5 '-UTR element for improving mRNA translation efficiency

Abstract

The invention discloses a 5 '-UTR element capable of improving the mRNA translation efficiency. The nucleotide sequence of the 5'-UTR element is 5 '-GGCCACCAACGCATTCGTTTTTTCGTT-3'. According to the 5 '-UTR element, the translation efficiency of mRNA is improved by at least 1.5 times through the new and optimized 5'-UTR element, and the 5 '-UTR element can be used as an element for enhancing the mRNA expression efficiency in nucleic acid treatment drugs or mRNA vaccines, so that more and better choices are provided for mRNA therapy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a 5'-UTR element for improving mRNA translation efficiency and its applications. Background Technology

[0002] mRNA technology is a biomedical technology that utilizes in vitro synthesized mRNA, coated with a delivery vector, to be transported into target cells and guide the synthesis of target proteins, thereby achieving prevention and treatment. mRNA technology is characterized by short development cycles, low production costs, and high safety, making it a complement and development of traditional medical technologies.

[0003] The functional efficiency of mRNA molecules is highly correlated with their structural elements. A complete mRNA consists of five parts: a 5' cap, a 5' untranslated region (5'-UTR), a coding sequence (CDS), a 3' untranslated region (3'-UTR), and a poly(A) tail. Among these, the 5'-UTR plays a decisive role in the translation level and half-life of mRNA. Studies have shown that the sequence characteristics, secondary structure, GC content, and presence of an upstream open reading frame (uORF) of the 5'-UTR element directly affect protein expression levels, making it a core regulatory node in mRNA technology.

[0004] The natural 5′UTR primarily serves "fine-tuning" during evolution, which is not well-suited to the high-yield expression requirements of mRNA drugs and vaccines. When the natural 5′UTR is directly applied to industrial or therapeutic scenarios, its shortcomings, such as excessive stability of secondary structures, high length and GC content, dense cryptic origin sites (uAUG / uORF), strong cell type dependence, and uncontrollable coupling with mRNA stability, can lead to uncontrollable protein yield.

[0005] CN116949041A only discloses the extraction of 5′UTR fragments from natural genes such as human HSP70, α-globin, and CYBA, and the improvement of expression through a "trial and error-screening" approach.

[0006] CN108949772A lists dozens of natural 5′UTRs and directly embeds them into expression cassettes. The claims only limit the content to "may contain one or more nucleotide additions / deletions", which is essentially still a simple patchwork of wild-type UTRs.

[0007] JP2020506189A (family WO2014 / 164253) inserts any wild-type UTR or its "variant" into the nucleic acid construct. The mutation method is only to add or subtract a few bases at the end or to make point mutations, which is a typical random mutation library strategy. Summary of the Invention

[0008] The purpose of this invention is to develop new and efficient 5'-UTR elements to improve the translation efficiency of mRNA, specifically providing a 5'-UTR element for improving mRNA translation efficiency.

[0009] The 5'-UTR element for improving mRNA translation efficiency of the present invention has a nucleotide sequence as shown in any of the following:

[0010] 5'-GGCCACCAACGACATTTTCCGTTTCGTT-3' (SEQ ID NO: 1);

[0011] 5'-GGCCACCAACGACATTCTCGTTTTCGTT-3' (SEQ ID NO: 2);

[0012] 5'-GGCCACCAACGACATTTTGCGTTTCGTT-3' (SEQ ID NO: 3);

[0013] 5'-GGCCACCAACGACATTCGTTTTTTCGTT-3' (SEQ ID NO: 4);

[0014] 5'-GGCCACCAACGACATTTTCGTTTTCGTT-3' (SEQ ID NO: 5);

[0015] 5'-GGCCACCAACGACATTGCCCCCCGCCGG-3' (SEQ ID NO: 9).

