CN118086288B - Polycistronic self-amplified RNA and preparation method thereof - Google Patents

Abstract

The invention provides a polycistronic self-amplified RNA and a preparation method thereof. The invention is based on the non-structural protein sequence and subgenomic promoter of the saRNA of alphavirus source, connects the first target gene behind the first subgenomic promoter partially overlapped with the nsP1234, and then repeatedly connects the subgenomic promoter and other target gene frameworks, thereby realizing the expression of a plurality of target genes and the effect of self-amplifying of a plurality of target genes.

Description

Polycistronic self-amplified RNA and preparation method thereof

Technical Field

The invention relates to the technical field of biology, in particular to a polycistron self-amplifying RNA and a preparation method thereof.

Background

MRNA vaccines are broadly classified into non-replicating mRNA vaccines and self-amplifying RNA vaccines. Compared with non-replicating mRNA vaccine, self-amplified RNA (Self-AMPLIFYING RNA, SARNA) can Self-replicate by using its own RNA sequence as a template. Lower doses of saRNA can achieve similar levels of immunogen protein expression and equivalent protective efficacy against viruses. Furthermore, the use of lower doses of saRNA will minimize the use of delivery materials (such as cationic liposomes), thereby helping to control costs and potential side effects.

The saRNA is longer than the traditional mRNA length, which can be as much as 9-12 kb. This is because, in addition to the most basic mRNA elements, such as caps, 5'UTR, 3' UTR, polyA tails, the sarNA also comprises a Non-structural protein (Non-structural protein) sequence from Alphavirus and a 26S subgenomic Promoter (Subgenomic Promoter, SG-Promoter) located upstream of the gene of interest (GOI), the Non-structural protein (Non-structural protein) sequence encoding the 4 Non-structural proteins (nsP 1, nsP2, nsP3, nsP 4) from the virus, the structure of which is shown in FIG. 4A. Deletion of viral structural proteins renders saRNA incapable of producing infectious viruses. Upon delivery into the cytosol of the cell, the released saRNA has translational capability and binds to host cell ribosomes to produce four functional components of an RNA-dependent RNA polymerase (RDRP) or viral genome replication device: nsP1, nsP2, nsP3 and nsP4.

As shown in fig. 1, translation of the nonstructural proteins of saRNA within the host cell is a multi-stage process. Early in the entry of saRNA into host cells, two precursor polyproteins P123 and P1234 are expressed using the cellular translation machinery, most of the translation products are P123, only a few are P1234, P1234 being cis-cleaved into P123 and nsP4 by the activity of nsP2 protease. As an early replication complex, P123+nsP4 was used to replicate negative strand RNA using positive strand RNA as template. Once the p123+nsp4 complex accumulates, P123 breaks down in trans to form nsP1, P23 and nsP4 complexes, enabling the synthesis of negative and positive strand RNAs. With cis-acting cleavage of P23 (production of nsP2 and nsP 3), a stable late replication complex consisting of all nonstructural proteins is formed. These four single component nonstructural proteins are then converted to RDRP, which is responsible for the synthesis of both positive-strand genomic and subgenomic mRNA. The synthesis of negative strand genomic RNA transcribes positive strand subgenomic mRNA via subgenomic promoters and then translates the gene of interest. Compared with the traditional mRNA technology, the saRNA has huge potential and can show better RNA method. There is currently increasing research on saRNA, but no attempt has been made to date to use saRNA for independent polycistronic simultaneous self-amplification and expression.

Polycistronic is often present in prokaryotes, and a transcript can be translated for multiple proteins. Eukaryotic organisms often utilize the monocistronic strategy. In molecular cloning, we can mimic this feature of prokaryotes, using polycistronic elements in eukaryotes, concatenating multiple Open reading frames (Open READING FRAME, ORF) and expressing multiple genes simultaneously. Currently, IRES (Internal Ribosome ENTRY SITE, IRES) elements or Self-cleaving polypeptide 2A (Self-cleaving A peptide, 2A) elements are widely used in eukaryotic cells. IRES elements have the function of independently recruiting ribosomes independently of cap structure, and thus can initiate translation of downstream genes. IRES has a major disadvantage, however, in that the expression level of the downstream cistron is relatively low (typically 10% -20% upstream) compared to the upstream cistron. To overcome some of the shortcomings of IRES elements, scientists engineered self-cleaving polypeptide 2A into polycistronic vectors. These short peptides are believed to act by skipping the synthesis of the C-terminal peptide bond of the 2A element by the ribosome, resulting in the final production of an upstream protein fused to the 2A peptide tail and a downstream protein with a proline at the N-terminus, respectively, due to their unique "cleavage" mechanism. The 2A element has the advantage that cistron belongs to the same coding frame, and the expression intensity tends to be consistent. However, the disadvantage is that an additional 2A peptide residue sequence is added to the N-terminal or C-terminal of the target protein, which may have a certain influence on the function of the target protein. However, due to the advantages and disadvantages of both IRES and 2A peptides, there is sometimes a need to find new methods for polycistronic expression in vitro experiments.

Disclosure of Invention

The invention provides polycistronic self-amplifying RNA, which has a structure shown in a formula (I):

（I）

Wherein the SGP-n unit and the GOI-n unit form a repeating frame, n is an integer greater than 1, and the UTR unit, the nsP1234/SGP-1 unit and the sequences of the SGP-n units in the repeating frame are all derived from alphaviruses.

The present invention provides a repeat framework, which refers to the combination of SGP units and GOI units, and the present invention provides self-amplifying RNA, wherein the 5 '-end of the first SGP-1 unit coincides with the 3' -end of nsP1234, i.e., the first SGP-1 unit has a partial overlapping sequence with the nsP1234 unit, and the SGP-n unit and the GOI-n unit are independent of each other and exist in tandem except for the SGP-1 overlapping with the nsP1234 and the GOI-1 unit connected thereto, so that the combination of SGP-n unit and GOI-n unit is called a repeat framework, but the specific sequence within each repeat framework may be different. In some embodiments, the self-amplifying RNA comprises one repeat framework, namely repeat framework 1 (comprising SGP+GOI-2), wherein GOI-1 and GOI-2 each express a different protein. In some embodiments, the self-amplifying RNA comprises two repeat frames, repeat frame 1 (comprising SGP+GOI-2) and repeat frame 2 (comprising SGP+GOI-3), respectively, wherein GOI-1, GOI-2 and GOI-3 express different proteins, respectively. In some embodiments, the self-amplifying RNA comprises three repeats, repeat 1 (containing SGP+GOI-2), repeat 2 (containing SGP+GOI-3), and repeat 3 (containing SGP+GOI-4), wherein GOI-1, GOI-2, GOI-3, and GOI-4 each express a different protein. The "-1" and "-n" in SGP-1 and SGP-n are used to mark the sequence number of the SGP unit in formula (I), as is the case for GOI units.

The polycistronic self-amplifying RNA (polycistronic/multicistronic self-AMPLIFYING RNA, PSARNA/msaRNA) provided by the invention, wherein the sequences of all units are independent of each other except that the 3 'end of the nsP1234 and the 5' end of the 1 st SGP unit are partially overlapped.

The alphavirus (alphavirus) is a kind of enveloped, single-stranded, sense RNA virus transmitted by arthropods such as mosquitoes, belongs to the togaviridae (Togaviridae), can widely infect various animals such as human, birds, mice, horses and the like and cause related diseases, such as Venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEEV), forest encephalitis virus (Semliki Forest virus, SFV), sindbis virus (SINV) and the like.

The polycistronic self-amplification RNA (polycistronic/multicistronic self-AMPLIFYING RNA, PSARNA/msaRNA) designed by the invention can independently and automatically amplify and express proteins of polycistronic RNA on one RNA chain while retaining the self-amplification effect of the conventional monocistronic saRNA. The method is simple and easy to implement, and can customize the expression of a plurality of target genes in one cell without distinction.

Further, n is any one integer from 2 to 5. Specifically, n may be 2, 3, 4, 5.

Further, the alphavirus in the self-amplifying RNA is selected from one or more of VEEV, SFV or SINV.

In some embodiments, the sequence of the UTR unit, nsP1234 unit, and each SGP unit of the self-amplifying RNA is derived from the same alphavirus. In some embodiments, the sequence of the UTR unit, the nsP1234/SGP-1 unit, and each SGP-n unit of the self-amplifying RNA is derived from VEEV. In some embodiments, the sequence of the UTR unit, the nsP1234/SGP-1 unit, and each SGP-n unit of the self-amplifying RNA is derived from SFV. In some embodiments, the sequence of the UTR unit, the nsP1234/SGP-1 unit, and each SGP-n unit of the self-amplifying RNA is derived from SINV.

In some embodiments, at least one of the UTR units, nsP1234/SGP-1 units, each SGP-n unit of self-amplifying RNA is of a different alphavirus source than the other units. In some embodiments, the UTR units of the self-amplifying RNA are of different alphavirus origin than the nsP1234/SGP-1 units, each SGP-n unit. In some embodiments, the nsP1234/SGP-1 unit of self-amplifying RNA is of a different alphavirus origin than the UTR unit, each SGP-n unit. In some embodiments, the self-amplifying RNA comprises two repeat frames, SGP-2 and SGP-3 within the two repeat frames are each from a different alphavirus.

