3'utr-4 and its use in increasing protein expression and in the preparation of mrna vaccines

Abstract

The application discloses 3'UTR-4 and application thereof in improving protein expression and mRNA vaccine preparation. The 3'UTR-4 disclosed by the application is a single-stranded RNA molecule shown in positions 2494-2558 of SEQ ID NO. 8 in the sequence listing. The 3'UTR-4 sequence of the application can obviously improve the protein expression after being constructed downstream of a firefly luciferase reporter gene and a green fluorescent protein gene or a gene coding a new coronavirus spike protein. Therefore, the 3'UTR-4 sequence can be used in protein production and novel mRNA drug molecule design to improve the expression of a target protein.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically 3'UTR-4 and its application in increasing protein expression levels and preparing mRNA vaccines. Background Technology

[0002] Based on the central dogma, genetic information in DNA is transmitted to proteins via messenger RNA (mRNA). The expression capacity of mRNA is closely related to the untranslated region (UTR) sequence, and the 3' untranslated region (3'UTR) can regulate protein expression levels by modulating mRNA stability and translation efficiency. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to increase the expression level of a protein.

[0004] To solve the above-mentioned technical problems, the present invention first provides an RNA molecule, named 3'UTR-4, which is as follows (a1) or (a2):

[0005] (a1) The single-stranded RNA molecule shown at positions 2494-2558 of SEQ ID NO.8 in the sequence listing;

[0006] (a2) A single-stranded RNA molecule in which positions 2494-2558 of SEQ ID NO.8 are substituted and / or deleted and / or added by one or more nucleotides and have the same function as positions 2494-2558 of SEQ ID NO.8.

[0007] The RNA molecule in (a2) above is a single-stranded RNA molecule that has 75% or more identity with (a1) and has the same function. The identity of 75% or more means 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0008] The present invention also provides a biomaterial (denoted as biomaterial 1) related to 3'UTR-4, wherein biomaterial 1 is any one of B1) to B3) below:

[0009] B1) Transcription of DNA molecules at 3'UTR-4;

[0010] B2) A recombinant vector containing the DNA molecule described in B1);

[0011] B3) Recombinant microorganisms containing the DNA molecule described in B1) or recombinant microorganisms containing the recombinant vector described in B2).

[0012] In the above-mentioned biological material 1, the DNA molecule described in B1) may be position 2941-3005 of SEQ ID NO.7.

[0013] The present invention also provides a set of RNA molecules, which consists of an RNA molecule named 3'UTR-4 and a 5'UTR, wherein the 5'UTR is the single-stranded RNA molecule shown in positions 4-48 of SEQ ID NO.2 or positions 4-73 of SEQ ID NO.15.

[0014] The complete set of RNA molecules can be used to produce proteins or to prepare mRNA vaccines.

[0015] The present invention also provides a kit comprising the biomaterial 1 and a biomaterial associated with the 5'UTR (denoted as biomaterial 2), wherein biomaterial 2 is any one of the following C1) to C3):

[0016] C1) Transcribe the DNA molecule described in the 5'UTR;

[0017] C2) A recombinant vector containing the DNA molecule described in C1);

[0018] C3) Recombinant microorganisms containing the DNA molecule described in C1) or recombinant microorganisms containing the recombinant vector described in C2).

[0019] In the above-mentioned complete set of products, the DNA molecule described in C1) may be the 451-495th position of SEQ ID NO.1 or the 451-520th position of SEQ ID NO.14.

[0020] The complete set of products can be used to produce proteins or to prepare mRNA vaccines.

[0021] The present invention also provides a method for preparing proteins, the method comprising 1) or 2):

[0022] 1) Link a 3'UTR-4 to the 3' end of the mRNA that translates the target protein to obtain recombinant RNA; express the recombinant RNA to obtain the target protein;

[0023] 2) Attach a 3'UTR-4 to the 3' end of the mRNA that translates the target protein and attach the 5'UTR to the 5' end to obtain recombinant RNA; express the recombinant RNA to obtain the target protein;

[0024] 3) Link the 3' end of the gene encoding the target protein to the DNA of the 3'UTR-4 transcription to obtain recombinant DNA; express the recombinant DNA to obtain the target protein;

[0025] 4) Link DNA transcribed from the 3' end of the target protein encoding gene to the 3' UTR-4 and DNA transcribed from the 5' end to the 5' UTR to obtain recombinant DNA; express the recombinant DNA to obtain the target protein.

