Arginine Fluorescent Probe, Method for Producing the Same, and Use

Abstract

The present invention provides an arginine fluorescent probe comprising an arginine-sensitive polypeptide B and a fluorescent protein A expressing arginine. The fluorescent protein A is inserted into polypeptide B, which is divided into upper and lower structural portions B1 and B2, forming a B1-A-B2 probe structure. Similarly, in optimized mutants obtained by cleavage or site-specific mutation at different sites, the fluorescent signal of fluorescent protein A is altered by the specific binding of polypeptide B to arginine, and polypeptide B is an arginine-binding protein and its mutants. The arginine fluorescent probe provided by the present invention has a relatively small protein molecular weight, is easy to express, exhibits large dynamic changes in fluorescence, and has good specificity. It can also be expressed in different suborganelles of cells by genetic engineering, allowing for high-throughput, quantitative detection of arginine both inside and outside the cell.

Description

Technical Field

[0001] The present invention relates to the technical field of optical probes, and in particular, to an arginine optical probe, a method for manufacturing the same, and its use.

Background Art

[0002] Arginine, as one of the 20 natural amino acids, was first isolated and extracted from the seedlings of the plant Lupinus by Schlus in 1886. At the beginning of the 20th century, its molecular structure was elucidated and it became possible to be artificially synthesized. Arginine plays a biological function in the form of L-arginine having physiological activity in vivo. Arginine is not only a component constituting the proteins of the living body, but also a precursor for synthesizing various bioactive substances such as polyamines and NO, and is involved in biological processes such as endocrine regulation and specific immune regulation of the living body by stimulating the secretion of some hormones. In addition, as an intermediate of the urea cycle, arginine prevents ammonia intoxication in the urea cycle, thereby avoiding metabolic disorders caused by excessive ammonia. In addition, arginine plays an important role in the homogenization metabolism of the living body and is utilized in various metabolic pathways such as arginase, nitric oxide synthase, arginine / glycine guanidinotransferase, and arginyl-tRNA synthetase. Therefore, at present, various biological functions of arginine have attracted wide attention from scientific researchers and have become the focus of amino acid research.

[0003] There are two main metabolic pathways of arginine in mammals. As the first metabolic pathway, arginine is decomposed into ornithine and urea by the action of arginase. Ornithine is a precursor for synthesizing polyamine substances. Polyamines have important significance for regulating cell growth and development. As the second metabolic pathway, arginine is decomposed into equimolecular citrulline and NO by the action of nitric oxide synthase. NO is a messenger molecule that acts intracellularly, intercellularly, and as a neurotransmitter, and is widely involved in intercellular and intracellular signal transduction. Also, as an amidine group donor in the amidine group transfer reaction, guanidinoacetic acid and phosphocreatine are synthesized from arginine, glycine, and methionine. In nerve tissue, γ-guanidinobutyric acid is also synthesized from arginine (Delforge J et al., Eur J Biochem. 1975, 57(1):231-239 (Non-Patent Document 1); Fernandez ML et al., J. Bacteriol. 2004, 186(18):6142-6149 (Non-Patent Document 2); Fernandez ML et al., J. Bacteriol. 2008, 190(18):3018-3025 (Non-Patent Document 3)).

[0004] Arginine is one of the three substrates for forming creatine. Creatine is an important nutrient (causing intellectual impairment due to creatine deficiency), is also used for the formation of ascites, and is also a signal molecule in the body. Arginine is an intermediate in the urea (including L-ornithine, L-citrulline, and argininosuccinic acid) and nitric oxide cycle (including ornithine and argininosuccinic acid), and can produce a polyamine structure that regulates cell function via ornithine. Deficiency of arginine (which can be induced by promoting the activity of arginase because arginase can convert arginine to ornithine) can affect B cell function (immunity), hair and muscle growth, and may also affect nerve and muscle function. It is already known that overexpression of arginase in type 2 diabetes patients is a factor in the progression of cardiovascular diseases in these patients, and that arginase is reduced in patients with renal insufficiency.

[0005] Since arginine has the important functions shown above, the detection of arginine content is particularly important. The usual detection methods for arginine include capillary electrophoresis (Li X-t et al., Chem Res Chin Univ 2013, 29(3):434-438 (Non-Patent Document 4); Meng J et al., The Analyst 2010, 135(7):1592-1599 (Non-Patent Document 5)), high performance liquid chromatography (Tateda N et al., Analytical sciences : the international journal of the Japan Society for Analytical Chemistry 2001, 17(6):775-778 (Non-Patent Document 6); Wadud S et al., Journal of chromatography B, Analytical technologies in the biomedical and life sciences 2002, 767(2):369-374 (Non-Patent Document 7)), enzyme-linked immunosorbent assay and ultraviolet-visible spectrophotometry (Hortpro MA et al., J Am Chem So 2003, 125(1):20-21 (Non-Patent Document 8); Pu F et al., Anal Chem 2010, 82(19):8211-8216 (Non-Patent Document 9); Du J et al., Chemical communications (Cambridge, England) 2013, 49(47):5399-5401 (Non-Patent Document 10); Engeser M et al., Chemical Communications 1999, (13):1191-1192 (Non-Patent Document 11)) as well as fluorescence spectroscopy (Engeser M et al., Chemical Communications 1999, (13):1191-1192 (Non-Patent Document 11)).

[0006] However, in the study of living cells, these detection methods have significant defects and require time-consuming sample processing procedures such as cell disruption, separation, extraction, and purification, and cannot detect in situ, in real-time, dynamically, with high throughput, and with high time resolution in living cells and sub-organelles. In this field, there is still a need for a method that can detect arginine in real-time, in situ, quantitatively, and with high throughput both inside and outside cells.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

[0008] In view of the above, an object of the present invention is to provide an arginine fluorescent probe for detecting arginine in real time, with high throughput and quantification both inside and outside cells. [Means for Solving the Problems]

[0009] In order to achieve the above object of the invention, the present invention provides the following technical solutions.

[0010] The present invention provides an arginine optical probe comprising an arginine-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 arginine-sensitive polypeptide or a functional variant thereof. The arginine-sensitive polypeptide or a functional variant thereof is divided into a first part and a second part by the optically active polypeptide or a functional variant thereof.

[0011] The present invention provides an arginine optical probe comprising an arginine-sensitive polypeptide B and an optically active polypeptide A, wherein the optically active polypeptide A is within the sequence of the arginine-sensitive polypeptide B, and the arginine-sensitive polypeptide B is divided into a first part B1 and a second part B2, forming a probe structure of the B1-A-B2 type.

[0012] In one embodiment, the arginine-sensitive polypeptide comprises the arginine-binding domain of an arginine-binding protein and mutants thereof. In one embodiment, the arginine-sensitive polypeptide is an arginine-binding protein or a functional fragment thereof. In one or more embodiments, the arginine-binding protein is the STM4351 protein. In one embodiment, the arginine-sensitive polypeptide has the sequence shown in SEQ ID NO:1, or 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%, at least 97%, at least 99% sequence identity thereto and retaining arginine sensitivity.

[0013] 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), saffron yellow fluorescent protein, red fluorescent protein, 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.

[0014] In one embodiment, the optical probe further includes one or more linkers linked on the side chain of the optically active polypeptide. In one embodiment, the side chain of the optically active polypeptide includes 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 of the side chain of the optically active polypeptide includes amino acid Y. In one embodiment, linker Y is at the N-terminus and / or C-terminus of the optically active polypeptide. In one embodiment, the optical probe is shown as follows, that is, the first part B1 - Y - optically active polypeptide A - the second part B2 of the arginine-sensitive polypeptide. In one embodiment, the optical probe of the present invention does not include a linker.

[0015] In one embodiment, the optically active polypeptide is between residues 103 - 111 and / or 197 - 209 of the arginine-sensitive polypeptide, and the numbers correspond to the full length of the arginine-binding protein. In one embodiment, the optically active polypeptide substitutes one or more amino acids between residues 102 - 112 and / or 196 - 210 of the arginine-sensitive polypeptide, and the numbers correspond to the full length of the arginine-binding protein.

[0016] In one embodiment, the optically active polypeptide is in one or more sites selected from the following of the arginine-sensitive polypeptide, namely, 103 / 104, 103 / 105, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 103 / 111, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 111, 106 / 107, 106 / 108, 106 / 109, 106 / 110, 106 / 111, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 110, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 207, 197 / 208, 197 / 209, 198 / 199, 198 / 200, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205, 198 / 206, 198 / 207, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 207, 199 / 208, 199 / 209, 200 / 201, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 200 / 207, 200 / 208, 200 / 209, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 201 / 209, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 202 / 209, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 203 / 209, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 204 / 209, 205 / 206, 205 / 207, 205 / 208, 205 / 209, 206 / 207, 206 / 208, 206 / 209, 207 / 208, 207 / 209 or 208 / 209.

