Lactic acid optical probe and method for preparing and using same

Abstract

The present invention relates to a lactate optical probe and a method for preparing and using the same. In one aspect, the present invention relates to an optical probe comprising a lactate-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is located within the sequence of the lactate-sensitive polypeptide or a functional variant thereof. The present invention also relates to a method for preparing the probe and its use in detecting lactate.

Description

[Technical Field]

[0001] The present invention relates to the technical field of optical probes, and in particular to a lactate optical probe and its preparation method and use. [Background technology]

[0002] Lactic acid is an α-hydroxy acid, which is a hydrogen ion in water. + Lactic acid is ionized to form the CH3CH(OH)COO-lactate ion. Lactic acid is a chiral molecule and consists of two enantiomers: D-(-)-lactate and L-(+)-lactate. Lactic acid can exist in two different configurations in the human body, with the majority being L-lactate. Lactic acidosis may occur when blood L-lactate concentrations exceed 5 mM and blood pH is below 7.35. Lactic acidosis can be caused by a lack of mitochondrial pyruvate transport, defects in the pyruvate dehydrogenase complex, citric acid cycle disorders, congenital mitochondrial respiratory chain disorders, or tissue hypoxia. Because lactate homeostasis is linked to glucose metabolism, diabetes is associated with impaired lactate metabolism. Compared to healthy controls, diabetic patients have a decreased proportion of glucose oxidation in their total body basal metabolism and an increased proportion of nonoxidative glycolysis, leading to excessive lactate production. Elevated blood L-lactate concentrations were previously thought to be due to waste products produced by skeletal muscle during anaerobic conditions via the glycolytic pathway. However, it is now widely recognized that L-lactate, produced in vivo by various cells under strictly aerobic conditions, is utilized as an energy source in most tissues via the peripheral circulation. Lactate is the primary energy source in the body, and its role in the tricarboxylic acid cycle is more important than glucose during lung tumor cell growth. Tumor cells consume large amounts of glucose, which provides energy for tumor cell growth and proliferation via the glycolytic pathway under aerobic conditions, while also producing large amounts of the metabolic product lactate. Lactate effectively inhibits immune cell function and survival, and also contributes to tumor cell immune evasion, demonstrating the important interaction between tumor-derived lactate and tumor-associated immune cells.

[0003] The metabolic pathways involved in lactate metabolism are important for understanding the physiological response to exercise and the pathogenesis of common diseases such as diabetes and cancer. Both lactate production and elimination depend on reversible redox reactions catalyzed by lactate dehydrogenase (LDH). During normal animal metabolism and exercise, glucose produces pyruvate via glycolysis, which then converts to L-lactate via lactate dehydrogenase (LDH). However, lactate concentrations do not rise until lactate production outpaces its elimination. During intense exercise, blood glucose rapidly synthesizes the ATP needed by the body through anaerobic respiration, producing large amounts of pyruvate, which is then quickly converted to lactate. In this case, lactate production outpaces its elimination, resulting in elevated lactate concentrations. Lactate in the body has two fate routes. One is regeneration to pyruvate, which then enters the aerobic respiratory chain and is oxidized to provide energy. The other pathway is converted to glucose by gluconeogenesis in the liver and enters the peripheral circulation. Lactate is the primary gluconeogenic precursor. During intense exercise, glycogen in muscle cells is converted to pyruvate by glycolysis. However, the absence of glucose-6-phosphatase in muscles prevents the catalysis of glucose-6-phosphate to produce glucose. Therefore, lactate penetrates the cell membrane and is released into the bloodstream. It then enters the liver, where it is converted to pyruvate by hepatic lactate dehydrogenase. Glucose is then produced via the gluconeogenic pathway, which then returns to the bloodstream as glucose needed for muscle and cerebral cortex. This cycle is known as the Cori cycle, or lactate cycle. Lactate upregulates hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF), playing an essential role in angiogenesis. Given its central role in lactate metabolism, lactate is now thought to be a component of primary and metastatic cancer metabolism, and several studies have shown that lactate plays a vital role in tumor growth and the progression of metastatic disease.

[0004] Because of the important role of lactic acid as described above, the detection of lactic acid content is particularly important. Typical methods for detecting lactic acid include NaOH solution titration and high-performance liquid chromatography (HPLC) (Shitoume et al., Analytical Chemistry, 2001, 29(4):413-415) (Non-Patent Document 1). Furthermore, because lactic acid is a chiral molecule and exhibits optical rotation, detection using a polarimeter, ultraviolet (UV)-enzyme analysis (Immonen, K., et al., Meat Sci, 2000, 54(2):163-7) (Non-Patent Document 2), and enzyme electrode analysis (Tanner, R.K. et al., Eur J Appl Physiol, 2010, 109(3):551-9) (Non-Patent Document 3) may also be used. However, these methods have many deficiencies, such as being unsuitable for studying living cells, requiring time-consuming sample processing steps such as cell disruption, separation, extraction, and purification, and lacking in situ, real-time, dynamic, high-throughput, and high-temporal resolution detection in living cells and sub-organelles. There remains a need in the art for real-time, in situ, quantitative, and high-throughput detection of lactate both inside and outside cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Shirofuyuume et al., Analytical Chemistry, 2001, 29(4):413-415 [Non-patent document 2] Immonen, K., et al., Meat Sci, 2000.54(2):p.163-7. [Non-patent document 3] Tanner, RK et al., Eur J Appl Physiol, 2010.109(3):p.551-9. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide probes and methods for real-time in situ, high-throughput, quantitative detection of lactate inside and outside cells. [Means for solving the problem]

[0007] In order to achieve the above object of the invention, the present invention provides the following technical solutions. The present invention provides a lactate optical probe comprising a lactate-sensitive polypeptide or a functional variant thereof and an optically active polypeptide or a functional variant thereof, wherein the optically active polypeptide or a functional variant thereof is within the sequence of the lactate-sensitive polypeptide or a functional variant thereof, and the lactate-sensitive polypeptide or a functional variant thereof is divided into a first portion and a second portion by the optically active polypeptide or a functional variant thereof.

[0008] The present invention provides a lactate optical probe comprising a lactate-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is within the sequence of the lactate-sensitive polypeptide B, and the lactate-sensitive polypeptide B is divided into a first portion B1 and a second portion B2, forming a probe structure of the formula B1-A-B2.

[0009] In one embodiment, the lactate-sensitive polypeptide comprises a lactate-binding domain of a lactate-binding protein. In one embodiment, the lactate-sensitive polypeptide is derived from Escherichia coli. In one embodiment, the lactate-sensitive polypeptide is a lactate-binding protein or a functional fragment thereof. In one or more embodiments, the lactate-binding protein is an LldR protein. In one embodiment, the lactate-sensitive polypeptide has the sequence set forth in SEQ ID NO:1, or a sequence having at least 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto and retaining lactate-binding function. In one embodiment, the lactate-sensitive polypeptide has amino acids 80 to 258 of the sequence set forth in SEQ ID NO:1, or a sequence having at least 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% sequence identity thereto and retaining lactate-binding function.

[0010] In one embodiment, the optically active polypeptide is a fluorescent protein or a functional fragment or variant thereof. In one embodiment, the fluorescent protein is selected from yellow fluorescent protein (cpYFP shown in SEQ ID NO:2), green fluorescent protein (cpGFP shown in SEQ ID NO:3), blue fluorescent protein (cpBFP shown in SEQ ID NO:4), and 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 NOs:2 to 5.

[0011] In one embodiment, the optical probe further comprises one or more linkers linked to the side chains of the optically active polypeptide. The linkers of the present invention may be of any length and any amino acid sequence. In one embodiment, the side chains of the optically active polypeptide comprise a linker of 5 or fewer amino acids, for example, a linker of 0, 1, 2, 3, or 4 amino acids. In one embodiment, the linker in the side chain of the optically active polypeptide comprises amino acid Y. In one embodiment, linker Y is located at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is shown as follows: first portion B1 of lactate-sensitive polypeptide - Y - optically active polypeptide A - second portion B2 of lactate-sensitive polypeptide. In one embodiment, the optical probe of the present invention does not comprise a linker.

[0012] In one embodiment, the optically active polypeptide is located at a site selected from the following in the lactate-sensitive polypeptide: residues 93-97, 119-121, 137-141, 158-161, 185-191, 208-210, and / or 230-232, and the lactate-sensitive polypeptide is a functional fragment of the lactate-binding protein represented by amino acids 80-258 of SEQ ID NO:1, where the numbers correspond to the full-length lactate-binding protein. In one embodiment, one or more amino acids at the following sites of the lactate-sensitive polypeptide, i.e., residues 93-97, 119-121, 137-141, 158-161, 185-191, 208-210, and / or 230-232, are substituted with an optically active polypeptide, and the lactate-sensitive polypeptide is a functional fragment of the lactate-binding protein represented by amino acids 80-258 of SEQ ID NO:1, where the numbers correspond to the full-length lactate-binding protein.

[0013] In one embodiment, the optically active polypeptide is selected from one or more of the following regions of a lactate sensitive polypeptide: 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 158 / 162, 158 / 163, 158 / 164, 158 / 165, 158 / 166, 158 / 167, 158 / 168, 158 / 169, 159 / 170, 159 / 171, 171 / 172, 172 / 173, 173 / 174, 174 / 175, 175 / 176, 176 / 177, 177 / 178, 178 / 179, 179 / 180, 180 / 181, 181 / 182, 182 / 183, 183 / 184, 184 / 185, 185 / 186, 186 / 187, 187 / 188, 188 / 189, 189 / 190, 190 / 191, 9 / 160, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 and / or 231 / 232. Preferably, the optically active polypeptide is located at one or more sites of the lactate-sensitive polypeptide selected from the following: 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191 or 190 / 191. In one or more embodiments, the B1-A-B2 optical probe of the present invention may be a probe in which cpYFP is located at the 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, or 190 / 191 site of a lactate binding protein or a functional fragment thereof.In an exemplary embodiment, the B1-A-B2 optical probe of the present invention may be a probe in which cpYFP is located at the 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 187 / 189, 189 / 191, and 190 / 191 sites of a lactate binding protein or a functional fragment thereof. In one or more embodiments, the functional fragment of the lactate binding protein is represented by positions 80 to 258 of SEQ ID NO: 1. In one embodiment, the optical probe of the present invention has or consists of the sequence represented by SEQ ID NO: 6 to 17.

[0014] The present invention further provides a mutant of a lactate-sensitive polypeptide having one or more mutations, in one embodiment, the mutations are at positions 185, 189 and / or 190 of the lactate-binding protein or functional fragment thereof.

