CN104211811A - Copper silver ion fluorescin probe, and making method and application thereof - Google Patents

Abstract

Provide a genetically encoded fluorescent probe for Cu/Ag ions, which is composed of a protein sensitive to Cu/Ag ions and a part that expresses Cu/Ag ions through changes in spectral properties, wherein the part that expresses Cu/Ag ions It is a fluorescent protein sequence or its derivatives, and the protein sensitive to Cu/Ag ions is a polypeptide or its functional fragments with the following characteristics: (1) domain B with Cu/Ag ion binding properties; and/or (2) source CueR protein, a transcriptional regulator sensitive to Cu/Ag ions. Also provided are fusion proteins comprising the fluorescent probe, nucleotide sequences encoding the fluorescent probe or fusion protein and expression vectors, methods for preparing the fluorescent probe or fusion protein, and methods for detecting Cu/Ag ions or screening Drug kit. The fluorescent probe of the present invention uses the CueR protein to sense the concentration and state changes of Cu/Ag ions in the environment, and to describe Cu/Ag ions intuitively and in real time through whether the fluorescent protein produces fluorescence or not and the intensity of the fluorescence, and can be used at physiological and sub- Cu/Ag ions are detected at the cellular level.

Description

Copper silver ion fluorescent protein probe and its preparation method and application

technical field

The invention relates to a Cu/Ag ion detection probe, in particular to a Cu/Ag ion recombinant fluorescent fusion protein detection probe. The present invention also relates to the preparation method of the detection probe and its application in the detection of Cu/Ag ions.

Background technique

The traditional methods for detecting metal ions mainly include atomic absorption spectroscopy, atomic emission spectroscopy, spectrophotometry, electrically coupled plasma mass spectrometry, electrochemical methods, and fluorescence analysis methods. There are certain requirements and it is easy to be limited, or the degree of discrimination of excessive metal radius is not high, so it is easy to be interfered in the detection. Fluorescence analysis is favored by researchers because of its high sensitivity and easy operation, and fluorescent small molecule chemical probes based on it have made great progress. Small molecule chemical probes represented by aromatic hydrocarbons and polyamines inevitably have the disadvantages of uneven sensitivity and single action medium. Moreover, the exogenously synthesized chemical small molecule probes need to characterize the activity of metal ions in the cell. There are many factors in the process of entering the cell membrane and functioning that prevent this process from going smoothly. For example, the membrane permeability of small molecule probes may Existing cytotoxicity, etc., thus cannot meet the requirements of life medical science research. Therefore, there is an urgent need in this field to develop a specific detection technology for copper and silver ions, especially a specific detection technology for copper and silver ions suitable for physiological and subcellular levels.

With the rapid development of molecular biology and protein science, the structure and properties of intracellular metal transcriptional regulatory proteins have become increasingly clear. These transcriptional regulatory proteins often have specific binding capabilities for metal ions, and their affinity for metal ions Can be used to infer the content of free metal ions in cells. These proteins serve as natural model proteins for the construction of intracellular metal ion probes using the fluorescent protein strategy.

Compared with the traditional small molecule dye detection technology and the rapidly developing quantum dot detection technology, the fluorescent protein detection technology has a unique overwhelming advantage in most live cell imaging. It can be genetically introduced into cells, tissues and even whole organs. Therefore, fluorescent proteins can be used as whole-cell markers or indicators of gene activation.

Green fluorescent protein was originally extracted from Aequorea victoria. The wild-type AvGFP consists of 238 amino acids and has a molecular weight of about 26kD. Current research has confirmed that the three amino acids Ser-Tyr-Gly at positions 65-67 in the natural GFP protein can spontaneously form a fluorescent chromophore: p-hydroxybenzylideneimidazolinone (ρ-hydroxybenzylideneimidazolinone), which is Its main luminous position. The spectral characteristics of wild-type AvGFP are very complex, the main peak of its fluorescence excitation is at 395nm, and there is another auxiliary peak at 475nm, the amplitude of the latter is about 1/3 of the former. Under standard solution conditions, excitation at 395nm produces emission at 508nm, while excitation at 475nm produces a maximum emission wavelength at 503nm (Heim, R. et al., Proc Natl Acad Sci U S A.1994, V.91 (26), pp. 12501-12504).

With the deepening of research on GFP protein mutations, using molecular biology techniques, a variety of outstanding GFP derivatives have been developed. By performing different single-point mutations or combinations on the basis of wild-type GFP, such as enhanced Type GFP (S65T, F64L), YFP (T203Y), CFP (Y66W), etc. With the help of the rearrangement of the GFP protein sequence, the original 145-238 amino acid part is used as the N-terminal of the new protein, and the original 1-144 amino acid is used as the C-terminal of the new protein. The peptide chains are connected to form a circular permutation fluorescent protein (circular permutation fluorescent protein) that is sensitive to spatial changes. On this basis, the point mutation of the original protein T203Y forms a circular permutation of yellow fluorescent protein cpYFP (Nagai, T etc., Proc Natl Acad Sci U S A.2001, V.98(6), pp.3197-3202).

Due to the increasingly in-depth research on fluorescent proteins, some related fluorescence-based analysis and detection technologies have also been further developed. For example, the currently commonly used fluorescence resonance energy transfer (FRET) technology, the main principle of this technology is that when two fluorescent chromophores are close enough, when the donor molecule absorbs photons of a certain frequency, it is excited to a higher electronic energy state. , before the electron returns to the ground state, the energy is transferred to the adjacent acceptor molecule through the dipole interaction (that is, energy resonance transfer occurs). FRET is a non-radiative energy transition. Through the electric dipole interaction between molecules, the energy of the excited state of the donor is transferred to the excited state of the acceptor, so that the fluorescence intensity of the donor is reduced, and the acceptor can emit stronger than itself The characteristic fluorescence (sensitized fluorescence) can also not fluoresce (fluorescence quenching). Further research on the current GFP found that cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), derived from GFP mutants, are a well-performing donor/acceptor pair. The emission spectrum of CFP overlaps considerably with the absorption spectrum of YFP. When they are close enough, the chromophore of CFP will resonantly transfer energy to the chromophore of YFP when excited by the absorption wavelength of CFP. , so the emission fluorescence of CFP will weaken or disappear, and the main emission will be the fluorescence of YFP. The energy conversion efficiency between two chromophores is inversely proportional to the sixth power of the spatial distance between them, and is very sensitive to changes in spatial position. Therefore, existing research reports use genetic engineering recombination methods to fuse the two ends of the desired protein gene with CFP and YFP to express a brand new fusion protein. Fluorescence changes are visualized visually.

Therefore, the fluorescent protein sequence used herein can be derived from the fluorescent protein of Aequoreavictoria and its derivatives, including but not limited to these mutants: yellow fluorescent protein (YFP), green fluorescent protein (GFP), The sequence of cyan fluorescent protein (CFP), etc., wherein the sequence of yellow fluorescent protein YFP is preferred, and the sequence of yellow fluorescent protein cpYFP arranged in a circle is more preferred.