[0017] Preferably, the optically active polypeptide is in one or more sites selected from the following of the arginine-sensitive polypeptide, namely, 103 / 104, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 104 / 105, 104 / 106, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 111, 106 / 107, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 208, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205, 198 / 206, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 208, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 200 / 207, 200 / 208, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 208, 201 / 209, 202 / 203, 202 / 205, 202 / 206, 203 / 204, 203 / 206, 204 / 205, 204 / 206, 204 / 208, 205 / 206, 205 / 207, 205 / 208 or 206 / 207. These probes have a response to arginine that exceeds that of the control group.

[0018] Preferably, the optically active polypeptide is in one or more sites selected from the following of the arginine-sensitive polypeptide, namely, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 110, 106 / 108, 106 / 109, 107 / 109, 107 / 111, 110 / 111, 197 / 199, 197 / 203, 197 / 204, 198 / 202, 198 / 203, 198 / 204, 199 / 200, 199 / 201, 199 / 202, 199 / 204, 199 / 205, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 205, 203 / 206, 204 / 205 or 204 / 206. These probes have a response to arginine that exceeds 1.5 times that of the control group.

[0019] Preferably, the optically active polypeptide is in one or more sites selected from the following of the arginine-sensitive polypeptide, namely, 104 / 110, 104 / 111, 105 / 106, 105 / 110, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 197 / 204, 198 / 202, 199 / 200, 199 / 202, 200 / 203, 200 / 205, 201 / 202, 201 / 205, 202 / 205, 203 / 206, 204 / 205 or 204 / 206. These probes have a response to arginine that exceeds 2 times that of the control group.

[0020] In an exemplary embodiment, the optical probe of the present invention may be a probe in which cpYFP is in the 104 / 110, 104 / 111, 105 / 106, 105 / 110, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 197 / 204, 198 / 202, 199 / 200, 199 / 202, 200 / 203, 200 / 205, 201 / 202, 201 / 205, 202 / 205, 203 / 206, 204 / 205 or 204 / 206 site of the arginine-binding protein. In one embodiment, the optical probe of the present invention has the sequence shown in SEQ ID NO: 6 to 39, or consists of these sequences.

[0021] The present invention further provides mutants of arginine-sensitive polypeptides having one or more mutations. The amino acid mutations include amino acid modification, substitution, deletion, or sequence cleavage. In one embodiment, the mutation is at one, two, or three of positions 30(S), 96(R), and 177(D) of the arginine-binding protein. Preferably, the mutation is selected from one, two, or three of S30N, D177N, R96M, and R96K.

[0022] The present invention further provides an optical probe comprising an arginine-sensitive polypeptide having one or more mutations. In one or more embodiments, the optical probe is any optical probe into which an optically active polypeptide is inserted as shown above, and the arginine-sensitive polypeptide in the optical probe has a mutation at one, two, or three sites selected from S30, R96, and D177. In one or more embodiments, the optical probe comprising the mutated arginine-sensitive polypeptide does not have a lower response to arginine than the non-mutated control. Preferably, the mutation is selected from one, two, or three of S30N, D177N, R96M, and R96K.

[0023] In an exemplary embodiment, the optical probe of the present invention may be a probe in which cpYFP is inserted at the 107 / 111 site of the arginine-binding protein and has one or more mutations selected from S30N, D177N, R96M, and R96K. Preferably, the optical probe of the present invention is a probe in which cpYFP is inserted at the 107 / 111 site of the arginine-binding protein and has a mutation selected from S30N or D177N. In one embodiment, the optical probe of the present invention has the sequence shown in SEQ ID NO: 40-41, or consists of these sequences.

[0024] The optical probe provided by the present invention contains any one of the amino acid sequences SEQ ID NO: 6 to 41 or a variant thereof. In one embodiment, the optical probe provided by the present invention contains 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%, at least 99% sequence identity with any one of the amino acid sequences SEQ ID NO: 6 to 41.

[0025] The present invention further provides a fusion polypeptide containing the optical probe described herein and other polypeptides. 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 contains a polypeptide that localizes the optical probe to a different organelle or sub-organelle, a purification tag, or an immunoblotting tag.

[0026] The present invention further provides a nucleic acid sequence containing the coding sequence of the polypeptide, probe, or protein described herein, or its complementary sequence or fragment. In one embodiment, the nucleic acid sequence of the present invention is selected from (1) the coding sequence of the amino acid sequence shown in any one of SEQ ID NO: 6 to 41 or its complementary sequence, (2) a sequence having at least 99%, 95%, 90%, 80%, 70%, or 50% identity with (1), (3) a fragment of (1) or (2). In one embodiment, the nucleic acid sequence of the present invention contains SEQ ID NO: 42 or a variant or fragment thereof. In one or more embodiments, the fragment is a primer.

[0027] The present invention further relates to the complementary sequence of the above nucleic acid sequence or a variant thereof, which contains a nucleic acid sequence encoding a fragment, analog, derivative, soluble fragment, and variant of the optical probe or fusion protein of the present invention, or its complementary sequence.

[0028] The present invention further provides a nucleic acid construct comprising a nucleic acid sequence described herein or its complementary sequence, wherein the nucleic acid sequence encodes an optical probe or a fusion polypeptide according to 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 cell expression vector, a eukaryotic cell expression vector and a viral vector.

[0029] The present invention further provides a cell comprising a nucleic acid sequence or an expression vector according to the present invention. In one or more embodiments, the cell expresses an optical probe or a fusion polypeptide described herein.

[0030] The present invention further provides a detection reagent kit comprising an optical probe, a fusion polypeptide, a polynucleotide described herein or an optical probe or a fusion polypeptide prepared by the method described herein.

[0031] The present invention provides a method for preparing an optical probe described herein, comprising providing a cell that expresses an optical probe or a fusion polypeptide described herein, culturing the cell under the expression conditions of the cell, and separating the optical probe or the fusion polypeptide.

[0032] The present invention further provides a method for detecting arginine in a sample, comprising contacting the sample with an optical probe or a fusion polypeptide described herein, or an optical probe or a fusion polypeptide prepared by the method described herein, and detecting a change in the optically active polypeptide. The detection may be performed in vivo, in vitro, sub-organelle or in situ. The sample is, for example, blood.

[0033] The present invention further provides a method for quantifying arginine in a sample, including contacting the sample with an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared by the method described herein, detecting a change in the optically active polypeptide, and quantifying arginine in the sample based on the change in the optically active polypeptide.

[0034] The present invention further provides a method for screening a compound (e.g., a drug), including contacting a candidate compound with an optical probe or fusion polypeptide described herein, or an optical probe or fusion polypeptide prepared by the method described herein, detecting a change in the optically active polypeptide, and screening the compound based on the change in the optically active polypeptide. The method can screen compounds with high throughput.

[0035] The present invention further provides the use of an arginine optical probe or fusion polypeptide described herein, or an arginine optical probe or fusion polypeptide prepared by the method described herein, in the intracellular / extracellular localization of arginine. In one or more embodiments, the localization is real-time localization.

Advantages of the Invention

[0036] The present invention has the following advantages. The arginine fluorescent probe provided by the present invention comprises a polypeptide B having arginine sensitivity and a fluorescent protein A. The fluorescent protein A is inserted into the polypeptide B, and B is divided into two parts, polypeptide B1 and polypeptide B2, to form a probe structure of the B1-A-B2 type. The B1-A-B2 type arginine fluorescent probe provided by the present invention is easy to mature, has a large dynamic change in fluorescence, good specificity, can be expressed in cells by genetic engineering methods, and can detect arginine in real-time localization, high-throughput, and quantification inside and outside cells. The processing steps of time-consuming samples are simplified. According to experimental results, the arginine fluorescent probe provided by the present application has a maximum response to arginine of more than 11 times, can detect the localization of cells in sub-organelle structures such as cytoplasm, mitochondria, cell nucleus, Golgi apparatus, peroxisome, and lysosome, and can perform high-throughput compound screening and quantitative detection of blood arginine.

[0037] The present invention further provides a method for preparing the above arginine optical probe, comprising the following steps: 1) incorporating a nucleic acid sequence encoding the arginine optical probe described herein into an expression vector; 2) transferring the expression vector into a host cell; 3) culturing the host cell under conditions suitable for the expression of the expression vector; and 4) separating the arginine optical probe.

Brief Description of the Drawings

[0038] Hereinafter, the present invention will be specifically described with reference to the drawings and examples.

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Modes for Carrying Out the Invention

[0039] When indicating a numerical value or range, the term "about" as used herein refers to the numerical value or range being within 20%, within 10%, and within 5% of a predetermined numerical value or range.

