5 'UTR (Untranslated Region) sequence and application thereof in improving mRNA (Messenger Ribonucleic Acid) translation efficiency

Abstract

The invention discloses a 5 'UTR sequence and application thereof in improving mRNA translation efficiency, and belongs to the technical field of biology, the designed 5' UTR sequence has an excellent mRNA expression promoting function, and compared with a common HBA1 gene 5 'UTR, the FLuc mRNA expression level fused with the 5' UTR can be improved to 2-3.5 times.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a 5'UTR sequence and an application thereof in improving mRNA translation efficiency. Background Art

[0002] In recent years, nucleic acid drugs represented by DNA and RNA have gradually become a hot spot in precision medicine and disease treatment. Compared with traditional drugs, nucleic acid drugs have the advantages of abundant candidate targets, simple design, short R&D cycle, strong targeting specificity, high expression efficiency, and long duration of action. Nucleic acid drugs can be roughly divided into two categories: DNA drugs and RNA drugs. Compared with DNA drugs, RNA drugs have lower risks of genomic integration and induced mutations, higher expression efficiency, and no risk of continuous cumulative toxicity, and have broader application prospects in disease prevention and treatment.

[0003] The complete mRNA molecule includes a 5' cap structure, a 5' untranslated region (5'UTR), an antigen coding sequence, a 3' untranslated region (3'UTR) and a polyadenylated tail. In addition to the antigen coding sequence, other structural elements are crucial to the stability and transcription efficiency of mRNA. Among them, 5'UTR is the main site for ribosome assembly during mRNA translation. The main obstacle to the development of UTR sequences is that it is difficult to predict the impact of any UTR sequence, because some of the cis-acting elements can affect multiple molecular processes through interactions with RNA-binding proteins and microRNAs, and the same UTR sequence even has differences in expression in different tissues. As the main determinant of translation efficiency, it is necessary to engineer and optimize 5'UTR. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0005] The present invention provides a 5'-UTR element, which is used to construct an mRNA template and improve the translation efficiency of a coding region in an mRNA transcribed from the mRNA template. The sequence of the 5'-UTR element is shown in SEQ ID NO: 1-9.

[0006] Furthermore, the sequence of the 5'-UTR element is shown in SEQ ID NO: 1, 2, 9.

[0007] The present invention provides a nucleotide construct, which comprises a nucleotide sequence encoding the above-mentioned 5'-UTR element.

[0008] Furthermore, the nucleotide construct further comprises any one or more of an enzyme cutting site and a promoter element.

[0009] In some embodiments, "restriction site" refers to a specific sequence of bases on DNA, which a restriction endonuclease can recognize and cut the DNA sequence into two segments.

[0010] In some embodiments, "promoter element", "promoter" or "promoter sequence" refers to a DNA sequence located at the 5' end (i.e., before) of the protein coding region of a DNA polymer. Most of the promoters known in nature are located before the transcription region. The promoter acts as a switch for gene expression and activates gene expression. If a gene is activated, it is called transcribed, or participates in transcription. Transcription involves the synthesis of mRNA from a gene. Therefore, the promoter acts as a transcriptional regulatory element and also provides a start site for gene transcription into mRNA. The promoter may be derived from a natural gene as a whole, or include different elements derived from different promoters found in nature, or even include synthetic DNA fragments. It is understood by those skilled in the art that different promoters can direct genes to be expressed in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is also recognized that, since the exact boundaries of regulatory sequences have not been fully defined in most cases, some variant DNA fragments may have the same promoter activity. Promoters that cause genes to be expressed in most cell types at most times are generally referred to as "constitutive promoters". New and various types of promoters that can be used in plant cells are constantly being discovered.

[0011] The present invention provides an mRNA construct, wherein the construct has the above-mentioned 5'-UTR element.

[0012] Furthermore, the mRNA construct has the following structure: a 5'-UTR element, an alternative coding region, and a 3'-UTR element are sequentially connected.

[0013] Furthermore, the mRNA construct further comprises one or more of an IRES sequence and a polyA element.

[0014] In some embodiments, an "IRES sequence" refers to an internal ribosome entry site that is capable of causing ribosomes to bind to the interior of an RNA in order to initiate translation.

