A circular RNA expression vector capable of translating protein and its construction method and expression method

Abstract

The present invention belongs to the field of molecular biology technology, and specifically relates to a circular RNA expression vector, a construction method, and an expression method for a translatable protein. The present invention provides a circular RNA overexpression skeleton, comprising the following structures connected in sequence: an upstream homology arm sequence, an upstream cyclization-promoting sequence, a first restriction enzyme cleavage site, a spacer sequence, an IRES sequence, a second restriction enzyme cleavage site, a downstream cyclization-promoting sequence, and a downstream homology arm sequence. The circular RNA overexpression skeleton of the present invention can enable the expressed circular RNA to have protein translation ability, and the linear sequence of the gene to be cyclized is recombined into a eukaryotic expression vector loaded with an expression skeleton through the restriction enzyme cleavage site, and then the recombinant plasmid is transfected into the cell to enable the expressed target circular RNA molecule to translate protein. The circular RNA overexpression skeleton and expression vector constructed by the present invention provide a tool for expressing circular RNA with protein translation function.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a circular RNA expression vector capable of translating proteins, and a construction method and an expression method thereof. Background Art

[0002] Circular RNA (circRNA) is a type of covalently closed single-stranded circular RNA molecule formed by backsplicing of pre-messenger RNA (pre-mRNA). CircRNAs have been shown to function as microRNA (miRNA) sponges, regulating the occurrence and metastasis of various tumor tissues, regulating transcription, and serving as tumor biomarkers.

[0003] circRNA does not have a 5' cap and a 3' polyA tail structure, so it is generally considered to have no coding function. However, with the deepening of circRNA-related research, more and more evidence shows that some circRNAs have protein or small peptide coding functions, and participate in the regulation of life activities through their translated functional products. The structural specificity of circRNA makes it less susceptible to degradation by nucleases, so it is more stable than linear RNA, and its ability to translate proteins is more lasting, making it an important means of replacing linear RNA translation. The translation of circRNA can only be achieved through a non-cap-dependent pathway, so the optimization of IRES sequences and other auxiliary sequences is conducive to improving the translation level of circRNA. Under natural conditions, the IRES or m 6 The activity of A sequence in initiating translation is generally weak. Summary of the Invention

[0004] The purpose of the present invention is to provide a circular RNA expression vector, construction method and expression method for translatable proteins, which further improves the protein translation efficiency of circRNA while ensuring the efficient circularization efficiency of circRNA, and can be applied to subsequent vaccine development and gene therapy.

[0005] The present invention provides a circular RNA overexpression skeleton, comprising the following structures connected in sequence: an upstream homology arm sequence, an upstream cyclization-promoting sequence, a first restriction enzyme cleavage site, a spacer sequence, an IRES sequence, a second restriction enzyme cleavage site, a downstream cyclization-promoting sequence, and a downstream homology arm sequence;

[0006] The upstream cyclization-promoting sequence and the downstream cyclization-promoting sequence are respectively selected from one of the sequences shown in SEQ ID No. 1 and SEQ ID No. 2;

[0007] The spacer sequence is shown as SEQ ID No.3.

[0008] Preferably, the first restriction enzyme cleavage site and the second restriction enzyme cleavage site are respectively selected from one of BamH I and Xho I.

[0009] Preferably, the IRES sequence is shown as SEQ ID No.4.

[0010] Preferably, the nucleotide sequence of the circular RNA overexpression backbone includes the sequence shown in SEQ ID No.5.

[0011] The present invention also provides a recombinant vector comprising the circular RNA overexpression skeleton.

[0012] Preferably, the basic vector of the recombinant vector includes a eukaryotic expression vector.

[0013] The present invention also provides a method for constructing the above-mentioned recombinant vector, comprising the following steps: connecting the above-mentioned circular RNA overexpression skeleton to a basic vector in a homologous recombination manner to obtain the recombinant vector.

[0014] Preferably, both ends of the circular RNA overexpression backbone contain a homology arm sequence, and amplification primers can be used to amplify the circular RNA overexpression backbone containing the homology arms and directly used for homologous recombination.

[0015] Preferably, the basic vector comprises pcDNA3.1(+).

[0016] The present invention also provides the use of the circular RNA overexpression skeleton or the recombinant vector in expressing target genes or circular RNA.

