A nanoparticle-fluorescent label fusion protein complex and a method for detecting single-stranded DNA

Abstract

The present invention provides a nanoparticle-fluorescent label fusion protein complex and a method for detecting single-stranded DNA, belonging to the technical field of nucleic acid detection. The present invention utilizes the property that the DGPprI protein in moderately thermophilic bacteria can specifically bind to single-stranded DNA, forms a complex NPs-eGFP-DGPprI by combining the eGFP-DGPprI fusion protein with nanoparticles, and adds it to the solution system to be detected. If single-stranded DNA (ssDNA) exists, the surface plasmon resonance absorption peak of the formed NPs-eGFP-DGPprI-ssDNA complex will shift. At the same time, since the complex is easy to precipitate by centrifugation, and the precipitate presents green due to carrying the GFP group, single-stranded DNA can be simply, visually, conveniently, and quickly identified and separated. The method for detecting single-stranded DNA provided by the present invention can be used to develop new molecular biology tools.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and in particular relates to a nanoparticle-fluorescently labeled fusion protein complex and a single-stranded DNA detection method thereof. Background Art

[0002] Single-stranded DNA is primarily found in organisms that use single-stranded DNA as their genetic material (such as a few viruses like bacteriophages), RNA reverse transcription products, and single-stranded fragments formed by damage to double-stranded DNA. Genomic DNA in organisms mostly exists in a stable double-stranded form. However, during various life processes, factors from both the external environment and within the organism can cause damage to the genomic DNA of cells. Commonly used techniques for detecting single-stranded DNA damage include electron microscopy, atomic force microscopy, and single-cell gel electrophoresis (comet assay). Microscopy techniques can directly observe single DNA fragments, even very short DNA fragments, but sample preparation is relatively complex. Comet electrophoresis can measure DNA damage in single cells, but it requires preparation, cell lysis, unwinding, electrophoresis, and slide reading, making analysis time-consuming.

[0003] The common single-strand DNA damage detection technologies mentioned above suffer from drawbacks such as complex sample preparation, time-consuming analysis, and reliance on specialized large-scale instruments. Furthermore, the detected single-stranded structures are difficult to separate. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a nanoparticle-fluorescently labeled fusion protein complex that not only has a strong property of binding to single-stranded DNA fragments but also allows single-stranded DNA fragments to be easily separated from the complex, thereby achieving the purpose of rapid detection of single-stranded DNA fragments in the sample to be tested.

[0005] The present invention provides a method for detecting single-stranded DNA, which utilizes the nanoparticle-fluorescent marker fusion protein complex to achieve simple, intuitive, convenient and rapid single-stranded DNA detection.

[0006] The present invention provides a nanoparticle-fluorescently labeled fusion protein complex, comprising a nanoparticle and a fluorescently labeled fusion protein;

[0007] The fluorescent marker fusion protein is formed by fusion expression of the fluorescent gene and the DGPprI protein;

[0008] The nanoparticles are combined with the fluorescent-labeled fusion protein through electrostatic forces.

[0009] Preferably, the amino acid sequence of the fluorescent-labeled fusion protein is as shown in SEQ ID NO: 1.

[0010] Preferably, the nanoparticles include negatively charged microparticles.

[0011] Preferably, the negatively charged microparticles include metal nanoparticles and / or nanoplastics.

[0012] Preferably, the metal nanoparticles include gold nanoparticles or silver nanoparticles.

[0013] The invention provides a single-stranded DNA detection kit, comprising the nanoparticle-fluorescent labeling fusion protein complex and a detection reagent.

[0014] Preferably, the detection reagent comprises a sample diluent.

[0015] The present invention provides a method for detecting single-stranded DNA, comprising the following steps:

[0016] The aqueous solution of the nanoparticle-fluorescently labeled fusion protein complex and the sample to be tested are mixed and incubated, and full-band scanning observation is performed. The red shift of the plasma resonance absorption peak indicates that the sample to be tested contains single-stranded DNA.

[0017] Preferably, the mass ratio of the nanoparticle-fluorescently labeled fusion protein complex to single-stranded DNA is 1 mg:20-170 ng.