[0016] Preferably, the 5'-UTR element has the nucleotide sequence 5'-GGCCACCAACGACATTCGTTTTTTCGTT-3'. This preferred 5'-UTR element is an artificially designed 5'-UTR element with short length, low immunogenicity, complete compatibility with 1-methylpseudouridine modification, and high translation efficiency potential (≥2 times higher than the positive control sequence), which is of great significance for the development of mRNA vaccines and drugs. This invention addresses the shortcomings of uncontrollable protein yield caused by the excessively long and complex secondary structure of natural 5'-UTR elements by providing an artificially designed 5'-UTR element. Its key technical features include: a length of 28 nt, a GC content of 46.4%, a stem-loop structure with a minimum free energy of -1.5 kcal / mol, and the inclusion of a Kozak shared sequence between the 5'-UTR element and the coding region; and high ribosome loading capacity. Compared to the 5'-UTR element of mRNA-1273 (ModernaSpikevax), the expression level of EGFP-Flag mRNA fused with the 5'-UTR element of this invention can be increased by 1.5-2 times. Furthermore, the 5'-UTR element of this invention is fully compatible with pseudouracil modification, providing a theoretical basis for its application in mRNA drugs and vaccines.

[0017] The present invention also provides an mRNA molecule whose 5'-UTR is the aforementioned 5'-UTR element. Preferably, the mRNA molecule is an mRNA molecule modified with pseudouracil.

[0018] Preferably, the mRNA molecule comprises a coding region encoding a polypeptide or protein and a fragment thereof, a 5'-UTR located upstream of the coding region, and a 3'-UTR located downstream of the coding region.

[0019] Furthermore, the coding region of the mRNA molecule is the EGFP-Flag fusion gene, the sequence of which is the nucleotide sequence shown in SEQ ID NO: 13. The EGFP gene allows for direct observation of the differences in fluorescence levels in cells between mRNAs containing different 5'-UTR elements, while the Flag gene was chosen based on the cost and practicality of antibodies incubated in Western blotting experiments.

[0020] In some embodiments, the mRNA molecule further includes a polyadenylated tail, and in a specific embodiment, the polyadenylated tail is poly(A).

[0021] Preferably, the length of the polyadenylate tail is 60-120 nt.

[0022] In the specific implementation scheme, we selected the polyadenylated tail as the nucleotide sequence shown in SEQ ID NO: 15.

[0023] The present invention also provides a nucleic acid molecule encoding the above-mentioned mRNA molecule, which can be used for in vitro transcription to produce the mRNA molecule.

[0024] The 5'-UTR element of this invention is compatible with pseudouracil modification. The two do not conflict with each other in function and can work synergistically to improve the overall performance of mRNA.

[0025] The present invention also provides a carrier comprising the above-mentioned nucleic acid molecules.

[0026] The vector used in this invention can be a plasmid vector commonly used in the art. By constructing the nucleic acid molecule encoding the mRNA molecule of this invention into the vector, efficient transcription of the mRNA molecule can be achieved. Preferably, a plasmid vector containing the T7 RNA polymerase promoter is used. The T7 RNA polymerase promoter sequence is the nucleotide sequence shown in SEQ ID NO: 12.

[0027] The present invention also provides a host cell comprising the above-described vector.

[0028] The host cell used in this invention is preferably a mammalian cell, such as human cervical cancer cells (HeLa).

[0029] The present invention also provides a kit containing the aforementioned 5'-UTR element for improving mRNA translation efficiency.

[0030] The present invention also provides the application of the 5'-UTR element, the mRNA molecule, the nucleic acid molecule, the vector, or the host cell in the preparation of nucleic acid therapeutic drugs or mRNA vaccines.

[0031] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0032] This invention improves mRNA translation efficiency by at least 1.5 times through a novel, optimized 5'-UTR element. It can be used as an element to enhance mRNA expression efficiency in nucleic acid therapeutics or mRNA vaccines, providing more and better options for mRNA therapy. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the pcDNA3.1-EGFP-Flag mRNA plasmid and its linear vector structure.

[0034] Figure 2 This is a PAGE gel image of RNA urea denaturation in Example 2;

[0035] Figure 3The images shown are bright-field and dark-field images and quantitative fluorescence graphs of HeLa cells after transfection with unmodified and modified mRNAs, respectively, from top to bottom. The images are: bright-field and dark-field images of HeLa cells 12 hours after transfection with unmodified mRNA; bright-field and dark-field images of HeLa cells 12 hours after transfection with modified mRNA; bright-field and dark-field images of HeLa cells 48 hours after transfection with unmodified mRNA; bright-field and dark-field images of HeLa cells 48 hours after transfection with modified mRNA; and quantitative fluorescence graphs of HeLa cells 48 hours after transfection with unmodified and modified mRNAs.

