nickel column affinity chromatography is then passed through a self-packed desalting column to be dissolved in 20mM MOPS buffer (pH 7.4) or phosphate Protein in buffer PBS.
  • the PdhR (96-254) gene in the E. coli gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and HindIII. At the same time, the pCDFDuet1 vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, the product was transformed into MachI, and the transformed MachI was spread on an LB plate (streptomycin sulfate 50ug/mL) and incubated at 37°C overnight. After the growing MachI transformants were subjected to plasmid extraction, PCR identification was performed. After the positive plasmid is sequenced correctly, the subsequent plasmid construction is carried out.
  • Example 2 Expression and detection of cpYFP optical probes at different insertion sites
  • the DNA fragment of cpYFP was generated by PCR, and the pCDFDuet-PdhR (96-254) linearized vector containing different break sites was generated by inverse PCR.
  • the linearized pCDFDuet-PdhR (96-254) and cpYFP fragments were Under the action of homologous recombinase, the recombinant plasmids were ligated to produce recombinant plasmids, and positive clones were selected by colony PCR, and the sequencing was completed by Shanghai Jie Li Biotechnology Co., Ltd.
  • the recombinant plasmid was transformed into JM109 (DE3) to induce expression, and the protein was purified.
  • the size was around 47.5Kda by SDS-PAGE electrophoresis. This size is consistent with the size of the PdhR(96-254)-cpYFP fusion protein expressed by pCDFDuet-PdhR(96-254)-cpYFP containing the His-tag purification tag. The result is shown in Figure 1.
  • the purified PdhR(96-254)-cpYFP fusion protein was subjected to pyruvate response screening, and the detection signal of the fusion fluorescent protein containing 100 mM pyruvate was divided by the detection signal of the fusion fluorescent protein without pyruvate.
  • the results are shown in Figure 2.
  • the test results show that the optical probes with a response of more than 2 times to pyruvate are 117/121, 141/143, 191/192, 191/193, 191/194, 191/195, 192/193 , 192/194, 192/195, 193/194, 193/195, 194/195 and 210/214 positions or the corresponding amino acid positions of their family proteins to implement the insertion of optical probes.
  • Example 3 Expression and detection of cpGFP optical probes at different insertion sites
  • cpYFP was replaced with cpGFP to construct a pyruvate green fluorescent protein fluorescent probe.
  • the detection results show that the optical probes that have a response of more than 2 times to pyruvate have optical probes inserted at positions 191/193 or the corresponding amino acid positions of their family proteins.
  • Example 4 Expression and detection of cpBFP optical probes at different insertion sites
  • cpYFP was replaced with cpBFP to construct a pyruvate blue fluorescent protein fluorescent probe.
  • the detection results show that the optical probes that have a response of more than 2 times to pyruvate have optical probes inserted at positions 193/194 and 194/195 or the corresponding amino acid positions of their family proteins.
  • Example 5 Expression and detection of cpmApple optical probes at different insertion sites
  • cpYFP was replaced with cpmApple to construct a pyruvate red fluorescent protein fluorescent probe.
  • the detection results show that the optical probes that have a response of more than 2 times to pyruvate have optical probes inserted at positions 191/195 and 192/193 or the corresponding amino acid positions of their family proteins.
  • the optical probe mutants were constructed on the basis of PdhR(96-254)-191/193-cpYFP, PdhR(96-254)-192/194-cpYFP and PdhR(96-254)-192/195-cpYFP.
  • the plasmid pCDFDuet-PdhR(96-254)-191/193-cpYFP was linearized by inverse PCR.
  • the primer contained the base sequence of the desired mutation site, and the PCR product obtained was subjected to the action of PNK, T4 DNA ligase and PEG4000 Phosphorus ligation was added to obtain the site-directed saturation mutant plasmids of Q138, R191, and E193.
  • Example 7 Titration curve of pyruvate optical probe
  • Example 8 Spectral performance and specificity of pyruvate optical probe
  • the purified pyruvate optical probe PdhR(96-254)-192/194-M194V-cpYFP is treated with 0 mM and 10 mM pyruvate for 10 minutes, respectively, and then the fluorescence spectrum is detected using a fluorescence spectrophotometer.
  • Determination of the excitation spectrum Record the excitation spectrum with an excitation range of 350nm to 500nm and an emission wavelength of 530nm, and read it every 5nm. The results show that the probe has two excitation peaks at about 420 and 490 nm, as shown in Figure 8A.
  • Example 9 Subcellular organelle positioning of optical probes
  • different positioning signal peptides are used to fuse the optical probes to locate the optical probes in different organelles.
  • the HeLa cells were transfected with optical probe plasmids fused with different positioning signal peptides for 36 hours, rinsed with PBS, placed in HBSS solution and used inverted fluorescence microscope for fluorescence detection under FITC channel. The result is shown in Figure 10.
  • the pyruvate optical probe can be localized to the subcellular organelles including cytoplasm, outer cell membrane, cell nucleus, endoplasmic reticulum, mitochondria, nuclear exclusion by fusion with different specific localization signal peptides. Different subcellular structures all show fluorescence, and the distribution and intensity of fluorescence are different.
  • Example 10 Study on the performance of pyruvate probe in cells
  • Example 11 High-throughput compound screening based on optical probes in living cells
  • the transfected HeLa cells were washed with PBS, placed in HBSS solution (without pyruvate) for 1 hour, and then treated with 10 ⁇ M compound for 1 hour. Pyruvic acid was added dropwise to each sample. Use a microplate reader to record the ratio of fluorescence intensity at 420nm excitation at 528nm emission and 485nm excitation at 528nm emission. A sample that was not treated with any compound was used as a control for normalization. The result is shown in Figure 12. Among the 2000 compounds used, most of the compounds have little effect on the entry of pyruvate into cells. There are 5 compounds that can increase the uptake of pyruvate by cells, and 8 other compounds can significantly reduce the uptake of pyruvate by cells.
  • Example 12 Quantitative detection of pyruvate in blood by optical probe
  • the pyruvate optical probe provided by the present invention has relatively small protein molecular weight, is easy to mature, has a large dynamic change in fluorescence, and has good specificity. It can be expressed in cells by genetic manipulation methods, and can be used inside and outside the cell. Real-time localization and quantitative detection of pyruvate; and high-throughput compound screening.

Abstract

Disclosed in the present invention are a pyruvic acid optical probe, a preparation method therefor, and an application thereof. One aspect of the present invention is the disclosure of an optical probe, which includes a pyruvic acid-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the pyruvic acid-sensitive polypeptide. The present invention also discloses a preparation method for the probe and an application of said probe in pyruvic acid measurement.

Description

丙酮酸光学探针及其制备方法和应用Pyruvate optical probe and its preparation method and application 技术领域Technical field
本发明涉及光学探针技术领域，尤其涉及一种丙酮酸光学探针及其制备方法和应用。The invention relates to the technical field of optical probes, in particular to a pyruvate optical probe and a preparation method and application thereof.
背景技术Background technique
丙酮酸是一种具有羰基和羧基两个官能团的有机酸，也是最简单的α-酮酸，其广泛存在于各类动植物中。丙酮酸和能量代谢密不可分，在糖酵解过程中消耗一分子葡萄糖或者糖原和两分子ATP，产生两分子NADH，四分子ATP和两分子丙酮酸。在细胞浆中丙酮酸能还原成供能，也能作为转运到线粒体成为三羧酸循环的主要燃料。丙酮酸在有氧条件下进入线粒体，经丙酮酸脱氢酶复合体催化氧化脱羧产生NADH、CO2和乙酰辅酶A，乙酰辅酶A进入三羧酸循环和氧化磷酸化彻底氧化为CO2和H2O，释放的能量在此过程中可产生大量ATP。这是糖的有氧氧化过程，是机体获得ATP的主要途径。Pyruvate is an organic acid with two functional groups, carbonyl and carboxyl, and is also the simplest α-keto acid, which is widely present in various animals and plants. Pyruvate is inseparable from energy metabolism. During glycolysis, one molecule of glucose or glycogen and two molecules of ATP are consumed to produce two molecules of NADH, four molecules of ATP and two molecules of pyruvate. In the cytoplasm, pyruvate can be reduced to supply energy, and can also be used as the main fuel for transport to the mitochondria to become the tricarboxylic acid cycle. Pyruvate enters the mitochondria under aerobic conditions. The pyruvate dehydrogenase complex catalyzes oxidative decarboxylation to produce NADH, CO2 and acetyl-CoA. Acetyl-CoA enters the tricarboxylic acid cycle and oxidative phosphorylation is completely oxidized to CO2 and H2O and released The energy of this process can produce a large amount of ATP. This is the aerobic oxidation process of sugar and the main way for the body to obtain ATP.
此外，丙酮酸可经转氨基作用生成丙氨酸，作为蛋白质合成的原料。丙酮酸还可通过乙酰辅酶A和三羧酸循环实现体内糖、脂肪和氨基酸间的相互转化。综上所述，丙酮酸在三大营养物质的代谢联系中起着重要的枢纽作用，是对真核和人体新陈代谢的许多方面至关重要的关键分子。In addition, pyruvate can generate alanine through transamination, which is used as a raw material for protein synthesis. Pyruvate can also realize the mutual conversion of sugars, fats and amino acids in the body through the acetyl-CoA and tricarboxylic acid cycle. In summary, pyruvate plays an important pivotal role in the metabolic connection of the three major nutrients, and is a key molecule that is vital to many aspects of eukaryotic and human metabolism.
有研究已表明，丙酮酸可直接通过非酶促的去碳酸基反应抑制过氧化氢，具有防止自由基损伤的作用，已在心脏再灌注损伤和急性肾衰竭中证实具有保护机体抗功能性损伤。补充丙酮酸可增强柠檬酸循环，柠檬酸产生增多后，抑制磷酸果糖激酶，从而进入磷酸戊糖旁路，产生还原型辅酶II(NADPH)，从而间接地增加谷胱甘肽(GSH)抗氧化系统的能力。Studies have shown that pyruvate can directly inhibit hydrogen peroxide through a non-enzymatic decanoation reaction, and has the effect of preventing free radical damage. It has been proven to protect the body against functional damage in cardiac reperfusion injury and acute renal failure. . Supplementing pyruvate can enhance the citric acid cycle. When the production of citric acid increases, it inhibits phosphofructokinase and enters the pentose phosphate bypass to produce reduced coenzyme II (NADPH), thereby indirectly increasing the antioxidant capacity of glutathione (GSH) System capabilities.
正是由于丙酮酸具有上述重要的作用，因此丙酮酸含量的检测也尤为重要。丙酮酸的常用检测方法包括紫外分光光度计法、高效液相色谱法、脱氢酶比色法等。但是这些方法并不适用于活细胞研究，存在很多缺陷：需要经过耗时的样品处理过程，例如细胞破碎、分离提取纯化等；不能在活细胞和亚细胞器中进行原位、实时、动态、高通量和高时空分辨率的检测。本领域仍需要能在细胞内、外实时定位、定量、高通 量检测丙酮酸的方法。It is precisely because of the above-mentioned important functions of pyruvate that the detection of pyruvate content is also particularly important. Commonly used detection methods for pyruvate include ultraviolet spectrophotometer, high performance liquid chromatography, dehydrogenase colorimetry and so on. However, these methods are not suitable for living cell research, and there are many shortcomings: time-consuming sample processing procedures are required, such as cell disruption, separation, extraction and purification, etc.; in situ, real-time, dynamic, and high-speed analysis in living cells and subcellular organelles cannot be performed. Detection of throughput and high temporal and spatial resolution. The art still needs a method capable of real-time positioning, quantitative, and high-throughput detection of pyruvate inside and outside the cell.
发明内容Summary of the invention
本发明的目的在于提供在细胞内、外实时定位、高通量、定量检测丙酮酸的探针和方法。The purpose of the present invention is to provide a probe and method for real-time positioning, high-throughput, and quantitative detection of pyruvate inside and outside the cell.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供一种丙酮酸光学探针，包含丙酮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丙酮酸敏感多肽或其功能变体的序列内。丙酮酸敏感多肽或其功能变体被光学活性多肽或其功能变体分为第一部分和第二部分。The present invention provides a pyruvate optical probe, comprising a pyruvate sensitive polypeptide or its functional variant and an optically active polypeptide or its functional variant, wherein the optically active polypeptide or its functional variant is located in the pyruvate sensitive polypeptide or its functional variant Within the sequence. The pyruvate sensitive polypeptide or its functional variant is divided into a first part and a second part by the optically active polypeptide or its functional variant.
本发明提供了一种丙酮酸光学探针，包括丙酮酸敏感多肽B和光学活性多肽A，其中光学活性多肽A位于丙酮酸敏感多肽B的序列内，将丙酮酸敏感多肽B分为第一部分B1和第二部分B2，形成B1-A-B2式的探针结构。The present invention provides a pyruvate optical probe, comprising pyruvate sensitive polypeptide B and optically active polypeptide A, wherein the optically active polypeptide A is located in the sequence of pyruvate sensitive polypeptide B, and the pyruvate sensitive polypeptide B is divided into the first part B1 And the second part B2, forming a probe structure of formula B1-A-B2.
在一个实施方式中，丙酮酸敏感多肽包括丙酮酸结合蛋白或其丙酮酸结合域。在一个实施方式中，丙酮酸敏感多肽源自大肠杆菌。在一个实施方式中，丙酮酸敏感多肽是丙酮酸结合蛋白或其功能片段。在一个或多个实施方案中，所述丙酮酸结合蛋白是PdhR蛋白。在一个实施方式中，丙酮酸敏感多肽具有SEQ ID NO：1所示的序列，或与其有至少35％、至少40％、至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少99％序列相同性并保留丙酮酸结合功能的序列。在一个实施方式中，丙酮酸敏感多肽的功能片段具有SEQ ID NO：1所示的序列第96-254位氨基酸，或与其有至少35％、至少40％、至少50％、至少60％、至少70％、至少80％、至少85％、至少90％、至少95％、至少99％序列相同性并保留丙酮酸结合功能的序列。In one embodiment, the pyruvate sensitive polypeptide includes a pyruvate binding protein or a pyruvate binding domain thereof. In one embodiment, the pyruvate sensitive polypeptide is derived from Escherichia coli. In one embodiment, the pyruvate sensitive polypeptide is a pyruvate binding protein or a functional fragment thereof. In one or more embodiments, the pyruvate binding protein is a PdhR protein. In one embodiment, the pyruvate-sensitive polypeptide has the sequence shown in SEQ ID NO: 1, or at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 85% of the sequence shown in SEQ ID NO: 1. %, at least 90%, at least 95%, at least 99% sequence identity and retain pyruvate binding function. In one embodiment, the functional fragment of the pyruvate-sensitive polypeptide has amino acids 96-254 of the sequence shown in SEQ ID NO:1, or at least 35%, at least 40%, at least 50%, at least 60%, or at least Sequences that are 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identical and retain pyruvate binding function.
在一个实施方式中，光学活性多肽是荧光蛋白或其功能变体。在一个实施方式中，荧光蛋白选自黄色荧光蛋白(如SEQ ID NO：2所示的cpYFP)、橘黄色荧光蛋白、红色荧光蛋白、绿色荧光蛋白(如SEQ ID NO：3所示的cpGFP)、蓝色荧光蛋白(如SEQ ID NO：4所示的cpBFP)、苹果红荧光蛋白(如SEQ ID NO：5所示的cpmApple)。优选地，光学活性多肽是cpYFP。在一个实施方式中，荧光蛋白具有SEQ ID NO：2-5中任一所示的序列。In one embodiment, the optically active polypeptide is a fluorescent protein or a functional variant thereof. In one embodiment, the fluorescent protein is selected from yellow fluorescent protein (cpYFP shown in SEQ ID NO: 2), orange fluorescent protein, red fluorescent protein, and green fluorescent protein (cpGFP shown in SEQ ID NO: 3) , Blue fluorescent protein (cpBFP shown in SEQ ID NO: 4), Apple red fluorescent protein (cpmApple shown in SEQ ID NO: 5). Preferably, the optically active polypeptide is cpYFP. In one embodiment, the fluorescent protein has a sequence shown in any one of SEQ ID NO: 2-5.
在一个实施方式中，光学探针还包含侧接所述光学活性多肽的一个或多个接头。本发明所述接头可以是任何长度的任何氨基酸序列。在一个实施方式中，光学活性多肽侧翼包含不超过5个氨基酸的接头，例如0、1、2、3、4个氨基酸的接头。在一个 实施方式中，光学活性多肽侧翼的接头包含氨基酸Y。在一个实施方式中，接头Y位于光学活性多肽的N端和/或C端。在一个实施方式中，光学探针如下所示：丙酮酸敏感多肽的第一部分B1-Y-光学活性多肽A-丙酮酸敏感多肽的第二部分B2。在一个实施方式中，本发明光学探针不包含接头。In one embodiment, the optical probe further comprises one or more linkers flanking the optically active polypeptide. The linker of the present invention can be any amino acid sequence of any length. In one embodiment, the optically active polypeptide flanking includes a linker of no more than 5 amino acids, such as a linker of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker flanking the optically active polypeptide comprises amino acid Y. In one embodiment, the linker Y is located at the N-terminus and/or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is as follows: the first part of the pyruvate sensitive polypeptide B1-Y-optically active polypeptide A-the second part of the pyruvate sensitive polypeptide B2. In one embodiment, the optical probe of the present invention does not include a joint.
本发明所述光学活性多肽可以位于丙酮酸敏感多肽的任何位置。在一个实施方式中，光学活性多肽位于丙酮酸敏感多肽的选自以下的一个或多个位置中：残基117-121、140-143、160-164、174-176、191-195和/或210-214，编号对应于丙酮酸结合蛋白的全长。在一个实施方式中，光学活性多肽置换丙酮酸敏感多肽的选自以下的一个或多个位置中的一个或多个氨基酸：残基117-121、140-143、160-164、174-176、191-195和/或210-214。The optically active polypeptide of the present invention can be located at any position of the pyruvate sensitive polypeptide. In one embodiment, the optically active polypeptide is located in one or more positions of the pyruvate sensitive polypeptide selected from the group consisting of residues 117-121, 140-143, 160-164, 174-176, 191-195, and/or 210-214, numbering corresponds to the full length of the pyruvate binding protein. In one embodiment, the optically active polypeptide replaces one or more amino acids in one or more positions of the pyruvate-sensitive polypeptide selected from the following: residues 117-121, 140-143, 160-164, 174-176, 191-195 and/or 210-214.
