Nucleic acid of Gaussian luciferase, recombinant expression vector and system, recombinant engineering bacteria, mRNA and preparation method, expression method, application and detection product

Abstract

The invention relates to nucleic acid of Gaussian luciferase, a recombinant expression vector and system, recombinant engineering bacteria, mRNA, a preparation method, an expression method, application and a detection product. The embodiment of the invention provides nucleic acid of Gaussian luciferase, the nucleic acid comprises a nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, a recombinant expression vector of the nucleic acid is constructed, and the Gaussian luciferase is efficiently expressed in human HEK293T cells through a transformation or transfection experiment. Therefore, the technical scheme in the disclosure can effectively improve the expression efficiency of Gaussian luciferase.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and biotechnology, and more specifically, to nucleic acids, recombinant expression vectors and systems, recombinant engineered bacteria, mRNAs of Gaussian luciferase, preparation methods, expression methods, applications, and detection products. Background Technology

[0002] Gaussian luciferase (GLuc) is the smallest and brightest known luciferase, derived from the marine copepod Gaussian princeps.

[0003] Gaussian luciferase possesses advantages such as small molecular weight, stable protein structure containing multiple disulfide bonds, and natural secretion into eukaryotic cells without the need for ATP as a cofactor. Therefore, it is used in fields such as the production of biomolecular probes through fusion with nucleic acids or proteins, tumor imaging through antibody fusion, and in vitro monitoring of cell growth, survival, and gene transduction in blood. Furthermore, Gaussian luciferase exhibits a unique luminescent effect, catalyzing the oxidation of coelenterazine and producing a strong blue light emission peak at 480 nm. The luminescence equation for Gaussian luciferase is as follows:

[0004]

[0005] Therefore, Gaussian luciferase plays an important role in bioluminescence systems, and as a reporter gene, it has broad application prospects in fields such as biology, biomedicine, and environmental monitoring.

[0006] HEK293 cells (human embryonic kidney 293 cells) are a cell line derived from human embryonic kidney cell tissue. They are easy to grow and transfect, and are commonly used cell cultures in academic research. However, Gaussian luciferase expression efficiency is low when applied to human HEK293 cells.

[0007] In the prior art, patent CN105779472A describes a method to improve Gaussian luciferase activity by altering the amino acid sequence of the Gaussian luciferase protein. However, in practical applications, the luminescence intensity of Gaussian luciferase still cannot meet market demands, and there is room for improvement in the expression intensity of Gaussian luciferase.

[0008] In summary, there is currently a lack of technical solutions to address the problem of low expression efficiency of Gaussian luciferase. Summary of the Invention

[0009] The purpose of this disclosure is to solve or improve the problems existing in the prior art, and more specifically, to provide nucleic acids, recombinant expression vectors and systems, recombinant engineered bacteria, mRNAs and preparation methods, expression methods, applications and detection products of Gaussian luciferase, with the aim of solving or improving the problem of low expression efficiency of Gaussian luciferase.

[0010] Specifically, the first aspect of this disclosure provides a nucleic acid for Gaussian luciferase, including:

[0011] (1) A nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; or

[0012] (2) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence described in (1) obtained by one or more base substitutions, insertions, deletions, or inversions; or

[0013] (3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence described in (1) or (2).

[0014] The nucleotide sequence of SEQ ID NO:1 is as follows:

