Rare earth ion fluorescence sensing protein and application thereof in rare earth ion detection

Abstract

The invention relates to the technical field of rare earth element detection, in particular to a fusion protein and application thereof in rare earth ion detection. The calcium ion binding protein, the fluorescent protein and the LanM protein are fused, the obtained fusion protein is used for quantitative detection of rare earth ions in a water sample, the detection limit is low, the accuracy is high, the rare earth ion concentration magnitude can be accurately measured, and interference of non-rare earth elements in the environment can be eliminated.

Description

Technical Field

[0001] The present invention relates to the technical field of rare earth element detection, in particular to rare earth ion fluorescent sensor protein and application thereof in rare earth ion detection. Background Art

[0002] Rare earth element recycling has become a global consensus: the cost of extracting rare earths from discarded electronic products is much lower than mining raw ores, and it can greatly reduce wastewater discharge, which is of strategic significance for achieving a resource circular economy.

[0003] When recovering rare earth elements, whether from mining wastewater or rare earth purification wastewater, the first step is to assess their value. Key to this assessment is the detection of rare earth elements and the precise measurement of their concentrations. Currently, rare earth detection methods include inductively coupled plasma mass spectrometry, laser-induced breakdown spectroscopy, and X-ray fluorescence spectroscopy. While ICP-MS offers ultra-high sensitivity (ppt-level), the equipment is expensive and requires a constant temperature and humidity laboratory and specialized operators, making it difficult to meet the demands of on-site testing at mining sites or in recycling lines. While laser-induced breakdown spectroscopy has achieved extensive research and development in portability and sensitivity, the equipment cost remains high, and it suffers from insufficient sensitivity and susceptibility to interference. While X-ray fluorescence spectroscopy offers non-destructive detection, its resolution for light rare earth elements (such as lanthanum and cerium) is insufficient, and matrix effects are significant, often leading to detection errors exceeding 15%. These methods suffer from common limitations, such as expensive equipment, complex pre-treatment, and demanding testing conditions, which have hindered the development of the rare earth recovery industry. Therefore, developing a simple, low-cost, and widely applicable method for detecting rare earth ions is of great significance. Detecting rare earth ions using fluorescence intensity is particularly advantageous due to its simplicity, speed, sensitivity, and low cost. Simply mixing the sample with a protein solution and using a microplate reader allows for rapid measurement and analysis. Summary of the Invention

[0004] In light of this, the technical problem addressed by the present invention lies in a fusion protein and its application in rare earth ion detection. This invention, for the first time, utilizes calcium-binding protein, fluorescent protein, and LanM protein to create a fusion protein for rare earth ion detection. This method is simple to operate and enables convenient and rapid detection of rare earth elements at a low cost. It is suitable for screening large numbers of samples and on-site monitoring, provides accurate measurement results for actual samples, and is sensitive to rare earth elements, enabling the detection of rare earth elements in water samples.

[0005] The fusion protein provided by the invention comprises calcium ion binding peptide ckkap, fluorescent protein and LanM protein.

[0006] In the fusion protein of the present invention, the calcium ion binding peptide has an amino acid sequence as shown in SEQ ID NO. 3. The nucleic acid sequence encoding the protein is shown in SEQ ID NO. 4.

[0007] In the fusion protein of the present invention, the LanM protein has an amino acid sequence as shown in SEQ ID NO. 7. The nucleic acid sequence encoding the protein is shown in SEQ ID NO. 8.

[0008] In the fusion protein of the present invention, the fluorescent protein is selected from EGFP, YFP, or RFP. In some specific embodiments, the fluorescent protein is EGFP (i.e., enhanced green fluorescent protein). Furthermore, the amino acid sequence of the EGFP protein is shown in SEQ ID NO. 5. The nucleic acid sequence encoding the EGFP protein is shown in SEQ ID NO. 6.

[0009] In some specific embodiments, the fusion protein includes calcium ion binding protein, EGFP protein and LanM protein from N-terminus to C-terminus, namely calcium ion binding protein (ckkap)-enhanced green fluorescent protein (EGFP)-LanM protein, referred to as fusion protein CEL.