In some embodiments, the sequence of the 5' utr unit is as set forth in SEQ ID NO: 1. 8 or 15; the sequence of the 3' UTR unit is shown as SEQ ID NO: 7. 14 or 21; the SGP unit has a sequence shown in SEQ ID NO: 6. 13 or 20; the sequences of the nsP1234/SGP-1 units are a series-connected nsP1234 sequence and a series-connected SGP unit sequence, except that the 5 '-end of the SGP unit sequence overlaps with the 3' -end of the nsP1234 sequence, wherein the nsP1234 sequence is an RNA coding sequence of a series-connected nsP1 protein, a series-connected nsP2 protein, a series-connected nsP3 protein and a series-connected nsP4 protein, and the RNA coding sequence of the nsP1 protein is shown as SEQ ID NO: 2. 9 or 16, wherein the RNA coding sequence of the nsP2 protein is shown as SEQ ID NO: 3. 10 or 17, wherein the RNA coding sequence of the nsP3 protein is shown as SEQ ID NO: 4. 11 or 18, wherein the RNA coding sequence of the nsP4 protein is shown as SEQ ID NO: 5. 12 or 19.

The present invention also provides a plasmid capable of transcribing any one of the self-amplifying RNAs as described above.

Further, the plasmid also comprises a T7 promoter. In some embodiments, the T7 promoter is located in front of (i.e., 5 'to) the coding sequence of the 5' utr unit.

In some embodiments, the plasmid further comprises a truncated human RNA polymerase I promoter. In some embodiments, the truncated human RNA polymerase I promoter is located before the T7 promoter.

In some embodiments, in the design and construction of plasmid, in T7 promoter design in front of truncated human RNA polymerase I promoter, the promoter directly transfected plasmid into eukaryotic cells after direct transcription from amplified RNA play a role. The T7 promoter can be used for in vitro transcription to artificially synthesize self-amplified RNA, followed by an alphavirus-derived 5'UTR and a nsP1234 sequence, wherein the 5' end part sequence of the first subgenomic promoter overlaps the 3 'end part of the nsP4 sequence, a target gene 1 (such as a green fluorescent protein reporter gene: neonGreen) is designed after the first subgenomic promoter, the sequence frame (repeatable) of the subgenomic promoter and other target genes (such as a red fluorescent protein reporter gene: mTagRFP) is again ligated after the last codon NeonGreen, then the alphavirus-derived 3' UTR and polyA tail are ligated, and a single cleavage site is designed after the polyA tail, followed by an RNA polymerase I terminator (SEQ ID NO: 28) and a T7 terminator (SEQ ID NO: 26).

SEQ ID NO:26: t7 terminator sequence.

tagcataaccccttggggcctctaaacgggtcttgaggggttttttg

SEQ ID NO:28: RNA polymerase I terminator sequence.

tcccccccaacttcggaggtcgaccagtactccgggcgacactttgttttttttttttcccccgatgctggaggtcgaccagatgtccgaaagtgtcccc In some embodiments, in the design and construction of plasmid, in T7 promoter design in front of truncated human RNA polymerase I promoter, the promoter directly transfected plasmid into eukaryotic cells after direct transcription from amplified RNA play a role. The T7 promoter can be used for in vitro transcription and artificial synthesis of self-amplified RNA, and is followed by a 5'UTR and a nsP1234 sequence derived from VEEV virus, wherein a first SGP partial sequence is overlapped with a 3' -end part of the nsP4 sequence, and a target gene 1 (such as blue fluorescent protein reporter gene: mTagBFP) is connected with the first SGP sequence; the last codon of mTagBFP is connected with a second VEEV virus-derived subgenomic promoter and other target genes (such as green fluorescent protein reporter gene: neonGreen), the last codon of NeonGreen is connected with a third VEEV virus-derived subgenomic promoter and other target genes (such as red fluorescent protein reporter gene: mTagRFP), then the 3' UTR of VEEV virus and the polyA tail are connected, and a single enzyme cutting site is designed behind the polyA tail, and then a mouse-derived RNA polymerase I terminator and a T7 terminator are arranged.

The invention also provides a preparation method of the self-amplified RNA, which comprises the following steps:

s1: constructing any one of the plasmids described above;

in some embodiments, when constructing a plasmid for polycistronic self-amplifying RNA, the nonstructural protein sequences on the plasmid are derived from VEEV and SFV.

In some embodiments, following the viral non-structural protein sequence, the gene of interest GOI-1 sequence is designed and then the viral subgenomic promoter and the gene of interest GOI-2 sequence are ligated.

In some embodiments, the sequence of the gene of interest GOI-1 is designed after the first SGP-1 sequence; then connecting a second SGP-2 sequence and a target gene GOI-2 sequence; then, the third SGP-3 sequence is ligated to the target gene GOI-3 sequence.

S2: replicating the plasmid and extracting;

s3: carrying out linearization cutting on the extracted plasmid to obtain a linearization plasmid;

In some embodiments, plasmid linearization is the enzymatic cleavage of a plasmid expressing polycistronic self-amplifying RNA following the polyA tail using a restriction endonuclease cleavage procedure.

S4: the linearized plasmid is transcribed to obtain self-amplifying RNA.

In some embodiments, the preparation of polycistronic self-amplifying RNA (psaRNA/msaRNA) is performed using an in vitro co-transcriptional capping method.

The polycistronic self-amplification RNA (psaRNA/msaRNA) designed by the invention has the structural formula shown in the formula (I), comprises a plurality of GOIs, and can independently and self-amplify polycistronic RNA and express protein on one RNA chain while retaining the self-amplification effect of the traditional monocistronic saRNA through SGP arranged in front of each GOI. The method is simple and easy to implement, and can customize the expression of a plurality of target genes in one cell without distinction.

Drawings

For a better understanding of the invention and to show more clearly how it may be carried into effect, features according to embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:

fig. 1: schematic representation of replication and expression of conventional saRNA in cells.

Fig. 2: psaRNA replication and expression in cells.

Fig. 3: schematic representation of replication and expression of resolved VEEVs 1234-psaRNA and VEEVsgp-repotersRNA in cells.

Fig. 4: a is a schematic diagram of a conventional saRNA structural element, B is a schematic diagram of a VEEV-psaRNA polycistronic self-amplifying element containing 2 reporter genes and only a VEEV sequence, C is a schematic diagram of an SFV-VEEV-psaRNA polycistronic self-amplifying element containing 2 reporter genes and containing an SFV and VEEV heterozygous sequence, D is a schematic diagram of a split VEEV1234-psaRNA + VEEVsgp-repotersRNA containing 2 reporter genes, and E is a schematic diagram of a VEEV-psaRNA containing 3 reporter genes.

Fig. 5: gel electrophoresis detection patterns of VEEV-PSaRNA, SFV-VEEV-PSaRNA, VEEV1234-psaRNA and VEEVsgp-repotersRNA.

Fig. 6: a is a fluorescence detection diagram (containing 2 reporter genes: green fluorescent protein and red fluorescent protein) of a plasmid molecule and an RNA molecule combined by VEEV-psaRNA, SFV-VEEV-psaRNA and VEEV1234-psaRNA + VEEVsgp-repotersRNA after transfection, and B is a fluorescence detection diagram (containing 3 reporter genes: blue fluorescent protein, green fluorescent protein and red fluorescent protein) of a plasmid-5 after transfection.

Detailed Description

Definition: in order to provide a clear and consistent understanding of the terms used in the description of the present invention, some definitions are provided below. Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The use of the word "a" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "an" but it is also known to the meaning of "one or more", "at least one" and "one or more". Similarly, the word "another" may mean at least a second or a plurality.

The word "comprising" (and any form of comprising, such as "comprising" and "comprises"), "having" (and any form of having, "having", "including" and "containing") as used in this specification and claims is inclusive and open-ended and does not exclude additional unrecited elements or process steps.

As used herein, "self-amplifying RNA," "self-replicating RNA," "self-amplifying RNA," and "saRNA" are used interchangeably, and are distinguished from common mRNA by the ability to self-replicate using their own RNA sequences as templates. Typically, mRNA encodes a protein that needs to be expressed, translation and protein production are accomplished using ribosomes in the cell, while saRNA carries sequences capable of expressing an RNA polymerase (known as RNA-dependent RNA polymerase, rdRP) in addition to the protein of interest. After the RNA polymerase is generated, more copies of the SARNA can be generated using the SARNA as a template.

As used herein, "polycistronic self-amplifying RNA," "polycistronic self-AMPLIFYING RNA," "multicistronic self-AMPLIFYING RNA," "psaRNA," and "msaRNA" are used interchangeably and refer to a saRNA possessing multiple expressed genes GOI.

As used herein, "SG Promoter", "SG-Promoter", "SGP", "SG-P" are used interchangeably and refer to subgenomic promoters.

As used herein, nsP1234 is a combined sequence encoding 4 nonstructural proteins nsP1, nsP2, nsP3 and nsP4 from a virus.

Examples: the invention will be more readily understood by reference to the following examples, which are provided to illustrate the invention and should not be construed to limit the scope of the invention in any way.

Unless defined otherwise or the context clearly indicates otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

Although the present invention has been described in detail with reference to the embodiments thereof, these embodiments are provided for the purpose of illustration and not limitation of the invention. Other embodiments that can be obtained according to the principles of the present invention fall within the scope of the invention as defined in the claims.

The experimental methods not specifically described in the present invention are carried out according to the methods described in the "molecular cloning Experimental guidelines (Fourth Edition) J.Sam Brooks (Joseph. Sambrook, molecular Cloning: A Laboratory Manual (Fourth Edition)), or according to the specifications of the related products. As used herein, all terms herein are to be understood in their ordinary sense as known in the art unless otherwise indicated. The biological agents used in the present invention, without specific description, are commercially available.

