Promoter with high strength and good orthogonality and application thereof in saRNA vaccine

Abstract

The invention discloses a promoter with high strength and good orthogonality and application of the promoter in saRNA vaccines, and belongs to the technical field of synthetic biology and biology. A library of alpha replicase protein sub-promoters is constructed through sequence randomization, 20 sub-promoters with different intensities and orthogonality are obtained through screening, then the system is applied to regulation and control of expression of different antigen genes in cells, the time for reaching the titer (6-8 months) which cannot be neutralized for variant viruses can be slowed down and reduced, and the method has the advantages that the method is simple and convenient to operate, and the cost is low. Meanwhile, the multivalent self-amplification mRNA vaccine capable of effectively regulating spatiotemporal expression of multiple genes is obtained, and the research and development bottleneck of the multivalent self-amplification mRNA vaccine is broken through.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of synthetic biology and biotechnology, in particular to a promoter with high strength and good orthogonality and its application in saRNA vaccine. BACKGROUND

[0002] mRNA vaccine is the preferred technology of many companies to fight the epidemic, and has great application value in the fields of AIDS, cancer, genetic metabolic diseases, etc. Self-amplifying mRNA (saRNA) vaccine as a new type of mRNA vaccine structure form not only retains the advantages of non-amplifying mRNA vaccine such as rapid development, modular design and cell-free synthesis, but also has the characteristics of inducing high initial level of antigen production and sustained immunogenicity with only a small amount of initial mRNA. It is considered as one of the main technologies for the development of next-generation mRNA drugs. However, studies have found that the protection efficiency of the vaccine will accelerate with the passage of time and shorten the time to maintain, which will accelerate the reduction of the titer (6-8 months) against variant viruses. This makes the development of booster (multivalent) self-amplifying mRNA vaccine a focus of research and development for major pharmaceutical companies, and receives widespread attention in the development of future nucleic acid drugs.

[0003] The gene expression regulation strategy is mainly based on the functional requirements to use various elements to accurately and efficiently regulate the expression of specific genes, so that the biological system can execute the artificially assigned biological functions. The timing of the regulation and the accuracy of the regulation level determine whether the designed gene circuit can accurately execute the artificially preset functions, and the most important thing for regulating multiple genes is the orthogonality of the regulation system. Therefore, how to use the orthogonality of the regulation elements to effectively regulate the spatiotemporal expression of multiple genes is one of the key points in the research and development of multivalent self-amplifying mRNA vaccine.

[0004] Alphavirus replicase protein as an important carrier of saRNA vaccine is composed of four non-structural proteins nsP1, nsP2, nsP3 and nsP4, which jointly execute the self-replication function of saRNA; and the subpromoter (SGP) is one of the important regulatory elements of saRNA vaccine, which can directly affect the synthesis efficiency of antigen mRNA, and then determine the expression level of antigen. Therefore, it is of great significance to study the alphavirus replicase protein as an important carrier of saRNA vaccine and the subpromoter as an important regulatory element. SUMMARY

[0005] The application aims to provide a promoter with high strength and good orthogonality and its application in saRNA vaccine to solve the problems in the prior art.

[0006] To achieve the above-mentioned purpose, the application provides the following solutions.

[0007] The application provides a promoter with high strength and good orthogonality, which comprises a nucleotide sequence as shown in any one of SEQ ID NO. 7-26.

[0008] The application also provides a recombinant vector comprising the promoter.

[0009] The application also provides a recombinant engineering bacterium comprising the recombinant vector.

[0010] The application also provides application of the promoter or the recombinant vector or the recombinant engineering bacterium in preparation of a product for accelerating expression of a target protein or increasing the expression amount of the target protein.

[0011] Preferably, the target protein comprises a fluorescent protein or an antigen.

[0012] The application also provides application of the promoter or the recombinant vector or the recombinant engineering bacterium in construction of a saRNA vaccine.

[0013] The application also provides a screening method of a promoter with high strength and good orthogonality, comprising the following steps.

[0014] Transferring genes encoding alphavirus replicase protein and fluorescent protein into a replicon transcription plasmid to construct a recombinant replicon transcription plasmid;

[0015] Taking the core sequence of the alphavirus replicase protein sub-promoter and the sequences upstream and downstream of the core sequence as research objects, a plurality of sets of PCR primers are designed, and then the recombinant replicon transcription plasmid is used as a template for PCR amplification, and the amplification product is transferred into eukaryotic cells to obtain an alphavirus replicase protein sub-promoter library;

[0016] The sub-promoters with different fluorescence intensities screened are sequenced by using a sequencing technology, and the original promoter sequence is compared to obtain a promoter with high strength and good orthogonality.