[0010] The applicant's research has found that the order in which proteins are linked has a significant impact on the sensitivity and accuracy of fusion protein detection. Experiments have shown that compared with other sequences, ckkap-EGFP-LanM is more effective. The fusion protein obtained by fusing calcium ion binding protein, EGFP protein and LanM protein, as well as full-length protein expression purification tags, calcium ion binding peptides, full-length LanM protein and fluorescent protein in the present invention is significantly less responsive to rare earth ions than the fusion protein CEL provided by the present invention. When used for rare earth ion detection, its sensitivity and accuracy are significantly reduced.

[0011] In some embodiments, the N-terminus of the fusion protein further comprises a protein purification tag. In some specific examples, the protein purification tag has an amino acid sequence as shown in SEQ ID NO: 1.

[0012] In the fusion protein of the present invention, the fusion protein includes a protein purification tag, ckkap, EGFP and LanM proteins connected in sequence, and the two adjacent proteins are connected by a linker. In some embodiments, the amino acid sequence of the linker is selected from the sequence shown in any one of SEQ ID NO.11 to 14. In a specific embodiment of the present invention, the fluorescent protein EGFP is a fusion protein of EGFP fragment 1 and EGFP fragment 2, and the linker includes linker 1, linker 2, linker 3 and linker 4; the EGFP fragment 1 and EGFP fragment 2 are connected through linker 3; the protein purification tag and ckkap protein are connected through linker 1; the ckkap protein and EGFP fragment 1 are connected through linker 2, and the EGFP fragment 2 and LanM are connected through linker 4, that is, the fusion protein is: protein purification tag-linker 1-ckkap-linker 2-EGFP fragment 1-linker 3-EGFP fragment 2-linker 4-LanM protein.

[0013] Specifically, linkers 1 to 4 are selected from the amino acid sequences set forth in SEQ ID NOs. 11 to 14, in order. The present invention has experimented with various linkers, and the results indicate that different linkers affect the detection performance of the fusion protein. Fusion proteins prepared using linkers 1 to 4 exhibit the strongest response to rare earth ions and achieve the highest sensitivity and accuracy for rare earth ion detection.

[0014] In some specific embodiments, the fusion protein has the amino acid sequence shown in SEQ ID NO.9.

[0015] The present invention also provides a biomaterial comprising a nucleic acid encoding the fusion protein described above. The nucleic acid may be a wild-type sequence or a codon-optimized sequence. The nucleic acid described herein may comprise only the ORF of the fusion protein or may include a nucleic acid sequence for restriction sites and / or a selection marker. In some specific embodiments, the nucleic acid has the sequence shown in SEQ ID NO. 10.

[0016] The present invention also provides a biomaterial comprising an expression cassette containing the nucleic acid. Furthermore, the expression cassette further comprises a promoter. Furthermore, the expression cassette further comprises a terminator. The nucleic acid is located between the promoter and the terminator. Furthermore, the expression cassette further comprises an enhancer. In some embodiments, the promoter of the expression cassette is a T7 promoter; and the terminator is a T7 terminator.

[0017] The present invention also provides a biomaterial, which is a recombinant vector containing the nucleic acid or the expression cassette. The backbone of the recombinant vector is a pET28 series or pET32 series vector. In a specific embodiment of the present invention, the backbone is a pET28 series vector, specifically pET28a(+).

[0018] The present invention also provides a biomaterial, which is a host, wherein the host transforms or transfects the nucleic acid recombinant vector, or the nucleic acid or the expression cassette is integrated into the host's genome. Transfection refers to the process of introducing exogenous nucleic acid (such as plasmid DNA, siRNA, etc.) into eukaryotic cells. Common transfection methods include liposome transfection, electroporation, viral vectors, etc. Transformation refers to the process of introducing exogenous DNA (such as plasmid) into prokaryotic cells (such as bacteria) or certain eukaryotic cells (such as yeast) through physical or chemical methods. Transformation methods include chemical methods, heat shock methods, electroporation methods, etc. In the present invention, the host can be a microorganism, a plant cell, or an animal cell. Furthermore, the microorganism is preferably a prokaryote. Furthermore, the prokaryote is preferably Escherichia coli. In some specific embodiments, the host is Escherichia coli BL21 (DE3). In the present invention, the recombinant vector is transferred into the host by electroporation.

