Nucleic acid, mRNA and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to nucleic acid, mRNA and application thereof. The invention provides nucleic acid for coding luciferase. The nucleic acid has a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2. According to the artificial codon optimization method provided by the invention, a plurality of version DNA sequences for highly expressing firefly luciferase are obtained by optimizing the proportion of each codon in a DNA sequence, the GC content range, the sequence repetition condition, the RNA secondary structure, the RNA free energy and the like. Compared with wild-type firefly luciferase protein, the expression quantity of other contrasts in a cell experiment is improved by 2-201 times, the fluorescent brightness is obviously improved in a mouse experiment, and the application value of firefly luciferase serving as a reporter gene is greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and particularly to nucleic acids, mRNAs and their applications. Background Art

[0002] The luciferase gene derived from Photinus pyralis in North America has a very wide range of applications. This gene can encode a luciferase protein of 550 amino acids, which is a 61 kDa monomeric enzyme. Without post-expression modification, it directly has full enzyme activity. Firefly luciferase can catalyze the oxidative decarboxylation of D-Luciferin in the presence of Mg 2+ , ATP, and O2, and simultaneously emit visible light with a wavelength of 550 - 580 nm. Firefly luciferase, like Green Fluorescent Protein (GFP), is widely used as a reporter gene and serves as a molecular marker. After multiple optimizations, GFP has been developed into Enhanced Green Fluorescent Protein (EGFP), which has achieved the highest protein expression efficiency. Therefore, the light intensity is extremely easy to observe, and it has a very wide range of applications at the cellular level. However, since the green fluorescence emitted by EGFP in mice is affected by the spontaneous green fluorescence of mice, the application of EGFP at the animal level requires dissecting mice to remove individual organs for section observation, and the mice cannot be reused.

[0003] The application of firefly luciferase at the cellular level is limited to a certain extent because it requires a series of operation processes, including cell harvesting, cell lysis, and supernatant extraction, to detect the fluorescence intensity. The detected values fluctuate due to the influence of multiple factors. However, since the yellow-green fluorescence emitted by firefly luciferase in mice is not affected by the spontaneous green fluorescence of mice, the application of firefly luciferase at the animal level is very extensive. The expression level and expression site of luciferase can be directly observed through in vivo animal imaging. The mice can still survive, and the degradation rate of luciferase in the mice over time can be tracked. Also, since animal fur blocks a certain amount of light intensity, if firefly luciferase can have a high protein expression level, it can be observed even at a low dose after a certain time in vivo. Therefore, firefly luciferase with a high protein expression level has a large application market. Currently, there is still room for improvement in the expression intensity of firefly luciferase. Summary of the Invention

[0004] In view of this, the present invention provides nucleic acids, mRNAs and their applications. The present invention provides an artificial codon optimization method. By optimizing the proportion of each codon, the GC content range, sequence repetition, RNA secondary structure, RNA free energy, etc. in the DNA sequence, multiple versions of DNA sequences with high expression of firefly luciferase are obtained. The firefly luciferase protein in the examples of the present invention has an expression level increased by 2 to 201 times compared with the wild type and other comparative examples in cell experiments, and has a significantly increased fluorescence brightness in mouse experiments, greatly enhancing the application value of firefly luciferase as a reporter gene. In the present invention, an in vitro transcription vector containing the firefly luciferase gene is constructed, and messenger RNA (mRNA) is obtained by in vitro transcription. Cell experiments are carried out to obtain the fluorescence expression data of firefly luciferase in 293T cells; the mRNA is encapsulated into lipid nanoparticles (LNP), and LNP-mRNA is injected into mice to obtain the fluorescence expression data of firefly luciferase in c57BL / 6J mice.

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

[0006] The present invention provides nucleic acids encoding luciferase, having:

[0007] (1) The nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:2; or

[0008] (2) The nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (1), and having the same or similar function as the nucleotide sequence shown in (1); or

[0009] (3) The nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (1) or (2).