[0026] In methods 1) and 2) above, the expression of the recombinant RNA can be achieved by transfecting the recombinant RNA into cells.

[0027] The DNA transcribed from the 3'UTR-4 may be positions 2941-3005 of SEQ ID NO. 7. The DNA transcribed from the 5'UTR may be positions 451-495 of SEQ ID NO. 1 or positions 451-520 of SEQ ID NO. 14.

[0028] In one embodiment of the present invention, the target protein is firefly luciferase (Fluc) and / or green fluorescent protein (GFP).

[0029] In another embodiment of the present invention, the target protein is the spike protein (Spike, S) of the SARS-CoV-2 virus.

[0030] The cells may be animal cells, plant cells, or microbial cells. The animal cells may be mammalian cells, such as HEK-293T cells.

[0031] 3'UTR-4, or the biological material 1, or the complete set of RNA molecules, or any of the following applications of the complete set of products:

[0032] Application of X1 in protein production;

[0033] Application of X2 in the preparation and production of protein products;

[0034] Application of X3 in the preparation of mRNA vaccines.

[0035] This invention identifies a novel 3'UTR sequence that can induce high expression of a target protein. When this 3'UTR sequence is constructed downstream of the Fluc reporter gene and the GFP gene or the SARS-CoV-2 S protein coding sequence, it can significantly increase protein expression. This 3'UTR sequence has not been reported in existing mRNA drugs and may be applied in the design of novel mRNA drug molecules in the future.

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0037] Figure 1 (A) Linearized plasmid detection. Plasmids pNR021 (containing control 3'UTR-BNT), pNR034 (containing 3'UTR-1), pNR035 (containing 3'UTR-2), pNR036 (containing 3'UTR-3), pNR037 (containing 3'UTR-4), or pNR038 (containing 3'UTR-5) were linearized by BsaI and the recovered products were analyzed by agarose gel electrophoresis. (B) Schematic diagram of the capped mRNA sequence (top) and agarose gel electrophoresis analysis results (bottom). The 5'-UTR sequence of the capped mRNA with different capped structures was fixed as 5'UTR-BNT, encoding Fluc and GFP proteins. The 3'-UTR sequences were 3'UTR-BNT (control sequence), 3'UTR-1, 3'UTR-2, 3'UTR-3, 3'UTR-4, or 3'UTR-5, respectively.

[0038] Figure 2 HEK-293T cells were either untransfected with mRNA (mock) or transfected with capped mRNA sequences of different sequences: including positive controls 3'UTR-BNT, 3'UTR-1, 3'UTR-2, 3'UTR-3, 3'UTR-4, and 3'UTR-5. Sixteen hours after transfection, GFP expression levels were compared by observing the intensity of green fluorescence (A), and firefly luciferase (Fluc) activity was detected (B). Scale bar: 200 μm; M = million (million, 10^6) 6 ).

[0039] Figure 3Effect of 3'UTR-4 on the expression level of SARS-CoV-2 S protein. (A) Schematic diagram of S protein mRNA sequence. The 5'-UTR sequence after the cap structure is fixed as 5'UTR-3, which encodes the SARS-CoV-2 S protein. The 3'-UTR sequence is either the control sequence 3'UTR-BNT or the candidate sequences 3'UTR-2 and 3'UTR-4. (B) Linearization plasmid detection. The products of plasmid pNR043 (containing 3'UTR-BNT), pNR053 (containing 3'UTR-2), or pNR054 (containing 3'UTR-4) after linearization with BsaI and recovery were analyzed by agarose gel electrophoresis. (C) Agarose gel electrophoresis analysis of capped mRNA. Different capped mRNAs contain the sequences 3'UTR-BNT, 3'UTR-2, or 3'UTR-4, respectively. (D) Effect of 3'-UTR sequence on S protein expression level. HEK-293T cells were either untransfected (mocked) or transfected with mRNA expressing the S protein with different 3'-UTR sequences. Cells were lysed 24 hours after transfection, and the expression levels of the S protein and the internal reference protein α-tubulin were detected by Western blot. Detailed Implementation

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Unless otherwise specified, in the following examples, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0041] In the sequence list, T in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, and SEQ ID NO.12 all represent U, and T in SEQ ID NO.15, SEQ ID NO.17, and SEQ ID NO.19 all represent m1Ψ.