Example 1: plasmid for designing and constructing polycistronic self-amplified RNA

The NCBI database is searched for the 5' UTR sequence, nsP1234 sequence, SG-Promoter sequence, and 3' UTR sequence of the alpha viruses such as VEEV, SFV and SINV, then other elements such as the UTR sequence, nsP1234 sequence, SG-Promoter sequence, neonGreen sequence and mTagRFP sequence of the VEEV are inserted into pUC plasmid skeleton, and two promoters are designed in front of the 5' UTR sequence, one is T7 Promoter for preparing the SARNA by in vitro transcription, and the other is truncated human RNA polymerase I Promoter for verifying the expression of the plasmid in eukaryotic cells.

Plasmid-1 is a homozygous plasmid of VEEV virus origin (shown as B in fig. 4). Wherein the 5' UTR sequence is as shown in SEQ ID NO:1, the RNA coding sequence of the nsP1 protein is shown as a sequence SEQ ID NO:2, the RNA coding sequence of the nsP2 protein is shown as a sequence SEQ ID NO:3, the RNA coding sequence of the nsP3 protein is shown as a sequence SEQ ID NO:4, the RNA coding sequence of the nsP4 protein is shown as a sequence SEQ ID NO:5, and wherein the RNA coding sequence end of nsP4 partially coincides with the SG-Promoter sequence of VEEV virus, and the independent SGP sequence is as set forth in SEQ ID NO:6, the 3' UTR sequence is shown as SEQ ID NO:7, the RNA coding sequence of NeoGreen protein is shown as SEQ ID NO:22, the RNA coding sequence of mTagRFP protein is shown as SEQ ID NO: 23.

SEQ ID NO:1: VEEV virus 5' utr sequence.

augggcggcgcaagagagaagcccaaaccaauuaccuacccaaa

SEQ ID NO:2: the RNA coding sequence of VEEV virus nsP 1.

auggagaaaguucacguugacaucgaggaagacagcccauuccucagagcuuugcagcggagcuucccgcaguuugagguagaagccaagcaggucacugauaaugaccaugcuaaugccagagcguuuucgcaucuggcuucaaaacugaucgaaacggagguggacccauccgacacgauccuugacauuggaagugcgcccgcccgcagaauguauucuaagcacaaguaucauuguaucuguccgaugagaugugcggaagauccggacagauuguauaaguaugcaacuaagcugaagaaaaacuguaaggaaauaacugauaaggaauuggacaagaaaaugaaggagcuggccgccgucaugagcgacccugaccuggaaacugagacuaugugccuccacgacgacgagucgugucgcuacgaagggcaagucgcuguuuaccaggauguauacgcgguugacggaccgacaagucucuaucaccaagccaauaagggaguuagagucgccuacuggauaggcuuugacaccaccccuuuuauguuuaagaacuuggcuggagcauauccaucauacucuaccaacugggccgacgaaaccguguuaacggcucguaacauaggccuaugcagcucugacguuauggagcggucacguagagggauguccauucuuagaaagaaguauuugaaaccauccaacaauguucuauucucuguuggcucgaccaucuaccacgagaagagggacuuacugaggagcuggcaccugccgucuguauuucacuuacguggcaagcaaaauuacacaugucggugugagacuauaguuaguugcgacggguacgucguuaaaagaauagcuaucaguccaggccuguaugggaagccuucaggcuaugcugcuacgaugcaccgcgagggauucuugugcugcaaagugacagacacauugaacggggagagggucucuuuucccgugugcacguaugugccagcuacauugugugaccaaaugacuggcauacuggcaacagaugucagugcggacgacgcgcaaaaacugcugguugggcucaaccagcguauagucgucaacggucgcacccagagaaacaccaauaccaugaaaaauuaccuuuugcccguaguggcccaggcauuugcuaggugggcaaaggaauauaaggaagaucaagaagaugaaaggccacuaggacuacgagauagacaguuagucaugggguguuguugggcuuuuagaaggcacaagauaacaucuauuuauaagcgcccggauacccaaaccaucaucaaagugaacagcgauuuccacucauucgugcugcccaggauaggcaguaacacauuggagaucgggcugagaacaagaaucaggaaaauguuagaggagcacaaggagccgucaccucucauuaccgccgaggacguacaagaagcuaagugcgcagccgaugaggcuaaggaggugcgugaagccgaggaguugcgcgcagcucuaccaccuuuggcagcugauguugaggagcccacucuggaggcagacgucgacuugauguuacaagaggcuggggcc

SEQ ID NO:3: the RNA coding sequence of VEEV virus nsP 2.

ggcucaguggagacaccucguggcuugauaaagguuaccagcuacgauggcgaggacaagaucggcucuuacgcugugcuuucuccgcaggcuguacucaagagugaaaaauuaucuugcauccacccucucgcugaacaagucauagugauaacacacucuggccgaaaagggcguuaugccguggaaccauaccaugguaaaguaguggugccagagggacaugcaauacccguccaggacuuucaagcucugagugaaagugccaccauuguguacaacgaacgugaguucguaaacagguaccugcaccauauugccacacauggaggagcgcugaacacugaugaagaauauuacaaaacugucaagcccagcgagcacgacggcgaauaccuguacgacaucgacaggaaacagugcgucaagaaagaacuagucacugggcuagggcucacaggcgagcugguggauccucccuuccaugaauucgccuacgagagucugagaacacgaccagccgcuccuuaccaaguaccaaccauagggguguauggcgugccaggaucaggcaagucuggcaucauuaaaagcgcagucaccaaaaaagaucuaguggugagcgccaagaaagaaaacugugcagaaauuauaagggacgucaagaaaaugaaagggcuggacgucaaugccagaacuguggacucagugcucuugaauggaugcaaacaccccguagagacccuguauauugacgaagcuuuugcuugucaugcagguacucucagagcgcucauagccauuauaagaccuaaaaaggcagugcucugcggggaucccaaacagugcgguuuuuuuaacaugaugugccugaaagugcauuuuaaccacgagauuugcacacaagucuuccacaaaagcaucucucgccguugcacuaaaucugugacuucggucgucucaaccuuguuuuacgacaaaaaaaugagaacgacgaauccgaaagagacuaagauugugauugacacuaccggcaguaccaaaccuaagcaggacgaucucauucucacuuguuucagagggugggugaagcaguugcaaauagauuacaaaggcaacgaaauaaugacggcagcugccucucaagggcugacccguaaagguguguaugccguucgguacaaggugaaugaaaauccucuguacgcacccaccucagaacaugugaacguccuacugacccgcacggaggaccgcaucguguggaaaacacuagccggcgacccauggauaaaaacacugacugccaaguacccugggaauuucacugccacgauagaggaguggcaagcagagcaugaugccaucaugaggcacaucuuggagagaccggacccuaccgacgucuuccagaauaaggcaaacguguguugggccaaggcuuuagugccggugcugaagaccgcuggcauagacaugaccacugaacaauggaacacuguggauuauuuugaaacggacaaagcucacucagcagagauaguauugaaccaacuaugcgugagguucuuuggacucgaucuggacuccggucuauuuucugcacccacuguuccguuauccauuaggaauaaucacugggauaacuccccgucgccuaacauguacgggcugaauaaagaagugguccgucagcucucucgcagguacccacaacugccucgggcaguugccacuggaagagucuaugacaugaacacugguacacugcgcaauuaugauccgcgcauaaaccuaguaccuguaaacagaagacugccucaugcuuuaguccuccaccauaaugaacacccacagagugacuuuucuucauucgucagcaaauugaagggcagaacuguccugguggucggggaaaaguuguccgucccaggcaaaaugguugacugguugucagaccggccugaggcuaccuucagagcucggcuggauuuaggcaucccaggugaugugcccaaauaugacauaauauuuguuaaugugaggaccccauauaaauaccaucacuaucagcagugugaagaccaugccauuaagcuuagcauguugaccaagaaagcuugucugcaucugaaucccggcggaaccugugucagcauagguuaugguuacgcugacagggccagcgaaagcaucauuggugcuauagcgcggcaguucaaguuuucccggguaugcaaaccgaaauccucacuugaagagacggaaguucuguuuguauucauuggguacgaucgcaaggcccguacgcacaauucuuacaagcuuucaucaaccuugaccaacauuuauacagguuccagacuccacgaagccggaugu

SEQ ID NO:4: the RNA coding sequence of VEEV virus nsP 3.

gcacccucauaucauguggugcgaggggauauugccacggccaccgaaggagugauuauaaaugcugcuaacagcaaaggacaaccuggcggaggggugugcggagcgcuguauaagaaauucccggaaagcuucgauuuacagccgaucgaaguaggaaaagcgcgacuggucaaaggugcagcuaaacauaucauucaugccguaggaccaaacuucaacaaaguuucggagguugaaggugacaaacaguuggcagaggcuuaugaguccaucgcuaagauugucaacgauaacaauuacaagucaguagcgauuccacuguuguccaccggcaucuuuuccgggaacaaagaucgacuaacccaaucauugaaccauuugcugacagcuuuagacaccacugaugcagauguagccauauacugcagggacaagaaaugggaaaugacucucaaggaagcaguggcuaggagagaagcaguggaggagauaugcauauccgacgacucuucagugacagaaccugaugcagagcuggugagggugcauccgaagaguucuuuggcuggaaggaagggcuacagcacaagcgauggcaaaacuuucucauauuuggaagggaccaaguuucaccaggcggccaaggauauagcagaaauuaaugccauguggcccguugcaacggaggccaaugagcagguaugcauguauauccucggagaaagcaugagcaguauuaggucgaaaugccccgucgaagagucggaagccuccacaccaccuagcacgcugccuugcuugugcauccaugccaugacuccagaaagaguacagcgccuaaaagccucacguccagaacaaauuacugugugcucauccuuuccauugccgaaguauagaaucacuggugugcagaagauccaaugcucccagccuauauuguucucaccgaaagugccugcguauauucauccaaggaaguaucucguggaaacaccaccgguagacgagacuccggagccaucggcagagaaccaauccacagaggggacaccugaacaaccaccacuuauaaccgaggaugagaccaggacuagaacgccugagccgaucaucaucgaagaggaagaagaggauagcauaaguuugcugucagauggcccgacccaccaggugcugcaagucgaggcagacauucacgggccgcccucuguaucuagcucauccugguccauuccucaugcauccgacuuugauguggacaguuuauccauacuugacacccuggagggagcuagcgugaccagcggggcaacgucagccgagacuaacucuuacuucgcaaagaguauggaguuucuggcgcgaccggugccugcgccucgaacaguauucaggaacccuccacaucccgcuccgcgcacaagaacaccgucacuugcacccagcagggccugcucgagaaccagccuaguuuccaccccgccaggcgugaauagggugaucacuagagaggagcucgaggcgcuuaccccgucacgcacuccuagcaggucggucucgagaaccagccuggucuccaacccgccaggcguaaauagggugauuacaagagaggaguuugaggcguucguagcacaacaacaaugacgguuugaugcgggugca

