Mutant of luciferase fusion protein, and bioluminescent nucleic acid probe constructed by the mutant and application thereof

Abstract

The application discloses a mutant of luciferase fusion protein, a bioluminescence nucleic acid probe constructed by the mutant and application of the mutant and the nucleic acid probe. The mutant is mutated at at least one of positions 144, 148, 339, 353 and 366 compared with the amino acid sequence shown in SEQ ID NO: 2. The mutant of the luciferase fusion protein can be coupled with the nucleic acid probe in a high-efficiency biological orthogonal manner, thereby constructing a bioluminescence nucleic acid probe, and effectively solving problems such as the need of professional equipment for signal excitation / reception, high background and false positive signal of the existing nucleic acid fluorescent probe. Moreover, the bioluminescence nucleic acid probe has a ratio type property and is not affected by factors such as environment and substrate consumption, thereby providing a new method and a new tool for the fields of environment monitoring and disease diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and biological detection technology, specifically relating to a mutant of a luciferase fusion protein and its constructed bioluminescent nucleic acid probe and its application. Background Technology

[0002] Nano Luc luciferase (NLuc) is a luciferase found in deep-sea shrimp that catalyzes the oxidation of furimazine, a compound of the imidazopyrazinone family, to produce luminescence. It is characterized by its small molecular weight, high luminescence efficiency, and good stability. In recent years, NLuc and its fusion proteins have been widely used in scientific research and industry.

[0003] Nucleic acid sensor (NAS) technology utilizes the principle of complementary nucleotide bases to identify a labeled single-stranded DNA (or RNA) molecule with a specific gene probe that targets a specific target. Its target range is very broad, applicable to the detection of metal ions, small molecules, nucleic acid molecules, proteins, cells, and even biological tissues. In recent years, NAS probes have played an important role in environmental monitoring (heavy metal ions), disease detection, and bioimaging. However, current NAS probes generally use fluorescence as a signal output strategy, requiring specialized equipment for signal excitation / reception. Furthermore, autofluorescence in complex biological samples can cause strong false-positive signals, hindering their analytical applications. Bioluminescence utilizes the interaction between luciferase and substrate to generate self-luminescence. Compared to fluorescence, bioluminescence does not require external excitation and effectively addresses the drawbacks of NAS probes, such as photobleaching, background signals, false-positive signals, and the need for specialized equipment. Therefore, developing bioluminescent NAS probes for quantitative detection of biological samples is essential. Summary of the Invention

[0004] The purpose of this invention is to provide a mutant of a luciferase fusion protein (a fusion protein of circular permutated Halo-tag and circular permutated luciferase, abbreviated as cpHNLuc) and its constructed bioluminescent nucleic acid probes and applications, thereby solving the problems of existing nucleic acid probes that require equipment for signal excitation / reception, high background, and false positive signals. This invention mutates certain amino acid sites in the luciferase fusion protein to obtain a novel luciferase fusion protein mutant, and then efficiently bio-orthogonally couples this protein with a nucleic acid probe, serving as a universal strategy for the construction of bioluminescent nucleic acid probes. The bioluminescent nucleic acid probes constructed by this invention exhibit significant and stable response signal changes and can be widely used in fields such as biosensing and molecular diagnostics.

[0005] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a mutant of a luciferase fusion protein, wherein the mutant is mutated at least one of the following positions: position 144, position 148, position 339, position 353, and position 366, compared with the amino acid sequence shown in SEQ ID NO:2.

[0007] The mutation at position 144 is preferably E144K; the mutation at position 148 is preferably E148K; the mutation at position 339 is preferably D339K; the mutation at position 353 is preferably E353K; and the mutation at position 366 is preferably E366K.

[0008] In this invention, E144K, i.e., the 144th amino acid, is mutated from E to K; E148K, i.e., the 148th amino acid, is mutated from E to K; D339K, i.e., the 339th amino acid, is mutated from D to K; E353K, i.e., the 353rd amino acid, is mutated from E to K; and E366K, i.e., the 366th amino acid, is mutated from E to K.