Another protein CueR involved in this technology is a bacterial transcriptional regulatory protein known in the art, which is involved in regulating the copper ion content in cells. According to Changela et al. (A.Changela, K.Chen, Y.Xue, J.Holschen, C.Outten, T.O'Halloran, A.Mondragon, Molecular basis of metal-ion selectivity and zeptomolarsensitivity by CueR.Science301 (2003 ) 1383-1387.) Structural and functional studies on CueR, which contains three domains: the C-terminal metal-binding domain, the N-terminal DNA-binding domain and the intermediate dimer region. Studies have shown that the key sites involved in metal binding in the CueR protein are cysteines at positions 112 and 120. When metal ions are bound to the metal binding region, the protein itself will be deformed, which is very conducive to the construction of intracellular cells combined with fluorescent proteins. Cu ⁺ probe.

Although the CueR protein itself is sensitive to the concentration and state of Cu/Ag ions in the environment, its own changes cannot be displayed intuitively and captured by the outside world. With the help of fluorescent protein as a tool, we can use the The two are fused and expressed to obtain a brand-new gene-encoded fluorescent probe, which uses the CueR protein to sense changes in the concentration and state of Cu/Ag ions in the environment and transmits this change to the fluorescent protein, which generates fluorescence through the fluorescent protein and Fluorescence intensity, real-time and intuitive description of Cu/Ag ion concentration and state changes in the environment.

In summary, we believe that the use of recombinant fluorescent fusion proteins containing CueR proteins can meet the urgent needs of detecting Cu/Ag ions at physiological and subcellular levels.

Citation or discussion of references described herein should not be construed as an admission that such references are prior art to the present invention.

Summary of the invention

The first object of the present invention is to provide a genetically encoded Cu/Ag ion fluorescent probe.

The second object of the present invention is to provide a fusion protein comprising a genetically encoded Cu/Ag ion fluorescent probe.

The third object of the present invention is to provide the nucleotide sequence encoding the fluorescent probe or the fusion protein or its complementary sequence.

The fourth object of the present invention is to provide an expression vector comprising said nucleotide sequence.

The fifth object of the present invention is to provide a method for preparing the fluorescent probe or the fusion protein.

The sixth object of the present invention is to provide a kit for detecting Cu/Ag ions or screening drugs.

The genetically encoded Cu/Ag ion fluorescent probe provided by the present invention is composed of a protein sensitive to Cu/Ag ions and a part that expresses Cu/Ag ions through changes in spectral properties, wherein the change in spectral properties affects Cu/Ag ions. The part where the /Ag ion is displayed is a fluorescent protein sequence or a derivative thereof, and the protein sensitive to the Cu/Ag ion is a polypeptide or a functional fragment thereof with the following characteristics:

(1) Domain B with Cu/Ag ion binding properties; and/or

(2) Derived from CueR protein, a transcriptional regulator sensitive to Cu/Ag ions.

In a preferred embodiment, the polypeptide sensitive to Cu/Ag ions may have the following characteristics:

(1) Containing the transcription regulator CueR protein from bacteria, the amino acid sequence of the protein is SEQ ID NO: 1; and

(2) Any homologous or non-homologous sequence having 70% identity with the sequence described in (1), preferably any sequence within at least 24 amino acid residues in the amino acid sequence having 70% identity with the sequence described in (1) Homologous or nonhomologous sequences of identity.

In another embodiment, the fluorescent probe of the present invention may comprise the Cys-Cys structure B shown in SEQ ID NO: 1 with Cu/Ag ion binding properties and the fluorescent probes shown in SEQ ID NO: 2, 3, and 4, respectively. Protein sequence A, A1 and/or A2, the combination thereof can be:

(1) B-A-B;

(2) A1-B-A2, wherein A1 and A2 can be the same or different, A1 can be the amino acid sequence of a fluorescent protein or a derivative thereof from Aequorea victoria, and A2 can be the amino acid sequence of a fluorescent protein from Aequorea victoria The amino acid sequence of another fluorescent protein or derivative thereof;

(3) First part of B-A-Second part of B, where A is inserted within the flexible region of B, thus dividing B into first part of B and second part of B, first part of B and second part of B constitute the complete B domain; or

(4) The first part of B-A-the second part of B-B, where A is inserted in the flexible area of B, thus dividing B into the first part of B and the second part of B, the first part of B and the second part of B The two parts constitute the complete B domain, and a B is connected to the end of the chimeric sequence.

The site where A is inserted into the flexible region of B may be any of the positions 113, 114, 115, 116, 117, 118 and 119 in the flexible region of the B domain, and the A inserted in the flexible region of B may be one or more.

In one embodiment, the fluorescent probe provided by the present invention comprises a fluorophore and a CueR protein or any fragment, derivative or analog of the CueR protein. In another embodiment, the fluorescent probe provided by the present invention comprises a fluorophore and a variant of CueR protein. In another embodiment, the fluorescent probe provided by the present invention comprises a fluorophore and a soluble fragment of CueR protein.

In a preferred embodiment, the Cu/Ag ion-sensitive protein in the fluorescent probe provided by the present invention includes any homologous or non-homologous sequence having 70% identity with the amino acid sequence SEQ ID NO: 1. In a preferred embodiment, the Cu/Ag ion-sensitive protein in the fluorescent probe provided by the present invention includes any homologous or non-homologous sequence substantially similar or identical to the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the Cu/Ag ion-sensitive protein in the fluorescent probe provided by the present invention comprises a variant or derivative of the amino acid sequence of SEQ ID NO:1.

In another aspect, the present invention provides a fusion protein comprising the fluorescent probe of the present invention. In one embodiment, the fusion protein comprises the fluorescent probe of the present invention and a specific subcellular localization signal, and the localization signal can localize the target protein in a designated subcellular organelle. The specific localization signal may be mitochondrial localization and cytoplasmic localization. In a preferred embodiment, the specific localization signal is the mitochondrial localization signal shown in SEQ ID NO:5.

In another aspect, the present invention provides a nucleotide sequence encoding the fluorescent probe or fusion protein of the present invention or its complementary sequence.

The nucleotide sequence provided by the present invention includes a nucleotide sequence encoding a fluorescent protein and a nucleotide sequence encoding a protein sensitive to Cu/Ag ions.

In a preferred embodiment, the nucleotide sequence encoding a protein sensitive to Cu/Ag ions is a nucleotide sequence encoding a polypeptide having the following characteristics or a functional fragment thereof:

(1) Domain B with Cu/Ag ion binding properties; and/or

(2) Derived from CueR protein, a transcriptional regulator sensitive to Cu/Ag ions.

In another preferred embodiment, the nucleotide sequence of the present invention comprises the coding sequence b of the structural domain B having Cu/Ag ion binding properties and the coding sequence a, a1 and/or a2 of the fluorescent protein, and its combination form can be:

(1) b-a-b;

(2) a1-b-a2, wherein a1 and a2 can be the same or different; a1 is from the coding sequence of a fluorescent protein of Aequorea victoria or its derivatives, and a2 is from another fluorescent protein of Aequorea victoria The coding sequence of its derivatives;

(3) The first part of b-a-the second part of b, where a is inserted in the flexible region of b, thus dividing b into the first part of b and the second part of b, the first part of b and the second part of b Partially constitutes the complete b domain;

(4) The first part of b-a-the second part of b-b, where a is inserted in the flexible region of b, thus dividing b into the first part of b and the second part of b, the first part of b and the The second part constitutes the complete b domain, and a b is attached at the end of the chimeric sequence.