[0015] In one or more embodiments, the mutations are selected from the group consisting of P189R and P190D, P189R and P190A, P189R and P190I, P189R and P190Q, P189R and P190N, P189D and P190D, P189D and P190E, P189D and P190V, P189D and P190L, P189D and P190F, P189D and P190I, P189D and P190Q, P189D and P190N, P189D and P190G, P189D and P190Y, P189D and P190W, P189E and P190R ... 89E and P190A, P189E and P190V, P189E and P190Q, P189A and P190L, P189A and P190F, P189A and P190M, P189A, P189A and P190N, P189A and P190G, P189A and P190H, P189A and P190T, P189V and P190D, P189V and P190E, P189V and P190A, P189V, P189V and P190N, P189V and P190H, P189V and P190Y, P189L and P190V, P189L and P190F, P189 L and P190M, P189L and P190G, P189L and P190H, P189F and P190D, P189F and P190L, P189F and P190F, P189F and P190I, P189F and P190N, P189F and P190H, P189F and P190Y, P189F and P190K, P189F and P190T, P189F and P190W, P189I and P190R, P189I and P190D, P189I and P190A, P189I and P190V, P189I and P190M, P189I and P190Q, P189I and P190G, P189I and P190Y, P189I and P190S, P189I and P190T, P189M and P190R, P189M and P190D, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189M and P190W, P189C and P190D, P189C and P190E, P189C and P190F, P189C and P190I, P189C and P190M, P189C and P190C, P189C, P189C and P190H, P189C and P190Y,P189C and P190S, P189C and P190W, P190L, P190F, P190I, P190Q, P190N, P190K, P190T, P189Q and P190E, P189Q and P190A, P189Q and P190V, P189Q and P190M, P189Q and P190C, P189Q and P190Q, P189Q and P190H, P189Q and P190S, P189N and P190R, P189N and P190D, P189N and P190L, P189N and P190F, P189N and P190C, P189N, P189N and P190N, P189N and P190G, P189N and P190H, P189N and P190Y, P189N and P190T, P189G and P190V, P189G and P190F, P189G and P190M, P189G and P190C, P189G and P190G, P189G and P190H, P189G and P190K, P189G and P190W, P189H and P190R, P189H and P190D, P189H and P190E, P189H and P190L, P189H and P190S, P189Y and 190R, P189Y and P19 0L, P189Y and P190N, P189Y and P190H, P189Y and P190S, P189Y and P190T, P189K and P190D, P189K and P190E, P189K and P190V, P189K and P190L, P189K and P190F, P189K and P190I, P189K and P190M, P189K, P189K and P190Q, P189K and P190N, P189K and P190Y, P189K and P190K, P189K and P190T, P189S and P190E, P189S and P190A, P189S and P190L, P189S and P190F, P189S and P190M, P189S and P190C, P189S, P189S and P190Q, 189S and P190Y, P189S and P190K, P189S and P190S, P189T and P190R, P189T and P190D, P189T and P190M, P189T and P190C, P189T, P189T and P190Q, P189T and P190N, P189T and P190H, P189T and P190Y, P189T and P190K, P189T and P190W, P189W and P190A,Includes P189W and P190V, P189W and P190F, P189W, P189W and P190Q, P189W and P190H, P189W and P190S, P189W and P190T, P189W and P190W. In one or more embodiments, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K, preferably, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K.

[0016] In one or more embodiments, the mutations are selected from the group consisting of P189R and P190A, P189D and P190D, P189D and P190E, P189D and P190Q, P189D and P190Y, P189A and P190N, P189A and P190G, P189V and P190H, P189F and P190I, P189F and P190N, P189F and P190K, P189 I and P190D, P189I and P190A, P189I and P190V, P189I and P190M, P189M and P190R, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189C and P190E, P190Q, P189Q and P190M, P189Q and P190C, P189N and P190N, P189G and P190F, P189H and P190L, P189H and P190S, P189Y and P190L, P189K and P190V, P189K and P190T, P189S and P190A, P189S and P190M, P189S and P190Q, P189S and P190K, P189S and P190S, P189T and P190D, P189W and P190A, P1 Includes P189W and P190T, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F, P189H and P190D, or P189S. In one or more embodiments, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K, preferably, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K.

[0017] In one or more embodiments, the mutations include P189S, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F, or P189H and P190D. In one or more embodiments, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K, preferably, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K.

[0018] In one or more embodiments, the mutations include (1) P189C and P190D, P189M and P190D, P189F and P190D, or P189H and P190D, and any of (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K.

[0019] In one or more embodiments, the mutations are any of (1) P189C and P190D, P189M and P190D, or P189H and P190D, and any of (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, ... or the mutations include (1) P189F and P190D, and any of (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K.

[0020] The lactate-sensitive polypeptide in the optical probe of the present invention may be a lactate-sensitive polypeptide having one or more mutations, as described above. In one or more embodiments, the optical probe containing the mutated lactate-sensitive polypeptide has a higher or lower response to lactate than its non-mutated counterpart.

[0021] In an exemplary embodiment, the optical probe of the present invention comprises a functional fragment of lactate binding protein having cpYFP inserted at the 185 / 189 position and one or more mutations selected from the following: P189R and P190D, P189R and P190A, P189R and P190I, P189R and P190Q, P189R and P190N, P189D and P190D, P189D and P190E, P189D and P190V, P189D and P190L, P189D and P190F, P189D and P190I, P189D and P190Q, P189D and P190N, P189D and P190G, P189D and P190Y, P189D and P190W, P189E and P190R, P189E and P190A, P189E and P190V, P189E and P190Q, P189A and P190L, P189A and P190F, P189A and P190M, P189A, P189A and P190N, P189A and P190G, P189A and P190H, P189A and P190T, P189V and P190D, P189V and P190E, P189V and P190A, P189V, P189V and P 190N, P189V and P190H, P189V and P190Y, P189L and P190V, P189L and P190F, P189L and P190M, P189L and P190G, P189L and P190H, P189F and P190D, P189F and P190L, P189F and P190F, P189F and P190I, P189F and P190N, P189F and P190H, P189F and P190Y, P189F and P190K, P189F and P190T, P189F and P190W, P189I and P190R, P189I and P19 0D, P189I and P190A, P189I and P190V, P189I and P190M, P189I and P190Q, P189I and P190G, P189I and P190Y, P189I and P190S, P189I and P190T, P189M and P190R, P189M and P190D, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189M and P190W, P189C and P190D, P189C and P190E, P189C and P190F, P189C and P190I,P189C and P190M, P189C and P190C, P189C, P189C and P190H, P189C and P190Y, P189C and P190S, P189C and P190W, P190L, P190F, P190I, P190Q, P190N, P190K, P190T, P189Q and P190E, P189Q and P190A, P189Q and P190V, P189Q and P190M, P189Q and P190C, P189Q and P190Q, P189Q and P190H, P189Q and P190S, P189N and P190R, P189N and P190D, P189N and P190L, P189N and P190F, P189N and P190C, P189N, P189N and P190N, P189N and P190G, P189N and P190H, P189N and P190Y, P189N and P190T, P189G and P190V, P189G and P190F, P189G and P190M, P189G and P190C, P189G and P190G, P189G and P190H, P189G and P190K, P189G and P190W, P189H and P190R, P189H and P190D, P 189H and P190E, P189H and P190L, P189H and P190S, P189Y and P190R, P189Y and P190L, P189Y and P190N, P189Y and P190H, P189Y and P190S, P189Y and P190T, P189K and P190D, P189K and P190E, P189K and P190V, P189K and P190L, P189K and P190F, P189K and P190I, P189K and P190M, P189K, P189K and P190Q, P189K and P190N, P189K and P1 90Y, P189K and P190K, P189K and P190T, P189S and P190E, P189S and P190A, P189S and P190L, P189S and P190F, P189S and P190M, P189S and P190C, P189S, P189S and P190Q, 189S and P190Y, P189S and P190K, P189S and P190S, P189T and P190R, P189T and P190D, P189T and P190M, P189T and P190C, P189T, P189T and P190Q, P189T and P190N,The probe may have P189T and P190H, P189T and P190Y, P189T and P190K, P189T and P190W, P189W and P190A, P189W and P190V, P189W and P190F, P189W, P189W and P190Q, P189W and P190H, P189W and P190S, P189W and P190T, or P189W and P190W. In a further embodiment, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E or M185K, preferably the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R or M185K.

[0022] In an exemplary embodiment, the optical probe of the present invention may be a probe having cpYFP inserted at the 185 / 189 position of a functional fragment of a lactate binding protein and one or more mutations selected from the following: P189S, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F, or P189H and P190D. In an exemplary embodiment, the functional fragment of the lactate binding protein is residues 80 to 258 of SEQ ID NO:1, and the mutations are P189N, P189S, P189C and P190F, P189N and P190F, P189N and P190Y, P189H and P190R, P189R and P190I, P189F and P190H, P189C and P190Y, P189C and P190D, P189M and P190D, P189H and P190D, or P189F and P190D. In a further embodiment, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E or M185K, preferably the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R or M185K.

[0023] In some specific embodiments, the optical probe of the present invention may be a probe having a mutation in which cpYFP is inserted at the 185 / 189 position of a functional fragment of a lactate binding protein, the functional fragment of the lactate binding protein being at positions 80 to 258 of SEQ ID NO: 1, and the mutation includes (1) P189C and P190D, P189M and P190D, P189F and P190D, or P189H and P190D, and any of (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K. Preferably, the mutations are (1) P189C and P190D, P189M and P190D, or P189H and P190D, and any of (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T ... 185R, M185E, or M185K, or the mutations include (1) P189F and P190D, and any of (2) M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K.

[0024] In one embodiment, the optical probe of the present invention has or consists of the sequence shown in SEQ ID NOs: 18-30, 34-40.

[0025] The optical probes provided by the present invention comprise any one of the amino acid sequences SEQ ID NOs: 6-30, 34-40, or variants thereof. In one embodiment, the optical probes provided by the present invention comprise a sequence having at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any one of the amino acid sequences SEQ ID NOs: 6-30, 34-40. In a preferred embodiment, the optical probes provided by the present invention consist of the sequence set forth in any one of SEQ ID NOs: 6-30, 34-40. In a more preferred embodiment, the optical probes provided by the present invention comprise or consist of SEQ ID NOs: 30, 34-40.

[0026] The present invention further provides fusion polypeptides comprising an optical probe described herein and another polypeptide. In some embodiments, the other polypeptide is at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide comprises a polypeptide that localizes the optical probe to a different organelle or suborganelle, and a purification tag or immunoblot tag.

[0027] The present invention further provides a nucleic acid sequence comprising a coding sequence for a polypeptide, probe, or protein described herein, or a complementary sequence or fragment thereof. In one embodiment, the nucleic acid sequence of the present invention is selected from (1) a coding sequence for an amino acid sequence set forth in any one of SEQ ID NOs: 6-30, 34-40, or a complementary sequence thereof, (2) a sequence having at least 99%, 95%, 90%, 80%, 70%, or 50% identity to (1), or (3) a fragment of (1) or (2). In one embodiment, the nucleic acid sequence of the present invention comprises SEQ ID NO: 34 or a variant or fragment thereof. In one or more embodiments, the fragment is a primer.

[0028] The present invention further relates to the complements of the above nucleic acid sequences or variants thereof, including nucleic acid sequences or complementary sequences thereof that encode fragments, analogs, derivatives, soluble fragments and variants of the optical probes or fusion proteins of the present invention.

[0029] The present invention further provides a nucleic acid construct comprising a nucleic acid sequence described herein or a complementary sequence thereof, wherein the nucleic acid sequence encodes an optical probe or fusion polypeptide of the present invention. In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or a recombinant vector. In one or more embodiments, the nucleic acid sequence is operably linked to an expression control sequence. In some embodiments, the expression vector is selected from a prokaryotic expression vector, a eukaryotic expression vector, and a viral vector.

[0030] The present invention further provides a cell comprising a nucleic acid sequence or nucleic acid construct of the present invention, in one or more embodiments, the cell expressing an optical probe or fusion polypeptide described herein.

[0031] The present invention further provides detection reagent kits comprising the optical probes, fusion polypeptides, polynucleotides described herein, or optical probes or fusion polypeptides prepared by the methods described herein.

[0032] The present invention provides a method for preparing an optical probe described herein, comprising providing a cell expressing an optical probe or fusion polypeptide described herein, culturing the cell under expression conditions for the cell, and isolating the optical probe or fusion polypeptide.

[0033] The present invention further provides a method for preparing the lactate optical probe described above, comprising the following steps: 1) transferring into a host cell an expression vector encoding the lactate optical probe described herein; 2) culturing the host cell under conditions suitable for expression of the expression vector; and 3) isolating the lactate optical probe.