[0017] Preferably, the coding gene sequence of the alphavirus replicase protein is as shown in SEQ ID NO. 28; the alphavirus replicase protein sub-promoter core sequence is as shown in SEQ ID NO. 27.

[0018] Preferably, the nucleotide sequences of the multiple sets of PCR primers are as shown in SEQ ID NO. 1-6.

[0019] The application also provides a saRNA vaccine comprising the promoter.

[0020] The application discloses the following technical effects:

[0021] The application develops a library of alphavirus replicase protein sub-promoters constructed by sequence randomization, and establishes a 96-well plate or 384-well plate high-throughput screening system for the strength of the sub-promoter, so as to efficiently screen the constructed promoter library and obtain sub-promoters with different strengths. Furthermore, the system is applied to the regulation of the expression of different antigen genes in cells, so as to slow down and reduce the time (6-8 months) for reaching the titer that cannot be neutralized by variant viruses, and obtain a multivalent self-amplifying mRNA vaccine for effectively regulating the spatiotemporal expression of multiple genes, break through the research and development bottleneck of the multivalent self-amplifying mRNA vaccine, and make the saRNA vaccine safer, more universal and more cost-effective in the future nucleic acid drug field. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described in the following only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0023] Figure 1 The figure is a map of the replicon transcription plasmid pSINrep5, which contains the alphavirus replicase protein and the sub-promoter core sequence and the upstream and downstream sequences;

[0024] Figure 2 The figure is the fluorescence intensity of the replicon transcription plasmid pSINrep5 in cells; A: the cell growth condition and fluorescence intensity after the replicon pSINrep5 is transfected for 6h; B: the cell growth condition and fluorescence intensity after the replicon pSINrep5 is transfected for 24h; C: the cell growth condition and fluorescence intensity after the replicon pSINrep5 is transfected for 72h;

[0025] Figure 3The sequence is a sub-promoter core sequence of Alphavirus replicase protein and the upstream and downstream sequences; the black square is the sub-promoter core sequence;

[0026] Figure 4 The fluorescence intensity of the replicon transcription plasmid pSINrep5(9G→T) in cells; A: the cell growth and fluorescence intensity after 6h of transfection of the replicon transcription plasmid pSINrep5(9G→T); B: the cell growth and fluorescence intensity after 24h of transfection of the replicon transcription plasmid pSINrep5(9G→T); C: the cell growth and fluorescence intensity after 72h of transfection of the replicon transcription plasmid pSINrep5(9G→T). DETAILED DESCRIPTION

[0027] The detailed description set forth below is intended as a description of various example embodiments of the application and is not intended to represent the only embodiments in which the application can be practiced. The detailed description is intended only to afford a broader range of options and alternatives to the practitioner of ordinary skill in the art.

[0028] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each individual value or range of values that falls within the range of the stated value or range of values. In any statement of a value or a range of values, the intermediate values and the smaller ranges within the stated range of values are also included in the application. The upper and lower limits of these smaller ranges can independently be included or excluded from the range.

[0029] Unless otherwise defined, 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 application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0030] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0031] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional, unrecited elements or method steps.

[0032] Example 1

[0033] 1. Alphavirus replicase protein encoding gene and replicon synthesis

[0034] Through literature review and experimental verification, an alphavirus replicase protein and replicon with intracellular replication antigen mRNA synthesis activity were successfully synthesized and obtained. Figure 1 The replicon contains relevant restriction sites that can be inserted into any expressible protein gene; it also has a subpromoter core sequence (5'-ACUAUUUAGGACCACCGUAGAGAU-3', SEQ ID NO.27).

[0035] 2. Construction of a fluorescence detection system for subpromoter intensity

[0036] Simultaneous transfection of hamster kidney fibroblasts (BHK-21) with genes encoding alphavirus replicase protein and fluorescent protein:

[0037] Genes encoding alphavirus replicase protein and fluorescent protein were synthesized separately.

[0038] Genes encoding alphavirus replicase protein and fluorescent protein were introduced into the replicon transcription plasmid pSINrep5 to obtain a recombinant plasmid; then, the recombinant plasmid, helper plasmid pSINrep5-CMV-nsP1-4, and transfection reagent (Thermo Fisher Scientific's Lipofectamine) were used. TM The 3000 reagent was mixed at a ratio of 0.5 μg: 0.5 μg: 1.00 μL and then transferred into BHK-21 cells to obtain the replicase cell line.