In another preferred embodiment, the present invention provides a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 6, 7 or 8. In another preferred embodiment, the present invention provides a nucleotide sequence comprising any nucleoside substantially similar or identical to the nucleotide sequence SEQ ID NO: 6, 7 or 8 within a length of at least 405 bases Acid sequence; In a preferred embodiment, the invention provides a nucleotide sequence comprising a variant or derivative of the nucleotide sequence SEQ ID NO: 6, 7 or 8.

The present invention also relates to complementary sequences and variants of the above-mentioned nucleotide sequences, which may include nucleotide sequences encoding fragments, analogs, derivatives, soluble fragments and variants of the fluorescent probe or fusion protein of the present invention or their complements sequence.

In yet another aspect, the present invention also provides an expression vector comprising the nucleotide sequence of the present invention operably linked to an expression control sequence. The expression control sequence may be an origin of replication, a promoter, an enhancer, an operator, a terminator, a ribosome binding site, and the like.

In yet another aspect, the present invention also provides a host cell comprising the expression vector of the present invention.

In yet another aspect, the present invention also provides a method for preparing the fluorescent probe or fusion protein of the present invention, comprising the following steps:

a. transferring the expression vector of the present invention into a host cell,

b. culturing said host cell under conditions suitable for expression by said host cell, and

c. Isolating said fluorescent probe or fusion protein from said host cell.

The present invention also provides the application of the fluorescent probe or fusion protein of the present invention in the detection of Cu/Ag ions. In one embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in detecting Cu/Ag ions in vitro or in vivo. In one embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in the detection of Cu/Ag ions at the subcellular level. In one embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in the in situ detection of Cu/Ag ions. In another embodiment, the present invention provides the application of the fluorescent probe or fusion protein of the present invention in screening drugs, which can be used to adjust the Cu/Ag ion level of the subject. In another embodiment, the present invention provides the use of the fluorescent probe or fusion protein of the present invention in the diagnosis of diseases that are related to Cu/Ag ion levels.

The present invention also provides a kit for detecting Cu/Ag ions or screening drugs, which contains the fluorescent probe or fusion protein of the present invention and instructions for use. The detection can be performed at the in vivo, in vitro, subcellular or in situ level. The present invention also provides a kit for screening drugs that can be used to regulate the Cu/Ag ion level of a subject, and the kit includes an effective amount of the fluorescent probe or fusion protein of the present invention. In use, those skilled in the art can conveniently determine the effective amount according to the activity of the fusion protein.

Detailed description of the invention

I. Definition:

As used herein, the term "about" when a value or range is given means that the value or range is within 20%, within 10%, and within 5% of the given value or range.

As used herein, the terms "comprising", "comprising" and their equivalents include the meanings of "containing" and "consisting of", for example a composition "comprising" X may consist solely of X or may contain other substances, such as X+ Y.

In the present invention, the term "CueR protein" refers to CueR protein, which is a bacterial transcriptional regulatory protein known in the art, with a molecular weight of 15kDa, which can regulate the copper ion content in Escherichia coli to maintain it in a relatively stable range Inside. It contains three structural domains: the metal-binding domain at the C-terminus, the DNA-binding domain at the N-terminus, and the dimer region in the middle. The key sites involved in metal binding in the CueR protein are cysteines at positions 112 and 120. The two cysteines are connected by a flexible region containing 7 amino acids. The "CueR protein" involved in the present invention may comprise the amino acid sequence encoded by the nucleotide sequence SEQ ID NO:1. The "flexible region" involved in the present invention refers to some specific structures such as Loop domains in the high-level structure of proteins. Compared with other high-level protein structures, these domains have higher mobility and flexibility, and can This leads to dynamic changes in the structural domains, and the protein also has a tendency to greatly change the spatial conformation in these regions. The flexible region involved in the claims mainly refers to the P113-D119 region in CueR

The term "Cu/Ag ion" used in this specification refers to monovalent and/or divalent copper ions, and monovalent silver ions.

The term "fluorescent probe" used in this specification refers to a protein that is sensitive to Cu/Ag ions in the environment and is fused with a fluorescent protein. The polypeptide that is sensitive to Cu/Ag ions in the environment can specifically be a CueR protein. The conformational change of the Cys-Cys structure combined with Cu/Ag ions combined with Cu/Ag ions causes the conformational changes of the fluorescent protein, which in turn leads to the generation or disappearance of fluorescence or the change of generated fluorescence, and with the help of different Cu/Ag ions The fluorescence of the fluorescent protein measured at the concentration is used to draw a standard curve to detect and analyze the presence and/or level of Cu/Ag ions.

The term "fusion protein" used in this specification is synonymous with "fluorescent fusion protein" and "recombinant fluorescent fusion protein", and refers to the amino acid sequence comprising the first polypeptide or protein or its fragment, analog or derivative, and heterologous polypeptide or A polypeptide or protein having an amino acid sequence different from a second polypeptide or protein or a fragment, analog or derivative thereof, ie a second polypeptide or protein or a fragment, analog or derivative thereof. In one embodiment, the fusion protein comprises a fluorescent protein fused to a heterologous protein, polypeptide or peptide. According to this embodiment, the heterologous protein, polypeptide or peptide may or may not be a different type of fluorescent protein. In one embodiment, the fusion protein maintains or increases the activity compared to the activity of the original polypeptide or protein prior to fusion to the heterologous protein, polypeptide or peptide. In a specific embodiment, the fusion protein comprises a fluorescent probe fused to a heterologous protein, polypeptide or peptide, which may be a specific subcellular localization signal.

The term "fluorophore" used in this specification is synonymous with "fluorescent protein" and refers to a protein that fluoresces itself or fluoresces under illumination. Fluorescent proteins are often used as detection methods, such as the green fluorescent protein GFP commonly used in the field of biotechnology and BFP, CFP, YFP, cpYFP, etc. derived from mutations of this protein.

The term "GFP" as used herein refers to green fluorescent protein, which is originally extracted from Aequoreavictoria. The wild-type AvGFP consists of 238 amino acids, has a molecular weight of about 26kD, and its amino acid sequence is SEQ ID No: 2. Current research has confirmed that the three amino acids Ser-Tyr-Gly at positions 65-67 in the natural GFP protein can spontaneously form a fluorescent chromophore: p-hydroxybenzylideneimidazolinone (ρ-hydroxybenzylideneimidazolinone), which is Its main luminous position. The spectral characteristics of wild-type AvGFP are very complex, the main peak of its fluorescence excitation is at 395nm, and there is another auxiliary peak at 475nm, the amplitude of the latter is about 1/3 of the former. Under standard solution conditions, excitation at 395nm produces emission at 508nm, while excitation at 475nm produces a maximum emission at 503nm.

The term "YFP" used in this manual refers to yellow fluorescent protein, which is derived from green fluorescent protein GFP, and its amino acid sequence is more than 90% homologous to GFP. Compared with GFP, the key change of YFP is that the 203rd amino acid is replaced by threonine Mutation to tyrosine (T203Y). Compared with the original AvGFP, the wavelength of the main excitation peak of YFP was red-shifted to 514nm and the emission wavelength was changed to 527nm. On this basis, site-directed mutation (S65T) of the 65th amino acid of YFP can obtain the fluorescence-enhanced yellow fluorescent protein EYFP, and the typical amino acid sequence of EYFP is SEQ ID No: 3. For the rearrangement of the EYFP protein sequence, the original 145-238 amino acid part is used as the N-terminal of the new protein, and the original 1-144 amino acid is used as the C-terminal of the new protein, and a small flexible short peptide is passed between the two fragments The chain VDGGSGGTG is connected to form a circular permutation yellow fluorescent protein cpYFP (circular permutation yellow fluorescent protein) that is sensitive to spatial changes. The typical amino acid sequence of cpYFP is SEQ ID No: 4.