[0018] Preferably, the length of the single-stranded DNA is 10 to 35 nt.

[0019] The nanoparticle-fluorescently labeled fusion protein complex provided by the present invention comprises a nanoparticle and a fluorescently labeled fusion protein; the fluorescently labeled fusion protein is formed by the fusion expression of a fluorescent gene and a DGPprI protein; the nanoparticle binds to the fluorescently labeled fusion protein via electrostatic forces. The DGPprI protein, derived from a moderately thermophilic bacterium, has the characteristic of specifically binding to single-stranded DNA. When single-stranded DNA is present in the system, the single-stranded DNA binds to the complex via the DGPprI protein to form an NPs-eGFP-DGPprI-ssDNA complex. Full-band scanning observation reveals that the plasmon resonance absorption peak of the NPs-eGFP-DGPprI-ssDNA complex undergoes a red shift. Simultaneously, the NPs-eGFP-DGPprI-ssDNA complex undergoes solid-liquid separation, and the resulting solid phase exhibits green eGFP protein, thereby achieving separation and detection of single-stranded DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the method for nanoparticle-fluorescent labeled protein binding and separation of single-stranded DNA;

[0021] Figure 2 This is the result of expression and purification of eGFP-DGPprI fusion protein;

[0022] Figure 3 The results show that eGFP-DGPprI specifically binds to single-stranded DNA;

[0023] Figure 4 The results of eGFP-DGPprI binding to single-stranded DNA of different lengths;

[0024] Figure 5 The shift results of the surface isoelectronic resonance peak of AgNP-eGFP-DGPprI before (left) and after (right) binding to ssDNA;

[0025] Figure 6 The shift results of the surface isoelectronic resonance peak of AgNP-eGFP-DGPprI before (left) and after (right) binding to ssDNA;

[0026] Figure 7 This is a curve diagram of the adsorption of single-stranded DNA by AgNPs-eGFP-DGPprI. DETAILED DESCRIPTION

[0027] The present invention provides a nanoparticle-fluorescently labeled fusion protein complex, comprising a nanoparticle and a fluorescently labeled fusion protein;

[0028] The fluorescent marker fusion protein is formed by fusion expression of the fluorescent gene and the DGPprI protein;

[0029] The nanoparticles are combined with the fluorescent-labeled fusion protein through electrostatic forces.

[0030] In the present invention, the fluorescently labeled fusion protein is preferably obtained by recombinantly expressing the genes encoding the GFP gene and the DGPprI protein, and the amino acid sequence is shown in SEQ ID NO: 1. The coding sequence of the fluorescently labeled fusion protein has a nucleotide sequence shown in SEQ ID NO: 2. The method for preparing the fluorescently labeled fusion protein preferably includes the following steps:

[0031] 1) Clone the coding sequence of the HMT-fluorescent marker fusion protein into a prokaryotic expression vector to obtain a recombinant prokaryotic expression vector;

[0032] 2) transforming the recombinant prokaryotic expression vector into host cells to obtain recombinant bacteria;

[0033] 3) The recombinant bacteria are subjected to resistance screening and IPTG induction culture, and the cells are collected;

[0034] 4) Isolating and purifying the recombinant protein from the bacteria, and digesting it with TEV enzyme to obtain eGFP-DGPprI fusion protein.

[0035] In the present invention, the nucleotide sequence encoding the HMT-fluorescent marker fusion protein is shown in SEQ ID NO:3. HMT is a tandem tag (His tag and MBP tag) for fusion protein expression, separated by a TEV cleavage site to facilitate subsequent purification of the recombinant protein. The amino acid sequence of the expressed HMT-fluorescent marker fusion protein is shown in SEQ ID NO:4.

[0036] In the present invention, the prokaryotic expression vector is preferably pET28-HMT. The cloning site of the prokaryotic expression vector is preferably NdeI / BamHI. The present invention has no particular limitation on the transformation method, and any transformation method known in the art, such as the CaCl2 heat shock method, can be used.

[0037] In the present invention, the resistance screening is preferably performed using LB medium containing kanamycin, and the IPTG induction culture conditions are preferably: IPTG with a final concentration of 0.2 mM, and expression induction at 30° C. for 5 h.