[0017] The present invention also provides a method for expressing a target gene using the above-mentioned recombinant vector, comprising the following steps: dividing the nucleotide sequence of the target gene into two segments, up and down, recombining the down segment into a first restriction enzyme cleavage site, and then recombining the up segment into a second restriction enzyme cleavage site to obtain a recombinant vector expressing the target gene;

[0018] The recombinant vector expressing the target gene is used to transform eukaryotic cells to express the target gene.

[0019] Beneficial effects: The present invention provides a circular RNA overexpression skeleton, comprising the following structures connected in sequence: an upstream homology arm sequence, an upstream cyclization-promoting sequence, a first restriction enzyme cleavage site, a spacer sequence, an IRES sequence, a second restriction enzyme cleavage site, a downstream cyclization-promoting sequence, and a downstream homology arm sequence. The circular RNA overexpression skeleton of the present invention can enable the expressed circular RNA to have protein translation ability, and the linear sequence of the gene to be cyclized is recombined into a eukaryotic expression vector loaded with the expression skeleton through the restriction enzyme cleavage site, and then the recombinant plasmid is transfected into the cell to enable the expressed target circular RNA molecule to translate protein. The circular RNA overexpression skeleton and expression vector constructed by the present invention provide a tool for expressing circular RNA with protein translation function. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the circular RNA overexpression skeleton;

[0021] Figure 2 is the plasmid map of pc-Scirc-protein;

[0022] Figure 3 This is a diagram showing the composition of the circular RNA expression vector into which the GFP gene is inserted;

[0023] Figure 4 This is the fluorescence quantitative detection result of the circular RNA expression vector inserted into the GFP gene;

[0024] Figure 5 This is the fluorescence observation result of the circular RNA expression vector inserted with the GFP gene. DETAILED DESCRIPTION

[0025] The present invention provides a circular RNA overexpression skeleton, comprising the following structures connected in sequence: an upstream homology arm sequence, an upstream cyclization-promoting sequence, a first restriction enzyme cleavage site, a spacer sequence, an IRES sequence, a second restriction enzyme cleavage site, a downstream cyclization-promoting sequence, and a downstream homology arm sequence;

[0026] The upstream cyclization-promoting sequence and the downstream cyclization-promoting sequence are respectively selected from one of the sequences shown in SEQ ID No.1 and SEQ ID No.2; in the embodiment of the present invention, SEQ ID No.1 (TGAAAACACGGGTTATTCCCCTCCTGGCAGGTATATAGGAGCCCTATCAA AGTCGAGCTAACGGAATGGGGTTTTCTTTCCCTCCTCTTCAG) is the upstream cyclization-promoting sequence, and SEQ ID No.2 (GTAAGACTTGCTTTTGTCAGTGGGGTGGCTCCCAGGATGCAGGGTCCCATAGGAGGGGAATAACCCGTGTTTTCA) is the downstream cyclization-promoting sequence.

[0027] The spacer sequence is shown in SEQ ID No. 3: GCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCC CTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGA.

[0028] The structure of the circular RNA overexpression backbone of the present invention is as follows Figure 1As shown, the generated circular RNA can have protein translation function. The first restriction enzyme cleavage site and the second restriction enzyme cleavage site of the present invention are preferably selected from one of BamH I and Xho I, respectively. In the embodiment, BamH I is used as the first restriction enzyme cleavage site and Xho I is used as the second restriction enzyme cleavage site as an example, but this is not the only example to be considered as the entire protection scope of the present invention. The IRES sequence of the present invention is preferably as shown in SEQ ID No. 4:

[0029] In the embodiment of the present invention, the nucleotide sequence of the circular RNA overexpression skeleton is preferably as shown in SEQ ID No. 5: TAAACTTAAGCTTGGTACCGAGCTCTGAAAACACGGGTTATTCCCCTCCTGGCAGGTATATAGGAGCCCTATCAAAGTCGAGCTAACGGAATGGGGTTTTCTTTCCCTCCTCTTCAGGGATCCGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGATTAAAACAGCGGATGGGTACCCCACCATCCGACCCACTGGGTGTAGTACTCTGGTACTTCGTACCTTTGTACGCCTGTTCTTCCCATTGTACCCTTCCTGAACTTCCAACCCAAGTAACGTTAGAAGCTCAACATTTAGTACAACAGGAAGCACCACATCCAGTGGTGTTTAGTACAAGCACTTCTGTTTCCCCGGAGCGAGGTATAGGCTGTACCCACTGCCAAAAACCTTTAACCGTTATCCGCCAACCAACTACGTAAAAGCTAGTAGTATTATGTTTTTAACTAGGCGTTCGATCAGGTGGATTTCCCCTCCACTAGTTTGGTCGATGAGGCTAGGAATTCCCCACGGGTGACCGTGTCCTAGCCTGCGTGGCGGCCAACCCAGCTTATGCTGGGACGCCTTTTTATAGACATGGTGTGAAGACTCGCATGTGCTTGGTTGTGATTCCTCCGGCCCCTGAATGCGGCTAACCTTAACCCTGGAGCCTTGTGTCACAAACCAGTGATGATAAGGTCGTAATGAGCAATTCCGGGACGGGACCGACTACTTTGGGTGTCCGTGTTTCTTATTTTTCTTATTATTGTCTTATGGTCACAGCATATATATAACATATACTGTGATCCTCGAGGTAAGACTTGCTTTTGTCAGTGGGGTGGCTCCCAGGATGCAGGGTCCCATAGGAGGGGAATAACCCGTGTTTTCA TCTAGAGGGCCCGTTTAAACCCGCTIn the sequence shown in SEQ ID No. 5 of the present invention, the 1st to 25th bp are upstream homology arm sequences for homologous recombination with the backbone plasmid, the 26th to 117th bp are upstream cyclization-promoting sequences, the 118th to 123th bp are restriction enzyme cleavage sites BamH I, the 124th to 224th bp are spacer sequences, the 225th to 850th bp are IRES sequences, the 851st to 856th bp are restriction enzyme cleavage sites Xho I, the 857th to 931th bp are downstream cyclization-promoting sequences, and the 932th to 956th bp are downstream homology arm sequences. The length of the adjusted new sequence is 956 bp.

[0030] The present invention also provides a recombinant vector comprising the circular RNA overexpression skeleton.

[0031] The basic vector of the recombinant vector of the present invention preferably includes a eukaryotic expression vector. pcDNA3.1(+) is used as an example in the embodiments, but it cannot be considered as the only vector in the entire protection scope of the present invention.

[0032] The present invention also provides a method for constructing the above-mentioned recombinant vector, comprising the following steps: using the above-mentioned circular RNA overexpression skeleton and a fragment (SEQ ID No. 5) with a length of 956 bp of homologous arm sequences at both ends as a template, after PCR amplification, recovering the target nucleic acid fragment, and connecting it to the basic vector by homologous recombination to obtain the recombinant vector.

[0033] The amplification primer design region of the present invention is the homology arm (underlined) portion, and the primer nucleotide sequence is as follows:

[0034] Frame-F (SEQ ID No. 6): 5' TAAACTTAAGCTTGGTACCGAGCTC 3', Frame-R (SEQ ID No. 7): 5' AGCGGGTTTAAACGGGCCCTCTAGA 3'.

[0035] The present invention preferably uses the synthesized SEQ ID No. 5 as a template to amplify a sequence containing homology arms at both ends. The amplification system preferably includes 25 μL of 2×Max buffer, 1 μL of dNTP, 2 μL of upstream and downstream primers (10 mM), and 1 μL of the synthesized backbone sequence DNA template (about 100 ng). 1 μL of Max Super-Fidelity DNA Polymerase and the balance of sterile water. The amplification reaction conditions of the present invention are preferably: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 1 minute, for a total of 30 cycles; further extension at 72°C for 5 minutes, followed by storage at 4°C.

[0036] The present invention preferably recovers the PCR product by gel tapping and purifies it using a gel recovery kit; the eukaryotic expression vector is double-enzyme-digested with an endonuclease at the first and second restriction sites, and then recovered and purified using a cleaning recovery kit; then the recovered PCR product is recombined into the double-enzyme-digested eukaryotic expression vector to obtain a recombinant vector containing a circular RNA overexpression skeleton.

[0037] The present invention also provides the use of the circular RNA overexpression skeleton or the recombinant vector in expressing target genes or circular RNA.