[0042] Example 1: 3'UTR-4 can increase the protein expression levels of Fluc and GFP.

[0043] In this embodiment, five naturally occurring 3'UTR sequences (denoted as 3'UTR-1, 3'UTR-2, 3'UTR-3, 3'UTR-4, and 3'UTR-5, respectively) were selected using bioinformatics methods, and 3'UTR sequences favorable for upstream target protein expression were screened experimentally. To this end, the inventors constructed the five 3'UTR sequences downstream of a plasmid capable of simultaneously expressing Fluor and GFP. Fluor and GFP were linked into an open reading frame via a self-cleaving polypeptide T2A. The expressed fusion protein Fluor-T2A-GFP was cleaved into two independent proteins, Fluor and GFP, by protease digestion. The optimal 3'UTR sequence was obtained by comparing the chemiluminescence level catalyzed by luciferase and the green fluorescence intensity of GFP. In addition, a positive control 3'UTR (BNT162b2 (Fubitai) 3'UTR), hereinafter referred to as 3'UTR-BNT, was also included, with its DNA sequence located at positions 2935-3229 of SEQ ID NO. 11 in the sequence listing.

[0044] 1. Preparation of linearized plasmid templates

[0045] Plasmids carrying Flux-GFP and candidate 3'UTRs or positive control 3'UTRs (i.e., pNR034, pNR035, pNR036, pNR037, pNR038, and pNR021) were synthesized by GenScript, and their sequences are SEQ ID NOs 1, 3, 5, 7, 9, and 11 in the sequence listing, respectively. After linearization by BsaI (nearshore protein) digestion, the plasmids were used... The linearized plasmid was purified using the SV Gel and PCR Clean-Up System (Promega) gel extraction kit. Analysis of the plasmid and its linearization product by agarose gel electrophoresis showed that the size of the linearized plasmid was consistent with the theoretical value. Figure 1 (A)

[0046] SEQ ID NO.1 is the DNA sequence of transcribed Fluc-GFP 3'UTR-1 mRNA. Positions 431-450 of SEQ ID NO.1 are the T7 promoter sequence, positions 451-495 are the 5'UTR-BNT DNA sequence, positions 502-2934 are the coding sequence for the Fluc-GFP fusion protein, and positions 2941-3013 are the 3'UTR-1 DNA sequence. The sequence of Fluc-GFP 3'UTR-1 mRNA is SEQ ID NO.2. Positions 4-48 of SEQ ID NO.2 are the 5'UTR-BNT sequence, and positions 2494-2566 are the 3'UTR-1 sequence.

[0047] SEQ ID NO.3 is the DNA sequence of transcribed Fluc-GFP 3'UTR-2 mRNA. Positions 431-450 of SEQ ID NO.3 are the T7 promoter sequence, positions 451-495 are the 5'UTR-BNT DNA sequence, positions 502-2934 are the coding sequence for the Fluc-GFP fusion protein, and positions 2941-3022 are the 3'UTR-2 DNA sequence. The sequence of Fluc-GFP 3'UTR-2 mRNA is SEQ ID NO.4. Positions 4-48 of SEQ ID NO.4 are the 5'UTR-BNT sequence, and positions 2494-2575 are the 3'UTR-2 sequence.

[0048] SEQ ID NO. 5 is the DNA sequence of transcribed Fluc-GFP 3'UTR-3 mRNA. Positions 431-450 of SEQ ID NO. 5 are the T7 promoter sequence, positions 451-495 are the 5'UTR-BNT DNA sequence, positions 502-2934 are the coding sequence for the Fluc-GFP fusion protein, and positions 2941-3034 are the 3'UTR-3 DNA sequence. The sequence of Fluc-GFP 3'UTR-3 mRNA is SEQ ID NO. 6. Positions 4-48 of SEQ ID NO. 6 are the 5'UTR-BNT sequence, and positions 2494-2587 are the 3'UTR-3 sequence.

[0049] SEQ ID NO.7 is the DNA sequence for transcribing Fluc-GFP 3'UTR-4 mRNA. Positions 431-450 of SEQ ID NO.7 are the T7 promoter sequence, positions 451-495 are the 5'UTR-BNT DNA sequence, positions 502-2934 are the coding sequence for the Fluc-GFP fusion protein, and positions 2941-3005 are the 3'UTR-4 DNA sequence. The sequence of Fluc-GFP 3'UTR-4 mRNA is SEQ ID NO.8. Positions 4-48 of SEQ ID NO.8 are the 5'UTR-BNT sequence, and positions 2494-2558 are the 3'UTR-4 sequence.