SEQ ID NO:5: the RNA coding sequence of the VEEV virus nsP4 (3' -end bold sequence is the part overlapping with the VEEV virus SG promoter sequence).

uacaucuuuuccuccgacaccggucaagggcauuuacaacaaaaaucaguaaggcaaacggugcuauccgaagugguguuggagaggaccgaauuggagauuucguaugccccgcgccucgaccaagaaaaagaagaauuacuacgcaagaaauuacaguuaaaucccacaccugcuaacagaagcagauaccaguccaggaagguggagaacaugaaagccauaacagcuagacguauucugcaaggccuagggcauuauuugaaggcagaaggaaaaguggagugcuaccgaacccugcauccuguuccuuuguauucaucuagugugaaccgugccuuuucaagccccaaggucgcaguggaagccuguaacgccauguugaaagagaacuuuccgacuguggcuucuuacuguauuauuccagaguacgaugccuauuuggacaugguugacggagcuucaugcugcuuagacacugccaguuuuugcccugcaaagcugcgcagcuuuccaaagaaacacuccuauuuggaacccacaauacgaucggcagugccuucagcgauccagaacacgcuccagaacguccuggcagcugccacaaaaagaaauugcaaugucacgcaaaugagagaauugcccguauuggauucggcggccuuuaauguggaaugcuucaagaaauaugcguguaauaaugaauauugggaaacguuuaaagaaaaccccaucaggcuuacugaagaaaacgugguaaauuacauuaccaaauuaaaaggaccaaaagcugcugcucuuuuugcgaagacacauaauuugaauauguugcaggacauaccaauggacagguuuguaauggacuuaaagagagacgugaaagugacuccaggaacaaaacauacugaagaacggcccaagguacaggugauccaggcugccgauccgcuagcaacagcguaucugugcggaauccaccgagagcugguuaggagauuaaaugcgguccugcuuccgaacauucauacacuguuugauaugucggcugaagacuuugacgcuauuauagccgagcacuuccagccuggggauuguguucuggaaacugacaucgcgucguuugauaaaagugaggacgacgccauggcucugaccgcguuaaugauucuggaagacuuagguguggacgcagagcuguugacgcugauugaggcggcuuucggcgaaauuucaucaauacauuugcccacuaaaacuaaauuuaaauucggagccaugaugaaaucuggaauguuccucacacuguuugugaacacagucauuaacauuguaaucgcaagcagaguguugagagaacggcuaaccggaucaccaugugcagcauucauuggagaugacaauaucgugaaaggagucaaaucggacaaauuaauggcagacaggugcgccaccugguugaauauggaagucaagauuauagaugcuguggugggcgagaaagcgccuuauuucuguggaggguuuauuuugugugacuccgugaccggcacagcgugccguguggcagacccccuaaaaaggcuguuuaagcuuggcaaaccucuggcagcagacgaugaacaugaugaugacaggagaagggcauugcaugaagagucaacacgcuggaaccgaguggguauucuuucagagcugugcaaggcaguagaaucaagguaugaaaccguaggaacuuccaucauaguuauggccaugacuacucuagcuagcaguguuaaaucauucagcuaccugagaggggccccuauaacucucuacggc

SEQ ID NO:6: VEEV virus SG promoter sequence.

ugacuacucuagcuagcaguguuaaaucauucagcuaccugagaggggccccuauaacucucuacggcuaaccugaauggacuacgacauagucuaguccgccaag

SEQ ID NO:7: VEEV virus 3' utr sequence.

ugaacauagcagcaauuggcaagcugcuuauauagaacuugcggcgauuggcaugccgcuuuaaaauuuuauuuuauuuucuuuucuuuuccgaaucggauuuuguuuuuaauauuuc

SEQ ID NO:22: neonGreen to the RNA coding sequence.

auggugagcaagggcgaggaggacaacauggccagccugcccgccacccacgagcugcacaucuucggcagcaucaacggcguggacuucgacauggugggccagggcaccggcaaccccaacgacggcuacgaggagcugaaccugaagagcaccaagggcgaccugcaguucagccccuggauccuggugccccacaucggcuacggcuuccaccaguaccugcccuaccccgacggcaugagccccuuccaggccgccaugguggacggcagcggcuaccaggugcacaggaccaugcaguucgaggacggcgccagccugaccgugaacuacagguacaccuacgagggcagccacaucaagggcgaggcccaggugaagggcaccggcuuccccgccgacggccccgugaugaccaacagccugaccgccgccgacuggugcaggagcaagaagaccuaccccaacgacaagaccaucaucagcaccuucaaguggagcuacaccaccggcaacggcaagagguacaggagcaccgccaggaccaccuacaccuucgcaaagcccauggccgccaacuaccugaagaaccagcccauguacguguucaggaagaccgagcugaagcacagcaagaccgagcugaacuucaaggaguggcagaaggccuucacc

SEQ ID NO:23: RNA coding sequence of mTagRFP.

auggugagcaagggcgaggagcugaucaaggagaacaugcacaugaagcuguacauggagggcaccgugaacaaccaccacuucaagugcaccagcgagggcgagggcaagcccuacgagggcacccagaccaugagaaucaaggugguggagggcggcccccugcccuucgccuucgacauccuggccaccagcuucauguacggcagcagaaccuucaucaaccacacccagggcauccccgacuucuucaagcagagcuuccccgagggcuucaccugggagagagugaccaccuacgaggacggcggcgugcugaccgccacccaggacaccagccugcaggacggcugccugaucuacaacgugaagaucagaggcgugaacuuccccagcaacggccccgugaugcagaagaagacccugggcugggaggccaacaccgagaugcuguaccccgccgacggcggccuggagggcagaagcgacauggcccugaagcuggugggcggcggccaccugaucugcaacuucaagaccaccuacagaagcaagaagcccgccaagaaccugaagaugcccggcguguacuacguggaccacagacuggagagaaucaaggaggccgacaaggagaccuacguggagcagcacgagguggccguggccagauacugcgaccugcccagcaagcugggccacaagcugaac

Plasmid-2 is a hybrid plasmid of SFV virus and VEEV virus origin (shown as C in FIG. 4), which is also composed of pUC plasmid backbone, which also contains T7 Promoter (SEQ ID NO: 25) and truncated human RNA polymerase I Promoter (SEQ ID NO: 27) before 5' UTR, 5' UTR sequence (SEQ ID NO: 8), RNA coding sequence of nsP1234 (SEQ ID NO: 9-12) is derived from SFV virus, and wherein the RNA coding sequence end of nsP4 overlaps partially with SG-Promoter sequence of SFV virus, followed by insertion of NeonGreen sequence (SEQ ID NO: 22) downstream thereof, followed by insertion of SG-Promoter sequence (SEQ ID NO: 6), mTagRFP sequence (SEQ ID NO: 23) of VEEV virus and 3' UTR sequence (SEQ ID NO: 7) of VEEV virus.

SEQ ID NO:25: t7 promoter sequence.

Ttaatacgactcactatag

SEQ ID NO:27: a human RNA polymerase I promoter sequence.

gtccccggcccggcgctgctcccgcgtgtgtcctggggttgaccagagggccccgggcgctccgtgtgtggctgcgatggtggcgtttttggggacaggtgtccgtgtcgcgcgtcgcctgggccggcggcgtggtcggtgacgcgacctcccggccccgggggaggtatatctttcgctccgagtcggcattttgggccgccgggttatt

SEQ ID NO:8: SFV virus 5' UTR sequence.

auggcggaugugugacauacacgacgccaaaagauuuuguuccagcuccugccaccucggcuacgcgagagauuaaccacccacg

SEQ ID NO:9: the RNA coding sequence of SFV virus nsP 1.