[0038] Under the premise of ensuring the efficient circularization efficiency of circRNA, the present invention optimizes the spacer sequence and IRES sequence required for its protein translation to further improve the protein translation efficiency of circRNA, in order to apply it to subsequent vaccine development and gene therapy.

[0039] The present invention also provides a method for expressing a target gene using the above-mentioned recombinant vector, comprising the following steps: dividing the nucleotide sequence of the target gene into two segments, up and down, recombining the down segment into a first restriction enzyme cleavage site, and recombining the up segment into a second restriction enzyme cleavage site, to obtain a recombinant vector expressing the target gene;

[0040] The recombinant vector expressing the target gene is used to transform eukaryotic cells to express the target gene.

[0041] The present invention preferably divides the nucleotide sequence of the target gene into two segments, up and down, and then uses BamH I enzyme to cut the vector at the BamH I position of the circular RNA expression backbone, and recombines the down segment into the circular RNA expression backbone; on this basis, continues to use Xho I enzyme to cut the vector at the Xho I position of the circular RNA expression backbone, and recombines the up segment into the circular RNA expression backbone in the previous step. The present invention segments the nucleotide sequence of the target gene, such as in the embodiment of the present invention, dividing the 720bp GFP gene into two segments of 285bp and 435bp.

[0042] To further illustrate the present invention, the circular RNA expression vector for a translatable protein, its construction method, and its expression method provided by the present invention are described in detail below in conjunction with the examples. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] Circular RNA overexpression backbone design

[0045] The circular RNA expression skeleton designed by the present invention includes an upstream homology arm sequence, an upstream cyclization-promoting sequence, an enzyme cleavage site BamH I, a spacer sequence, an IRES sequence, an enzyme cleavage site Xho I, a downstream cyclization-promoting sequence and a downstream homology arm sequence. The base sequence of the skeleton is shown in SEQ ID No. 5. The 1st to 25bp are upstream homology arm sequences, which are used for homologous recombination with the skeleton plasmid, the 26th to 117bp are upstream cyclization-promoting sequences, the 118th to 123bp are enzyme cleavage sites BamH I, the 124th to 224bp are spacer sequences, the 225th to 850bp are IRES sequences, the 851st to 856bp are enzyme cleavage sites Xho I, the 857th to 931bp are downstream cyclization-promoting sequences, and the 932th to 956bp are downstream homology arm sequences. The skeleton composition diagram is shown in FIG. Figure 1 shown.

[0046] Example 2

[0047] Obtaining circular RNA expression backbone sequences

[0048] The complete backbone sequence of the circular RNA overexpression backbone described in Example 1 was chemically synthesized. Using the synthesized backbone sequence DNA as a template, upstream and downstream amplification primers of the backbone sequence were designed. After PCR amplification, the full-length sequence was ligated into a plasmid vector using homologous recombination.

[0049] 1. Design PCR amplification primers

[0050] Frame-F (SEQ ID No. 6) and Frame-R (SEQ ID No. 7), the amplified fragment size is 956 bp.

[0051] 2. PCR amplification of circular RNA expression backbone

[0052] High-fidelity enzyme Max Super-Fidelity DNA Polymerase (Vazyme) and the above primers were used to prepare a 50 μL PCR reaction system, 2×Max buffer 25 μL, dNTP 1 μL, 2 μL of upstream and downstream primers (10 mM), and 1 μL of the synthesized backbone sequence DNA template (about 100 ng). Max Super-Fidelity DNA Polymerase 1 μL was added and the volume was made up to 50 μL with sterile water. The reaction conditions were: 95°C pre-denaturation for 5 min; 30 cycles of 95°C denaturation for 30 s, 56°C annealing for 30 s, and 72°C extension for 1 min; a further extension at 72°C for 5 min, and then storage at 4°C.

[0053] The PCR product was recovered by gel extraction and purified using a gel recovery kit; the eukaryotic expression vector pcDNA3.1(+) was double-digested with BamH I and Xho I and then recovered and purified using a clean recovery kit. The recovered backbone DNA was then recombined into the BamH I / Xho I double-digested pcDNA3.1(+) vector to obtain a new plasmid pc-Scirc-protein containing the circular RNA overexpression framework. Its schematic diagram can be found in Figure 2 .