[0036] Figure 4 This image shows the Western Blot results and relative grayscale quantitative statistics of the unmodified and modified mRNAs expressed in HeLa cells for 12 hours and 48 hours, as shown in Example 3. From top to bottom, the images represent two replicates of the Western Blot results and relative grayscale quantitative statistics of the unmodified mRNA expressed in HeLa cells for 12 hours, 48 ​​hours, 12 hours, and 48 hours, respectively.

[0037] Figure 5 The images show the Western Blot results, polysome calibration peak diagram, polysome raw peak diagram, and gene expression statistics for each component of the modified mRNA in Example 4. From top to bottom, they show the Western Blot results and grayscale quantitative statistics of mRNAs containing Top4, Bottom2, and mRNA-1273 expressed in HeLa cells for 24 hours, the calibration peak diagram used to detect whether the experiment was performed normally, the raw peak diagram dividing different components, and the bar chart showing the content of target mRNA in each component. Detailed Implementation

[0038] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0039] Example 1: 5'-UTR Component Design

[0040] The 5'-UTR regulates the initiation of mRNA translation and is crucial for protein synthesis. Optimization of the 5'-UTR can improve mRNA translation efficiency, increase antigen yield, and enhance immune responses. The strategy employed in this invention is to screen for target 5'-UTR elements using a large language model (LLM), generate mRNA containing the target 5'-UTR element through in vitro transcription, and then transfect cells for mRNA translation efficiency testing and subsequent identification.

[0041] This experiment designed 12-nt randomly synthesized 5'-UTR elements and used an algorithm to screen for elements with high and low scores. For subsequent experiments, a fixed 16-nt sequence was added before the 12-nt base to ensure the normal generation of the mRNA construct. After designing the 5'-UTR element using a fixed sequence + 12-nt randomly synthesized base, the algorithm screened for high-scoring (Top) and low-scoring (Bottom) 5'-UTR elements. The obtained 5'-UTR element information is shown in Table 1.

[0042] Table 1. Optimized design of 5'-UTR components

[0043] The component names mentioned in the table are abbreviated in subsequent charts: Top1, 2, 3, 4, 5 are abbreviated as T1, T2, T3, T4, T5; Bottom1, 2, 3, 4, 5 are abbreviated as B1, B2, B3, B4, B5; and mRNA-1273 is abbreviated as 1273.

[0044] Example 2: Preparation and quality control of nucleic acid for synthesizing mRNA containing the 5'-UTR to be detected

[0045] 2-1. Linearization of template DNA

[0046] The 5'-UTR designed in Example 1 was constructed into a linear vector containing the T7 RNA polymerase promoter, Kozak, EGFP-Flag coding region, 3'-UTR, and polyadenylated tail. The sequences used for each part are shown in Table 2.

[0047] Table 2 Sequences of each part of the mRNA construct

[0048] In preparing the linear vector for the 5'-UTR to be detected, we used direct PCR amplification, and obtained the linear vector after gel extraction. We designed the 5'-UTR elements to be detected onto the forward primers for amplification. The primer sequences consisted of the T7 RNA polymerase promoter sequence, the 5'-UTR element to be detected, the Kozak conserved element, and the base sequence complementary to the vector plasmid. The 5'-UTR to be detected in the forward primers were selected from the nucleotide sequences shown in Table 1, resulting in 11 different forward primers. The reverse primer was the nucleotide sequence shown in SEQ ID NO: 16. The enzyme used for PCR amplification had to be a high-fidelity enzyme. We prepared 11 different forward primers and reverse primers to form forward and reverse primer mixtures, and added vectors (i.e., cloning the T7 RNA polymerase promoter-5'-UTR element-Kozak-EGFP-Flag-3'-UTR-polyadenylate tail between the multiple cloning sites of the pcDNA3.1 vector to obtain linear vectors, synthesized by Guangzhou Aiji Biotechnology Co., Ltd.), high-fidelity enzymes and matching reagents, and amplified according to the amplification program shown in Table 3. The products were separated using 1% agarose gel, and fragments of the target size were excised and recovered using an agarose gel DNA recovery kit. The purity and concentration of the recovered products were measured using a UV spectrophotometer to ensure that the purity and concentration of the linear vectors were up to standard.