auggccgccaaagugcauguugauauugaggcugacagcccauucaucaagucuuugcagaaggcauuuccgucguucgagguggagucauugcaggucacaccaaaugaccaugcaaaugccagagcauuuucgcaccuggcuaccaaauugaucgagcaggagacugacaaagacacacucaucuuggauaucggcagugcgccuuccaggagaaugaugucuacgcacaaauaccacugcguaugcccuaugcgcagcgcagaagaccccgaaaggcucguaugcuacgcaaagaaacuggcagcggccuccgggaaggugcuggauagagagaucgcaggaaaaaucaccgaccugcagaccgucauggcuacgccagacgcugaaucuccuaccuuuugccugcauacagacgucacgugucguacggcagccgaaguggccguauaccaggacguguaugcuguacaugcaccaacaucgcuguaccaucaggcgaugaaaggugucagaacggcguauuggauuggguuugacaccaccccguuuauguuugacgcgcuagcaggcgcguauccaaccuacgccacaaacugggccgacgagcagguguuacaggccaggaacauaggacugugugcagcauccuugacugagggaagacucggcaaacuguccauucuccgcaagaagcaauugaaaccuugcgacacagucauguucucgguaggaucuacauuguacacugagagcagaaagcuacugaggagcuggcacuuacccuccguauuccaccugaaagguaaacaauccuuuaccuguaggugcgauaccaucguaucaugugaaggguacguaguuaagaaaaucacuaugugccccggccuguacgguaaaacgguaggguacgccgugacguaucacgcggagggauuccuagugugcaagaccacagacacugucaaaggagaaagagucucauucccuguaugcaccuacguccccucaaccaucugugaucaaaugacuggcauacuagcgaccgacgucacaccggaggacgcacagaaguuguuagugggauugaaucagaggauaguugugaacggaagaacacagcgaaacacuaacacgaugaagaacuaucugcuuccgauuguggccgucgcauuuagcaagugggcgagggaauacaaggcagaccuugaugaugaaaaaccucuggguguccgagagaggucacuuacuugcugcugcuugugggcauuuaaaacgaggaagaugcacaccauguacaagaaaccagacacccagacaauagugaaggugccuucagaguuuaacucguucgucaucccgagccuauggucuacaggccucgcaaucccagucagaucacgcauuaagaugcuuuuggccaagaagaccaagcgagaguuaauaccuguucucgacgcgucgucagccagggaugcugaacaagaggagaaggagagguuggaggccgagcugacuagagaagccuuaccaccccucguucccaucgcgccggcggagacgggagucgucgacgucgacguugaagaacuagaguaucacgcaggugca

SEQ ID NO:10: the RNA coding sequence of SFV virus nsP 2.

ggggucguggaaacaccucgcagcgcguugaaagucaccgcacagccgaacgacguacuacuaggaaauuacguaguucuguccccgcagaccgugcucaagagcuccaaguuggcccccgugcacccucuagcagagcaggugaaaauaauaacacauaacgggagggccggccguuaccaggucgacggauaugacggcaggguccuacuaccauguggaucggccauuccggucccugaguuucaagcuuugagcgagagcgccacuaugguguacaacgaaagggaguucgucaacaggaaacuauaccauauugccguucacggaccgucgcugaacaccgacgaggagaacuacgagaaagucagagcugaaagaacugacgccgaguacguguucgacguagauaaaaaaugcugcgucaagagagaggaagcgucggguuugguguuggugggagagcuaaccaaccccccguuccaugaauucgccuacgaagggcugaagaucaggccgucggcaccauauaagacuacaguaguaggagucuuugggguuccgggaucaggcaagucugcuauuauuaagagccucgugaccaaacacgaucuggucaccagcggcaagaaggagaacugccaggaaauagucaacgacgugaagaagcaccgcggacuggacauccaggcaaaaacaguggacuccauccugcuaaacgggugucgucgugccguggacauccuauauguggacgaggcuuucgcuugccauuccgguacucugcuagcccuaauugcucuuguuaaaccucggagcaaagugguguuaugcggagaccccaagcaaugcggauucuucaauaugaugcagcuuaaggugaacuucaaccacaacaucugcacugaaguaugucauaaaaguauauccagacguugcacgcguccagucacggccaucgugucuacguugcacuacggaggcaagaugcgcacgaccaacccgugcaacaaacccauaaucauagacaccacaggacagaccaagcccaagccaggagacaucguguuaacaugcuuccgaggcuggguaaagcagcugcaguuggacuaccguggacacgaagucaugacagcagcagcaucucagggccucacccgcaaagggguauacgccguaaggcagaaggugaaugaaaaucccuuguaugccccugcgucggagcacgugaauguacugcugacgcgcacugaggauaggcugguguggaaaacgcuggccggcgaucccuggauuaagguccuaucaaacauuccacaggguaacuuuacggccacauuggaagaauggcaagaagaacacgacaaaauaaugaaggugauugaaggaccggcugcgccuguggacgcguuccagaacaaagcgaacguguguugggcgaaaagccuggugccuguccuggacacugccggaaucagauugacagcagaggaguggagcaccauaauuacagcauuuaaggaggacagagcuuacucuccagugguggccuugaaugaaauuugcaccaaguacuauggaguugaccuggacaguggccuguuuucugccccgaaggugucccuguauuacgagaacaaccacugggauaacagaccugguggaaggauguauggauucaaugccgcaacagcugccaggcuggaagcuagacauaccuuccugaaggggcaguggcauacgggcaagcaggcaguuaucgcagaaagaaaaauccaaccgcuuucugugcuggacaauguaauuccuaucaaccgcaggcugccgcacgcccugguggcugaguacaagacgguuaaaggcaguaggguugaguggcuggucaauaaaguaagaggguaccacguccugcuggugagugaguacaaccuggcuuugccucgacgcagggucacuugguugucaccgcugaaugucacaggcgccgauaggugcuacgaccuaaguuuaggacugccggcugacgccggcagguucgacuuggucuuugugaacauucacacggaauucagaauccaccacuaccagcagugugucgaccacgccaugaagcugcagaugcuugggggagaugcgcuacgacugcuaaaacccggcggcagccucuugaugagagcuuacggauacgccgauaaaaucagcgaagccguuguuuccuccuuaagcagaaaguucucgucugcaagaguguugcgcccggauugugucaccagcaauacagaaguguucuugcuguucuccaacuuugacaacggaaagagacccucuacgcuacaccagaugaauaccaagcugagugccguguaugccggagaagccaugcacacggccgggugu

SEQ ID NO:11: the RNA coding sequence of SFV virus nsP 3.

gcaccauccuacagaguuaagagagcagacauagccacgugcacagaagcggcugugguuaacgcagcuaacgcccguggaacuguaggggauggcguaugcagggccguggcgaagaaauggccgucagccuuuaagggagaagcaacaccagugggcacaauuaaaacagucaugugcggcucguaccccgucauccacgcuguagcgccuaauuucucugccacgacugaagcggaaggggaccgcgaauuggccgcugucuaccgggcaguggccgccgaaguaaacagacugucacugagcagcguagccaucccgcugcuguccacaggaguguucagcggcggaagagauaggcugcagcaaucccucaaccaucuauucacagcaauggacgccacggacgcugacgugaccaucuacugcagagacaaaaguugggagaagaaaauccaggaagccauagacaugaggacggcuguggaguugcucaaugaugacguggagcugaccacagacuuggugagagugcacccggacagcagccuggugggucguaagggcuacaguaccacugacgggucgcuguacucguacuuugaagguacgaaauucaaccaggcugcuauugauauggcagagauacugacguuguggcccagacugcaagaggcaaacgaacagauaugccuauacgcgcugggcgaaacaauggacaacaucagauccaaauguccggugaacgauuccgauucaucaacaccucccaggacagugcccugccugugccgcuacgcaaugacagcagaacggaucgcccgccuuaggucacaccaaguuaaaagcauggugguuugcucaucuuuuccccucccgaaauaccauguagauggggugcagaagguaaagugcgagaagguucuccuguucgacccgacgguaccuucagugguuaguccgcggaaguaugccgcaucuacgacggaccacucagaucggucguuacgaggguuugacuuggacuggaccaccgacucgucuuccacugccagcgauaccaugucgcuacccaguuugcagucgugugacaucgacucgaucuacgagccaauggcucccauaguagugacggcugacguacacccugaacccgcaggcaucgcggaccuggcggcagaugugcauccugaacccgcagaccauguggaccucgagaacccgauuccuccaccgcgcccgaagagagcugcauaccuugccucccgcgcggcggagcgaccggugccggcgccgagaaagccgacgccugccccaaggacugcguuuaggaacaagcugccuuugacguucggcgacuuugacgagcacgaggucgaugcguuggccuccgggauuacuuucggagacuucgacgacguccugcgacuaggccgcgcgggugca

SEQ ID NO:12: the RNA coding sequence of SFV virus nsP4 (3' -end bold sequence is the part overlapping with the VEEV virus SG promoter sequence).

Uauauuuucuccucggacacuggcagcggacauuuacaacaaaaauccguuaggcagcacaaucuccagugcgcacaacuggaugcggucgaggaggagaaaauguacccgccaaaauuggauacugagagggagaagcuguugcugcugaaaaugcagaugcacccaucggaggcuaauaagagucgauaccagucucgcaaaguggagaacaugaaagccacggugguggacaggcucacaucgggggccagauuguacacgggagcggacguaggccgcauaccaacauacgcgguucgguacccccgccccguguacuccccuaccgugaucgaaagauucucaagccccgauguagcaaucgcagcgugcaacgaauaccuauccagaaauuacccaacaguggcgucguaccagauaacagaugaauacgacgcauacuuggacaugguugacgggucggauaguugcuuggacagagcgacauucugcccggcgaagcuccggugcuacccgaaacaucaugcguaccaccagccgacuguacgcagugccgucccgucacccuuucagaacacacuacagaacgugcuagcggccgccaccaagagaaacugcaacgucacgcaaaugcgagaacuacccaccauggacucggcaguguucaacguggagugcuucaagcgcuaugccugcuccggagaauauugggaagaauaugcuaaacaaccuauccggauaaccacugagaacaucacuaccuaugugaccaaauugaaaggcccgaaagcugcugccuuguucgcuaagacccacaacuugguuccgcugcaggagguucccauggacagauucacggucgacaugaaacgagaugucaaagucacuccagggacgaaacacacagaggaaagacccaaaguccagguaauucaagcagcggagccauuggcgaccgcuuaccugugcggcauccacagggaauuaguaaggagacuaaaugcuguguuacgcccuaacgugcacacauuguuugauaugucggccgaagacuuugacgcgaucaucgccucucacuuccacccaggagacccgguucuagagacggacauugcaucauucgacaaaagccaggacgacuccuuggcucuuacagguuuaaugauccucgaagaucuagggguggaucaguaccugcuggacuugaucgaggcagccuuuggggaaauauccagcugucaccuaccaacuggcacgcgcuucaaguucggagcuaugaugaaaucgggcauguuucugacuuuguuuauuaacacuguuuugaacaucaccauagcaagcaggguacuggagcagagacucacugacuccgccugugcggccuucaucggcgacgacaacaucguucacggagugaucuccgacaagcugauggcggagaggugcgcgucgugggucaacauggaggugaagaucauugacgcugucaugggcgaaaaacccccauauuuuugugggggauucauaguuuuugacagcgucacacagaccgccugccguguuucagacccacuuaagcgccuguucaaguuggguaagccgcuaacagcugaagacaagcaggacgaagacaggcgacgagcacugagugacgagguuagcaagugguuccggacaggcuugggggccgaacuggagguggcacuaacaucuagguaugagguagagggcugcaaaaguauccucauagccauggccaccuuggcgagggacauuaaggcguuuaagaaauugagaggaccuguuauacaccucuacggcgguccuagauuggugcgu