[0010]

[0011] In some embodiments of the present invention, the sequence of SEQ ID NO:2 is: ATGGAGGACG CCAAGAACATCAAGAAGGGGCCCGCCCCTTTCTACCCACTGGAGGACGGCACAGCCGGCGAACAGCTGCACAAGGCAATGAAACGGTACGCCCTGGTGCCCGGCACCATCGCCTTCACCGACGCCCACATCGAGGTGGACATCA

[0012] CCTACGCCGAGTACTTCGAGATGAGCGTGCGCCTGGCCGAGGCCATGA

[0013] AGAGATATGGCCTGAATACCAACCACCGGATCGTGGTGTGCAGCGAGA

[0014] ACAGCCTGCAGTTCTTCATGCCCGTGCTGGGCGCCCTGTTCATCGGCGT

[0015] GGCCGTGGCACCTGCCAACGACATCTACAACGAGCGGGAGCTGCTGAA

[0016] CAGCATGGGCATCAGCCAGCCCACAGTGGTCTTCGTGAGCAAGAAAGG

[0017] CCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCCATCATCCAGAA

[0018] GATCATCATCATGGACAGCAAGACCGACTACCAGGGCTTCCAGAGCAT

[0019] GTACACCTTCGTGACCAGCCACCTGCCCCCTGGCTTCAACGAGTACGAC

[0020] TTCGTGCCCGAGTCCTTCGACCGGGACAAGACCATTGCCCTGATCATGA

[0021] ACAGCTCCGGCAGCACCGGACTGCCTAAGGGCGTGGCTCTGCCCCACC

[0022] GGACCGCCTGCGTGCGGTTCAGCCACGCCCGGGACCCCATCTTCGGCA

[0023] ACCAGATCATCCCCGACACCGCTATTCTGAGCGTGGTGCCTTTTCATCA

[0024] CGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATCTGCGGCTTCAGA

[0025] GTGGTCCTGATGTACCGGTTCGAGGAGGAGCTGTTCCTGCGGAGCCTGC

[0026] AGGACTACAAGATCCAGAGCGCCCTGCTGGTGCCCACCCTGTTCTCCTT

[0027] CTTCGCCAAGTCCACACTGATCGACAAATACGACCTGAGCAACCTGCA

[0028] CGAGATCGCCAGCGGCGGCGCCCCCCTGAGCAAGGAGGTGGGCGAGG

[0029] CCGTGGCCAAGCGGTTCCACCTGCCTGGCATCCGGCAGGGCTATGGGC

[0030] TGACAGAGACCACCAGCGCCATCCTGATCACACCCGAAGGCGATGACA

[0031] AGCCCGGCGCTGTGGGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTGG

[0032] TCGACCTGGACACAGGCAAGACACTCGGCGTGAATCAAAGAGGCGAAC

[0033] TGTGTGTGCGGGGCCCCATGATCATGAGCGGCTACGTGAACAACCCTG

[0034] AGGCCACCAACGCCCTGATCGACAAGGACGGCTGGCTGCACAGCGGCG

[0035] ACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGC

[0036] TGAAGTCCCTGATTAAGTACAAGGGCTACCAGGTGGCTCCCGCCGAGC

[0037] TGGAGAGCATCCTGCTGCAGCACCCCAACATTTTCGATGCCGGCGTGGC

[0038] TGGACTGCCAGACGACGACGCCGGCGAACTGCCCGCCGCCGTGGTGGT

[0039] GCTGGAGCACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTACGT

[0040] GGCCAGCCAGGTGACCACCGCCAAGAAGCTGAGGGGCGGCGTGGTGTT

[0041] CGTGGACGAGGTGCCTAAGGGCCTGACCGGCAAGCTGGACGCCCGGAA

[0042] GATCAGAGAGATCCTGATCAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTGATAATAG。(No.9)

[0043] The present invention also provides a recombinant vector, comprising: the above nucleic acid and an acceptable genetic element.

[0044] The present invention also provides a host, which is transformed and / or transfected with the above recombinant vector.