[0038] In the present invention, the purification method preferably uses an AKTA protein purification system (GE Company), and performs purification in sequence using a nickel column, an MBP column, TEV enzyme digestion, a desalting column, an MBP column, a nickel column, a desalting column, an ion exchange column, and a molecular sieve column to finally obtain a relatively pure target protein.

[0039] In the present invention, the nanoparticles preferably include negatively charged microparticles. The negatively charged microparticles preferably include metal nanoparticles and / or nanoplastics. The metal nanoparticles preferably include gold nanoparticles or silver nanoparticles. The nanoplastic preferably has a particle size of 50 to 200 nm, more preferably 100 nm. In the present embodiment, the nanoplastic was purchased from Daye (Tianjin) Technology Co., Ltd.

[0040] In the present invention, the method for preparing the nanoparticle-fluorescently labeled fusion protein complex preferably includes a method for preparing a metal nanoparticle-fluorescently labeled fusion protein complex and a method for preparing a nanoplastic-fluorescently labeled fusion protein complex.

[0041] In the present invention, the method for preparing the metal nanoparticle-fluorescently labeled fusion protein complex is preferably as follows: mixing a solution containing metal ions with a solution of the fluorescently labeled fusion protein, allowing the solution to react, observing the red shift of the plasmon resonance absorption peak using a full-band scan, centrifuging, resuspending in ultrapure water, and filtering with a filter membrane to obtain the metal nanoparticle-fluorescently labeled fusion protein complex. During the reaction, eGFP-DGPprI reduces the metal ions to form metal NPs-eGFP-DGPprI particles. The pH is preferably 6.8-7.2, more preferably 7; the reaction temperature is preferably 20-27°C, more preferably 25°C. The concentration of eGFP-DGPprI is preferably 1.8-2.2 mg / mL, more preferably 2 mg / mL. The concentration of the metal ions is preferably 1.8-2.2 mM, more preferably 2 mM. The volume ratio of the eGFP-DGPprI fusion protein to the metal ion-containing solution is preferably 1:2-1:6, more preferably 1:3. The reaction time is preferably 5-30 minutes, more preferably 15 minutes. The centrifugal conditions are preferably as follows: 12,000×g, 20 min. The membrane pore size during the membrane filtration is preferably 0.22 μm.

[0042] In the present invention, the method for preparing the nanoplastic-fluorescently labeled fusion protein complex preferably comprises the following steps: mixing a solution containing the nanoplastic with a solution containing the fluorescently labeled fusion protein, allowing the solution to react, observing the red shift of the plasmon resonance absorption peak using full-band scanning, centrifuging, resuspending in ultrapure water, and filtering with a filter membrane to obtain the nanoplastic-fluorescently labeled fusion protein complex. The concentration of the nanoplastic solution is preferably 10-50 μg / mL, more preferably 20 μg / mL. The concentration of the fluorescently labeled fusion protein solution is preferably 1-5 μg / mL, more preferably 2 μg / mL. The solvent for the static reaction is a buffer solution containing 150 mM NaCl and 20 mM Tris-HCl, pH 7.5. The static reaction time is preferably 9-12 minutes, more preferably 10 minutes. The centrifugation conditions are preferably 12,000 × g for 20 minutes.

[0043] The invention provides a single-stranded DNA detection kit, comprising the nanoparticle-fluorescent labeling fusion protein complex and a detection reagent.

[0044] In the present invention, the detection reagent preferably includes a sample diluent. The sample diluent provides a buffer environment for separation. The sample diluent is preferably a buffer containing 150mM NaCl, pH 7.5, and 20mM Tris-HCl.

[0045] In the present invention, in order to further separate single-stranded DNA, the detection reagent preferably further includes proteinase K. Proteinase K enzymatically hydrolyzes DGPprI to separate single-stranded DNA from the NPs-eGFP-DGPprI-ssDNA complex, so that the sequence of the single-stranded DNA can be further detected.