[0049] Table 3. PCR amplification program for linear vector preparation.

[0050] 2-2 In vitro transcription

[0051] The qualified linear vector obtained in step 2-1 was reacted with T7 RNA polymerase, buffer solution, NTPs (including natural and chemically modified NTPs) and other essential elements for IVT at 37 °C for 2 or 4 hours, as shown in Table 4 (EasyCapT7 Co-transcription Kit with CAG Trimer, Vazyme, DD4203, China).

[0052] Table 4 IVT Reaction System

[0053] After the reaction was complete, 2 μL of DNase I was added to each tube, mixed well, and incubated at 37 °C for at least 15 minutes to digest the linear vector. After digestion, LiCl was added to a final concentration of 2.5 M, and the mixture was allowed to stand at -20 °C for at least 30 minutes. After standing, the mixture was centrifuged at 12,000 rpm for 15 minutes at 4 °C, the supernatant was discarded, and the tube was washed twice with 70% ethanol, then once with 100% anhydrous ethanol. The supernatant was removed, and the tube was dried at room temperature. An appropriate amount of RNase-free water was added to dissolve the supernatant, the concentration was measured, and the tubes were aliquoted. One experimental volume was used, and the remainder was stored at -80 °C. Two types of mRNA were prepared: pseudouridine-modified mRNA and unmodified mRNA. The pseudouridine-modified mRNA was selected for RNA urea denaturation PAGE electrophoresis to identify the banding pattern. The identification results are as follows: Figure 2 As shown in the diagram. In subsequent experiments, the modified mRNAs were all prefixed with an "N".

[0054] from Figure 2 The results show that the mRNAs obtained from in vitro transcription are of uniform size and have a single band.

[0055] Example 3: Quantitative Detection of Translation Efficiency

[0056] 3-1. Sample Preparation and Processing

[0057] Immortalized human cervical cancer cells (HeLa) were used for cell transfection experiments. Cells in good growth condition were collected, digested, resuspended, and counted. Cells were then divided into groups of 3 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and incubated overnight at 37 °C in a 5% CO2 incubator. The next day, cells were harvested and transfected using a LipoRNA transfection reagent. TM (Beyotime, C0535, China) Transfected cells with the mRNA molecules prepared in Example 2 at a dosage of 2 μg mRNA per well. After 6 hours of transfection, the medium was changed, and the cells were incubated at 37 °C in a 5% CO2 incubator for 12 hours and 48 hours, respectively. The fluorescence signal of each well was detected at these two time points. The results of the cell fluorescence signal observation are as follows: Figure 3 As shown.

[0058] from Figure 3The cellular fluorescence signal images show that at the 12-hour time point, the cellular fluorescence signal is weak and the difference is not significant. At the 48-hour time point, the cellular fluorescence signal of the modified group mRNA is stronger than that of the unmodified group mRNA. Furthermore, quantitative fluorescence results show that the cellular fluorescence signal of mRNAs with different 5'-UTR elements modified with pseudouracil is stronger than that of the unmodified group mRNA. In the development of mRNA vaccines and mRNA drugs, pseudouracil is added to modify mRNA, thereby reducing the immunogenicity of mRNA vaccines and drugs. The cellular fluorescence signal results demonstrate that our 5'-UTR element mRNA, after pseudouracil modification, has better expression-promoting effects. In addition, at the 48-hour time point, the cellular fluorescence signal of mRNA containing the Top series 5'-UTR elements is stronger than that of mRNA containing the Bottom series 5'-UTR elements.