[0045] The present invention also provides a method for preparing mRNA. After linearizing the above recombinant vector, in vitro transcription, high-temperature denaturation, capping reaction are carried out, then DNA is degraded, and after purification, the mRNA is obtained.

[0046] The present invention also provides the mRNA obtained by the above preparation method.

[0047] The present invention also provides an mRNA liposome, comprising: the above mRNA and lipids.

[0048] The present invention also provides the use of the above nucleic acid, the above recombinant vector, the above host, the above mRNA and / or the above mRNA liposome in in vitro transcription of mRNA.

[0049] The present invention also provides the use of the above nucleic acid, the above recombinant vector, the above host, the above mRNA and / or the above mRNA liposome in the preparation of a luciferase detection product.

[0050] In some embodiments of the present invention, in the above use, the objects of action of the luciferase detection product include: cells and / or mammals.

[0051] In some embodiments of the present invention, in the above use, the cells include: 293T cells.

[0052] In some embodiments of the present invention, in the above use, the mammals include: c57BL / 6J mice.

[0053] The present invention also provides a luciferase detection product, comprising: the above nucleic acid, the above recombinant vector, the above host, the above mRNA and / or the above mRNA liposome, and acceptable auxiliaries, carriers or devices.

[0054] The present invention provides an artificial codon optimization method. By optimizing the proportion of each codon, the GC content range, sequence repetition, RNA secondary structure, RNA free energy, etc. in the DNA sequence, multiple versions of DNA sequences with high expression of firefly luciferase are obtained. The firefly luciferase protein in the examples of the present invention has an expression level increased by 2 to 201 times compared with the wild type and other comparative examples in cell experiments, and there is a significant increase in fluorescence brightness in mouse experiments, greatly enhancing the application value of firefly luciferase as a reporter gene. In the present invention, an in vitro transcription vector containing the firefly luciferase gene is constructed, and messenger RNA (mRNA) is obtained by in vitro transcription, and cell experiments are carried out to obtain the fluorescence expression data of firefly luciferase in 293T cells; the mRNA is encapsulated into lipid nanoparticles (Lipid Nanoparticle, LNP), and LNP-mRNA is injected into mice to obtain the fluorescence expression data of firefly luciferase in c57BL / 6J mice. Description of the Drawings

[0055] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0056] Figure 1 Schematic diagram showing the codon representation of the "Homo sapiens" species;

[0057] Figure 2 Schematic diagram showing the GC content ratio analysis of the first version of the luciferase DNA sequence;

[0058] Figure 3Schematic diagram showing the sequence repeat analysis of the first version of the luciferase DNA sequence;

[0059] Figure 4 Schematic diagram showing the RNA secondary structure of the first version of the luciferase DNA sequence;

[0060] Figure 5 Schematic diagram showing the standard codon;

[0061] Figure 6 Schematic diagram showing the GC content ratio analysis of No.8;

[0062] Figure 7 Schematic diagram showing the sequence repeat analysis of No.8;

[0063] Figure 8 Schematic diagram showing the RNA secondary structure of No.8;

[0064] Figure 9 Schematic diagram showing the GC content ratio analysis of No.9;

[0065] Figure 10 Schematic diagram showing the sequence repeat analysis of No.9;

[0066] Figure 11 Schematic diagram showing the RNA secondary structure of No.9;

[0067] Figure 12 Schematic diagram showing the in vitro transcription vector of the firefly luciferase gene;

[0068] Figure 13 Schematic diagram showing the cell experiment data of the firefly luciferase gene;

[0069] Figure 14 Schematic diagram showing the in vivo imaging results 6 h after injecting LNP-mRNA; among them: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right are: No.1, No.3, No.5, No.8, No.9;

[0070] Figure 15 Schematic diagram showing the in vivo imaging results 24 h after injecting LNP-mRNA; among them: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right are: No.1, No.3, No.5, No.8, No.9;

[0071] Figure 16 Schematic diagram showing the in vivo imaging results 48 h after injecting LNP-mRNA; among them: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right are: No.1, No.3, No.5, No.8, No.9;

[0072] Figure 17Shows the in vivo imaging results 72 hours after injecting LNP-mRNA; among them: the left shows the 10 μg dose group; the right shows the 30 μg dose group, from left to right are: No.1, No.3, No.5, No.8, No.9. Detailed implementation manners

[0073] The present invention discloses nucleic acids, mRNAs and their applications.