[0046] The present invention provides a method for detecting single-stranded DNA, the principle diagram of which is shown in FIG. Figure 1 , including the following steps:

[0047] The aqueous solution of the nanoparticle-fluorescently labeled fusion protein complex and the sample to be detected are mixed and incubated, and full-band scanning observation is performed. The red shift of the plasma resonance absorption peak indicates that the sample to be detected contains single-stranded DNA.

[0048] In the present invention, the mass ratio of the nanoparticle-fluorescently labeled fusion protein complex to single-stranded DNA is preferably 1 mg:20-170 ng, more preferably 1 mg:40-150 ng, further preferably 1 mg:70-120 ng, and most preferably 1 mg:100 ng. The length of the single-stranded DNA is preferably 10-35 nt, more preferably 10-35 nt, and most preferably 29-35 nt. Experiments in the present invention have shown that the longer the ssDNA, the stronger the binding ability of the nanoparticle-fluorescently labeled fusion protein complex. The detection method can detect the presence of single-stranded DNA for non-diagnostic purposes.

[0049] In the present invention, it is preferred that a separation method is also included, preferably, the system in which the plasma resonance absorption peak undergoes red shift is subjected to solid-liquid separation, the solid phase is collected to obtain a nanoparticle-fluorescent marker protein-single-stranded DNA complex, and the nanoparticle-fluorescent marker protein-single-stranded DNA complex is treated with a proteinase K solution to obtain an aqueous solution containing single-stranded DNA.

[0050] The following is a detailed description of a nanoparticle-fluorescently labeled fusion protein complex and a single-stranded DNA detection method thereof provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] In vitro expression and purification of eGFP-DGPprI fusion protein

[0053] (1) The recombinant bacteria containing the HMT-eGFP-DGPprI vector were inoculated into 5 ml of LB medium containing kanamycin and cultured at 37°C overnight. The next day, the bacteria were transferred to 500 ml of LB medium and cultured at 37°C in a shaking incubator until the OD 600About 0.8, IPTG was added to a final concentration of 0.2mM, and expression was induced at 30°C for 5 hours. Because it carries the eGFP group, protein expression can be visually identified, and SDS-PAGE is used to further confirm it before subsequent purification.

[0054] (2) Using the AKTA protein purification system (GE), the target protein was purified by nickel column, MBP column, TEV enzyme digestion, desalting column, MBP column, nickel column, desalting column, ion exchange column, and molecular sieve column in sequence to obtain a relatively pure target protein. Nickel column A solution used for protein purification: 1M NaCl, 20mM Tris-HCl pH 7.5, 5% glycerol; Nickel column B solution: 1M NaCl, 20mM Tris-HCl pH 7.5, 500mM imidazole, 5% glycerol; MBP column A solution: 1M NaCl, 20mM Tris-HCl pH 7.5, 5% glycerol; MBP column B solution: 1M NaCl, 20mM Tris-HCl pH 7.5, 20mM maltose, 5% glycerol; desalting buffer (ion exchange A solution): 150mM NaCl, 20mM Tris-HCl pH 7.5, 5% glycerol; ion exchange B solution: 1M NaCl, 20mM Tris-HCl pH 7.5, 5% glycerol; molecular sieve buffer: 150mM NaCl, 20mM Tris-HCl pH 7.5. The TEV enzyme digestion system was: 0.5 mM EDTA, 1 mM DTT, 5% glycerol, and digestion was performed at 4°C for 12 h.

[0055] The results of fusion protein expression and purification are shown in Figure 2 .from Figure 2 As can be seen from the strips, the present invention successfully recombined and expressed the eGFP-DGPprI fusion protein (SEQ ID NO: 1).

[0056] Example 2

[0057] eGFP-DGPprI-ssDNA binding assay

[0058] The substrates were 5′FAM-labeled single-stranded DNA (19 nt (SEQ ID NO: 5), 23 nt (SEQ ID NO: 6), 29 nt (SEQ ID NO: 7), and 35 nt (SEQ ID NO: 8) nonspecific sequences, and 35 bp double-stranded DNA (SEQ ID NO: 8). The reaction system was prepared using the following buffer conditions: 150 mM NaCl, 20 mM Tris-HCl 7.5, 100 nM 5′FAM-labeled DNA, and 2 μM eGFP-DGPprI protein. The mixture was mixed and incubated at room temperature for 30 min. After completion of the reaction, the samples were separated using an 8% polyacrylamide gel in 1× TB buffer and electrophoresed at 140 V for 40 min, followed by scanning and imaging using a Typhoon FLA9500 (GE).