在一个实施方式中，光学活性多肽插入丙酮酸敏感多肽的选自以下的一个或多个位点：117/118，117/119，117/120，117/121，118/119，118/120，118/121，119/120，119/121，120/121，140/141，140/142，140/143，141/142，141/143，142/143，160/161，160/162，160/163，160/164，161/162，161/163，161/164，162/163，162/164，163/164，174/175，174/176，175/176，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195，210/211，210/212，210/213，210/214，211/212，211/213，211/214，212/213，212/214和/或213/214。优选地，光学活性多肽位于丙酮酸敏感多肽的选自以下的一个或多个位点：117/121，141/143，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195和210/214。In one embodiment, the optically active polypeptide is inserted into one or more positions of the pyruvate sensitive polypeptide selected from the following: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 140/141, 140/142, 140/143, 141/142, 141/143, 142/143, 160/161, 160/162, 160/ 163, 160/164, 161/162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191/192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/211, 210/212, 210/213, 210/214, 211/ 212, 211/213, 211/214, 212/213, 212/214 and/or 213/214. Preferably, the optically active polypeptide is located at one or more positions of the pyruvate sensitive polypeptide selected from the following: 117/121, 141/143, 191/192, 191/193, 191/194, 191/195, 192/193 , 192/194, 192/195, 193/194, 193/195, 194/195 and 210/214.
在一个或多个实施方式中，本发明光学探针可为当cpYFP位于丙酮酸结合蛋白或其功能片段的选自以下的一个或多个位点的探针：117/120，117/121，118/119，118/120，118/121，119/120，119/121，120/121，140/143，141/142，141/143，142/143，160/161，160/162，160/163，160/164，161/163，161/164，162/163，162/164，163/164，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195，210/211，210/212，210/214，211/212，211/213，211/214，212/213，212/214。在示例性实施方式中，本发明光学探针可为当cpYFP位于丙酮酸结合蛋白或其功能片段的选自以下的一个或多个位点的探针：117/121，119/120，119/121，120/121，140/143，141/142，141/143，160/161，160/164，161/163，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195，210/212，210/214，211/213和211/214。优选地，本发明光学探针可为当cpYFP位于丙酮酸结合蛋白或其功能片段的选自以下的一个或多个位点的探针：117/121，141/143，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195和210/214 时形成的探针。所述丙酮酸结合蛋白的功能片段是SEQ ID NO：1的第96-254位。In one or more embodiments, the optical probe of the present invention may be a probe selected from one or more of the following sites: 117/120, 117/121, when cpYFP is located in the pyruvate binding protein or its functional fragment. 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 140/143, 141/142, 141/143, 142/143, 160/161, 160/162, 160/ 163, 160/164, 161/163, 161/164, 162/163, 162/164, 163/164, 191/192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/211, 210/212, 210/214, 211/212, 211/213, 211/214, 212/213, 212/214. In an exemplary embodiment, the optical probe of the present invention may be a probe selected from one or more of the following sites when cpYFP is located in a pyruvate binding protein or a functional fragment thereof: 117/121, 119/120, 119/ 121, 120/121, 140/143, 141/142, 141/143, 160/161, 160/164, 161/163, 191/192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/212, 210/214, 211/213 and 211/214. Preferably, the optical probe of the present invention may be a probe selected from one or more of the following positions when cpYFP is located in a pyruvate binding protein or a functional fragment thereof: 117/121, 141/143, 191/192, 191/ 193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195 and 210/214. The functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
在一个或多个实施方式中，本发明光学探针可为当cpGFP位于丙酮酸结合蛋白功能片段的选自以下的一个或多个位点的探针：117/120，118/119，119/120，140/141，140/142，141/142，141/143，142/143，160/161，161/163，161/164，162/163，174/175，191/193，210/212，211/212，211/214，212/213，212/214和/或213/214。在优选的实施方式中，本发明光学探针可为当cpGFP位于丙酮酸结合蛋白功能片段的选自以下的一个或多个位点的探针：118/119，140/141，160/161，191/193，210/212，212/213，212/214和/或213/214。所述丙酮酸结合蛋白的功能片段是SEQ ID NO：1的第96-254位。In one or more embodiments, the optical probe of the present invention may be a probe selected from one or more of the following sites when cpGFP is located in the functional fragment of pyruvate binding protein: 117/120, 118/119, 119/ 120, 140/141, 140/142, 141/142, 141/143, 142/143, 160/161, 161/163, 161/164, 162/163, 174/175, 191/193, 210/212, 211/212, 211/214, 212/213, 212/214 and/or 213/214. In a preferred embodiment, the optical probe of the present invention may be a probe selected from one or more of the following positions when cpGFP is located in the functional fragment of pyruvate binding protein: 118/119, 140/141, 160/161, 191/193, 210/212, 212/213, 212/214 and/or 213/214. The functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
在示例性实施方式中，本发明光学探针可为当cpBFP位于丙酮酸结合蛋白或其功能片段的选自以下的一个或多个位点的探针：117/118，117/120，119/120，119/121，140/141，141/142，160/162，160/163，161/163，174/176，191/192，191/194，192/193，192/195，193/194，193/195，194/195，210/212，211/213或212/213。在优选的实施方式中，本发明光学探针可为当cpBFP位于丙酮酸结合蛋白或其功能片段的选自以下的一个或多个位点的探针：141/142，160/163，192/193，192/195，193/194，193/195，194/195，210/212，211/213。所述丙酮酸结合蛋白的功能片段是SEQ ID NO：1的第96-254位。In an exemplary embodiment, the optical probe of the present invention may be a probe selected from one or more of the following sites when cpBFP is located in a pyruvate binding protein or a functional fragment thereof: 117/118, 117/120, 119/ 120, 119/121, 140/141, 141/142, 160/162, 160/163, 161/163, 174/176, 191/192, 191/194, 192/193, 192/195, 193/194, 193/195, 194/195, 210/212, 211/213 or 212/213. In a preferred embodiment, the optical probe of the present invention may be a probe selected from one or more of the following sites when cpBFP is located in a pyruvate binding protein or a functional fragment thereof: 141/142, 160/163, 192/ 193, 192/195, 193/194, 193/195, 194/195, 210/212, 211/213. The functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
在示例性实施方式中，本发明光学探针可为当cpmApple位于丙酮酸结合蛋白或其功能片段的选自以下的一个或多个位点的探针：117/119，117/120，118/119，118/120，119/121，140/141，140/142，141/142，160/161，160/162，160/164，162/163，174/176，175/176，191/194，191/195，192/193，192/194，192/195，193/194，210/212，211/213，211/214，212/214。在优选的实施方式中，本发明光学探针可为当cpmApple位于丙酮酸结合蛋白或其功能片段的选自以下的一个或多个位点的探针：117/120，118/120，160/162，162/163，191/195，192/193，192/194，192/195，210/212，211/213。所述丙酮酸结合蛋白的功能片段是SEQ ID NO：1的第96-254位。In an exemplary embodiment, the optical probe of the present invention may be a probe selected from one or more of the following positions when cpmApple is located in a pyruvate binding protein or a functional fragment thereof: 117/119, 117/120, 118/ 119, 118/120, 119/121, 140/141, 140/142, 141/142, 160/161, 160/162, 160/164, 162/163, 174/176, 175/176, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 210/212, 211/213, 211/214, 212/214. In a preferred embodiment, the optical probe of the present invention can be a probe selected from one or more of the following positions when cpmApple is located in a pyruvate binding protein or a functional fragment thereof: 117/120, 118/120, 160/ 162, 162/163, 191/195, 192/193, 192/194, 192/195, 210/212, 211/213. The functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1.
在一个实施方式中，本发明光学探针具有SEQ ID NO：6-18所示序列或由其组成。In one embodiment, the optical probe of the present invention has or consists of the sequence shown in SEQ ID NO: 6-18.
本发明还提供具有一个或多个突变的丙酮酸敏感多肽。所述突变位于丙酮酸结合蛋白或其功能片段的Q138，S190，R191，R192，E193，M194，L195中的1个、2个、3个、4个、5个、6个或7个位点。示例性地，所述突变选自以下的1个、2个、3个、4个、5个、6个或7个：Q138S，Q138Y，Q138C，Q138L，Q138P，Q138H，Q138R，Q138W，Q138I，Q138T，Q138N，Q138K，Q138F，Q138V，Q138A，Q138D，Q138E，Q138M，Q138A，S190E，R191S，R191Y，R191C，R191L，R191P，R191H， R191Q，R191W，R191I，R191T，R191N，R191K，R191F，R191V，R191A，R191D，R191E，R191M，R191A，R192D，E193S，E193Y，E193C，E193L，E193P，E193H，E193Q，E193W，E193R，E193I，E193T，E193N，E193K，E193M，E193V，E193F，E193D，E193A，E193G，M194S，M194Y，M194C，M194L，M194P，M194H，M194Q，M194W，M194R，M194I，M194A，M194N，M194K，M194T，M194V，M194F，M194D，M194E，T102G，L195S，L195Y，L195C，L195D，L195P，L195H，L195Q，L195W，L195I，L195T，L195N，L195K，L195R，L195V，L195A，L195F，L195E，L195M和/或L195A。The present invention also provides pyruvate sensitive polypeptides with one or more mutations. The mutation is located at 1, 2, 3, 4, 5, 6, or 7 positions in Q138, S190, R191, R192, E193, M194, and L195 of the pyruvate binding protein or its functional fragments . Exemplarily, the mutation is selected from 1, 2, 3, 4, 5, 6, or 7 of the following: Q138S, Q138Y, Q138C, Q138L, Q138P, Q138H, Q138R, Q138W, Q138I, Q138T, Q138N, Q138K, Q138F, Q138V, Q138A, Q138D, Q138E, Q138M, Q138A, S190E, R191S, R191Y, R191C, R191L, R191P, R191H, R191Q, R191W, R191I, R191T, R191N, R191K, R191F, R191F, R191A, R191D, R191E, R191M, R191A, R192D, E193S, E193Y, E193C, E193L, E193P, E193H, E193Q, E193W, E193R, E193I, E193T, E193N, E193K, E193M, E193V, E193G193F, E193D, E193A, E193A M194S, M194Y, M194C, M194L, M194P, M194H, M194Q, M194W, M194R, M194I, M194A, M194N, M194K, M194T, M194V, M194F, M194D, M194E, T102G, L195S, L195Y, L195C, L195D, L195P, L195P L195Q, L195W, L195I, L195T, L195N, L195K, L195R, L195V, L195A, L195F, L195E, L195M and/or L195A.
本发明还提供包含具有一个或多个突变的丙酮酸敏感多肽的光学探针。在一个或多个实施方案中，光学探针是如上所述的插入有光学活性多肽的任意光学探针，并且所述光学探针中的丙酮酸敏感多肽在选自Q138，S190，R191，R192，E193，M194，L195中的1个、2个、3个、4个、5个、6个或7个位点具有突变。在一个或多个实施方案中，包含突变的丙酮酸敏感多肽的光学探针对丙酮酸的响应不低于未突变的对应物。在一个或多个实施方案中，所述突变选自以下的1个、2个、3个、4个、5个、6个或7个：Q138S，Q138Y，Q138C，Q138L，Q138P，Q138H，Q138R，Q138W，Q138I，Q138T，Q138N，Q138K，Q138F，Q138V，Q138A，Q138D，Q138E，Q138M，Q138A，S190E，R191S，R191Y，R191C，R191L，R191P，R191H，R191Q，R191W，R191I，R191T，R191N，R191K，R191F，R191V，R191A，R191D，R191E，R191M，R191A，R192D，E193S，E193Y，E193C，E193L，E193P，E193H，E193Q，E193W，E193R，E193I，E193T，E193N，E193K，E193M，E193V，E193F，E193D，E193A，E193G，M194S，M194Y，M194C，M194L，M194P，M194H，M194Q，M194W，M194R，M194I，M194A，M194N，M194K，M194T，M194V，M194F，M194D，M194E，T102G，L195S，L195Y，L195C，L195D，L195P，L195H，L195Q，L195W，L195I，L195T，L195N，L195K，L195R，L195V，L195A，L195F，L195E，L195M和/或L195A。The present invention also provides an optical probe comprising a pyruvate sensitive polypeptide with one or more mutations. In one or more embodiments, the optical probe is any optical probe inserted with an optically active polypeptide as described above, and the pyruvate-sensitive polypeptide in the optical probe is selected from Q138, S190, R191, R192 , E193, M194, and L195 have mutations at 1, 2, 3, 4, 5, 6, or 7 positions. In one or more embodiments, the optical probe containing the mutant pyruvate sensitive polypeptide has no less response to pyruvate than its non-mutated counterpart. In one or more embodiments, the mutation is selected from 1, 2, 3, 4, 5, 6, or 7 of the following: Q138S, Q138Y, Q138C, Q138L, Q138P, Q138H, Q138R , Q138W, Q138I, Q138T, Q138N, Q138K, Q138F, Q138V, Q138A, Q138D, Q138E, Q138M, Q138A, S190E, R191S, R191Y, R191C, R191L, R191P, R191H, R191Q, R191R191N, R191T, R191K , R191F, R191V, R191A, R191D, R191E, R191M, R191A, R192D, E193S, E193Y, E193C, E193L, E193P, E193H, E193Q, E193W, E193R, E193I, E193T, E193N, E193K, E193M, E193V, E193V , E193A, E193G, M194S, M194Y, M194C, M194L, M194P, M194H, M194Q, M194W, M194R, M194I, M194A, M194N, M194K, M194T, M194V, M194F, M194D, M194E, T102L195C, L195D, L195 , L195P, L195H, L195Q, L195W, L195I, L195T, L195N, L195K, L195R, L195V, L195A, L195F, L195E, L195M and/or L195A.
在示例性实施方式中，本发明B1-A-B2式光学探针可为丙酮酸合蛋白功能片段的141/143，191/193，192/194或192/195位点插入有cpYFP并具有Q138P，Q138L，R191Y，R191F，R191L，R191P，E193Q，E193L，M194D，M194V，M194H，M194W、M194V/S190E/R191N/R192D、M194V/S190D/R191Y/R192T、S190P/R191H/R192P、S190R/R191S/R192P、S190L/R191V、S190T/R191Q/R192E和/或R191S/R192T突变的探针，编号对应于丙酮酸结合蛋白的全长。在一个或多个实施方案中，本发明光学探针是在不同插入位点基础上的突变体，其中插入位点和突变的组合选自： 191/193-Q138P，191/193-Q138L，191/193-R191Y，191/193-R191F，191/193-R191L，191/193-R191P，191/193-E193Q，191/193-E193L，192/194-M194D，192/194-M194V，192/194-M194H，192/194-M194W、192/194-M194V-S190E/R191N/R192D、192/194-M194V/S190D/R191Y/R192T、141/143-S190P/R191H/R192P、141/143-S190R/R191S/R192P、141/143-S190L/R191V、141/143-S190T/R191Q/R192E和141/143-R191S/R192T。在示例性实施方式中，所述丙酮酸结合蛋白功能片段是SEQ ID NO：1的第96-254位。优选地，本发明光学探针具有SEQ ID NO：19-30所示序列或由其组成。In an exemplary embodiment, the optical probe of the B1-A-B2 type of the present invention can be a pyruvate synthin functional fragment with cpYFP inserted at positions 141/143, 191/193, 192/194 or 192/195 and having Q138P. , Q138L, R191Y, R191F, R191L, R191P, E193Q, E193L, M194D, M194V, M194H, M194W, M194V/S190E/R191N/R192D, M194V/S190D/R191Y/R192T, S190P/R191H/R192P, S190R/R191S/R192P , S190L/R191V, S190T/R191Q/R192E and/or R191S/R192T mutant probes, numbering corresponds to the full length of the pyruvate binding protein. In one or more embodiments, the optical probe of the present invention is a mutant based on different insertion sites, wherein the combination of insertion site and mutation is selected from: 191/193-Q138P, 191/193-Q138L, 191 /193-R191Y, 191/193-R191F, 191/193-R191L, 191/193-R191P, 191/193-E193Q, 191/193-E193L, 192/194-M194D, 192/194-M194V, 192/194 -M194H, 192/194-M194W, 192/194-M194V-S190E/R191N/R192D, 192/194-M194V/S190D/R191Y/R192T, 141/143-S190P/R191H/R192P, 141/143-S190R/R191S /R192P, 141/143-S190L/R191V, 141/143-S190T/R191Q/R192E and 141/143-R191S/R192T. In an exemplary embodiment, the functional fragment of the pyruvate binding protein is positions 96-254 of SEQ ID NO:1. Preferably, the optical probe of the present invention has or consists of the sequence shown in SEQ ID NO: 19-30.
本发明提供的光学探针包含氨基酸序列SEQ ID NO：6-30中任一或其变体。在一个实施方式中，本发明提供的光学探针包含与氨基酸序列SEQ ID NO：6-30中任一有35％、40％、50％、60％、70％、80％、85％、90％、95％、99％序列相同性的序列。在优选实施方式中，本发明提供的光学探针包含与氨基酸序列SEQ ID NO：6-30中任一实质上相似或相同的序列。在更优选的实施方式中，本发明提供的光学探针包含SEQ ID NO：19-30中任一或由其组成。The optical probe provided by the present invention comprises any one of the amino acid sequence SEQ ID NO: 6-30 or a variant thereof. In one embodiment, the optical probe provided by the present invention contains 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90% of the amino acid sequence SEQ ID NO: 6-30. %, 95%, 99% sequence identity. In a preferred embodiment, the optical probe provided by the present invention contains a sequence substantially similar or identical to any one of the amino acid sequence SEQ ID NO: 6-30. In a more preferred embodiment, the optical probe provided by the present invention comprises or consists of any one of SEQ ID NO: 19-30.
本发明还提供融合多肽，包含本文所述光学探针和其它多肽。在一些实施方式中，其他多肽位于所述光学探针的N端和/或C端。在一些实施方式中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。The present invention also provides fusion polypeptides, including the optical probes described herein and other polypeptides. In some embodiments, other polypeptides are located at the N-terminus and/or C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize optical probes to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting.