[0074] It should be understood that the expression "one or more of..." individually includes each of the objects recited after said expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0075] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unrecited elements or steps, unless specifically stated otherwise or understood from the context.

[0076] It should be understood that the order of steps or the order of performing certain actions is not important as long as the present invention remains operable. In addition, two or more steps or actions can be carried out simultaneously.

[0077] The use of any and all examples or exemplary language such as "for example" or "including" herein is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0078] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0079] The sequences involved in the present invention are as follows:

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] The present invention will be further described below in conjunction with embodiments:

[0087] Example 1 Codon Optimization

[0088] The DNA sequence optimization method in the present invention is as follows.

[0089] (1) Amino acid sequence analysis

[0090] Two versions of the amino acid sequence of North American firefly luciferase are involved in the present invention, as shown in Table 1.

[0091] Table 1

[0092]

[0093] Amino acid sequence 1: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVP GTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL***. (As shown in SEQ ID NO:3)

[0094] Amino acid sequence 2: MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVP GTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV***. (as shown in SEQ ID NO:4)

[0095] (2) Conversion of amino acid sequence to DNA sequence

[0096] Convert all amino acid sequences to the most commonly used codon sequences of the species to be applied. For example, for the "Homosapiens" species, change all "alanine" to "GCC". Obtain the first version of the DNA sequence. As Figure 1 shown.

[0097] Link URL:

[0098] https: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=9606

[0099] https: / / www.kazusa.or.jp / codon /

[0100]

[0101] (3), DNA sequence analysis

[0102] Perform the operation in step (2) with amino acid sequence 2, and the obtained DNA sequence is the first version of the DNA sequence.

[0103] Perform GC content ratio analysis on the DNA sequence of the first version. As Figure 2 shown.

[0104] Link URL: https: / / www.vectorbuilder.cn / tool / gc-content-calculator.html

[0105] Perform sequence repeat analysis on the DNA sequence of the first version.

[0106] Link URL: https: / / www.vectorbuilder.cn / tool / sequence-dot-plot.html

[0107] Perform sequence RNA secondary structure and free energy analysis on the DNA sequence of the first version. The minimum free energy (MFE) of this sequence RNA is -687.5 kcal / mol, and the RNA secondary structure is as Figure 4 shown.

[0108] Link URL: http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi

[0109] (4), DNA sequence adjustment

[0110] Combining parameters such as codon preference, GC content ratio, sequence repeat situation, RNA secondary structure, and RNA free energy, an optimized version of the DNA sequence is obtained.

[0111] The optimization description of each parameter is as follows.

[0112] 1), Codon preference: After obtaining the DNA sequence of the first version according to the most frequently used codons, sequentially replace the codons with the first proportion with the codons with the second, third, and fourth proportions. The general principle is that the proportion of the codons with the first, second, third, and fourth usage frequencies in the total sequence of this protein is controlled within the range of: 40 - 100%, 0 - 50%, 0 - 25%, 0 - 15%. As Figure 5 shown, distinguish the codons corresponding to amino acids according to the standard codon table, and then combine with Figure 1The codon table of the Homo sapiens species was obtained to obtain codon preference. In the "Homo sapiens" species, the proportion of GGC for glycine is 40-100%, the proportion of GGA is 0-50%, the proportion of GGG is 0-25%, and the proportion of GGT is 0-15%.

[0113] Link: https: / / zh.wikipedia.org / wiki / DNA%E5%AF%86%E7%A0%81%E5%AD%90%E8%A1%A8

[0114] 2) GC content ratio: Higher than 0% - 15% of the total GC content ratio of this species. The total GC content of the "Homo sapiens" species is 52.27%, so the total GC content ratio of the optimized DNA sequence should be in the range of "52.27% - 67.27%". For different species, their GC content should be different. For example, the total GC content of the "Macaca fascicularis" species is 49.64%, so the total GC content ratio of the optimized DNA sequence should be in the range of "49.64% - 64.64%". The local GC content of the DNA sequence should be controlled between 30 - 95%.