[0040] The terms "comprising", "including", "containing", and their equivalent forms including "consisting of" as used herein mean that, for example, in the case of a composition "comprising" X, it may consist only of X, or it may contain other substances, for example, like X + Y.

[0041] The term "arginine - sensitive polypeptide" or "arginine - responsive polypeptide" as used herein refers to a polypeptide that responds to arginine. The response includes any response of the chemical, biological, electrical, or physiological parameters of the polypeptide regarding the interaction of the sensitive polypeptide. The response includes, for example, small changes such as a change in the orientation of the amino acids or peptide fragments of the polypeptide, and changes in the primary, secondary, or tertiary structure of the polypeptide, for example, changes such as protonation, electrochemical potential, and / or conformational changes. "Conformation" is the three - dimensional arrangement of the primary, secondary, and tertiary structures of a molecule with pendant groups. When the three - dimensional structure of a molecule changes, the conformation changes. Examples of conformational changes include the conversion from an α - helix to a β - sheet or from a β - sheet to an α - helix. It may be understood that even if the detected change is not a conformational change, the fluorescence of the fluorescent protein moiety may be changed. The arginine - sensitive polypeptides described herein may further include functional variants thereof. Functional variants of arginine - sensitive polypeptides include, but are not limited to, variants that can undergo homologous or similar changes to the parent arginine - sensitive polypeptide upon interaction with arginine.

[0042] The arginine-sensitive polypeptide according to the present invention includes, but is not limited to, the arginine-binding protein STM4351 derived from Salmonella Typhimurium or the arginine periplasmic-binding protein derived from Escherichia coli, or a variant having 90% or more homology therewith. These binding proteins consist of two structural domains, and the two structural domains are linked by a peptide chain of two flexible amino acids. The arginine-binding protein can sense changes in arginine concentration, and the conformation of the arginine-binding protein changes significantly during the dynamic change process of arginine concentration. STM4351 consists of an arginine-binding / regulatory domain and a DNA-binding domain. An exemplary STM4351 protein is shown in SEQ ID NO:1. In one or more embodiments, the arginine-sensitive polypeptide includes the arginine-binding domain of the STM4351 protein but does not include the DNA-binding domain.

[0043] As used herein, the term "optical probe" refers to an arginine-sensitive polypeptide that is fused with an optically active polypeptide. The inventors have found that a conformational change of an optically active polypeptide (e.g., a fluorescent protein) occurs due to a conformational change generated by the specific binding of an arginine-sensitive polypeptide, such as an arginine-binding protein, to physiological concentrations of arginine, and further a change in the optical properties of the optically active polypeptide occurs. By creating a calibration curve based on the fluorescence of the fluorescent protein measured at different concentrations of arginine, the presence and / or level of arginine can be detected and analyzed. The amino acid residue numbers referred to when describing the optical probe of the present invention (e.g., when describing the insertion site or mutation site) refer to SEQ ID NO:1.

[0044] In the optical probe of the present invention, an optically active polypeptide (for example, a fluorescent protein) is operably inserted into an arginine-sensitive polypeptide. The "optically active polypeptide" based on a protein is a polypeptide having a fluorescence emission ability. Fluorescence is an optical property of the optically active polypeptide and may be used as a detection means for the responsiveness of the optical probe of the present invention. Preferably, a protein substrate is selected so as to have fluorescence characteristics that can be easily distinguished between the inactivated and activated conformational states. The optically active polypeptide described in the present specification may be a functional variant thereof. The functional variant of the optically active polypeptide includes, but is not limited to, a variant capable of generating a change in fluorescence characteristics that is homologous or similar to that of the parent optically active polypeptide.

[0045] As used herein, the term "fluorescent protein" refers to a protein that emits fluorescence upon irradiation with excitation light. Fluorescent proteins are basic detection means in the biological field, for example, green fluorescent protein GFP commonly used in the biotechnology field, and circularly permuted blue fluorescent protein (cpBFP), circularly permuted green fluorescent protein (cpGFP), circularly permuted yellow fluorescent protein (cpYFP) induced by mutations of the protein, etc. can be mentioned. Furthermore, red fluorescent protein RFP commonly used in this technical field, and circularly permuted proteins induced by the protein such as cpmApple, cpmOrange, cpmKate, etc. can be mentioned. In this field, fluorescent proteins and their sequences that can be used in the present invention are known. Exemplarily, cpYFP is shown in SEQ ID NO:2, cpGFP is shown in SEQ ID NO:3, cpBFP is shown in SEQ ID NO:4, and cpmApple is shown in SEQ ID NO:5.

[0046] "Linker" or "linker region" refers to an amino acid or nucleotide sequence that links two parts in the polypeptide, protein or nucleic acid of the present invention. Exemplarily, in the present invention, the number of amino acids at the amino group terminus of the linker region between the arginine-sensitive polypeptide and the optically active polypeptide is selected from 0 to 3, and the number of amino acids at the carboxyl group terminus is selected from 0 to 2. However, when the recombinant optical probe is linked to a functional protein as a basic unit, it may be fused to the amino acid or carboxyl group terminus of the recombinant optical probe. The linker sequence may be, for example, a short-chain peptide consisting of one or more soft amino acids, such as Y.

[0047] The arginine optical probe according to the present invention includes, for example, an arginine-sensitive polypeptide B which is an arginine-binding protein or a variant thereof, and an optically active polypeptide A which is, for example, a fluorescent protein. The optically active polypeptide A is inserted into the arginine-sensitive polypeptide B, and B is divided into two parts, B1 and B2, to form a probe structure of the B1-A-B2 type. When the arginine-sensitive polypeptide B interacts with arginine, the optical signal of the optically active polypeptide A becomes stronger.

[0048] In the optical probe of the present invention, the optically active polypeptide may be at any site of the arginine-sensitive polypeptide. In one embodiment, the optically active polypeptide is at any site of the arginine-sensitive polypeptide in the N-C direction in the N-C direction. Specifically, the optically active polypeptide is in the flexible region of the arginine-sensitive polypeptide. The flexible region refers to some special structures existing in the higher-order structure of a protein, such as a cyclic structure domain. These structural domains have higher mobility and flexibility than other higher-order structures of the protein, and after the protein binds to a ligand, the conformation of the three-dimensional structure of the region dynamically changes. The flexible region according to the present invention mainly refers to a region having an insertion site in an arginine-binding protein, for example, the regions of amino acid residues 102 to 112 and 196 to 210.In one embodiment, the optically active polypeptide is in one or more sites selected from the following of the arginine-sensitive polypeptide, namely, 103 / 104, 103 / 105, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 103 / 111, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 111, 106 / 107, 106 / 108, 106 / 109, 106 / 110, 106 / 111, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 110, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 207, 197 / 208, 197 / 209, 198 / 199, 198 / 200, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205, 198 / 206, 198 / 207, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 207, 199 / 208, 199 / 209, 200 / 201, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 200 / 207, 200 / 208, 200 / 209, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 201 / 209, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 202 / 209, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 203 / 209, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 204 / 209, 205 / 206, 205 / 207, 205 / 208, 205 / 209, 206 / 207, 206 / 208, 206 / 209, 207 / 208, 207 / 209 or 208 / 209.In this specification, when two numbers of a site represented in the form of "X / Y" are consecutive integers, it indicates that the optically active polypeptide is between the amino acids indicated by the numbers. For example, the insertion site 93 / 94 indicates that the optically active polypeptide is between amino acids 93 and 94 of the arginine-sensitive polypeptide. When two numbers of a site represented in the form of "X / Y" are not consecutive integers, it indicates that the amino acids between the amino acids indicated by the numbers are substituted with the optically active polypeptide. For example, the insertion site 93 / 97 indicates that amino acids 94 to 96 of the arginine-sensitive polypeptide are substituted with the optically active polypeptide. Preferably, the optically active polypeptide is in one or more sites selected from the following of the arginine-sensitive polypeptide, namely, 104 / 110, 104 / 111, 105 / 106, 105 / 110, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 197 / 204, 198 / 202, 199 / 200, 199 / 202, 200 / 203, 200 / 205, 201 / 202, 201 / 205, 202 / 205, 203 / 206, 204 / 205 or 204 / 206.