[0019] In the biomaterial of the present invention, the nucleic acid involved is the nucleic acid encoding the fusion protein of the present invention. The nucleic acid is as described above and will not be described in detail here.

[0020] The present invention also provides a method for preparing the fusion protein, which comprises culturing the host and obtaining the fusion protein through induction.

[0021] The present invention does not specifically limit the specific methods of culture and induction; culture and induction conditions can be selected based on the host cell type. Parameters such as culture temperature, medium composition, pH, osmotic pressure, and inducer type, concentration, and timing of addition can all be optimized and adjusted based on the characteristics and needs of the host cell.

[0022] The present invention also provides a method for detecting rare earth ions, comprising the following steps:

[0023] The fusion protein of the present invention is prepared by using the fusion protein of the present invention, or the biomaterial of the present invention;

[0024] mixing the fusion protein and a buffer to obtain a fusion protein solution;

[0025] Mixing the fusion protein solution, the sample to be tested and a buffer to obtain a mixed system to be tested;

[0026] The fluorescence intensity of the mixed system is measured to determine the concentration level of rare earth ions in the sample to be tested.

[0027] In the present invention, the final concentration of the fusion protein in the mixed system is 0.5 mmol / L.

[0028] In the present invention, the buffer used in the preparation of the fusion protein solution and the mixed system to be tested is selected from at least one of MOPS buffer and HEPES, and chelating buffers such as Tris should be avoided throughout the process. In a specific embodiment, the buffer is MOPS buffer.

[0029] The detection method of the present invention further comprises the step of drawing a standard curve of rare earth ion concentration-fluorescence intensity.

[0030] In some embodiments, the method for drawing the standard curve comprises:

[0031] mixing the fusion protein solution, a buffer solution, and a series of standard rare earth ion solutions to obtain a standard mixed system containing different rare earth ion concentrations;

[0032] The fluorescence intensity of the standard mixed system is measured, and a standard curve of fluorescence intensity and rare earth ion concentration is drawn.

[0033] The excitation wavelength of the fluorescence is 450–490 nm, and the emission wavelength is 520–550 nm.

[0034] In the present invention, the rare earth ion standard solution contains Nd 3+ 、Dy 3+ At least one of the following, the concentration is 1M, and it is diluted to 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , 10 -3 In a specific embodiment, the rare earth ion standard solution contains Nd 3+ 、Dy 3+ .

[0035] The present invention also provides a reagent for detecting rare earth ions, which comprises any of the biological materials described in the present invention:

[0036] (1) A nucleic acid encoding the fusion protein of the present invention;

[0037] (2) an expression cassette containing the nucleic acid described in (1);

[0038] (3) A recombinant vector containing the nucleic acid described in (1) or the expression cassette described in (2);

[0039] (4) Transfect or transform the host with the recombinant vector (3), or the host with the nucleic acid (1) or the expression cassette (2) integrated into its genome.

[0040] The fusion protein prepared by the present invention has the characteristic of a strong fluorescent response to rare earth ions. When added to a water sample containing rare earth ions, it binds to the rare earth ions and, by measuring fluorescence using an enzyme marker, can rapidly detect the concentration of rare earth ions. The fusion protein provided by the present invention can effectively assess the concentration of rare earth elements in water. The present invention also discloses a method for constructing the rare earth ion fluorescent sensor protein used. The sensor has a detection limit as low as 1.0 nanomole per liter and a wide detection range of 1 nanomole per liter to 100 micromoles per liter. It also has high sensitivity and can quantify the total rare earth ion concentration in the system. The present invention is simple to operate, can achieve convenient and rapid detection of rare earth elements, and has low detection costs. It is suitable for screening large numbers of samples and on-site monitoring, and its measurement results for actual samples are accurate. It has a sensitive response to rare earth elements and can detect rare earth elements in water samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the SDS-PAGE electrophoresis diagram of the fusion protein (rare earth ion fluorescent sensor protein) prepared by the present invention;