Plasmid-3 and plasmid-4 were split from plasmid 1 (shown as D in FIG. 4). Plasmid-3 contains the VEEV virus nonstructural protein (VEEV-P1234) expression element, is obtained by enzyme cutting NeonGreen-SGP-mTagRFP sequence from plasmid-1 and self-ligating, only the VEEV virus nsP1234 expression frame and front and rear UTR and polyA tail sequences are reserved, namely plasmid-3 is VEEV1234-psaRNA plasmid; plasmid-4 is obtained by cleavage of the nsP1234 protein sequence from plasmid-1 by enzyme digestion and self-ligation, and only the T7 promoter-5 'UTR-SGP-NeonGreen-SGP-mTagRFP-3' UTR-PolyA sequence is reserved, i.e., plasmid-4 is VEEVsgp-repoters plasmid, and is also constructed by a seamless cloning method.

Plasmid-5 (shown as E in FIG. 4) was prepared by adding a repeat unit of the SG promoter of VEEV (SEQ ID NO: 6) + mTagBFP sequence (SEQ ID NO: 24) to plasmid-1 to verify that three target proteins can be expressed.

SEQ ID NO:24: mTagBFP to the RNA coding sequence.

Augagcgagcugauuaaggagaacaugcacaugaagcucuacauggagggcaccguggacaaccaccacuucaagugcacauccgagggcgaaggcaagcccuacgagggcacccagaccaugagaaucaagguggucgagggcggcccucuccccuucgccuucgacauccuggcuaccagcuuccucuacggcagcaagaccuucaucaaccacacccagggcauccccgacuucuucaagcaguccuuccccgagggcuucacaugggagagagucaccacauacgaagacgggggcgugcugaccgcuacccaggacaccagccuccaggacggcugccucaucuacaacgucaagaucagaggggugaacuucacaagcaacggcccugugaugcagaagaaaacacucggcugggaggccuucacagagacccuguaccccgcugacggcggccuggaaggcagaaacgacauggcccugaagcucgugggcgggagccaccugaucgcaaacaucaagaccacauacagauccaagaaacccgcuaagaaccucaagaugcccggcgucuauuauguggacuacagacuggaaagaaucaaggaggccaacaacgagaccuacguggagcagcacgagguggcaguggccagauacugcgaccucccuagcaaacuggggcacaagcucaau

After the plasmid design is completed, the plasmid can be obtained through gene synthesis.

Example 2: method for preparing capped psaRNA by co-transcription

Plasmid-1, plasmid-2, plasmid-3, plasmid-4 and plasmid-5 were transformed into DH5alpha strain, respectively, and were selected and cultured overnight at 37℃in a shaking table to collect bacteria, and a large amount of plasmids were obtained by the endotoxin-free plasmid extraction kit (Beijing Tiangen Biochemical Co., ltd.). The enzyme digestion system is prepared according to the instruction, namely, the plasmid is uniformly mixed with the specific restriction enzyme and the buffer solution thereof, and the mixture reacts for 2 hours at the working temperature, so that the plasmid is thoroughly linearized. After complete linearization, the recovery of the linearized plasmid was performed using a general DNA product purification recovery kit (beijing tiangen biochemical technologies limited).

After linearizing the transcription template plasmid with restriction enzymes, in vitro transcription of RNA was initiated by T7 RNA polymerase, co-transcription capping of psaRNA was performed using a co-transcription capping T7 in vitro transcription kit (marchan big desert new enzyme biotechnology limited). The reaction system is shown in Table 1.

TABLE 1 Co-transcriptional capping reaction System

37. After the reaction was completed by incubating at 2h ℃and adding 2. Mu.l of DNase I, the DNA plasmid template was removed by digestion at 37℃for 30 min. And then carrying out post-transcriptional purification according to the following steps:

(1) Mixing the IVT reaction solution and the 5M LiCl solution according to the volume ratio of 1:1, standing for 30min at-20 ℃, centrifuging at 12000 rpm for 10 min, observing the sediment at the bottom, and removing the supernatant;

(2) Placing the sample on ice, adding 1ml of precooled 70% ethanol, vibrating or blowing to suspend the precipitate, centrifuging at 4deg.C, 12000 rpm for 10 min, discarding the supernatant, and repeating the operation for 2 times;

(3) Under the condition of not interfering with precipitation, residual ethanol is sucked as much as possible, the cover is opened and dried in a biosafety cabinet, and a proper amount of water without nuclease is added to dissolve the precipitate after the precipitate is dried.

The RNA obtained by in vitro transcription of the linearized plasmid is subjected to agarose electrophoresis to obtain a single band, and the single band reaches the purity standard. As shown in fig. 5, M represents a molecular weight Marker, and bands 1,2,3, and 4 are respectively: VEEV-PSaRNA, SFV-VEEV-PSaRNA, VEEV1234-psaRNA and VEEVsgp-repotersRNA samples.

Example 3: cell transfection verifies the function of polycistronic self-amplifying RNA

In order to verify whether the polycistronic self-amplified RNA obtained by the invention can express 2 target genes in the same cell, because a truncated human RNA polymerase I promoter is designed when constructing plasmids, the promoter can start the transcription function of the plasmids in eukaryotic cells, the successfully constructed plasmids and the prepared saRNA are subjected to cell transfection, and whether the 2 target genes or 3 target genes are expressed is observed under a fluorescence microscope.

The specific implementation process is as follows: HEK-293T cells (ATCC No. CRL-3216) were seeded in 24 well plates at an advance of 16 h at 2X 10 ⁶ cells/well, cultured at 37℃with 5% CO ₂, all reagents involved were derived from commercial products. When the cell confluency reached 70 to 80%, the prepared plasmid and saRNA were introduced into the cells by transfection, and the transfection method may be selected, for example, liposome transfection, PEI transfection, etc., and the uBrigene PEI kit (Yiming organism) transfection was selected in this example. 48 hours after transfection, fluorescence microscopy was performed.

As a result, see FIG. 6, A, both the green fluorescent protein and the red fluorescent protein were expressed in the same cell, whether transfected with plasmid-1, plasmid-2, or VEEV-psaRNA, SFV-VEEV-psaRNA. FIG. 6A shows that the plasmid or RNA containing the complete VEEV-psaRNA or SFV-VEEV-psaRNA self-amplifying element is capable of generating complete negative strand after entering the cell, so that the positive strand RNA containing only red fluorescent protein can be amplified independently (see schematic diagram of FIG. 2), thereby expressing the corresponding red color. The above results demonstrate that psaRNA provided by the present invention is capable of expressing two different proteins, while the expression of red fluorescent protein itself also demonstrates that psaRNA provided by the present invention is capable of polycistronic self-amplification.

For the homozygous VEEV-psaRNA of VEEV virus source, the expression intensity of the green fluorescent protein and the red fluorescent protein is relatively uniform, which shows that the two cistrons are successfully expressed after independent self-amplification, and the expression intensity of the two proteins is similar as the front SGP and the rear SGP are both of VEEV virus sources and are matched with the nonstructural proteins of four VEEV sources.

For the hybrid SFV-VEEV-psaRNA of SFV virus and VEEV virus sources, the green fluorescent protein is expressed more strongly, the red fluorescent protein is expressed slightly weakly, probably because the first SG promoter before the green fluorescent protein is of SFV virus source, is naturally matched with the four SFV-source nonstructural proteins contained in the plasmid, has stronger capability of driving the SFV-source SG promoter to self-amplify, and obtains higher expression. The second SG promoter in the plasmid is of VEEV source, and can interact with four non-structural proteins of SFV virus to play a self-amplifying function, but the self-amplifying strength is relatively low, so that the expression of red fluorescent protein is weak.

Therefore, the invention discovers that when various matched known alphavirus nonstructural proteins, subgenomic promoters and UTRs are selected (such as SFV, VEEV, SINV, etc., related sequences of SINV are shown in SEQ ID NO: 15-21), the phenomenon of non-uniform polycistronic expression caused by similar IRES can be overcome, and the polycistronic synchronous and uniform expression can be realized without adding any additional sequences at two ends of a target gene like 2A peptide, thereby having good application prospect in the aspect of synchronous and uniform expression of polycistronic genes without additional sequences.

