CN113025586B - Modified luciferase mutant protein, bioluminescent probe, probe set, preparation method and detection method - Google Patents

Modified luciferase mutant protein, bioluminescent probe, probe set, preparation method and detection method Download PDF

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CN113025586B
CN113025586B CN202110367329.1A CN202110367329A CN113025586B CN 113025586 B CN113025586 B CN 113025586B CN 202110367329 A CN202110367329 A CN 202110367329A CN 113025586 B CN113025586 B CN 113025586B
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倪维
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Abstract

The invention relates to a modified luciferase mutant protein, a bioluminescent probe, a probe set, a preparation method and a detection method, belonging to the technical field of molecular biology. The amino acid sequence of the modified luciferase mutant protein provided by the invention is shown in SEQ ID NO. 1. The modified luciferase mutant protein provided by the invention is obtained by mutating cysteine at 166 th site of the luciferase protein into serine and adding cysteine at C-terminal of luciferase, and the controllable and equal-proportion covalent coupling of the modified luciferase protein and DNA molecules is realized through modification of the luciferase protein, so that a high-signal-to-noise ratio bioluminescence probe capable of responding to nucleic acid is constructed, the accurate sensing of the nucleic acid molecules is realized, and the problem that the bioluminescence sensing technology is difficult to apply to the nucleic acid molecules is solved.

Description

Modified luciferase mutant protein, bioluminescent probe, probe set, preparation method and detection method
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a modified luciferase mutant protein, a bioluminescent probe, a probe set, a preparation method and a detection method.
Background
MicroRNAs (miRNAs) are small-molecule RNAs with the length of 22-25 nt, participate in important biological processes such as cell differentiation and are key marker molecules of major diseases such as tumors. At present, miRNAs detection methods based on electrochemical, fluorescent, chemiluminescent and other sensing principles have been established. However, the existing method still has the problems of complex operation, easy interference from environmental factors and high cost of detection equipment.
Compared with the traditional detection technology, the bioluminescence sensor has the advantages of high signal-to-noise ratio, low phototoxicity and no interference of an external excitation light source and autofluorescence, so that the bioluminescence sensor has certain application in the field of biomedical analysis. At present, bioluminescence sensing technology has been successfully applied to the detection of biomarkers such as proteins and cells, but the application of bioluminescence sensing technology in the detection of nucleic acid markers such as microRNAs is still very limited. The main reasons for the above problems are: bioluminescent luciferases are unable to respond directly to changes in nucleic acid content and structure in real time and accurately. The above problems restrict the use of bioluminescence in the detection of nucleic acid markers.
Disclosure of Invention
In view of the above, the present invention aims to provide an engineered luciferase mutant protein, a bioluminescent probe, a probe set, a preparation method and a detection method. The modified luciferase mutant protein provided by the invention can be prepared into a bioluminescent probe based on DNA strand displacement, and can realize accurate sensing of nucleic acid molecules, so that a detection technology with high sensitivity and high accuracy for miRNAs is established.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides an engineered luciferase mutant protein, the amino acid sequence of which is shown in SEQ ID No. 1.
The present invention provides a method for preparing an engineered luciferase mutant protein described in the above technical scheme, comprising the steps of: mutating the 166 th cysteine of the luciferase into serine, and adding a connecting region containing a cysteine position at the C-terminal of the protein to obtain DNA encoding the modified luciferase mutant protein; inserting DNA encoding the modified luciferase mutant protein into a plasmid vector to obtain a recombinant plasmid; transferring the obtained recombinant plasmid into escherichia coli to obtain recombinant escherichia coli; culturing the recombinant escherichia coli to obtain the modified luciferase mutant protein.
Preferably, the plasmid vector comprises one of pET-25 (b +), pET-26 (b +), and pET-30 (b +).
Preferably, the E.coli comprises one of BL21 (DE 3), BL21 (DE 3) pLyS, rosetta (DE 3), and Origima (DE 3).
The invention provides a bioluminescent probe for detecting nucleic acid molecules, wherein raw materials for preparing the bioluminescent probe comprise the modified luciferase mutant protein and the double-stranded DNA molecule in the technical scheme;
the double-stranded DNA molecule comprises a first strand and a second strand, wherein the nucleotide sequence of the first strand is shown as SEQ ID NO.3, and the nucleotide sequence of the second strand is shown as SEQ ID NO. 4; the 5 'end of the first chain is modified with an amino group, and the 3' end of the second chain is modified with a luminescence quenching group Dabcyl.