[0042] Figure 2 The detection of different concentrations of Ca in Example 2 of the present invention 2+ 、Nd 3+ 、Dy 3+ Fluorescence intensity graph. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] The following preparation steps are described in some detail for the purpose of illustration and based on the comparability of each example. Those skilled in the art can fully summarize the method for preparing the product of the present invention based on existing knowledge.

[0045] The following preparation steps are described in some detail for the purpose of illustration and based on the comparability of each example. Those skilled in the art can fully summarize the method for preparing the product of the present invention based on existing knowledge.

[0046] In the present invention, the nucleotide sequence and amino acid sequence information involved are as follows:

[0047] The amino acid sequence of the protein expression and purification tag is shown in SEQ ID NO.1:

[0048] SEQ ID NO. 1: MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLAT MVD.

[0049] The nucleic acid sequence encoding the protein expression purification tag is shown in SEQ ID NO. 2, and its sequence is as follows:

[0050] ATGGGGTTCTCATCATCATCATCATGGTATGGCTAGCATGACTGGT GGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCTCG CCACCATGGTCGAC.

[0051] The amino acid sequence of calcium ion binding peptide (ckkap) is shown in SEQ ID NO.3:

[0052] SEQ ID NO. 3: VKHIPSLTTVILVKSMLRKRSFGNPF.

[0053] The nucleic acid sequence encoding ckkap is shown in SEQ ID NO.4, and its sequence is as follows:

[0054] GTCAAGCACATCCCCAGCCTGACCACTGTGATCCTGGTCAAGTCTAT GCTGAGAAAGCGTTCCTTTGGAAACCCATTTAGGGAC.

[0055] The amino acid sequence of the EGFP fusion protein is SEQ ID NO.5, and its sequence is as follows:

[0056] NVYIMADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGPVLLP

[0057] DNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK GGTGGS M

[0058] VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTG

[0059] KLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYN .

[0060] Note: The two underlined parts are the amino acid sequences of EGFP fragment 1 and EGFP fragment 2, respectively. The two protein fragments are connected by linker 3 (GGTGGS).

[0061] The nucleic acid sequence of the EGFP fusion gene is shown in SEQ ID NO.6, and the sequence is as follows:

[0062] AACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCGA

[0063] ACTTCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTA

[0064] CCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC

[0065] GACAACCACTACCTGAGCACCCAGTCCGTACTTTCGAAAGACCCCAACG

[0066] AGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGAT

[0067] CACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGGAGGGAGCAT

[0068] GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

[0069] CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGCGCGGCGA

[0070] GGGTGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTG

[0071] CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTG

[0072] ACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGC

[0073] ACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCAC

[0074] CATCTTCTTCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAA

[0075] GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC.

[0076] The amino acid sequence of the LanM protein is SEQ ID NO.7, and its sequence is as follows:

[0077] ASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTTLESGETKG

[0078] RLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDS KAGQGVLVMIMK.

[0079] The nucleic acid sequence encoding the LanM protein is shown in SEQ ID NO.8, and its sequence is as follows:

[0080] GCAAGTGGCGCGGATGCTTTGAAGGCGCTTAACAAAGACAATGACG

[0081] ATTCGCTGGAAATTGCAGAGGTAATCCACGCAGGCGCAACTACGTTCAC

[0082] GGCAATCAACCCGGACGGAGACACAACTTTGGAGAGCGGAGAGACGA

[0083] AAGGACCTTGACAGAAAAGGATTGGGCTAGAGCTAATAAAGACGGGG

[0084] ACCAGACGTTGGAAATGGACGAATGGCTGAAGATCCTGCGTACTAGATT

[0085] TAAAAGAGCCGATGCTAATAAGGATGGCAAATTAACGGCTGCGGAGTTGGATTCCAAAGCGGGGCAAGGGGTATTGGTCATGATCATGAAA.