In the present invention, the CueR protein fused with the fluorophore can be the full length or fragment thereof isolated from Escherichia coli CueR protein, preferably the amino acid 1-135 of the natural Escherichia coli CueR protein, more preferably the amino acid of the Escherichia coli CueR protein 1-135.

"Linker" refers to an amino acid or nucleotide sequence that joins two parts in a polypeptide, protein or nucleic acid of the invention. When connecting in the polypeptide or protein of the present invention, the length of the linker is not more than 6 amino acids, preferably not more than 4 amino acids, more preferably 3 amino acids. When linking is performed in the nucleic acid of the present invention, the length of the linker is not more than 18 nucleotides, preferably not more than 12 nucleotides, more preferably 9 nucleotides.

When referring to a certain polypeptide or protein, the term "variant" used in the present invention includes variants that have the same function of the polypeptide or protein but have different sequences. These variants include (but are not limited to): one or more deletions, insertions and/or substitutions (usually 1-30, preferably 1-20, more preferably 1-10, preferably 1-5) amino acids, and one or several (usually within 20, preferably within 10, more preferably 5) added at its C-terminal and/or N-terminal within 1) amino acid sequence obtained. For example, in this field, substitutions with amino acids with similar or similar properties generally do not change the function of the polypeptide or protein. In this field, amino acids with similar properties often refer to amino acid families with similar side chains, which have been 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 side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g. threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the polypeptide or protein. Those skilled in the art know that in gene cloning operations, it is often necessary to design suitable restriction sites, which inevitably introduces one or more irrelevant residues at the end of the expressed polypeptide or protein, which does not affect the purpose Activity of polypeptide or protein. Another example is to construct a fusion protein, promote the expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate the purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminal, C-terminal or the recombinant protein. Within other suitable regions within the protein, for example, including but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, glutathione S-transferase (GST), maltose E binding protein, protein A, such as 6His or Flag tag, or proteolytic enzyme site for factor Xa or thrombin or enterokinase. Variants of polypeptides or proteins may include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, those capable of hybridizing to the DNA of the polypeptide or protein under high or low stringency conditions Polypeptide or protein encoded by DNA, and polypeptide or protein obtained by using antiserum against said polypeptide or protein. These variants may also comprise at least about 70%, at least about 75%, at least about 80%, at least about 24%, at least about 90%, at least about 95%, at least about 98% sequence identity to the polypeptide or protein. %, at least about 99%, or 100% of the polypeptide or protein.

In two or more polypeptide or nucleic acid molecule sequences, the term "identity" or "identity percentage" refers to the comparison window or specified region, using methods known in the art such as sequence comparison algorithms, by manual alignment and visual inspection When examined to compare and align for maximum correspondence, two or more sequences or subsequences are identical or have a certain percentage of amino acid residues or nucleotides identical over a specified region (for example, 60%, 65%, 70%, 75%, 80%, 24%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% the same). For example, preferred algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST2.0 algorithms, see Altschul et al. (1977) Nucleic Acids Res. 25:3389 and Altschul et al. (1990) J. Mol. Biol, respectively. .215:403.

The term "soluble fragment" as used herein generally refers to at least about 10 contiguous amino acids, usually at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, of the full-length sequence of the protein. Amino acids, optimally stretches of at least about 100 contiguous amino acids.

The terms "functional fragment", "derivative" and "analogue" as used herein refer to a protein that substantially maintains the same biological function or activity as the native CueR of the present invention. The functional fragments, derivatives or analogs of CueR of the present invention may be (i) proteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acids The residue may or may not be encoded by the genetic code, or (ii) the protein has a substitution group in one or more amino acid residues, or (iii) the mature protein is combined with another compound (such as a compound that extends the half-life of the protein , such as polyethylene glycol), or (iv) an additional amino acid sequence fused to the protein sequence (such as a leader or secretory sequence or a sequence or proprotein sequence used to purify the protein, Or the fusion protein formed with the antigen IgG fragment). Based on the teaching herein, these functional fragments, derivatives and analogs are within the scope of those skilled in the art.

The difference between the analogue and the natural CueR protein may be a difference in amino acid sequence, or a modification that does not affect the sequence, or both. These proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology.

The analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the CueR protein of the present invention is not limited to the representative proteins, fragments, derivatives and analogs listed above. Modified (usually without altering primary structure) forms include: chemically derivatized forms of proteins such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications during protein synthesis and processing or during further processing steps. This modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are proteins that have been modified to increase their resistance to proteolysis or to optimize solubility.

The term "nucleic acid" as used in the present invention may be in the form of DNA or RNA. Forms of DNA include cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either the coding strand or the non-coding strand. The coding region sequence encoding the mature protein may be the same as the coding region sequence shown in SEQ ID NO: 6, 7 or 8 or a degenerate variant thereof. As used herein, "degenerate variant" in the present invention refers to a nucleotide sequence that encodes the fluorescent fusion protein of the present invention, but differs from the sequence of the coding region shown in SEQ ID NO: 6, 7 or 8.

As used herein, the term "variant" when referring to a nucleic acid 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 known in the art, an allelic variant is an alternative form of a nucleic acid, which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of its encoded protein. Nucleic acids of the invention may comprise at least about 70%, at least about 75%, at least about 80%, at least about 24%, at least about 90%, at least about 95%, at least about 98% sequence identity to said nucleotide sequence. %, at least about 99%, or 100% of the nucleotide sequence.

As used herein, the term "hybridizes under stringent conditions" is used to describe hybridization and wash conditions typically under which nucleotide sequences that are at least 60% homologous to each other can still hybridize to each other. Preferably, stringent conditions are those under which sequences that are at least 65%, more preferably at least 70%, and even more preferably at least 80% or more homologous to each other will generally still hybridize to each other. Such stringent conditions are well known to those of ordinary skill in the art. A preferred, non-limiting example of stringent conditions is: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 0°C; or (2) hybridization with With denaturant, 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, better Hybridization occurs only when it is more than 95%. Moreover, the protein encoded by the hybridizable nucleic acid has the same biological function and activity as the mature protein.

The present invention also relates to nucleic acid fragments that hybridize to the above-mentioned sequences. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides in length, more preferably at least 50 nucleotides in length, most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques (eg, PCR).

The full-length sequence or fragments of the fluorescent probe or fusion protein of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant polypeptide or protein is obtained by isolating and purifying from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragments, derivatives, analogs or variants) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art. Mutations can be introduced into the protein sequence of the present invention by methods such as mutation PCR or chemical synthesis.

As used herein, the terms "expression vector" and "recombinant vector" are used interchangeably and refer to prokaryotic or eukaryotic vectors well known in the art, such as bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, Retroviral or other vectors, which are capable of replicating and stabilizing in the host, an important feature of these recombinant vectors is that they usually contain expression control sequences.