[0015] In some embodiments, the polyA element refers to a "polyA tail", i.e., a polyA sequence, which includes the structure of the polyA tail region on the mRNA or the structure corresponding to the coding sequence on the DNA template. The addition of the polyA sequence contributes to the stability and transport of the mRNA, prevents its degradation, and plays an important role in the post-transcriptional modification process. The poly (A) sequence can be a continuous chain of pure adenine nucleotides, or it can contain non-adenine nucleotides. In any form, as long as its function is equivalent to that of a traditional poly (A) sequence, that is, it can provide similar biological functions to traditional poly (A) sequences, such as affecting the stability of mRNA, translation efficiency, or ribosome binding, the sequence is identified as a poly (A) sequence. This includes but is not limited to known variants such as the human growth hormone (hGH) poly (A) sequence and the simian virus 40 (SV40) poly (A) sequence, which may differ in nucleotide composition but are functionally identified as equivalent to traditional poly (A) sequences. In the present disclosure, the poly A sequence has a length of 20-500 adenine nucleotides, for example, a length of 25, 50, 100, 150, 175, 200, 300, 400 or 500 adenine nucleotides.

[0016] In some embodiments, the polypeptide or protein encoded by the replaceable coding region is used to prevent and / or treat infectious diseases, rare genetic diseases, neurodegenerative diseases, retinopathy, cancer or tumors.

[0017] The term "infectious disease" in the present invention includes diseases caused by various organisms, such as bacteria, mycoplasmas, protozoa, fungi and viruses (such as HIV, hepatitis viruses, especially HBV or HCV).

[0018] In some embodiments, the cancer or tumor type includes but is not limited to colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, endometrial uterine cancer, ependymoma, epithelioid hemangioendothelioma (EHE), esophageal cancer, Ewing's sarcoma family of tumors, Ewing's sarcoma in the Ewing's tumor family, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, gallbladder cancer, gastric (stomach) Cancer, Gastric Carcinoid, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST), Gestational Trophoblastic Tumor, Brain Stem Glioma, Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer, Hypothalamic and Visual Pathway Glioma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi's Sarcoma, Kidney Cancer (Renal Cell Carcinoma), Laryngeal Cancer, Acute Lymphoblastic Leukemia (also known as Acute Lymphocytic Leukemia), Acute Myeloid Leukemia (also known as Acute Myeloid Leukemia) , chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), leukemia (leukaemia), chronic myeloid leukemia (also known as chronic myeloid leukemia), hairy cell leukemia, lip and oral cancer, liposarcoma, liver cancer (primary), non-small cell lung cancer, small cell lung cancer, lymphoma (AIDS-related), lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma , squamous cell carcinoma of the neck with occult metastatic primary lesions, oral cancer, multiple endocrine neoplasm syndrome, multiple myeloma (bone marrow cancer) in children, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative diseases, chronic myeloid leukemia, myxoma, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer.

[0019] In some embodiments, the neurodegenerative disease includes but is not limited to neurological disorders (e.g., migraine; epilepsy; Alzheimer's disease; Parkinson's disease; brain injury; stroke; cerebrovascular disease (including cerebral arteriosclerosis, cerebral amyloid angiopathy, hereditary cerebral hemorrhage and cerebral hypoxia-ischemia); spinal muscular atrophy; lateral sclerosis; multiple sclerosis; cognitive disorders (including amnesia, senile dementia, HIV-related dementia, Alzheimer's disease-related dementia, Huntington's disease-related dementia, Lewy body dementia, vascular dementia, drug-related dementia, delirium and mild cognitive impairment); cognitive dysfunction associated with Parkinson's disease and depression; mental deficits (including Down syndrome and fragile X syndrome); psychosis (including schizophrenia (e.g., continuous or episodic, paranoid, juvenile, catatonic, poorly differentiated and residual schizophrenia), schizoaffective disorder, schizophreniform psychosis and delusional disorder.

[0020] In some embodiments, the construct or sequence may be modified at the level of the bonds of the nucleic acid (e.g., phosphorothioate, H-phosphate, alkyl phosphate), at the level of the backbone (e.g., α-oligonucleotides) or PNA or 2′-O-alkyl ribose). Each of these modifications may occur in combination, as long as at least one phosphate is present in the nucleic acid. The nucleic acid may be natural or synthetic, an oligonucleotide, a polynucleotide, a nucleic acid fragment, a ribosomal RNA, a messenger RNA, a transfer RNA, a nucleic acid obtained by enzymatic amplification techniques.