[0049] When a particular polypeptide or protein is mentioned, the terms "variant" or "mutant" as used in the present invention include variants having a function homologous to the polypeptide or protein but different in sequence. Variants of a polypeptide or protein may include homologous sequences, conservative variants, allelic variants, naturally occurring mutants, and induced mutants. These variants have one or more (usually 1 to 30, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5) amino acids deleted, inserted and / or substituted in the sequence of the polypeptide or protein, and one or more (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids added to the carboxyl-terminal and / or amino-terminal thereof, but are not limited thereto. These variants may further include polypeptides or proteins having a sequence identity with the polypeptide or protein of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100%. Without being bound by theory, it is preferred that even if the amino acid residues change, the overall conformation and function of the polypeptide or protein do not change, i.e., functional conserved mutations. For example, in this field, when substituting with amino acids having similar properties or similar characteristics, usually the function of the polypeptide or protein does not change. In this field, amino acids having similar characteristics are usually amino acid families having similar side chains and are clearly defined in this field.These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, arginine, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Also, for example, even when one or more amino acids are added to the amino group terminus and / or carboxy group terminus, usually the function of the polypeptide or protein does not change. Conservative amino acid substitutions of various non-genetically encoded amino acids that are generally known are already known in the art. Other conservative substitutions of non-coded amino acids are determined by comparison of their physical properties with those of genetically encoded amino acids.

[0050] In two or more polypeptide or nucleic acid molecule sequences, the terms "identity" or "percentage of identity" are the same when comparing and aligning for the greatest match over a comparison window or specified region, as measured using methods known in the art such as sequence comparison algorithms, either by manual alignment or by visual inspection, of two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same over a particular region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical). For example, preferred algorithms suitable for measuring sequence identity percentage and sequence similarity percentage 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.

[0051] In gene cloning operations, since it is always necessary to design appropriate restriction enzyme cleavage sites, one or more irrelevant residues are introduced at the ends of the expressed polypeptide or protein. It is well known to those skilled in the art that this does not affect the activity of the target polypeptide or protein. Also, for example, for the construction of fusion proteins, the promotion of recombinant protein expression, the acquisition of recombinant proteins that are automatically secreted outside the host cell, or the assistance of recombinant protein purification, it is always necessary to add specific amino acids to the N-terminus, C-terminus, or other appropriate regions of the recombinant protein. Other appropriate regions of the 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 proteolytic enzyme sites such as Factor Xa, thrombin, or enterokinase.

[0052] The optical probe of the present invention may contain an arginine-sensitive polypeptide having a mutation. In one embodiment, the mutation is at positions 30(S), 96(R), and 177(D) of the arginine-binding protein. Exemplarily, the mutation is S30N, D177N, R96M, R96K. The arginine-sensitive polypeptide in the optical probe of the present invention may be the mutant. In an exemplary embodiment, the optical probe of the present invention may be a probe in which cpYFP is inserted at the 107 / 111 site of the arginine-binding protein and has one or more mutations selected from S30N, D177N, R96M, and R96K.

[0053] As used herein, the terms "functional fragment", "derivative", and "analogue" refer to proteins that substantially retain the same biological function or activity as the original polypeptide or protein (e.g., arginine-binding protein or fluorescent protein). Functional variants, derivatives, or analogues of the polypeptide or protein of the present invention (e.g., arginine-binding protein or fluorescent protein) include (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and the substituted amino acid residues may or may not be encoded by the genetic code; (ii) proteins having substituents on one or more amino acid residues; (iii) proteins formed by fusing the mature protein with other compounds (e.g., compounds that extend the half-life of the protein, such as polyethylene glycol); or (iv) proteins formed by fusing an added amino acid sequence to the protein sequence (e.g., a secretion sequence, a sequence for purifying the protein, or a protein-constituting sequence, or a fusion protein formed with an antigen IgG fragment). As taught herein, these functional variants, derivatives, and analogues are well known to those skilled in the art.

[0054] The differences between the analogue and the original polypeptide or protein may be differences in the amino acid sequence, differences in modified forms that do not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, for example, by radiation or exposure to a mutagen to induce random mutations, or by site-directed mutagenesis or other known molecular biology techniques.

[0055] The analogs further include analogs having residues different from natural L - amino acids (e.g., D - amino acids), and analogs having non - naturally occurring amino acids or synthetic amino acids (e.g., β,γ - amino acids). It should be understood that the arginine - sensitive polypeptide of the present invention is not limited to the representative proteins, variants, derivatives, and analogs listed above. Modified (usually without changing the primary structure) forms include chemically induced forms of proteins in vivo or in vitro, such as acetylation or carboxylation. The modification further includes glycosylation, for example, proteins produced by performing glycosylation modification during the synthesis and processing of proteins or during further processing steps. The modification is carried out by exposing the protein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). The modified forms further include sequences having residues of amino acids that are phosphorylated (e.g., phosphorylated tyrosine, phosphorylated serine, phosphorylated threonine). The modified forms further include proteins whose proteolysis - preventing properties are improved or whose solubility is improved by being modified.

[0056] The fusion polypeptide of the present invention includes the optical probe described herein and other polypeptides. In some embodiments, the optical probe described herein further includes other polypeptides that are fused thereto. The other polypeptides described herein do not affect the properties of the optical probe. The other polypeptides may be at the N - terminus and / or C - terminus of the optical probe. In some embodiments, the other polypeptides include polypeptides that localize the optical probe to different organelles or sub - organelles, and purification tags or immunoblotting tags. A linker may be present between the optical probe and the other polypeptides in the fusion polypeptide described herein.

[0057] The sub-organelles described in this specification include cytoplasm, mitochondria, cell nucleus, endoplasmic reticulum, cell membrane, Golgi apparatus, lysosome, peroxisome, etc. In some embodiments, the tags for purification or for immunoblotting include 6 histidines (6*His), glutathione-S-transferase (GST), Flag.

[0058] The expression vector of the present invention includes the nucleic acid sequence according to the present invention or its complementary sequence operably linked to an expression control sequence, and the nucleic acid sequence encodes the optical probe or fusion polypeptide according to the present invention. The terms "nucleic acid", "nucleotide", "polynucleotide" or "nucleic acid sequence" used in the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. When a nucleic acid is referred to, the term "variant" used in this specification may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include degenerate variants, substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is an alternative form of a nucleic acid and may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the protein encoded thereby. The nucleic acid of the present invention may include a nucleotide sequence having at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% sequence identity with the above nucleic acid sequence. The present invention further relates to nucleic acid fragments that hybridize with the above sequences. The length of the "nucleic acid fragment" used in this specification includes at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. The nucleic acid fragment can be used in nucleic acid amplification techniques (e.g., PCR).

[0059] The full-length sequence or fragment of the optical probe or fusion protein of the present invention is usually obtained by PCR amplification method, artificial synthesis method or recombinant method. For the PCR amplification method, primers are designed according to the nucleotide sequences disclosed in the present invention, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art is used as a template for amplification to obtain related sequences. When the nucleotide sequence is larger than 2500 bp, preferably after performing PCR amplification 2 to 6 times, the amplified fragments are ligated in the correct order. The present invention does not particularly limit the process and system of the above-mentioned PCR amplification, and the ordinary PCR amplification process and system in this field may be used. Furthermore, related sequences can be obtained in large quantities by the recombinant method. Usually, it is cloned into a vector, introduced into cells, and then separated and purified from host cells grown by a conventional method to obtain related polypeptides or proteins. Also, especially when the length of the fragment is short, the related sequence may be synthesized by the artificial synthesis method. In the present invention, when the nucleotide sequence of the optical probe is smaller than 2500 bp, it may be synthesized by the artificial synthesis method. The artificial synthesis method is a conventional DNA artificial synthesis method in this field, and there are no other special requirements. Usually, after synthesizing a plurality of small fragments, these are ligated to obtain a fragment of a long sequence. Currently, the DNA sequence encoding the protein (or its functional variant, derivative or analog) of the present invention can be obtained only by chemical synthesis. Thereafter, the DNA sequence is introduced into cells with various existing DNA molecules (for example, vectors) known in this field. Mutations may be introduced into the protein sequence of the present invention by methods such as mutant PCR or chemical synthesis.

[0060] The present invention also relates to polynucleotides described herein and nucleic acid constructs comprising one or more regulatory sequences operably linked to these sequences. The polynucleotides according to the present invention may be manipulated in various forms in order to ensure the expression of the polypeptide or protein. Before inserting 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 changing polynucleotide sequences by recombinant DNA methods are known in the art.

[0061] 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. The cloning vector can be used to provide the coding sequence of the protein or polypeptide of the present invention. The expression vector may be provided to cells in the form of a bacterial vector or a viral vector. Usually, the polynucleotide of the present invention is operably linked to a promoter and the construct is introduced into an expression vector, thereby realizing the expression of the polynucleotide of the present invention. The vector is suitable for replication and integration in eukaryotic cells. Representative 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. The homologous recombination vector is used to integrate the expression cassette described herein into the host genome.