[0050] SEQ ID NO.9 is the DNA sequence for transcribed Fluc-GFP 3'UTR-5 mRNA. Positions 431-450 of SEQ ID NO.9 are the T7 promoter sequence, positions 451-495 are the 5'UTR-BNT DNA sequence, positions 502-2934 are the coding sequence for the Fluc-GFP fusion protein, and positions 2941-3028 are the 3'UTR-5 DNA sequence. The sequence of Fluc-GFP 3'UTR-5 mRNA is SEQ ID NO.10. Positions 4-48 of SEQ ID NO.10 are the 5'UTR-BNT sequence, and positions 2494-2581 are the 3'UTR-5 sequence.

[0051] SEQ ID NO. 11 is the DNA sequence for transcribed Fluc-GFP 3'UTR-BNT mRNA. Positions 431-450 of SEQ ID NO. 11 are the T7 promoter sequence, positions 451-495 are the 5'UTR-BNT DNA sequence, positions 502-2934 are the coding sequence for the Fluc-GFP fusion protein, and positions 2935-3229 are the 3'UTR-BNT DNA sequence. The sequence of Fluc-GFP 3'UTR-BNT mRNA is SEQ ID NO. 12. Positions 4-48 of SEQ ID NO. 12 are the 5'UTR-BNT sequence, and positions 2488-2782 are the 3'UTR-BNT sequence.

[0052] pNR034, pNR035, pNR036, pNR037, pNR038, and pNR021 all express the Fluc-T2A-GFP protein sequence shown in SEQ ID NO.13. Positions 1-550 of SEQ ID NO.13 represent the Fluc protein sequence, positions 551-571 represent the T2A sequence, and positions 572-809 represent the GFP protein sequence.

[0053] 2. In vitro transcription and capping

[0054] Using the linearized plasmid obtained in step 1 as a template, in vitro transcription was performed according to the HiScribe T7 High Yield RNA Synthesis Kit (NEB) instructions. After incubation at 37°C for 3 hours, DNase I (nearshore protein) was added and incubated at 37°C for 15 minutes to remove residual DNA template. After the reaction, the uncapped mRNA was recovered by lithium chloride (Thermo) precipitation. Finally, capping was performed using the Cap 1 Capping System (nearshore protein) kit. The capping product was recovered by lithium chloride precipitation to obtain Cap1-capped mRNA.

[0055] The obtained Fluc-GFP3'UTR-1 mRNA, Fluc-GFP3'UTR-2 mRNA, Fluc-GFP3'UTR-3 mRNA, Fluc-GFP3'UTR-4 mRNA, Fluc-GFP3'UTR-5 mRNA, and Fluc-GFP3'UTR-BNT mRNA can be prepared according to the above method or directly synthesized.

[0056] To preliminarily confirm the quality of the mRNA product, the inventors performed agarose gel electrophoresis analysis. The results showed that the capped mRNA product exhibited a single band, indicating high mRNA quality. Figure 1 (B)

[0057] 3. Detection of intracellular luciferase and GFP expression levels

[0058] The prepared capped mRNA was obtained through MessengerMAX TM (Thermo) was transfected into HEK-293T cells. Sixteen hours after transfection, the intensity of green fluorescence in the cells after transfection with mRNA was observed under a fluorescence microscope, and the result was analyzed using ONE-Globe. TM The EX Luciferase Assay System (Promega) kit was used to determine the intracellular luciferase activity.

[0059] The results showed that cells transfected with mRNA containing 3'UTR-4 exhibited the strongest green fluorescence signal. Figure 2 (A). Similarly, cells transfected with mRNA containing 3'UTR-4 showed the highest intracellular luciferase activity, which was 1.5 times (3.29 x 10⁻⁶) higher than that transfected with the positive control 3'UTR mRNA (3'UTR-BNT). 6 / 2.20x10 6 ()( Figure 2 (B)

[0060] Based on the results of luciferase expression detection and GFP expression observation, 3'UTR-4 can increase the expression level of upstream target protein.

[0061] Example 2: 3'UTR-4 can increase the expression level of the SARS-CoV-2 S protein.