[0059] Depend on Figure 3 It can be seen that DGPprI protein can bind to single-stranded DNA but not double-stranded DNA.

[0060] Depend on Figure 4 It can be seen that DGPprI protein can bind to single-stranded DNA of different lengths, and the longer the single-stranded DNA, the higher the binding property.

[0061] Example 3

[0062] Preparation method of AgNPs-eGFP-DGPprI protein complex

[0063] In a clean 250 mL beaker, a 2 mM Ag(I) solution was thoroughly mixed with a 2 mg / mL eGFP-DGPprI protein solution. The pH was adjusted to 7.0, and the reaction was allowed to stand at room temperature. A multifunctional microplate reader (Spectramax M5, MD) was used to monitor the formation of AgNPs in the solution by observing the shift in the plasmon resonance absorption peak. The resulting silver nanoparticles were centrifuged at high speed (12,000 × g, 20 min) and resuspended in ultrapure water to obtain relatively pure AgNPs-eGFP-DGPprI. The AgNPs-eGFP-DGPprI protein solution was then collected by filtration using a 0.22 μm needle filter. The AgNPs-eGFP-DGPprI system was then scanned for plasmon resonance absorption peaks using a multifunctional microplate reader. ssDNA was then added to the AgNPs-eGFP-DGPprI system, and the plasmon resonance absorption peak shift was observed using a full-band scan.

[0064] See the results Figure 5 After AgNP-eGFP-DGPprI binds to single-stranded DNA, the surface isoelectronic resonance peak red-shifts.

[0065] Example 4

[0066] Detection method of nanoparticle-fluorescent labeled protein binding to single-stranded DNA

[0067] eGFP-DGPprI at a final concentration of 20 μg / mL was incubated with negatively charged Ag nanoparticles in 150 mM NaCl, 20 mM Tris-HCl (pH 7.5) buffer at room temperature for 10 minutes. Excess eGFP-DGPprI was removed by centrifugation at 12,000 × g for 20 minutes to obtain pure NPs-eGFP-DGPprI. The shift in the plasmon resonance absorption peak was observed using a multifunctional microplate reader for full-band scanning. The shift in the plasmon resonance absorption peak was also observed when ssDNA was added to the NPs-eGFP-DGPprI system.

[0068] Figure 6 is the displacement of the surface isoelectronic resonance peak before and after NP-eGFP-DGPprI binds to ssDNA. Figure 6 It can be seen that after AgNP-eGFP-DGPprI binds to single-stranded DNA, the surface isoelectronic resonance peak red-shifts.

[0069] A mixture of ssDNA substrates at different concentrations of 1 to 10 mg / mL was added to the NPs-eGFP-DGPprI system, incubated at room temperature for 30 minutes, and then centrifuged at 12,000 × g for 30 minutes to obtain the NPs-eGFP-DGPprI-ssDNA complex. eGFP can be used to conveniently observe the presence of protein nanoparticle complexes. Full-band scanning is used to observe the shift of the plasmon resonance absorption peak and monitor the formation process of the complex in the solution.

[0070] Depend on Figure 7 It can be seen that the level of single-stranded DNA adsorbed by AgNP-eGFP-DGPprI is basically proportional to its concentration.

[0071] The nanoparticle-fluorescent marker protein-single-stranded DNA complex was treated with proteinase K at a final concentration of 20 μg / mL to obtain an aqueous solution containing single-stranded DNA. The aqueous solution containing single-stranded DNA was used for subsequent studies.