[0054] Example 3

[0055] pc-Scirc-GFP overexpresses circular RNA

[0056] PCR amplification primers were designed based on the GFP gene (SEQ ID No. 16), and the GFP gene sequence was divided into two segments, up (G) and down (FP), with nucleotide sequence lengths of 285 bp and 435 bp, respectively. The GFP-down and GFP-up sequences were then sequentially recombined into the circular RNA overexpression backbone pc-Scirc-protein constructed in Example 2 of the present invention through the BamH I and Xho I restriction sites to construct the pc-Scirc-GFP overexpression plasmid. The schematic diagram of its backbone composition is shown in FIG. Figure 3 Finally, the vector was transfected into HEK 293 cells to detect the expression efficiency.

[0057] The constructed circular RNA overexpression backbone was tested by SYBR dye fluorescence quantitative PCR to efficiently overexpress the target circular RNA. The specific steps are as follows:

[0058] 1. Design of primers for circular RNA GFP PCR amplification

[0059] Primers were designed using Primer Premier 5.0, and 15 bp homology arms were added to the 5' ends of the forward and reverse primers for homologous recombination with the pcDNA3.1(+) plasmid. The primer sequences are as follows:

[0060] circGFP-up-F (SEQ ID No. 8): 5'ACATATACTGTGATCATGGTGAGCA AGGGCGAGGAG 3',

[0061] circGFP-up-R (SEQ ID No. 9): 5'AAAAGCAAGTCTTACCTGGACGTA GCCTTCGGGCAT 3';

[0062] circGFP-down-F (SEQ ID No.10): 5'TTCCCTCCTCTTCAGGAGCGCA CCATCTTCTTCAAG3',

[0063] circGFP-down-R (SEQ ID No. 11)5'CACCGAGGCTCCAGCCTACTTGTA CAGCTCGTCCATGC3'.

[0064] 2. PCR amplification of circular RNA GFP sequence

[0065] High-fidelity enzyme Prepare 50 μL PCR reaction system with Max Super-Fidelity DNA Polymerase and the above primers, 25 μL 2×Max buffer, 1 μL dNTP, 2 μL each of upstream and downstream primers (10 mM), and 1 μL DNA template (about 100 ng). Max Super-Fidelity DNA Polymerase (1 μL) was added and the volume was brought to 50 μL with sterile water. The reaction conditions were: 95°C pre-denaturation for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds; an additional extension at 72°C for 5 minutes, followed by storage at 4°C. The PCR product was recovered by gel tapping and recombined into the pc-Scirc-protein vector using the restriction enzymes BamHI and XhoI. The new plasmid was named pc-Scirc-GFP.

[0066] 3. Fluorescence quantitative PCR detection of circular RNA overexpressed in pc-Scirc-GFP

[0067] pc-Scirc-protein and pc-Scirc-GFP were transfected into HEK 293 cells using Biobest transfection reagent at a plasmid transfection concentration of 1 μg / mL. Circular RNA expression was detected by fluorescence quantitative PCR 24 h after transfection.

[0068] Primer Premier 5.0 was used to design back-to-back primers for amplification containing the circular RNA GFP linker sequence. The primer sequences are as follows:

[0069] q-circGFP-F (SEQ ID No. 12): 5'CGACCACATGAAGCAGCACGACT 3', q-circGFP-R (SEQ ID No. 13): 5'TCAGCTCGATGCGGTTCACCA3'.

[0070] GAPDH gene was selected as the internal reference gene and used as a correction control for the fluorescence quantitative results. The primer sequences are as follows:

[0071] q-GAPDH-F(SEQ ID No.14)5'TGGTGAAGGTCGGAGTGAAC 3',

[0072] q-GAPDH-R (SEQ ID No. 15) 5'GGAAGAT GGTGATGGGATTTC 3'.

[0073] The specific detection method is as follows.

[0074] (1) Extraction of total cellular RNA. Total cellular RNA was extracted from HEK 293 cells transfected with pc-Scirc-protein and pc-Scirc-GFP plasmids strictly according to the instructions of RNA isolater Total RNA Extraction Reagent.