[0115] 3) Sequence repetition: Optimize the number of repetitive sequences as few as possible in relation to the sequence length. For example, replace a certain codon in a sequence segment with more than 20 base repetitive sequences with a synonymous codon to reduce the number of bases in the repetitive sequence. Example: Before optimization, the repetitive sequence "ATGGAGGACGCCAAG AACATCAAG" with a total of 24 bases appears 3 times. Two codons at different positions can be selected to mutate into synonymous codons, obtaining two different DNA sequences, "ATGGAAGACGCCAAGAACATCAAG" and "ATGGAGGATGCCAAGAACATCAAG". The amino acids of the three DNA sequences are all "MEDAKNIK".

[0116] 4) RNA secondary structure and free energy: Reduce the continuous base pairing region and lower the minimum free energy. If it is not possible to take both into account, prioritize reducing the continuous base pairing region.

[0117] According to the above optimization method, No. 8 and No. 9 in the present invention are obtained.

[0118] Such as Figures 6 - 8As shown, the usage frequency ratio of No.8: the proportion ranges of the first, second, third, and fourth codons in the total sequence of this protein are controlled at: 68 - 100%, 0 - 29%, 0 - 7%, 0 - 7%. The minimum free energy (MFE) of the RNA sequence of No.8 is -639.90 kcal / mol.

[0119] As Figures 9 - 11 shown, the usage frequency ratio of No.9: the proportion ranges of the first, second, third, and fourth codons in the total sequence of this protein are controlled at: 44 - 100%, 0 - 50%, 0 - 14%, 0 - 3%. The minimum free energy of the RNA sequence of No.9 is -655.40 kcal / mol.

[0120] Table 2

[0121]

[0122] Example 4 Vector Construction

[0123] Insert the DNA sequence of North American firefly luciferase PpyLuc into the linearized backbone through the Gibson reaction to obtain an in vitro transcription vector containing the target gene. The DNA sequences of other elements in the comparative example and the example are the same except for the DNA sequence of PpyLuc.

[0124] Synthesize a plasmid containing the target gene fragment, obtain the target gene fragment through PCR amplification, and obtain the purified target gene fragment through a gel recovery kit.

[0125] Digest the in vitro transcription vector pmRVac plasmid with the restriction enzyme BsaI, and obtain the purified linearized pmRVac backbone through a gel recovery kit.

[0126] Connect the purified target gene fragment and the purified linearized pmRVac backbone through the Gibson reaction to obtain a reaction system of the in vitro transcription vector containing the target gene.

[0127] Transfer the Gibson reaction system into VB UltraStable competent cells by the chemical transformation method. Transformation steps: Thaw the competent cells on ice for 30 min, add the Gibson reaction system to the competent cells, incubate on ice for 30 min, heat in a 42°C water bath for 1 min, incubate on ice for 2 min, add LB medium and shake culture at 37°C for 1 h, then take an appropriate amount of bacterial liquid and spread it on an LB solid plate containing kanamycin, and incubate it upside down at 37°C for 16 h.

[0128] Example 5 Clone Identification

[0129] Randomly pick several single colonies on the plate in Example 4 into a small amount of sterile water. Using this bacterial solution as a template, perform bacterial solution PCR reaction with the designed specific forward and reverse primers and PCR enzyme. Verify the bacterial solution PCR results by gel electrophoresis. Inoculate the clones with bacterial solution PCR results meeting the expectations into LB liquid medium containing kanamycin and culture at 37 °C with shaking for 16 h. Take a part of the cultured bacterial solution and store it in glycerol, and extract the plasmid from the remaining bacterial solution using a plasmid extraction kit. Perform enzyme digestion and Sanger sequencing verification on the plasmid.