本发明还提供编码本文所述多肽、探针或蛋白的核酸序列或其互补序列。在一个实施方案中，本发明的核酸序列选自(1)SEQ ID NO：6-30中任一所示氨基酸序列的编码序列或其互补序列，(2)与(1)具有至少99％、至少95％、至少90％、至少80％、至少70％或至少50％相同性的序列，(3)(1)或(2)的片段。在一个或多个实施方案中，所述片段是引物。在一个实施方案中，本发明核酸序列包含核苷酸序列SEQ ID NO：31或其变体，所述变体编码的氨基酸序列具有检测色氨酸的功能。在优选实施方案中，本发明提供一种核酸序列，包含与核苷酸序列SEQ ID NO：31具有至少99％、至少95％、至少90％、至少80％、至少70％或至少50％相同性的序列。本发明还涉及上述核酸序列的互补序列或其变体，其可包含编码本发明光学探针或融合多肽的片段、类似物、衍生物、可溶性片段和变体的核酸序列或其互补序列。The present invention also provides nucleic acid sequences encoding the polypeptides, probes or proteins described herein or their complementary sequences. In one embodiment, the nucleic acid sequence of the present invention is selected from (1) the coding sequence of any amino acid sequence shown in SEQ ID NO: 6-30 or its complementary sequence, and (2) and (1) have at least 99%, Sequences that are at least 95%, at least 90%, at least 80%, at least 70%, or at least 50% identical, fragments of (3) (1) or (2). In one or more embodiments, the fragment is a primer. In one embodiment, the nucleic acid sequence of the present invention comprises the nucleotide sequence SEQ ID NO: 31 or a variant thereof, and the amino acid sequence encoded by the variant has the function of detecting tryptophan. In a preferred embodiment, the present invention provides a nucleic acid sequence comprising at least 99%, at least 95%, at least 90%, at least 80%, at least 70% or at least 50% identical to the nucleotide sequence SEQ ID NO: 31 The sequence of sex. The present invention also relates to a complementary sequence of the above-mentioned nucleic acid sequence or a variant thereof, which may comprise a nucleic acid sequence or a complementary sequence thereof encoding a fragment, analog, derivative, soluble fragment and variant of the optical probe or fusion polypeptide of the present invention.
本发明还提供包含本文所述核酸序列或其互补序列的核酸构建物，该核酸序列编码本发明所述光学探针或融合多肽。在一个或多个实施方案中，所述核酸构建物是克隆载体、表达载体或重组载体。在一个或多个实施方案中，所述核酸序列与表达控制序列操作性连接。在一些实施方式中，表达载体选自原核表达载体、真核表达载体 和病毒载体。The present invention also provides a nucleic acid construct comprising the nucleic acid sequence described herein or its complementary sequence, which nucleic acid sequence encodes the optical probe or fusion polypeptide of the present invention. In one or more embodiments, the nucleic acid construct is a cloning vector, expression vector or recombinant vector. In one or more embodiments, the nucleic acid sequence is operably linked to an expression control sequence. In some embodiments, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors and viral vectors.
本发明还提供包含本发明所述核酸序列或核酸构建物的细胞。在一个或多个实施方案中，所述细胞表达本文所述光学探针或融合多肽。The present invention also provides a cell comprising the nucleic acid sequence or nucleic acid construct of the present invention. In one or more embodiments, the cell expresses the optical probe or fusion polypeptide described herein.
本发明还提供包括本文所述丙酮酸光学探针或融合多肽或如本文所述方法制备的丙酮酸光学探针或融合多肽的检测试剂盒。The present invention also provides a detection kit comprising the pyruvate optical probe or fusion polypeptide described herein or the pyruvate optical probe or fusion polypeptide prepared by the method described herein.
本发明提供制备本文所述光学探针的方法，包括：提供表达本文所述光学探针或融合多肽的细胞，在所述细胞表达的条件下培养所述细胞，和分离光学探针或融合多肽。在一个实施方案中，制备本文所述色氨酸光学探针或融合多肽的方法包括：1)将编码本文所述丙酮酸光学探针的表达载体转移到宿主细胞中；2)在适合所述表达载体表达的条件下培养所述宿主细胞，3)分离光学探针。The present invention provides a method for preparing the optical probe described herein, including: providing a cell expressing the optical probe or fusion polypeptide described herein, culturing the cell under the condition of expression of the cell, and isolating the optical probe or fusion polypeptide . In one embodiment, the method for preparing the tryptophan optical probe or fusion polypeptide described herein includes: 1) transferring the expression vector encoding the pyruvate optical probe described herein into a host cell; 2) The host cell is cultured under the condition of expression vector expression, and 3) the optical probe is isolated.
本发明还提供检测样品中丙酮酸的方法，包括：使本文所述光学探针或融合多肽或如本文所述方法制备的光学探针或融合多肽与样品接触，和检测光学活性多肽的变化。所述检测可以在体内、体外、亚细胞或原位进行。所述样品例如血液。The present invention also provides a method for detecting pyruvate in a sample, including: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, and detecting changes in the optically active polypeptide. The detection can be performed in vivo, in vitro, subcellular or in situ. The sample is for example blood.
本文还提供定量样品中丙酮酸的方法，包括：使本文所述光学探针或融合多肽或如本文所述方法制备的光学探针或融合多肽与样品接触，检测光学活性多肽的变化，和根据光学活性多肽的变化定量样品中的丙酮酸。This article also provides a method for quantifying pyruvate in a sample, including: contacting the optical probe or fusion polypeptide described herein or the optical probe or fusion polypeptide prepared by the method described herein with the sample, detecting changes in the optically active polypeptide, and The change in the optically active peptide quantifies the pyruvate in the sample.
本发明还提供筛选化合物(例如药物)的方法，包括：使本文所述光学探针或融合多肽或如本文所述方法制备的光学探针或融合多肽与候选化合物接触，检测光学活性多肽的变化，和根据光学活性多肽的变化筛选化合物。所述方法可以高通量地筛选化合物。The present invention also provides a method for screening a compound (such as a drug), comprising: contacting an optical probe or fusion polypeptide as described herein or an optical probe or fusion polypeptide prepared as described herein with a candidate compound to detect changes in the optically active polypeptide , And screening compounds based on changes in optically active polypeptides. The method can screen compounds with high throughput.
本发明还提供本文所述丙酮酸光学探针或融合多肽或如本文所述方法制备的丙酮酸光学探针或融合多肽在丙酮酸细胞内/外定位中的应用。在一个或多个实施方案中，所述定位是实时定位。The present invention also provides the application of the pyruvate optical probe or fusion polypeptide described herein or the pyruvate optical probe or fusion polypeptide prepared by the method described herein in the intracellular/extracellular localization of pyruvate. In one or more embodiments, the positioning is real-time positioning.
本发明的有益效果：本发明提供的丙酮酸光学探针易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、高通量、定量检测丙酮酸，省去了耗时的处理样品步骤。实验效果表明本申请所提供的丙酮酸光学探针对丙酮酸的最高响应达到对照的10倍以上，并且可以在细胞浆、线粒体、细胞核、内质网、溶酶体和高尔基体等亚细胞结构中对细胞进行定位、定性、定量检测，并且可以进行高通量的化合物筛选以及血液中丙酮酸定量检测。The beneficial effects of the present invention: the pyruvate optical probe provided by the present invention is easy to mature, has large dynamic changes in fluorescence, good specificity, and can be expressed in cells by genetic manipulation methods, and can be located inside and outside cells in real time, with high throughput, Quantitative detection of pyruvate saves time-consuming sample processing steps. The experimental results show that the highest response of the pyruvate optical probe provided in this application to pyruvate is more than 10 times that of the control, and it can be used in subcellular structures such as cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosome, and Golgi apparatus. It can locate, qualitatively and quantitatively detect cells, and can perform high-throughput compound screening and quantitative detection of pyruvate in blood.
附图说明Description of the drawings
下面结合附图和实施例对本发明作进一步说明。The present invention will be further described below with reference to the drawings and embodiments.
图1为实施例1所述的示例性丙酮酸光学探针的SDS-PAGE图；Fig. 1 is an SDS-PAGE chart of the exemplary pyruvate optical probe described in Example 1;
图2为实施例2所述的示例性的包含cpYFP和丙酮酸结合蛋白的丙酮酸光学探针对丙酮酸响应变化图；2 is a graph showing the response change of the exemplary pyruvate optical probe containing cpYFP and pyruvate binding protein to pyruvate described in Example 2;
图3为实施例3所述的示例性的包含cpGFP和丙酮酸结合蛋白的丙酮酸光学探针对丙酮酸响应变化图；3 is a graph showing the response change of the exemplary pyruvate optical probe containing cpGFP and pyruvate binding protein to pyruvate described in Example 3;
图4为实施例4所述的示例性的包含cpBFP和丙酮酸结合蛋白的丙酮酸光学探针对丙酮酸响应变化图；4 is a graph showing the response of the exemplary pyruvate optical probe containing cpBFP and pyruvate binding protein to pyruvate described in Example 4;
图5为实施例5所述的示例性的包含cpmApple和丙酮酸结合蛋白的丙酮酸光学探针对丙酮酸响应变化图；5 is a graph showing the response change of the exemplary pyruvate optical probe containing cpmApple and pyruvate binding protein to pyruvate described in Example 5;
图6为实施例6所述的示例性的在丙酮酸结合蛋白的位点191/193，192/194或192/195插入cpYFP基础上具有突变的丙酮酸光学探针对丙酮酸的响应；6 shows the response of an exemplary pyruvate optical probe with mutations to pyruvate based on the insertion of cpYFP at positions 191/193, 192/194 or 192/195 of the pyruvate binding protein described in Example 6;
图7A-B为实施例7所述的示例性的丙酮酸光学探针对不同浓度丙酮酸的滴定曲线；7A-B are titration curves of the exemplary pyruvate optical probe described in Example 7 to different concentrations of pyruvate;
图8A-C为实施例8所述的示例性丙酮酸光学探针的荧光光谱性质图；8A-C are graphs of the fluorescence spectrum properties of the exemplary pyruvate optical probe described in Example 8;
图9为实施例8所述的示例性丙酮酸光学探针的特异性检测的柱状图；9 is a histogram of the specific detection of the exemplary pyruvate optical probe described in Example 8;
图10为实施例9所述的示例性丙酮酸光学探针在哺乳动物细胞中的亚细胞器定位照片；10 is a photograph of the subcellular organelle location of the exemplary pyruvate optical probe described in Example 9 in mammalian cells;
图11为实施例10所述的对示例性丙酮酸光学探针在哺乳动物细胞中对外源丙酮酸响应的示意图；11 is a schematic diagram of the response of the exemplary pyruvate optical probe described in Example 10 to exogenous pyruvate in mammalian cells;
图12为实施例11所述的示例性丙酮酸光学探针在活细胞水平进行高通量化合物筛选的点图；Fig. 12 is a point diagram of the exemplary pyruvate optical probe described in Example 11 for high-throughput compound screening at the level of living cells;
图13为实施例12所述的示例性丙酮酸光学探针对小鼠和人血液中的丙酮酸进行定量的柱状图。13 is a bar graph of the exemplary pyruvate optical probe described in Example 12 for quantifying pyruvate in the blood of mice and humans.
具体实施方式Detailed ways
在给出数值或范围时，本文所用术语“约”指该数值或范围在给定数值或范围的20％以内、10％以内和5％以内。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.
本文所用术语“丙酮酸敏感多肽”或“丙酮酸响应多肽”指对丙酮酸产生响应的多肽，所述响应包括与敏感多肽的相互作用相关的多肽的化学，生物学，电学或生理学参数的任何响应。响应包括小的变化，例如，多肽的氨基酸或肽片段的方向的变化以及例如多肽的一级，二级或三级结构的变化，包括例如质子化，电化学势和/或构象的变化。“构象”是分子中包含侧基的分子的一级，二级和三级结构的三维排列；当分 子的三维结构发生变化时，构象发生变化。构象变化的实例包括从α-螺旋转变为β-折叠或从β-折叠转变为α-螺旋。可以理解的是，只要荧光蛋白部分的荧光被改变，可检测到的改变不需要是构象改变。本文所述丙酮酸敏感多肽还可包括其功能变体。丙酮酸敏感多肽的功能变体包括但不限于可以与丙酮酸相互作用从而发生与亲本丙酮酸敏感多肽相同或相似变化的变体。As used herein, the term "pyruvate sensitive polypeptide" or "pyruvate responsive polypeptide" refers to a polypeptide that responds to pyruvate, and the response includes any of the chemical, biological, electrical or physiological parameters of the polypeptide related to the interaction of the sensitive polypeptide. response. The response includes small changes, for example, changes in the orientation of the amino acids or peptide fragments of the polypeptide and, for example, changes in the primary, secondary, or tertiary structure of the polypeptide, including, for example, changes in protonation, electrochemical potential, and/or conformation. "Conformation" refers to the three-dimensional arrangement of the primary, secondary and tertiary structure of the molecule containing side groups; when the three-dimensional structure of the molecule changes, the conformation changes. Examples of conformational changes include conversion from α-helix to β-sheet or from β-sheet to α-helix. It is understandable that as long as the fluorescence of the fluorescent protein portion is changed, the detectable change need not be a conformational change. The pyruvate sensitive polypeptides described herein may also include functional variants thereof. Functional variants of the pyruvate-sensitive polypeptide include, but are not limited to, variants that can interact with pyruvate to undergo the same or similar changes as the parent pyruvate-sensitive polypeptide.
本发明所述丙酮酸敏感多肽包括但不限于丙酮酸结合蛋白PdhR或与其有90％以上同源性的变体。本发明所述示例性丙酮酸结合蛋白PdhR来源于大肠杆菌。PdhR是细菌转录因子由丙酮酸结合/调节域和DNA结合域组成。示例性PdhR蛋白如SEQ ID NO：1所示。在一个或多个实施方案中，丙酮酸敏感多肽包含PdhR蛋白的丙酮酸结合域，即96-254位氨基酸，而不包括DNA结合域。当描述本发明光学探针或丙酮酸结合蛋白时(例如描述插入位点或突变位点时)，提及氨基酸残基编号均参考SEQ ID NO：1。The pyruvate sensitive polypeptides of the present invention include but are not limited to pyruvate binding protein PdhR or variants with more than 90% homology. The exemplary pyruvate binding protein PdhR of the present invention is derived from Escherichia coli. PdhR is a bacterial transcription factor composed of a pyruvate binding/regulatory domain and a DNA binding domain. An exemplary PdhR protein is shown in SEQ ID NO:1. In one or more embodiments, the pyruvate sensitive polypeptide comprises the pyruvate binding domain of the PdhR protein, that is, amino acids 96-254, but does not include the DNA binding domain. When describing the optical probe or pyruvate binding protein of the present invention (for example, when describing the insertion site or the mutation site), all references to the amino acid residue numbers refer to SEQ ID NO:1.
本文所用术语“光学探针”是指与光学活性多肽融合的丙酮酸敏感多肽。发明人发现，丙酮酸敏感多肽例如丙酮酸结合蛋白专一性地对生理浓度的丙酮酸结合后所产生的构象变化会引起光学活性多肽(例如荧光蛋白)的构象变化，进而导致光学活性多肽的光学性质发生改变。借助不同丙酮酸浓度下测定的荧光蛋白的荧光绘制标准曲线，可以检测并分析丙酮酸的存在和/或水平。The term "optical probe" as used herein refers to a pyruvate sensitive polypeptide fused to an optically active polypeptide. The inventors found that the conformational changes produced by pyruvate-sensitive polypeptides such as pyruvate-binding proteins that specifically bind to physiological concentrations of pyruvate will cause conformational changes of optically active polypeptides (such as fluorescent proteins), which in turn leads to optically active polypeptides. The optical properties have changed. By drawing a standard curve with the fluorescence of fluorescent proteins measured at different pyruvate concentrations, the presence and/or level of pyruvate can be detected and analyzed.
在本发明的光学探针中，光学活性多肽(例如荧光蛋白)可操作地插入丙酮酸敏感多肽中。基于蛋白质的“光学活性多肽”是具有发射荧光能力的多肽。荧光是光学活性多肽的一种光学性质，其可用作检测本发明的光学探针的响应性的手段。优选地，选择蛋白质底物以具有在未激活和活化的构象状态下容易区分的荧光特性。本文所述光学活性多肽还可包括其功能变体。光学活性多肽的功能变体包括但不限于可以发生与亲本光学活性多肽相同或相似荧光性质变化的变体。In the optical probe of the present invention, the optically active polypeptide (for example, fluorescent protein) is operably inserted into the pyruvate-sensitive polypeptide. Protein-based "optically active polypeptides" are polypeptides that have the ability to emit fluorescence. Fluorescence is an optical property of optically active polypeptides, which can be used as a means to detect the responsiveness of the optical probe of the present invention. Preferably, the protein substrate is selected to have fluorescent properties that are easily distinguishable in the unactivated and activated conformational states. The optically active polypeptides described herein may also include functional variants thereof. Functional variants of an optically active polypeptide include, but are not limited to, variants that can have the same or similar changes in fluorescence properties as the parent optically active polypeptide.
“接头”或“连接区”指在本发明多肽、蛋白质或核酸中连接两个部分的氨基酸或核苷酸序列。示例性地，本发明中丙酮酸敏感多肽与光学活性多肽的连接区氨基端的氨基酸数目选择的是0-3个，羧基端的氨基酸数目选择的是0-2个；当重组光学探针作为基本单元与功能蛋白连接时，可以融合在重组光学探针的氨基酸或羧基端。接头序列可为一个或多个柔性氨基酸组成的短肽链，如Y。"Linker" or "linking region" refers to an amino acid or nucleotide sequence that connects two parts in the polypeptide, protein or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino terminus of the connecting region of the pyruvate sensitive polypeptide and the optically active polypeptide is selected from 0 to 3, and the number of amino acids at the carboxy terminus is selected from 0 to 2; when the recombinant optical probe is used as the basic unit When connected to a functional protein, it can be fused to the amino acid or carboxyl end of the recombinant optical probe. The linker sequence can be a short peptide chain composed of one or more flexible amino acids, such as Y.
本文所用术语“荧光蛋白”指在激发光照射下发出荧光的蛋白质。例如绿色荧光蛋白GFP及由该蛋白突变衍生出的环状重排的蓝色荧光蛋白(cpBFP)、环状重排的绿色荧光蛋白(cpGFP)、环状重排的黄色荧光蛋白(cpYFP)等；还有本技术领域常用的红色荧光蛋白RFP，及由该蛋白衍生出来的环状重排的蛋白，如cpmApple， cpmOrange，cpmKate等。本领域知晓可用于本发明的荧光蛋白及其序列。示例性地，cpYFP如SEQ ID NO：2所示；cpGFP如SEQ ID NO：3所示；cpBFP如SEQ ID NO：4所示；cpmApple如SEQ ID NO：5所示。The term "fluorescent protein" as used herein refers to a protein that fluoresces under excitation light. For example, green fluorescent protein GFP and circularly rearranged blue fluorescent protein (cpBFP) derived from the mutation of the protein, circularly rearranged green fluorescent protein (cpGFP), circularly rearranged yellow fluorescent protein (cpYFP), etc. There are also red fluorescent protein RFP commonly used in the art, and circular rearranged proteins derived from this protein, such as cpmApple, cpmOrange, cpmKate, etc. The fluorescent protein and its sequence that can be used in the present invention are known in the art. Exemplarily, cpYFP is shown in SEQ ID NO: 2; cpGFP is shown in SEQ ID NO: 3; cpBFP is shown in SEQ ID NO: 4; cpmApple is shown in SEQ ID NO: 5.