The invention provides a preparation method of the bioluminescent probe in the technical scheme, which comprises the following steps: covalently coupling the modified luciferase mutant protein in the technical scheme with a double-stranded DNA molecule to obtain the bioluminescent probe.
Preferably, the covalently coupled cross-linking agent comprises SMCC.
The invention provides a probe group for detecting nucleic acid molecules, which comprises the bioluminescent probe and the single-stranded DNA probe in the technical scheme; the single-stranded DNA probe comprises a sequence for triggering bioluminescence response and a sequence for identifying a target nucleic acid molecule, wherein the nucleotide sequence of the sequence for triggering bioluminescence response is shown as SEQ ID NO.5.
The invention provides a detection kit for detecting nucleic acid molecules, which comprises the probe set in the technical scheme.
The invention provides a detection method for detecting nucleic acid molecules, which comprises the steps of mixing target nucleic acid molecules and a single-stranded DNA probe for amplification to obtain an amplification product; co-incubating the amplification product with the bioluminescent probe in the technical scheme to obtain an incubated product; adding a luciferase substrate into the obtained incubated product, and completing bioluminescence detection by an enzyme-labeling instrument to realize quantitative analysis of target nucleic acid molecules.
Has the advantages that:
the invention provides an improved luciferase mutant protein, and the amino acid sequence of the improved luciferase mutant protein is shown as SEQ ID NO. 1. The modified luciferase mutant protein provided by the invention is obtained by mutating cysteine at 166 th site of the luciferase protein into serine and adding cysteine at C-terminal of luciferase, and the controllable covalent coupling of the modified luciferase protein and DNA molecules is realized by modifying the luciferase protein, so that a high signal-to-noise ratio bioluminescence probe capable of responding to nucleic acid is constructed, the accurate sensing of the nucleic acid molecules is realized, and the problem that a bioluminescence sensing technology is difficult to apply to the nucleic acid molecules is solved. The results of the embodiments show that the bioluminescent probe constructed by the modified luciferase mutant protein provided by the invention can realize the quantitative detection of the tumor marker miR-21, and the detection limit can reach 12.7fM.
Drawings
FIG. 1 is a schematic structural diagram of a bioluminescent probe provided by the present invention;
FIG. 2 shows the results of the validation of the feasibility and effectiveness of the bioluminescent probe of the present invention;
FIG. 3 shows the detection result of the nucleic acid molecule miR-21 of the invention.
Detailed Description
The invention provides an engineered luciferase mutant protein, the amino acid sequence of which is shown in SEQ ID No. 1. The modified luciferase mutant protein provided by the invention is obtained by mutating cysteine at 166 th site of the luciferase protein into serine and adding cysteine at C-terminal of luciferase, and the controllable and equal-proportion covalent coupling of the modified luciferase protein and DNA molecules is realized through modification of the luciferase protein, so that a high-signal-to-noise ratio bioluminescent probe capable of responding to nucleic acid is constructed, the accurate sensing of the nucleic acid molecules is realized, and the problem that the bioluminescent sensing technology is difficult to apply to the nucleic acid molecules is solved.
The present invention provides a method for preparing an engineered luciferase mutant protein described in the above technical scheme, comprising the steps of: mutating the 166 th cysteine of the luciferase into serine, and adding a connecting region containing the cysteine position at the C-terminal end of the luciferase to obtain a DNA for coding the modified luciferase mutant protein, wherein the nucleotide sequence of the DNA for coding the modified luciferase mutant protein is shown as SEQ ID NO. 2; inserting DNA encoding the modified luciferase mutant protein into a plasmid vector to obtain a recombinant plasmid; transferring the obtained recombinant plasmid into escherichia coli to obtain recombinant escherichia coli; and culturing the recombinant escherichia coli to obtain the modified luciferase mutant protein.
The invention mutates the 166 th cysteine of luciferase into serine, and adds a connecting region containing the cysteine position at the C-terminal of luciferase to obtain DNA for coding modified luciferase mutant protein. In the present invention, the method of mutation is preferably performed using an overlap PCR technique. The addition of a linking region comprising a cysteine site at the C-terminus of luciferase enables controlled, proportional covalent coupling of the linking region to DNA molecules. The nucleotide sequence of the DNA for coding the modified luciferase mutant protein is shown in SEQ ID NO. 2.