[0021] The present invention provides a cell, wherein the cell contains the nucleotide construct as described above.

[0022] The term "cell" as used herein refers to a cell into which a vector containing a polynucleotide sequence encoding an antibody can be introduced for cloning or gene expression. Cells suitable for cloning or expressing the DNA in the vector of the present invention are prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., Serratia marcescans; and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

[0023] In addition to prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for expressing anti-coronavirus N proteins. Saccharomyces cerevisiae or common baker's yeast is the most commonly used among lower eukaryotic host microorganisms. However, a variety of other genera, species and strains are generally available and suitable for use in the present invention, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotoleran and K. marxianus; Yarrowia (EP 402 402); ,226); Pichia pastoris (EP 183 ,070); Candida; Trichoderma reesia (EP 244 ,234); Neurosporacrassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium and Aspergillus hosts, such as A. nidulans and A. niger.

[0024] Vertebrate cells have also attracted great interest, and propagating vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines are monkey kidney CV1 strain transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR; mouse Sertoli cells (TM4); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human liver tumor line (Hep G2).

[0025] The present invention provides a method for producing optimized mRNA for preparing mRNA drugs, the method comprising the steps of: culturing the cells as described above to obtain a cell culture containing a nucleotide construct of a 5'-UTR element; isolating the nucleotide construct from the cell culture, and transcribing the nucleotide construct to obtain optimized mRNA.

[0026] Furthermore, the optimized mRNA is purified and / or modified.

[0027] The present invention provides an optimized mRNA, which is prepared by the method described above, and has the following structure: a 5'-UTR element, a replaceable coding region, and a 3'-UTR element are sequentially connected.

[0028] Furthermore, the optimized mRNA also includes elements and / or modifications that stabilize the mRNA.

[0029] The present invention provides a method for preparing an mRNA pharmaceutical composition, the method comprising: mixing the optimized mRNA as described above with a pharmaceutically acceptable adjuvant to obtain the mRNA pharmaceutical composition.

[0030] The present invention provides a pharmaceutical composition, which comprises the above-mentioned mRNA construct or the above-mentioned optimized mRNA, and a pharmaceutically acceptable adjuvant.

[0031] Pharmaceutically acceptable adjuvants used in the compositions of the present invention may include, but are not limited to, for example, pharmaceutically acceptable liquids, gels or solid carriers, aqueous vehicles (e.g., sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection or Ringer's dextrose and lactate injection), non-aqueous vehicles (e.g., non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil), antimicrobial agents, isotonic agents (e.g., sodium chloride or dextrose), buffers (e.g., phosphate or citrate buffers), antioxidants (e.g., sodium bisulfate), anesthetics (e.g., procaine hydrochloride), suspending / dispersing agents (e.g., sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agents (e.g., EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifiers (e.g., polysorbate 80 (Tween-80)), diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof. Suitable components may include, for example, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants or emulsifiers.

[0032] The term "pharmaceutically acceptable" as used in the present invention refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, within the scope of reasonable medical judgment, and in accordance with a reasonable benefit / risk ratio.

[0033] The present invention provides the use of the above-mentioned 5'-UTR element, wherein the 5'-UTR element is used to construct an mRNA construct, thereby improving the mRNA translation efficiency.

[0034] The present invention provides a method for preparing a polypeptide or protein, the method comprising: replacing the 5'-UTR element of an mRNA transcription template of the polypeptide or protein with the aforementioned 5'-UTR element, translating the replaced mRNA transcription template, and obtaining the polypeptide or protein.