[0015] ATGGGCGTGAAGGTGCTGTTCGCCCTGATTTGCATCGCCGTGGCCGAGGCCAAGCCCACAGAGAACAACGAGGACTTCAACATCGTGGCCGTGGCCAGCAATTTCGCCACCACCGACCTGGACGCTGACCGGGGCAAGCTGCCTGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAAATGGAGGCCAACGCCCGGAAAGCCGGCTGCACCAGAGGCTGTCTGATCTGCCTGAGCCACATCAAGTGCACCCCCAAGATGAAGAAGTTCATCCCCGGCCGGTGCCACACCTACGAGGGCGACAAGGAGAGCGCCCAAGGCGGCATCGGGGAGGCTATCGTGGACATCCCAGAGATCCCCGGCTTCAAGGACCTGGAACCTATGGAGCAGTTCATCGCCCAGGTCGACCTGTGTGTGGATTGCACCACAGGCTGCCTGAAGGGCCTGGCCAACGTGCAGTGCAGCGACCTGCTGAAGAAGTGGCTGCCCCAGAGGTGTGCCACCTTCGCATCCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGCGCCGGCGGAGAC。<00000​​​​ATGGGCGTGAAGGTGCTGTTCGCCCTGATTTGCATCGCCGTGGCCGAGGCCAAGCCTACAGAAAACAACGAGGACTTCAACATCGTGGCCGTGGCCAGCAATTTCGCCACCACCGACCTGGACGCTGATCGGGGCAAGCTGCCTGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAAATGGAGGCCAACGCCCGGAAAGCCGGCTGCACCAGAGGCTGTCTGATCTGCCTGTCTCACATCAAGTGCACACCTAAGATGAAGAAGTTCATCCCCGGCAGATGCCACACCTACGAGGGAGACAAGGAGAGCGCCCAGGGCGGCATTGGGGAGGCTATCGTGGACATCCCAGAGATCCCCGGCTTTAAGGATCTGGAACCTATGGAGCAGTTCATCGCCCAGGTCGACCTGTGTGTGGATTGCACCACAGGCTGCCTGAAGGGCCTGGCCAACGTGCAGTGCAGCGACCTGCTGAAGAAATGGCTGCCCCAGAGATGTGCCACCTTCGCATCCAAGATCCAGGGCCAGGTGGACAAGATCAAAGGCGCCGGCGGAGAC。

[0018] The nucleotide sequence of SEQ ID NO:3 is as follows:

[0019] ATGGGCGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCCGAGGCCAAGCCCACCGAGAACAACGAGGACTTCAACATCGTGGCCGTGGCCAGCAACTTCGCCACCACCGACCTGGACGCCGACCGGGGCAAGCTGCCCGGCAAGAAGCTGCCCCTGGAGGTGCTGAAGGAGATGGAGGCCAACGCCCGGAAGGCCGGCTGCACCCGGGGCTGCCTGATCTGCCTGAGCCACATCAAGTGCACCCCCAAGATGAAGAAGTTCATCCCCGGCCGGTGCCACACCTACGAGGGCGACAAGGAGAGCGCCCAGGGCGGCATCGGCGAGGCCATCGTGGACATCCCCGAGATCCCCGGCTTCAAGGACCTGGAGCCCATGGAGCAGTTCATCGCCCAGGTGGACCTGTGCGTGGACTGCACCACCGGCTGCCTGAAGGGCCTGGCCAACGTGCAGTGCAGCGACCTGCTGAAGAAGTGGCTGCCCCAGCGGTGCGCCACCTTCGCCAGCAAGATCCAGGGCCAGGTGGACAAGATCAAGGGCGCCGGCGGCGAC。

[0020] ​​​.

[0022] Given the need for high-quality proteins with proper folding and modification in biomedical and biotechnology research and industrial production, exploring and summarizing potential beneficial rules and patterns reflecting codon usage preferences of highly expressed genes is crucial for improving protein expression levels. However, protein expression is a multi-step process involving regulation at the levels of transcription, mRNA turnover, translation, and post-translational modifications to enable the formation of stable products. Even a single synonymous codon substitution can increase transgenic expression by more than 1000-fold. Therefore, codon optimization lays the foundation for optimal expression of synthetic genes in recombinant hosts.

[0023] When Gaussian luciferase was applied to human HEK293T cells, its expression efficiency was low. The inventors independently developed an artificial codon optimization method. By optimizing the proportion of different codons for the same amino acid, GC content, and sequence repetition in the wild-type Gaussian luciferase DNA sequence, they achieved high expression of the Gaussian luciferase version of the DNA sequence. The expression efficiency after codon optimization was 1.4-16.2 times higher than that of the wild-type sequence.

[0024] The second aspect of this disclosure provides a recombinant expression vector for Gaussian luciferase, including the nucleic acid of Gaussian luciferase and other acceptable elements.

[0025] Preferably, the recombinant expression vector includes the Gaussian luciferase gene and the pmRVac backbone.

[0026] Preferably, the recombinant expression vector is an mRNA expression vector.

[0027] This disclosure describes the construction of an in vitro transcription vector containing a Gaussian luciferase DNA sequence, through which messenger RNA (mRNA) is obtained via in vitro transcription. This mRNA can be used in cell experiments to achieve highly efficient fluorescent expression. With the same amount of mRNA, the mRNA using the artificially codon-optimized sequence described in this invention exhibits higher expression levels in HEK293T cells compared to the wild-type sequence.

