5 'UTR element for improving protein expression quantity and application of 5' UTR element

Abstract

The invention provides a 5 'UTR (Untranslated Region) element for improving protein expression quantity and application of the 5' UTR element, and belongs to the technical field of biology. The sequence of the 5 'UTR element is shown as SEQ ID NO.1. Modified mRNA is obtained by adding the 5' UTR element between an initiation codon of unmodified mRNA and 5 'UTR. Compared with the unmodified mRNA, the target protein expression quantity of the modified mRNA is remarkably improved, and meanwhile, the 5' UTR element is good in universality and suitable for improving the expression quantity of most mRNA, so that the 5 'UTR element has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a 5'UTR element for improving protein expression and an application thereof. Background Art

[0002] The 5' untranslated region (5'UTR) of mRNA refers to the region of mature mRNA located upstream of the coding region (CDS) and downstream of the 5' end cap that is not translated into protein. It contains important regulatory elements, including the internal ribosome entry site (IRES), 5'UTR secondary structure, G-quadruplexes (G4), 5' cap structure, upstream open reading frame (uORF), Kozak sequence, upstream start codon AUG (upstream AUG, uAUG) and 5'UTR introns (5UIs). The regulation of gene expression by 5'UTR involves multiple levels and plays an important role in maintaining the stability of mRNA, nuclear transport, RNA splicing and processing, and cell proliferation. Optimizing the sequence of 5'UTR is expected to significantly improve the expression efficiency of mRNA, thereby increasing the expression of the target protein. How to find a broad-spectrum 5'UTR element that can significantly increase the expression of the target protein requires further study.

[0003] Enhanced Green Fluorescent Protein (EGFP) is a fluorescent reporter protein widely used in biological research. It is a mutant of green fluorescent protein (GFP) isolated from Aequoreavictoria, obtained through amino acid mutation and codon optimization. EGFP has stronger fluorescence intensity and higher photostability than wild-type GFP, making it the main choice for live cell imaging and other applications. Choosing EGFP as the target protein makes it easier to detect protein expression levels, so using EGFP to evaluate 5'UTR sequences is a common method in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a 5'UTR element for increasing protein expression and its application.

[0005] The present invention provides a 5'UTR element of mRNA, and the sequence of the 5'UTR element is shown as SEQ ID NO.1.

[0006] The present invention also provides the use of the aforementioned 5'UTR element in increasing the expression level of mRNA for a target protein.

[0007] The present invention also provides an mRNA, wherein the 5'UTR of the mRNA comprises the aforementioned 5'UTR element.

[0008] Furthermore, the aforementioned 5'UTR element is located before the start codon of mRNA.

[0009] Furthermore, the 5'UTR of the aforementioned mRNA is connected to the start codon via the aforementioned 5'UTR element.

[0010] Furthermore, the sequence of the aforementioned mRNA is shown as SEQ ID NO.2.

[0011] The present invention also provides a vector, wherein the vector contains the aforementioned mRNA.

[0012] The present invention also provides a cell, which comprises the aforementioned 5'UTR element, the aforementioned mRNA or the aforementioned vector.

[0013] The present invention also provides the use of the aforementioned mRNA, the aforementioned vector or the aforementioned cell in increasing the expression amount of the target protein.

[0014] Furthermore, the target protein is enhanced green fluorescent protein.

[0015] The present invention has achieved the following beneficial effects:

[0016] The present invention provides a 5'UTR element, which is added between the start codon and the 5'UTR of an unmodified mRNA to obtain a modified mRNA. Compared with the unmodified mRNA, the expression amount of the target protein in the modified mRNA is significantly improved. At the same time, the 5'UTR element has good universality and is suitable for increasing the expression amount of most mRNAs. Therefore, the 5'UTR element has good application prospects.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1The target protein (EGFP) expression results of the existing mRNA (Ctrl) and the mRNA optimized by the 5'UTR element of the present invention (Optimized): A is the WB result diagram, in which ACTB is β-actin (protein detection internal reference); B is the quantitative result of protein expression. DETAILED DESCRIPTION

[0020] The raw materials and equipment used in the specific implementation manner of the present invention are all known products, which are obtained by purchasing commercially available products.