The term "expression control sequence" as used herein refers to elements that regulate the transcription, translation and expression of the gene of interest that can be operably linked to the gene of interest, and can be an origin of replication, a promoter, a marker gene or a translation control element, including enhancers, operators , terminator, ribosome binding site, etc., the choice of expression control sequence depends on the host cell used. The expression control sequences mainly include: ① There is a TATA box about 20-30 nucleotides upstream of the 5′-terminal transcription initiation point; ② About 70-80 nucleotides upstream of the 5′-terminal transcription initiation point, There is a CAAT box; ③ At the position about 100 nucleotides upstream of the 5′ terminal transcription start point, some sequences can enhance the transcriptional activity, which can enhance the transcriptional activity hundreds of times, so it is called an enhancer ; ④ There is a nucleotide sequence of AATAAA downstream of the 3′-terminal stop codon, which may play an important role in the tailing of mRNA (adding poly A to the tail of mRNA).

Recombinant vectors suitable for use in the present invention include, but are not limited to, bacterial plasmids. In a recombinant expression vector, "operably linked" means that a nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence. Those skilled in the art are familiar with methods that can be used to construct expression vectors containing the fusion protein coding sequences of the present invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, reverse LTRs of transcription viruses and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

Those of ordinary skill in the art will appreciate that the design of a recombinant expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, and the like. In addition, the recombinant expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as chloramphenicol or ampicillin resistance for E. coli.

In one embodiment, the coding sequence of the fluorescent probe or fusion protein of the present invention is double-digested with NheI and HindIII and then ligated with the pRSET _b vector cut with NheI and HindIII to obtain a recombinant expression vector for Escherichia coli. Expression vectors of the present invention can be transferred into host cells to produce proteins or peptides, including fusion proteins. This transfer process can be carried out by conventional techniques such as transformation or transfection, which are well known to those skilled in the art.

The term "host cell" used herein is also called recipient cell, which refers to a cell capable of receiving and accommodating recombinant DNA molecules, and is a place for recombinant gene amplification. Ideal recipient cells should meet the two conditions of easy acquisition and proliferation. The "host cell" of the present invention may include prokaryotic cells and eukaryotic cells, specifically bacterial cells, yeast cells, insect cells and mammalian cells.

The expression vector of the present invention can be used to express the fluorescent probe or fusion protein of the present invention in prokaryotic or eukaryotic cells. Thus, the present invention relates to a host cell, preferably Escherichia coli, into which the expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell, representative examples are: bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, plant cells, insect cells of Drosophila S2 or Sf9, CHO, COS , 293 cells, or animal cells of Bowes melanoma cells, etc., including but not limited to those host cells mentioned above. The host cells are preferably various cells that are conducive to gene product expression or fermentative production, and such cells are well known and commonly used in the art, such as various Escherichia coli cells and yeast cells. In one embodiment of the present invention, Escherichia coli BL21 is selected to construct a host cell expressing the fusion protein of the present invention. Those of ordinary skill in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

As used herein, the terms "transformation" and "transfection", "conjugation" and "transduction" mean various transformations of exogenous nucleic acid (e.g., linear DNA or RNA (e.g., linearized vector or vector-free vector) known in the art. Techniques for introducing a single gene construct)) or a nucleic acid in the form of a vector (for example, a plasmid, cosmid, phage, phagemid, phagemid, transposon, or other DNA) into a host cell, including calcium phosphate or calcium chloride co- Precipitation, DEAE-mannan-mediated transfection, lipofection, native competence, chemical-mediated transfer, or electroporation. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host cells are eukaryotic cells, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformed cells can be cultured by conventional methods suitable for the expression of the host cells to express the fusion protein of the present invention. The medium used in the culture may be various conventional mediums depending on the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant protein in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated or purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

In one embodiment, the fluorescent probe or fusion protein of the present invention is produced by fermentation of Escherichia coli comprising the coding sequence of the fusion protein of the present invention, and purified by ammonium sulfate precipitation, ion exchange chromatography and gel chromatography to obtain the present invention in pure form. Invent fluorescent probes or fusion proteins.

The use of fluorescent probe or fusion protein of the present invention includes but not limited to: detection of Cu/Ag ions, detection of Cu/Ag ions in physiological state, detection of Cu/Ag ions at subcellular level, detection of Cu/Ag ions in situ, screening Drugs, diagnosis and diseases related to Cu/Ag ion levels, etc.

Concentrations, amounts, percentages, and other numerical values may be expressed herein in the form of ranges. It should also be understood that the use of this range format is for convenience and brevity only and should be construed flexibly to include the values expressly recited by the upper and lower limits of the range, and also to include all individual values or subranges subsumed within that range, as if expressly Individual values and subranges are referred to as such.

Brief description of the drawings

Figure 1 shows the SDS-PAGE identification results of the expression product of the recombinant plasmid pRSETb-CueRa-cpYFP-CueRb.

Figure 2 shows the spectroscopic properties of the recombinant plasmid CueRa-cpYFP-CueRb.

Figure 3 shows the fluorescence response of each mutant sample of the CueRa-cpYFP-CueRb derivative series to Cu/Ag ions.

Figure 4 shows the fluorescence response of acb-C1N5 fluorescent protein truncated samples to copper ions.

Figure 5 shows the metal selectivity of acb-C1N5C129/130S.

Figure 6 shows the metal selectivity of acb-C1N5A118P.

Figure 7 shows the detection results of acb-C1N5C129/130S expression in mammals.

Figure 8 shows the detection results of acb-C1N5A118P expression in mammals.

Figure 9 shows the results of confocal imaging after the expression of acb-C1N5A118P in Escherichia coli.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

The experimental method that does not indicate specific conditions in the following examples, generally according to conventional conditions such as Sambrook etc., "Molecular Cloning Laboratory Guide" (New York State, USA: Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), 1989); or Follow the conditions recommended by the manufacturer. Herein, percentages and parts are by weight unless otherwise indicated.

I. Experimental Materials and Reagents

Reagents: Unless otherwise noted, all others were from Shanghai Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Taq enzyme, buffer, dNTP used in PCR amplification, protease, buffer, T4DNA ligase, T4DNA ligase buffer, T4 polynucleotide kinase (PNK), T4PNK buffer used in molecular biology experiments , all from Fermentas (Fermentas, Vilnius, Lithuania).

Construction and prokaryotic expression of embodiment 1pRSET _b -CueR(a)-cpYFP-CueR(b)

1. Amplify the nucleic acid sequence of cpYFP:

Using pMD19-cpYFP (Nagai, T. et al., Proc Natl Acad Sci U S A.2001, V.98(6), pp.3197-3202) (obtained from the Protein Chemistry Laboratory of East China University of Science and Technology (Shanghai, China) as a template , using primers P1 and P2 to amplify the coding sequence of yellow fluorescent protein (cpYFP), the primer sequence (primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd. (Shanghai, China)) is as follows:

P1: TCTGCAGGCTACAACAGCGACAACGTCTATATC (sequence 9)

P2: GCC GGTACC GTTGTACTCCAGCTTGTGCC (sequence 10)

The PCR amplification product was electrophoresed in 1% agarose gel for 30 minutes to obtain a cpYFP fragment of about 750bp. The cpYFP fragment was recovered and purified from the gel using the Shanghai Sangon DNA Fragment Recovery and Purification Kit (Shanghai Bioengineering Co., Ltd., Shanghai, China) according to the manufacturer's instructions.