[0034] The present invention further provides a method for detecting lactate in a sample, comprising contacting the sample with an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared by the methods described herein, and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, suborganelle, or in situ. The sample may be, for example, blood.

[0035] The present invention further provides a method for quantifying lactate in a sample, comprising contacting the sample with an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared by the methods described herein, detecting a change in the optically active polypeptide, and quantifying lactate in the sample based on the change in the optically active polypeptide.

[0036] The present invention further provides a method for screening compounds (e.g., drugs) comprising contacting a candidate compound with an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared by the methods described herein, detecting a change in the optical activity of the polypeptide, and screening the compound based on the change in the optical activity of the polypeptide, which method allows for high-throughput screening of compounds.

[0037] The present invention further provides the use of a lactate optical probe or fusion polypeptide described herein, or a lactate optical probe or fusion polypeptide prepared by a method described herein, in intracellular / extracellular localization of lactate. In one or more embodiments, the localization is real-time localization.

[0038] The present invention has the advantages that the lactate optical probe provided by the present invention is easy to mature, has a large dynamic change in fluorescence, has good specificity, and can be expressed in cells by genetic engineering, allowing for real-time in situ, high-throughput, and quantitative detection of lactate both inside and outside the cells, thereby simplifying time-consuming sample processing procedures. Experimental results have shown that the lactate optical probe provided by the present invention has a maximum response to lactate that is more than 13 times that of the control group, and can be used for cellular localization, qualitative and quantitative detection of sub-organelle structures such as the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, lysosomes and Golgi apparatus, as well as for high-throughput compound screening and quantitative detection of blood lactate. [Brief explanation of the drawings]

[0039] The present invention will be specifically described below with reference to the drawings and examples. [Figure 1] FIG. 1 is an SDS-PAGE diagram of an exemplary lactate optical probe according to Example 1. [Figure 2] FIG. 2 is a graph showing changes in the response to lactate of an exemplary lactate optical probe comprising cpYFP and a lactate binding protein according to Example 2. [Figure 3] FIG. 3 is a graph showing changes in the response to lactate of an exemplary lactate optical probe comprising cpGFP and lactate-binding protein according to Example 3. [Figure 4] FIG. 4 is a graph showing the change in response to lactate of an exemplary lactate optical probe comprising cpBFP and lactate binding protein according to Example 4. [Figure 5] FIG. 5 is a graph showing changes in the response to lactate of an exemplary lactate optical probe comprising cpmApple and lactate-binding protein according to Example 5. [Figure 6] FIG. 6 shows the response to lactate of an exemplary mutant lactate optical probe in which cpYFP is inserted into the 185 / 189 site of lactate binding protein according to Example 6. [Figure 7A]FIG. 7A shows titration curves of an exemplary mutant lactate optical probe in which cpYFP is inserted into the 185 / 189 site of lactate binding protein against different concentrations of lactate, according to Example 7. [Figure 7B] FIG. 7B shows titration curves of mutant lactate optical probes according to Example 8 against different concentrations of lactate. [Figure 8] FIG. 8 is a fluorescence spectrum diagram of an exemplary lactate optical probe according to Example 9. [Figure 9] FIG. 9 is a histogram of specificity detection of an exemplary lactate optical probe according to Example 9. [Figure 10] FIG. 10 is an image of sub-organellar localization in mammalian cells of an exemplary lactate optical probe according to Example 10. [Figure 11] FIG. 11 is a schematic diagram of the dynamic monitoring of lactate transmembrane transport in mammalian cells with an exemplary lactate optical probe according to Example 11. [Figure 12] FIG. 12 is a scatter plot of a high-throughput compound screen at the live cell level for an exemplary lactate optical probe according to Example 12. [Figure 13] FIG. 13 is a histogram of quantification of lactate in mouse and human blood for an exemplary lactate optical probe according to Example 13. DETAILED DESCRIPTION OF THE INVENTION

[0040] As used herein, when referring to a numerical value or range, the term "about" means that the numerical value or range is within 20%, within 10%, and within 5% of the stated numerical value or range.

[0041] As used herein, the terms "comprise," "comprise," "contain," and their equivalents, including "consist of," mean that, for example, in the case of a composition "comprising" X, the composition may consist solely of X, or may include other substances, e.g., X+Y.

[0042] As used herein, the term "lactate-sensitive polypeptide" or "lactate-responsive polypeptide" refers to a polypeptide that responds to lactate. The response includes any response of a chemical, biological, electrical, or physiological parameter of a polypeptide related to the interaction of the sensitive polypeptide. Responses include small changes, such as changes in the orientation of amino acids or peptide fragments of a polypeptide, and changes in the primary, secondary, or tertiary structure of a polypeptide, such as changes in protonation, electrochemical potential, and / or conformation. "Conformation" refers to the configuration of the primary, secondary, and tertiary structure of a molecule with pendant groups. A change in molecular conformation results in a change in conformation. Examples of conformational changes include a conversion from alpha helix to beta sheet or a conversion from beta sheet to alpha helix. It may be understood that the detected change does not necessarily have to be a conformational change, as long as the fluorescence of the fluorescent protein moiety is altered. The lactate-sensitive polypeptides described herein may also include functional variants thereof. Functional variants of lactate-sensitive polypeptides include, but are not limited to, variants that can undergo homologous or similar changes to the parent lactate-sensitive polypeptide upon interaction with lactate.

[0043] Lactate-sensitive polypeptides of the present invention include, but are not limited to, the lactate-binding protein LldR or variants having 90% or more homology thereto. An exemplary lactate-binding protein LldR of the present invention is derived from Escherichia coli JM109. LldR is a bacterial transcription factor and consists of a lactate-binding / regulatory domain and a DNA-binding domain. An exemplary LldR protein is set forth in SEQ ID NO:1. In one or more embodiments, the lactate-sensitive polypeptide comprises a functional fragment of a lactate-binding protein (lactate-binding domain), i.e., amino acids 80 to 258. When describing the optical probe or lactate-binding protein of the present invention (e.g., when describing an insertion site or mutation site), the amino acid residue numbers referred to refer to SEQ ID NO:1.

[0044] The term "optical probe" as used herein refers to a lactate-sensitive polypeptide fused to an optically active polypeptide. The inventors discovered that the conformational change caused by the specific binding of a lactate-sensitive polypeptide, such as a lactate-binding protein, to physiological concentrations of lactate induces a conformational change in the optically active polypeptide (e.g., a fluorescent protein), which in turn induces a change in the optical properties of the optically active polypeptide. The presence and / or level of lactate can be detected and analyzed by creating a calibration curve based on the fluorescence of the fluorescent protein measured at different concentrations of lactate.

[0045] In the optical probes of the present invention, an optically active polypeptide (e.g., a fluorescent protein) is operably inserted into a lactate-sensitive polypeptide. A protein-based "optically active polypeptide" is a polypeptide capable of emitting fluorescence. Fluorescence is an optical property of an optically active polypeptide and may be used as a means of detecting the responsiveness of the optical probes of the present invention. Preferably, the protein substrate is selected to have readily distinguishable fluorescent properties between the unactivated and activated conformational states. The optically active polypeptides described herein may further include functional variants thereof. Functional variants of optically active polypeptides include, but are not limited to, variants that can exhibit changes in fluorescent properties that are homologous or similar to those of the parent optically active polypeptide.

[0046] The term "fluorescent protein" as used herein refers to a protein that emits fluorescence upon irradiation with excitation light. Fluorescent proteins are fundamental detection tools in the biological field, including, for example, green fluorescent protein (GFP), which is commonly used in the biotechnology field, and circularly rearranged blue fluorescent protein (cpBFP), circularly rearranged green fluorescent protein (cpGFP), and circularly rearranged yellow fluorescent protein (cpYFP), which are induced by mutations of the green fluorescent protein. Fluorescent proteins also include red fluorescent protein (RFP), which is commonly used in the technical field, and circularly rearranged proteins induced by the red fluorescent protein, such as cpmApple, cpmOrange, and cpmKate. Fluorescent proteins and their sequences that can be used in the present invention are known in the art. For example, cpYFP is represented by SEQ ID NO:2, cpGFP is represented by SEQ ID NO:3, cpBFP is represented by SEQ ID NO:4, and cpmApple is represented by SEQ ID NO:5.

[0047] The term "linker" or "linker region" refers to an amino acid or nucleotide sequence that links two parts of the polypeptide, protein, or nucleic acid of the present invention. For example, in the present invention, the linker region between the lactate-sensitive polypeptide and the optically active polypeptide has 0 to 3 amino acids at the amino terminus and 0 to 2 amino acids at the carboxy terminus. However, when the recombinant optical probe is linked to a functional protein as a basic unit, the linker sequence may be fused to the amino acid or carboxy terminus of the recombinant optical probe. The linker sequence may be, for example, a short peptide consisting of one or more flexible amino acids, such as Y.

[0048] The lactate optical probe of the present invention comprises lactate-sensitive polypeptide B, which is, for example, a lactate-binding protein or its lactate-binding domain (residues 80 to 258) or a variant thereof, and optically active polypeptide A, which is, for example, a fluorescent protein. Optically active polypeptide A is inserted into lactate-sensitive polypeptide B, which is then divided into two parts, B1 and B2, to form a B1-A-B2 probe structure. The interaction of lactate-sensitive polypeptide B with lactate intensifies the optical signal of optically active polypeptide A.

[0049] In the optical probe of the present invention, the optically active polypeptide may be located at any site of the lactate-sensitive polypeptide. In one embodiment, the optically active polypeptide is located at any site of the lactate-sensitive polypeptide in the N-C direction. Specifically, the optically active polypeptide is located in a flexible region of the lactate-sensitive polypeptide. The flexible region refers to some special structures present in the higher-order structure of a protein, such as a cyclic structural domain. These structural domains have higher mobility and flexibility than other higher-order structures of the protein, and this region dynamically changes its three-dimensional conformation after the protein binds to a ligand. The flexible region of the present invention mainly refers to the region containing the insertion site in the lactate-binding protein, for example, the regions of amino acid residues 93-97, 119-121, 137-141, 158-161, 185-191, 208-210, and / or 230-232. Exemplary optically active polypeptides may be selected from the following: 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 1 Located at sites 59 / 160, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 or 231 / 232. In this specification, when the two numbers in a site represented by the format "X / Y" are consecutive integers, it indicates that the optically active polypeptide is located between the amino acids represented by the numbers.For example, insertion site 93 / 94 indicates that the optically active polypeptide is located between amino acids 93 and 94 of the lactate-sensitive polypeptide. If the two numbers in the "X / Y" format are not consecutive integers, the amino acids between the amino acids indicated by the numbers are substituted with the optically active polypeptide. For example, insertion site 93 / 97 indicates that amino acids 94 to 96 of the lactate-sensitive polypeptide are substituted with the optically active polypeptide. In one embodiment, the optically active polypeptide is located at one or more sites selected from the following of the lactate binding protein or functional fragment thereof: 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, or 190 / 191. In one embodiment, the optically active polypeptide is located at one or more sites selected from the following of the lactate binding protein or a functional fragment thereof: 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 187 / 189, 189 / 191, and 190 / 191. In one embodiment, the optically active polypeptide is located at one or more sites selected from the following of the lactate binding protein or a functional fragment thereof: 185 / 189, 186 / 189, or 187 / 189. Preferably, the optical probe of the present invention may be a probe in which cpYFP is located at the 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191 or 190 / 191 site of the lactate binding protein or a functional fragment thereof.In an exemplary embodiment, the optical probe of the present invention may be a probe in which cpYFP is located at positions 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 187 / 189, 189 / 191, and 190 / 191 of the lactate binding protein or a functional fragment thereof. In one or more embodiments, the functional fragment of the lactate binding protein is set forth in positions 80 to 258 of SEQ ID NO: 1. In one embodiment, the optical probe of the present invention has or consists of the sequence set forth in SEQ ID NOs: 6 to 17.