[0062] 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, which may be an origin of replication, a promoter, a marker gene, or a translation control element, and includes enhancers, operons, terminators, ribosome binding sites, etc. The selection of the expression control sequence is determined by the host cell used. In a recombinant expression vector, "operably linked" means that the target nucleotide sequence is linked to the regulatory sequence in a form capable of expressing the nucleotide sequence. Methods for constructing an expression vector containing the coding sequence of the fusion protein of the present invention and appropriate transcription / translation control signals are well known to those skilled in the art. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, etc. To direct mRNA synthesis, the DNA sequence may be effectively linked to an appropriate promoter in the expression vector. Representative examples of these promoters include the lac or trp promoter of Escherichia coli; the λ phage PL promoter; and eukaryotic cell promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses, and promoters of other known controllable genes expressed in prokaryotic cells, eukaryotic cells, or their viruses. The expression vector further includes a ribosome binding site used for translation initiation and a transcription terminator. In one embodiment, a commercially available pET28a vector can be used as the expression vector, and there are no other special requirements. Exemplarily, after double-digesting the nucleotide sequence encoding the optical probe and the expression vector with BamHI and EcoRI, respectively, the digestion products of both are ligated to obtain a recombinant expression vector. The present invention is not particularly limited to the specific steps and parameters of digestion and ligation, and ordinary steps and parameters in this field may be used.

[0063] To produce a protein or peptide containing a fusion protein, after obtaining a recombinant expression vector, the vector is transformed into a host cell. Such an introduction process can be carried out by ordinary techniques well-known to those skilled in the art, such as transformation or transfection. The host cell according to the present invention refers to a cell that can receive and accommodate a recombinant DNA molecule and is a place for amplifying a recombinant gene. Desirable recipient cells should satisfy two conditions: being easily obtainable and proliferable. The "host cell" of the present invention includes prokaryotic cells and eukaryotic cells, specifically including bacterial cells, yeast cells, insect cells, and mammalian cells. Specifically, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells such as Drosophila S2 or Sf9, animal cells such as CHO, COS, HEK293, HeLa cells, or Bowes melanoma cells, etc. are included, and include but are not limited to the above-mentioned host cells. The above host cells are preferably various cells that are advantageous for the expression of gene products or fermentative production. These cells are well-known and commonly used in this field. An exemplary host cell used in the examples of the present invention is the Escherichia coli JM109-DE3 strain. Those skilled in the art clearly know the method of selecting appropriate vectors, promoters, enhancers, and host cells.

[0064] The method of introducing into the host cell according to the present invention is an ordinary method in this field, including calcium phosphate or calcium chloride coprecipitation method, DEAE-mannan-mediated transfection, lipofection, natural competence, chemically mediated introduction, or electroporation. When the host is a prokaryotic cell such as Escherichia coli, preferably, the method is to treat by the CaCl2 method or the MgCl2 method, and the steps used are well-known in this field. When the host cell is a eukaryotic cell, DNA transfection methods such as the calcium phosphate coprecipitation method, or ordinary mechanical methods such as microinjection, electroporation, and liposome packaging may be selected.

[0065] After introducing the expression vector into the host cell, the host cell introduced with the expression vector is amplified, expressed, cultured, and further separated to obtain an arginine optical probe. For the amplification, expression, and culture of the host cell, ordinary methods may be used. The medium used for culture may be various ordinary media depending on the type of host cell used. Culture is carried out under conditions suitable for the growth of the host cell.

[0066] 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 by various separation methods using its physical, chemical, and other properties. The separation method of the arginine fluorescent protein in the present invention is not particularly limited, and ordinary separation methods for fusion proteins in this field may be used. These methods are well-known to those skilled in the art and include ordinary regeneration treatment, salting-out method, centrifugation, osmotic sterilization, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography, adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, as well as combinations of these methods, but are not limited thereto. In one embodiment, the optical probe is separated by His-tag affinity chromatography.

[0067] The present invention further provides the use of the arginine optical probe in the real-time localization, quantitative detection of arginine, and high-throughput compound screening. In one embodiment, the arginine optical probe is preferably linked to signal peptides at different sites of cells, introduced into the cells, and by detecting the intensity of the fluorescence signal in the cells, real-time localization of arginine is performed, and quantitative detection of the corresponding arginine is performed according to the arginine standard titration curve. The arginine standard titration curve according to the present invention is created based on the fluorescence signals of the arginine optical probe in the presence of different concentrations of arginine. Since the arginine optical probe according to the present invention is directly introduced into cells, in the process of real-time localization and quantitative detection of arginine, a time-consuming sample treatment process is not required, and it is more accurate. When the arginine optical probe of the present invention performs high-throughput compound screening, different compounds are added to the cell culture medium, and by measuring the change in the arginine content, compounds that affect the change in the arginine content are screened. The uses of the arginine optical probe according to the present invention in the real-time localization, quantitative detection of arginine, and high-throughput compound screening are all not for diagnostic and therapeutic purposes and have nothing to do with the diagnosis and treatment of diseases.

[0068] In this specification, concentrations, contents, percentages, and other numerical values can be expressed in range form. Considering convenience and brevity, the range form is adopted. It should be understood that the range form includes the numerical values clearly indicated as the upper and lower limits of the range, and further includes all single numerical values or sub-ranges included in the range.

Examples

[0069] Hereinafter, the arginine fluorescence probe provided by the present invention will be described in detail with reference to examples, but it should be understood that the protection scope of the present invention is not limited.

[0070] I. Experimental Materials and Reagents In the examples, methods such as ordinary genetic engineering molecular biology cloning methods, cell culture, and imaging methods are mainly used. These methods are well-known to those skilled in the art. For example, Jane Roskams et al., "Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench"; Joseph Sambrook, David W. Russell, translated by Huang Peitang et al., "Molecular Cloning: A Laboratory Manual" (3rd Edition, August 2002, published by Science Press, Beijing); R.I. Freshney et al., "Culture of Animal Cells: a Manual of Basic Technique" (5th Edition), translated by Zhang Jingbo, Xu Cunshuan et al.; Juan S. Bonifacino, M. Dassault et al., "Short Protocols in Cell Biology", translated by Zhang Jingbo et al.

[0071] The plasmids pET28a-cpYFP and pET28a-arginine-binding protein used in the examples were constructed by the Protein Laboratory of East China University of Science and Technology, and the pET28a plasmid vector was purchased from Invitrogen. All primers used in PCR were synthesized and purified by Shanghai Jierui Biotechnology Co., Ltd., and the results of identification by mass spectrometry were appropriate. The expression plasmids constructed in the examples were sequenced. Sequencing was performed by BGI and Jie Li Sequencing Co., Ltd. The Taq DNA polymerase used in each example was purchased from Dongsheng Biology, 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 accompanied by the corresponding polymerase buffer and dNTP at the time of purchase. Restriction endonucleases such as BamHI, BglII, HindIII, NdeI, XhoI, EcoRI, SpeI, T4 ligase, and T4 phosphorylase (T4 PNK) were purchased from Fermentas and were accompanied by the corresponding buffer, etc. at the time of purchase. The transfection reagent Lip2000 Kit was purchased from Invitrogen. Arginine, etc. were all purchased from Sigma. Unless otherwise specified, all chemical reagents such as inorganic salts were purchased from Sigma-Aldrich. HEPES salt, ampicillin (Amp), and puromycin were purchased from Ameresco. The 96-well test plates (black) and 384-well fluorescence test plates (black) were purchased from Grenier.

[0072] The DNA purification reagent kit used in the examples was purchased from BBI (Canada), and the general plasmid miniprep kit was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The clone strain Mach1 was purchased from Invitrogen. The packing of the Ni-NTA affinity column and desalting column was purchased from GE healthcare.

[0073] The main equipment used in the examples includes a Biotek Synergy 2 multimode microplate reader (Bio-Tek, USA), an X-15R high-speed refrigerated centrifuge (Beckman, USA), a Microfuge22R desktop high-speed refrigerated centrifuge (Beckman, USA), a PCR amplification device (Biometra, Germany), an ultrasonic crusher (Ningbo Xinzhi Company), a nucleic acid electrophoresis device (Shenneng Bojin Company), a fluorescence spectrophotometer (Varian, USA), a CO2 incubator (SANYO), and an inverted fluorescence microscope (Nikon, Japan).

[0074] II. Molecular Biology Methods and Cell Experiment Methods II.1 Polymerase Chain Reaction (PCR): 1. Target Fragment Amplification PCR: This method is mainly used for the amplification of gene fragments and the identification of positive clones by colony PCR. The reaction system for the PCR amplification is as follows: that is, 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 dNTP (10 mM), 41.5 - 42 μL of sterile ultrapure water (ddH2O), and the total volume is 50 μL. The PCR amplification process is as follows: that is, denaturation at 95°C for 2 - 10 minutes, followed by 30 cycles (holding at 94 - 96°C for 30 - 45 seconds, holding at 50 - 65°C for 30 - 45 seconds, holding at 72°C for a certain time (600 bp / min)), and extension at 72°C for 10 minutes.