本发明所述的丙酮酸光学探针包括丙酮酸敏感多肽(B)的丙酮酸结合域96-254位氨基酸，例如丙酮酸结合蛋白或其变体，和光学活性多肽(A)，例如荧光蛋白。光学活性多肽(A)插入到丙酮酸敏感多肽(B)的丙酮酸结合域96-254位氨基酸中，将B分为B1和B2两个部分，形成B1-A-B2式的探针结构；丙酮酸敏感多肽B和丙酮酸相互作用导致光学活性多肽(A)的光学信号变强。The pyruvate optical probe of the present invention includes amino acids 96-254 of the pyruvate binding domain of a pyruvate sensitive polypeptide (B), such as pyruvate binding protein or its variants, and an optically active polypeptide (A), such as fluorescent protein . The optically active polypeptide (A) is inserted into the 96-254 amino acids of the pyruvate binding domain of the pyruvate sensitive polypeptide (B), and B is divided into two parts, B1 and B2, to form a probe structure of formula B1-A-B2; The interaction between the pyruvate sensitive polypeptide B and pyruvate causes the optical signal of the optically active polypeptide (A) to become stronger.
在本发明的光学探针中，光学活性多肽可以位于丙酮酸敏感多肽的任何位置。在一个实施方式中，光学活性多肽以N-C方向位于N-C方向的丙酮酸敏感多肽的任何位置。具体地，光学活性多肽位于丙酮酸敏感多肽的柔性区域，所述的柔性区域是指蛋白质高级结构中存在的一些特定的如环状结构域等结构，这些结构域相比于蛋白质的其他高级结构具有更高的移动性和柔性，并且该区域可以在该蛋白质和配体结合后，空间结构构象发生动态变化。本发明中所述的柔性区域主要指丙酮酸结合蛋白中的插入位点所在区域，如氨基酸残基117-121、140-143、160-164、174-176、191-195和210-214区域。示例性地，光学活性多肽位于丙酮酸结合蛋白的氨基酸序列的选自以下的一个或多个位点：117/118，117/119，117/120，117/121，118/119，118/120，118/121，119/120，119/121，120/121，140/141，140/142，140/143，141/142，141/143，142/143，160/161，160/162，160/163，160/164，161/162，161/163，161/164，162/163，162/164，163/164，174/175，174/176，175/176，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195，210/211，210/212，210/213，210/214，211/212，211/213，211/214，212/213，212/214或213/214。在优选实施方式中，光学活性多肽位于丙酮酸结合蛋白或其功能片段的氨基酸序列的选自以下的一个或多个位点：117/121，141/143，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195或210/214处。本文中，如果以“X/Y”形式表示的位点中的两个数字是连续的整数，则表示光学活性多肽位于该数字所述的氨基酸之间。例如插入位点117/118表示光学活性多肽位于丙酮酸敏感多肽或其功能片段的氨基酸117与118之间。如果以“X/Y”形式表示的位点中的两个数字不是连续的整数，则表示光学活性多肽置换该数字所示氨基酸之间的氨基酸。例如插入位点191/195表示光学活性多肽置换丙酮酸敏感多肽或其功能片段的氨基酸192-194。在优选实施方式中，光学活性多肽位于丙酮酸结合蛋白或其功能片段的氨基酸序列的选自以下的一个或多个位点：117/118，117/119，117/120，117/121，118/119，118/120， 118/121，119/120，119/121，120/121，140/141，140/142，140/143，141/142，141/143，142/143，160/161，160/162，160/163，160/164，161/162，161/163，161/164，162/163，162/164，163/164，174/175，174/176，175/176，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195，210/211，210/212，210/213，210/214，211/212，211/213，211/214，212/213，212/214或213/214，所述功能片段包含丙酮酸结合蛋白的第96-254位氨基酸。在更优选的实施方式中，光学活性多肽位于包含第96-254位氨基酸的丙酮酸结合蛋白功能片段的氨基酸序列的选自以下的一个或多个位点：117/121，141/143，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195或210/214处，如SEQ ID NO：6-18所示。In the optical probe of the present invention, the optically active polypeptide can be located at any position of the pyruvate sensitive polypeptide. In one embodiment, the optically active polypeptide is located at any position of the pyruvate sensitive polypeptide in the N-C direction in the N-C direction. Specifically, the optically active polypeptide is located in the flexible region of the pyruvate-sensitive polypeptide. The flexible region refers to some specific structures such as loop domains that exist in the high-level structure of the protein. These domains are compared to other high-level structures of the protein. It has higher mobility and flexibility, and the region can dynamically change its spatial structure and conformation after the protein and ligand are combined. The flexible region described in the present invention mainly refers to the region where the insertion site in the pyruvate binding protein is located, such as the region of amino acid residues 117-121, 140-143, 160-164, 174-176, 191-195 and 210-214 . Exemplarily, the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120 ,118/121,119/120,119/121,120/121,140/141,140/142,140/143,141/142,141/143,142/143,160/161,160/162,160 /163, 160/164, 161/162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191/192, 191/193 , 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/211, 210/212, 210/213, 210/214, 211 /212, 211/213, 211/214, 212/213, 212/214 or 213/214. In a preferred embodiment, the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein or functional fragment thereof: 117/121, 141/143, 191/192, 191/193, 191 /194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195 or 210/214. Herein, if the two numbers in the position expressed in the form of "X/Y" are consecutive integers, it means that the optically active polypeptide is located between the amino acids described in the number. For example, the insertion site 117/118 indicates that the optically active polypeptide is located between amino acids 117 and 118 of the pyruvate sensitive polypeptide or its functional fragment. If the two numbers in the position expressed in the form of "X/Y" are not consecutive integers, it means that the optically active polypeptide replaces the amino acids between the amino acids indicated by the numbers. For example, the insertion site 191/195 indicates that the optically active polypeptide replaces amino acids 192-194 of the pyruvate sensitive polypeptide or a functional fragment thereof. In a preferred embodiment, the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein or functional fragment thereof: 117/118, 117/119, 117/120, 117/121, 118 /119, 118/120, 118/121, 119/120, 119/121, 120/121, 140/141, 140/142, 140/143, 141/142, 141/143, 142/143, 160/161 , 160/162, 160/163, 160/164, 161/162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191 /192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/211, 210/212, 210/213 , 210/214, 211/212, 211/213, 211/214, 212/213, 212/214 or 213/214, the functional fragments comprise amino acids 96-254 of pyruvate binding protein. In a more preferred embodiment, the optically active polypeptide is located at one or more positions selected from the following in the amino acid sequence of the pyruvate binding protein functional fragment containing amino acids 96-254: 117/121, 141/143, 191 /192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195 or 210/214, such as SEQ ID NO: 6 -18 shown.
提到某多肽或蛋白时，本发明所用术语“变体”或“突变体”包括具有所述多肽或蛋白相同功能、但序列不同的变体。多肽或蛋白的变体可包括：同源序列、保守性变体、等位变体、天然突变体、诱导突变体。这些变体包括但并不限于：在所述多肽或蛋白的序列中缺失、插入和/或取代一个或多个(通常为1-30个，较佳地1-20个，更佳地1-10个，最佳地1-5个)氨基酸，以及在其羧基末端和/或氨基末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸获得的序列。这些变体还可包含与所述多肽或蛋白的序列相同性为至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的多肽或蛋白。不希望受理论限制，氨基酸残基发生改变而不改变多肽或蛋白质的总体构型和功能，即功能保守突变。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变多肽或蛋白的功能。在本领域中，性能相似的氨基酸往往指具有相似侧链的氨基酸家族，在本领域已有明确定义。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、精氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。又比如，在氨基末端和/或羧基末端添加一个或数个氨基酸通常也不会改变多肽或蛋白的功能。对于许多常见已知非遗传性编码氨基酸的保守氨基酸取代本领域已知。其他非编码氨基酸的保守取代可基于其物理性质与遗传上编码的氨基酸的性质的比较来确定。When referring to a certain polypeptide or protein, the term "variant" or "mutant" used in the present invention includes variants that have the same function of the polypeptide or protein but have different sequences. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants. These variants include but are not limited to: deletion, insertion and/or substitution of one or more (usually 1-30, preferably 1-20, more preferably 1- 10, preferably 1-5) amino acids, and one or more (usually within 20, preferably within 10, more preferably within 5) at the carboxyl and/or amino terminus ) Sequence obtained from amino acids. These variants may also comprise sequence identity with the polypeptide or protein of 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% polypeptide or protein. Without wishing to be limited by theory, amino acid residues are changed without changing the overall configuration and function of the polypeptide or protein, that is, functional conservative mutations. For example, in the art, when amino acids with similar or similar properties are substituted, the functions of the polypeptide or protein are usually not changed. In the art, amino acids with similar properties often refer to amino acid families with similar side chains, which have been clearly defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), and uncharged polar Side chain amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, Leucine, isoleucine, arginine, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g. threonine, valine, isoleucine) ) And amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). For another example, adding one or several amino acids to the amino terminus and/or carboxy terminus usually does not change 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-encoded amino acids can be determined based on the comparison of their physical properties with those of genetically encoded amino acids.
在两种或多种多肽或核酸分子序列中，术语“相同性”或“相同性百分数”指在比 较窗口或指定区域上，采用本领域已知方法如序列比较算法，通过手工比对和目测检查来比较和比对最大对应性时，两个或多个序列或子序列相同或其中在指定区域有一定百分数的氨基酸残基或核苷酸相同(例如，至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％相同)。例如，适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法，分别可参见Altschul等(1977)Nucleic Acids Res.25：3389和Altschul等(1990)J.Mol.Biol.215：403。In the sequence of two or more polypeptides or nucleic acid molecules, the term "identity" or "percent identity" refers to the comparison window or designated area, using methods known in the art such as sequence comparison algorithms, through manual alignment and visual inspection When checking to compare and align the maximum correspondence, two or more sequences or subsequences are the same or a certain percentage of amino acid residues or nucleotides in the specified region are the same (for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , At least 99% or 100% the same). For example, the preferred algorithms suitable for determining the percentage of sequence identity and the percentage of sequence similarity are BLAST and BLAST 2.0 algorithms, respectively, see Altschul et al. (1977) Nucleic Acids Res. 25: 3389 and Altschul et al. (1990) J. Mol. Biol. 215: 403.
本领域技术人员公知，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的多肽或蛋白末端引入了一个或多个不相干的残基，而这并不影响目的多肽或蛋白的活性。又如为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签，或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。Those skilled in the art know that in gene cloning operations, it is often necessary to design suitable restriction sites, which inevitably introduce one or more irrelevant residues at the end of the expressed polypeptide or protein, and this does not affect the purpose. The activity of a polypeptide or protein. For another example, in order to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted out of the host cell, or facilitate the purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus or the recombinant protein. Within other suitable regions within the protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, such as 6His or Flag tag, or factor Xa or proteolytic enzyme site of thrombin or enterokinase.
本发明光学探针可包含具有突变的丙酮酸敏感多肽。所述突变例如Q138，S190，R191，R192，E193，M194，和/或L195等位点的突变。示例性地，所述突变选自以下的一个或多个：Q138S，Q138Y，Q138C，Q138L，Q138P，Q138H，Q138R，Q138W，Q138I，Q138T，Q138N，Q138K，Q138F，Q138V，Q138A，Q138D，Q138E，Q138M，Q138A，S190E，R191S，R191Y，R191C，R191L，R191P，R191H，R191Q，R191W，R191I，R191T，R191N，R191K，R191F，R191V，R191A，R191D，R191E，R191M，R191A，R192D，E193S，E193Y，E193C，E193L，E193P，E193H，E193Q，E193W，E193R，E193I，E193T，E193N，E193K，E193M，E193V，E193F，E193D，E193A，E193G，M194S，M194Y，M194C，M194L，M194P，M194H，M194Q，M194W，M194R，M194I，M194A，M194N，M194K，M194T，M194V，M194F，M194D，M194E，T102G，L195S，L195Y，L195C，L195D，L195P，L195H，L195Q，L195W，L195I，L195T，L195N，L195K，L195R，L195V，L195A，L195F，L195E，L195M和/或L195A。在优选实施方式中，所述突变选自以下的一个或多个：Q138P，Q138L，S190E，R191Y，R191N，R191F，R191L，R191P，R192D，E193Q，E193L，M194D，M194V，M194H，M194W，M194V和S190E和R191N和R192D(M194V/S190E/R191N/R192D)，M194V和S190D和R191Y和R192T (M194V/S190D/R191Y/R192T)、S190P和R191H和R192P(S190P/R191H/R192P)、S190R和R191S和R192P(S190R/R191S/R192P)、S190L和R191V(S190L/R191V)、S190T和R191Q和R192E(S190T/R191Q/R192E)或R191S和R192T(R191S/R192T)。The optical probe of the present invention may comprise a pyruvate sensitive polypeptide with mutation. Such mutations are, for example, Q138, S190, R191, R192, E193, M194, and/or L195 allele mutations. Exemplarily, the mutation is selected from one or more of the following: Q138S, Q138Y, Q138C, Q138L, Q138P, Q138H, Q138R, Q138W, Q138I, Q138T, Q138N, Q138K, Q138F, Q138V, Q138A, Q138D, Q138E, Q138M, Q138A, S190E, R191S, R191Y, R191C, R191L, R191P, R191H, R191Q, R191W, R191I, R191T, R191N, R191K, R191F, R191V, R191A, R191D, R191E, R191M, R191A, R192D, E193S, E193S E193C, E193L, E193P, E193H, E193Q, E193W, E193R, E193I, E193T, E193N, E193K, E193M, E193V, E193F, E193D, E193A, E193G, M194S, M194Y, M194C, M194L, M194P, M194H, M194Q, M194Q M194R, M194I, M194A, M194N, M194K, M194T, M194V, M194F, M194D, M194E, T102G, L195S, L195Y, L195C, L195D, L195P, L195H, L195Q, L195W, L195I, L195T, L195N, L195K, L195R, L195R L195A, L195F, L195E, L195M and/or L195A. In a preferred embodiment, the mutation is selected from one or more of the following: Q138P, Q138L, S190E, R191Y, R191N, R191F, R191L, R191P, R192D, E193Q, E193L, M194D, M194V, M194H, M194W, M194V and S190E and R191N and R192D (M194V/S190E/R191N/R192D), M194V and S190D and R191Y and R192T (M194V/S190D/R191Y/R192T), S190P and R191H and R192P (S190P/R191H/R192P), S190R and R191S and R192 (S190R/R191S/R192P), S190L and R191V (S190L/R191V), S190T and R191Q and R192E (S190T/R191Q/R192E) or R191S and R192T (R191S/R192T).
在示例性实施方式中，本发明B1-A-B2式光学探针可为PdhR(96-254)的选自117/121，141/143，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195，210/214的一个或多个位点插入有cpYFP并具有选自以下的一个或多个突变的探针：Q138P，Q138L，R191Y，R191F，R191L，R191P，E193Q，E193L，M194D，M194V，M194H，M194W，M194V和S190E和R191N和R192D(M194V/S190E/R191N/R192D)，M194V和S190D和R191Y和R192T(M194V/S190D/R191Y/R192T)、S190P和R191H和R192P(S190P/R191H/R192P)、S190R和R191S和R192P(S190R/R191S/R192P)、S190L和R191V(S190L/R191V)、S190T和R191Q和R192E(S190T/R191Q/R192E)或R191S和R192T(R191S/R192T)。优选地，所述光学探针为PdhR(96-254)的191/193位点插入有cpYFP并且具有Q138P，Q138L，R191Y，R191F，R191L，R191P，E193Q或E193L突变的探针，或PdhR(96-254)的192/194位点插入有cpYFP并且具有M194D，M194V，M194H或M194W突变的探针。在一个实施方式中，丙酮酸敏感多肽中光学活性多肽的插入位点与丙酮酸敏感多肽的突变的组合选自以下一种或多种：191/193-Q138P，191/193-Q138L，191/193-R191Y，191/193-R191F，191/193-R191L，191/193-R191P，191/193-E193Q，191/193-E193L，192/194-M194D，192/194-M194V，192/194-M194H，192/194-M194W192/194-M194V/S190E/R191N/R192D，192/194-M194V/S190D/R191Y/R192T、141/143-S190P/R191H/R192P、141/143-S190R/R191S/R192P、141/143-S190L/R191V、141/143-S190T/R191Q/R192E和/或141/143-R191S/R192T。这些光学探针的序列如SEQ ID NO：19-30所示。In an exemplary embodiment, the B1-A-B2 optical probe of the present invention may be PdhR (96-254) selected from 117/121, 141/143, 191/192, 191/193, 191/194, 191 /195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/195, 210/214 are inserted into one or more positions of cpYFP and have one or more selected from the following Mutation probes: Q138P, Q138L, R191Y, R191F, R191L, R191P, E193Q, E193L, M194D, M194V, M194H, M194W, M194V and S190E and R191N and R192D (M194V/S190E/R191N/R192D), M194V and S190D and R191Y and R192T (M194V/S190D/R191Y/R192T), S190P and R191H and R192P (S190P/R191H/R192P), S190R and R191S and R192P (S190R/R191S/R192P), S190L and R191V (S190L/R191V), S190T and R191Q and R192E (S190T/R191Q/R192E) or R191S and R192T (R191S/R192T). Preferably, the optical probe is a probe with Q138P, Q138L, R191Y, R191F, R191L, R191P, E193Q or E193L mutation inserted into the 191/193 position of PdhR(96-254), or PdhR(96 -254) has a cpYFP inserted at position 192/194 and has a M194D, M194V, M194H or M194W mutation probe. In one embodiment, the combination of the insertion site of the optically active polypeptide in the pyruvate-sensitive polypeptide and the mutation of the pyruvate-sensitive polypeptide is selected from one or more of the following: 191/193-Q138P, 191/193-Q138L, 191/ 193-R191Y, 191/193-R191F, 191/193-R191L, 191/193-R191P, 191/193-E193Q, 191/193-E193L, 192/194-M194D, 192/194-M194V, 192/194- M194H, 192/194-M194W192/194-M194V/S190E/R191N/R192D, 192/194-M194V/S190D/R191Y/R192T, 141/143-S190P/R191H/R192P, 141/143-S190R/R191S/R192P, 141/143-S190L/R191V, 141/143-S190T/R191Q/R192E and/or 141/143-R191S/R192T. The sequence of these optical probes is shown in SEQ ID NO: 19-30.