[0050] When referring to a specific polypeptide or protein, the term "variant" or "mutant" as used herein includes variants that have homologous functions to the polypeptide or protein but differ in sequence. These variants include, but are not limited to, sequences obtained by deleting, inserting, and / or substituting one or more amino acids (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5) from the polypeptide or protein sequence, and adding one or more amino acids (usually 20 or less, preferably 10 or less, and more preferably 5 or less) to the carboxy-terminus and / or amino-terminus. Without being bound by theory, functionally conservative mutations are preferred, in which the overall configuration and function of the polypeptide or protein are not altered even when the amino acid residue is changed. For example, substitution with an amino acid with similar or similar properties generally does not alter the function of the polypeptide or protein. In the art, amino acids with similar properties are generally considered to be a family of amino acids with similar side chains, which are clearly defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, lactate, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Furthermore, the addition of one or more amino acids, for example, to the amino and / or carboxy termini, generally does not alter the function of the polypeptide or protein. Conservative amino acid substitutions for various commonly known non-genetically encoded amino acids are known in the art.Conservative substitutions for other non-encoded amino acids are determined by comparing their physical properties with the properties of the genetically encoded amino acids.

[0051] In the context of two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "percentage identity" refers to two or more sequences or subsequences that are the same, or have a specified percentage of amino acid residues or nucleotides that are the same over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using methods known in the art, such as sequence comparison algorithms, by manual alignment or by visual inspection. For example, preferred algorithms suitable for determining percentage sequence identity and percentage sequence similarity are the BLAST and BLAST 2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol. 215:403, respectively.

[0052] Those skilled in the art are aware that gene cloning always requires the design of appropriate restriction enzyme cleavage sites, resulting in the introduction of one or more extraneous residues at the termini of the expressed polypeptide or protein without affecting the activity of the target polypeptide or protein. Furthermore, specific amino acids must always be added to the N-terminus, C-terminus, or other appropriate regions of a recombinant protein, for example, to construct a fusion protein, promote recombinant protein expression, obtain a recombinant protein that is automatically secreted outside of host cells, or aid in recombinant protein purification. Examples of other appropriate regions of a protein include, but are not limited to, appropriate linker peptides, signal peptides, leader peptides, terminal extensions, tags such as glutathione S-transferase (GST), maltose E-binding protein, protein A, 6His, or Flag, or protease sites for Factor Xa, thrombin, or enterokinase.

[0053] The optical probe of the present invention may comprise a lactate-sensitive polypeptide having a mutation, which may be at the P189 and / or P190 site and optionally at the M185 site of the lactate-binding protein or a functional fragment thereof.

[0054] Exemplarily, in one or more embodiments, the mutations are selected from the group consisting of P189R and P190D, P189R and P190A, P189R and P190I, P189R and P190Q, P189R and P190N, P189D and P190D, P189D and P190E, P189D and P190V, P189D and P190L, P189D and P190F, P189D and P190I, P189D and P190Q, P189D and P190N, P189D and P190G, P189D and P190Y, P189D and P190W, P189E and P190R , P189E and P190A, P189E and P190V, P189E and P190Q, P189A and P190L, P189A and P190F, P189A and P190M, P189A, P189A and P190N, P189A and P190G, P189A and P190H, P189A and P190T, P189V and P190D, P189V and P190E, P189V and P190A, P189V, P189V and P190N, P189V and P190H, P189V and P190Y, P189L and P190V, P189L and P190F, P1 89L and P190M, P189L and P190G, P189L and P190H, P189F and P190D, P189F and P190L, P189F and P190F, P189F and P190I, P189F and P190N, P189F and P190H, P189F and P190Y, P189F and P190K, P189F and P190T, P189F and P190W, P189I and P190R, P189I and P190D, P189I and P190A, P189I and P190V, P189I and P190M, P189I and P190Q, P189 I and P190G, P189I and P190Y, P189I and P190S, P189I and P190T, P189M and P190R, P189M and P190D, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189M and P190W, P189C and P190D, P189C and P190E, P189C and P190F, P189C and P190I, P189C and P190M, P189C and P190C, P189C, P189C and P190H, P189C and P190Y,P189C and P190S, P189C and P190W, P190L, P190F, P190I, P190Q, P190N, P190K, P190T, P189Q and P190E, P189Q and P190A, P189Q and P190V, P189Q and P190M, P189Q and P190C, P189Q and P190Q, P189Q and P190H, P189Q and P190S, P189N and P190R, P189N and P190D, P189N and P190L, P189N and P190F, P189N and P190C, P189N, P189N and P190N, P189N and P190G, P189N and P190H, P189N and P190Y, P189N and P190T, P189G and P190V, P189G and P190F, P189G and P190M, P189G and P190C, P189G and P190G, P189G and P190H, P189G and P190K, P189G and P190W, P189H and P190R, P189H and P190D, P189H and P190E, P189H and P190L, P189H and P190S, P189Y and 190R, P189Y and P19 0L, P189Y and P190N, P189Y and P190H, P189Y and P190S, P189Y and P190T, P189K and P190D, P189K and P190E, P189K and P190V, P189K and P190L, P189K and P190F, P189K and P190I, P189K and P190M, P189K, P189K and P190Q, P189K and P190N, P189K and P190Y, P189K and P190K, P189K and P190T, P189S and P190E, P189S and P190A, P189S and P190L, P189S and P190F, P189S and P190M, P189S and P190C, P189S, P189S and P190Q, 189S and P190Y, P189S and P190K, P189S and P190S, P189T and P190R, P189T and P190D, P189T and P190M, P189T and P190C, P189T, P189T and P190Q, P189T and P190N, P189T and P190H, P189T and P190Y, P189T and P190K, P189T and P190W, P189W and P190A,In some specific embodiments, the mutation is selected from P189W and P190V, P189W and P190F, P189W, P189W and P190Q, P189W and P190H, P189W and P190S, P189W and P190T, P189W and P190W. In some specific embodiments, the mutation is selected from P189R and P190A, P189D and P190D, P189D and P190E, P189D and P190Q, P189D and P190Y, P189A and P190N, P189A and P190G, P189V and P190H, P189F and P190I, P189F and P190N, P189F and P190K, P189 I and P190D, P189I and P190A, P189I and P190V, P189I and P190M, P189M and P190R, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189C and P190E, P190Q, P189Q and P190M, P189Q and P190C, P189N and P190N, P1 89G and P190F, P189H and P190L, P189H and P190S, P189Y and P190L, P189K and P190V, P189K and P190T, P189S and P190A, P189S and P190M, P189S and P190Q, P189S and P190K, P189S and P190S, P189T and P190D, P189W and P190A, P189W and P190T, or P189S, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F, or P189H and P190D. The response of the optical probe containing the above mutations to lactate is 1.5 times greater than that of the control group. In some embodiments, the mutations are, i.e., P189S, P189C and P190D, P189C and P190Y, P189N and P190Y, P189R and P190I, P189M and P190D, P189H and P190R, P189N, P189F and P190D, P189F and P190H, P189N and P190F, P189C and P190F,or P189H and P190D. Optical probes containing the above mutations respond to lactate more than twice as well as controls. In a preferred embodiment, the mutations are P189C and P190D, P189M and P190D, P189H and P190D, or P189F and P190D.

[0055] In a further embodiment, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E, or M185K. In a further preferred embodiment, the mutations further comprise M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, or M185K.

[0056] As used herein, the terms "functional variant," "derivative," and "analog" refer to a protein that essentially retains a biological function or activity homologous to that of the original polypeptide or protein (e.g., lactate-binding protein or fluorescent protein). A functional variant, derivative, or analog of a polypeptide or protein (e.g., lactate-binding protein or fluorescent protein) of the present invention may be (i) a protein in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, and the substituted amino acid residues may or may not be encoded by the genetic code; (ii) a protein having a substitution at one or more amino acid residues; (iii) a protein formed by fusing a mature protein to another compound (e.g., a compound that extends the half-life of the protein, such as polyethylene glycol); or (iv) a protein formed by fusing an additional amino acid sequence to the protein sequence (e.g., a secretory sequence, a sequence for purifying the protein, or a protein-constituting sequence, or a fusion protein formed with an antigenic IgG fragment). As taught herein, these functional variants, derivatives, and analogs are well known to those skilled in the art.

[0057] The analogs may differ from the original polypeptide or protein by differences in amino acid sequence, by modifications that do not affect sequence, or by both. These proteins include naturally occurring or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutations induced by radiation or exposure to mutagenic agents, or by site-directed mutagenesis or other known molecular biology techniques.

[0058] Analogs also include those with residues other than natural L-amino acids (e.g., D-amino acids) and those with non-naturally occurring or synthetic amino acids (e.g., β- and γ-amino acids). It should be understood that the lactate-sensitive polypeptides of the present invention are not limited to the representative proteins, variants, derivatives, and analogs listed above. Modified forms (which generally do not alter the primary structure) include chemically derivatized forms of proteins in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, including proteins produced by glycosylation modifications during protein synthesis and processing or during further processing. Such modifications are performed by exposing the protein to a glycosylation enzyme (e.g., a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Further included are proteins that have been modified to enhance their anti-proteolytic properties or improve their solubility.

[0059] The fusion polypeptides of the present invention comprise an optical probe described herein and another polypeptide. In some embodiments, the optical probe described herein further comprises another polypeptide fused thereto. The other polypeptide described herein does not affect the properties of the optical probe. The other polypeptide may be located at the N-terminus and / or C-terminus of the optical probe. In some embodiments, the other polypeptide includes a polypeptide that localizes the optical probe to a different organelle or suborganelle, and a tag for purification or immunoblotting. A linker may be present between the optical probe and the other polypeptide in the fusion polypeptide described herein.

[0060] Suborganelles described herein include the cytoplasm, mitochondria, nucleus, endoplasmic reticulum, plasma membrane, Golgi apparatus, lysosomes, and peroxisomes. In some embodiments, tags for purification or immunoblotting include six histidines (6*His), glutathione-S-transferase (GST), and Flag.

[0061] As used herein, the terms "nucleic acid," "nucleotide," "polynucleotide," or "nucleic acid sequence" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be the coding strand or the non-coding strand. When referring to nucleic acids, the term "variant" as used herein may refer to naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include degenerate variants, substitution variants, deletion variants, and insertion variants. As known in the art, allelic variants are interchangeable forms of a nucleic acid, which may result in the substitution, deletion, or insertion of one or more nucleotides, but do not substantially alter the function of the encoded protein. The nucleic acids of the present invention may comprise nucleotide sequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% sequence identity to the nucleic acid sequences. The present invention further relates to nucleic acid fragments that hybridize to the above sequences. In an exemplary embodiment, the nucleic acid sequence is shown in SEQ ID NO:31, which shows the coding sequence of a probe with a P189F / P190D mutation and a cpYFP insertion at the 185 / 189 position of a functional fragment of lactate binding protein. As used herein, the length of a "nucleic acid fragment" comprises at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR).