[0035] The term "5'UTR" used in the present invention generally refers to the sequence between the 5' end of the mRNA molecule and the translation start codon, which can recruit the ribosome complex and initiate the translation of the mRNA. 5'UTR includes the 5'UTR region structure on the mRNA or the structure corresponds to the coding sequence on the DNA template. 5'UTR regulates post-transcriptional modification, the formation and stability of the translation initiation complex by interacting with transcription factors, ribosomes and other transcription regulatory proteins. The sequence design and optimization of this region are crucial to improving the efficiency of post-transcriptional modification and protein expression. As used herein, the terms "5'UTR structure", "5'UTR", "5'UTR sequence" and "5'UTR element" are used interchangeably, all of which refer to the 5'UTR element that can enhance the expression of the target gene obtained after a large number of screening by the inventors, and the 5'UTR sequence has a sequence selected from the following group: a nucleic acid sequence shown in any one of SEQ ID NO: 1-9 or its complementary sequence, or a nucleic acid sequence having at least 80% homology with a nucleotide sequence shown in any one of SEQ ID NO: 1-9 or its complementary sequence. Furthermore, the 5'UTR sequence of the present invention has the nucleic acid sequence shown in SEQ ID NO: 1, 2, 9. The 5'UTR element of the present invention can be used for mRNA molecular structure and DNA molecular template design of mRNA therapy, mRNA vaccine and personalized immunotherapy to improve translation efficiency and enhance the expression of target genes.

[0036] The terms "polypeptide", "peptide", and "protein" used in the present invention are used interchangeably in the present invention to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", "amino acid sequence" and "protein" may also include modified forms, including but not limited to glycosylation, lipid linkage, sulfation, γ-carboxylation, hydroxylation and ADP-ribosylation of glutamic acid residues. The polypeptide may be a polypeptide of eukaryotic, prokaryotic or viral origin. In certain embodiments, the polypeptide may be any polypeptide for therapeutic, preventive or diagnostic purposes. For example, the polypeptide may be an antigen, an antibody, a gene editing enzyme such as a CRISPR nuclease, etc. The polypeptide may also be a chimeric antigen receptor, an immunomodulatory protein, a transcription factor, etc. Examples of polypeptides include, but are not limited to: luciferase, red / green fluorescent protein, human erythropoietin, β-galactosidase.

[0037] As used herein, the term "purified" as used herein refers to a polypeptide removed, isolated or separated from its natural environment or from a recombinantly produced source, which is at least 60%, more preferably at least 80% free from other components with which they are naturally associated, such as membranes and microsomes.

[0038] The term "genetic modification" or "modification" used in the present invention refers to any insertion, deletion or mutation of a cell's native nucleotide sequence or nucleic acid sequence.

[0039] Advantages and beneficial effects of the present invention:

[0040] The present invention utilizes the existing 5'UTR sequence optimization design model combined with a specific CDS sequence, optimizes multiple key parameters through model prediction, and thus designs some 5'UTR sequences with higher translation efficiency and better stability. The nucleic acid molecules of the present invention can be used as elements for enhancing RNA expression levels in nucleic acid therapeutic drugs or mRNA vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram of the Fluc mRNA in vitro transcription backbone vector.

[0042] Figure 2 The figure is the result of detecting the expression level of FLuc mRNA containing various 5'UTR in different cells transfected. DETAILED DESCRIPTION

[0043] Example 1, 5'UTR sequence design

[0044] Although 5'UTR does not encode proteins, it regulates the translation initiation of mRNA and is crucial for protein synthesis. Optimizing 5'UTR can improve the translation efficiency of mRNA, increase tumor antigen production, enhance immune response, and at the same time improve the stability of mRNA and prolong the duration of action, which is crucial to improving the efficacy of tumor therapeutic mRNA vaccines. The conventional 5'UTR optimization strategy is to screen from the 5'UTR of highly expressed genes in cells, such as the 5'UTR of the more commonly used α-globin gene HBA1. With the development of computer technology, some algorithm models have been used to predict the translation efficiency and stability of mRNA sequences, and there are even some programs that can generate 5'UTR sequences from scratch that can improve translation efficiency.

[0045] These algorithm models generally optimize the 5'UTR sequence through the following strategies: (1) adding KOZAK sequences that can improve translation initiation efficiency; (2) by predicting and optimizing the minimum free energy (MFE) of the 5'UTR sequence, ensuring that the 5'UTR has a moderately stable secondary structure, thereby improving the stability of mRNA; (3) based on the overall functional optimization concept of mRNA, a joint optimization algorithm is used, while considering the interaction between 5'UTR and CDS. The optimization process includes balancing parameters such as translation initiation efficiency (TIE), codon adaptation index (CAI) and minimum free energy (MFE), and achieving global sequence optimization through multiple rounds of iterations; (4) with the help of artificial intelligence models such as UTR-LM, combined with large-scale training data sets, the sequence characteristics of 5'UTR and CDS (such as GC content, AUG context structure and secondary structure characteristics) are analyzed, and multiple key parameters (such as TIE, CAI and MFE) are optimized through model prediction, thereby generating 5'UTR sequences with higher translation efficiency and better stability.