After obtaining the DNA encoding the engineered luciferase mutant protein, the present invention inserts the DNA encoding the engineered luciferase mutant protein into a plasmid vector to obtain a recombinant plasmid. In the present invention, the plasmid vector preferably includes one of pET-25 (b +), pET-26 (b +), and pET-30 (b +), and more preferably pET-25 (b +).
After the recombinant plasmid is obtained, the obtained recombinant plasmid is transferred into escherichia coli to obtain the recombinant escherichia coli. In the present invention, the E.coli preferably includes one of BL21 (DE 3), BL21 (DE 3) pLyS, rosetta (DE 3), and Origima (DE 3); further preferred is BL21 (DE 3).
After obtaining the recombinant escherichia coli, the recombinant escherichia coli is cultured to obtain the modified luciferase mutant protein. In the present invention, the culture medium preferably includes an LB medium including the following components in the following concentrations: 2.5-5 g/L yeast extract, 5-10 g/L peptone and 5-10 g/L NaCl, and more preferably 5g/L yeast extract, 10g/L peptone and 10g/L NaCl. In the present invention, the temperature of the culture is preferably 28 to 37 ℃, and more preferably 30 ℃. In the present invention, the time for the culture is preferably 8 to 12 hours, and more preferably 12 hours. The invention preferably adds IPTG with the final concentration of 0.1-0.6 mM in the culture process to induce the generation of the modified luciferase mutant protein. After culturing, the present invention preferably breaks and purifies the culture to obtain the modified luciferase mutant protein. The invention preferably adopts an ultrasonic crushing method to crush the culture; the crushing condition is preferably 50-100W, the ultrasonic crushing is carried out for 2-5 min at the frequency of 1s and 1s for each time, and the crushing is repeated for 5-8 times; more preferably, the power is 100W, and the disruption is repeated 5 times for 2min each time at a frequency of 1s of ultrasound and 1s stop. In the present invention, the purification is preferably performed using a nickel affinity column. After purification, the engineered luciferase mutant protein is obtained.
The invention provides a bioluminescent probe for detecting nucleic acid molecules, wherein raw materials for preparing the bioluminescent probe comprise the modified luciferase mutant protein and the double-stranded DNA molecule in the technical scheme; the double-stranded DNA molecule comprises a first strand and a second strand, wherein the nucleotide sequence of the first strand is shown as SEQ ID NO.3, and the nucleotide sequence of the second strand is shown as SEQ ID NO. 4; the 5 'end of the first chain is modified with an amino group, and the 3' end of the second chain is modified with a luminescence quenching group Dabcyl. In the invention, the amino group modified at the 5' end of the first chain can be coupled with cysteine at the C-terminal end of the modified luciferase mutant protein through an amide bond, and double-stranded DNA is connected with the modified luciferase mutant protein, so that controllable and equal-proportion covalent coupling of the modified luciferase mutant protein and DNA molecules can be realized, and the condition that one modified luciferase mutant protein is connected with a plurality of DNA molecules or one DNA molecule is connected with a plurality of modified luciferase mutant proteins can be prevented, thus leading to poor signal-to-noise ratio and result reproducibility of a probe. In the present invention, the 3' -end of the second strand is modified with a luminescence quenching group Dabcyl, so that luminescence of the modified luciferase mutant protein can be quenched. In the invention, after the first strand and the second strand are complementarily paired, the first strand extends out of a single strand of 8nt at the 3 'end and can identify a specific single-strand DNA probe of a target nucleic acid molecule, so that a strand displacement matrix is triggered, the second strand modified with a luminescence quenching group Dabcyl at the 3' end falls off, and finally a specific bioluminescent signal is generated to realize the quantitative detection of the target nucleic acid molecule.
The invention provides a preparation method of the bioluminescent probe in the technical scheme, which comprises the following steps: covalently coupling the modified luciferase mutant protein in the technical scheme with a double-stranded DNA molecule to obtain the bioluminescent probe. In the present invention, the covalent coupling step comprises the steps of: functionalizing the double-stranded DNA with a crosslinking agent; reduction of an engineered luciferase mutant protein; covalently coupling the reduced engineered luciferase mutant protein to a functionalized double stranded DNA molecule. In the present invention, the crosslinking agent comprises SMCC. In the present invention, the engineered luciferase mutant protein reduction is preferably achieved using TECP.