2. Extract the target gene sequence from Escherichia.coli Origami cells:

a. Sample processing

(i) Escherichia coli (Escherichia.coli) Origami cells were obtained from strains preserved in the Protein Chemistry Laboratory of East China University of Science and Technology (Shanghai, China).

(ii) Take 100 μl of cultured Origami cells and measure the optical density of the culture medium at 600 nm. When OD ₆₀₀ =0.1, the cell density is 1×10 ⁷ to 5×10 ⁷ cells/ml, and calculate the actual number of cells based on this. Add 1 ml of TRIzol reagent (from Invitrogen, California, USA) per 1×10 ⁷ E. coli cells for treatment.

(iii) Take a certain amount of bacterial liquid, centrifuge at 5000 rpm for 10 minutes at 4°C, and discard the supernatant.

(iv) The cell pellet was washed with 100 μl of 1×TE buffer (10 mM Tris-HCl, 1 mM EDTA pH 8.0, reagents from Amresco, Ohio, USA), centrifuged at 5000 rpm for 10 minutes, and the supernatant was discarded.

(v) Resuspend the bacterial pellet with 100 μl of 1×TE buffer (containing 2 mg/ml lysozyme (from Meiji Biotech, Shanghai, China)), and incubate at 37° C. for 30 minutes.

b. Phase separation

(i) Add 1ml TRIzol reagent (Invitrogen), mix well by blowing with a pipette, and let stand at room temperature for 5 minutes.

(ii) Add 200 μl of chloroform, shake and mix for 15 seconds, let stand at room temperature for 2-3 minutes, and centrifuge at 12,000 g for 15 minutes at 4°C.

(iii) The solution is separated after centrifugation. The upper aqueous phase accounts for about 40% and contains RNA, and the lower organic phase accounts for about 60% and contains DNA and protein. Carefully aspirate and remove the upper aqueous phase.

c. Remove impurities

Add 50 μl of 10% SDS and 250 μl of saturated saline to the organic phase, shake and mix, centrifuge at 12,000 g for 5 minutes at 4°C, and discard the upper aqueous phase.

d. DNA ethanol precipitation

Add 750 μl pre-cooled 95% ethanol to the organic phase, invert and mix well, and let stand at -80°C for 15 minutes to precipitate DNA.

e. DNA cleaning

(i) Pour off the upper organic phase.

(ii) The precipitate was washed several times with 1ml of 0.1M sodium citrate/10% ethanol solution, and centrifuged at 12,000g for 5 minutes at 4°C each time.

(iii) Finally, wash once with 75% ethanol, and centrifuge at 12,000 g for 5 minutes at 4°C.

(iv) Naturally air-dry ethanol at room temperature.

f. Dissolving DNA

Precipitate DNA with 50μl 8mM NaOH solution and store at 4°C or -20°C.

Using the genome extracted as described above as a template, primers P3 and P4, P3 and P6, and P5 and P4 were used to amplify the full length of Escherichia coli CueR gene, CueR fragment a and CueR fragment b, respectively. The primers P3 and P4 amplified to obtain a full-length fragment of the CueR protein gene containing an NheI restriction site at the N-terminal and a HindIII restriction site at the C-terminus. The sequences of primers P3, P4, P5 and P6 are as follows:

P3: CCCCTA GCTAGC ATGAACATCAGCGATGTAGCAAAAA (Sequence 11)

P4: CTTTTT AAGCTT TCACCCTGCCCGATGATGACAGCAGCCG (sequence 12)

P5: GTACAACGGTACCGGCAGCGCCGACTGCCCGATTATCGAAAA (sequence 13)

P6:GTTGTAGCCTGCAGAGTCATCGCCAGGGC (sequence 14)

The PCR amplified product was electrophoresed in 1% agarose gel for 30 minutes to obtain a CueR fragment with a size of about 400bp, a CueR fragment a with a size of about 350bp, and a CueR fragment b with a size of about 60bp. The CueR fragment was recovered and purified using the Shanghai Sangon DNA Fragment Recovery and Purification Kit (Shanghai Bioengineering Co., Ltd., Shanghai, China) according to the manufacturer's instructions.

3. Ligation of the target gene fragment and the vector

Use overlap extension PCR technology to perform PCR with CueRa, cpYFP, and CueRb as templates and P3 and P4 as primers. The PCR system is:

The PCR conditions are:

(1) Purify the template fragment.

(2) The forward and reverse primers were not included in the initial reaction system, and the primers were added to the reaction system after 10 cycles of reaction.

The PCR amplified product was electrophoresed in 1% agarose gel for 40 minutes to obtain a CueRa-cpYFP-CueRb fragment with a size of about 1200bp. The recovered and purified PCR fragment CueRa-cpYFP-CueRb and the vector plasmid pRSET _b were subjected to double enzyme digestion respectively. The system is as follows:

Reaction conditions: 37°C, 5 hours.

After the reaction, 10 μl of 6× loading buffer was added to the 50 μl reaction system to terminate the reaction. Then, the target fragment was separated by agarose gel electrophoresis, and the fragment was recovered and purified using the Shanghai Sangon DNA Fragment Recovery and Purification Kit (Shanghai Bioengineering Co., Ltd., Shanghai, China) according to the manufacturer's instructions.

Ligate the recovered CueRa-cpYFP-CueRb double digestion product with the carrier plasmid pRSET _b double digestion product, the system is as follows:

Reaction conditions: overnight at 16°C. Thus the ligation product _pRSETb -CueRa-cpYFP-CueRb was formed.

The clones identified as positive by colony PCR were sequenced with universal primers and sequenced by Beijing Liuhe Huada Gene Technology Co., Ltd. Shanghai Branch. The determined sequences were compared and analyzed with Vector NTI8.0. The result shows that the nucleotide sequence of CueRa-cpYFP-CueRb (as shown in SEQ ID NO: 6 in the sequence listing) is indeed inserted in the plasmid.

4. Conversion

The recombinant plasmid pRSETb-CueRa-cpYFP-CueRb was transformed into competent Escherichia coli (E.coli) BL21(DE3)pLys (preserved by the Protein Chemistry Laboratory of East China University of Science and Technology (Shanghai, China), originally purchased from Tiangen Biochemical, (China Beijing)), to obtain recombinant bacteria, the specific method is as follows:

(i) Under clean conditions, take 1 μl of plasmid or 10 μl of ligation product and add it to 100 μl of competent cells, and place in ice bath for 45 minutes;

(ii) After ice bathing, quickly heat shock in a water bath at 42°C for 90-120 seconds;

(iii) ice bath for 5 minutes;

(iv) Add 600ul LB liquid medium, recover on a shaker at 37°C and 150rpm for 1 hour;

(v) After centrifuging at 4000rpm and normal temperature for 5 minutes, discard the supernatant;

(vi) Resuspend the pellet with a small amount of fresh LB, then evenly spread all the bacterial solution on the desired LB plate, and culture it upside down at 37°C overnight.

5. Plasmid identification and prokaryotic protein expression

Positive clones were screened by conventional colony PCR method, and transferred to 5 ml of LB liquid medium containing the corresponding resistance, and cultured overnight at 37°C and 220rpm. The recombinant bacteria transformed with the target plasmid were cultured in LB medium at 37°C until the bacterial cell concentration OD was 0.6, added 1mM IPTG, induced expression at 18°C for 20 hours, and used Ni ²⁺ affinity chromatography column (General Electric Company, Uzbekistan, Sweden) Pusala) isolates and purifies the recombinant protein from the thalline lysate, and the SDS-PAGE identification result is shown in Figure 1, there is only one protein band at about 43kD, which is a recombinant protein.