[0062] The full-length sequence or fragments of the optical probe or fusion protein of the present invention can be obtained typically by PCR amplification, artificial synthesis, or recombinant techniques. For PCR amplification, primers are designed based on the nucleotide sequence disclosed herein, and related sequences are obtained by amplifying a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. If the nucleotide sequence is longer than 2500 bp, PCR amplification is preferably performed 2 to 6 times, and the amplified fragments are then ligated in the correct order. The present invention does not particularly limit the PCR amplification process or system; conventional PCR amplification processes and systems in the field can be used. Furthermore, large quantities of related sequences can be obtained by recombinant techniques. Typically, the sequence is cloned into a vector, introduced into cells, and then isolated and purified from host cells grown using conventional methods to obtain related polypeptides or proteins. Furthermore, particularly when the fragments are short, related sequences can be synthesized by artificial synthesis. In the present invention, if the nucleotide sequence of the optical probe is shorter than 2500 bp, it can be synthesized by artificial synthesis. The artificial synthesis method is a conventional DNA artificial synthesis method in the field, and no other requirements apply. Typically, multiple small fragments are synthesized and then joined to obtain a longer fragment. Currently, DNA sequences encoding the proteins of the present invention (or functional variants, derivatives, or analogs thereof) can be obtained solely by chemical synthesis. The DNA sequences are then introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art. Mutations may also be introduced into the protein sequences of the present invention by methods such as mutagenic PCR or chemical synthesis.

[0063] The present invention further provides a detection reagent kit comprising the optical probe, fusion polypeptide, or polynucleotide described herein, or an optical probe or fusion polypeptide prepared by the methods described herein, and optionally further comprising other reagents for detecting lactate with the optical probe, such other reagents being conventional and known in the art.

[0064] The present invention also relates to nucleic acid constructs comprising the polynucleotides described herein and one or more regulatory sequences operably linked to these sequences. The polynucleotides of the present invention may be manipulated in various ways to ensure expression of the polypeptide or protein. Prior to insertion of the nucleic acid construct into a vector, the nucleic acid construct may be manipulated according to the differences or requirements of the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are known in the art.

[0065] In certain embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombination vector. The polynucleotides of the present invention may be cloned into various types of vectors, such as plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors can be used to provide the coding sequence for the protein or polypeptide of the present invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Expression of the polynucleotide of the present invention is typically achieved by operably linking the polynucleotide of the present invention to a promoter and introducing the construct into an expression vector. Such vectors are suitable for replication and integration in eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence. In one or more embodiments, the cloning vector and the expression vector are the same vector, i.e., a cloning expression vector. A homologous recombination vector is used to integrate the expression cassettes described herein into a host genome.

[0066] As used herein, the term "expression control sequence" refers to an element that regulates the transcription, translation, and expression of a target gene and is operably linked to the target gene. It may be a replication origin, promoter, marker gene, or translation control element, including an enhancer, operon, terminator, ribosome binding site, etc. The choice of expression control sequence is determined by the host cell to be used. In a recombinant expression vector, "operably linked" refers to the target nucleotide sequence being linked to a regulatory sequence in a form that allows the nucleotide sequence to be expressed. Methods for constructing expression vectors containing the coding sequence of the fusion protein of the present invention and appropriate transcriptional / translational control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence may be operatively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include the Escherichia coli lac or trp promoter; the λ phage PL promoter; and eukaryotic promoters, including the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters of controllable genes expressed in prokaryotic or eukaryotic cells or their viruses. The expression vector further comprises a ribosome binding site used for translation initiation and a transcription terminator. In one embodiment, the expression vector can be the commercially available pET28a vector, with no other requirements. For example, the nucleotide sequence encoding the optical probe and the expression vector are double-digested with BamHI and EcoRI, respectively, and the digest products are then ligated to obtain a recombinant expression vector. The present invention is not limited to specific steps and parameters for digestion and ligation; conventional steps and parameters in the art can be used.

[0067] To produce a protein or peptide, including a fusion protein, a recombinant expression vector is obtained and then transformed into a host cell. This introduction process can be carried out by conventional techniques, such as transformation or transfection, familiar to those skilled in the art. The term "host cell" as used herein refers to a cell capable of receiving and accommodating a recombinant DNA molecule and a site for amplifying the recombinant gene. Desirable recipient cells satisfy two conditions: they must be easily obtainable and easily propagated. The "host cell" of the present invention includes prokaryotic and eukaryotic cells, specifically bacterial cells, yeast cells, insect cells, and mammalian cells. Specific examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fruit fly S2 or Sf9; and animal cells such as CHO, COS, HEK293, HeLa, or Bowes melanoma cells, but are not limited to these. The host cell may be any of a variety of cells advantageous for the expression or fermentation of a gene product. These cells are well known and commonly used in the art. Those skilled in the art will know clearly how to select appropriate vectors, promoters, enhancers and host cells.

[0068] The method for introducing the DNA into the host cell 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, chemically mediated transfection, or electroporation. When the host is a prokaryotic cell such as E. coli, the preferred method is treatment with CaCl or MgCl, and the procedures used are well known in the art. When the host cell is a eukaryotic cell, a DNA transfection method such as calcium phosphate co-precipitation, or a conventional mechanical method such as microinjection, electroporation, or liposome packaging may be selected.

[0069] In the present invention, after introducing an expression vector into a host cell, the host cell introduced with the expression vector is amplified, expressed, and cultured, and then separated to obtain a lactate optical probe. Conventional methods can be used for amplifying, expressing, and culturing the host cell. The culture medium used for the culture may be any of a variety of conventional media depending on the type of host cell used. The host cell is cultured under conditions suitable for its growth.

[0070] In the present invention, the optical probe is expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein may be separated or purified using various separation methods based on its physical, chemical, and other properties. The present invention is not particularly limited to the method for separating the lactate fluorescent protein, and conventional separation methods for fusion proteins in the art may be used. These methods are well known to those skilled in the art and include, but are not limited to, conventional renaturation treatments, salting-out methods, centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods. In one embodiment, the optical probe is separated by affinity chromatography of the His tag.

[0071] The present invention also provides uses of the lactate optical probe in real-time localization and quantitative detection of lactate and high-throughput compound screening. In one embodiment, the lactate optical probe is preferably linked to signal peptides at different locations in the cell and introduced into the cell. The intensity of the fluorescent signal in the cell is detected to perform real-time localization of lactate, and the corresponding quantitative detection of lactate is performed using a lactate standard titration curve. The lactate standard titration curve of the present invention is generated based on the fluorescent signal of the lactate optical probe at different concentrations of lactate. Because the lactate optical probe of the present invention is directly introduced into the cell, the real-time localization and quantitative detection of lactate does not require time-consuming sample processing, making it more accurate. In high-throughput compound screening, the lactate optical probe of the present invention is used to add different compounds to a cell culture medium and measure changes in lactate content to select compounds that affect the changes in lactate content. The use of the lactate optical probe of the present invention in real-time localization and quantitative detection of lactate and high-throughput compound screening is not intended for diagnostic or therapeutic purposes and is not related to the diagnosis or treatment of diseases.

[0072] [Specific embodiment] 1. An optical probe comprising a lactate-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is within the sequence of the lactate-sensitive polypeptide.

[0073] 2. The optical probe according to embodiment 1, wherein the lactate-sensitive polypeptide has the sequence shown in SEQ ID NO: 1 or a functional fragment thereof, or a sequence having at least 70% sequence identity thereto; Preferably, the lactate-sensitive polypeptide has a sequence as set forth in amino acids 80 to 258 of SEQ ID NO: 1, or as set forth in SEQ ID NO: 1, or a sequence having at least 70% sequence identity thereto; Preferably, the lactate-sensitive polypeptide comprises mutations at the following sites: (1) P189 and / or P190, and optionally (2) a mutation at M185; more preferably, the mutations are: (1) P189R and P190D, P189R and P190A, P189R and P190I, P189R and P190Q, P189R and P190N, P189D and P190D, P189D and P190E, P189D and P190V, P189D and P190L, P189D and P190F, P189D and P190I, P189D and P 190Q, P189D and P190N, P189D and P190G, P189D and P190Y, P189D and P190W, P189E and P190R, P189E and P190A, P189E and P190V, P189E and P190Q, P189A and P190L, P189A and P190F, P189A and P190M, P189A, P189A and P190N, P189A and P190G, P189A and P190H, P189A and P190T, P189V and P190D, P189V and P190E, P189V and P190A, P18 9V, P189V and P190N, P189V and P190H, P189V and P190Y, P189L and P190V, P189L and P190F, P189L and P190M, P189L and P190G, P189L and P190H, P189F and P190D, P189F and P190L, P189F and P190F, P189F and P190I, P189F and P190N, P189F and P190H, P189F and P190Y, P189F and P190K, P189F and P190T, P189F and P190W, P189I and P190 R, P189I and P190D, P189I and P190A, P189I and P190V, P189I and P190M, P189I and P190Q, P189I and P190G, P189I and P190Y, P189I and P190S, P189I and P190T, P189M and P190R, P189M and P190D, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189M and P190W, P189C and P190D, P189C and P190E, P189C and P190F,P189C and P190I, P189C and P190M, P189C and P190C, P189C, P189C and P190H, P189C and P190Y, P189C and P190S, P189C and P190W, P190L, P190F, P190I, P190Q, P190N, P190K, P190T, P189Q and P190E, P189Q and P190A, P189Q and P190V, P189Q and P190M, P189Q and P190C, P189Q and P190Q, P189Q and P190H, P189Q and P190S, P189N and P190R, P189N and P190D, P189N and P190L, P189N and P190F, P189N and P190C, P189N, P189N and P190N, P189N and P190G, P189N and P190H, P189N and P190Y, P189N and P190T, P189G and P190V, P189G and P190F, P189G and P190M, P189G and P190C, P189G and P190G, P189G and P190H, P189G and P190K, P189G and P190W, P189H and P190R, P 189H and P190D, P189H and P190E, P189H and P190L, P189H and P190S, P189Y and P190R, P189Y and P190L, P189Y and P190N, P189Y and P190H, P189Y and P190S, P189Y and P190T, P189K and P190D, P189K and P190E, P189K and P190V, P189K and P190L, P189K and P190F, P189K and P190I, P189K and P190M, P189K, P189K and P190Q, P189K and P1 90N, P189K and P190Y, P189K and P190K, P189K and P190T, P189S and P190E, P189S and P190A, P189S and P190L, P189S and P190F, P189S and P190M, P189S and P190C, P189S, P189S and P190Q, 189S and P190Y, P189S and P190K, P189S and P190S, P189T and P190R, P189T and P190D, P189T and P190M, P189T and P190C, P189T, P189T and P190Q,P189T and P190N, P189T and P190H, P189T and P190Y, P189T and P190K, P189T and P190W, P189W and P190A, P189W and P190V, P189W and P190F, P189W, P189W and P190Q, P189W and P190H, P189W and P190S, P189W and P190T, P189W and P190W, and any (2) optical probes including M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E or M185K.

[0074] 3. The optical probe according to embodiment 1 or 2, wherein the optically active polypeptide is located in a region of the lactate-sensitive polypeptide selected from the following: 93-97, 119-121, 137-141, 158-161, 185-191, 208-210, and / or 230-232. Preferably, the optically active polypeptide is located in one or more regions of the lactate-sensitive polypeptide selected from the following: 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 121, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 13 8 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 160, 159 / 161, 160 / 161, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 190, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232 and / or 231 / 232, optical probes.

[0075] 4. An optical probe according to embodiment 3, wherein the optically active polypeptide is located at residues 185 to 191 of the lactate-sensitive polypeptide, and preferably, the optical probe of the present invention has or consists of the sequence shown in SEQ ID NOs: 6 to 30, 34 to 40.

[0076] 5. A nucleic acid sequence comprising: (1) A polynucleotide encoding the optical probe according to any one of embodiments 1 to 4; (2) Fragment of (1), (3) A nucleic acid sequence selected from the complementary sequence of (1) or (2).

[0077] 6. A nucleic acid construct comprising the nucleic acid sequence of embodiment 5, which is preferably an expression vector.