[0009] Preferably, the mutant includes either E144K or E148K, or any two of E144K, E148K, and D339K.

[0010] More preferably, the mutant contains E144K, E148K and D339K simultaneously, or contains E144K, E148K, D339K and E353K simultaneously, or contains E144K, E148K, D339K, E353K and E366K simultaneously.

[0011] The present invention also provides an isolated nucleic acid that encodes a mutant as described in the first aspect of the present invention; in some embodiments, the base sequence of the nucleic acid is shown as any one of SEQ ID NO:3 to 10.

[0012] The present invention also provides a recombinant expression vector comprising the isolated nucleic acid described herein. In some embodiments, the backbone of the recombinant expression vector is the pET28a plasmid.

[0013] The present invention also provides a transformant containing the isolated nucleic acid described in this invention, or the recombinant expression vector described in this invention. In some embodiments, the host cell used in constructing the transformant is *Escherichia coli*. In some specific embodiments, the *Escherichia coli* is *E. coli* BL21(DE3).

[0014] The present invention also provides a method for preparing the mutant of the present invention, which includes culturing the transformant of the present invention to obtain a fermentation product, and obtaining the mutant from the fermentation product.

[0015] Secondly, the present invention provides a method for preparing a bioluminescent nucleic acid probe, comprising the following steps:

[0016] S1. The deoxyribonuclease (DNAzyme) is modified to obtain the modified deoxyribonuclease;

[0017] S2. The deoxyribozyme modified in step S1 is orthogonally coupled with the mutant as described in the first aspect of the present invention to obtain the coupling product;

[0018] S3. Hybridize the coupling product from step S2 with the deoxyribozyme substrate chain to obtain the bioluminescent nucleic acid probe.

[0019] In some embodiments, in step S1, the deoxyribozyme has metal ion-specific response properties.

[0020] In some embodiments, in step S1, the sequence (5'-3') of the deoxyribozyme is: CACGT CCATCTCTTCTCCGAGCCGGTCGAAATAGTGAGTAGT-NH2, as shown in SEQ ID NO: 21.

[0021] In some embodiments, in step S1, the deoxyribozyme is modified with a HaloTag ligand molecule.

[0022] In some embodiments, the structure of the HaloTag ligand molecule is shown in the following formula:

[0023]

[0024] Where n = 2 or 4, n = 4 is preferred.

[0025] In some embodiments, the HaloTag ligand molecule is commercially available or synthesized using existing techniques.

[0026] In some specific embodiments, the specific process of modifying deoxyribozyme with HaloTag ligand molecules in step S1 is as follows: HaloTag ligand molecules and deoxyribozyme are dissolved in borate buffer solution and reacted overnight at 25-40°C and 160-200 rpm to obtain the modified deoxyribozyme.

[0027] In some specific embodiments, the borate buffer solution contains 50 mM sodium borate and has a pH of 8.5.

[0028] In some embodiments, the specific process of orthogonal coupling of the modified deoxyribozyme and the mutant in step S2 is as follows: the modified deoxyribozyme and the mutant are mixed in Tris-NaCl buffer solution at a concentration ratio of (1-3):1 μM and incubated at room temperature for 1-2 h to obtain the coupling product.

[0029] In some embodiments, the Tris-NaCl buffer solution contains 50 mM Tris and 100 mM NaCl, and has a pH of 7.4.

[0030] In some embodiments, in step S3, the deoxyribonuclease substrate chain sequence (5'-3') is: ACTCACTATrAGGAAGAGATGGACGTG (where rA is adenosine), as shown in SEQ ID NO: 22.

[0031] In some embodiments, in step S3, the 5' end of the deoxyribozyme substrate chain is modified with a fluorophore, which may be FITC (fluorescein), Cy3, or TAMRA (dimethylrhodamine), preferably Cy3.

[0032] In some embodiments, the specific implementation process of hybridization between the coupling product and the deoxyribonuclease substrate chain in step S3 is as follows: after diluting the coupling product with the Tris-NaCl buffer solution, the deoxyribonuclease substrate chain is added and incubated at room temperature for 0.5 to 1 h to obtain the bioluminescent nucleic acid probe.