Example 2. Spectral properties of recombinant protein CueRa-cpYFP-CueRb

According to the spectral properties of fluorescent proteins, the recombinant protein CueRa‐cpYFP‐CueRb(acb) has two absorption peaks around 400nm and 500nm. With 530nm emission and 485nm excitation as fixed conditions respectively, the study of excitation and emission spectra was carried out, and the obtained normalized data were plotted, and the results are shown in Figure 2. Therefore, in the actual detection process, combined with the laboratory's own test conditions, 390nm and 485nm were selected as dual excitation wavelengths, and 528nm was used as the emission wavelength to detect the fluorescence changes of the probe under different conditions.

Example 3. Construction of CueRa-cpYFP-CueRb derived series probes, prokaryotic expression and detection

The principle of probe construction: using pRSET _b -CueRa-cpYFP-CueRb as a template, and according to the principle of site-directed mutagenesis, a series of derivative probes were constructed. The truncated mutant sequence is shown as follows:

1. The original sequence model:

CueRa-CCTGGCGATGAC TCTGCAGGC -cpYFP- GGTACCGGCAGCGCCGAC -CueRb

N1: CueRa-CCTGGCGATGAC-cpYFP -GGTACCGGCAGCGCCGAC -CueRb

N2: CueRa-CCTGGCGAT-cpYFP -GGTACCGGCAGCGCCGAC -CueRb

N3: CueRa-CCTGGC-cpYFP -GGTACCGGCAGCGCCGAC -CueRb

N4: CueRa-CCT-cpYFP -GGTACCGGCAGCGCCGAC -CueRb

N5: CueRa-cpYFP- GGTACCGGCAGCGCCGAC -CueRb

C1:CueRa-CCTGGCGATGACTCTGCAGGC - cpYFP-AGCGCCGAC-CueRb

C2:CueRa- CCTGGCGATGACTCTGCAGGC- cpYFP-GCCGAC-CueRb

C3:CueRa-CCTGGCGATGACTCTGCAGGC - cpYFP-GAC-CueRb

C4:CueRa- CCTGGCGATGACTCTGCAGGC -cpYFP-CueRb

Construction of mutant library

1. Primer design (Shanghai Sangong)

2. PCR amplification

Truncation and site-directed mutagenesis were performed using site-directed mutagenesis PCR.

Mutant PCR amplification system (primers, enzymes, dNTPs, etc. are from Fuzyces Co.):

3. DNA fragment isolation, purification, connection, transformation

Concrete experimental operation is the same as embodiment 1.

4. Build the Probe Series

According to the above method, the following probe series were further obtained, and the amino acid numbers were as shown in the table:

6. Plasmid identification and prokaryotic protein expression

Plasmid identification and prokaryotic protein expression were performed using the same method as in Example 1, and the SDS-PAGE identification results showed only one protein band.

7. Routine live bacteria test

Transform Escherichia coli BL21(DE3)pLys cells with the target plasmid, pick a single clone from the plate and place it in an EP tube for primary culture, inoculate it in a test tube for secondary culture, when the OD ₆₀₀ of the bacteria reaches about 0.6, induce with 1mM IPTG , 18°C for 20 hours. Take 1 mM bacterial solution, centrifuge at 4000 rpm for 5 minutes at room temperature, and discard the supernatant. Resuspend the precipitate with buffer solution, adjust the OD ₆₀₀ of the bacteria to 0.05, take 80 μl of the bacterial solution in a 384-well fluorescent plate, and use a microplate reader to measure the fluorescence values of each sample before and after adding different concentrations of copper and silver ion solutions. The results Do normalization. The responses of the above mutation samples to copper ions and silver ions are shown in Figure 3.

Example 4.Construction of acb-C1N5 saturation mutation probe, prokaryotic expression and detection

Probe construction principle:

Using the pRSETb-acb-C1N5 intermediate transition plasmid as a template, saturation mutations were performed on the amino acids near the binding metal.

Primer design

The NNN part in the synthesized primer represents the codon sequence of the amino acid to be mutated.

The molecular biology operation of concrete process is with embodiment 1, and product is as following table:

Build a list of plasmids

The test results of the above samples are shown in the table below:

77 bits

81 bits

117 bits

118 bits

119 bits

121 bits

122 bits

123 bits

Example 5.Construction of truncated probe for acb-C1N5 fluorescent protein, prokaryotic expression and detection

Probe construction principle:

Using the pRSETb-acb-C1N5 intermediate transition plasmid as a template, the amino acids near the metal binding domain were subjected to saturation mutation.

1. Primer Design

2. Inverse PCR amplification

For the specific amplification system, refer to the PCR amplification in Example 2, and cut the gel to recover the PCR product.

3. DpnI digestion of PCR products

Reaction conditions: 37°C, 3h. Heat inactivation at 80°C for 20 minutes.

4. Add phosphorus to the target fragment

Reaction conditions: 37°C for 1h, heat inactivation at 75°C for 20 minutes.

5. Carrier self-connection

Reaction condition: 22°C1h

6. Ligation product transformation clone strain

For specific experimental steps, refer to the conversion in Example 1.

7. Plasmid construction list

8. Plasmid identification

With embodiment 1.

9. Response results of samples to metal ions

The response results of the acb-C1N5 fluorescent protein truncated probe sample to copper ions are shown in Figure 4.

Example 6. Construction of C129/130S mutant fluorescent probe, prokaryotic expression and detection

Probe construction principle:

The mutation site was designed in the primer, and the cysteine at positions 129 and 130 of acb-C1N5 was mutated into serine (SEQ ID NO.77) by self-ligating after inverse PCR amplification of the vector.

The specific construction method is the same as in Example 5. The test results are shown in Figure 5.

Embodiment 7.Cu/Ag ion fluorescent probe is to the response of other metal ions

The fluorescent probe plasmid prepared as above was transformed into Escherichia coli BL21(DE3)pLys cells, and after induction of expression for 20 hours, it was washed and resuspended with assay buffer (100mM HEPES, pH7.4). Ag, Cu, K, Na, Ca, Mg, Zn, Fe, Au, Mn, Al, Ni, and Hg were prepared into different concentrations of stock solutions with assay buffer (100 mM HEPES, pH 7.4), and diluted to 21 μM to be prepared. use.

Take 80 μl OD600=0.05 fluorescent probe bacterial suspension, add 4 μl 21 μM Ag, Cu, K, Na, Ca, Mg, Zn, Fe, Au, Mn, Al, Ni and Hg dropwise to the bacterial solution respectively, measure 30 Fluorescence change of fluorescence intensity emitted at 528nm after 424nm or 420nm fluorescence excitation within 1 minute, the reading interval is 1.5 minutes, shake for 3 seconds before each reading to fully react. The fluorescence excitation and emission measurement of the samples were completed by using a multifunctional fluorescent microplate reader (Collaboration 2, Boten Company).

The measurement results (Fig. 5, Fig. 6) showed that at the same concentration (1 μM) the Cu/Ag ion fluorescent probe only had a significant response to Cu ions or Ag ions, but had no significant response to other metal ions.