[0028] A third aspect of this disclosure provides a recombinant expression system for Gaussian luciferase, comprising:

[0029] Recombinant expression vector for Gaussian luciferase;

[0030] The host is used to transfect and / or transform the recombinant expression vector.

[0031] Preferably, the host includes host bacteria and host cells.

[0032] Preferably, the host bacteria include Escherichia coli.

[0033] Preferably, the host cell includes HEK293T cells.

[0034] HEK293T cells are one of the most commonly used types of HEK293 cells. Compared to other HEK293 cells, HEK293T cells can express the temperature-sensitive SV40 large T antigen, thereby promoting the amplification of vectors containing the SV40 replication origin site, which increases protein expression levels. Therefore, using HEK293T cells as the host cell for the recombinant expression system of Gaussian luciferase can further improve expression efficiency.

[0035] The fourth aspect of this disclosure provides a recombinant engineered bacterium of Gaussian luciferase, comprising a recombinant expression vector of Gaussian luciferase and a host bacterium transforming the recombinant expression vector.

[0036] Preferably, the host bacterium includes Escherichia coli.

[0037] The fifth aspect of this disclosure provides a method for preparing Gaussian luciferase mRNA, including:

[0038] The recombinant expression vector of Gaussian luciferase was processed through linearization, in vitro transcription, capping, and purification steps to obtain the mRNA.

[0039] The sixth aspect of this disclosure provides mRNA obtained by a method for preparing Gaussian luciferase mRNA.

[0040] Gaussian luciferase mRNA expression vectors can translate into the protein Gaussian luciferase within cells, which reacts with its substrate coelenterate to emit fluorescence. The intensity of the chemiluminescence can be detected using a microplate chemiluminescence assay and converted into a numerical value. Based on the numerical value, the expression levels of Gaussian luciferase mRNA expression vectors with different DNA sequences can be compared within cells, i.e., the strength of the expression effect.

[0041] This method for preparing Gaussian luciferase mRNA can achieve sufficiently strong luminescence intensity and obtain good experimental results.

[0042] The seventh aspect of this disclosure provides a method for Gaussian luciferase expression, comprising the following steps:

[0043] The recombinant expression vector of Gaussian luciferase was prepared into mRNA;

[0044] The mRNA was transfected into host cells to induce the expression of the Gaussian luciferase.

[0045] The eighth aspect of this disclosure provides the use of the above-mentioned nucleic acid, the above-mentioned recombinant expression vector, the above-mentioned recombinant expression system, the above-mentioned recombinant engineered bacteria and / or the above-mentioned mRNA in the preparation of Gaussian luciferase detection products.

[0046] The ninth aspect of this disclosure provides a Gaussian luciferase detection product, comprising the aforementioned nucleic acid, the aforementioned recombinant expression vector, the aforementioned recombinant expression system and / or the aforementioned mRNA, as well as acceptable adjuvants, vectors or devices.

[0047] Through the above-mentioned solutions, this disclosure has at least the following advantages and benefits: the mRNA expression vector constructed from the Gaussian luciferase nucleic acid in this invention has a higher expression efficiency in HEK293T cells than the mRNA expression vector constructed from the wild-type Gaussian luciferase DNA sequence. Attached Figure Description

[0048] The above and other features, advantages, and aspects of the embodiments of this disclosure will become more apparent from the accompanying drawings and the following detailed description. In the drawings, the same or similar reference numerals denote the same or similar elements, wherein:

[0049] Figure 1 This is a schematic diagram of the mRNA expression vector provided in the embodiments of this disclosure;

[0050] Figure 2 This is a schematic diagram of the most commonly used cryptographic subsequences involved in this disclosure;

[0051] Figure 3 This is a graph showing the GC content percentage analysis of the DNA sequence in Example 3 of this disclosure;

[0052] Figure 4 This is a sequence duplication analysis diagram of the DNA sequence involved in Example 3 of this disclosure;

[0053] Figure 5 This is a schematic diagram of the RNA secondary structure of the DNA sequence involved in Example 3 of this disclosure;

[0054] Figure 6 This is a schematic diagram of the standard genetic codons involved in this disclosure;