[0059] 3-2. Western Blot and Expression Results Analysis

[0060] Following the procedure in section 3-1, HeLa cells were transfected with unmodified and modified mRNAs of different 5'-UTR elements. The medium was changed after 6 hours, and the transfection continued for 12 and 48 hours, respectively. Cells were collected at both time points using 1% SDS (containing bromophenol blue). After treatment at 95 °C for 15 minutes, the cells were immediately cooled on ice. Protein separation was performed using a 5% upper gel and a 10% lower gel PAGE gel. Formal electrophoresis was conducted at 80 V for 10 minutes and 120 V for 60 minutes. A pre-electrophoresis at 120 V for 20 minutes was typically performed before the formal electrophoresis. Wet transfer was performed using a 0.45 uM PVDF membrane (Immobilon®-P PVDFFMembrane, Merck KGaA, IPVH00010, US). After incubation at 250 mA for 2 hours, the membrane was blocked with 5% milk for at least 45 minutes. The membrane was then washed three times with the prepared TBST washing buffer for 5 minutes each time. Because our target protein EGFP-Flag is 28 kDa and Actin is 43 kDa, we cut membrane regions of the target size and incubated them separately with mouse-derived Flag (monoclonal antibody-FLAG® M2 mouse antibody, Sigma-Aldrich, F1804, US) and mouse-derived Actin (betaActin antibody-C4, Santa Cruz Biotechnology, sc-47778, US) at 4 °C overnight. The next day, the membrane was washed three times with TBST washing agent for 5 minutes each time, and then incubated with mouse-derived secondary antibody at 4 °C for 2 hours. After three washes with the washing agent, Western blot development was performed. Image J was used to quantify the band gray values. The results are as follows: Figure 4 As shown.

[0061] from Figure 4 The Western blot results show that, in the two Western blot results at 12 hours, the target protein band of the mRNA containing the 5'-UTR element shown in SEQ ID NO: 4 was darker. Quantitative analysis using ImageJ software revealed that the relative grayscale of the target protein containing the 5'-UTR element shown in SEQ ID NO: 4 was approximately twice that of the control group. Figure 4 The results showed that in two Western blotting results at 48 hours, the target protein band of mRNA containing the 5'-UTR element shown in SEQ ID NO: 4 was darker. Image J quantification revealed that the relative grayscale of the target protein in mRNA containing the 5'-UTR element shown in SEQ ID NO: 4 was approximately 1.5 times higher than that of the control group. Therefore, the preliminary conclusion is that the element shown in SEQ ID NO: 4 can promote mRNA translation. Furthermore, from... Figure 4 The Western Blot results and relative gray-scale quantitative statistics show that the two replicate results of the unmodified group mRNA at 12 hours and 48 hours are unstable.

[0062] Example 4: Analysis of Translation Regulation Mechanisms

[0063] 4-1 Sample Preparation and Processing

[0064] Further testing was performed on the 5'-UTR element shown in SEQ ID NO:4, and the 5'-UTR elements shown in SEQ ID NO:7 and SEQ ID NO11 were selected as transfection control groups.

[0065] Take HeLa cells in good growth condition, digest them with trypsin, and then add 1×10⁻⁶ cells per dish. 6Ten 10 cm dishes were seeded with a number of cells and incubated overnight at 37 °C in a 5% CO2 incubator. The next day, mRNA containing SEQ ID NO: 4, SEG ID NO: 7, and SEQ ID NO: 11 was transfected into the cells using a transfection reagent (LipoRNAi™, Beyotime, C0535, China) at a dosage of 10 μg per dish. Each mRNA was transfected into two dishes of cells. The remaining four dishes and the six dishes of cells transfected with mRNA were then incubated at 37 °C in a 5% CO2 incubator. After 24 hours of culture, two plates of cells were added to anisomycin (MCE, HY-18982, US) at a final concentration of 1 μg / ml and incubated at 37 ℃ in a 5% CO2 incubator for 10 minutes. After 10 minutes, ten plates of cells were removed and cycloheximide (MCE, HY-12320, US) at a final concentration of 100 μg / ml were added and incubated at 37 ℃ in a 5% CO2 incubator for another 5 minutes. The culture medium was aspirated, and each plate was washed with 1 ml of DPBS. The DPBS was aspirated, and 0.25% trypsin was added to digest the cells until they began to detach. The cells were neutralized with 5 times the volume of trypsin in cell culture medium, centrifuged at 100 rpm for 3 minutes, and the supernatant was aspirated. The cells were resuspended in 1 ml of pre-chilled DPBS. 35 μl of the cell suspension was diluted and counted. The remaining suspension was placed on ice. Centrifuge at 100 °C for 3 minutes, remove the supernatant, and flash-freeze the precipitate with liquid nitrogen at -80 °C.