Example 8. Localization and expression of Cu/Ag ion fluorescent probes in different subcellular organelles

Using pRSETb-acb-C1N5A118P and pRSETb-acb-C1N5C129/130S as templates, use BamHI-containing upstream primer P35: TATGGATCCCGCCACCATGAACATCAGCGATGTAGCAAAAA (sequence 80) and the original downstream primer to re-amplify the target fragment, using (BamHI and HindIII double enzyme cut Cu/ The Ag ion fluorescent probe gene was recovered and connected to the pcDNA3.1-Hygro-Cyto and pcDNA3.1-Hygro-Mito vectors (the protein chemistry laboratory of East China University of Science and Technology in Shanghai, China from the commercial vector pcDNA3.1 -Hygro modified) on.

Preparation method: Unless otherwise stated, all primers in this method were synthesized by Shanghai Sangong (Shanghai Sangong, Shanghai, China). First construct the pcDNA3.1-Hygro-Cyto vector, based on pcDNA3.1-Hygro(+) (Invitrogen, California, USA), design two primers P36 and P37,

P36: CTAGCATGGCGGATCCACTAGTAAGCTTAAGC (SEQ ID NO: 81)

P37: TCGAGCTTAAGCTTACTAGTGGATCCGCCATG (Sequence 82)

This set of primers contains restriction restriction sites and the initiation codon ATG, and the structure is "NheI-ATG-GC-BamHI-HindIII-XhoI". The obtained primers are operated as follows for double primer annealing: the dry powder of the primers is Buffer (10mM Tris, pH7.5-8.0; 50mM NaCl, 1mM EDTA) was diluted to 100μM, the primer pair to be annealed was mixed equimolarly, the total volume was no more than 500μl, heated to 95°C, and then slowly cooled to room temperature ( lower than 30°C), and the product was stored at -20°C for use. For the positioning signal Mito, the method of artificially synthesizing the sequence was adopted. After setting the NheI restriction site at the N-terminal of the positioning signal and the BamHI restriction site at the C-terminal, the positioning signal was inserted into pcDNA3.1-Hygro ( +) in the carrier.

Construction of recombinant plasmids pcDNA3.1-Hygro-Cyto-acb-C1N5A118P, pcDNA3.1-Hygro-mito-acb-C1N5A118P, pcDNA3.1-Hygro-Cyto-acb-C1N5C129/130S, pcDNA3.1-Hygro-mito-acb -C1N5C129/130S. Sequencing was carried out, and the sequencing results showed that the amino acid sequence of the target fragment was shown in SEQ ID NO: 77 and SEQ ID NO: 83. HEK293 cells were transfected with the obtained recombinant plasmids.

Example 9. Fusion probes indicate changes in Cu/Ag ions in mammalian cells

Cu/Ag ion fluorescent probe real-time measurement of Cu/Ag ions transmembrane into cells leads to the increase of Cu/Ag ion levels in different subcellular compartments.

According to Example 8, Cu/Ag ion fluorescent probes were expressed in different subcellular organelles of HEK293 cells, and the results showed that this series of probes can detect the effect of adding Cu/Ag ions in the cell culture medium on the intracellular Cu/Ag ion levels in real time (Figure 7, Figure 8). We further use this series of probes to detect the effect of adding Cu/Ag ions on the level of Cu/Ag ions in other organelles, and the results show that adding Cu ⁺ can also lead to the increase of Cu ⁺ levels in mitochondria. The above results show that the present invention Cu/Ag ion fluorescent probes can well indicate that Cu/Ag ions transmembrane into mammalian cells leading to an increase in intracellular Cu/Ag ion levels.

Example 10. Live cell imaging of silver ion fluorescent probes

In prokaryotic cells, the Ag fluorescent probe is used to detect the increase of Ag ion level caused by the transmembrane entry of Ag ion into the cell in real time. The Ag ion fluorescent probe was expressed in E.coli BL21(DE3)pLys cells. When Ag ions were added to the cell culture medium, the expression of the target probe was observed in real time with a confocal laser microscope (from Nikon, Japan). The transmembrane migration status of Ag ions in the cells of the needle. The live cell imaging results (Fig. 9) show that the emission fluorescence at 528nm of the probe under 488nm fluorescence excitation is significantly stronger than that of the control group, and Ag ⁺ can enter the cell across the membrane, resulting in There is an immediate increase in intracellular Ag ⁺ levels.

Claims (10)

1. A genetically encoded Cu/Ag ion fluorescent probe, composed of Cu/Ag ion-sensitive proteins and a part that performs Cu/Ag ions through changes in spectral properties, wherein the change in spectral properties affects The part where Cu/Ag ions are expressed is a fluorescent protein sequence or a derivative thereof, and the protein sensitive to Cu/Ag ions is a polypeptide or a functional fragment thereof with the following characteristics: (1) Domain B with Cu/Ag ion binding properties; and/or (2) Derived from CueR protein, a transcriptional regulator sensitive to Cu/Ag ions. 2. Cu/Ag ion fluorescent probe as claimed in claim 1, wherein said protein sensitive to Cu/Ag ion is selected from: (1) the transcription regulator CueR protein derived from bacteria, the amino acid sequence of the CueR protein is SEQ ID NO: 1; and (2) Any homologous or non-homologous sequence having 70% identity with the sequence described in (1). 3. Cu/Ag ion fluorescent probe as claimed in claim 1 or 2, is characterized in that, comprises the Cys-Cys structure with Cu/Ag ion binding characteristic and is shown in by SEQ ID NO:2,3,4 respectively Fluorescent protein sequences A, A1 and/or A2 of which in combination are selected from: (1) B-A-B; (2) A1-B-A2, wherein A1 and A2 are the same or different amino acid sequences of fluorescent proteins or derivatives thereof from Aequorea victoria; (3) any one of positions 113, 114, 115, 116, 117, 118, and 119 within the flexible region of the B domain is followed by one or more A's; and (4) Any one of the positions 113, 114, 115, 116, 117, 118, and 119 in the flexible region of the B domain is followed by chimeric one or more A, and a B is connected at the end of the chimeric sequence. 4. A fusion protein comprising the fluorescent probe of claim 1. 5. The fusion protein according to claim 4, wherein the fusion protein is fused with the fluorescent probe according to claim 1 and a specific subcellular localization signal, and the localization signal can bind the target protein Localized within designated subcellular organelles. 6. The fusion protein according to claim 5, wherein the specific subcellular localization signal is a mitochondrial localization signal shown in SEQ ID NO:5. 7. The nucleotide sequence encoding the fluorescent probe according to any one of claims 1-3 or the fusion protein according to any one of claims 4-6 or its complementary sequence. 8. An expression vector comprising the nucleotide sequence of claim 7 operably linked to an expression control sequence. 9. A method for preparing the fluorescent probe according to any one of claims 1-3 or the fusion protein according to any one of claims 4-6, comprising the following steps: (1) transferring the expression vector according to claim 8 into a host cell, (2) culturing said host cell under conditions suitable for expression by said host cell, and (3) Isolating the fluorescent probe or fusion protein from the host cell. 10. A test kit for detecting Cu/Ag ions or screening drugs, comprising the fluorescent probe according to any one of claims 1-3 or the fusion protein according to any one of claims 4-6 , and instruction manual.