[0075] 2. Long Fragment (>2500 bp) Amplification PCR: The long - fragment amplification used in the present invention is mainly an amplification vector for inverse PCR, and is a technique for obtaining site - specific mutations in the following examples. Inverse PCR primers are designed for the mutation site, and the mutated nucleotide sequence is included at the 5' end of one of the primers. The amplified product contains the corresponding mutation site. The long - fragment amplification PCR reaction system is as follows: that is, 1 μL of template sequence (10 pg - 1 ng), 0.5 μL of forward primer (25 μM), 0.5 μL of reverse primer (25 μM), 10 μL of 5×PrimerSTAR buffer, 0.5 μL of PrimerSTAR DNA polymerase, 4 μL of dNTP (2.5 mM), 33.5 μL of sterile ultrapure water (ddH2O), with a total volume of 50 μL. The PCR amplification process is as follows: that is, denaturation at 95°C for 5 minutes, followed by 30 cycles (holding at 98°C for 10 seconds, holding at 50 - 68°C for 5 - 15 seconds, holding at 72°C for a certain time (1000 bp / min)), extension at 72°C for 10 minutes, or denaturation at 95°C for 5 minutes, followed by 30 cycles (holding at 98°C for 10 seconds, holding at 68°C for a certain time (1000 bp / min)), and extension at 72°C for 10 minutes.

[0076] II.2 Digestive reaction with endonuclease: The system for performing double - digestion on the plasmid vector is as follows: that is, 20 μL of plasmid vector (about 1.5 μg), 5 μL of 10× buffer, 1 - 2 μL of restriction endonuclease 1, 1 - 2 μL of restriction endonuclease 2, and sterile ultrapure water is supplemented to make the total volume 50 μL. The reaction condition is to react at 37°C for 1 - 7 hours.

[0077] II.3 Phosphorylation reaction at the 5' end of the DNA fragment Plasmids or genomic ends extracted from microorganisms contain phosphate groups, but PCR products do not. If the ends of a DNA molecule do not contain phosphate groups, ligation cannot occur. Therefore, a phosphate addition reaction is performed on the bases at the 5' end of the PCR product. The phosphorylation reaction system is as follows: that is, 5 - 8 μL of the DNA sequence of the PCR product fragment, 1 μL of 10×T4 ligase buffer, 1 μL of T4 polynucleotide kinase (T4 PNK), 0 - 3 μL of sterile ultrapure water, with a total volume of 10 μL. After reacting at 37°C for 30 minutes to 2 hours, it is inactivated at 72°C for 20 minutes.

[0078] II.4 Ligation reaction of the target fragment and the vector There are differences in the ligation methods with the vector depending on the different fragments. In the present invention, three ligation methods were used.

[0079] 1. Blunt - end ligation of a short blunt - ended fragment and a linear vector As the mechanism of this method, after the blunt - ended product obtained by PCR undergoes a phosphorylation reaction on the 5' end of the DNA fragment under the action of T4 PNK, it undergoes the action of PEG4000 and T4 DNA ligase and is ligated to the linearized vector to obtain a recombinant plasmid. The homologous recombination ligation system is as follows: that is, 4 μL of the DNA fragment treated with T4 PNK, 4 μL of the linear vector fragment, 1 μL of PEG4000, 1 μL of 10×T4 ligase buffer, 1 μL of T4 DNA ligase, with a total of 10 μL. The reaction conditions are to react at 22°C for 30 minutes.

[0080] 2. Ligation of a DNA fragment containing sticky ends and a vector fragment containing sticky ends DNA fragments cleaved by restriction endonucleases usually produce protruding sticky ends, so they can be ligated to vector fragments containing sticky ends complementary to the sequence to form recombinant plasmids. The ligation reaction system is as follows: that is, 1-7 μL of DNA of the PCR product fragment after digestion, 0.5-7 μL of the plasmid after digestion, 1 μL of 10×T4 ligase buffer, and 1 μL of T4 DNA ligase. Sterile ultrapure water is supplemented to make the total volume 10 μL. The reaction is carried out at 16 °C for 4-8 hours.

[0081] 3. After site-specific mutations are introduced into inverse PCR, ligation reaction by self-cyclization of the DNA fragment product with phosphorylated 5' ends Perform a ligation reaction by self-cyclization on the DNA fragment with phosphorylated 5' ends, and perform a ligation reaction on the 3' and 5' ends of the linear vector to obtain a recombinant plasmid. The self-cyclization ligation reaction system is as follows: that is, 10 μL of the phosphorylation reaction system, 0.5 μL of T4 ligase (5 U / μL), and the total volume is 10.5 μL. The reaction is carried out at 16 °C for 4-16 hours.

[0082] II.5 Preparation and transformation of competent cells Preparation of competent cells: 1. Select a single colony (for example, Mach1), inoculate it into 5 mL of LB medium, and place it on a shaker at 37 °C overnight. 2. Take 0.5-1 mL of the overnight cultured bacterial solution, transfer it to 50 mL of LB medium, and culture it at 37 °C and 220 rpm for 3-5 hours until the OD600 reaches 0.5. 3. Pre-cool the cells in an ice bath for 2 hours. 4. Centrifuge at 4 °C and 4000 rpm for 10 minutes. 5. Discard the supernatant, resuspend the cells with 5 mL of pre-cooled buffer, and after it becomes uniform, add the resuspension buffer to make the final volume 50 mL. 6. Place it in an ice bath for 45 minutes. 7. Centrifuge at 4 °C and 4000 rpm for 10 minutes, and resuspend the bacteria with 5 mL of storage buffer pre-cooled on ice. 8. Put 100 μL of the bacterial solution into each EP tube and store it frozen at -80 °C or in liquid nitrogen. Resuspension buffer: CaCl2 (100 mM), MgCl2 (70 mM), NaAc (40 mM) Storage buffer: 0.5 mL DMSO, 1.9 mL 80% glycerol, 1 mL 10×CaCl2 (1 M), 1 mL 10×MgCl2 (700 mM), 1 mL 10×NaAc (400 mM), 4.6 mL ddH2O

[0083] Transformation of competent cells: 1. Take 100 μL of competent cells and let them thaw in an ice bath. 2. Add an appropriate volume of the ligation product, gently pipette to mix evenly, and place in an ice bath for 30 minutes. Usually, the volume of the added ligation product is less than 1 / 10 of the volume of the competent cells. 3. Place the bacterial solution in a 42 °C water bath, apply a heat shock for 90 seconds, then quickly transfer it to an ice bath and let it stand for 5 minutes. 4. Add 500 μL of LB and culture at 200 rpm for 1 hour using a 37 °C constant temperature shaker. 5. Centrifuge the bacterial solution at 4000 rpm for 3 minutes, leave 200 μL of the supernatant, resuspend the cell pellet evenly, spread it evenly on the surface of an agar plate containing an appropriate antibiotic, invert the plate, and place it in a 37 °C constant temperature incubator overnight.

[0084] II.6 Protein Expression, Purification and Fluorescent Detection 1. Transform the arginine probe plasmid based on pET28a into JM109(DE3), invert and culture overnight, select from the plate, clone it into a 250 ml Erlenmeyer flask, place it on a 37 °C shaker, culture at 220 rpm until OD = 0.4 - 0.8, add 1 / 1000 (v / v) of IPTG (1 M), and induce expression at 18 °C for 24 - 36 hours. 2. After the induction of expression is completed, centrifuge at 4000 rpm for 30 minutes to harvest the cells, add 50 mM phosphate buffer to resuspend the cell pellet, and disrupt the cells by sonication until the cells become transparent. Centrifuge at 9600 rpm at 4 °C for 20 minutes. 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 with a self-loading desalting column to obtain the protein dissolved in 20 mM MOPS buffer (pH 7.4) or phosphate buffer PBS. 4. After identifying the purified arginine-binding protein mutant protein by SDS-PAGE, the probe was diluted to a protein solution with a final concentration of 5 - 10 μM in a measurement buffer (100 mM HEPES, 100 mM NaCl, pH 7.3) or phosphate buffer PBS. Arginine was prepared in a stock solution with a final concentration of 1 M in a measurement buffer (20 mM MOPS, pH 7.4) or phosphate buffer PBS. 5. Take 100 μl of 5 μM protein solution, incubate at 37 °C for 5 minutes, add arginine respectively to make the final concentration 100 mM and mix uniformly, and measure the absorbance of the protein at 340 nm using a multimode fluorescence microplate reader. 6. Take 100 μl of 1 μM fluorescent probe solution, incubate at 37 °C for 5 minutes, titrate with arginine, and measure the intensity of the fluorescence emitted at 528 nm when the protein is excited by 485 nm fluorescence. The measurement of the excitation and emission fluorescence of the sample is carried out using a multimode fluorescence microplate reader. 7. Take 100 μl of 1 μM fluorescent probe solution, incubate at 37 °C for 5 minutes, add arginine, and measure the absorption spectrum and fluorescence spectrum of the probe-protein. The measurement of the absorption spectrum and fluorescence spectrum of the sample is carried out using a spectrophotometer and a fluorescence spectrophotometer.