[0055] Figure 7 This is a graph showing the GC content percentage analysis of the DNA sequence involved in Example 1 of this disclosure;

[0056] Figure 8 This is a sequence duplication analysis diagram of the DNA sequence involved in Example 1 of this disclosure;

[0057] Figure 9 This is a schematic diagram of the RNA secondary structure of the DNA sequence involved in Example 1 of this disclosure;

[0058] Figure 10 This is a graph showing the GC content percentage analysis of the DNA sequence in Example 2 of this disclosure;

[0059] Figure 11 This is a sequence duplication analysis diagram of the DNA sequence involved in Example 2 of this disclosure;

[0060] Figure 12 This is a schematic diagram of the RNA secondary structure of the DNA sequence involved in Example 2 of this disclosure;

[0061] Figure 13 This is a graph showing the GC content percentage analysis of the DNA sequence in Example 4 of this disclosure;

[0062] Figure 14 This is a sequence duplication analysis diagram of the DNA sequence involved in Example 4 of this disclosure;

[0063] Figure 15 This is a schematic diagram of the RNA secondary structure of the DNA sequence involved in Example 4 of this disclosure;

[0064] Figure 16 This is a graph showing the expression intensity of four Gaussian luciferase genes involved in this disclosure in HEK293T cells. Detailed Implementation

[0065] This disclosure will be described more fully below with reference to the accompanying drawings, in which various embodiments are illustrated. However, this disclosure may be implemented in many different ways and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be exhaustive and complete, and will fully convey the scope of this disclosure to those skilled in the art.

[0066] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage.

[0067] The terms “including,” “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 unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0068] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0069] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0070] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0071] It should be understood that, in this disclosure, a reporter gene is a molecular biology concept, which refers to a class of genes that are expressed in cells, tissues / organs or individuals under specific conditions, causing them to produce traits that are easily detectable and would not otherwise be produced by the experimental material, and whose expression products can be quantitatively determined.

[0072] Codon optimization is a novel technique that enhances protein expression levels in organisms by increasing the translation efficiency of target genes. It typically involves redesigning genes by avoiding rare codons, utilizing preferred codons, simplifying mRNA secondary structure, optimizing repetitive sequences, eliminating restriction enzyme sites, and adjusting GC content to improve translation efficiency and thus increase protein expression levels.

[0073] Codons exhibit degeneracy, meaning that amino acids can be designated by different synonymous codons. These synonymous codons are used at unequal frequencies in most sequenced genomes. This phenomenon is known as codon usage bias.

[0074] Unless otherwise specified, all terms used herein, including technical and scientific terms, shall have the same meaning as commonly understood by one of ordinary skill in the art. Terms defined in commonly used dictionaries shall be interpreted as having the same meaning as in the relevant technical context, and shall not be construed as having a formal meaning in an idealized or overly formal sense unless expressly defined in the specification.

[0075] In the embodiments disclosed herein, all raw materials and reagents used are commercially available.

[0076] The sequences involved in this disclosure embodiment are as follows:

[0077] Comparative Example 1 (Sequence ID:AY015993.1):

[0078] ATGGGAGTGAAAGTTCTTTTTGCCCTTATTTGTATTGCTGTGGCCGAGGCCAAACCAACTGAAAACAATGAAGATTTCAACATTGTAGCTGTAGCTAGCAACTTTGCTACAACGGATCTCGATGCTGACCGTGGTAAATTGCCCGGAAAAAAATTACCACTTGAGGTACTCAAAGAAATGGAAGCCAATGCTAGGAAAGCTGGCTGCACTAGGGGATGTCTGATATGCCTGTCACACATCAAGTGTACACCCAAAATGAAGAAGTTTATCCCAGGAAGATGCCACACCTATGAAGGAGACAAAGAAAGTGCACAGGGAGGAATAGGAGAGGCTATTGTTGACATTCCTGAAATTCCTGGGTTTAAGGATTTGGAACCCATGGAACAATTCATTGCACAAGTTGACCTATGTGTAGACTGCACAACTGGATGCCTCAAAGGTCTTGCCAATGTGCAATGTTCTGATTTACTCAAGAAATGGCTGCCACAAAGATGTGCAACTTTTGCTAGCAAAATTCAAGGCCAAGTGGACAAAATAAAGGGTGCCGGTGGTGAT (as shown in SEQ ID NO:5);

[0079] Comparative Example 2 (product of other company):

[0080] (as shown in SEQ ID NO:6);

[0081] The present invention will be further illustrated below with reference to the embodiments:

[0082] Obtain the four Gaussian luciferase sequences from Examples 1 to 4.