The invention provides a probe group for detecting nucleic acid molecules, which comprises the bioluminescent probe and the single-stranded DNA probe in the technical scheme; the single-stranded DNA probe comprises a sequence for triggering bioluminescence response and a sequence for identifying a target nucleic acid molecule, wherein the nucleotide sequence of the sequence for triggering bioluminescence response is shown as SEQ ID NO.5. In the invention, the sequence for identifying the target nucleic acid molecule can realize amplification of the target nucleic acid molecule, so that a bioluminescent signal is improved, and the sensitivity is improved. The sequence triggering bioluminescence response can enable the amplification product of the target nucleic acid molecule to identify the 8nt single strand extended from the bioluminescence probe, so that strand displacement reaction is triggered, the second strand modified with a luminescence quenching group Dabcyl at the 3' end is separated, a specific bioluminescence signal is finally generated, and quantitative detection of the target nucleic acid molecule is realized.
The invention provides a detection kit for detecting nucleic acid molecules, which comprises the probe set in the technical scheme. The detection kit provided by the invention can realize quantitative detection of nucleic acid molecules. In the invention, the nucleic acid molecule is preferably miRNA, which solves the difficult problem that the bioluminescence sensing technology is difficult to be applied to nucleic acid molecules.
The invention provides a detection method for detecting nucleic acid molecules, which comprises the steps of mixing target nucleic acid molecules with a single-stranded DNA probe for rolling circle amplification to obtain an amplification product; co-incubating the amplification product with the bioluminescent probe in the technical scheme to obtain an incubated product; and adding a luciferase substrate into the incubated product, and completing bioluminescence detection by using an enzyme labeling instrument to realize quantitative analysis of the target nucleic acid molecules. The detection method provided by the invention can realize the quantitative detection of nucleic acid molecules, particularly miRNA molecules, has simple operation steps, high sensitivity and strong specificity, and solves the problem that the bioluminescence sensing technology is difficult to apply to the nucleic acid molecules.
To further illustrate the present invention, the following examples are provided to describe in detail the engineered luciferase mutant protein, bioluminescent probe, probe set, preparation method and detection method provided by the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparation of engineered luciferase mutant proteins
(1) Construction of engineered luciferase mutant expression vectors: respectively amplifying N-terminal and C-terminal coding sequences of luciferase by PCR, mutating cysteine codon to serine codon at a 166 site of the luciferase, and simultaneously adding a Linker coding sequence containing cysteine behind the C-terminal coding sequence; recovering fragments generated by PCR amplification through gel electrophoresis, amplifying the complete coding sequence of the luciferase mutant through overlapping PCR, and respectively adding NdeI and HindIII enzyme cutting sites at the 5 'end and the 3' end; the fragment is subjected to electrophoretic recovery and enzyme digestion, then is connected with a pET-25 (b +) vector subjected to enzyme digestion by NdeI and HindIII, and the correctness of cloning is verified by sequencing; the primers used in PCR are shown in SEQ ID NO. 6-SEQ ID NO. 8.
(2) Expression and purification of engineered luciferase mutants: transferring the expression vector with correct sequencing into an escherichia coli BL21 (DE 3) strain; picking the obtained transformant to a liquid LB culture medium containing 50 mu g/mL ampicillin, and carrying out shake culture overnight at 30 ℃ and 200 rpm; sucking 50 μ L of overnight culture into fresh 50mL of liquid LB medium, adding ampicillin with a final concentration of 100 μ g/mL, and shake-culturing at 28 deg.C and 150rpm for 2h; adding IPTG with the final concentration of 0.5mM into the bacterial liquid, and continuing shaking culture at 28 ℃ and 150rpm for overnight; the resulting overnight cultures were harvested by centrifugation at 10,000rpm for 5min and resuspended in 10mL PBS solution (pH = 7.0) and disrupted on a sonicator using the conditions: the power is 100W, the crushing is carried out for 2min at the frequency of 1s of ultrasound and 1s stop each time, and the process is repeated for 5 times; centrifuging at 12,000rpm for 20min to obtain lysate supernatant, adding to the top of the nickel affinity column, after the solution flows out, washing the nickel column with 100mL of washing buffer (1 × PBS,40mM imidazole, pH = 7.0), and finally eluting the target protein with 5mL of elution buffer (1 × PBS,500mM imidazole, pH = 7.0); the resulting protein solution was dialyzed against PBS buffer for 4 hours and stored in a refrigerator at-80 ℃.