本文所用术语“功能片段”、“衍生物”和“类似物”是指基本上保持与原始多肽或蛋白(例如PdhR蛋白或荧光蛋白)相同的生物学功能或活性的蛋白。本发明的多肽或蛋白(例如PdhR蛋白或荧光蛋白)的功能变体、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的蛋白，或(iii)成熟蛋白与另一个化合物(比如延长蛋白半衰期的化合物，例如聚乙二醇)融合所形成的蛋白，或(iv)附加的氨基酸序列融合到此蛋白序列而形成的蛋白(如分泌序列或用来纯化此蛋白的序列或蛋白原序列，或 与抗原IgG片段的形成的融合蛋白)。根据本文的教导，这些功能变体、衍生物和类似物属于本领域熟练技术人员公知的范围。所述类似物与原始多肽或蛋白的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。这些蛋白包括天然或诱导的遗传变体。诱导变体可以通过各种技术得到，如通过辐射或暴露于诱变剂而产生随机诱变，还可通过定点诱变法或其他已知分子生物学的技术得到。The terms "functional fragment", "derivative" and "analog" as used herein refer to a protein that substantially maintains the same biological function or activity as the original polypeptide or protein (for example, PdhR protein or fluorescent protein). A functional variant, derivative or analogue of the polypeptide or protein (e.g., PdhR protein or fluorescent protein) of the present invention may be (i) having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) A protein that is substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins with substitution groups in one or more amino acid residues, or (iii) mature proteins A protein formed by fusion with another compound (such as a compound that prolongs the half-life of a protein, such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the protein sequence to form a protein (such as a secretory sequence or used to purify the protein). The sequence of the protein or the proprotein sequence, or the fusion protein formed with the antigen IgG fragment). According to the teachings herein, these functional variants, derivatives and analogs belong to the scope well known to those skilled in the art. The difference between the analog and the original polypeptide or protein may be a difference in the amino acid sequence, a difference in the modified form that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques.
本发明融合多肽包含本文所述光学探针和其它多肽。在一些实施方式中，本文所述光学探针还包含与之融合的其它多肽。本文所述其他多肽不影响光学探针的性质。其他多肽可位于所述光学探针的N端和/或C端。在一些实施方式中，其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。本文所述融合多肽中的光学探针和其它多肽之间可具有接头。本文所述亚细胞器包括细胞浆、线粒体、细胞核、内质网、细胞膜、高尔基体、溶酶体和过氧化物酶体等。在一些实施方式中，用于纯化的标签或者用于免疫印迹的标签包括6组氨酸(6*His)、谷胱甘肽硫转移酶(GST)、Flag。The fusion polypeptide of the present invention includes the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further includes other polypeptides fused to it. The other polypeptides described herein do not affect the properties of the optical probe. Other polypeptides can be located at the N-terminus and/or C-terminus of the optical probe. In some embodiments, other polypeptides include polypeptides that localize optical probes to different organelles or subcellular organelles, tags for purification, or tags for immunoblotting. The optical probe in the fusion polypeptide described herein may have a linker between the other polypeptide. The subcellular organelles described herein include cytoplasm, mitochondria, nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosomes, peroxisomes, and the like. In some embodiments, the tag used for purification or the tag used for immunoblotting includes 6 histidine (6*His), glutathione sulfur transferase (GST), and Flag.
本发明表达载体包含与表达控制序列操作性连接的本发明所述的核酸序列或其互补序列，该核酸序列编码本发明所述光学探针或融合多肽。在一些实施方式中，表达载体选自原核表达载体、真核表达载体和病毒载体。在一些实施方式中，原核表达载体优选由质粒pCDFDuet-1与本文所述的核酸序列操作性连接得到。在一些实施方式中，表达控制序列包括复制起点、启动子、增强子、操纵子、终止子、核糖体结合位点。The expression vector of the present invention comprises the nucleic acid sequence of the present invention or its complementary sequence operably linked to an expression control sequence, and the nucleic acid sequence encodes the optical probe or fusion polypeptide of the present invention. In some embodiments, the expression vector is selected from prokaryotic expression vectors, eukaryotic expression vectors and viral vectors. In some embodiments, the prokaryotic expression vector is preferably obtained by operably linking the plasmid pCDFDuet-1 with the nucleic acid sequence described herein. In some embodiments, the expression control sequence includes an origin of replication, promoter, enhancer, operator, terminator, ribosome binding site.
本发明还提供了上述丙酮酸光学探针的制备方法，包括以下步骤：1)将编码本文所述丙酮酸光学探针的核酸序列纳入表达载体；2)将表达载体转移到宿主细胞中；2)在适合所述表达载体表达的条件下培养所述宿主细胞，3)分离丙酮酸光学探针。The present invention also provides a method for preparing the above-mentioned pyruvate optical probe, including the following steps: 1) incorporating the nucleic acid sequence encoding the pyruvate optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 2 ) Culturing the host cell under conditions suitable for the expression of the expression vector, 3) Isolating the pyruvate optical probe.
本发明所用术语“核酸”或“核苷酸”或“多核苷酸”或“核酸序列”可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。提到核酸时，本文所用术语“变体”可以是天然发生的等位变体或非天然发生的变体。这些核苷酸变体包括简并变体、取代变体、缺失变体和插入变体。本发明核酸可包含与所述核酸序列的序列相同性为至少约50％、至少约60％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或100％的核苷酸序列。本发明还涉及与上述的序列杂交的核酸片段。在示例性实施方式中，核酸序列如SEQ ID NO：31所示，其表示丙酮酸合蛋白功能片段的192/194位点插入有cpYFP 并具有M194V突变的探针的编码序列。如本文所用，“核酸片段”的长度至少含15个核苷酸，较好是至少30个核苷酸，更好是至少50个核苷酸，最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR)。The term "nucleic acid" or "nucleotide" or "polynucleotide" or "nucleic acid sequence" used in the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. When referring to nucleic acids, the term "variant" as used herein may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. The nucleic acid of the present invention may comprise a sequence identity with the nucleic acid sequence of at least about 50%, at least about 60%, 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% of the nucleotide sequence. The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. In an exemplary embodiment, the nucleic acid sequence is shown in SEQ ID NO: 31, which represents the coding sequence of the probe with cpYFP inserted at position 192/194 of the pyruvate synthin functional fragment and with the M194V mutation. As used herein, the "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. Nucleic acid fragments can be used in nucleic acid amplification techniques (such as PCR).
本发明光学探针或融合蛋白的全长序列或其片段通常可以用PCR扩增法、人工合成法或重组法获得。对于PCR扩增法，可根据本发明所公开的核苷酸序列设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当核苷酸序列大于2500bp时，优选的进行2～6次PCR扩增，然后将各次扩增的片段按正确次序拼接在一起。本发明对所述的PCR扩增的程序和体系没有特殊限定，采用本领域常规的PCR扩增程序和体系即可。还可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离和纯化得到有关多肽或蛋白。此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。在本发明中，在光学探针的核苷酸序列小于2500bp时，可采用人工合成方法来合成。所述人工合成方法为本领域常规的DNA的人工合成方法，无其他特殊要求。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。本发明多肽的DNA序列也可完全通过化学合成来得到。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(如载体)和细胞中。可通过突变PCR或化学合成等方法将突变引入本发明蛋白序列中。The full-length sequence or fragments of the optical probe or fusion protein of the present invention can usually be obtained by PCR amplification method, artificial synthesis method or recombination method. For the PCR amplification method, primers can be designed according to the nucleotide sequence disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as a template, and the relevant amplification can be obtained. sequence. When the nucleotide sequence is greater than 2500 bp, it is preferable to perform 2-6 PCR amplifications, and then splice the amplified fragments together in the correct order. The present invention does not specifically limit the PCR amplification program and system, and the conventional PCR amplification program and system in this field can be used. The recombination method can also be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then separating and purifying it from the proliferated host cell by conventional methods to obtain the relevant polypeptide or protein. In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is short. In the present invention, when the nucleotide sequence of the optical probe is less than 2500 bp, it can be synthesized by artificial synthesis. The artificial synthesis method is a conventional DNA synthesis method in the field, and there are no other special requirements. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences. The DNA sequence of the polypeptide of the present invention can also be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. The mutation can be introduced into the protein sequence of the present invention by mutation PCR or chemical synthesis.
本发明还提供包括本文所述光学探针或融合多肽或多核苷酸或如本文所述方法制备的光学探针或融合多肽的检测试剂盒。该试剂盒还任选包含利用光学探针检测丙酮酸所需的其他试剂。本领域知晓常规的所述其他试剂。The present invention also provides a detection kit comprising the optical probe or fusion polypeptide or polynucleotide described herein or the optical probe or fusion polypeptide prepared by the method described herein. The kit also optionally contains other reagents required for the detection of pyruvate using an optical probe. Such other reagents are conventionally known in the art.
本发明也涉及核酸构建物，该核酸构建物含有本文所述的多核苷酸，以及与这些序列操作性连接的一个或多个调控序列。本发明所述的多核苷酸可以多种方式被操作以保证所述多肽或蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。The present invention also relates to nucleic acid constructs, which contain the polynucleotides described herein, and one or more regulatory sequences operably linked to these sequences. The polynucleotide of the present invention can be manipulated in a variety of ways to ensure the expression of the polypeptide or protein. The nucleic acid construct can be manipulated according to the different or requirements of the expression vector before inserting the nucleic acid construct into the vector. Techniques for using recombinant DNA methods to alter polynucleotide sequences are known in the art.
在某些实施方案中，所述核酸构建物是载体。载体可以是克隆载体，也可以是表达载体，或者是基因敲入载体，例如同源重组载体。本发明的多核苷酸可被克隆入许多类型的载体，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。克隆载体可用于提供本发明蛋白或多肽的编码序列。表达载体可以以细菌载体或病毒载体形式提供给细胞。通常通过可操作地连接本发明的多核苷酸至启动子，并将构建体并入表达载体，实现本发明多核苷酸的表达。该载体对于复制和整合真核细胞可为合适的。典型的表达载体包含可用于调节期望核酸序列表达的表达控制序列。基因敲入载体用 于将本文所述的表达框敲入或整合到宿主基因组中。In certain embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector, such as a homologous recombination vector. The polynucleotide of the present invention can be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide coding sequences for the proteins or polypeptides of the invention. The expression vector can be provided to the cell in the form of a bacterial vector or a viral vector. The expression of the polynucleotide of the present invention is usually achieved by operably linking the polynucleotide of the present invention to a promoter, and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. A typical expression vector contains expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence. Gene knock-in vectors are used to knock-in or integrate the expression cassettes described herein into the host genome.
本文所用术语“表达控制序列”指调控目的基因的转录、翻译和表达的可以与目的基因操作性连接的元件，可以是复制起点、启动子、标记基因或翻译控制元件，包括增强子、操纵子、终止子、核糖体结合位点等，表达控制序列的选择取决于所用的宿主细胞。在重组表达载体中，“操作性连接”是指目的的核苷酸序列与调节序列以允许核苷酸序列表达的方式连接。本领域的技术人员熟知能用于构建含本发明融合蛋白编码序列和合适的转录/翻译控制信号的表达载体的方法。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。这些启动子的代表性例子有：大肠杆菌的lac或trp启动子；λ噬菌体PL启动子；真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTR和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。在一个实施方式中，表达载体可采用市售的pRSETb载体，无其他特殊要求。示例性地，采用BamHI和EcoRI分别对编码所述光学探针的核苷酸序列和表达载体进行双酶切，然后将二者的酶切产物连接得到重组表达载体。本发明对酶切和连接的具体步骤和参数没有特殊限定，采用本领域常规的步骤和参数即可。As used herein, the term "expression control sequence" refers to an element that can be operatively linked to the target gene that regulates the transcription, translation, and expression of a target gene. It can be an origin of replication, a promoter, a marker gene, or a translation control element, including enhancers and operons. , Terminator, ribosome binding site, etc. The choice of expression control sequence depends on the host cell used. In a recombinant expression vector, "operably linked" means that the target nucleotide sequence and the regulatory sequence are linked in a manner that allows the expression of the nucleotide sequence. Those skilled in the art are familiar with methods that can be used to construct an expression vector containing the fusion protein coding sequence of the present invention and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters are: Escherichia coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, anti The LTR of the transcription virus and some other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. In one embodiment, the expression vector can be a commercially available pRSETb vector without other special requirements. Exemplarily, the nucleotide sequence encoding the optical probe and the expression vector are double digested with BamHI and EcoRI respectively, and then the digested products of the two are connected to obtain a recombinant expression vector. In the present invention, the specific steps and parameters of restriction digestion and ligation are not particularly limited, and conventional steps and parameters in this field can be used.
在获得重组表达载体后，将该载体转化到宿主细胞中，以产生包括融合蛋白的蛋白或肽。此种转移过程可用转化或转染等本领域技术人员熟知的常规技术进行。本发明所述的宿主细胞是指能够接收和容纳重组DNA分子的细胞，是重组基因扩增的场所，理想的受体细胞应该满足易于获取和增殖两个条件。本发明的“宿主细胞”可包括原核细胞和真核细胞，具体包括细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞。所述宿主细胞优选各种利于基因产物表达或发酵生产的细胞，此类细胞已为本领域熟知并常用。本领域一般技术人员都清楚如何选择适当的载体、启动子、增强子和宿主细胞。After obtaining the recombinant expression vector, the vector is transformed into a host cell to produce a protein or peptide including a fusion protein. Such transfer process can be carried out by conventional techniques known to those skilled in the art, such as transformation or transfection. The host cell in the present invention refers to a cell capable of receiving and accommodating recombinant DNA molecules, and is a place for recombinant gene amplification. The ideal recipient cell should meet the two conditions of easy acquisition and proliferation. The "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells and mammalian cells. The host cell is preferably a variety of cells that are conducive to gene product expression or fermentation production, and such cells are well-known and commonly used in the art. Those of ordinary skill in the art know how to select appropriate vectors, promoters, enhancers and host cells.
本发明所述的转移到宿主细胞的方法为本领域常规的方法，包括磷酸钙或氯化钙共沉淀、DEAE-甘露聚糖-介导的转染、脂转染、天然感受态、化学介导的转移或电穿孔。当宿主为原核生物如大肠杆菌时，所述方法优选的为CaCl 2法或MgCl 2法处理，所用的步骤为本领域公知。当宿主细胞是真核细胞时，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。 The method of transferring to host cells according to the present invention is a conventional method in the art, including calcium phosphate or calcium chloride co-precipitation, DEAE-mannan-mediated transfection, lipofection, natural competence, chemical mediation Guided transfer or electroporation. When the host is a prokaryotic organism such as Escherichia coli, the method is preferably the CaCl 2 method or the MgCl 2 method, and the steps used are well-known in the art. When the host cell is a eukaryotic cell, the following DNA transfection methods can be selected: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
本发明在将表达载体转入宿主细胞后，对转入表达载体的宿主细胞进行扩增表达培养，分离得到丙酮酸光学探针。所述宿主细胞扩增表达培养采用常规的方法即可。 根据所用的宿主细胞种类，培养中所用的培养基可以是各种常规培养基。在适于宿主细胞生长的条件下进行培养。In the present invention, after the expression vector is transferred into the host cell, the host cell transferred into the expression vector is subjected to amplification and expression culture, and the pyruvate optical probe is isolated. Conventional methods may be used for the amplification and expression culture of the host cell. Depending on the type of host cell used, the medium used in the culture may be various conventional mediums. The culture is carried out under conditions suitable for the growth of the host cell.
在本发明中，光学探针在细胞内、细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离或纯化重组的蛋白。本发明对分离所述丙酮酸荧光蛋白的方法没有特殊限定，采用本领域常规的融合蛋白的分离方法即可。这些方法是本领域技术人员所熟知的，包括但并不限于：常规的复性处理、盐析方法、离心、渗透破菌、超声处理、超离心、分子筛层析、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。在一个实施方式中，利用His标签的亲和层析法进行光学探针的分离。In the present invention, the optical probe is expressed in the cell, on the cell membrane, or secreted out of the cell. If necessary, the recombinant protein can be separated or purified by various separation methods using its physical, chemical and other characteristics. The present invention does not specifically limit the method for separating the pyruvate fluorescent protein, as long as the conventional method for separating the fusion protein in the art can be used. These methods are well known to those skilled in the art, including but not limited to: conventional renaturation treatment, salting-out methods, centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange layer Analysis, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. In one embodiment, the separation of optical probes is performed using affinity chromatography of His tag.
本发明还提供了所述丙酮酸光学探针在丙酮酸实时定位、定量检测以及高通量化合物筛选中的应用。在一个方面，所述的丙酮酸光学探针优选与细胞不同部位的信号肽连接，转入到细胞中，通过检测细胞中荧光信号的强弱，进行丙酮酸的实时定位；通过丙酮酸标准滴加曲线进行相应丙酮酸的定量检测。本发明所述的丙酮酸标准滴加曲线是根据丙酮酸光学探针在不同浓度丙酮酸的情况下的荧光信号绘制而成。本发明所述丙酮酸光学探针直接转入细胞中，在丙酮酸实时定位和定量检测过程中，不需要耗时的样品处理过程，更加准确。本发明丙酮酸光学探针在进行高通量化合物筛选时，将不同的化合物添加到细胞培养液中，测定丙酮酸含量的变化，从而筛选出对丙酮酸含量变化有影响的化合物。在本发明中所述的丙酮酸光学探针在丙酮酸实时定位、定量检测以及高通量化合物筛选中的应用，均是非诊断和治疗目的，不涉及疾病的诊断和治疗。The invention also provides the application of the pyruvate optical probe in the real-time positioning, quantitative detection and high-throughput compound screening of pyruvate. In one aspect, the pyruvate optical probe is preferably connected to signal peptides in different parts of the cell and transferred to the cell. The real-time localization of pyruvate is performed by detecting the strength of the fluorescent signal in the cell; Add the curve for the quantitative detection of the corresponding pyruvate. The standard dropping curve of pyruvate according to the present invention is drawn according to the fluorescence signal of the pyruvate optical probe under different concentrations of pyruvate. The pyruvate optical probe of the present invention is directly transferred into cells, and in the process of real-time positioning and quantitative detection of pyruvate, the time-consuming sample processing process is not required, which is more accurate. When the pyruvate optical probe of the present invention performs high-throughput compound screening, different compounds are added to the cell culture solution to measure the change in the content of pyruvate, thereby screening compounds that have an effect on the change in the content of pyruvate. The application of the pyruvate optical probe described in the present invention in the real-time positioning, quantitative detection and high-throughput compound screening of pyruvate is for non-diagnostic and therapeutic purposes, and does not involve the diagnosis and treatment of diseases.