[0078] 7. A host cell comprising: (1) The optical probe according to any one of embodiments 1 to 4 is produced, (2) comprising the nucleic acid sequence of embodiment 5; or (3) The nucleic acid construct according to embodiment 6, Preferably, the host cell wherein said nucleic acid construct is an expression vector.

[0079] 8. A method for preparing the optical probe according to any one of embodiments 1 to 4, comprising culturing the host cell according to embodiment 7, and isolating the optical probe from the culture.

[0080] 9. Use of an optical probe according to any one of embodiments 1 to 4, a nucleic acid sequence according to embodiment 5, or a nucleic acid construct according to embodiment 6 in detecting lactate in a sample or selecting a compound, preferably wherein the detection is localized or quantitative detection of lactate.

[0081] 10. A detection reagent kit, comprising: (1) An optical probe according to any one of embodiments 1 to 4 or an optical probe prepared by the method according to embodiment 8; (2) the nucleic acid sequence of embodiment 5; (3) The nucleic acid construct according to embodiment 6, or (4) The cell of embodiment 7, and and other reagents for detecting lactate with an optical probe.

[0082] Concentrations, contents, percentages, and other numerical values ​​may be expressed in range format herein. While range formats have been adopted for convenience and brevity, they should be understood to include the numerical values ​​expressly stated as the upper and lower limits of the range, and to include, as appropriate, all individual numerical values ​​or subranges contained within the range. [Example]

[0083] The lactate optical probe provided by the present invention will be described in detail below with reference to examples, but it should be understood that the scope of the present invention is not limited thereto.

[0084] I. Experimental materials and reagents The examples mainly use conventional genetic engineering, molecular biology cloning methods, cell culture, and imaging methods, etc. These methods are familiar to those skilled in the art. For example, see Jane Roskams et al., "Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench"; Joseph Sambrook and David W. Russell, translated by Huang Pedang et al., "Molecular Cloning: A Laboratory Manual" (3rd ed., August 2002, published by Science Press, Beijing); R.I. Freshney et al., "Culture of Animal Cells: a Manual of Basic Technique" (5th ed.), translated by Zhang Jingbo, Xu Cunqian et al.; Juan S. Bonifacino, M. Dassault et al., "Short Protocols in Cell Biology" translated by Zhang Jingbo et al.

[0085] The pCDFDuet-cpYFP and pCDFDuet-lactate-binding protein-based plasmids used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pCDFDuet plasmid vector was purchased from Novagen. All primers used in PCR were synthesized and purified by Shanghai Jierui Bioengineering Technology Co., Ltd. and verified to be appropriate by mass spectrometry. The expression plasmids constructed in the examples were sequenced. Sequencing was performed by China University Genetics Company and Jie Li Sequencing Company. The Taq DNA polymerase used in each example was purchased from Dongsheng Biological, the pfu DNA polymerase was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the primeSTAR DNA polymerase was purchased from TaKaRa. The three polymerases were purchased with the corresponding polymerase buffer and dNTPs. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, and SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas, and the corresponding buffers were included with the purchase. The transfection reagent Lip2000 Kit was purchased from Invitrogen. All compounds, including lactic acid, were purchased from Sigma. Unless otherwise noted, all chemical reagents, including inorganic salts, were purchased from Sigma-Aldrich. HEPES salt, streptomycin sulfate, and puromycin were purchased from Ameresco. The 96-well test plate (black) and 384-well fluorescent test plate (black) were purchased from Grenier.

[0086] The DNA purification reagent kit used in the examples was purchased from BBI (Canada), and the general plasmid small-scale extraction kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The clone strain Mach1 was purchased from Invitrogen. The Ni-NTA affinity column and desalting column packing were purchased from GE Healthcare.

[0087] The main equipment used in the examples includes a Biotek Synergy 2 multi-mode microplate reader (Bio-Tek, USA), an X-15R high-speed refrigerated centrifuge (Beckman, USA), a Microfuge22R tabletop high-speed refrigerated centrifuge (Beckman, USA), a PCR amplification device (Biometra, Germany), an ultrasonic homogenizer (Ningbo Xinzhi Co., Ltd.), a nucleic acid electrophoresis device (Shen Neng Bocai Co., Ltd.), a fluorescence spectrophotometer (Varian, USA), a CO2 incubator (SANYO), and an inverted fluorescence microscope (Nikon, Japan).

[0088] II. Molecular Biology and Cell Experimental Methods II.1 Polymerase Chain Reaction (PCR): 1. Target fragment amplification PCR: This method is primarily used for amplifying gene fragments and identifying positive clones by colony PCR. The PCR amplification reaction system is as follows: 0.5-1 μL of template sequence, 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 5 μL of 10× pfu buffer, 0.5 μL of pfu DNA polymerase, 1 μL of 10 mM dNTPs, and 41.5-42 μL of sterile ultrapure water (ddH2O), for a total volume of 50 μL. The PCR amplification process is as follows: denaturation at 95°C for 2-10 minutes, 30 cycles (hold at 94-96°C for 30-45 seconds, hold at 50-65°C for 30-45 seconds, hold at 72°C for a fixed time (600 bp / min)), and extension at 72°C for 10 minutes.

[0089] 2. Long fragment (>2500bp) amplification PCR: The long fragment amplification used in this invention primarily involves an inverse PCR amplification vector, a technique used to obtain site-specific mutations in the examples below. Inverse PCR primers are designed for the mutation site, with one primer containing the mutated nucleotide sequence at its 5' end. The amplified product contains the corresponding mutation site. The long fragment amplification PCR reaction system is as follows: 1 μL of template sequence (10 pg to 1 ng), 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 10 μL of 5x PrimerSTAR buffer, 0.5 μL of PrimerSTAR DNA polymerase, 4 μL of dNTPs (2.5 mM), and 33.5 μL of sterile ultrapure water (ddH2O), for a total volume of 50 μL. The PCR amplification process was as follows: denaturation at 95°C for 5 minutes, followed by 30 cycles (hold at 98°C for 10 seconds, hold at 50-68°C for 5-15 seconds, hold at 72°C for a certain period of time (1000 bp / min)), and extension at 72°C for 10 minutes; alternatively, denaturation at 95°C for 5 minutes, followed by 30 cycles (hold at 98°C for 10 seconds, hold at 68°C for a certain period of time (1000 bp / min)), and extension at 72°C for 10 minutes.

[0090] II.2 Endonuclease digestion: The double digest system for the plasmid vector is as follows: 20 μL (approximately 1.5 μg) of plasmid vector, 5 μL of 10x buffer, 11-2 μL of restriction endonuclease 1, and 1-2 μL of restriction endonuclease 2, supplemented with sterile ultrapure water to a total volume of 50 μL. The reaction conditions are 37°C and the reaction is allowed to proceed for 1-7 hours.

[0091] II.3 Phosphorylation of the 5' ends of DNA fragments The termini of plasmids or genomes extracted from microorganisms contain phosphate groups, but PCR products do not. Because ligation reactions are not possible without phosphate groups at the ends of DNA molecules, a phosphate group addition reaction is performed on the 5' end of the PCR product. The phosphorylation reaction system is as follows: 5-8 μL of PCR product fragment DNA sequence, 1 μL of 10x T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), and 0-3 μL of sterile ultrapure water, for a total volume of 10 μL. The reaction is carried out at 37°C for 30 minutes to 2 hours, followed by inactivation at 72°C for 20 minutes.

[0092] II.4 Ligation of target fragment and vector There are different methods for ligating different fragments to vectors, and three ligation methods were used in this invention.

[0093] 1. Blunt-end ligation of a blunt-ended short fragment with a linear vector The mechanism of this method is as follows: the blunt-ended PCR product is subjected to the action of T4 PNK to phosphorylate the 5' end of the DNA fragment, and then ligated to the linearized vector using PEG4000 and T4 DNA ligase to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: 4 μL of T4 PNK-treated DNA fragment, 4 μL of linear vector fragment, 1 μL of PEG4000, 1 μL of 10x T4 ligase buffer, and 1 μL of T4 DNA ligase, for a total of 10 μL. The reaction is carried out at 22°C for 30 minutes.

[0094] 2. Ligation of a DNA fragment containing a sticky end with a vector fragment containing a sticky end DNA fragments digested with restriction endonucleases typically produce protruding sticky ends, which can be ligated to vector fragments containing complementary sticky ends to form recombinant plasmids. The ligation reaction system is as follows: 1-7 μL of digested PCR product DNA, 0.5-7 μL of digested plasmid, 1 μL of 10x T4 ligase buffer, and 1 μL of T4 DNA ligase, supplemented with sterile ultrapure water to a total volume of 10 μL. The reaction is carried out at 16°C for 4-8 hours.

[0095] 3. After site-specific mutations are introduced by inverse PCR, the 5'-terminal phosphorylated DNA fragments are ligated by self-cyclization. The 5'-terminal phosphorylated DNA fragment is ligated via self-cyclization to the 3' and 5' ends of the linear vector, yielding a recombinant plasmid. The self-cyclization ligation reaction system is as follows: 10 μL of phosphorylation reaction system, 0.5 μL of T4 ligase (5 U / μL), and a total volume of 10.5 μL. The reaction is carried out at 16°C for 4 to 16 hours.

[0096] II.5 Preparation and transformation of competent cells Preparation of competent cells: 1. Select a single colony (e.g., Mach1), inoculate 5 mL of LB medium, and place in a shaker at 37°C overnight. 2. Transfer 0.5 to 1 mL of the overnight cultured bacterial solution to 50 mL of LB medium and culture at 37°C, 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 and resuspend the cells in 5 mL of pre-chilled buffer until homogenous, then add resuspension buffer to bring the final volume to 50 mL. 6. Place in an ice bath for 45 minutes. 7. Centrifuge at 4000 rpm for 10 min at 4°C and resuspend the bacteria in 5 mL of storage buffer pre-chilled on ice. 8. Add 100 μL of bacterial suspension to each EP tube and store frozen at -80°C or in liquid nitrogen. Resuspension buffer: CaCl2 (100mM), MgCl2 (70mM), NaAc (40mM) Storage buffer: 0.5mL DMSO, 1.9mL 80% glycerin, 1mL 10X CaCl2 (1M), 1mL 10X MgCl2 (700mM), 1mL 10X NaAc (400mM), 4.6mL ddH2O

[0097] Transformation of competent cells: 1. Take 100 μL of competent cells and thaw them in an ice bath. 2. Add an appropriate volume of ligation product, blow gently and evenly, and place in an ice bath for 30 minutes. The volume of the ligation product added is usually less than 1 / 10 of the volume of the competent cells. 3. Place the bacterial solution in a 42°C water bath for 90 seconds to heat shock it, then quickly transfer it to an ice bath and leave it for 5 minutes. 4. Add 500 μL of LB and culture in a 37°C thermostatic shaker at 200 rpm for 1 hour. 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, retain 200 μL of the supernatant, spray the bacteria evenly, and spread it evenly on the surface of an agar plate containing the appropriate antibiotic. Invert the plate and place it in a 37°C incubator overnight.