[0075] (2) RNA was reverse transcribed into cDNA. Total RNA was reverse transcribed using HiScript IIQ RT SuperMix for qPCR (+gDNA wiper). The reaction system and reaction conditions were as follows: 950 ng RNA, 4 μL 4× gDNA wiper Mix, 16 μL ddH2O, mixed and incubated at 42°C in a water bath for 2 min, then 4 μL 5× HiScript II qRT SuperMix II was added to the system, mixed and incubated at 50°C for 15 min, and then at 85°C for 5 s for reverse transcription. The obtained cDNA was stored in a -40°C refrigerator for subsequent qPCR detection.

[0076] (3) Fluorescence quantitative detection of circular RNA GFP. The expression level of circular RNA GFP in the cDNA obtained in the previous step was detected using the AceQ qPCR SYBR Green MasterMix kit (Vazyme). The detection system was as follows: 2×AceQ qPCR SYBR Green Master Mix 10μL, 0.4μL each of 10μM upstream and downstream primers, cDNA 4μL, ddH2O 5.2μL. The qPCR program was 1 cycle of pre-denaturation at 95℃ for 5min; 40 cycles of 95℃ for 10s, 60℃ for 30s, and 95℃ for 15s; and 1 cycle of 60℃ for 30s and 95℃ for 15s.

[0077] Fluorescence quantitative results such as Figure 4 As shown in Figure 3, the expression level of circular RNA in HEK 293 cells transfected with the pc-Scirc-GFP plasmid was more than 5000 times that in HEK 293 cells transfected with the backbone plasmid pc-Scirc-protein.

[0078] 4. Fluorescence Observation of Circular RNA Overexpression with pc-Scirc-GFP

[0079] pc-Scirc-protein and pc-Scirc-GFP were transfected into HEK 293 cells using Biobest transfection reagent at a plasmid transfection concentration of 2 μg / mL. 72 hours after transfection, protein translation of the circular RNA GFP was observed using a fluorescence microscope. No fluorescent signal was observed in HEK 293 cells transfected with the pc-Scirc-protein plasmid, but a clear green fluorescent signal was observed in HEK 293 cells transfected with the pc-Scirc-GFP plasmid. This demonstrates that the circular RNA expression backbone constructed in this invention can be used to express circular RNAs capable of protein translation.

[0080] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A circular RNA overexpression backbone, characterized in that The method comprises the following structures connected in sequence: an upstream homology arm sequence, an upstream cyclization-promoting sequence, a first restriction enzyme cleavage site, a spacer sequence, an IRES sequence, a second restriction enzyme cleavage site, a downstream cyclization-promoting sequence, and a downstream homology arm sequence; The upstream cyclization-promoting sequence and the downstream cyclization-promoting sequence are respectively selected from one of the sequences shown in SEQ ID No. 1 and SEQ ID No. 2; The spacer sequence is shown in SEQ ID No. 3; The first restriction enzyme cleavage site and the second restriction enzyme cleavage site are respectively selected from one of BamH I and Xho I; The IRES sequence is shown as SEQ ID No.

4.

2. The circular RNA overexpression backbone according to claim 1, wherein The nucleotide sequence of the circular RNA overexpression skeleton includes the sequence shown in SEQ ID No.

5.

3. A recombinant vector comprising the circular RNA overexpression backbone according to claim 1 or 2.

4. The recombinant vector according to claim 3, characterized in that The basic vector of the recombinant vector includes a eukaryotic expression vector.

5. The method for constructing the recombinant vector according to claim 3 or 4, characterized in that: The method comprises the following steps: connecting the circular RNA overexpression skeleton according to claim 1 or 2 to a basic vector in a homologous recombination manner to obtain the recombinant vector.

6. The construction method according to claim 5, characterized in that: The basic vector includes pcDNA3.1(+).

7. Use of the circular RNA overexpression backbone according to claim 1 or 2 or the recombinant vector according to claim 3 or 4 in expressing a target gene or circular RNA.

8. A method for expressing a target gene using the recombinant vector according to claim 3 or 4, characterized in that: The following steps are involved: The nucleotide sequence of the target gene is divided into two segments, up and down, and the down segment is recombined into the first restriction enzyme cutting site, and then the up segment is recombined into the second restriction enzyme cutting site to obtain a recombinant vector expressing the target gene; The recombinant vector expressing the target gene is used to transform eukaryotic cells to express the target gene.

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