[0021] The sequence of the 5'UTR element of the optimized mRNA of the present invention is acguagg (SEQ ID NO. 1).

[0022] The sequence of the optimized mRNA of the present invention (the target protein encoded is EGFP) is:

[0023]

[0024] The sequence of the mRNA for producing EGFP in the prior art is:

[0025]

[0026] The nucleotide sequences shown in the above SEQ ID NO.2 and SEQ ID NO.3 were commissioned to Guangzhou Paizhen Biosynthesis.

[0027] Preparing mRNA into lipid nanoparticles is one of the conventional steps in the art of producing proteins using mRNA. The nucleotide sequences shown in the above SEQ ID NO.2 and SEQ ID NO.3 are prepared into lipid nanoparticles (LNP) according to conventional methods in the art, and then subsequent target protein expression experiments are carried out. The preparation of lipid nanoparticles (LNP) is also entrusted to Guangzhou Paizhen Biosynthesis. The raw material components of lipid nanoparticles are mainly cationic lipids, auxiliary phospholipids, cholesterol and PEG lipids. The raw material components of lipid nanoparticles will not affect the expression of the target protein.

[0028] Example 1: Protein expression of mRNA after optimization of the 5'UTR element of the present invention

[0029] 1. mRNA transfection

[0030] Bone marrow-derived dendritic cells (BMDCs) were plated for 2 hours, and LNPs containing the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3 were added to the cells, respectively, and samples were collected after 48 hours of culture.

[0031] 2. WB Verification

[0032] 1) Cell protein extraction

[0033] A. Take cells cultured in a 6-well plate for 48 hours and remove the old solution. Gently wash twice with PBS, add 100 μL of RIPA lysis buffer containing protease and phosphatase inhibitors to each well, react on ice for 5 minutes, scrape the cells back and forth with a cell scraper to fully lyse them, and transfer the cell lysate to a pre-cooled EP tube.

[0034] B. Vortex the lysate at high speed every 10 minutes and place on ice. Repeat 3 times.

[0035] C. Centrifuge at 13300 rpm for 30 min at 4°C. Gently aspirate the supernatant and transfer it to a new EP tube. It can be stored at -80°C.

[0036] 2) Determination of cell protein concentration

[0037] A. Prepare a protein standard solution (25 mg / mL), then dilute to a 0.5 mg / mL working solution.

[0038] B. Prepare standard curves of various concentrations using the protein standard working solution.

[0039] C. Prepare color-developing liquid (A:B=50:1), mix well and store in a dark place.

[0040] D. Take 2 μL of protein sample supernatant and add it to 38 μL PBS to dilute it 20 times.

[0041] E. Add protein standards and diluted protein samples to a 96-well plate in descending order of protein concentration, 10 μL per well. Take three replicate wells for the sample, then add 100 μL of colorimetric solution to each well, adding along the lower side wall for final mixing.

[0042] F. Incubate at 37°C in dark room for 25 min, measure the absorbance and record the protein concentration.

[0043] G. Add buffer to each tube of protein at a volume ratio of 4:1 and cook at 95℃ at 300rpm for 10min.

[0044] 3) Protein electrophoresis

[0045] Rinse the electrophoresis tank with running water and pure water, wipe it dry and set aside. Calculate the loading volume based on the total amount of protein per well of 20μg, slowly remove the comb on the upper layer of the colloid and fix it, and add protein marker and protein sample to each well in sequence. After spotting, first perform electrophoresis at a constant voltage of 150V for 45min until the bromophenol blue indicator runs to the bottom of the colloid.