[0098] II.6 Protein expression, purification and fluorescence detection 1. Transform JM109(DE3) cells with the expression vector, invert the cells, and culture overnight. Select from the plate and clone into a 250 ml Erlenmeyer flask. Place in a shaker at 37°C and culture at 220 rpm until the OD reaches 0.4-0.8. Add 1 / 1000 (v / v) IPTG (1M) and induce expression at 18°C ​​for 24-36 hours. 2. After induction and expression are complete, harvest the cells by centrifugation at 4000 rpm for 30 minutes, resuspend the cell pellet in 50 mM phosphate buffer, and sonicate until the cells are transparent. Centrifuge at 9600 rpm for 20 minutes at 4°C. 3. The supernatant was centrifuged and purified using a self-loading Ni-NTA affinity column to obtain the protein. The protein treated by Ni-NTA affinity chromatography was further treated using a self-loading desalting column to obtain the protein dissolved in 20 mM MOPS buffer (pH 7.4) or phosphate buffer PBS. 4. After the purified protein is analyzed by SDS-PAGE, dilute the probe in assay buffer (100 mM HEPES, 100 mM NaCl, pH 7.3) or phosphate buffer PBS to a final concentration of 5-10 μM. Prepare a stock solution containing lactate at a final concentration of 1 M in assay buffer (20 mM MOPS, pH 7.4) or phosphate buffer PBS. 5. Take 100 μl of the 5 μM protein solution, incubate at 37°C for 5 minutes, add lactic acid to the solution to a final concentration of 100 mM, and mix thoroughly. Then measure the absorbance of the protein at 340 nm using a multi-mode fluorescence microplate reader. 6. Take 100 μl of 1 μM protein solution, incubate at 37°C for 5 minutes, add lactate to titrate, and measure the intensity of 528 nm fluorescence emitted by the protein when excited by 485 nm fluorescence. The excitation and emission fluorescence of the sample are measured using a multi-mode fluorescence microplate reader. 7. Take 100 μl of 1 μM protein solution, incubate at 37 °C for 5 minutes, add lactic acid, and measure the absorption spectrum and fluorescence spectrum of the protein. The absorption spectrum and fluorescence spectrum of the sample are measured using a spectrophotometer and a spectrofluorometer.

[0099] II.7 Mammalian cell transfection and fluorescence detection 1. Transfect HeLa cells with the pCDNA3.1+-based lactate optical probe plasmid using the transfection reagent Lipofectamine 2000 (Invitrogen) and culture them in a 37°C, 5% CO2 incubator. Fluorescence detection is performed after sufficient expression of the foreign gene for 24-36 hours. 2. After the induction expression was completed, the adherent cultured HeLa cells were washed three times with PBS and placed in HBSS solution for fluorescence microscopy and microplate reader detection, respectively.

[0100] Example 1. Lactate-binding protein plasmid The LldR (80-258) gene in E. coli was amplified by PCR. The PCR product was subjected to gel electrophoresis, recovered, and then digested with BamHI and EcoRI. At the same time, the corresponding double digest was performed on the pCDFDuet1 vector. After ligation with T4 DNA ligase, the product was transformed with MachI. The transformed MachI plates were plated on LB plates (streptomycin sulfate 50 μg / mL) and cultured overnight at 37°C. Plasmids were extracted from the grown MachI transformants and analyzed by PCR. Positive plasmids were sequenced, and if confirmed, further plasmid construction was performed.

[0101] Example 2. Expression and detection of cpYFP optical probes with different insertion sites In this example, based on pCDFDuet-LldR(80-258), the following sites were identified according to the crystal structure of lactate-binding protein: 93 / 94, 93 / 95, 93 / 96, 93 / 97, 94 / 95, 94 / 96, 94 / 97, 95 / 96, 95 / 97, 96 / 97, 119 / 120, 119 / 12 1, 120 / 121, 137 / 138, 137 / 139, 137 / 140, 137 / 141, 138 / 139, 138 / 140, 138 / 141, 139 / 140, 139 / 141, 140 / 141, 158 / 159, 158 / 160, 158 / 161, 159 / 160, 159 / 161, 160 / 161 1, 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 188, 187 / 189, 187 / 190, 187 / 191, 188 / 189, 188 / 19 0, 188 / 191, 189 / 190, 189 / 191, 190 / 191, 208 / 209, 208 / 210, 209 / 210, 230 / 231, 230 / 232, or 231 / 232 were selected for cpYFP insertion, resulting in the corresponding pCDFDuet-LldR(80-258)-cpYFP plasmid. The amino acid sequences of exemplary optical probes are shown in Table 1.

[0102] [Table 1]

[0103] The cpYFP DNA fragment was generated by PCR, and the pCDFDuet-LldR(80~258) linear vector containing different cleavage sites was generated by inverse PCR amplification. The linearized pCDFDuet-LldR(80~258) was ligated with the cpYFP fragment using homologous recombination recombinase to generate a recombinant plasmid. Positive clones were selected by colony PCR and sequenced by Shanghai Jie Li Biotechnology Co., Ltd.

[0104] If the sequencing results were accurate, the recombinant plasmid was transformed into BL21(DE3) and induced for expression. The protein was purified and subjected to SDS-PAGE electrophoresis, revealing a size of approximately 48 kda. This size was consistent with that of the LldR(80-258)-cpYFP fusion protein containing a His-tag purification tag expressed from pCDFDuet-LldR(80-258)-cpYFP. The results are shown in Figure 1.

[0105] The lactate response of purified LldR(80-258)-cpYFP fusion protein and the control protein cpYFP was measured. The detection signal of the fusion fluorescent protein containing 10 mM lactate was divided by the detection signal of the fusion fluorescent protein without lactate. The results are shown in Figure 2. The detection results showed that the optical probes with a lactate response of more than 1.5-fold were the 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 187 / 189, 189 / 191, and 190 / 191 sites or the corresponding amino acid sites in their family proteins.

[0106] Example 3. Expression and detection of cpGFP optical probes at different insertion sites A lactate-related green fluorescent protein fluorescent probe was constructed by replacing cpYFP with cpGFP according to the method of Example 2, and expression and detection were carried out according to the method of Example 2. As shown in Figure 3, the detection results showed that the optical probes with insertions at the 188 / 190 and 189 / 190 sites showed a greater than 1.5-fold response to lactate.

[0107] Example 4. Expression and detection of cpBFP optical probes at different insertion sites Using the method described in Example 2, cpYFP was replaced with cpBFP to construct a lactate blue fluorescent protein fluorescent probe, which was then expressed and detected using the method described in Example 2. As shown in Figure 4, the detection results showed that the optical probes with insertions at the 187 / 190 and 187 / 191 sites showed a greater than 1.5-fold response to lactate.

[0108] Example 5: Expression and detection of cpmApple optical probes at different insertion sites Using the method described in Example 2, cpYFP was replaced with cpmApple to construct a lactate-sensitive red fluorescent protein fluorescent probe, which was then expressed and detected using the method described in Example 2. This is shown in Figure 5. The detection results showed that the optical probes with a response to lactate of more than 1.5 times were those inserted at the 185 / 190, 185 / 191, 186 / 190, 186 / 191, 187 / 189, and 188 / 191 sites.

[0109] Example 6. Expression and detection of two-site mutants of optical probes Two mutants, 189 and 190, were constructed using LldR(80-258)-185 / 189-cpYFP. The inverse PCR linearization plasmid pCDFDuet-LldR(80-258)-185 / 189-cpYFP and the mutated sequences with the mutation sites introduced into the primers were used to ligate the resulting PCR products via phosphate group addition using PNK, T4 DNA ligase, and PEG4000. BL21(DE3) was transformed and screened. The detection signals of the fusion protein and control protein cpYFP in the presence of 10 mM lactate were divided by the detection signals in the absence of lactate. The lactate response of each mutant is shown in Table 2. Samples with a greater than two-fold response to lactate were P189 / P190 (WT), P189S (12H4), P189C / P190D (14B5), P189C / P190Y (14D2), P189N / P190Y (3D1), P189R / P190I (14H5), P189M / P190D (10F4), P189H / P190R (13H6), P189N (2F5), P189F / P190D (3C2), P189F / P190H (2G3), P189N / P190F (9B6), P189C / P190F (2A5), and P189H / P190D (1A3). The sequences of exemplary optical probe mutants are shown in Table 3. An exemplary nucleic acid sequence is shown in SEQ ID NO:31 (LldR(80-258)-185 / 189-P189F / P190D-cpYFP).

[0110] [Table 2]

[0111] [Table 3]

[0112] Example 7. Titration curves of two-site mutant optical probes The optical probes obtained in Example 6, which showed a response to lactate of more than two times, namely, P189 / P190 (WT), P189N (2F5), P189C / P190F (2A5), P189N / P190F (9B6), P189N / P190Y (3D1), P189H / P190R (13H6), P189R / P190I ( Lactate detection was performed using a concentration gradient for the following mutants: 2F5, 2A5, 9B6, 3D1, 13H6, 14H5, 2G3, 12H4, 14D2, 14B5, 10F4, 1A3, and 3C2. The ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was measured. The wild-type WT and mutants 2F5, 2A5, 9B6, 3D1, 13H6, 14H5, 2G3, 12H4, 14D2, 14B5, 10F4, 1A3, and 3C2 were used. d The binding constants were 112 μM, 67 μM, 114 μM, 46 μM, 95 μM, 194 μM, 70 μM, 152 μM, 88 μM, 41 μM, 57 μM, 234 μM, 100 μM, and 95 μM, respectively, and the rangeabilities were 3.4-fold, 2.1-fold, 2.3-fold, 2.5-fold, 3.1-fold, 3.2-fold, 3.5-fold, 3.7-fold, 3.8-fold, 4.3-fold, 5.3-fold, 7.0-fold, 8.9-fold, and 13.4-fold, respectively. The results are shown in Figure 7A.

[0113] Example 8. Expression and detection of three-site mutants of optical probes Among the mutants based on the two sites 189 and 190 in Example 7, the optical probes with a response to lactate of more than five times higher are P189C / P190D (14B5), P189M / P190D (10F4), P189F / P190D (3C2), and P189H / P190D (1A3). Based on these four mutants, saturation mutagenesis was performed on the M185 site, and the response of all mutants to lactate is shown in Table 4.Samples with a greater than two-fold response to lactate were M185F / P189F / P190D, M185I / P189F / P190D, M185G / P189F / P190D, M185H / P189F / P190D, M185A / P189F / P190D, M185S / P189F / P190D, M185V / P189F / P190D, M185F / P189H / P190D, M185Y / P189H / P190D, M185L / P189H / P190D, M185I / P189H / P190D, M185G / P189H / P190D, and M185Q / P189F / P190D. 189H / P190D, M185N / P189H / P190D, M185C / P189H / P190D, M185W / P189H / P1 90D, M185S / P189H / P190D, M185V / P189H / P190D, M185D / P189H / P190D, M18 5T / P189H / P190D, M185E / P189H / P190D, M185F / P189M / P190D, M185Y / P189 M / P190D, M185L / P189M / P190D, M185I / P189M / P190D, M185G / P189M / P190D, M185Q / P189M / P190D, M185H / P189M / P190D, M185A / P189M / P190D, M185C / P 189M / P190D, M185W / P189M / P190D, M185S / P189M / P190D, M185V / P189M / P1 90D, M185T / P189M / P190D, M185E / P189M / P190D, M185F / P189C / P190D, M18 5Y / P189C / P190D, M185L / P189C / P190D, M185I / P189C / P190D, M185G / P189 C / P190D, M185Q / P189C / P190D, M185H / P189C / P190D, M185A / P189C / P190D , M185P / P189C / P190D, M185N / P189C / P190D, M185C / P189C / P190D, M185W / P189C / P190D, M185S / P189C / P190D, M185V / P189C / P190D, M185D / P189C / P 190D, M185T / P189C / P190D, M185E / P189C / P190D, M185K / P189C / P190D.Samples with a greater than five-fold response to lactate were M185F / P189H / P190D, M185L / P189H / P190D, M185I / P189H / P190D, M185S / P189H / P190D, M185V / P189H / P190D, M185L / P189M / P190D, and M185A / P189M / P190D.