[0083] 1. The Gaussian luciferase sequence codons in the examples are optimized as follows:

[0084] Wild-type CDS sequences from the genus *Gaussia princeps* (Sequence ID: AY015993.1) were used as experimental material. Four versions of the target gene sequence for Gaussian luciferase were optimized using a self-developed artificial codon optimization method. The DNA sequence optimization method in this invention is as follows.

[0085] (1) Amino acid sequence analysis

[0086] The amino acid sequence of the Gaussian luciferase protein involved in this invention is shown in Table 1.

[0087] Table 1

[0088]

[0089] Amino acid sequence: MGVKVLFALICIAVAEAKPTENNEDFNIVAVASNFATTD LDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMK KFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCV DCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD. (As shown in SEQ ID NO:7)

[0090] (2) Conversion of amino acid sequences into DNA sequences

[0091] The entire amino acid sequence is converted to the most commonly used codon sequence for the species to which it is to be applied, such as... Figure 2 As shown. This most commonly used codon sequence can be obtained from the following URL:

[0092] https: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=9606.

[0093] For example, in the species "Homo sapiens", all "alanine" is replaced with "GCC". This yields the first version of the DNA sequence.

[0094] First version of the DNA sequence: ATGGGCGTGAAGGTGCTGTTCGCCCTGAT (SEQ ID NO:3)

[0095] (3) DNA sequence analysis

[0096] The first version of the DNA sequence was analyzed for GC content percentage. For example... Figure 3 As shown

[0097] Link to GC content percentage analysis:

[0098] https: / / www.vectorbuilder.cn / tool / gc-content-calculator.html

[0099] The first version of the DNA sequence was analyzed for sequence repetition. For example... Figure 4 As shown

[0100] Link to DNA sequence duplication analysis:

[0101] https: / / www.vectorbuilder.cn / tool / sequence-dot-plot.html

[0102] The first version of the DNA sequence was analyzed for RNA secondary structure and free energy. The minimum free energy (MFE) of this RNA sequence was -241.70 kcal / mol. The RNA secondary structure is as follows: Figure 5 As shown.

[0103] Link to RNA secondary structure and free energy analysis:

[0104] http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi

[0105] (4) DNA sequence adjustment

[0106] By combining parameters such as codon bias, GC content, sequence repetition, RNA secondary structure, and RNA free energy, an optimized version of the DNA sequence is obtained.

[0107] The optimization of each parameter is described below.

[0108] 1) Codon bias: After obtaining the first version of the DNA sequence based on the most frequently used codons, the first-most frequently used codons are replaced sequentially with the second, third, and fourth most frequently used codons. The general principle is that the percentages of the first, second, third, and fourth most frequently used codons in the total sequence of the protein should be controlled within the following ranges: 40–100%, 0–50%, 0–25%, and 0–15%, respectively. Figure 6 As shown, the codons corresponding to amino acids are distinguished according to the standard codon table, and then combined with... Figure 2 The codon tables of Homo sapiens were used to obtain codon preferences. In the Homo sapiens species, the proportion of glycine GGC was 40-100%, GGA was 0-50%, GGG was 0-25%, and GGT was 0-15%.

[0109] Standard cipher table link:

[0110] https: / / zh.wikipedia.org / wiki / DNA%E5%AF%86%E7%A0%81%E5%AD%90%E8%A1%A8

[0111] 2) GC content percentage: The GC content percentage should be higher than 0%–15% of the total GC content of the species. For example, the total GC content of *Homo sapiens* is 52.27%, so the optimized DNA sequence should have a total GC content percentage within the range of 52.27%–67.27%. Different species may have different GC contents. For instance, the total GC content of *Macaca fascicularis* is 49.64%, so the optimized DNA sequence should have a total GC content percentage within the range of 49.64%–64.64%. The local GC content of the DNA sequence should be controlled between 30% and 95%.