[0130] The primer sequences are shown in Table 3 and Table 4:

[0131] Table 3

[0132] Serial number Sequencing primer sequence SeqF1 CGCAGCCTACCGTAGTGTTTGTTTCC (as shown in SEQ ID NO:13) SeqF2 CGAAAGAAGTCGGGGAAGCGGTTGC (as shown in SEQ ID NO:14) SeqF3 CGAAAGGTCTTACCGGAAAACTCGACG (as shown in SEQ ID NO:15) SeqF4 CGGGAGCTGCTGAACAGCATGAATAT (as shown in SEQ ID NO:16) SeqF5 CACCCTGATCGACAAGTACGACCTGTCG (as shown in SEQ ID NO:17) SeqF6 AGATCGTCGACTACGTGGCCAG (as shown in SEQ ID NO:18) SeqF7 CTACAACGAGCGCGAGCTGCTGA (as shown in SEQ ID NO:19) SeqF8 AACTTGCACGAGATCGCCAGCG (as shown in SEQ ID NO:20) SeqF9 GGAGATCGTGGACTATGTGGCCAG (as shown in SEQ ID NO:21) SeqF10 ATCTACAACGAGCGGGAGCTGCTGAAC (as shown in SEQ ID NO:22) SeqF11 GAGCGGGAGCTGCTGAACAGCATG (as shown in SEQ ID NO:23) SeqF12 CCTGAGCAAGGAGGTGGGCGAG (as shown in SEQ ID NO:24) SeqF13 GGACGAGGTGCCTAAGGGCC (as shown in SEQ ID NO:25)

[0133] Table 4

[0134]

[0135]

[0136] Plasmid extraction in Example 6

[0137] Inoculate the glycerol bacteria corresponding to the plasmid verified correctly in Example 5 into 100 mL of LB medium containing kanamycin and culture at 37 °C with shaking for 16 h. Extract the plasmid from the cultured bacterial solution using an endotoxin-free plasmid extraction kit. Perform enzyme digestion and Sanger sequencing verification on the extracted plasmid.

[0138] Plasmid linearization in Example 7

[0139] Take the plasmid in Example 6 and perform single enzyme digestion reaction with SapI. Purify the linearized plasmid using a DNA purification and recovery kit, elute and store the linearized DNA template with nuclease-free water, and identify the sample by non-denaturing gel agarose gel electrophoresis.

[0140] mRNA preparation and purification in Example 8

[0141] Using the linearized plasmid in Example 7 as a transcription template, obtain mRNA by in vitro transcription using an mRNA in vitro transcription kit. First, obtain a crude mRNA sample, perform capping reaction after high-temperature denaturation, and then add DNase I to degrade the residual DNA in the system. Purify the mRNA using carboxyl magnetic beads and store it in sodium citrate solution. Identify the concentration and mRNA integrity of the sample.

[0142] Cell experiment in Example 9

[0143] Two groups of experiments were set up to compare the dosages, and the amounts of mRNA were 0.25 μg / well and 0.5 μg / well respectively. The mRNA obtained in Example 8 was added to the 293T cells in a 12-well plate (the amount of 293T cells used was 10 6 / well), cultured at 37 °C, and the samples were processed at the time points of 6 h, 24 h, 48 h, and 72 h. The cells transfected with mRNA were taken out of the incubator, the cell waste liquid was removed in a laminar flow hood, the cell lysate was added, and after reacting at room temperature for 1 min, the cells were gently shaken to make them fall off, and the cell lysate was aspirated into a clean 1.5 mL centrifuge tube and centrifuged at 12000 rpm at 4 °C for 3 min. An equal amount of the supernatant of the centrifuged cell lysate was added to a 96-well plate containing D-Luciferin solution, gently pipetted and mixed evenly, placed in a microplate reader in the dark, and the fluorescence intensity value was read.

[0144] The results showed that the expression level of Luciferase reached the peak at 24 h, and the corresponding chemiluminescence intensities of Samples No. 8 and No. 9 (i.e., Example 2 and Example 3) were relatively high.