[0033] Thirdly, the present invention provides a bioluminescent nucleic acid probe prepared by the preparation method described in the second aspect of the present invention.

[0034] Fourthly, the present invention provides the application of the bioluminescent nucleic acid probe as described in the third aspect of the present invention in the construction of a bioluminescent nucleic acid probe detection kit.

[0035] Fifthly, the present invention provides an application of a bioluminescent nucleic acid probe as described in the third aspect of the present invention in the instantaneous detection of metal ions.

[0036] In some embodiments, the metal ion is a zinc ion.

[0037] In some specific embodiments, the application of the bioluminescent nucleic acid probe in the instantaneous detection of zinc ions includes the following steps: reacting the sample to be tested with the bioluminescent nucleic acid probe at room temperature, then adding Furimazine luminescent substrate to the reaction solution, and then taking a picture with a smartphone in a dark room. If the sample contains zinc ions, the light emission is blue; if it does not contain zinc ions, the light emission of the corresponding fluorophore is displayed.

[0038] The principle behind the bioluminescent nucleic acid probe of this invention for the real-time detection of zinc ions is as follows: Figure 2 As shown. Specifically, in the absence of zinc ions, the fluorophore on the deoxyribozyme substrate chain of the bioluminescent nucleic acid probe is close to the cpHNLuc mutant. When furimazine luminescent substrate is added to the reaction solution, the cpHNLuc mutant catalyzes the oxidation of the furimazine luminescent substrate, emitting blue bioluminescence with a wavelength of 400-500 nm. This blue bioluminescence can undergo bioluminescent energy resonance transfer (BRET) with the fluorophore on the deoxyribozyme substrate chain, thereby causing the detection solution to produce the corresponding fluorophore's luminescence. That is, in the absence of metal ions, the probe displays the fluorophore's luminescence. When zinc ions are present, the deoxyribozyme chain binds to the zinc ions and subsequently cleaves the rA (adenosine) site on the substrate chain, breaking the substrate chain into two fragments. Since the hybridization of the substrate chain fragment containing the fluorophore with the deoxyribozyme chain is unstable at this time, it dissociates and moves away from the cpHNLuc mutant, thereby reducing the bioluminescent energy resonance transfer effect between the mutant and the fluorophore, causing the solution to produce blue bioluminescence. Since the cleavage of substrate chains by deoxyribozymes is zinc ion concentration-dependent, this can be used to detect zinc ions.

[0039] The beneficial effects of this invention are:

[0040] 1) The cpHNLuc fusion protein mutant provided by this invention possesses the ability to orthogonally couple nucleic acid probes, making it suitable for constructing bioluminescent nucleic acid probes. The bioluminescent nucleic acid probes constructed by this invention effectively solve the problems of existing nucleic acid fluorescent probes requiring specialized equipment for signal excitation / reception, high background, and false positive signals, thus improving the performance of existing nucleic acid probes in biological detection. The bioluminescent nucleic acid probes constructed by this invention can be used to construct bioluminescent nucleic acid probe detection kits, providing new methods and tools for fields such as environmental monitoring and disease diagnosis.

[0041] 2) The bioluminescent nucleic acid probe of the present invention has ratiometric properties and is not affected by environmental factors or substrate consumption. Furthermore, the bioluminescent nucleic acid probe of the present invention does not require device excitation, and its luminescence signal can be acquired by taking pictures with a camera or mobile phone, making it suitable for point-of-care testing (POCT). Attached Figure Description

[0042] Figure 1 This is the mass spectrum of the product covalently coupled between the DNAzyme sequence and the HaloTag ligand molecule.

[0043] Figure 2 This is a schematic diagram illustrating the principle of using the bioluminescent nucleic acid probe of this invention for the real-time detection of zinc ions.

[0044] Figure 3This is a plasmid map of the cpHNLuc fusion protein.

[0045] Figure 4 This is an SDS-PAGE image of the purified cpHNLuc fusion protein mutant.

[0046] Figure 5 This is an SDS-PAGE image of the cpHNLuc fusion protein mutant after conjugation with DNAzyme.