[0066] 4-2 Preparation of sucrose gradient

[0067] Table 5 General Buffer Recipes

[0068] Prepare the general-purpose buffer in advance according to Table 5. Weigh 25 g of solid sucrose, dissolve it in the general-purpose buffer, and bring the volume to 50 ml to obtain a 50% sucrose solution. Dilute a portion of the 50% sucrose solution to a 10% concentration. Filter the prepared sucrose solution through a 0.45 μM filter. Add RNase inhibitor, protease inhibitor, and actinomycete ketone to the filtered sucrose solution. First, add an appropriate amount of 10% sucrose solution to a centrifuge tube (such as a 14×89 mm ultra-clean tube compatible with SW41). Then, inject the 50% sucrose solution from the bottom of the centrifuge tube using a long syringe, avoiding the generation of air bubbles during injection. Seal the tube and prepare a continuous linear density gradient of 10%-50% using a gradient preparer. The gradient preparation requires pre-cooling at 4 °C to avoid convection interference and should be placed on ice for 30 to 60 minutes.

[0069] 4-3 Cell lysis

[0070] Table 6 Lysis Buffer

[0071] Prepare Lysis Buffer according to Table 6, and add actinomycete ketone at a rate of 1×10⁻⁶. 7 Add lysis buffer at a ratio of 500 μL per cell, and lyse at 4 °C using a vertical mixer for 15-20 minutes. Centrifuge the lysate at 4 °C at maximum speed for 10 minutes, discard the pellet, and transfer the cytoplasmic lysate to a new RNase-free tube. Add 10 μL of 5×SDS-PAGE protein loading buffer to 40 μL of the cytoplasmic lysate for Western blotting to detect the effect of different components on mRNA expression efficiency. Add 500 μL of Trizol to 50 μL of the cytoplasmic lysate as a control.

[0072] 4-4 Ultracentrifugation

[0073] Carefully spread 450 μL of cytoplasmic lysis buffer onto the top layer of the gradient buffer, avoiding disturbance of the gradient interface. Centrifuge using an SW41 horizontal rotor at 4 °C and 40,000 RPM (approximately 250,000 centrifugal force) for 2 hours to separate ribosome complexes of different sizes according to their sedimentation coefficients. The centrifuge should be pre-cooled to 4 °C to prevent temperature fluctuations from causing complex dissociation.

[0074] 4-5 Component Collection and Detection

[0075] After centrifugation, the gradient tubes are placed in a density gradient fractionation system, and the samples are collected from top to bottom via puncture. The system must be equipped with a UV detector (260 nm wavelength) to monitor nucleic acid absorbance in real time. A typical spectrum sequentially presents the free RNA peak (at the very top), the 40S small subunit peak, the 60S large subunit peak, the 80S monoribosome peak, and a series of polysome peaks. Based on the peak spectrum, 15 fractions are automatically collected, approximately 780 μL per tube, ensuring that each fraction corresponds to a different density of ribosome complex.

[0076] 4-6 RNA Extraction

[0077] According to the peak patterns, the 15 tubes were divided into free RNA peaks, 40S-60S-80S peaks, and polyribosome series peaks. An equal volume of sucrose lysis buffer (containing 10 mM HEPES, 100 mM KCl, and 5 mM MgCl2) was added to each fraction, and proteinase K was added to a final concentration of 0.2 mg / mL. Digestion was performed at 37 °C for 30 minutes. An equal volume of acidic phenol-chloroform-isoamyl alcohol (25:24:1) was added for extraction. After vigorous shaking, the mixture was centrifuged at 4 °C at maximum speed for 15 minutes. The aqueous phase was collected, and 2.5 volumes of anhydrous ethanol and 0.1 volumes of 3 M sodium acetate (pH 5.2) were added. Precipitation was carried out at -20 °C for 2 hours. Centrifugation was performed at 4 °C at maximum speed for 30 minutes, and the supernatant was discarded. The precipitate was washed twice with 75% ethanol and once with 100% anhydrous ethanol, dried, and dissolved in DEPC water. RNA concentration and purity were measured.