Example 2
Construction of bioluminescent probes for detection of nucleic acid molecules
The structure of the bioluminescent probe for detecting nucleic acid molecules provided by the invention is shown in figure 1, and the construction method is as follows:
(1) Coupling of double-stranded DNA:
a) Functionalization of DNA by SMCC: incubating 1mM of a first strand single-stranded DNA with amino group modification at the 5 'end and 1.2mM of a second strand single-stranded DNA with Dabcyl group modification at the 3' end in a PBS solution for 1h, wherein the nucleotide sequence of the first strand is shown as SEQ ID NO.2, and the nucleotide sequence of the second strand is shown as SEQ ID NO. 3; adding SMCC with the final concentration of 20mM into the solution, reversing the solution, uniformly mixing, and incubating at 25 ℃ for 2h; further 1/10 volume of 3M NaAc (pH = 5.3) was added, and 1-fold volume of isopropanol was added, and incubated at room temperature for 30min; the resulting double-stranded DNA was precipitated by centrifugation at 20,000rpm for 20min and washed 3 times with 1mL of 75% (v/v) ethanol; drying the washed double-stranded DNA precipitate at 50 ℃ for 15min, finally resuspending the double-stranded DNA precipitate by PBS, and adjusting the concentration of the double-stranded DNA to 100 mu M;
b) Reduction of engineered luciferase mutants: incubating 10 μ M of the engineered luciferase mutant protein with 1mM TECP in PBS (pH = 7.0) for 1h, placing the resulting solution in a 3kDa ultrafiltration tube, centrifuging at 3000rpm for 10min, and repeating the above centrifugation step 3 times;
c) Mixing the reduced modified luciferase mutant protein with SMCC functionalized DNA according to a molar ratio of 1:4, and incubating for 2h at room temperature; resuspending 1mg of nickel-compatible magnetic beads using the above solution, incubating at room temperature for 30min, separating on a magnetic rack, and discarding the supernatant; the resulting magnetic bead pellet was washed 8 times with wash buffer (1 × PBS,20mM imidazole, pH = 7.0) and then resuspended with 1mL of elution buffer (1 × PBS,500mM imidazole, pH = 7.0); the resulting solution was desalted by dialysis, loaded onto the top of a centrifugal anion exchange column, centrifuged at 2000rpm for 2min, rinsed 5 times with an ion exchange rinsing buffer (20 mM Tris-HCl,200mM NaCl, pH = 7.0), and finally eluted with an ion exchange elution buffer ((20 mM Tris-HCl,1MNaCl, pH = 7.0)), and desalted by dialysis and stored in a refrigerator at-80 ℃.
And (3) verifying the feasibility and effectiveness of the bioluminescence probe: adding 10nM of the purified bioluminescent probe and 5nM single-stranded DNA target into 50. Mu.L of reaction buffer, wherein the nucleic acid sequence of the single-stranded DNA target is shown in SEQ ID NO.9, the concentration of each component of the reaction buffer is 50mM Tris-HCl,150mM NaCl,0.2mM TCEP,0.01mg/mL BSA, and the pH of the reaction buffer is =7.0, and incubating for 5min at room temperature; to the above solution, 50. Mu.L of luciferase substrate buffer was added, and analysis was performed using a fluorescence spectrometer. The results are shown in FIG. 2. From the results of fig. 2, it is understood that the positive sample with the single-stranded DNA target added produced a significant increase in bioluminescence compared to the negative control without the single-stranded DNA target added.
Example 3
Method for detecting miRNAs based on bioluminescent probe of the present invention
(1) Probe construction for target amplification of miRNAs: designing a single-stranded DNA probe for amplification according to the sequence of the target miRNAs, and adding a phosphate group at the 5' end of the single-stranded DNA probe; in the embodiment, miR-21 is used as a target, a specific single-stranded DNA probe is designed, and the nucleotide sequence of the probe is shown in SEQ ID NO. 10; the single-stranded DNA probe consists of a sequence for triggering bioluminescence response and a sequence for identifying a target nucleic acid molecule, wherein the nucleotide sequence of the sequence for triggering bioluminescence response is shown as SEQ ID NO.5.
(2) Amplification of miRNAs targets: mixing miR-21 with the 1nM single-stranded DNA probe, 1mM dithiothreitol, 1U RNase inhibitor and 5U T4 DNA ligase, and reacting at 16 ℃ for 1h; further, 1mM dNTPs and 0.5 Upihi-29 DNA polymerase were added thereto, and the reaction was carried out at 30 ℃ for 1 hour to complete the amplification reaction.