[0047] Figure 6 Bioluminescent energy transfer efficiency of the DNAzyme probe constructed for the cpHNLuc fusion protein mutant.

[0048] Figure 7 (a) shows the emission spectra of the bioluminescent DNA zyme probe in Example 6 in response to different zinc ions; (b) shows the intensity ratio of the 450 nm emission peak to the 565 nm emission peak of the bioluminescent DNA zyme probe in Example 6 in response to different zinc ions; (c) shows the linear range of the bioluminescent DNA zyme probe in Example 6 in response to different zinc ions; and (d) shows the selectivity of the bioluminescent DNA zyme probe in Example 6 for different ions.

[0049] Figure 8 This is a mobile phone image showing the response of the bioluminescent DNAzyme probe to zinc ions in the blood in Example 7. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. It should be understood that the embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention. The parameters, proportions, etc., of the embodiments can be selected according to local conditions without substantially affecting the results.

[0051] In some specific embodiments of this invention: the cpHNLuc fusion protein gene used in this invention was synthesized by Shanghai Sangon Biotech Co., Ltd. This invention uses Gibson assembly to construct the cpHNLuc base sequence into the prokaryotic expression vector pET28a, obtaining a recombinant plasmid named pET28a-cpHNLuc, as shown in the diagram. Figure 3As shown, this plasmid was used as a template for directed evolution. A mutant library of the cpHNLuc fusion protein was obtained through single-point or combined mutations using site-directed mutagenesis. This mutant library was transformed into BL21(DE3) competent cells, and single clones were selected for mutant expression and purification. The cpHNLuc fusion protein mutant can orthogonally couple with nucleic acid probes modified with HaloTag ligands, thereby constructing bioluminescent nucleic acid probes. After adding the catalytic substrate Furimazine, the bioluminescence generated by the mutant protein produces an energy transfer signal with the fluorophore on the nucleic acid probe. This energy transfer signal is correlated with the target concentration, thus enabling the detection of the target.

[0052] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0053] Example 1: Construction of a cpHNLuc fusion protein mutant library

[0054] Using pET28a-cpHNLuc as a template, where the base sequence of cpHNLuc is shown in SEQ ID NO: 1, site-directed or combined mutations were performed on five sites of the cpHNLuc fusion protein: E144, E148, D339, E353, and E366. These mutations included, but were not limited to, the following sites / combinations: E144K, E148K, D339K, E353K, and E366K. Primers were designed for different mutation sites, specifically as follows:

[0055] The primer sequences corresponding to E144K are shown in SEQ ID NO: 11-12; the primer sequences corresponding to E148K are shown in SEQ ID NO: 13-14; the primer sequences corresponding to D339K are shown in SEQ ID NO: 15-16; the primer sequences corresponding to E353K are shown in SEQ ID NO: 17-18; and the primer sequences corresponding to E366K are shown in SEQ ID NO: 19-20.

[0056] Site-directed mutants were prepared using PrimeSTAR Max Premix, and the PCR reaction system and PCR reaction were performed according to its instructions. Taking the preparation of E144K mutant as an example, the PCR reaction system and PCR reaction conditions are shown in Table 1 and Table 2, respectively.

[0057] Table 1 PCR reaction system

[0058]

[0059]

[0060] Table 2 PCR reaction conditions

[0061]

[0062] After the reaction was complete, 0.5 μL of Dpn I enzyme was added to the system to digest the template. The mixture was then transformed into DH5α and plated on a plate containing kanamycin resistance at a final concentration of 50 μg / mL. The next day, single colonies were picked from the plate, cultured overnight at 37°C, and plasmids were extracted. Sequencing results confirmed that the sequence had mutated.

[0063] Example 2 Expression and purification of cpHNLuc fusion protein mutant

[0064] The plasmid was transferred into BL21(DE3) competent cells, plated, and incubated overnight at 37°C. Single colonies were picked from the plates and incubated overnight at 37°C. Then, the cells were transferred to fresh medium, diluted 1:100, and cultured until the OD value reached 0.6-0.8. IPTG was added to a final concentration of 1 mM, and the cells were incubated overnight at 16°C to induce protein expression.