[0062] To further confirm the translation efficiency of 3'UTR-4, this example further analyzed the effects of the positive control sequence (3'UTR-BNT), 3'UTR-2, and 3'UTR-4 sequences on the expression level of the SARS-CoV-2 spike protein (Spike, S). A schematic diagram of the constructed mRNA is shown below. Figure 3 A.

[0063] 1. Preparation of linearized plasmid templates

[0064] Plasmids carrying the S protein and candidate 3'UTR sequences (i.e., pNR043, pNR053, and pNR054) were synthesized and cloned by GenScript, and their sequences are SEQ ID NO. 14, 16, and 18 in the sequence listing. After linearization by BsaI (nearshore protein) digestion, the plasmids were used... The linearized plasmid was purified using the SV Gel and PCR Clean-Up System (Promega) gel extraction kit. Analysis of the plasmid and its linearization product by agarose gel electrophoresis showed that the size of the linearized plasmid was consistent with the theoretical value. Figure 3 (B)

[0065] SEQ ID NO. 14 is the DNA sequence for transcribed S 3'UTR-2 mRNA. Positions 431-450 of SEQ ID NO. 14 are the T7 promoter sequence, positions 451-520 are the 5'UTR-3 DNA sequence, positions 530-4354 are the coding sequence for the S protein, and positions 4361-4442 are the 3'UTR-2 DNA sequence. The sequence of S3'UTR-2 mRNA is SEQ ID NO. 15. Positions 4-73 of SEQ ID NO. 15 are the 5'UTR-3 sequence, and positions 3914-3995 are the 3'UTR-2 sequence.

[0066] SEQ ID NO.16 is the DNA sequence for transcription of S 3'UTR-4 mRNA. Positions 431-450 of SEQ ID NO.16 are the T7 promoter sequence, positions 451-520 are the 5'UTR-3 DNA sequence, positions 530-4354 are the coding sequence for the S protein, and positions 4361-4425 are the 3'UTR-4 DNA sequence. The sequence of S 3'UTR-4 mRNA is SEQ ID NO.17. Positions 4-73 of SEQ ID NO.17 are the 5'UTR-3 sequence, and positions 3914-3978 are the 3'UTR-4 sequence.

[0067] SEQ ID NO.18 is the DNA sequence for transcribed S 3'UTR-BNT mRNA. Positions 431-450 of SEQ ID NO.18 are the T7 promoter sequence, positions 451-520 are the 5'UTR-3 DNA sequence, positions 530-4354 are the coding sequence for the S protein, and positions 4355-4649 are the 3'UTR-BNT DNA sequence. The sequence of S 3'UTR-BNT mRNA is SEQ ID NO.19. Positions 4-73 of SEQ ID NO.19 are the 5'UTR-3 sequence, and positions 3908-4202 are the 3'UTR-BNT sequence.

[0068] pNR053, pNR054, and pNR043 can all express the S protein shown in SEQ ID NO.20.

[0069] 2. In vitro transcription and capping

[0070] Using the linearized plasmid obtained in step 1 as a template, in vitro transcription was performed according to the HiScribe T7 High Yield RNA Synthesis Kit (NEB) instructions, with uracil (UTP) replaced by 1-methylpseudouracil (m1ψ). After incubation at 37°C for 3 hours, DNase I (nearshore protein) was added and incubated at 37°C for 15 minutes to remove residual DNA template. After the reaction, the uncapped mRNA was recovered by lithium chloride (Thermo) precipitation. Finally, capping was performed using the Cap 1 Capping System (nearshore protein) kit. The capped product was recovered by lithium chloride precipitation to obtain Cap1-capped mRNA.

[0071] The obtained S3'UTR-2 mRNA, S3'UTR-4 mRNA, and S3'UTR-BNT mRNA can be prepared according to the above method or synthesized directly.

[0072] To preliminarily confirm the quality of the mRNA product, the inventors performed agarose gel electrophoresis analysis. The results showed that the capped mRNA product exhibited a single band. Figure 3 (C)

[0073] 3. Detection of intracellular S protein expression levels

[0074] The prepared capped mRNA was obtained through HEK-293T cells were transfected using the Transfection Kit (Mirus). Twenty-four hours after transfection, the expression level of the intracellular S protein was detected by Western blot. The antibody used for S protein detection was the SARS-CoV-2 (2019-nCoV) Spike Antibody (SinoBiological), a polyclonal antibody against the S1 subunit of the SARS-CoV-2 spike protein, and the internal control antibody was the α-Tubulin Mouse Monoclonal Antibody (proteintech).