[0086] The amino acid sequence of the fusion protein of the present invention is shown in SEQ ID NO.9, and its sequence is as follows:

[0087] MGSHHHHHHGMASMTGGQQMGRDLYDDDDK DLATMVD VKHIPSLT

[0088] TVILVKSMLRKRSFGNPF RD NVYIMADKQKNGIKANFKIRHNIEDGGVQLA

[0089] YHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGI

[0090] TLGMDELYK GGTGGS MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEG

[0091] EGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFF

[0092] KSAMPEGYIQERTIFFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNI

[0093] LGHKLEYN TK ASGADALKALNKDNDDSLEIAEVIHAGATTFTAINPDGDTT

[0094] LESGETKGRLTEKDWARANKDGDQTLEMDEWLKILRTRFKRADANKDGKLTAAELDSKAGQGVLVMIMK.

[0095] Note: In SEQ ID NO.9, the underlined sequences are the amino acid sequences of linker 1, linker 2, linker 3, and linker 4, respectively.

[0096] The nucleic acid sequence encoding the fusion protein of the present invention is shown in SEQ ID NO.10, and its sequence is as follows:

[0097] ATGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGT

[0098] GGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGATCTCG

[0099] CCACCATGGTCGACGTCAAGCACATCCCCAGCCTGACCACTGTGATCCT

[0100] GGTCAAGTCTATGCTGAGAAAGCGTTCCTTTGGAAACCCATTTAGGGAC

[0101] AACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGCGAACT

[0102] TCAAGATCCGCCACAACATCGAGGACGGCGGCGTGCAGCTCGCCTACC

[0103] ACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCG

[0104] ACAACCACTACCTGAGCACCCAGTCCGTACTTTCGAAAGACCCCAACGA

[0105] GAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATC

[0106] ACTCTCGGCATGGACGAGCTGTACAAGGGCGGTACCGGAGGGAGCATG

[0107] GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTC

[0108] GAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGCGCGGCGAG

[0109] GGTGAGGGCGATGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGC

[0110] ACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGA

[0111] CCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCA

[0112] CGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACATCCAGGAGCGCACC

[0113] ATCTTCTTCAAGGACGACGGCACCTACAAGACCCGCGCCGAGGTGAAG

[0114] TTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGAC

[0115] TTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACACC

[0116] AAGGCAAGTGGCGCGGATGCTTTGAAGGCCGCTTAACAAAGACAATGAC

[0117] GATTCGCTGGAAATTGCAGAGGTAATCCACGCAGGCGCAACTACGTTCA

[0118] CGGCAATCAACCCGGACGGAGACACAACTTTGGAGAGCGGAGAGACG

[0119] AAAGGACCTTGACAGAAAAGGATTGGGCTAGAGCTAATAAAGACGGG

[0120] GACCAGACGTTGGAAATGGACGAATGGCTGAAGATCCTGCGTACTAGAT

[0121] TTAAAAGAGCCGATGCTAATAAGGATGGCAAATTAACGGCTGCGGAGTTGGATTCCAAAGCGGGGCAAGGGGTATTGGTCATGATCATGAAA.

[0122] The amino acid sequence of linker 1 is SEQ ID NO. 11, and the sequence is as follows: DLATMVD.

[0123] The amino acid sequence of linker 2 is SEQ ID NO. 12, and the sequence is as follows: RD.

[0124] The amino acid sequence of linker 3 is SEQ ID NO. 13, and the sequence is as follows: GGTGGS.

[0125] The amino acid sequence of linker 4 is SEQ ID NO. 14, and the sequence is as follows: TK.

[0126] Herein, “fusion protein of the present invention”, “rare earth ion fluorescent sensor protein” and “fluorescent sensor protein” have the same meaning and can be used interchangeably.