[0078] 4-7 Results Analysis

[0079] RT-qPCR was performed on the RNA fractions to calculate the relative abundance of the target mRNA in the polysome-enriched fractions and assess its translation efficiency. Results are as follows: Figure 5 As shown.

[0080] from Figure 5 As can be seen from the Western Blot results, the target protein band of mRNA containing the 5'-UTR element shown in SEQ ID NO: 4 is darker. Image J quantification showed that the relative gray level of the target protein mRNA containing the 5'-UTR element shown in SEQ ID NO: 4 was 1.5 times higher than that of the control group, consistent with the trend of previous translation efficiency quantification results, further confirming the conclusion that the element shown in SEQ ID NO: 4 can promote mRNA translation. The polysome peak curve is smooth and the peaks are obvious, indicating excellent separation of the sucrose density gradient components. Quantification results of each component show that the relative expression levels of mRNA containing the 5'-UTR element shown in SEQ ID NO: 4 are higher in the 40S-60S-80S and polysome regions. This indicates that the ribosome loading of mRNA containing the 5'-UTR element shown in SEQ ID NO: 4 is high, thereby promoting mRNA translation in cells.

[0081] mRNAs containing the 5'-UTR element shown in SEQ ID NO: 4 have high ribosome loading, and higher ribosome loading can provide higher translation efficiency. Compared with other groups of mRNAs, the only difference is the 5'-UTR element. Therefore, this indirectly indicates that the 5'-UTR element shown in SEQ ID NO: 4 can improve the ribosome loading capacity of mRNA, and is expected to be applied in the development of mRNA vaccines and mRNA drugs.

Claims

1. A 5'-UTR element for improving mRNA translation efficiency, characterized in that, The nucleotide sequence is any of the following: 5'-GGCCACCAACGACATTTTCCGTTTCGTT-3'; 5'-GGCCACCAACGACATTCTCGTTTTCGTT-3'; 5'-GGCCACCAACGACATTTTGCGTTTCGTT-3'; 5'-GGCCACCAACGACATTCGTTTTTTCGTT-3'; 5'-GGCCACCAACGACATTTTCGTTTTCGTT-3'; 5'-GGCCACCAACGACATTGCCCCCCGCCGG-3'.

2. The 5'-UTR element according to claim 1, characterized in that, The 5'-UTR element has the nucleotide sequence 5'-GGCCACCAACGACATTCGTTTTTTCGTT-3'.

3. An mRNA molecule, characterized in that, The 5'-UTR it contains is the 5'-UTR element as described in claim 1 or 2, preferably, the mRNA molecule is an mRNA molecule modified with pseudouracil.

4. The mRNA molecule according to claim 3, characterized in that, The mRNA molecule contains a coding region encoding a polypeptide or protein and its fragments, a 5'-UTR upstream of the coding region and a 3'-UTR downstream of the coding region. Preferably, the coding region of the mRNA molecule is an EGFP-Flag fusion gene, the sequence of which is the nucleotide sequence shown in SEQ ID NO:

13.

5. The mRNA molecule according to claim 3, characterized in that, The mRNA molecule further includes a polyadenylated tail, preferably poly(A), more preferably the polyadenylated tail is 60-120 nt in length, and even more preferably the polyadenylated tail is the nucleotide sequence shown in SEQ ID NO:

15.

6. A nucleic acid molecule encoding the mRNA molecule of claim 3, 4 or 5.

7. A vector comprising the nucleic acid molecule of claim 6, preferably, the vector being a plasmid vector containing a T7 RNA polymerase promoter, and more preferably, the T7 RNA polymerase promoter sequence being the nucleotide sequence shown in SEQ ID NO:

12.

8. A host cell comprising the vector of claim 7, preferably a mammalian cell, and more preferably a human cervical cancer cell.

9. A reagent kit, characterized in that, It contains the 5'-UTR element for improving mRNA translation efficiency as described in claim 1.

10. The use of the 5'-UTR element of claim 1 or 2, the mRNA molecule of claim 3, the nucleic acid molecule of claim 6, the vector of claim 7, or the host cell of claim 8 in the preparation of nucleic acid therapeutic drugs or mRNA vaccines.

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