SEQ ID NO:13: SFV virus SG promoter sequence.

ccauggccaccuuggcgagggacauuaaggcguuuaagaaauugagaggaccuguuauacaccucuacggcgguccuagauuggugcguuaauacacagaauucugauuauagcgcacuauuauagcacc

SEQ ID NO:14: SFV virus 3' -UTR sequence.

guuaggguaggcaauggcauugauauagcaagaaaauugaaaacagaaaaaguuaggguaagcaauggcauauaaccauaacuguauaacuuguaacaaagcgcaacaagaccugcgcaauuggccccgugguccgccucacggaaacucggggcaacucauauugacacauuaauuggcaauaauuggaagcuuacauaagcuuaauucgacgaauaauuggauuuuuauuuuauuuugcaauugguuuuuaauauuucc

SEQ ID NO:15: SINV virus 5' -UTR sequence.

auugacggcguaguacacacuauugaaucaaacagccgaccaauugcacuaccaucaca

SEQ ID NO:16: RNA coding sequence of SINV virus nsP 1.

auggagaagccaguaguaaacguagacguagacccccagaguccguuugucgugcaacugcaaaaaagcuucccgcaauuugagguaguagcacagcaggucacuccaaaugaccaugcuaaugccagagcauuuucgcaucuggccaguaaacuaaucgagcuggagguuccuaccacagcgacgaucuuggacauaggcagcgcaccggcucguagaauguuuuccgagcaccaguaucauugugucugccccaugcguaguccagaagacccggaccgcaugaugaaauacgccaguaaacuggcggaaaaagcgugcaagauuacaaacaagaacuugcaugagaagauuaaggaucuccggaccguacuugauacgccggaugcugaaacaccaucgcucugcuuucacaacgauguuaccugcaacaugcgugccgaauauuccgucaugcaggacguguauaucaacgcucccggaacuaucuaucaucaggcuaugaaaggcgugcggacccuguacuggauuggcuucgacaccacccaguucauguucucggcuauggcagguucguacccugcguacaacaccaacugggccgacgagaaaguccuugaagcgcguaacaucggacuuugcagcacaaagcugagugaagguaggacaggaaaauugucgauaaugaggaagaaggaguugaagcccgggucgcggguuuauuucuccguaggaucgacacuuuauccagaacacagagccagcuugcagagcuggcaucuuccaucgguguuccacuugaauggaaagcagucguacacuugccgcugugauacaguggugaguugcgaaggcuacguagugaagaaaaucaccaucagucccgggaucacgggagaaaccgugggauacgcgguuacacacaauagcgagggcuucuugcuaugcaaaguuacugacacaguaaaaggagaacggguaucguucccugugugcacguacaucccggccaccauaugcgaucagaugacugguauaauggccacggauauaucaccugacgaugcacaaaaacuucugguugggcucaaccagcgaauugucauuaacgguaggacuaacaggaacaccaacaccaugcaaaauuaccuucugccgaucauagcacaaggguucagcaaaugggcuaaggagcgcaaggaugaucuugauaacgagaaaaugcuggguacuagagaacgcaagcuuacguauggcugcuugugggcguuucgcacuaagaaaguacauucguuuuaucgcccaccuggaacgcagaccugcguaaaagucccagccucuuuuagcgcuuuucccaugucguccguauggacgaccucuuugcccaugucgcugaggcagaaauugaaacuggcauugcaaccaaagaaggaggaaaaacugcugcaggucucggaggaauuagucauggaggccaaggcugcuuuugaggaugcucaggaggaagccagagcggagaagcuccgagaagcacuuccaccauuaguggcagacaaaggcaucgaggcagccgcagaaguugucugcgaaguggaggggcuccaggcggacaucggagca

SEQ ID NO:17: RNA coding sequence of SINV virus nsP 2.

gcauuaguugaaaccccgcgcggucacguaaggauaauaccucaagcaaaugaccguaugaucggacaguauaucguugucucgccaaacucugugcugaagaaugccaaacucgcaccagcgcacccgcuagcagaucagguuaagaucauaacacacuccggaagaucaggaagguacgcggucgaaccauacgacgcuaaaguacugaugccagcaggaggugccguaccauggccagaauuccuagcacugagugagagcgccacguuaguguacaacgaaagagaguuugugaaccgcaaacuauaccacauugccaugcauggccccgccaagaauacagaagaggagcaguacaagguuacaaaggcagagcuugcagaaacagaguacguguuugacguggacaagaagcguugcguuaagaaggaagaagccucaggucugguccucucgggagaacugaccaacccucccuaucaugagcuagcucuggagggacugaagacccgaccugcggucccguacaaggucgaaacaauaggagugauaggcacaccggggucgggcaagucagcuauuaucaagucaacugucacggcacgagaucuuguuaccagcggaaagaaagaaaauugucgcgaaauugaggccgacgugcuaagacugagggguaugcagauuacgucgaagacaguagauucgguuaugcucaacggaugccacaaagccguagaagugcuguacguugacgaagcguucgcgugccacgcaggagcacuacuugccuugauugcuaucgucaggccccgcaagaagguaguacuaugcggagaccccaugcaaugcggauucuucaacaugaugcaacuaaagguacauuucaaucacccugaaaaagacauaugcaccaagacauucuacaaguauaucucccggcguugcacacagccaguuacagcuauuguaucgacacugcauuacgauggaaagaugaaaaccacgaacccgugcaagaagaacauugaaaucgauauuacaggggccacaaagccgaagccaggggauaucauccugacauguuuccgcggguggguuaagcaauugcaaaucgacuaucccggacaugaaguaaugacagccgcggccucacaagggcuaaccagaaaaggaguguaugccguccggcaaaaagucaaugaaaacccacuguacgcgaucacaucagagcaugugaacguguugcucacccgcacugaggacaggcuaguguggaaaaccuugcagggcgacccauggauuaagcagcccacuaacauaccuaaaggaaacuuucaggcuacuauagaggacugggaagcugaacacaagggaauaauugcugcaauaaacagccccacuccccgugccaauccguucagcugcaagaccaacguuugcugggcgaaagcauuggaaccgauacuagccacggccgguaucguacuuaccgguugccaguggagcgaacuguucccacaguuugcggaugacaaaccacauucggccauuuacgccuuagacguaauuugcauuaaguuuuucggcauggacuugacaagcggacuguuuucuaaacagagcaucccacuaacguaccaucccgccgauucagcgaggccgguagcucauugggacaacagcccaggaacccgcaaguauggguacgaucacgccauugccgccgaacucucccguagauuuccgguguuccagcuagcugggaagggcacacaacuugauuugcagacggggagaaccagaguuaucucugcacagcauaaccuggucccggugaaccgcaaucuuccucacgccuuaguccccgaguacaaggagaagcaacccggcccggucaaaaaauucuugaaccaguucaaacaccacucaguacuugugguaucagaggaaaaaauugaagcuccccguaagagaaucgaauggaucgccccgauuggcauagccggugcagauaagaacuacaaccuggcuuucggguuuccgccgcaggcacgguacgaccugguguucaucaacauuggaacuaaauacagaaaccaccacuuucagcagugcgaagaccaugcggcgaccuuaaaaacccuuucgcguucggcccugaauugccuuaacccaggaggcacccucguggugaaguccuauggcuacgccgaccgcaacagugaggacguagucaccgcucuugccagaaaguuugucagggugucugcagcgagaccagauugugucucaagcaauacagaaauguaccugauuuuccgacaacuagacaacagccguacacggcaauucaccccgcaccaucugaauugcgugauuucguccguguaugaggguacaagagauggaguuggagcc

SEQ ID NO:18: RNA coding sequence of SINV virus nsP 3.

gcgccgucauaccgcaccaaaagggagaauauugcugacugucaagaggaagcaguugucaacgcagccaauccgcuggguagaccaggcgaaggagucugccgugccaucuauaaacguuggccgaccaguuuuaccgauucagccacggagacaggcaccgcaagaaugacugugugccuaggaaagaaagugauccacgcggucggcccugauuuccggaagcacccagaagcagaagccuugaaauugcuacaaaacgccuaccaugcaguggcagacuuaguaaaugaacauaacaucaagucugucgccauuccacugcuaucuacaggcauuuacgcagccggaaaagaccgccuugaaguaucacuuaacugcuugacaaccgcgcuagacagaacugacgcggacguaaccaucuauugccuggauaagaaguggaaggaaagaaucgacgcggcacuccaacuuaaggagucuguaacagagcugaaggaugaagauauggagaucgacgaugaguuaguauggauccauccagacaguugcuugaagggaagaaagggauucaguacuacaaaaggaaaauuguauucguacuucgaaggcaccaaauuccaucaagcagcaaaagacauggcggagauaaagguccuguucccuaaugaccaggaaaguaaugaacaacugugugccuacauauugggugagaccauggaagcaauccgcgaaaagugcccggucgaccauaacccgucgucuagcccgcccaaaacguugccgugccuuugcauguaugccaugacgccagaaaggguccacagacuuagaagcaauaacgucaaagaaguuacaguaugcuccuccaccccccuuccuaagcacaaaauuaagaauguucagaagguucagugcacgaaaguaguccuguuuaauccgcacacucccgcauucguucccgcccguaaguacauagaagugccagaacagccuaccgcuccuccugcacaggccgaggaggcccccgaaguuguagcgacaccgucaccaucuacagcugauaacaccucgcuugaugucacagacaucucacuggauauggaugacaguagcgaaggcucacuuuuuucgagcuuuagcggaucggacaacucuauuacuaguauggacaguuggucgucaggaccuaguucacuagagauaguagaccgaaggcagguggugguggcugacguucaugccguccaagagccugccccuauuccaccgccaaggcuaaagaagauggcccgccuggcagcggcaagaaaagagcccacuccaccggcaagcaauagcucugagucccuccaccucucuuuuggugggguauccaugucccucggaucaauuuucgacggagagacggcccgccaggcagcgguacaaccccuggcaacaggccccacggaugugccuaugucuuucggaucguuuuccgacggagagauugaugagcugagccgcagaguaacugaguccgaacccguccuguuuggaucauuugaaccgggcgaagugaacucaauuauaucgucccgaucagccguaucuuuuccacuacgcaagcagagacguagacgcaggagcaggaggacugaauacuga