在本文中，浓度、含量、百分数和其它数值均可用范围的形式表示。也应理解，使用这种范围形式只是为了方便和简洁，应该被弹性地解读为包括范围上下限所明确提及的数值，还应包括该范围内包括的所有单个数值或子范围。In this article, concentration, content, percentage and other values can be expressed in the form of ranges. It should also be understood that the use of this range format is only for convenience and brevity, and should be flexibly interpreted as including the values explicitly mentioned in the upper and lower limits of the range, as well as all individual values or sub-ranges included in the range.
实施例Example
下面结合实施例对本发明提供的丙酮酸光学探针进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。The pyruvate optical probes provided by the present invention will be described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present invention.
I.实验材料和试剂I. Experimental materials and reagents
实施例中主要采用常规的基因工程分子生物学克隆方法和细胞培养以及成像方法等，这些方法是本领域普通技术人员所熟知的，例如：简·罗斯凯姆斯等的《分子生物学实验参考手册》，J.萨姆布鲁克，D.W.拉塞尔著，黄培堂等译：《分子克隆实验指南》(第三版，2002年8月，科学出版社出版，北京)；费雷谢尼等的《动物 细胞培养：基本技术指南》(第五版)，章静波，徐存拴等译；J.S.博尼费斯农，M.达索等的《精编细胞生物学实验指南》，章静波等译。The examples mainly adopt conventional genetic engineering molecular biology cloning methods, cell culture and imaging methods, etc. These methods are well known to those of ordinary skill in the art, for example: "Molecular Biology Experiment Reference" by Jane Roskemes Handbook, written by J. Sambrook, DW Russell, translated by Huang Peitang et al.: "Molecular Cloning Experiment Guide" (third edition, August 2002, published by Science Press, Beijing); "Frescheni et al." Animal Cell Culture: Basic Techniques Guide (Fifth Edition), translated by Zhang Jingbo, Xu Cunshuan, etc.; JS Boni Fesnon, M. Dassault et al., "Fine Compiling Cell Biology Experiment Guide", translated by Zhang Jingbo, etc.
实施例中所用的基于pRSETb-cpYFP，pRSETb-丙酮酸结合蛋白质粒由华东理工大学蛋白质实验室构建，pRSETb质粒载体购自Invitrogen公司。所有用于PCR的引物均由上海捷瑞生物工程技术有限公司合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定，序列测定由华大基因公司和杰李测序公司完成。各实施例所用的Taq DNA聚合酶购自东盛生物，pfu DNA聚合酶购自天根生化科技(北京)有限公司，primeSTAR DNA聚合酶购自TaKaRa公司，三种聚合酶购买时都附带赠送对应聚合酶缓冲液和dNTP。BamHI、BglII、HindIII、NdeI、XhoI、EcoRI、SpeI等限制性内切酶、T4连接酶、T4磷酸化酶(T4 PNK)购自Fermentas公司，购买时附带有相对应的缓冲液等。转染试剂Lip2000Kit购于Invitrogen公司。丙酮酸等氨基酸均购自Sigma公司。除非特别声明，无机盐类等化学试剂均购自Sigma-Aldrich公司。HEPES盐，氨苄青霉素(Amp)和嘌呤霉素购自Ameresco公司。96孔检测黑板、384孔荧光检测黑板购自Grenier公司。Based on pRSETb-cpYFP, the pRSETb-pyruvate binding protein particles used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pRSETb plasmid vector was purchased from Invitrogen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Jierui Bioengineering Technology Co., Ltd. The expression plasmids constructed in the examples have all undergone sequence determination, which was completed by Huada Gene Company and Jie Li Sequencing Company. The Taq DNA polymerase used in each example was purchased from Dongsheng Bio, pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., primeSTAR DNA polymerase was purchased from TaKaRa Company, and the three polymerases were purchased with a corresponding gift Polymerase buffer and dNTP. Restriction enzymes such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and the corresponding buffer was included with the purchase. The transfection reagent Lip2000Kit was purchased from Invitrogen. Amino acids such as pyruvate were purchased from Sigma. Unless otherwise stated, chemical reagents such as inorganic salts are purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp) and puromycin were purchased from Ameresco. 96-well detection blackboard and 384-well fluorescence detection blackboard were purchased from Grenier Company.
实施例中所用的DNA纯化试剂盒购自BBI公司(加拿大)，普通质粒小抽试剂盒购自天根生化科技(北京)有限公司。克隆菌株Mach1购自Invitrogen公司。镍柱亲和层析柱和脱盐柱填料均来自GE healthcare公司。The DNA purification kit used in the examples was purchased from BBI Company (Canada), and the common plasmid mini-purge kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The cloned strain Mach1 was purchased from Invitrogen. Both the nickel column affinity chromatography column and the desalting column packing materials are from GE Healthcare.
实施例中用到的主要仪器包括：Biotek Synergy 2多功能酶标仪(美国Bio-Tek公司)，X-15R高速冷冻离心机(美国Beckman公司)，Microfuge22R台式高速冷冻离心机(美国Beckman公司)，PCR扩增仪(德国Biometra公司)，超声破碎仪(宁波新芝公司)，核酸电泳仪(申能博彩公司)，荧光分光光度计(美国Varian公司)，CO 2恒温细胞培养箱(SANYO)，倒置荧光显微镜(日本尼康公司)。 The main instruments used in the examples include: Biotek Synergy 2 multifunctional microplate reader (Bio-Tek, USA), X-15R high-speed refrigerated centrifuge (Beckman, USA), and Microfuge 22R desktop high-speed refrigerated centrifuge (Beckman, USA) , PCR amplification instrument (Germany Biometra company), ultrasonic disruptor (Ningbo Xinzhi company), nucleic acid electrophoresis system (Sheneng Group), fluorescence spectrophotometer (U.S. Varian company), CO 2 constant temperature cell incubator (SANYO) , Inverted fluorescence microscope (Nikon, Japan).
II.分子生物学方法和细胞实验方法II. Molecular biology methods and cell experiment methods
II.1聚合酶链式反应(PCR)：II.1 Polymerase chain reaction (PCR):
1.目的片段扩增PCR：1. Target fragment amplification PCR:
该方法主要用于基因片段扩增和菌落PCR鉴定阳性克隆。所述PCR扩增的反应体系如下：模板序列0.5-1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，10×pfu缓冲液5μL，pfu DNA聚合酶0.5μL，dNTP(10mM)1μL，灭菌超纯水(ddH2O)41.5-42μL，总体积50μL。PCR扩增程序如下：95℃变性2-10分钟，30轮循环(94-96℃持续30-45秒，50-65℃持续30-45秒，72℃持续一定时间(600bp/min))，72℃延伸10分钟。This method is mainly used for gene fragment amplification and colony PCR to identify positive clones. The PCR amplification reaction system is as follows: template sequence 0.5-1μL, forward primer (25μM) 0.5μL, reverse primer (25μM) 0.5μL, 10×pfu buffer 5μL, pfu DNA polymerase 0.5μL, dNTP( 10mM) 1μL, sterile ultrapure water (ddH2O) 41.5-42μL, total volume 50μL. The PCR amplification procedure is as follows: denaturation at 95°C for 2-10 minutes, 30 cycles (94-96°C for 30-45 seconds, 50-65°C for 30-45 seconds, 72°C for a certain time (600bp/min)), Extend at 72°C for 10 minutes.
2.长片段(＞2500bp)扩增PCR：2. Long fragment (>2500bp) amplification PCR:
本发明中使用的长片段扩增，主要是反向PCR扩增载体，在下述实施例中用于获得定点突变的一种技术。在变异部位设计反向PCR引物，其中一条引物的5’端包含变异的核苷酸序列。扩增后的产物就含有相应的突变位点。长片段扩增PCR反应体系如下：模板序列(10pg-1ng)1μL，正向引物(25μM)0.5μL，反向引物(25μM)0.5μL，5×PrimerSTAR缓冲液10μL，PrimerSTAR DNA聚合酶0.5μL，dNTP(2.5mM)4μL，灭菌超纯水(ddH2O)33.5μL，总体积50μL。PCR扩增程序如下：95℃变性5分钟，30轮循环(98℃持续10秒，50-68℃持续5-15秒，72℃持续一定时间(1000bp/min))，72℃延伸10分钟；或者95℃变性5分钟，30轮循环(98℃持续10秒，68℃持续一定时间(1000bp/min))，72℃延伸10分钟。The long fragment amplification used in the present invention is mainly an inverse PCR amplification vector, which is a technique for obtaining site-directed mutation in the following examples. Design the reverse PCR primers at the mutation site, and one of the primers contains the mutation nucleotide sequence at the 5'end. The amplified product contains the corresponding mutation site. The PCR reaction system for long-range amplification is as follows: template sequence (10pg-1ng) 1μL, forward primer (25μM) 0.5μL, reverse primer (25μM) 0.5μL, 5×PrimerSTAR buffer 10μL, PrimerSTAR DNA polymerase 0.5μL, dNTP (2.5mM) 4μL, sterilized ultrapure water (ddH2O) 33.5μL, total volume 50μL. The PCR amplification procedure is as follows: denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 50-68°C for 5-15 seconds, 72°C for a certain time (1000bp/min)), 72°C for 10 minutes; Or denaturation at 95°C for 5 minutes, 30 cycles (98°C for 10 seconds, 68°C for a certain time (1000bp/min)), 72°C for 10 minutes extension.
II.2核酸内切酶酶切反应：II.2 Endonuclease digestion reaction:
对质粒载体进行双酶切的体系如下：质粒载体20μL(约1.5μg)，10×缓冲液5μL，限制性内切酶11-2μL，限制性内切酶21-2μL，用灭菌超纯水补至总体积50μL。反应条件37℃，1-7小时。The system for double digestion of plasmid vector is as follows: plasmid vector 20μL (about 1.5μg), 10× buffer 5μL, restriction endonuclease 11-2μL, restriction endonuclease 21-2μL, sterilized ultrapure water Make up to a total volume of 50μL. The reaction conditions were 37°C, 1-7 hours.
II.3 DNA片段5’端磷酸化反应II.3 Phosphorylation reaction at the 5'end of DNA fragments
从微生物中抽提出的质粒或者基因组末端都含有磷酸基团，而PCR产物没有，故需对PCR产物的5’端碱基进行磷酸基团加成反应，只有末端含有磷酸基团DNA分子才能发生连接反应。磷酸化反应体系如下：PCR产物片段DNA序列5-8μL，10×T4连接酶缓冲液1μL，T4多聚核苷酸激酶(T4 PNK)1μL，灭菌超纯水0-3μL，总体积10μL。反应条件37℃，30分钟-2小时后72℃灭活20分钟。Plasmids extracted from microorganisms or genome ends contain phosphate groups, but PCR products do not, so it is necessary to perform phosphate group addition reaction on the 5'end bases of PCR products. Only DNA molecules with phosphate groups at the end can occur. Connection reaction. The phosphorylation reaction system is as follows: PCR product fragment DNA sequence 5-8μL, 10×T4 ligase buffer 1μL, T4 polynucleotide kinase (T4 PNK) 1μL, sterilized ultrapure water 0-3μL, total volume 10μL. The reaction conditions were 37°C, and inactivation at 72°C for 20 minutes after 30 minutes to 2 hours.
II.4目的片段和载体的连接反应II.4 The ligation reaction between the target fragment and the vector
不同的片段和载体之间的连接方法有所差异，本发明中使用了三种连接方法The connection methods between different fragments and vectors are different. Three connection methods are used in the present invention.
1.平末端短片段和线性化载体的平末端连接1. The blunt-end short fragment and the blunt-end connection of the linearized vector
该方法的原理是PCR获得的平末端产物在T4 PNK作用下对DNA片段的5’末端进行磷酸化反应后，与线性化的载体在PEG4000和T4 DNA连接酶的作用下连接获得重组质粒。同源重组连接体系如下：T4 PNK处理的DNA片段4μL，线性化载体片段4μL，PEG4000 1μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，总计10μL。反应条件22℃，30分钟。The principle of this method is that the blunt-end product obtained by PCR undergoes phosphorylation reaction on the 5'end of the DNA fragment under the action of T4 PNK, and is ligated with the linearized vector under the action of PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: T4 PNK treated DNA fragment 4μL, linearized vector fragment 4μL, PEG4000 1μL, 10×T4 ligase buffer 1μL, T4 DNA ligase 1μL, total 10μL. The reaction conditions were 22°C, 30 minutes.
2.含有粘性末端的DNA片段和含有粘性末端载体片段的连接2. Connection of DNA fragments with sticky ends and vector fragments with sticky ends
通过限制性内切酶切割的DNA片段通常会产生突出的粘性末端，因此可以和含有序列互补的粘性末端载体片段连接，形成重组质粒。连接反应体系如下：酶切后的PCR产物片段DNA 1-7μL，酶切后的质粒0.5-7μL，10×T4连接酶缓冲液1μL，T4 DNA连接酶1μL，灭菌超纯水补至总体积10μL。反应条件16℃，4-8小时。 其中PCR产物片段与载体双酶切产物的质量比大致在2∶1-6∶1之间。DNA fragments cleaved by restriction endonucleases usually produce protruding sticky ends, so they can be ligated with vector fragments containing complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7μL of digested PCR product fragment DNA, 0.5-7μL of digested plasmid, 1μL of 10×T4 ligase buffer, 1μL of T4 DNA ligase, sterilized ultrapure water to make up to the total volume 10μL. The reaction conditions were 16°C, 4-8 hours. The mass ratio of PCR product fragments and vector double enzyme digestion products is roughly between 2:1-6:1.
3.反向PCR引入定点突变后5’端磷酸化的DNA片段产物自身环化的连接反应3. The ligation reaction in which the 5'end phosphorylated DNA fragment product self-circularizes after the introduction of site-directed mutation by inverse PCR
将5’端磷酸化的DNA片段通过自身环化连接反应将线性化载体的3’端和5’端连接反应得到重组质粒。自身环化连接反应体系如下：磷酸化反应体系10μL，T4连接酶(5U/μL)0.5μL，总体积10.5μL。反应条件16℃，4-16小时。The 5'end phosphorylated DNA fragment is ligated with the 3'end and 5'end of the linearized vector through self-circularization ligation reaction to obtain a recombinant plasmid. The self-cyclization ligation reaction system is as follows: phosphorylation reaction system 10μL, T4 ligase (5U/μL) 0.5μL, total volume 10.5μL. The reaction conditions were 16°C, 4-16 hours.
II.5感受态细胞的制备与转化II.5 Preparation and transformation of competent cells
感受态细胞的制备：Preparation of competent cells:
1.挑取单菌落(如Mach1)接种于5mL LB培养基中，37℃摇床过夜。2.取0.5-1mL过夜培养的菌液转种到50mL LB培养基中，37℃，220rpm培养3至5小时，直到OD600达到0.5。3.冰浴预冷细胞2小时。4. 4℃，4000rpm离心10分钟。5.弃上清，用5mL预冷的缓冲液重悬细胞，待均匀后再加入重悬缓冲液至终体积为50mL。6.冰浴45分钟。7. 4℃4000rpm离心10分钟，用5mL冰预冷的储存缓冲液重悬细菌。8.每个EP管中放100μL菌液，-80℃或液氮冻存。1. Pick a single colony (such as Mach1) and inoculate it in 5mL LB medium, shake it overnight at 37°C. 2. Transfer 0.5-1 mL of the overnight cultured bacterial solution to 50 mL LB medium, and incubate at 37°C and 220 rpm for 3 to 5 hours until the OD600 reaches 0.5. 3. Pre-cool the cells in an ice bath for 2 hours. 4. Centrifuge at 4000 rpm for 10 minutes at 4°C. 5. Discard the supernatant, resuspend the cells with 5 mL of pre-cooled buffer, and add resuspension buffer to a final volume of 50 mL after uniformity. 6. Ice bath for 45 minutes. 7. Centrifuge at 4000 rpm at 4°C for 10 minutes, and resuspend the bacteria in 5 mL ice-cold storage buffer. 8. Put 100μL of bacterial solution in each EP tube, freeze at -80℃ or liquid nitrogen.
其中，重悬缓冲液：CaCl 2(100mM)、MgCl 2(70mM)、NaAc(40mM)。储存缓冲液：0.5mL DMSO、1.9mL 80％甘油、1mL 10×CaCl 2(1M)、1mL 10×MgCl 2(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH 2O。 Among them, the resuspension buffer: CaCl 2 (100 mM), MgCl 2 (70 mM), NaAc (40 mM). Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl 2 (1M), 1 mL 10×MgCl 2 (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH 2 O.
感受态细胞的转化：Transformation of competent cells:
1.取100μL感受态细胞于冰浴上融化。2.加入适当体积的连接产物，轻轻吹打混匀，冰浴30分钟。通常加入的连接产物的体积少于感受态细胞体积的1/10。3.将菌液放入42℃水浴中热激90秒，迅速转移至冰浴中放置5分钟。4.加入500μL LB，于37℃恒温摇床上200rpm培养1小时。5.将菌液4000rpm离心3分钟，留200μL上清将菌体吹匀，均匀涂布于含适当抗生素的琼脂平板表面，平板于37℃恒温培养箱内倒置过夜。1. Take 100μL of competent cells to melt on an ice bath. 2. Add appropriate volume of ligation product, gently pipette to mix, and ice bath for 30 minutes. Usually the volume of the ligation product added is less than 1/10 of the volume of the competent cells. 3. Put the bacterial solution in a 42°C water bath for 90 seconds, heat shock, and quickly transfer to an ice bath for 5 minutes. 4. Add 500 μL of LB, and incubate for 1 hour at 200 rpm on a 37°C constant temperature shaker. 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, leave 200 μL of supernatant to blow evenly, and evenly spread on the surface of an agar plate containing appropriate antibiotics, and invert the plate overnight in a constant temperature incubator at 37°C.
II.6蛋白质的表达，纯化和荧光检测II.6 Protein expression, purification and fluorescence detection
1.将表达载体(例如以pRSETb为基础的丙酮酸光学探针表达载体)转化到JM109(DE3)细胞中，倒置培养过夜，从平板上挑取克隆到250ml锥形瓶中，置于37℃摇床，220rpm培养至OD＝0.4-0.8，加入1/1000(v/v)的IPTG(1M)，18℃诱导表达24-36小时。1. Transform the expression vector (such as pRSETb-based pyruvate optical probe expression vector) into JM109(DE3) cells, invert the culture overnight, pick the clones from the plate into a 250ml Erlenmeyer flask, and place them at 37°C Incubate on a shaker at 220 rpm to OD = 0.4-0.8, add 1/1000 (v/v) IPTG (1M), and induce expression at 18°C for 24-36 hours.