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention. Sequence Listing <110> Zhejiang University <120> A nanoparticle-fluorescently labeled fusion protein complex and single-stranded DNA detection method thereof <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 522 <212> PRT <213> Artificial Sequence <400> 1 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn 145 150 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ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc 300 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt 360 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat 420 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc 480 acctggccgc tgattgctgc tgacggggt tatgcgttca agtatgaaaa cggcaagtac 540 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt 600 gacctgatta aaaaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc 660 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac 720 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa 780 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca 840 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac 900 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt 960 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg 1020 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact 1080 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac 1140 aataacaaca acctcgggat cgaggaaaac ctgtattttc agggccatat ggtgagcaag 1200 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 1260 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc 1320 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1380 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 1440 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgacgac 1500 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1560 gagctgaagg gcatcgactt caaggagcac ggcaacatcc tggggcacaa gctggagtac 1620 1680 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 1740 cagaaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 1800 cagtccgcccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 1860 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggagg cggtggctca 1920 ggaggcggtg gctcgccgga gactggagcg ctggccccgg ccaaggcgcg tatgcgggaa 1980 ctggcgactg cctacgctcg ccgtttgccg gggctggata cccacagcct gatgagcggg 2040 ctggacgcga ccctcacctt tatgccgatg ggtgaccgtg acggagccta tgaccccgag 2100 caccgcgtgg tgctgatcaa cagtcgggta cgcccggaac gccagcgctt cacactggcc 2160 cacgagatca gccatgccct cctgctgggc gacgacgact tgctcagcga tctgcacgac 2220 gcctacgagg gagagcggtt ggagcaggtc atagagacac tttgcaatgt gggggcagca 2280 gcgatcctga tgcccgagac cctgattgac gagctgctcg cgcgctttgg gccgagtggg 2340 cgtgcgctgg ctgagctggc gcggcgggca gacgtgagtg ccagcagtgc cctctatgcc 2400 ctggcggagc gaacctcagt gccggtgctg tacgcggtgt gcgcggtcag ccggctggaa 2460 gcagaatccg gagaggaacg gctccccgaa aaggcgctta ctgttcgggc cagtgcggga 2520 tcacccggcg tgaagtacag cctgcgcccc ggcacgctca tcccagatga ccacccggtc 2580 gccgttgcgc tggaaacgcg gctgcccatc acccaggaga gttacgtgcc cttccgttcg 2640 gggcggcgga tgcccgccta tgtcgacgcc tttcctgagc gtcagcgggt gctggtgagt 2700 tttgccctct tgcccaaagc gacgaaggga ggcgagcagg atgagtcggg tgtc 2754 <210> 4 <211> 918 <212> PRT <213> Artificial Sequence <400> 4 Gly Ser Ser Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn 1 5 10 15 Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu 20 25 30 Lys Asp Thr Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu 35 40 45 Glu Lys Phe Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile 50 55 60 Phe Trp Ala His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu 65 70 75 80 Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe 85 90 95 Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile 100 105 110 Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn 115 120 125 Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys 130 135 140 Ala Lys Gly Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe 145 150 155 160 Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu 165 170 175 Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala 180 185 190 Lys Ala Gly Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met 195 200 205 Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly 210 215 220 Glu Thr Ala Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp 225 230 235 240 Thr Ser Lys Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly 245 250 255 Gln Pro Ser Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala 260 265 270 Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu 275 280 285 Leu Thr Asp Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly 290 295 300 Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg 305 310 315 320 Ile Ala Ala Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn 325 330 335 Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile 340 345 350 Asn Ala Ala Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala 355 360 365 Gln Thr Asn Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn 370 375 380 Leu Gly Ile Glu Glu Asn Leu Tyr Phe Gln Gly His Met Val Ser Lys 385 390 395 400 Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp 405 410 415 Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly 420 425 430 Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly 435 440 445 Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly 450 455 460 Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe 465 470 475 480 Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe 485 490 495 Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu 500 505 510 Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys 515 520 525 Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser 530 535 540 His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val 545 550 555 560 Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala 565 570 575 Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu 580 585 590 Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro 595 600 605 Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala 610 615 620 Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Gly Gly Ser 625 630 635 640 Gly Gly Gly Gly Ser Pro Glu Thr Gly Ala Leu Ala Pro Ala Lys Ala 645 650 655 Arg Met Arg Glu Leu Ala Thr Ala Tyr Ala Arg Arg Leu Pro Gly Leu 660 665 670 Asp Thr His Ser Leu Met Ser Gly Leu Asp Ala Thr Leu Thr Phe Met 675 680 685 Pro Met Gly Asp Arg Asp Gly Ala Tyr Asp Pro Glu His Arg Val Val 690 695 700 Leu Ile Asn Ser Arg Val Arg Pro Glu Arg Gln Arg Phe Thr Leu Ala 705 710 715 720 His Glu Ile Ser His Ala Leu Leu Leu Gly Asp Asp Asp Leu Leu Ser 725 730 735 Asp Leu His Asp Ala Tyr Glu Gly Glu Arg Leu Glu Gln Val Ile Glu 740 745 750 Thr Leu Cys Asn Val Gly Ala Ala Ala Ile Leu Met Pro Glu Thr Leu 755 760 765 Ile Asp Glu Leu Leu Ala Arg Phe Gly Pro Ser Gly Arg Ala Leu Ala 770 775 780 Glu Leu Ala Arg Arg Ala Asp Val Ser Ala Ser Ser Ala Leu Tyr Ala 785 790 795 800 Leu Ala Glu Arg Thr Ser Val Pro Val Leu Tyr Ala Val Cys Ala Val 805 810 815 Ser Arg Leu Glu Ala Glu Ser Gly Glu Glu Arg Leu Pro Glu Lys Ala 820 825 830 Leu Thr Val Arg Ala Ser Ala Gly Ser Pro Gly Val Lys Tyr Ser Leu 835 840 845 Arg Pro Gly Thr Leu Ile Pro Asp Asp His Pro Val Ala Val Ala Leu 850 855 860 Glu Thr Arg Leu Pro Ile Thr Gln Glu Ser Tyr Val Pro Phe Arg Ser 865 870 875 880 Gly Arg Arg Met Pro Ala Tyr Val Asp Ala Phe Pro Glu Arg Gln Arg 885 890 895 Val Leu Val Ser Phe Ala Leu Leu Pro Lys Ala Thr Lys Gly Gly Glu 900 905 910 Gln Asp Glu Ser Gly Val 915 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence(Artificial Sequence) <400> 5 gttatgctct tgacgtaac 19 <210> 6 <211> twenty three <212> DNA <213> Artificial Sequence <400> 6 tcgttatgct cttgacgtaa caa 23 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <400> 7 tcttcgttat gctcttgacg taacaaact 29 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <400> 8 cgctcttcgt tatgctcttg acgtaacaaa cttgg 35