[0127] In this article, "expression cassette" has the same meaning as "expression cassette", "transcription unit" and "expression unit" mentioned in the field of genetic engineering and can be used interchangeably.

[0128] In this article, the descriptions of "including", "comprising" and "having" include both closed technical solutions consisting of the listed features and open technical solutions containing the listed features.

[0129] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0130] As used herein, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every point value between such minimum and maximum values. Furthermore, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. Unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0131] The present invention has no particular restrictions on the sources of all raw materials. Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

[0132] Example 1 Preparation of rare earth ion fluorescent sensor protein

[0133] The present invention utilizes a rare earth ion binding protein (LanM) that rapidly and specifically binds to rare earth ions through EF Hands, triggering conformational changes in the rare earth ion fluorescent sensor protein and, consequently, changes in fluorescence intensity. This allows for rapid detection of rare earth ion concentrations in analytes, thus enabling its application in rare earth ion detection. Since the rare earth ion fluorescent sensor protein produces a significant fluorescence response only upon binding to rare earth ions, it can be used to detect the presence of rare earth ions in water samples.

[0134] The gene sequence of the chemically synthesized rare earth ion fluorescent sensor protein (SEQ ID NO.8) includes the nucleotide sequence of calcium ion binding sequence (ckkap)-green fluorescent protein (EGFP)-LanM. The amino acid sequence of the fusion protein is shown in SEQ ID NO.7.

[0135] The pET28a(+) expression vector was used for ligation to obtain a recombinant vector, which was then transformed into Escherichia coli BL21(DE3). The recombinant strain was cultured for 12 h to obtain recombinant Escherichia coli. Expression was induced for 16 h, and the cells were disrupted by ultrasonication. The protein was purified using the His-tag protein purification method, eluted with 0.02 mol / L Tris-HCl (pH = 8.6) (containing 0.4 mol / L imidazole), and then desalted with 0.02 mol / L MOPS (pH = 7.2) and Sephadex G25. After lyophilization, the rare earth ion fluorescent sensor protein was obtained. The electrophoresis pattern of the obtained fluorescent sensor protein is shown in FIG. Figure 1 .

[0136] Example 2: Detection of rare earth ions using rare earth ion fluorescent sensor proteins

[0137] 1. Preparation of Rare Earth Ion Fluorescent Sensor Protein Standard Solution

[0138] The rare earth ion fluorescent sensor protein obtained in Example 1 was dissolved in MOPS buffer solution (pH=7.2) to prepare a fusion protein solution, and MOPS buffer solution was added to make the final concentration of the fluorescent sensor protein 0.020 mol / L.

[0139] 2. Develop a rare earth ion standard curve

[0140] The rare earth ion fluorescent sensor protein standard solution, rare earth ion standard solution (1M) and MOPS buffer solution were mixed to make the final concentration of rare earth ions 10 -9 , 10 -8 , 10 -7 , 10 -6 , 10 -5 , 10 -4 , 10 -3 M, the final concentration of protein is 0.5mmol / L, and the fluorescence intensity is quickly measured in a microplate reader; a graph showing the relationship between fluorescence intensity and the concentrations of rare earth and non-rare earth ions is drawn, as shown in FIG. Figure 2 The rare earth ion fluorescent sensor protein Ca 2+ 、Nd 3+ and Dy 3+ The standard curve of

[0141] 3. Testing of samples to be tested

[0142] Mix the rare earth ion fluorescent sensor protein standard solution, the sample to be tested, and MOPS buffer solution to a final protein concentration of 0.5 mmol / L. Quickly measure the fluorescence intensity using a microplate reader. Estimate the rare earth ion concentration in the sample based on the standard curve obtained in step 3.

[0143] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

[0144] Example 3: Detecting actual water environment samples using rare earth ion fluorescent sensor proteins

[0145] 1. Preparation of rare earth ion fluorescent sensor protein standard solution and rare earth ion standard curve

[0146] According to the method of Example 2, a standard solution of rare earth ion fluorescent sensor protein and a standard curve of rare earth ions were prepared.