[0085] II.7 Transfection and Fluorescence Detection of Mammalian Cells 1. Transfect HeLa cells with an arginine probe plasmid based on PAAV using the transfection reagent Lipofectamine 2000 (Invitrogen), and culture them in an incubator at 37°C with 5% CO2. After the foreign gene is sufficiently expressed in 24 - 36 hours, fluorescence detection is performed. 2. After the induced expression is completed, wash the adherently cultured HeLa cells three times with PBS, place them in HBSS solution, and perform fluorescence microscopy and microplate reader detection respectively.

[0086] Example 1: Arginine - binding protein plasmid Amplify the STM4351 gene in the Agrobacterium tumefaciens gene by PCR. After performing gel electrophoresis on the PCR product, recover it and digest it with BamHI and EcoRI. At the same time, perform double digestion on the pET28a vector in the same way. After ligation with T4 DNA ligase, ligate the products, transform Trans5a, and spread the transformed Trans5a on an LB plate (kanamycin 100 μg / mL) and culture it overnight at 37°C. After extracting the plasmid from the grown Trans5a transformant, perform PCR identification. If the sequencing of the positive plasmid shows accuracy, subsequent plasmid construction is carried out.

[0087] Example 2: Expression and detection of cpYFP optical probes with different insertion sites In this example, based on pET28a-STM4351 and according to the crystal structure of the arginine-binding protein, the following sites were selected to insert cpYFP, namely, 103 / 104, 103 / 105, 103 / 106, 103 / 107, 103 / 108, 103 / 109, 103 / 110, 103 / 111, 104 / 105, 104 / 106, 104 / 107, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 109, 105 / 110, 105 / 111, 106 / 107, 106 / 108, 106 / 109, 106 / 110, 106 / 111, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 109, 108 / 110, 108 / 111, 109 / 110, 109 / 111, 110 / 111, 197 / 198, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 197 / 206, 197 / 207, 197 / 208, 197 / 209, 198 / 199, 198 / 200, 198 / 201, 198 / 202, 198 / 203, 198 / 204, 198 / 205, 198 / 206, 198 / 207, 198 / 208, 198 / 209, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 199 / 207, 199 / 208, 199 / 209, 200 / 201, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 200 / 207, 200 / 208, 200 / 209, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 201 / 207, 201 / 208, 201 / 209, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 202 / 207, 202 / 208, 202 / 209, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 203 / 208, 203 / 209, 204 / 205, 204 / 206, 204 / 207, 204 / 208, 204 / 209, 205 / 206, 205 / 207, 205 / 208, 205 / 209, 206 / 207, 206 / 208, 206 / 209, 207 / 208, 207 / 209 or 208 / 209, and the corresponding plasmids were obtained.The acid sequences of exemplary optical probes are shown in Table 1.

[0088]

Table 1

[0089] The DNA fragment of cpYFP was produced by PCR, and the DNA fragment was inactivated by adding a phosphate group at the 5'-end. At the same time, a pET28a-arginine-binding protein linear vector containing different cleavage sites was produced by inverse PCR amplification. Under the action of PEG4000 and T4 DNA ligase, the linearized pET28a-STM4351 and the cpYFP fragment with phosphorylated 5'-end were ligated to produce a recombinant plasmid. These plates were placed in a Kodak multifunctional bioluminescence imaging system, and clones that were excited by the FITC channel and emitted yellow fluorescence were selected. Sequencing was performed by Shanghai Branch of Beijing Liuhe Huada Gene Technology Co., Ltd.

[0090] As a result of sequencing, if it was accurate, the recombinant plasmid was transformed into JM109(DE3) for induction expression, and the protein was purified. As a result of SDS-PAGE electrophoresis, the size was about 55 Kda. This size was consistent with the size of the STM4351-cpYFP fusion protein containing the His-tag purification tag expressed by pET28a-STM4351-cpYFP. The results are shown in Figure 1.

[0091] Arginine response screening of the purified STM4351-cpYFP fusion protein was performed. The detection signal of the fusion fluorescent protein containing 100 mM arginine was divided by the detection signal of the fusion fluorescent protein without arginine. The results are shown in Figure 2. According to the detection results, the responses to arginine exceeding 2-fold are at sites 104 / 110, 105 / 106, 105 / 109, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 198 / 201, 199 / 200, 199 / 202, 199 / 203, 199 / 204, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 205, 204 / 205.

[0092] Example 3. Expression and detection of cpGFP optical probes at different insertion sites cpYFP was changed to green fluorescent protein cpGFP by the method of Example 1 and fused to an arginine-binding protein to construct an arginine green fluorescent protein fluorescence probe, and expression and detection were performed by the method of Example 2. The results are shown in Figure 3. According to the fluorescence detection results, the responses to arginine exceeding 2-fold are at sites 104 / 110, 105 / 106, 105 / 109, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 198 / 201, 199 / 200, 199 / 202, 199 / 203, 199 / 204, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 205, 204 / 205.

[0093] Example 4. Expression and detection of cpBFP optical probes at different insertion sites By the method of Example 1, cpYFP was changed to blue fluorescent protein cpBFP, fused with arginine-binding protein to construct an arginine blue fluorescent protein fluorescence probe, and expression and detection were carried out by the method of Example 2. The results are shown in Figure 4. According to the fluorescence detection results, the responses to arginine exceeding 2-fold are at the sites of 104 / 110, 105 / 106, 105 / 109, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 198 / 201, 199 / 200, 199 / 202, 199 / 203, 199 / 204, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 205, 204 / 205.

[0094] Example 5. Expression and Detection of cpmApple Optical Probes at Different Insertion Sites By the method of Example 1, cpYFP was changed to apple red fluorescent protein cpmApple, fused with arginine-binding protein to construct an arginine red fluorescent protein fluorescence probe, and expression and detection were carried out by the method of Example 2. The results are shown in Figure 5. According to the fluorescence detection results, the responses to arginine exceeding 2-fold are at the sites of 104 / 110, 105 / 106, 105 / 109, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 109, 107 / 111, 197 / 203, 198 / 201, 199 / 200, 199 / 202, 199 / 203, 199 / 204, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 205, 204 / 205.

[0095] Example 6. Expression and Detection of Mutated cpYFP Optical Probes Inverse PCR linearization plasmid pET28a-STM4351-107 / 111-cpYFP, ​​primer contains the base sequence of the site to be mutated, and the PCR product obtained is ligated by phosphate group addition with the action of PNK, T4 DNA ligase and PEG4000 to obtain the site-specific mutation plasmid of three sites S30, R96 and D177, which was sequenced by Beijing Liuhe Hua Da Gene Technology Co., Ltd. Shanghai Branch. Expression and detection were performed according to the method of Example 2. The amino acid sequence of the partially mutated optical probe is shown in SEQ ID NO: 40 (107 / 111-S30N) and SEQ ID NO: 41 (107 / 111-D177N), and the nucleic acid sequence is shown in SEQ ID NO: 42 (S30N-107 / 111). The results are shown in FIG. 6, where the fluorescence detection results show that the S30N-107 / 111 and D177N-107 / 111 mutants respond more than three-fold to arginine.

[0096] Example 7. Probe titration curves and specificity 28 STM4351-cpYFP fusion proteins 104 / 110, 105 / 106, 105 / 110, 106 / 108, 106 / 109, 107 / 109, 197 / 199, 197 / 203, 197 / 204, 198 / 201, 198 / 202, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 205, 200 / 202, 20 Arginine detection was performed with a concentration gradient by selecting 0 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 205, 202 / 205, 203 / 206, 204 / 205, and 204 / 206, and the change in 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. The different insertion sites 105 / 110, 199 / 200, 200 / 203, and 200 / 205 arginine probes were K d(Binding constants) are 5.4 μM, 11.7 μM, 58.5 μM, and 15 μM respectively, and the rangeabilities are 4.3 times, 11.0 times, 8.6 times, and 3.0 times respectively. The results are shown in FIGS. 7A - B. Specificity detection was performed on the insertion sites 104 / 110, 107 / 109, 107 / 111, 197 / 203, 199 / 200, 199 / 201, 199 / 202, 200 / 202, 200 / 203, 200 / 204, 200 / 205, 201 / 202 with relatively large response multiples. According to the results, as shown in FIG. 8, the probe has excellent specificity for arginine.