Claims

1. A nanoparticle-fluorescent label fusion protein complex, characterized in that, It includes nanoparticles and a fluorescently labeled fusion protein; The fluorescently labeled fusion protein is formed by the fusion expression of a fluorescent gene and a DGPprI protein; The nanoparticles are bound to the fluorescently labeled fusion protein through electrostatic forces; The amino acid sequence of the fluorescently labeled fusion protein is as shown in SEQ ID NO:

1.

2. The nanoparticle-fluorescent label fusion protein complex according to claim 1, wherein The nanoparticles include negatively charged microparticles.

3. The nanoparticle-fluorescent label fusion protein complex according to claim 2, wherein The negatively charged microparticles include metal nanoparticles and / or nanoplastics.

4. The nanoparticle-fluorescent label fusion protein complex according to claim 3, characterized in that, The metal nanoparticles include gold nanoparticles or silver nanoparticles.

5. A single-stranded DNA detection kit, characterized in that, It includes the nanoparticle-fluorescently labeled fusion protein complex according to any one of claims 1 to 4 and a detection reagent.

6. The kit according to claim 5, characterized in that, The detection reagent includes a sample diluent.

7. A method for detecting single-stranded DNA, characterized in that, It includes the following steps: Mix and incubate an aqueous solution of the nanoparticle-fluorescently labeled fusion protein complex according to any one of claims 1 to 4 with a sample to be detected, and observe by full-band scanning. If the surface plasmon resonance absorption peak undergoes a red shift, it indicates that single-stranded DNA is contained in the sample to be detected; The length of the single-stranded DNA is 29 to 35 nt.

8. The detection method according to claim 7, characterized in that, The mass ratio of the nanoparticle-fluorescently labeled fusion protein complex to the single-stranded DNA is 1 mg: 20 to 170 ng.

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