SEQ ID NO:19: the RNA coding sequence of SINV virus nsP4 (3' -end bold sequence is the part overlapping with the VEEV virus SG promoter sequence).

cuaaccgggguagguggguacauauuuucgacggacacaggcccugggcacuugcaaaagaaguccguucugcagaaccagcuuacagaaccgaccuuggagcgcaauguccuggaaagaauucaugccccggugcucgacacgucgaaagaggaacaacucaaacucagguaccagaugaugcccaccgaagccaacaaaaguagguaccagucucguaaaguagaaaaucagaaagccauaaccacugagcgacuacugucaggacuacgacuguauaacucugccacagaucagccagaaugcuauaagaucaccuauccgaaaccauuguacuccaguagcguaccggcgaacuacuccgauccacaguucgcuguagcugucuguaacaacuaucugcaugagaacuauccgacaguagcaucuuaucagauuacugacgaguacgaugcuuacuuggauaugguagacgggacagucgccugccuggauacugcaaccuucugccccgcuaagcuuagaaguuacccgaaaaaacaugaguauagagccccgaauauccgcagugcgguuccaucagcgaugcagaacacgcuacaaaaugugcucauugccgcaacuaaaagaaauugcaacgucacgcagaugcgugaacugccaacacuggacucagcgacauucaaugucgaaugcuuucgaaaauaugcauguaaugacgaguauugggaggaguucgcucggaagccaauuaggauuaccacugaguuugucaccgcauauguagcuagacugaaaggcccuaaggccgccgcacuauuugcaaagacguauaauuuggucccauugcaagaagugccuauggauagauucgucauggacaugaaaagagacgugaaaguuacaccaggcacgaaacacacagaagaaagaccgaaaguacaagugauacaagccgcagaaccccuggcgacugcuuacuuaugcgggauucaccgggaauuagugcguaggcuuacggccgucuugcuuccaaacauucacacgcuuuuugacaugucggcggaggauuuugaugcaaucauagcagaacacuucaagcaaggcgacccgguacuggagacggauaucgcaucauucgacaaaagccaagacgacgcuauggcguuaaccggucugaugaucuuggaggaccuggguguggaucaaccacuacucgacuugaucgagugcgccuuuggagaaauaucauccacccaucuaccuacggguacucguuuuaaauucggggcgaugaugaaauccggaauguuccucacacuuuuugucaacacaguuuugaaugucguuaucgccagcagaguacuagaagagcggcuuaaaacguccagaugugcagcguucauuggcgacgacaacaucauacauggaguaguaucugacaaagaaauggcugagaggugcgccaccuggcucaacauggagguuaagaucaucgacgcagucaucggugagagaccaccuuacuucugcggcggauuuaucuugcaagauucgguuacuuccacagcgugccgcguggcggauccccugaaaaggcuguuuaaguuggguaaaccgcucccagccgacgacgagcaagacgaagacagaagacgcgcucugcuagaugaaacaaaggcgugguuuagaguagguauaacaggcacuuuagcaguggccgugacgacccgguaugagguagacaauauuacaccuguccuacuggcauugagaacuuuugcccagagcaaaagagcauuccaagccaucagaggggaaauaaagcaucucuacggugguccuaaa

SEQ ID NO:20: SINV virus SG promoter sequence.

ugagaacuuuugcccagagcaaaagagcauuccaagccaucagaggggaaauaaagcaucucuacggugguccuaaauagucagcauaguacauuucaucugacuaauacuacaacaccaccacc

SEQ ID NO:21: SINV virus 3' UTR sequence.

ugaccgcuacgccccaaugauccgaccagcaaaacucgauguacuuccgaggaacugaugugcauaaugcaucaggcugguacauuagauccccgcuuaccgcgggcaauauagcaacacuaaaaacucgauguacuuccgaggaagcgcagugcauaaugcugcgcaguguugccacauaaccacuauauuaaccauuuaucuagcggacgccaaaaacucaauguauuucugaggaagcguggugcauaaugccacgcagcgucugcauaacuuuuauuauuucuuuuauuaaucaacaaaauuuuguuuuuaacauuuc

In contrast, co-transfection of either the combination of split plasmid-3 and plasmid-4 or the combination of VEEV1234-psaRNA and VEEVsgp-repotersRNA, only a weaker green fluorescent protein was observed, and no red fluorescent protein was observed. This suggests that plasmid-3 after resolution has independent expression elements in cells for 4 nonstructural proteins of the VEEV virus, but VEEV1234-psaRNA and VEEVsgp-repotersRNA combine to be ineffective in producing negative-strand RNA containing the reporter gene, and thus cannot be further amplified to produce positive-strand RNA containing only red fluorescent protein (see schematic diagram of fig. 3), so red fluorescence is not expressed, while green fluorescence is expressed from RNA transcribed directly from the human RNA polymerase I promoter on the VEEVsgp-repoters plasmid. The RNA is positive-strand RNA sequentially comprising a green fluorescent protein expression frame and a red fluorescent protein expression frame. Ribosomes can initiate translation of the first coding region (i.e., the green fluorescent protein) near the 5' end of the positive stranded RNA (but not amplified, so that the green fluorescent protein can be expressed but of low intensity). Since NeonGreen terminal contains stop codon, the ribosome encounters the stop codon, translation is stopped, and the red fluorescent protein is not expressed.

For transfected 3 cistron plasmid-5, the B results in fig. 6 show that blue, green and red fluorescent proteins can be expressed in the same cell.

In summary, the invention skillfully uses the SG promoters of VEEV virus and SFV virus on the basis of the traditional saRNA, not only maintains the expression and self-amplification functions of the GOI adjacent to the nsP1234 unit in the traditional saRNA, but also enables the expression and self-amplification of other GOIs not adjacent to the nsP1234 unit, successfully carries out independent self-amplification and protein expression of a plurality of target Genes (GOIs)/cistron in cells, and does not introduce additional other sequences on the amino acid sequence of the target genes. The present invention further provides the discovery that polycistronic synchronous uniform expression can be achieved when matched various known alphavirus nonstructural proteins, subgenomic promoters, and UTRs are selected.

Although the present invention has been described in detail with reference to the embodiments thereof, these embodiments are provided for the purpose of illustration and not limitation of the invention. Other embodiments that can be obtained according to the principles of the present invention fall within the scope of the invention as defined in the claims.

Claims (9)

1. The polycistronic self-amplifying RNA is characterized in that the structure is shown as a formula (I):

（I）

Wherein the SGP-n units and the GOI-n units form a repeating framework, n is an integer greater than 1, and the sequences of the 5'UTR unit, the 3' UTR unit, the nsP1234/SGP-1 unit, and each SGP-n unit within the repeating framework are derived from an alphavirus selected from the group consisting of VEEV and SFV,

Wherein the sequence of the 5' UTR unit is shown in SEQ ID NO:1 or 8;

the sequence of the 3' UTR unit is shown as SEQ ID NO:7 or 14;

the SGP unit has a sequence shown in SEQ ID NO:6 or 13;

the sequences of the nsP1234/SGP-1 units are a series-connected nsP1234 sequence and a series-connected SGP unit sequence, except that the 5 '-end of the SGP unit sequence overlaps with the 3' -end of the nsP1234 sequence, wherein the nsP1234 sequence is an RNA coding sequence of a series-connected nsP1 protein, a series-connected nsP2 protein, a series-connected nsP3 protein and a series-connected nsP4 protein, and the RNA coding sequence of the nsP1 protein is shown as SEQ ID NO:2 or 9, wherein the RNA coding sequence of the nsP2 protein is shown as SEQ ID NO:3 or 10, the RNA coding sequence of the nsP3 protein is shown as SEQ ID NO:4 or 11, wherein the RNA coding sequence of the nsP4 protein is shown as SEQ ID NO:5 or 12.

2. The self-amplifying RNA of claim 1, wherein n is any integer from 2 to 5.

3. The self-amplifying RNA of claim 1, wherein the UTR unit, the nsP1234/SGP-1 unit, and the sequences of the SGP units are all derived from the same alphavirus.

4. The self-amplifying RNA of claim 1, wherein at least one of said UTR unit, said nsP1234/SGP-1 unit, and said SGP units has a different alphavirus source than the other units.

5. The self-amplifying RNA of claim 1, wherein each GOI-1 unit within the repeat frame is a different gene of interest from each GOI-n unit within the repeat frame.

6. A plasmid comprising the coding sequence of self-amplifying RNA of any one of claims 1 to 5.

7. The plasmid of claim 6 further comprising a T7 promoter upstream of the coding sequence of the self-amplifying RNA.

8. The plasmid of claim 7 further comprising a truncated human RNA polymerase I promoter upstream of the coding sequence of the self-amplifying RNA.

9. The method for producing self-amplified RNA as claimed in any one of claims 1 to 5, comprising the steps of:

s1: constructing a plasmid according to any one of claims 6 to 8;

s2: replicating the plasmid and extracting;

S3: carrying out linearization cutting on the extracted plasmid to obtain a linearization plasmid;

s4: and (3) transcribing the linearized plasmid to obtain the self-amplified RNA.