2.诱导表达完成后，4000rpm，30分钟离心收菌，加入50mM的磷酸盐缓冲液重悬菌体沉淀，超声破碎至菌体澄清。9600rpm，4℃离心20分钟。2. After the induction of expression is completed, the bacteria are harvested by centrifugation at 4000 rpm for 30 minutes, and 50 mM phosphate buffer is added to resuspend the bacterial pellet and ultrasonically broken until the bacterial cells are clear. Centrifuge at 9600 rpm for 20 minutes at 4°C.
3.离心上清通过自装的镍柱亲和层析柱纯化获得蛋白，镍柱亲和层析后的蛋白再通过自装的脱盐柱获得溶解在20mM MOPS缓冲液(pH 7.4)或者磷酸盐缓冲液PBS 中的蛋白。3. The centrifugal supernatant is purified by a self-packed nickel column affinity chromatography column to obtain protein, and the protein after nickel column affinity chromatography is then passed through a self-packed desalting column to be dissolved in 20mM MOPS buffer (pH 7.4) or phosphate Protein in buffer PBS.
4.纯化的蛋白经过SDS-PAGE鉴定后，使用测定缓冲液(100mM HEPES，100mM NaCl，pH 7.3)或者磷酸盐缓冲液PBS稀释探针成终浓度为5-10μM的蛋白溶液。用测定缓冲液(20mM MOPS，pH 7.4)或者磷酸盐缓冲液PBS将丙酮酸配制成终浓度为1M的储液。4. After the purified protein is identified by SDS-PAGE, use assay buffer (100mM HEPES, 100mM NaCl, pH 7.3) or phosphate buffer PBS to dilute the probe to a protein solution with a final concentration of 5-10μM. Use assay buffer (20mM MOPS, pH 7.4) or phosphate buffered saline PBS to prepare pyruvate into a stock solution with a final concentration of 1M.
5.取100μl 5μM的蛋白溶液，37℃温育5分钟，分别加入丙酮酸混匀后至终浓度为100mM，利用多功能荧光酶标仪测定蛋白在340nm下的光吸收。5. Take 100μl of 5μM protein solution, incubate at 37°C for 5 minutes, add pyruvate and mix until the final concentration is 100mM, use a multifunctional fluorescent microplate reader to measure the light absorption of the protein at 340nm.
6.取100μl 1μM的蛋白溶液，37℃温育5分钟，加入丙酮酸滴定，测定蛋白的485nm荧光激发后528nm发射的荧光强度。对样品的荧光激发、发射测定利用多功能荧光酶标仪完成。6. Take 100μl of 1μM protein solution, incubate at 37°C for 5 minutes, add pyruvate to titrate, and measure the fluorescence intensity emitted by the protein at 528nm after 485nm fluorescence excitation. The fluorescence excitation and emission measurement of the sample are completed by a multifunctional fluorescence microplate reader.
7.取100μl 1μM的蛋白溶液，37℃温育5分钟，加入丙酮酸，测定蛋白的吸收光谱和荧光光谱。对样品的吸收光谱和荧光光谱的测定通过分光光度计和荧光分光光度计完成。7. Take 100μl of 1μM protein solution, incubate at 37°C for 5 minutes, add pyruvate, and measure the absorption spectrum and fluorescence spectrum of the protein. The measurement of the absorption spectrum and fluorescence spectrum of the sample is completed by a spectrophotometer and a fluorescence spectrophotometer.
II.7哺乳动物细胞的转染和荧光检测II.7 Transfection and fluorescence detection of mammalian cells
1.将pCDNA3.1+为基础的丙酮酸光学探针质粒通过转染试剂Lipofectamine2000(Invitrogen)转染到HeLa中，置于37℃，5％CO 2的细胞培养箱中培养。待外源基因充分表达24～36h后进行荧光检测。 1. Transfect the pCDNA3.1+-based pyruvate optical probe plasmid into HeLa with the transfection reagent Lipofectamine2000 (Invitrogen), and place it in a 37°C, 5% CO 2 cell incubator. Fluorescence detection was performed after the foreign gene was fully expressed for 24 to 36 hours.
2.诱导表达完成后，将贴壁的HeLa细胞，用PBS冲洗三次，置于HBSS溶液中分别进行荧光显微镜和酶标仪检测。2. After the induction of expression is complete, wash the adherent HeLa cells with PBS three times, and place them in the HBSS solution for fluorescence microscopy and microplate reader detection.
实施例1：丙酮酸结合蛋白质粒Example 1: Pyruvate binding protein pellets
通过PCR扩增大肠杆菌基因中的PdhR(96-254)基因，PCR产物凝胶电泳后回收后用BamHI和HindIII酶切，同时对pCDFDuet1载体进行相应的双酶切。用T4 DNA连接酶连接后，将产物转化MachI，转化的MachI涂布于LB平板(硫酸链霉素50ug/mL)，置于37℃培养过夜。将生长MachI转化子进行质粒抽提后，进行PCR鉴定。阳性质粒经过测序正确后进行后续的质粒构建。The PdhR (96-254) gene in the E. coli gene was amplified by PCR, and the PCR product was recovered after gel electrophoresis and digested with BamHI and HindIII. At the same time, the pCDFDuet1 vector was subjected to corresponding double digestion. After ligation with T4 DNA ligase, the product was transformed into MachI, and the transformed MachI was spread on an LB plate (streptomycin sulfate 50ug/mL) and incubated at 37°C overnight. After the growing MachI transformants were subjected to plasmid extraction, PCR identification was performed. After the positive plasmid is sequenced correctly, the subsequent plasmid construction is carried out.
实施例2：不同插入位点的cpYFP光学探针的表达和检测Example 2: Expression and detection of cpYFP optical probes at different insertion sites
本实施例中，以pCDFDuet-PdhR(96-254)为基础根据丙酮酸结合蛋白晶体结构选择了下述位点插入cpYFP，得到相应pCDFDuet-PdhR(96-254)-cpYFP质粒：117/118，117/119，117/120，117/121，118/119，118/120，118/121，119/120，119/121，120/121，140/141，140/142，140/143，141/142，141/143，142/143，160/161，160/162，160/163，160/164，161/162，161/163，161/164，162/163，162/164，163/164，174/175，174/176，175/176，191/192，191/193，191/194，191/195，192/193，192/194，192/195， 193/194，193/195，194/195，210/211，210/212，210/213，210/214，211/212，211/213，211/214，212/213，212/214或213/214。示例性的序列如表1所示。In this example, based on pCDFDuet-PdhR(96-254), the following sites were selected to insert cpYFP according to the pyruvate-binding protein crystal structure to obtain the corresponding pCDFDuet-PdhR(96-254)-cpYFP plasmid: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120, 119/121, 120/121, 140/141, 140/142, 140/143, 141/ 142, 141/143, 142/143, 160/161, 160/162, 160/163, 160/164, 161/162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191/192, 191/193, 191/194, 191/195, 192/193, 192/194, 192/195, 193/194, 193/195, 194/ 195, 210/211, 210/212, 210/213, 210/214, 211/212, 211/213, 211/214, 212/213, 212/214 or 213/214. Exemplary sequences are shown in Table 1.
表1，光学探针的序列Table 1. The sequence of the optical probe
序列sequence 插入位点Insertion site
SEQ ID NO：6SEQ ID NO: 6 117/121117/121
SEQ ID NO：7SEQ ID NO: 7 141/143141/143
SEQ ID NO：8SEQ ID NO: 8 191/192191/192
SEQ ID NO：9SEQ ID NO: 9 191/193191/193
SEQ ID NO：10SEQ ID NO: 10 191/194191/194
SEQ ID NO：11SEQ ID NO: 11 191/195191/195
SEQ ID NO：12SEQ ID NO: 12 192/193192/193
SEQ ID NO：13SEQ ID NO: 13 192/194192/194
SEQ ID NO：14SEQ ID NO: 14 192/195192/195
SEQ ID NO：15SEQ ID NO: 15 193/194193/194
SEQ ID NO：16SEQ ID NO: 16 193/195193/195
SEQ ID NO：17SEQ ID NO: 17 194/195194/195
SEQ ID NO：18SEQ ID NO: 18 210/214210/214
利用PCR产生cpYFP的DNA片段，同时通过反向PCR扩增产生含有不同断裂位点的pCDFDuet-PdhR(96-254)线性化载体，将线性化的pCDFDuet-PdhR(96-254)和cpYFP片段在同源重组酶的作用下连接产生重组质粒，通过菌落PCR，选取阳性克隆，由上海杰李生物技术有限公司完成测序。The DNA fragment of cpYFP was generated by PCR, and the pCDFDuet-PdhR (96-254) linearized vector containing different break sites was generated by inverse PCR. The linearized pCDFDuet-PdhR (96-254) and cpYFP fragments were Under the action of homologous recombinase, the recombinant plasmids were ligated to produce recombinant plasmids, and positive clones were selected by colony PCR, and the sequencing was completed by Shanghai Jie Li Biotechnology Co., Ltd.
经过测序正确后，将重组质粒转化到JM109(DE3)中诱导表达，并纯化蛋白质，通过SDS-PAGE电泳大小在47.5Kda附近。该大小符合pCDFDuet-PdhR(96-254)-cpYFP表达出的含His-tag纯化标签的PdhR(96-254)-cpYFP融合蛋白质的大小。结果如图1所示。After the correct sequencing, the recombinant plasmid was transformed into JM109 (DE3) to induce expression, and the protein was purified. The size was around 47.5Kda by SDS-PAGE electrophoresis. This size is consistent with the size of the PdhR(96-254)-cpYFP fusion protein expressed by pCDFDuet-PdhR(96-254)-cpYFP containing the His-tag purification tag. The result is shown in Figure 1.
将纯化的PdhR(96-254)-cpYFP融合蛋白质进行丙酮酸响应筛选，将含有100mM丙酮酸的融合荧光蛋白质的检测信号除以无丙酮酸的融合荧光蛋白质的检测信号。结果如图2所示，检测结果显示对丙酮酸响应超过2倍的光学探针有在117/121，141/143，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195和210/214位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。The purified PdhR(96-254)-cpYFP fusion protein was subjected to pyruvate response screening, and the detection signal of the fusion fluorescent protein containing 100 mM pyruvate was divided by the detection signal of the fusion fluorescent protein without pyruvate. The results are shown in Figure 2. The test results show that the optical probes with a response of more than 2 times to pyruvate are 117/121, 141/143, 191/192, 191/193, 191/194, 191/195, 192/193 , 192/194, 192/195, 193/194, 193/195, 194/195 and 210/214 positions or the corresponding amino acid positions of their family proteins to implement the insertion of optical probes.
实施例3：不同插入位点的cpGFP光学探针的表达和检测Example 3: Expression and detection of cpGFP optical probes at different insertion sites
按照实施例2中的方法将cpYFP替换为cpGFP，构建丙酮酸绿色荧光蛋白荧光探针。如图3所示，检测结果显示对丙酮酸响应超过2倍的光学探针有在191/193位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。According to the method in Example 2, cpYFP was replaced with cpGFP to construct a pyruvate green fluorescent protein fluorescent probe. As shown in Figure 3, the detection results show that the optical probes that have a response of more than 2 times to pyruvate have optical probes inserted at positions 191/193 or the corresponding amino acid positions of their family proteins.
实施例4：不同插入位点的cpBFP光学探针的表达和检测Example 4: Expression and detection of cpBFP optical probes at different insertion sites
按照实施例2中的方法将cpYFP替换为cpBFP，构建丙酮酸蓝色荧光蛋白荧光探针。如图4所示，检测结果显示对丙酮酸响应超过2倍的光学探针有在193/194和194/195位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。According to the method in Example 2, cpYFP was replaced with cpBFP to construct a pyruvate blue fluorescent protein fluorescent probe. As shown in Figure 4, the detection results show that the optical probes that have a response of more than 2 times to pyruvate have optical probes inserted at positions 193/194 and 194/195 or the corresponding amino acid positions of their family proteins.
实施例5：不同插入位点的cpmApple光学探针的表达和检测Example 5: Expression and detection of cpmApple optical probes at different insertion sites
按照实施例2中的方法将cpYFP替换为cpmApple，构建丙酮酸红色荧光蛋白荧光探针。如图5所示，检测结果显示对丙酮酸响应超过2倍的光学探针有在191/195和192/193位点或者其家族蛋白的对应氨基酸位点实施插入的光学探针。According to the method in Example 2, cpYFP was replaced with cpmApple to construct a pyruvate red fluorescent protein fluorescent probe. As shown in Fig. 5, the detection results show that the optical probes that have a response of more than 2 times to pyruvate have optical probes inserted at positions 191/195 and 192/193 or the corresponding amino acid positions of their family proteins.
综合实施例2-5的结果，表明通过简单地更换荧光蛋白cpYFP、cpGFP、cpBFP或者cpmApple以获得不同颜色的对丙酮酸响应的探针是行不通的。Combining the results of Examples 2-5, it is shown that it is not feasible to obtain pyruvate-responsive probes of different colors by simply replacing the fluorescent proteins cpYFP, cpGFP, cpBFP or cpmApple.
实施例6：突变的cpYFP光学探针的表达和检测Example 6: Expression and detection of mutant cpYFP optical probe
在PdhR(96-254)-191/193-cpYFP、PdhR(96-254)-192/194-cpYFP和PdhR(96-254)-192/195-cpYFP的基础上构建光学探针突变体。通过反向PCR线性化质粒pCDFDuet-PdhR(96-254)-191/193-cpYFP引物中含有所要突变位点的碱基序列，对得到的PCR产物在PNK、T4 DNA连接酶和PEG4000的作用下加磷连接，得到Q138、R191、E193这3个位点的定点饱和突变质粒。采用相同的方法，我们得到pCDFDuet-PdhR(96-254)-192/194-cpYFP基础上M194位点的定点饱和突变质粒，以及pCDFDuet-PdhR(96-254)-192/195-cpYFP基础上的L195位的定点饱和突变质粒。并在PdhR(96-254)-192/194-M194V-cpYFP和PdhR(96-254)-141/143-cpYFP的基础上分别构建190、191、192三个位点的随机突变库。并由上海杰李生物技术有限公司完成测序。部分突变的光学探针的序列如表2所示。示例性的核酸序列如SEQ ID NO：31(192/194-M194V)所示。The optical probe mutants were constructed on the basis of PdhR(96-254)-191/193-cpYFP, PdhR(96-254)-192/194-cpYFP and PdhR(96-254)-192/195-cpYFP. The plasmid pCDFDuet-PdhR(96-254)-191/193-cpYFP was linearized by inverse PCR. The primer contained the base sequence of the desired mutation site, and the PCR product obtained was subjected to the action of PNK, T4 DNA ligase and PEG4000 Phosphorus ligation was added to obtain the site-directed saturation mutant plasmids of Q138, R191, and E193. Using the same method, we obtained pCDFDuet-PdhR(96-254)-192/194-cpYFP based on the site-directed saturation mutation plasmid at position M194, and pCDFDuet-PdhR(96-254)-192/195-cpYFP based on Site-directed saturation mutation plasmid at position L195. And on the basis of PdhR(96-254)-192/194-M194V-cpYFP and PdhR(96-254)-141/143-cpYFP, three random mutation libraries of 190, 191 and 192 were constructed respectively. The sequencing was completed by Shanghai Jie Li Biotechnology Co., Ltd. The sequence of the partially mutated optical probe is shown in Table 2. An exemplary nucleic acid sequence is shown in SEQ ID NO: 31 (192/194-M194V).
表2，突变的光学探针的序列Table 2. The sequence of the mutant optical probe
序列sequence 插入位点Insertion site 突变mutation
SEQ ID NO：19SEQ ID NO: 19 191/193191/193 Q138突变为A，N，D，G，H，L，K，M，P，S或TQ138 mutations to A, N, D, G, H, L, K, M, P, S or T
SEQ ID NO：20SEQ ID NO: 20 191/193191/193 R191突变为A，N，D，C，Q，E，G，H，I，L，K，M，F，P，S，T，W，Y或VMutation of R191 to A, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y or V
SEQ ID NO：21SEQ ID NO: 21 191/193191/193 E193突变为A，R，D，C，Q，I，L，K，M，F，P，S，T，Y或VE193 mutations to A, R, D, C, Q, I, L, K, M, F, P, S, T, Y or V
SEQ ID NO：22SEQ ID NO: 22 192/194192/194 M194突变为A，R，N，D，C，Q，E，G，L，K，F，P，S，T，W，Y或V)M194 mutation is A, R, N, D, C, Q, E, G, L, K, F, P, S, T, W, Y or V)
SEQ ID NO：23SEQ ID NO: 23 192/195192/195 L195突变为D，H，I或YL195 mutation to D, H, I or Y
SEQ ID NO：24SEQ ID NO: 24 192/194192/194 M194V/S190E/R191N/R192DM194V/S190E/R191N/R192D
SEQ ID NO：25SEQ ID NO: 25 192/194192/194 M194V/S190D/R191Y/R192TM194V/S190D/R191Y/R192T
SEQ ID NO：26SEQ ID NO: 26 141/143141/143 S190P/R191H/R192PS190P/R191H/R192P
SEQ ID NO：27SEQ ID NO: 27 141/143141/143 S190R/R191S/R192PS190R/R191S/R192P
SEQ ID NO：28SEQ ID NO: 28 141/143141/143 S190L/R191VS190L/R191V
SEQ ID NO：29SEQ ID NO: 29 141/143141/143 S190T/R191Q/R192ES190T/R191Q/R192E
SEQ ID NO：30SEQ ID NO: 30 141/143141/143 R191S/R192TR191S/R192T
结果如图6所示，荧光检测结果显示Q138，R191，E193，M194和L195这5个位点饱和突变后的多个突变体对丙酮酸响应增强，表明这5个位点对丙酮酸的结合非常重要，此外，S190、R191和R192三个位点的随机突变结果表明这三个位点的有些突变体增强了对丙酮酸的响应，因此这三个位点对丙酮酸结合也很重要。The results are shown in Figure 6. Fluorescence detection results show that multiple mutants after saturation mutations at the 5 sites of Q138, R191, E193, M194 and L195 have an enhanced response to pyruvate, indicating that these 5 sites bind to pyruvate It is very important. In addition, the random mutation results of S190, R191 and R192 indicate that some mutants of these three sites enhance the response to pyruvate, so these three sites are also important for pyruvate binding.