[0065] The cpHNLuc fusion protein mutant was purified using a His-tagged protein purification kit (purchased from Beyotime Biotechnology Co., Ltd., product number P2226). 50 ml of bacterial cells were collected, and 3 ml of non-denaturing lysis buffer (product number P2226-2) was added. The cells were sonicated (10 s on 10 s off, 50% power) for 8 min, and then centrifuged at 12000 rpm for 30 min at 4°C to separate the supernatant (cell lysis buffer) and precipitate. 1 ml of BeyoGold was added to an empty affinity chromatography column (product number P2226-6). TM His-tag Purification Resin was used to equilibrate the packing material by rinsing with 3 ml of binding buffer. Then, 3 ml of filtered cell lysis buffer was added. The packing material was rinsed 6 times (1 ml / rinse) with non-denaturing wash buffer (product number P2226-3), and then eluted with non-denaturing elution buffer (product number P2226-4) 4–5 times (0.5 mL / rinse). The eluted protein was collected.

[0066] The eluted protein was dialyzed overnight at 4°C with dialysis buffer (25 mM Tris, pH 8.0, 250 mM NaCl). Protein concentration was determined using a BCA protein quantification kit (purchased from Beyotime Biotechnology Co., Ltd., product number P0010S). The eluted protein was analyzed by 12% SDS-PAGE. Figure 4 (As shown). Figure 4In the table, 1 represents the original cpHNLuc fusion protein; 2 represents the E144K mutant; 3 represents the E148K mutant; 4 represents the D339K mutant; 5 represents the E148K / D339K mutant; 6 represents the E144K / D339K mutant; 7 represents the E144K / E148K mutant; 8 represents the E144K / E148K / D339K mutant; 9 represents the E144K / E148K / D339K / E353K mutant; and 10 represents the E144K / E148K / D339K / E353K / E366K mutant. All mutants were successfully expressed with a purity higher than 95%.

[0067] Example 3: Modification and purification of HaloTag ligands on DNAzyme sequences

[0068] The HaloTag ligand molecule used in this embodiment was synthesized using existing techniques, and its structure is as follows:

[0069]

[0070] 1 mg of HaloTag ligand and 14OD amino-modified DNAzyme (purchased from Shanghai Sangon Biotech Co., Ltd., sequence shown in SEQ ID NO: 21) were dissolved in 200 μL of borate buffer (50 mM sodium borate, pH 8.5) and reacted overnight at 37°C with shaking at 180 rpm. The reaction solution was filtered through a 3 kDa ultrafiltration tube. Ultrafiltration (purchased from Sigma-Aldrich, product number: UFC5003) was used to remove excess HaloTag ligand molecules. 300 μL of ultrapure water was added to 200 μL of reaction solution to bring the total volume to 500 μL. This volume was then added to the inner tube of the ultrafiltration tube and centrifuged at 8500 rpm for 5 min at room temperature. The filtrate in the outer tube was discarded, and the reaction solution in the inner tube was added to bring the volume to 500 μL. The mixture was centrifuged again, and the washing was repeated five times to obtain the cross-linking product of DNAzyme and HaloTag ligand. The UV absorbance of the sample at 260 nm was measured, and the molar extinction coefficient of this sequence was found to be 398000 L / (mole·cm). The product concentration was calculated to be 19.25 μM according to Beer-Lambert's law. The obtained product was characterized by mass spectrometry, and the spectrum is shown below. Figure 1 As shown, the calculated molecular weight was 13415.48, and the detected molecular weight was 13411.2, which proves that DNAzyme and HaloTag ligand were successfully cross-linked.