[0112] 3) Sequence repetition: Optimize the number and length of repetitive sequences as little as possible. For example, replace a codon in a repetitive sequence segment longer than 20 bases with a synonymous codon to reduce the number of bases in the repetitive sequence. For example, the repetitive sequence "ATGGAGGACGCCAA GAACATCAAG" appears 3 times with 24 bases. Two codons at different positions can be mutated to synonymous codons to obtain two other different DNA sequences, "ATGGAAGACGCCAAGAACATCAAG" and "ATGGAGGATGCCAAGAACATCAAG". All three DNA sequences contain the amino acid "MEDAKNIK".

[0113] 4) RNA secondary structure and free energy: Reduce the number of consecutive base-pairing regions to lower the minimum free energy. If both cannot be achieved simultaneously, prioritize reducing the number of consecutive base-pairing regions.

[0114] Based on the above optimization method, embodiments 1, 2, 3, and 4 of the present invention are obtained.

[0115] like Figures 7-9 As shown, the percentage of use of codons 1, 2, 3, and 4 in the total sequence of this protein was controlled within the following ranges: 68-100%, 0-29%, 0-7%, and 0-7%, respectively. The minimum free energy (MFE) of the RNA sequence in Example 1 is -226.96 kcal / mol.

[0116] like Figures 10-12 As shown, the percentage of use of codons 1, 2, 3, and 4 in the total sequence of this protein in Example 2 was controlled within the following ranges: 44–100%, 0–50%, 0–14%, and 0–3%, respectively. The minimum free energy of the RNA sequence in Example 2 is -202.70 kcal / mol.

[0117] like Figures 3-5As shown, the percentage of use of codons 1, 2, 3, and 4 in the total sequence of this protein in Example 3 was controlled within the following ranges: 70-100%, 0-40%, 0-15%, and 0-5%, respectively. The minimum free energy of the RNA sequence in Example 3 was -241.70 kcal / mol.

[0118] like Figures 13-15 As shown, the percentage of use of codons 1, 2, 3, and 4 in the total sequence of this protein in Example 4 was controlled within the following ranges: 44–100%, 0–40%, 0–12%, and 0%, respectively. The minimum free energy of the RNA sequence in Example 4 was -210.10 kcal / mol.

[0119] Using a DNA sequence with four optimized codons as an example, and comparing it with a wild-type DNA sequence and a DNA sequence from a vector obtained from another company, cell experiments were conducted to validate the results. The names are shown in Table 2.

[0120] Table 2 Comparative analysis of Gaussian luciferase protein and DNA sequences

[0121]

[0122] 2. Synthesis of Gaussian luciferase sequence

[0123] The DNA fragment of the target gene is synthesized using chemical synthesis methods. Alternatively, the DNA fragment of the target gene is amplified by PCR based on an existing template.

[0124] Construction of recombinant expression vectors

[0125] The DNA of the Gaussian luciferase gene obtained through codon optimization in Examples 1, 2, 3, and 4, and the DNA sequences of Comparative Examples 1 and 2, were applied to, for example, the pmRVac backbone. The mRNA expression vector contained the Gaussian luciferase gene, for example... pmRVac The backbone and other components are used to construct corresponding mRNA expression vectors through genetic engineering methods.

[0126] Recombinant engineered bacteria

[0127] (1) Bacterial culture: The constructed recombinant expression vector was transformed into Escherichia coli, and the transformed E. coli were grown on LB plates containing agar and kanamycin. Single colonies were picked, inoculated, and cultured overnight in LB liquid medium containing kanamycin.

[0128] (2) Plasmid extraction: Plasmids were extracted using a plasmid extraction kit. The correctness of the base sequence from the T7 promoter to the target region of the plasmid was verified by Sanger sequencing. Glycerol bacteria corresponding to the plasmids whose sequences were verified to be correct by Sanger sequencing were inoculated into LB liquid medium containing kanamycin and cultured overnight. The bacterial culture was preserved with glycerol, and the remaining bacterial culture was used to extract plasmids using an endotoxin-free plasmid extraction kit.

[0129] mRNA preparation

[0130] (1) Plasmid linearization: The aforementioned plasmid is cut with restriction endonuclease to linearize it.

[0131] (2) Purification of linearized plasmids: The linearized plasmids were purified using a DNA purification and recovery kit. After purification, the samples were identified by agarose gel electrophoresis to confirm that the enzyme digestion was complete.