[0147] 2. Testing of samples to be tested

[0148] Mix the rare earth ion fluorescence sensor protein standard solution, tap water sample and MOPS buffer solution to make the final concentration of the protein 0.5mmol / L. Quickly measure the fluorescence intensity in a microplate reader and calculate the ratio of the fluorescence intensity to the fluorescence intensity of the blank sample, F t / F0=1.08 (error is negligible, close to the range of 1.00-1.10). According to the standard curve obtained in step 3, the concentration of rare earth ions in tap water is estimated to be 0, that is, no rare earth ions are detected in the tap water sample.

[0149] The samples to be tested were measured three times in parallel, and the F t / F0 is between 0.95 and 1.08, indicating that the detection method of the embodiment of the present invention is relatively reliable and meets the analysis requirements.

[0150] The results show that the rare earth ion fluorescent sensor protein of the present invention is used for the quantitative detection of rare earth ions in water samples, has a low detection limit, has high accuracy, can accurately measure the concentration magnitude of rare earth ions, and can eliminate the interference of non-rare earth elements in the environment.

[0151] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A fusion protein, characterized in that Including calcium ion binding peptide ckkap, fluorescent protein and LanM protein.

2. The fusion protein according to claim 1, characterized in that The calcium ion binding protein has an amino acid sequence as shown in SEQ ID NO.3; The LanM protein has an amino acid sequence as shown in SEQ ID NO.7; The fluorescent protein is selected from EGFP, YFP or RFP.

3. The fusion protein according to claim 1 or 2, characterized in that The N-terminus of the fusion protein also includes a protein purification tag; Preferably, the protein purification tag has an amino acid sequence as shown in SEQ ID NO: 1; Preferably, the fusion protein comprises a protein purification tag, ckkap, EGFP and LanM protein connected sequentially; two adjacent proteins are connected by a linker; the linker is selected from the amino acid sequence shown in any one of SEQ ID NOs. 11 to 14; Preferably, the fusion protein has the amino acid sequence shown in SEQ ID NO.

9.

4. Biomaterial, characterized in that It is any of the following: (1) A nucleic acid encoding the fusion protein according to any one of claims 1 to 3; (2) an expression cassette containing the nucleic acid described in (1); (3) A recombinant vector containing the nucleic acid described in (1) or the expression cassette described in (2); (4) Transfect or transform the host with the recombinant vector (3), or the host with the nucleic acid (1) or the expression cassette (2) integrated into its genome.

5. The biomaterial according to claim 4, characterized in that The nucleic acid has the sequence shown in SEQ ID NO.

10.

6. A method for preparing a fusion protein, characterized in that: The host in the biological material according to claim 4 or 5 is cultured and the fusion protein is obtained by induction.

7. A method for detecting rare earth ions, characterized in that: The fusion protein is prepared using the fusion protein according to any one of claims 1 to 3, or using the biological material according to any one of claims 4 to 5; mixing the fusion protein and a buffer to obtain a fusion protein solution; Mixing the fusion protein solution, the sample to be tested and a buffer to obtain a mixed system to be tested; The fluorescence intensity of the mixed system is measured, and the concentration level of the rare earth ions in the sample to be tested is determined based on the fluorescence intensity.

8. The detection method according to claim 7, characterized in that The final concentration of the fusion protein in the mixed system is 0.5 mmol / L; The buffer is selected from at least one of MOPS buffer and HEPES buffer.

9. The detection method according to claim 7, characterized in that The method also includes the steps of drawing a standard curve of rare earth ion concentration-fluorescence intensity; The method for drawing the standard curve comprises: mixing the fusion protein solution, a buffer solution, and a series of standard rare earth ion solutions to obtain a standard mixed system containing different rare earth ion concentrations; The fluorescence intensity of the standard mixed system is measured, and a standard curve of fluorescence intensity and rare earth ion concentration is drawn.

10. A reagent for detecting rare earth ions, characterized in that: It comprises the biomaterial according to claim 4 or 5.

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