[0071] Example 4: Preparation of bioluminescent DNA zyme probes

[0072] The DNAzyme and HaloTag ligand cross-linking products obtained in Example 3 were mixed with proteins numbered 1-10 in Example 2 at a concentration ratio of 1:1 μM in buffer solution (50 mM Tris, 100 mM NaCl, pH 7.4) and incubated at room temperature for 2 h to obtain NLuc-DNAzyme conjugates with different cpH values. Samples were taken every 10 min during the process, and the orthogonal coupling efficiency was detected using 12% SDS-PAGE. The loading volume of the conjugates used for electrophoresis was 10 μM, the electrophoresis time was 30 min, and the voltage was 160 V. After electrophoresis, the proteins were stained with Coomassie Brilliant Blue rapid staining solution for 1 h, then destained with ultrapure water for 2 h, and finally analyzed using a gel imaging system. The results are shown below. Figure 5 And as shown in Table 3.

[0073] Different cpHNLuc-DNAzyme conjugates were diluted to 20 nM with buffer solution (50 mM Tris, 150 mM NaCl, pH 7.4), and 30 nM of substrate nucleic acid sequence containing Cy3 modification (sequence shown in SEQ ID NO: 22) was added. The mixture was incubated at room temperature for 1 h to prepare different bioluminescent DNAzyme probes.

[0074] Table 3 Orthogonal coupling efficiency of the original cpHNLuc fusion protein and its mutants

[0075]

[0076] Example 5: Performance Verification of Bioluminescent DNAzyme Probe

[0077] The performance of the bioluminescent DNA zyme probes (probes constructed from all mutants) prepared in Example 4 was investigated:

[0078] Different bioluminescent DNAzyme probes were diluted in 100 μL of reaction buffer (50 mM Tris, 150 mM NaCl, pH 7.4) to obtain working solutions with a concentration of 20 nM. 1 μL of 1 μg / mL furimazine luminescent substrate was added to the working solutions, and the bioluminescence emission spectra were then measured using a fluorescence spectrometer (Edinburgh Instruments Ltd, FS5, UK). The excitation source was turned off, the excitation slit width was 0 nm, the emission slit width was 10 nm, and the emission signal scanning range was from 400 nm to 700 nm. Simultaneously, bioluminescence images of each sample were captured using a mobile phone (HUAWEI Mate 40 Pro) at ISO 1600 and an exposure time of 4 s. The results are as follows: Figure 6As shown, bioluminescent DNAzyme probes constructed from mutants containing E144K / E148K, E144K / E148K / D339K, E144K / E148K / D339K / E353K, and E144K / E148K / D339K / E353K / E366K exhibit better bioluminescent energy transfer performance. Among them, the bioluminescent DNAzyme probe constructed from the mutant containing E144K / E148K / D339K shows the best performance, with an energy transfer ratio as high as about 3 times (565nm / 450nm).

[0079] Example 6: Bioluminescent DNAzyme probe for metal ion detection

[0080] The bioluminescent DNAzyme probe constructed using the E144K / E148K / D339K mutant in Example 4 was used to detect metal ions:

[0081] A series of 100 μL reaction buffer solutions (50 mM Tris, 150 mM NaCl, pH 7.4) containing 20 nM bioluminescent DNAzyme probe and different concentrations of zinc ions (0.2, 0.4, 0.5, 1, 2, 3, 5, 10 μM) were incubated at room temperature for 1 h. Then, 1 μL of 1 μg / mL Furimazine luminescent substrate was added to the reaction solution. The bioluminescence emission spectrum was then measured using a fluorescence spectrometer with the excitation source turned off. The excitation slit width was 0 nm, the emission slit width was 10 nm, and the emission signal scan range was from 400 nm to 700 nm. The results are as follows: Figure 7 As shown in (a), as the zinc ion concentration increased from 0.2 μM to 10 μM, the luminescence intensity at 450 nm increased, while the luminescence intensity at 565 nm decreased. The ratio R450 nm / 565 nm increased from 0.27 to 1.4, as shown in Figure (a). Figure 7 As shown in (b), the probe exhibits good linearity in the range of 0.6–4.0 μM (R² = 0.995), and its detection limit for zinc ions is calculated to be 202 nM. Figure 7 As shown in (c).

[0082] In the selective experiment, Na + K + Mg 2+ Ba 2+ Ni 2+ Ca 2+ Mn 2+ Co 2+ As a control, the response of the bioluminescent DNAzyme probe to these ions at 100 μM was examined. The results are as follows: Figure 7 As shown in (d), only Zn2+ The probe can elicit a significant change in the luminescence signal, demonstrating its good selectivity for zinc ions.