实施例7：丙酮酸光学探针的滴定曲线Example 7: Titration curve of pyruvate optical probe
对实施例2和实施例6中所得的部分丙酮酸光学探针，即141/143、191/193-E193Q、191/193-R191Y、191/193-R191F、191/193-R191L、192/194-M194V、191/193-Q138P、192/194-M194D、192/194-M194H、191/193-R191P、191/193-Q138L、192/194-M194W、191/193-E193L、192/194-M194V/S190E/R191N/R192D、192/195、141/143-S190P/R191H/R192P、141/143-S190R/R191S/R192P、141/143-S190L/R191V、141/143-S190T/R191Q/R192E、141/143-R191S/R192T、141/143-S190D/R191Y/R192T进行浓度梯度的丙酮酸检测，检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。141/143、191/193-E193Q、191/193-R191Y、191/193-R191F、191/193-R191L、192/194-M194V、191/193-Q138P、192/194-M194D、192/194-M194H、191/193-R191P、191/193-Q138L、192/194-M194W、191/193-E193L、192/194-M194V-S190E/R191N/R192D、192/195的K d(结合常数)分别为28μM、195μM、222μM、422μM、463μM、475μM、599μM、664μM、728μM、799μM、1253μM、5142μM、9255μM、1066μM、19944μM、84μM、158μM、185μM、186μM、164μM和134μM，变化幅度分别为2.0倍、4.1倍、4.3倍、5.3倍、5.0倍、9.7倍、6.4倍、5.3倍、6.1倍、6.5倍、5.1倍、7.2倍、3.4倍、13.1倍、8.3倍、3.2倍、3.1倍、4.2倍、2.7倍、3.9倍和6.4倍，结果如图7A-B所示。 For some of the pyruvate optical probes obtained in Example 2 and Example 6, namely 141/143, 191/193-E193Q, 191/193-R191Y, 191/193-R191F, 191/193-R191L, 192/194 -M194V, 191/193-Q138P, 192/194-M194D, 192/194-M194H, 191/193-R191P, 191/193-Q138L, 192/194-M194W, 191/193-E193L, 192/194-M194V /S190E/R191N/R192D, 192/195, 141/143-S190P/R191H/R192P, 141/143-S190R/R191S/R192P, 141/143-S190L/R191V, 141/143-S190T/R191Q/R192E, 141 /143-R191S/R192T, 141/143-S190D/R191Y/R192T carry out concentration gradient detection of pyruvate to detect the ratio of fluorescence intensity at 420nm excitation at 528nm emission and 485nm excitation at 528nm emission. 141/143, 191/193-E193Q, 191/193-R191Y, 191/193-R191F, 191/193-R191L, 192/194-M194V, 191/193-Q138P, 192/194-M194D, 192/194- The K d (binding constants) of M194H, 191/193-R191P, 191/193-Q138L, 192/194-M194W, 191/193-E193L, 192/194-M194V-S190E/R191N/R192D, and 192/195 are respectively 28μM, 195μM, 222μM, 422μM, 463μM, 475μM, 599μM, 664μM, 728μM, 799μM, 1253μM, 5142μM, 9255μM, 1066μM, 19944μM, 84μM, 158μM, 185μM, 186μM, 164μM and 134μM, respectively Times, 4.3 times, 5.3 times, 5.0 times, 9.7 times, 6.4 times, 5.3 times, 6.1 times, 6.5 times, 5.1 times, 7.2 times, 3.4 times, 13.1 times, 8.3 times, 3.2 times, 3.1 times, 4.2 times, 2.7 times, 3.9 times and 6.4 times, the results are shown in Figure 7A-B.
实施例8：丙酮酸光学探针的光谱性能和特异性Example 8: Spectral performance and specificity of pyruvate optical probe
示例性地，将纯化的丙酮酸光学探针PdhR(96-254)-192/194-M194V-cpYFP分别进行0mM和10mM丙酮酸处理10分钟后，使用荧光分光光度计进行荧光谱的检测。Illustratively, the purified pyruvate optical probe PdhR(96-254)-192/194-M194V-cpYFP is treated with 0 mM and 10 mM pyruvate for 10 minutes, respectively, and then the fluorescence spectrum is detected using a fluorescence spectrophotometer.
对激发光谱的测定：以350nm至500nm的激发范围和530nm的发射波长记录激发光谱，每5nm读取一次。结果显示，探针在约420和490nm处有两个激发峰，如图8A所示。Determination of the excitation spectrum: Record the excitation spectrum with an excitation range of 350nm to 500nm and an emission wavelength of 530nm, and read it every 5nm. The results show that the probe has two excitation peaks at about 420 and 490 nm, as shown in Figure 8A.
对发射光谱的测定：固定激发波长分别为420nm和490nm，记录505-600nm的发射光谱，每5nm读取一次。结果显示，探针在添加10mM丙酮酸后，在420nm激发下荧光强度降低为添加0mM丙酮酸的0.6倍；在490nm激发下荧光强度增强为添加0mM丙酮酸的5.8倍。如图8B和8C所示。Measurement of emission spectra: fixed excitation wavelengths of 420nm and 490nm, record emission spectra of 505-600nm, and read it every 5nm. The results showed that after the probe was added with 10 mM pyruvate, the fluorescence intensity under excitation at 420 nm decreased to 0.6 times that of 0 mM pyruvate; under excitation at 490 nm, the fluorescence intensity increased to 5.8 times that of 0 mM pyruvate. As shown in Figures 8B and 8C.
对纯化的丙酮酸光学探针PdhR(96-254)-141/143-cpYFP、PdhR(96-254)-192/194-M194V/S190D/R191Y/R192T-cpYFP、PdhR(96-254)-192/194-M194V-cpYFP和PdhR(96-254)-192/194-M194V/S190E/R191N/R192D-cpYFP测定其特异性，结果表明探针具有很好的特异性，如图9所示。For purified pyruvate optical probes PdhR(96-254)-141/143-cpYFP, PdhR(96-254)-192/194-M194V/S190D/R191Y/R192T-cpYFP, PdhR(96-254)-192 The specificity of /194-M194V-cpYFP and PdhR(96-254)-192/194-M194V/S190E/R191N/R192D-cpYFP were determined, and the results showed that the probe has good specificity, as shown in Figure 9.
实施例9：光学探针的亚细胞器定位本实施例中，使用不同的定位信号肽与光学探针融合，将光学探针定位到不同的细胞器中。Example 9: Subcellular organelle positioning of optical probes In this example, different positioning signal peptides are used to fuse the optical probes to locate the optical probes in different organelles.
用融合不同定位信号肽的光学探针质粒转染HeLa细胞36小时后，使用PBS冲洗，置于HBSS溶液中使用倒置荧光显微镜在FITC通道下进行荧光检测。结果如图10所示。丙酮酸光学探针通过与不同的特异定位信号肽融合能够定位到包括细胞浆、细胞外膜、细胞核、内质网、线粒体、核排阻等亚细胞器中。不同的亚细胞结构中都显示有荧光，并且荧光的分布和强度各不相同。The HeLa cells were transfected with optical probe plasmids fused with different positioning signal peptides for 36 hours, rinsed with PBS, placed in HBSS solution and used inverted fluorescence microscope for fluorescence detection under FITC channel. The result is shown in Figure 10. The pyruvate optical probe can be localized to the subcellular organelles including cytoplasm, outer cell membrane, cell nucleus, endoplasmic reticulum, mitochondria, nuclear exclusion by fusion with different specific localization signal peptides. Different subcellular structures all show fluorescence, and the distribution and intensity of fluorescence are different.
实施例10：丙酮酸探针在细胞内的性能研究Example 10: Study on the performance of pyruvate probe in cells
选择蛋白上ratio485/420大于2倍的样品，用胞浆表达的光学探针质粒转染HeLa细胞36小时后，使用PBS冲洗，置于HBSS溶液中，然后添加10mM丙酮酸，检测30min后420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。结果如图11所示，外源加入丙酮酸会引起HeLa细胞胞浆中探针的快速响应。Select samples with ratio485/420 greater than 2 times on the protein, transfect HeLa cells with the optical probe plasmid expressed in the cytoplasm for 36 hours, rinse with PBS, place in HBSS solution, and then add 10mM pyruvate, detect 30min after 420nm excitation The ratio of the fluorescence intensity at 528nm emission to that of 485nm excitation at 528nm emission. The results are shown in Figure 11. The addition of exogenous pyruvate will cause a rapid response of the probe in the cytoplasm of HeLa cells.
实施例11：在活细胞中基于光学探针进行高通量化合物筛选Example 11: High-throughput compound screening based on optical probes in living cells
本实施例中，我们使用胞浆表达丙酮酸探针PdhR(96-254)-192/194-M194V-cpYFP的HeLa细胞进行高通量化合物筛选。In this example, we used HeLa cells expressing the pyruvate probe PdhR(96-254)-192/194-M194V-cpYFP in the cytoplasm for high-throughput compound screening.
经转染的HeLa细胞使用PBS冲洗，置于HBSS溶液中(无丙酮酸)处理1小时，然后使用10μM的化合物处理1小时。各样品中分别滴加丙酮酸。使用酶标仪记录420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值变化。以未用任何化合物处理的样品作为对照进行标准化。结果如图12所示。在使用的2000种化合物中，绝大部分的化合物对丙酮酸进入细胞影响极小。有5种化合物能够提高细胞对丙酮酸的摄取能力，另外有8种化合物能够明显降低细胞对丙酮酸的摄取。The transfected HeLa cells were washed with PBS, placed in HBSS solution (without pyruvate) for 1 hour, and then treated with 10 μM compound for 1 hour. Pyruvic acid was added dropwise to each sample. Use a microplate reader to record the ratio of fluorescence intensity at 420nm excitation at 528nm emission and 485nm excitation at 528nm emission. A sample that was not treated with any compound was used as a control for normalization. The result is shown in Figure 12. Among the 2000 compounds used, most of the compounds have little effect on the entry of pyruvate into cells. There are 5 compounds that can increase the uptake of pyruvate by cells, and 8 other compounds can significantly reduce the uptake of pyruvate by cells.
实施例12：光学探针定量检测血液中的丙酮酸Example 12: Quantitative detection of pyruvate in blood by optical probe
在本实施例中，我们使用纯化的丙酮酸探针PdhR(96-254)-141/143-cpYFP对小鼠和人的血液上清中的丙酮酸进行分析。In this example, we used the purified pyruvate probe PdhR(96-254)-141/143-cpYFP to analyze the pyruvate in the blood supernatant of mice and humans.
将丙酮酸探针PdhR(96-254)-141/143-cpYFP与稀释的血液上清混合处理10分钟 后，使用酶标仪检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值。结果如图13所示，小鼠血液中的丙酮酸含量在270μM左右，人血液中的丙酮酸含量在130μM左右。After mixing the pyruvate probe PdhR(96-254)-141/143-cpYFP with the diluted blood supernatant for 10 minutes, use a microplate reader to detect the ratio of the fluorescence intensity at 420nm excitation and 528nm emission at 485nm excitation . The results are shown in Figure 13, the pyruvate content in the blood of mice is about 270 μM, and the content of pyruvate in human blood is about 130 μM.
由以上实施例可知，本发明提供的丙酮酸光学探针，蛋白分子量相对较小且易于成熟，荧光动态变化大，特异性好，并且能够通过基因操作的方法在细胞中表达，可在细胞内外实时定位、定量检测丙酮酸；并且能够进行高通量的化合物筛选。It can be seen from the above examples that the pyruvate optical probe provided by the present invention has relatively small protein molecular weight, is easy to mature, has a large dynamic change in fluorescence, and has good specificity. It can be expressed in cells by genetic manipulation methods, and can be used inside and outside the cell. Real-time localization and quantitative detection of pyruvate; and high-throughput compound screening.
其它实施方式Other embodiments
本说明书描述了许多实施方式。然而应理解，本领域技术人员通过阅读本说明书获知的不背离本发明的构思和范围的各种改进，也应包括在所附权利要求书的范围内。This specification describes many implementations. However, it should be understood that various improvements that are known to those skilled in the art by reading this specification without departing from the concept and scope of the present invention should also be included in the scope of the appended claims.

Claims (10)

  1. 一种光学探针，包含丙酮酸敏感多肽和光学活性多肽，其中光学活性多肽位于丙酮酸敏感多肽的序列内。An optical probe comprising a pyruvate sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is located in the sequence of the pyruvate sensitive polypeptide.
  2. 如权利要求1所述的光学探针，其中，丙酮酸敏感多肽具有SEQ ID NO：1所示的序列或其功能片段，或与其有35％、40％、50％、60％、70％、80％、85％、90％、95％、99％序列相同性的序列，优选地，丙酮酸敏感多肽具有SEQ ID NO：1所示序列的第96-254位氨基酸，或与其有35％、40％、50％、60％、70％、80％、85％、90％、95％、99％序列相同性的序列。The optical probe of claim 1, wherein the pyruvate sensitive polypeptide has the sequence shown in SEQ ID NO:1 or its functional fragment, or 35%, 40%, 50%, 60%, 70%, Sequences with 80%, 85%, 90%, 95%, 99% sequence identity, preferably, the pyruvate-sensitive polypeptide has amino acids 96-254 of the sequence shown in SEQ ID NO:1, or 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% sequence identity.
  3. 如权利要求1或2所述的光学探针，其中，光学活性多肽位于丙酮酸敏感多肽的选自以下的位置中：残基117-121、140-143、160-164、174-176、191-195和/或210-214，The optical probe of claim 1 or 2, wherein the optically active polypeptide is located in a position of the pyruvate sensitive polypeptide selected from the group consisting of residues 117-121, 140-143, 160-164, 174-176, 191 -195 and/or 210-214,
    优选地，光学活性多肽位于丙酮酸敏感多肽的选自以下的位点：117/118，117/119，117/120，117/121，118/119，118/120，118/121，119/120，119/121，120/121，140/141，140/142，140/143，141/142，141/143，142/143，160/161，160/162，160/163，160/164，161/162，161/163，161/164，162/163，162/164，163/164，174/175，174/176，175/176，191/192，191/193，191/194，191/195，192/193，192/194，192/195，193/194，193/195，194/195，210/211，210/212，210/213，210/214，211/212，211/213，211/214，212/213，212/214和/或213/214，Preferably, the optically active polypeptide is located at a site selected from the group consisting of the pyruvate-sensitive polypeptide: 117/118, 117/119, 117/120, 117/121, 118/119, 118/120, 118/121, 119/120 , 119/121, 120/121, 140/141, 140/142, 140/143, 141/142, 141/143, 142/143, 160/161, 160/162, 160/163, 160/164, 161 /162, 161/163, 161/164, 162/163, 162/164, 163/164, 174/175, 174/176, 175/176, 191/192, 191/193, 191/194, 191/195 ,192/193,192/194,192/195,193/194,193/195,194/195,210/211,210/212,210/213,210/214,211/212,211/213,211 /214, 212/213, 212/214 and/or 213/214,
    优选地，所述丙酮酸敏感多肽包含选自下述一个或多个位点处的突变：Q138，S190，R191，R192，E193，M194，L195。Preferably, the pyruvate sensitive polypeptide contains mutations at one or more positions selected from the group consisting of: Q138, S190, R191, R192, E193, M194, L195.
  4. 如权利要求3所述的光学探针，其中，光学活性多肽位于丙酮酸敏感多肽的选自以下的位置中：残基117-121、140-143、191-195和210-214。The optical probe according to claim 3, wherein the optically active polypeptide is located in a position of the pyruvate-sensitive polypeptide selected from the group consisting of residues 117-121, 140-143, 191-195, and 210-214.
  5. 一种核酸序列，选自A nucleic acid sequence selected from
    (1)编码权利要求1-4中任一项所述的光学探针的多核苷酸；(1) A polynucleotide encoding the optical probe of any one of claims 1 to 4;
    (2)(1)的片段；(2) Fragments of (1);
    (3)(1)或(2)的互补序列。(3) The complementary sequence of (1) or (2).
  6. 一种核酸构建物，其包含权利要求5所述的核酸序列，优选地，所述核酸构建物是表达载体。A nucleic acid construct comprising the nucleic acid sequence of claim 5, preferably, the nucleic acid construct is an expression vector.
  7. 一种宿主细胞，所述宿主细胞A host cell, the host cell
    (1)表达权利要求1-4中任一项所述的光学探针；(1) Express the optical probe of any one of claims 1-4;
    (2)包含权利要求5所述的核酸序列；或(2) Comprising the nucleic acid sequence of claim 5; or
    (3)包含权利要求6所述的核酸构建物。(3) Comprising the nucleic acid construct of claim 6.
  8. 一种制备权利要求1-4中任一项所述的光学探针的方法，包括培养权利要求7所述的宿主细胞，和由培养物分离所述光学探针。A method for preparing the optical probe according to any one of claims 1 to 4, comprising culturing the host cell according to claim 7, and separating the optical probe from the culture.
  9. 权利要求1-4中任一项所述的光学探针、权利要求5所述的核酸序列或权利要求6所述的核酸构建物在检测样品中的丙酮酸或筛选化合物中的应用，优选地，所述检测是丙酮酸定位或定量检测。The use of the optical probe according to any one of claims 1 to 4, the nucleic acid sequence according to claim 5 or the nucleic acid construct according to claim 6 in detecting pyruvate in a sample or screening compounds, preferably The detection is pyruvate localization or quantitative detection.
  10. 一种检测试剂盒，其包含A detection kit comprising
    (1)权利要求1-4中任一项所述的光学探针或权利要求8所述方法制备的光学探针；(1) The optical probe according to any one of claims 1 to 4 or the optical probe prepared by the method according to claim 8;
    (2)权利要求5所述的核酸序列；(2) The nucleic acid sequence of claim 5;
    (3)权利要求6所述的核酸构建物；或(3) The nucleic acid construct of claim 6; or
    (4)权利要求7所述的细胞；和(4) The cell of claim 7; and
    任选的利用光学探针检测丙酮酸所需的其他试剂。Optional use of optical probes to detect other reagents required for pyruvate.
PCT/CN2021/076317 2020-02-18 2021-02-09 Pyruvic acid optical probe, preparation method therefor, and application thereof WO2021164668A1 (en)

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Title
DANA C. NADLER, STACY-ANNE MORGAN, AVI FLAMHOLZ, KAITLYN E. KORTRIGHT, DAVID F. SAVAGE: "Rapid construction of metabolite biosensors using domain-insertion profiling", NATURE COMMUNICATIONS, vol. 7, pages 12266, XP055480386, DOI: 10.1038/ncomms12266 *
DATABASE PROTEIN 31 January 2014 (2014-01-31), ANONYMOUS: "Pyruvate dehydrogenase complex repressor [Escherichia coli CFT073]", XP055839657, retrieved from NCBI Database accession no. AAN78636 *

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