[0046] 4) Protein transfer

[0047] Use a ruler to cut a PVDF membrane of appropriate size, soak it in a certain amount of methanol for 15 seconds, and then add pure water to keep it moist. Place the clamp and filter paper in order, with the sponge on the outermost side. Place the glue and membrane on the filter paper in turn under the environment of electrotransfer solution, then cover the filter paper and one side of the sponge, clamp and fix it. After each step, moisten it with electrotransfer solution and use a roller to completely drive out the bubbles. Connect the positive and negative poles of the power supply, put the clamp into the electrotransfer tank and add enough electrotransfer solution. Fill the electrotransfer tank with ice as much as possible to keep it low, set the cross flow to 220mA, 90min.

[0048] 5) Closure

[0049] Dissolve 2.5 g BSA in 50 mL TBST to prepare a 5% BSA blocking solution. Transfer the electroporated PVDF membrane to the blocking solution and block at room temperature for 1 hour on a shaker at 60 rpm.

[0050] 6) Primary antibody incubation

[0051] Thaw the primary antigen solution at 4°C, add it to 1000 times volume of TBST, and incubate it at 4°C for 12-16 hours. The next day, recover the primary antibody dilution solution and wash 3 times.

[0052] 7) Secondary antibody incubation

[0053] Use blocking solution to prepare secondary antibody diluent (1:10000), and incubate the PVDF membrane on a shaker at room temperature for 1 hour. Discard the used secondary antibody, wash the membrane 3 times with TBST, shake on a shaker at 130 times / min, 10 minutes each time.

[0054] 8) Exposure

[0055] Prepare ECL luminescent liquid according to the volume ratio of A liquid: B liquid in ECL reagent = 1:1, mix well and keep away from light for later use. Use tweezers to clamp the membrane and place it on a glass plate. Use filter paper to absorb TBST on the membrane, evenly add luminescent liquid, and then expose it in a Bio-Rad chemiluminometer. Obtain WB results, and quantify the expression of the target protein by the grayscale of the WB results.

[0056] 3. Results

[0057] The protein expression results of mRNA before and after optimization are as follows Figure 1 As shown. Figure 1 It can be seen that compared with the protein expression level of mRNA before optimization, the expression level of EGFP increased significantly after optimization.

[0058] In summary, the present invention provides a 5'UTR element, which is added between the start codon and the 5'UTR of an unmodified mRNA to obtain a modified mRNA. Compared with the unmodified mRNA, the expression level of the target protein in the modified mRNA is significantly improved. At the same time, the 5'UTR element has good universality and is suitable for increasing the expression level of most mRNAs. Therefore, the 5'UTR element has good application prospects.

Claims

1. A 5'UTR element of mRNA, characterized in that: The sequence of the 5'UTR element is shown in SEQ ID NO.

1.

2. Use of the 5'UTR element according to claim 1 in increasing the expression level of a target protein by mRNA.

3. An mRNA, characterized in that: The 5'UTR of the mRNA comprises the 5'UTR element of claim 1.

4. The mRNA according to claim 3, characterized in that: The 5'UTR element of claim 1 is located before the start codon of mRNA.

5. The mRNA according to claim 4, characterized in that: The 5'UTR of the mRNA is connected to the start codon via the 5'UTR element of claim 1.

6. The mRNA according to claim 5, characterized in that: The sequence of the mRNA is shown in SEQ ID NO.

2.

7. A carrier, characterized in that: The vector contains the mRNA according to any one of claims 3 to 6.

8. A cell, characterized in that: The cell comprises the 5'UTR element according to claim 1, the mRNA according to any one of claims 3 to 6, or the vector according to claim 7.

9. Use of the mRNA according to any one of claims 3 to 6, the vector according to claim 7 or the cell according to claim 8 in increasing the expression level of a target protein.

10. The use according to claim 9, characterized in that: The target protein is enhanced green fluorescent protein.

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