[0114] The sequences of exemplary optical probe mutants are shown in Table 5. Lactate detection was performed on these nine samples at a concentration gradient. The ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was detected. M185F / P189H / P190D, M185L / P189H / P190D(G9), M185I / P189H / P190D, M185S / P189H / P190D, M185V / P189H / P190D, M185L / P189M / P190D, and M185A / P189M / P190D(G25) were used. d The binding constants were 61 μM, 154 μM, 42 μM, 660 μM, 210 μM, 62 μM, and 1000 μM, respectively, and the rangeabilities were 8-fold, 15-fold, 5.6-fold, 7-fold, 6.2-fold, 5.5-fold, and 8.4-fold, respectively. The results are shown in Figure 7B.

[0115] [Table 4]

[0116] [Table 5]

[0117] Example 9. Spectral properties and specificity of optical probes For example, the purified lactate optical probes 3C2, 10F4 and G9 were treated with 0 mM and 10 mM lactate, respectively, for 10 minutes, and then the fluorescence spectra were detected by a fluorescence spectrophotometer.

[0118] Excitation spectra were recorded with an excitation range of 350 nm to 515 nm and an emission wavelength of 530 nm, with readings taken in 5 nm increments. The results show that probes 3C2, 10F4, and G9 have two excitation peaks at approximately 420 and 500 nm, as shown in Figures 8A, 8D, and 8G.

[0119] For emission spectrum measurements, the designated excitation wavelengths were 420 nm and 490 nm, respectively. The emission spectrum was recorded from 500 to 600 nm, and the readings were taken in 5 nm increments. The results showed that after the addition of 10 mM lactate, the fluorescence intensity at 420 nm excitation for probe 3C2 decreased to 0.31-fold that of 0 mM lactate, and the fluorescence intensity at 490 nm excitation increased to 4.2-fold that of 0 mM lactate. These results are shown in Figures 8B and 8C. For probe 10F4, the fluorescence intensity at 420 nm excitation decreased to 0.37-fold that of 0 mM lactate, and the fluorescence intensity at 490 nm excitation increased to 3-fold that of 0 mM lactate. These results are shown in Figures 8E and 8F. After the addition of 10 mM lactate, the fluorescence intensity of probe G9 at 420 nm excitation decreased to 0.18-fold compared to the case where 0 mM lactate was added, and the fluorescence intensity at 490 nm excitation increased to 2.8-fold compared to the case where 0 mM lactate was added, as shown in Figures 8H and 8I.

[0120] The specificity of the purified lactate optical probes 3C2, 10F4, G9 and G25 was measured and the results are shown in Figure 9, which show that the probes have good specificity.

[0121] Example 10. Suborganellar localization of optical probes In this example, different localization signal peptides are fused to the optical probes to localize the optical probes to different organelles. HeLa cells were transfected with optical probe plasmids fused to different localization signal peptides for 36 hours, washed with PBS, placed in HBSS solution, and subjected to fluorescence detection in the FITC channel using an inverted fluorescence microscope. The results are shown in Figure 10. By fusing the lactate optical probe to different specific localization signal peptides, it localizes to suborganelles, such as the cytoplasm, extracellular membrane, nucleus, endoplasmic reticulum, mitochondria, and nucleus excluded region. Fluorescence was observed in all suborganelle structures, with varying distribution and intensity of fluorescence.

[0122] Example 11. Dynamic monitoring of lactate transmembrane transport HeLa cells were transfected with the cytoplasmically expressed optical probe plasmid for 36 hours, washed with PBS, placed in HBSS, and then 10 mM lactate was added. The change in the ratio of fluorescence intensity at 420 nm excitation and 528 nm emission to fluorescence intensity at 485 nm excitation and 528 nm emission was measured within 30 minutes. The results are shown in Figure 11. The samples with added lactate showed a gradual increase in 485 / 420, with 3C2 and 10F4 reaching a maximum of 4.8- and 3.1-fold increases, respectively. In contrast, the control group without added lactate showed no change in 485 / 420, remaining at 1.

[0123] Example 12. Optical probe-based high-throughput compound screening in live cells In this example, a high-throughput compound screen was performed in HeLa cells for the cytoplasmically expressed lactate probe 3C2. Transfected HeLa cells were washed with PBS, placed in HBSS solution (without lactate), and treated for 1 hour, followed by treatment with 10 μM compounds for 1 hour. Lactate was added dropwise to each sample. The ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was recorded using a microplate reader. Samples not treated with any compound were used as a control group for normalization. The results are shown in Figure 12. Of the 2,000 compounds used, most had little effect on lactate entry into cells. Twelve compounds improved the cell's ability to uptake lactate, and six compounds significantly reduced the cell's lactate uptake.

[0124] Example 13. Quantitative detection of blood lactate using an optical probe In this example, lactate was analyzed in mouse and human blood supernatants with the purified lactate probe 3C2. After mixing the lactate probe 3C2 with the diluted blood supernatant for 10 minutes, the ratio of the fluorescence intensity at 420 nm excitation and 528 nm emission to the fluorescence intensity at 485 nm excitation and 528 nm emission was measured using a microplate reader. The results are shown in Figure 13. The lactate content in mouse blood is approximately 2.4 mM, and the lactate content in human blood is approximately 1.7 mM. According to the above examples, the lactate optical probe provided by the present invention has a relatively small protein molecular weight, is easy to mature, has large dynamic changes in fluorescence, and has good specificity. It can be expressed in cells by genetic engineering methods, and can perform real-time localization and quantitative detection of lactate inside and outside the cells, and can also be used for high-throughput compound screening.

[0125] [Other embodiments] Although several embodiments have been described herein, it should be understood that various modifications known from this specification are within the scope of the appended claims, provided that they do not depart from the spirit and scope of the present invention.

Claims

1. An optical probe comprising a lactate-sensitive polypeptide or a mutant thereof and an optically active polypeptide, wherein the optically active polypeptide is located within the sequence of the lactate-sensitive polypeptide; The lactate-sensitive polypeptide comprises a sequence set forth in amino acids 80 to 258 of SEQ ID NO: 1, or a sequence having at least 90% sequence identity thereto; The optically active polypeptide comprises a sequence set forth in any one of SEQ ID NOs: 2 to 5, or a sequence having at least 90% sequence identity thereto; the optically active polypeptide is located at one site of a lactate-sensitive polypeptide selected from the group consisting of 185 / 186, 185 / 187, 185 / 188, 185 / 189, 185 / 190, 185 / 191, 186 / 187, 186 / 188, 186 / 189, 186 / 190, 186 / 191, 187 / 189, 187 / 190, 187 / 191, 188 / 190, 188 / 191, 189 / 190, 189 / 191 and 190 / 191; The mutants of the lactate-sensitive polypeptide are P189R and P190D, P189R and P190A, P189R and P190I, P189R and P190Q, P189R and P190N, P189D and P190D, P189D and P190E, P189D and P190V, P189D and P190L, P189D and P190F, P189D and P190I, P189D and P190Q, P189D and P190N, P189D and P190G, P189D and P190Y, P189D and P190W, P189E and P190R, P189E and P190A, P189E and P190V, P189E and P19 0Q, P189A and P190L, P189A and P190F, P189A and P190M, P189A, P189A and P190N, P189A and P190G, P189A and P190H, P189A and P190T, P189V and P190D, P189V and P190E, P189V and P190A, P 189V, P189V and P190N, P189V and P190H, P189V and P190Y, P189L and P190V, P189L and P190F, P189L and P190M, P189L and P190G, P189L and P190H, P189F and P190D, P189F and P190L, P189F and P190F, P189F and P190I, P189F and P190N, P189F and P190H, P189F and P190Y, P189F and P190K, P189F and P190T, P189F and P190W, P189I and P190R, P189I and P190D, P189I and P190A, P189I and P190V, P189I and P190M, P189I and P190Q, P189I and P190G, P189I and P190Y, P189I and P190S, P189I and P190T, P189M and P190R, P189M and P190D, P189M and P190E, P189M and P190F, P189M and P190G, P189M and P190S, P189M and P190W, P189C and P190D, P189C and P190E, P189C and P190F, P189C and P190I, P189C and P190M, P189C and P190C, P189C, P189C and P190H, P189C and P190Y, P189C and P190S, P189C and P190W, P190L, P190F, P190I, P190Q, P190N, P190K, P190T, P 189Q and P190E, P189Q and P190A, P189Q and P190V, P189Q and P190M, P189Q and P190C, P189Q and P190Q, P189Q and P190H, P189Q and P190S, P189N and P190R, P189N and P190D, P189N and P190L, P189N and P190F, P189N and P190C, P189N, P189N and P190N, P189N and P190G, P189N and P190H, P189N and P190Y, P189N and P190T, P189G and P19 0V, P189G and P190F, P189G and P190M, P189G and P190C, P189G and P190G, P189G and P190H, P189G and P190K, P189G and P190W, P189H and P190R, P189H and P190D, P189H and P190E, P189H and P190L, P189H and P190S, P189Y and P190R, P189Y and P190L, P189Y and P190N, P189Y and P190H, P189Y and P190S, P189Y and P190T, P189K and P190D,P189K and P190E, P189K and P190V, P189K and P190L, P189K and P190F, P189K and P190I, P189K and P190M, P189K, P189K and P190Q, P189K and P190N, P189K and P190Y, P189K and P190K, P189K and P190T, P189S and P190E, P189S and P190A, P189S and P190L, P189S and P190F, P189S and P190M, P189S and P190C, P189S, P189S and P190Q, 189S and P190Y, P189S and P190K, P 189S and P190S, P189T and P190R, P189T and P190D, P189T and P190M, P189T and P190C, P189T, P189T and P190Q, P189T and P190N, P189T and P190H, P189T and P190Y, P189T and P190K, P Optical probes comprising the mutations P189T and P190W, P189W and P190A, P189W and P190V, P189W and P190F, P189W, P189W and P190Q, P189W and P190H, P189W and P190S, P189W and P190T, or P189W and P190W.

2. 2. The optical probe of claim 1, wherein the mutant lactate-sensitive polypeptide further comprises a mutation at the following site: M185F, M185Y, M185L, M185I, M185Q, M185G, M185H, M185A, M185P, M185N, M185C, M185W, M185S, M185V, M185D, M185T, M185R, M185E or M185K.

3. 2. The optical probe of claim 1, wherein the optical probe has a sequence shown in any one of SEQ ID NOs: 6 to 30, 34 to 40, or consists of any one of these sequences.

4. A nucleic acid, (1) A polynucleotide encoding the optical probe according to any one of claims 1 to 3; (2) A nucleic acid selected from the group consisting of a complementary sequence of (1).

5. A nucleic acid construct comprising the nucleic acid of claim 4.

6. The nucleic acid construct of claim 5, wherein the nucleic acid construct is an expression vector.

7. A host cell comprising: (1) The optical probe according to any one of claims 1 to 3 is developed, (2) A nucleic acid according to claim 4, or (3) A host cell comprising the nucleic acid construct according to claim 5 or 6.

8. A method for preparing the optical probe according to any one of claims 1 to 3, comprising culturing the host cell according to claim 7, and isolating the optical probe from the culture.

9. Use of an optical probe according to any one of claims 1 to 3, a nucleic acid according to claim 4, or a nucleic acid construct according to claim 5 or 6 in the manufacture of a kit for detecting lactic acid in a sample or for screening compounds.

10. 10. The use according to claim 9, wherein the detection is lactate localization or quantitative detection.

11. A detection reagent kit, comprising: (1) An optical probe according to any one of claims 1 to 3 or an optical probe prepared by the method according to claim 8; (2) The nucleic acid according to claim 4. (3) The nucleic acid construct according to claim 5 or 6, or (4) The host cell according to claim 7, and and other reagents for detecting lactate with an optical probe.

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