(3) And (3) bioluminescence detection: mu.L of the amplification product was mixed with 100nM of the bioluminescent probe of example 2 and incubated for 5min at room temperature in assay reaction buffer with 50mM Tris-HCl,150mM NaCl,0.2mM TCEP,0.01mg/mL BSA, pH =7.0; after 50. Mu.L of luciferase substrate buffer was added, the assay was performed on a spectrometer or microplate reader, and the results are shown in FIG. 3, with the detection limits: 12.7fM.
The results of the above examples show that the invention realizes the controllable covalent coupling of luciferase protein and DNA molecules by modifying the luciferase protein, thereby constructing a bioluminescent probe with high signal-to-noise ratio and capable of responding to nucleic acid, realizing the accurate sensing of nucleic acid molecules, and solving the problem that the bioluminescent sensing technology is difficult to be applied to nucleic acid molecules.
Although the present invention has been described in detail with reference to the above embodiments, it is to be understood that the present invention is not limited to the details of the embodiments, and that various other embodiments may be devised without departing from the spirit and scope of the present invention.
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cagtgtgg 8
<210> 6
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
accagtttcg catatggttt tcaccctgga agacttcg 38
<210> 7
<211> 53
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcaaccctgg aagtacaggt tttcagccag gatacgttca gacagacgcc aac 53
<210> 8
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gactgaaagc ttgcaaccct ggaagtacag gttttc 36
<210> 9
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccacactgcg tacctctcct gtctctcttc tcacc 35
<210> 10
<211> 57
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctgataagct aggtgagaag agagacagga gaggtacgca gtgtggtcaa catcagt 57

Claims (10)

1. An engineered luciferase mutant protein, wherein the engineered luciferase mutant protein
The amino acid sequence of white is shown in SEQ ID NO. 1.
2. The method of making an engineered luciferase mutant protein of claim 1, comprising the steps of: mutating the 166 th cysteine of the luciferase into serine, and adding a connecting region containing the cysteine position at the C-terminal end of the luciferase to obtain a DNA for coding the modified luciferase mutant protein, wherein the nucleotide sequence of the DNA for coding the modified luciferase mutant protein is shown as SEQ ID NO. 2; inserting DNA encoding the modified luciferase mutant protein into a plasmid vector to obtain a recombinant plasmid; transferring the obtained recombinant plasmid into escherichia coli to obtain recombinant escherichia coli; culturing the recombinant escherichia coli to obtain the modified luciferase mutant protein.
3. The method according to claim 2, wherein the plasmid vector comprises one of pET-25b (+), pET-26b (+), and pET-30b (+).
4. The method according to claim 2, wherein the Escherichia coli comprises BL21 (DE 3), BL21
(DE 3) pLysS, rosetta (DE 3) and Origami (DE 3).
5. A bioluminescent probe for detecting a nucleic acid molecule, which is a raw material for producing the bioluminescent probe
Comprising the engineered luciferase mutant protein of claim 1 and a double stranded DNA molecule;
the double-stranded DNA molecule comprises a first strand and a second strand, wherein the nucleotide sequence of the first strand is shown in SEQ ID
The nucleotide sequence of the second chain is shown as SEQ ID NO. 4; the 5 'end of the first chain is modified with an amino group, and the 3' end of the second chain is modified with a luminescence quenching group Dabcyl.
6. The method for preparing a bioluminescent probe according to claim 5, comprising the steps of: covalently coupling the engineered luciferase mutant protein of claim 1 to a double stranded DNA molecule to yield the bioluminescent probe.
7. The method of claim 6, wherein the covalently coupled cross-linking agent comprises SMCC.
8. A set of probes for detecting a nucleic acid molecule, comprising the bioluminescent probe of claim 5 and a single-stranded DNA probe; the single-stranded DNA probe comprises a sequence for triggering bioluminescence response and a sequence for identifying a target nucleic acid molecule, wherein the nucleotide sequence of the sequence for triggering bioluminescence response is shown as SEQ ID NO.5.
9. An assay kit for detecting a nucleic acid molecule comprising the probe set of claim 8.
10. A detection method for detecting nucleic acid molecules is characterized in that target nucleic acid molecules and single-stranded DNA probes are mixed and amplified to obtain amplification products; incubating the amplification product with the bioluminescent probe of claim 5 to obtain an incubated product; adding a luciferase substrate into the obtained incubated product, and completing bioluminescence detection by an enzyme-labeling instrument to realize quantitative analysis of target nucleic acid molecules.
CN202110367329.1A 2021-04-06 2021-04-06 Modified luciferase mutant protein, bioluminescent probe, probe set, preparation method and detection method Expired - Fee Related CN113025586B (en)

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