[0046] Using the above algorithm strategy, our laboratory designed 5'UTR sequences of about 30 bp, 50 bp, 70 bp and 80 bp in length, and screened out some 5'UTR sequences with higher scores. The sequence information of the obtained 5'UTR is shown in Table 1.

[0047] Table 1 Optimized 5'UTR sequence information

[0048]

[0049] Example 2: 5'UTR vector construction and mRNA molecule preparation

[0050] The designed 5'UTR sequence was obtained in full length by gene synthesis (GenScript Biotech Co., Ltd.) and ligated to the pUC57 vector backbone containing the T7 promoter sequence, HBA1 5'UTR sequence, FLuc CDS sequence, hBg 3'UTR sequence and poly(A) sequence, i.e. Figure 1 The pIPMKC4-FLuc backbone in the plasmid was replaced with the original HBA1 5'UTR sequence. The inserted 5'UTR sequence was sequenced and confirmed to be correct. The endotoxin-free plasmid extraction kit (Viglas Biotechnology Co., Ltd.) was used for large-scale extraction and purification, and the concentration was determined using the ultra-micro spectrophotometer Nanodrop. The obtained plasmid was used to prepare mRNA by in vitro co-transcription.

[0051] Take 5µg of the extracted plasmid, use the restriction endonuclease BspQI (NEB) to linearize the plasmid template, add 2µL of the endonuclease, add 10×Reaction Buffer and RNase-free water to prepare a total volume of 50µL, shake well and mix, then centrifuge briefly to allow the liquid to accumulate at the bottom of the test tube, react at 50℃ for 3h, then inactivate at 80℃ for 20min, and the obtained product is further purified and recovered using magnetic beads.

[0052] Prepare DNA magnetic beads (VAHTSⓇDNA Clean Beads) in advance, balance to room temperature, invert or vortex to mix the beads thoroughly, add an equal volume of magnetic beads according to the volume of the enzyme digestion product, use a pipette to mix thoroughly, and then purify the template plasmid according to the magnetic bead recovery steps. After purification, take 1µL of the purified product and use the ultra-micro spectrophotometer Nanodrop to measure the concentration, and then take 50ng of the purified product for agarose gel electrophoresis (1% TAE) to confirm whether the plasmid template is sufficiently linear.

[0053] In this example, T7 RNA polymerase co-transcription kit (Novozyme Biotech Co., Ltd.) was used for in vitro transcription. After adding all components according to the instructions, the mixture was fully pipetted and mixed, and the liquid was collected to the bottom of the tube by brief centrifugation. The mixture was incubated at 37°C for 2h, and then 5µL of DNaseI was added to the reaction product to digest the DNA template, and the reaction was carried out at 37°C for 15min. The mRNA in the transcription product was purified and recovered using RNA magnetic beads (VAHTS RNA Clean Beads). The amount of magnetic beads used was 2 to 4 times that of the co-transcription system. After fully mixing, the operation was carried out according to the instructions for use of the magnetic beads. Finally, an appropriate amount of Nuclease-free water was used for elution. 1µL of the purified RNA was taken and the concentration was detected using an ultra-micro spectrophotometer (RNA mode). 50ng of the purified RNA was taken for denaturing agarose gel electrophoresis (1% TBE + 6% urea) to detect the integrity and purity of the RNA product.

[0054] Example 3: Effects of different 5'UTRs on the expression level of firefly luciferase in HepG2 and DC2.4 cells

[0055] In order to verify the effect of the designed 5'UTR sequence on the expression level of firefly luciferase in different cells, the present invention selected immortalized human liver cancer cell line (HepG2) and mouse bone marrow derived dendritic cell line (DC2.4) for transfection test. Take HepG2 or DC2.4 cells with good growth status, digest and resuspend them, and count them. Take 2×10 6Add 10 cells to a 50 mL centrifuge tube and add RPMI 1640 complete medium (Thermo Fisher Scientific) to make up to 20 mL. Adjust the cell density to 1 × 10 5 After thorough mixing, the cells were evenly distributed into a 96-well white plate (Corning Company), 100 μL / well, and the number of cells plated in each well was 1×10 4 , pat gently to mix, and then place it in a 37℃ incubator for overnight culture (18-24h). The next day, the cells were taken out and Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) was used to add the FLuc mRNA containing different 5'UTRs prepared by the above transcription into the cells at a dose of 0.1μg / well for transfection. The commonly used HBA1 gene 5'UTR (HBA1 group) was used as the control group, and the transfection steps were carried out according to the instructions.