[0145] Example 10 Mouse Experiment

[0146] The corresponding mRNAs of No. 1, No. 3, No. 5, No. 8, and No. 9 were selected for LNP encapsulation, and then in vivo experiments on mice were carried out. Female c57BL / 6J mice aged 6 - 8 weeks were selected. The total injection doses were 10 μg and 30 μg (the sum of the doses in both legs). The sample concentrations and injection volumes are as follows in the table. For injection volumes less than 50 μL, the total injection volume was made up to 50 μL with 1x PBS solution.

[0147] Table 5

[0148]

[0149] Before injecting the samples, the mice were marked, grabbed and fixed, and after disinfecting the injection area with 70% alcohol, the samples were injected into the muscle of both hind legs respectively. After injection, the mice were put back into the original cage and raised normally. In vivo imaging was carried out at 6 h, 24 h, 48 h, and 72 h after injection. Before in vivo imaging, a 15 mg / mL D-Luciferin solution (substrate) was prepared with D-PBS, filtered and sterilized with a 0.22 μm filter membrane, and kept in the dark for standby. Before injecting the substrate, each mouse was weighed and recorded. The D-Luciferin solution was intraperitoneally injected into the mice at 150 mg / kg body weight. After injecting the substrate, the mice were placed in an induction anesthesia chamber (isoflurane gas anesthesia) to fully anesthetize the mice. Wait for about 15 minutes after injecting the luciferin substrate, and then carry out in vivo imaging.

[0150] From Figures 14 - 17, it can be seen from the results in Tables 6 to 9 that the in vivo expression effects of Samples No. 8 and No. 9 (i.e., Example 2 and Example 3) are relatively strong.

[0151] Table 6 Chemiluminescence detection data of Firefly Luciferase transfection for 6 h

[0152]

[0153] Table 7 Chemiluminescence detection data of Firefly Luciferase transfection for 24 h

[0154]

[0155] Table 8 Chemiluminescence detection data of Firefly Luciferase transfection for 48 h

[0156]

[0157]

[0158] Table 9 Chemiluminescence detection data of Firefly Luciferase transfection for 72 h

[0159]

[0160]

[0161] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A nucleic acid encoding luciferase, characterized in that, Comprising: (1) a nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:2; or (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence as shown in (1), and having the same or similar function as the nucleotide sequence as shown in (1); or (3) a nucleotide sequence having at least 80% identity with the nucleotide sequence as shown in (1) or (2).

2. Recombinant vector, characterized in that, Including: the nucleic acid as claimed in claim 1 and an acceptable genetic element.

3. Host, characterized in that, transforming and / or transfecting the recombinant vector as claimed in claim 2.

4. A method for preparing mRNA, characterized in that, After linearizing the recombinant vector as claimed in claim 2, performing in vitro transcription, high-temperature denaturation, capping reaction, degrading DNA, and purifying, the mRNA is obtained.

5. The mRNA obtained by the preparation method as claimed in claim 4.

6. mRNA liposome, characterized in that, Including: the mRNA as claimed in claim 5 and lipids.

7. Use of the nucleic acid as claimed in claim 1, the recombinant vector as claimed in claim 2, the host as claimed in claim 3, the mRNA as claimed in claim 5 and / or the mRNA liposome as claimed in claim 6 in in vitro transcription of mRNA.

8. Use of the nucleic acid as claimed in claim 1, the recombinant vector as claimed in claim 2, the host as claimed in claim 3, the mRNA as claimed in claim 5 and / or the mRNA liposome as claimed in claim 6 in the preparation of a luciferase detection product.

9. The application according to claim 8, wherein The detection object of the luciferase detection product includes: cells and / or mammals.

10. Luciferase detection product. It is characterized in that, Including: the nucleic acid as claimed in claim 1, the recombinant vector as claimed in claim 2, the host as claimed in claim 3, the mRNA as claimed in claim 5 and / or the mRNA liposome as claimed in claim 6, and acceptable adjuvants, carriers or devices.

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