[0083] Example 7: Bioluminescent DNAzyme probe for the detection of zinc ions in blood

[0084] The bioluminescent DNA zyme probe constructed using the E144K / E148K / D339K mutant in Example 4 was used to detect zinc ions in the blood.

[0085] A 20 μL blood sample was obtained via finger-prick blood collection and then diluted 50-fold with a buffer solution (50 mM Tris, 150 mM NaCl, pH 7.4). 100 μL of the diluted blood and 40 nM bioluminescent DNAzyme probe were added to two centrifuge tubes, one with 100 μM zinc ions and the other without. The reaction was carried out at room temperature for 1 h. 1 μL of 1 μg / mL Furimazine luminescent substrate was added to each reaction solution, and the images were then taken in a dark room using a smartphone (Huawei Mate 40 Pro) at ISO 1600 and an exposure time of 4 s. The results are as follows: Figure 8 As shown, the sample containing zinc ions emits blue light, while the sample without zinc ions emits red light, proving that this probe can be used to detect metal ions in blood.

Claims

1. A mutant of a luciferase fusion protein, characterized in that, The mutant, compared to the amino acid sequence shown in SEQ ID NO: 2, has mutations only at positions 144, 148, and 339; the mutation at position 144 is E144K; the mutation at position 148 is E148K; and the mutation at position 339 is D339K.

2. An isolated nucleic acid encoding the mutant as described in claim 1.

3. A method for preparing a bioluminescent nucleic acid probe, comprising the following steps: S1. The deoxyribozyme was modified to obtain the modified deoxyribozyme; S2. The deoxyribozyme modified in step S1 is orthogonally coupled with the mutant as described in claim 1 to obtain the coupling product; S3. Hybridize the coupling product of step S2 with the deoxyribozyme substrate chain to obtain the bioluminescent nucleic acid probe; In step S1, the 5'-3' sequence of the deoxyribozyme is: CACGTCCATCTCTTCTCCGAGCCGGTCGAAATAGTGAGTAGT-NH2; the modification is performed using a HaloTag ligand molecule, the structure of which is shown below: in, n = 2 or 4; The specific process of modifying deoxyribozyme with the HaloTag ligand molecule is as follows: the HaloTag ligand molecule and deoxyribozyme are dissolved in borate buffer solution and reacted overnight at 25~40℃ and 160~200 rpm with shaking to obtain the modified deoxyribozyme; the borate buffer solution contains 50mM sodium borate and the pH is 8.

5. In step S3, the 5'-3' sequence of the deoxyribozyme substrate chain is: ACTCACTATrAGGAAGAGATGGACGTG.

4. The preparation method according to claim 3, characterized in that, In step S2, the specific process of orthogonal coupling between the modified deoxyribozyme and the mutant is as follows: the modified deoxyribozyme and the mutant are mixed in a Tris-NaCl buffer solution at a concentration ratio of (1~3):1µM, and incubated at room temperature for 1~2 h to obtain the coupling product; the Tris-NaCl buffer solution contains 50 mM Tris and 100 mM NaCl, and the pH is 7.

4.

5. The preparation method according to claim 3, characterized in that, In step S3, the 5' end of the deoxyribozyme substrate chain is modified with a fluorophore, which is one of FITC, Cy3, or TAMRA. The specific implementation process of hybridization between the conjugation product and the deoxyribozyme substrate chain is as follows: the conjugation product is diluted with Tris-NaCl buffer solution, and then the deoxyribozyme substrate chain is added. The mixture is incubated at room temperature for 0.5-1 h to obtain the bioluminescent nucleic acid probe. The Tris-NaCl buffer solution contains 50 mM Tris and 100 mM NaCl, and the pH is 7.

4.

6. A bioluminescent nucleic acid probe prepared by the method described in claim 3.

7. The application of the bioluminescent nucleic acid probe as described in claim 6 in the construction of a bioluminescent nucleic acid probe detection kit; the kit is used for zinc ion detection.

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