[0132] (3) Transcription: Using linearized DNA as a template, mRNA containing a 5' cap structure is obtained by in vitro transcription to synthesize mRNA and then performing a capping reaction.

[0133] (4) mRNA purification: mRNA was purified using an mRNA purification kit. The concentration of mRNA samples was determined, and the integrity of the mRNA was assessed by gel electrophoresis on denaturing agarose gel.

[0134] It should be understood that the specific experimental parameters and reagent selections in the aforementioned steps of constructing expression vectors, bacterial culture, and mRNA preparation are no different from those in the prior art. Those skilled in the art can make specific selections according to actual needs. Due to space limitations, this disclosure will not elaborate further.

[0135] High-efficiency expression of Gaussian luciferase

[0136] The prepared mRNA was transfected into HEK293T cells using mRNA transfection reagents. 1 μg of mRNA was transfected into each well of a 12-well plate, and the cells were incubated at 37°C with 5% CO2. At four time points post-transfection (6 h, 24 h, 48 h, and 72 h), 20 μL of cell culture supernatant was mixed with 0.5 μL of the substrate coelentin and 50 μL of reaction buffer. The expression product, Gaussian luciferase, reacted with its substrate coelentin to produce an enzymatic reaction, resulting in chemiluminescence. The intensity of the chemiluminescence was measured using a microplate chemiluminescence analyzer and converted into numerical values, indicating the expression efficiency of the product. The intensity of the chemiluminescence reaction corresponds to the expression efficiency of mRNAs optimized with different codons. The experimental results are shown in Table 3. Figure 16 As shown.

[0137] Table 3. Comparison of expression intensity between the examples and comparative examples in cell experiments.

[0138]

[0139] The results showed that Examples 2 and 3 were significantly better than Comparative Example 1 and slightly better than Comparative Example 2. Examples 1 and 4 were slightly better than Comparative Example 1. The mRNA expression vector constructed from the Gaussian luciferase DNA sequence of this invention showed higher expression efficiency in HEK293T cells than the mRNA expression vector constructed from the wild-type Gaussian luciferase DNA sequence.

[0140] Based on the foregoing, those skilled in the art will understand that the technical solutions claimed in this disclosure and their equivalents will be readily apparent. Furthermore, those skilled in the art can make appropriate modifications and alterations to the disclosed technical solutions as needed, and these modifications and improvements are also within the scope of protection of the claims in this disclosure.

Claims

1. The nucleic acid of Gaussian luciferase, characterized in that, include: (1) A nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; or (2) A nucleotide sequence that is functionally identical or similar to the nucleotide sequence described in (1) obtained by one or more base substitutions, insertions, deletions, or inversions; or (3) A nucleotide sequence that has at least 80% homology with the nucleotide sequence described in (1) or (2).

2. A recombinant expression vector for Gaussian luciferase, characterized in that, Includes nucleic acids, vectors, and other acceptable elements as described in claim 1.

3. A recombinant expression system for Gaussian luciferase, characterized in that, include: The recombinant expression vector as described in claim 2; The host is used for transfection and / or transformation of the recombinant expression vector.

4. A recombinant engineered bacterium of Gaussian luciferase, characterized in that, It includes the recombinant expression vector as described in claim 2 and the host bacteria that transform the recombinant expression vector.

5. A method for preparing Gaussian luciferase mRNA, characterized in that, include: The recombinant expression vector as described in claim 2 is processed by steps including linearization, in vitro transcription, capping, and purification to obtain the mRNA.

6. The mRNA obtained by the preparation method according to claim 5.

7. A method for Gaussian luciferase expression, characterized in that, Includes the following steps: The recombinant expression vector as described in claim 2 is prepared into mRNA; The mRNA was transfected into host cells to induce the expression of the Gaussian luciferase.

8. The use of the nucleic acid as described in claim 1, the recombinant expression vector as described in claim 2, the recombinant expression system as described in claim 3, the recombinant engineered bacteria as described in claim 4, and / or the mRNA as described in claim 6 in the preparation of Gaussian luciferase detection products.

9. A Gaussian luciferase assay product, characterized in that, include: The nucleic acid as claimed in claim 1, the recombinant expression vector as claimed in claim 2, the recombinant expression system as claimed in claim 3, and / or the mRNA as claimed in claim 6, as well as acceptable adjuvants, vectors, or devices.

Images