[0075] The results showed that, compared with the blank control (Mock), the full-length S protein and S1 protein subunits were detected in all samples. The expression levels of 3'UTR-2 and 3'UTR-4 were slightly higher than those in the positive control, with 3'UTR-4 showing a significantly higher expression level. Figure 3 (D).

[0076] In summary, the experimental results of Examples 1 and 2 show that 3'UTR-4 can increase the expression level of upstream target proteins.

[0077] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A 3'UTR RNA molecule, which is the single-stranded RNA molecule shown at positions 2494-2558 of SEQ ID NO.8 in the sequence listing, where T in SEQ ID NO.8 represents U.

2. The biological material associated with the RNA molecule of claim 1 is any one of B1) to B3) below: B1) Transcribe the DNA molecule of the RNA molecule as described in claim 1; B2) A recombinant vector containing the DNA molecule described in B1); B3) Recombinant microorganisms containing the DNA molecule described in B1) or recombinant microorganisms containing the recombinant vector described in B2).

3. The biomaterial according to claim 2, characterized in that: B1) The DNA molecule described is position 2941-3005 of SEQ ID NO.

7.

4. A set of RNA molecules, comprising the RNA molecule of claim 1 and an RNA molecule named 5'UTR, wherein the 5'UTR is the single-stranded RNA molecule shown in positions 4-48 of SEQ ID NO. 2 or positions 4-73 of SEQ ID NO. 15, and T in SEQ ID NO. 2 and 15 both represent U.

5. A complete product comprising biomaterials associated with the 3'UTR and biomaterials associated with the 5'UTR, wherein the biomaterials associated with the 3'UTR are any one of B1) to B3) below: B1) Transcribe the DNA molecule of the 3'UTR RNA molecule as described in claim 1; B2) A recombinant vector containing the DNA molecule described in B1); B3) Recombinant microorganisms containing the DNA molecule described in B1), or recombinant microorganisms containing the recombinant vector described in B2); The biomaterial associated with the 5'UTR is any one of the following C1) to C3): C1) Transcription of a DNA molecule with a 5'UTR; wherein the 5'UTR is a single-stranded RNA molecule represented by positions 4-48 of SEQ ID NO.2 or positions 4-73 of SEQ ID NO.15, where T in SEQ ID NO.2 and 15 both represent U; C2) A recombinant vector containing the DNA molecule described in C1); C3) Recombinant microorganisms containing the DNA molecule described in C1) or recombinant microorganisms containing the recombinant vector described in C2).

6. The complete set of products according to claim 5, characterized in that: B1) The DNA molecule described is positions 2941-3005 of SEQ ID NO.7; C1) The DNA molecule is position 451-495 of SEQ ID NO.1 or position 451-520 of SEQ ID NO.

14.

7. A method for preparing a protein, comprising 1) or 2) or 3) or 4): 1) The RNA molecule of claim 1 is ligated to the 3' end of the mRNA that translates the target protein to obtain recombinant RNA; the recombinant RNA is expressed to obtain the target protein. 2) The RNA molecule of claim 1 is ligated to the 3' end of the mRNA that translates the target protein, and the 5' UTR of claim 5 is ligated to the 5' end to obtain recombinant RNA; the recombinant RNA is expressed to obtain the target protein; 3) Link the DNA of the RNA molecule described in claim 1 to the 3' end of the gene encoding the target protein to obtain recombinant DNA; express the recombinant DNA to obtain the target protein; 4) Connect the DNA of the RNA molecule of claim 1 to the 3' end of the gene encoding the target protein and connect the DNA of the 5'UTR of claim 5 to the 5' end to obtain recombinant DNA; express the recombinant DNA to obtain the target protein.

8. The method according to claim 7, characterized in that: In 1) and 2), the expression of the recombinant RNA is achieved by transfecting the cells with the recombinant RNA.

9. Any of the following applications of the RNA molecule of claim 1, the biomaterial of claim 2 or 3, the complete set of RNA molecules of claim 4, or the complete set of products of claim 5 or 6: X1) Applications in protein production; X2) Applications in the preparation and production of protein products; Application of X3 in the preparation of mRNA vaccines.

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