[0056] 24h and 48h after transfection, take out the corresponding well plates for luciferase activity detection. Take out the Bio-LiteLuciferase Assay System kit (Novozyme Biotech Co., Ltd.) in advance and restore it to room temperature. Prepare it according to its instructions. Take out the well plate to be tested, use a pipette to remove the culture medium supernatant, then add 100μL of detection reagent to each well, tap to mix, let it stand for 1-2min to allow the reaction to proceed fully, and then place it on the microplate reader (BioTek, model Synergy H1) for measurement. The test results are as follows: Figure 2 shown.

[0057] from Figure 2 The results show that in HepG2 and DC2.4 cells, the expression levels of FLuc mRNA in most of the fused 5'UTR sequences designed by the present invention were higher than those in the control group, and the best expression effect was denovo80-4, whose expression levels in HepG2 and DC2.4 cells were 2.2 times and 3.5 times that of the control group, respectively. Among these de novo designed 5'UTR sequences, the transfection results did not find a significant correlation between the length of the UTR and the expression effect, indicating that the effect of 5'UTR on mRNA translation efficiency is affected by multiple factors. Figure 2 The significant difference analysis is shown in Table 2.

[0058] Table 2 Figure 2 Results of data difference analysis

Claims

1. A 5'-UTR element, characterized in that The 5'-UTR element is used to construct an mRNA template to improve the translation efficiency of the coding region in the mRNA transcribed from the mRNA template, and the sequence of the 5'-UTR element is shown in SEQ ID NO: 1-9; Preferably, the sequence of the 5'-UTR element is shown in SEQ ID NO: 1, 2, 9.

2. A nucleotide construct, characterized in that The nucleotide construct comprises a nucleotide sequence encoding the 5'-UTR element of claim 1; Preferably, the nucleotide construct further comprises any one or more of an enzyme cleavage site and a promoter element.

3. An mRNA construct, characterized in that The construct has the 5`-UTR element of claim 1; Preferably, the mRNA construct has the following structure: Sequential connection of 5'-UTR element, alternative coding region, and 3'-UTR element.

4. A cell, characterized in that The cell contains the nucleotide construct of claim 2.

5. A method for producing optimized mRNA for preparing mRNA drugs, characterized in that The method comprises the steps of: Cultivating the cell according to claim 4 to obtain a cell culture containing a nucleotide construct of a 5'-UTR element; isolating the nucleotide construct from the cell culture, and transcribing the nucleotide construct to obtain an optimized mRNA; Preferably, the optimized mRNA is purified and / or modified.

6. An optimized mRNA, characterized in that The optimized mRNA is prepared by the method according to claim 5, and the structure of the optimized mRNA is as follows: Sequential connection of 5`-UTR element, alternative coding region, and 3`-UTR element; Preferably, the optimized mRNA further comprises elements and / or modifications that stabilize the mRNA.

7. A method for preparing an mRNA pharmaceutical composition, characterized in that: The method comprises: mixing the optimized mRNA as claimed in claim 6 with a pharmaceutically acceptable adjuvant to obtain the mRNA pharmaceutical composition.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the mRNA construct according to claim 3 or the optimized mRNA according to claim 6, and a pharmaceutically acceptable adjuvant.

9. The use of the 5'-UTR element according to claim 1, characterized in that: The 5'-UTR element is used to construct an mRNA construct, thereby improving the mRNA translation efficiency.

10. A method for preparing a polypeptide or protein, characterized in that: The method comprises: replacing the 5'-UTR element of the mRNA transcription template of the polypeptide or protein with the 5'-UTR element of claim 1, translating the replaced mRNA transcription template, and obtaining the polypeptide or protein.

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