[0097] Example 8. Spectral Characteristics of the Probe The mutant STM4351 - 199 / 200 - cpYFP with relatively high response multiple and excellent specificity was purified. After the purified arginine fluorescent probe was treated with 0 mM and 100 mM arginine for 10 minutes respectively, the fluorescence spectrum was detected with a fluorescence spectrophotometer. As the measurement of the emission spectrum, the predetermined excitation wavelengths are 420 nm and 485 nm respectively, and the emission spectrum from 360 to 540 nm was recorded and the numerical values were read at 5 - nm intervals. The spectral curve of the arginine fluorescent probe STM4351 - 104 / 110 - cpYFP is shown in the first figure of FIG. 9A. The probe shows that the decrease in fluorescence intensity at 420 - nm excitation after adding 500 mM arginine is 1.4 times that of adding 0 mM arginine, and the increase in fluorescence intensity at 485 - nm excitation is 2.0 times that of adding 0 mM arginine. Under the same conditions, the same treatment was performed on the probes 105 / 106, 105 / 110, 107 / 111, 197 / 199, 197 / 203, 197 / 204, 198 / 202, 198 / 203, 199 / 200, 199 / 202, 199 / 205, 200 / 203, 200 / 204, 200 / 205, 201 / 202, 201 / 204, 201 / 205, 201 / 206, 202 / 205, and 204 / 205, and their spectral curves are shown in FIGS. 9A - B.

[0098] Example 9. Sub - organellar Localization of the Probe and Characteristics in Sub - organelles In this example, different localization signal peptides were fused to the C-terminus or N-terminus of the arginine fluorescent probe STM4351-199 / 200-cpYFP, and the arginine fluorescent probe STM4351-199 / 200-cpYFP was localized to different organelles. After the plasmid of the arginine fluorescent probe STM4351-199 / 200-cpYFP gene fused with different localization signal peptides was transfected into HeLa cells for 36 hours, it was washed with PBS, placed in HBSS solution, and fluorescence detection was performed in the FITC channel with an inverted fluorescence microscope. By fusing the arginine fluorescent probe FLIPpro with different specific localization signal peptides, its localization to sub-organelles such as the cytoplasm, mitochondria, nucleus, Golgi apparatus, endoplasmic reticulum, and cell membrane was expressed. The results are shown in Figure 10, where fluorescence is shown in different sub-organelle structures, and the distribution and intensity of the fluorescence are various.

[0099] Example 10. Dynamic Monitoring of Arginine Transmembrane Transport After the plasmid of the STM4351-199 / 200-cpYFP gene expressed in the cytoplasm was transfected into HeLa cells for 36 hours, it was washed with PBS, placed in HBSS solution, and then the change in 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 within 40 minutes. The results are shown in Figure 11. After 2 hours of starvation, 10 mM arginine was added and detected for 20 minutes, and ratio485 / 420 gradually increased by 2.2 times. The same was true for the STM4351-105 / 110-cpYFP probe and the STM4351-200 / 203-cpYFP probe.

[0100] Example 11. High-Throughput Compound Screening Based on Probes at the Live-Cell Level In this example, high-throughput compounds were screened using HeLa cells expressing the arginine probe STM4351-199 / 200-cpYFP in the cytoplasm. HeLa cells transfected with the STM4351-199 / 200-cpYFP gene were washed with PBS and then placed in HBSS solution (without arginine) and treated for 1 hour, followed by treatment with 10 μM compound for 1 hour. Arginine was added dropwise respectively. The change in 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. A sample not treated with any compound was used as a standard. The results are shown in Figure 12. After treating cells with 2000 compounds, most compounds had little effect on arginine entry into cells. There were 16 compounds that improved the uptake ability of cells for arginine, and 11 compounds that significantly decreased the uptake of arginine by cells.

[0101] Example 12. Quantitative detection of the probe for arginine in blood In this example, the purified arginine fluorescent probe STM4351-199 / 200-cpYFP protein was used to analyze arginine in the blood supernatants of mice and humans. After mixing the arginine fluorescent probe STM4351-199 / 200-cpYFP fluorescent protein 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 detected using a microplate reader. The results are shown in Figure 13. The arginine content in mouse blood was about 56 μM, and the arginine content in human blood was about 68 μM. According to the above examples, the arginine fluorescent probe provided by the present invention has a relatively small protein molecular weight, is easy to mature, has a large dynamic change in fluorescence, good specificity, can be expressed in cells by genetic engineering methods, can perform real-time localization and quantitative detection of arginine inside and outside cells, and can further perform high-throughput compound screening.

[0102] What is described above is a preferred embodiment of the present invention. Those skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are regarded as falling within the protection scope of the present invention.

Claims

1. An optical probe comprising an arginine-sensitive polypeptide and an optically active polypeptide, wherein the optically active polypeptide is within the sequence of the arginine-sensitive polypeptide, the arginine-sensitive polypeptide has the sequence shown in SEQ ID NO: 1, the optically active polypeptide is a fluorescent protein, the optically active polypeptide is cpYFP, and this cpYFP is located at one position of the arginine-sensitive polypeptide selected from the group consisting of 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 111, 109 / 110, 110 / 111, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 197 / 205, 198 / 201, 198 / 202, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 203 / 204, 203 / 205, 203 / 206, 203 / 207, 204 / 205, 204 / 206, 204 / 207, 205 / 206, 206 / 207, 207 / 208, and 208 / 209. The optically active polypeptide is cpGFP, and this cpGFP is located at one position of an arginine-sensitive polypeptide selected from the group consisting of 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 108 / 111, 110 / 111, 197 / 199, 197 / 200, 197 / 201, 197 / 202, 197 / 203, 197 / 204, 198 / 201, 198 / 202, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 204, 199 / 205, 199 / 206, 200 / 202, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 203 / 205, 203 / 206, 203 / 207, 204 / 205, 204 / 206, and 205 / 206. The optically active polypeptide is cpBFP, and this cpBFP is located at one position of an arginine-sensitive polypeptide selected from the group consisting of 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 109, 107 / 110, 107 / 111, 108 / 111, 110 / 111, 197 / 199, 197 / 201, 197 / 203, 197 / 204, 198 / 201, 198 / 202, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 203, 202 / 204, 202 / 205, 202 / 206, 203 / 205, 203 / 206, 203 / 207, 204 / 205, 204 / 206, 205 / 206, 206 / 207, and 207 / 208, or The optically active polypeptide is cpmApple, and this cpmApple is located at one position of an arginine-sensitive polypeptide selected from the group consisting of 103 / 104, 104 / 108, 104 / 109, 104 / 110, 104 / 111, 105 / 106, 105 / 107, 105 / 108, 105 / 110, 105 / 111, 106 / 108, 106 / 109, 107 / 108, 107 / 109, 107 / 110, 107 / 111, 108 / 111, 109 / 110, 110 / 111, 197 / 199, 197 / 201, 197 / 203, 197 / 204, 197 / 205, 198 / 201, 198 / 202, 198 / 203, 199 / 200, 199 / 201, 199 / 202, 199 / 203, 199 / 204, 199 / 205, 199 / 206, 200 / 203, 200 / 204, 200 / 205, 200 / 206, 201 / 202, 201 / 203, 201 / 204, 201 / 205, 201 / 206, 202 / 203, 202 / 205, 202 / 206, 203 / 204, 203 / 206, 203 / 207, 204 / 205, 204 / 206, 205 / 206, 206 / 207, and 207 / 208, and is an optical probe.

2. The optical probe according to claim 1, wherein cpYFP has the sequence shown in SEQ ID NO: 2, cpGFP has the sequence shown in SEQ ID NO: 3, cpBFP has the sequence shown in SEQ ID NO: 4, and cpmApple has the sequence shown in SEQ ID NO:

5.

3. The optical probe according to claim 1 or claim 2, wherein the arginine-sensitive polypeptide contains a mutation selected from S30N, D177N, R96M, and R96K.

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 the complementary sequences of (1).

5. A nucleic acid construct comprising the nucleic acid according to claim 4.

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

7. A host cell, (1) expressing the optical probe according to any one of claims 1 to 3, (2) containing the nucleic acid according to claim 4, or A host cell comprising the nucleic acid construct according to claim 5 or 6. **Claim 8** A method for preparing an optical probe according to any one of claims 1 to 3, comprising culturing the host cell according to claim 7 and separating the optical probe from the culture. **Claim 9** Use of the optical probe according to any one of claims 1 to 3, the nucleic acid according to claim 4, or the nucleic acid construct according to claim 5 or 6 for the detection of arginine or the selection of a compound in a sample, wherein the detection comprises qualitative, localization or quantitative detection of arginine. **Claim 10** 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 cell according to claim 7, and other reagents for detecting arginine with the optical probe.

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