[0039] Internal calibration: Co-transfection of 0.1 μg mScarlet-CAG plasmid (red fluorescence, λ_ex = 561 nm, λ_em = 610 nm) was used as an internal control. The relative promoter activity P was defined by the mNeonGreen / mScarlet fluorescence intensity ratio. a .

[0040] The alphavirus replicase protein was expressed using the host cell's transcription-translation system. This replicase protein specifically recognizes a subpromoter to synthesize mRNA encoding the fluorescent protein EGFP. Finally, the fluorescent protein was translated again using the host cell's translation system. The fluorescence intensity was directly proportional to the expression concentration of the fluorescent protein and also to the intensity of the alphavirus replicase protein subpromoter. The changes in fluorescence intensity over reaction times (6 h, 24 h, and 72 h) were measured using a fluorescence microplate reader (Biotek, Synergy H1) in black 96-well plates.

[0041] The results are as follows Figure 2As shown, a fluorescence detection system and a high-throughput screening system targeting subpromoter intensity have been established, which can greatly enhance the retention in host cells while maintaining fluorescence performance, thereby significantly improving the accuracy of RNA product fluorescence detection and screening; Figure 2 As shown in section A, cells transfected with the replicon transcription plasmid pSINrep5 for 6 hours showed significant growth, but almost no fluorescence intensity, indicating that the fluorescent protein had not yet been expressed. Figure 2 As shown in section B, cells transfected with the replicon transcription plasmid pSINrep5 showed significant growth 24 hours later and began to exhibit fluorescence intensity, indicating that the fluorescent protein had been expressed at this time; Figure 3 As shown in Figure C, cells transfected with the replicon transcription plasmid pSINrep5 for 72 hours showed greater growth and stronger fluorescence intensity than before, indicating that the fluorescent protein was already highly expressed and had not degraded at this time.

[0042] The nucleotide sequence (SEQ ID NO.28) of the alphavirus replicase protein is as follows:

[0043]

[0044] The amino acid sequence of the alphavirus replicase protein (SEQ ID NO.29) is as follows:

[0045]

[0046] The nucleotide sequence of the fluorescent protein (SEQ ID NO. 30) is as follows:

[0047] ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGT

[0048] TAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTA

[0049] AATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGT

[0050] TCAATGCTTTTCCCGTTATCCGGATCATATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGG

[0051] TTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGT

[0052] TTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTC

[0053] TCGGACACAAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAA

[0054] TGGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCAGACCATT

[0055] ATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAAT

[0056] CTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA.

[0057] 3. Perform high-throughput screening on the startup library.

[0058] With a 24nt subpromoter core sequence (5'-ACUAUUUAGGACCACCGUAGAGAU-3') Primer name Using alphavirus replicase protein subpromoters as the research object, degenerate PCR primers were redesigned (Table 1), and a randomized subpromoter library based on PCR primers and sequences was constructed. By introducing the previously designed fluorescence detection system for alphavirus replicase protein subpromoters, high-throughput screening of this promoter library can be performed. This lays a solid foundation for obtaining promoters with different intensities and good orthogonality. Specific experimental steps:

[0059] The plasmid pSINrep5 containing the fluorescent protein EGFP was used as a template, and multiple sets of synthetic primers were designed (as shown in Table 1). MutanBEST PCR (TaKaRa) was performed in a 50 μL reaction system using the manufacturer's protocol, containing 0.25 μL of enzyme, 2 ng / μL pSINrep5-EGFP template, and 0.5 μM of each primer. PCR amplification was then performed with an initial denaturation at 94 °C for 30 s, followed by 30 thermal cycles, each consisting of annealing at 55 °C for 30 s and extension at 72 °C for 12 min. The PCR product was then digested with DMT enzyme at 37 °C for 1 h. To screen for positive transformants and obtain a large number of cloned target genes, the PCR product obtained by digesting the methylated template with DMT enzyme was transfected into BHK-21 cells for subsequent observation of the fluorescent protein expression intensity.

[0060] Table 1. Thermal cycling primers used to replace specific sites during random mutation processes.

[0061] Sequence number Primer sequence (5'-3') Primer 1F SEQ ID NO. 1 GGTGAAATAAAGCATNNNNNNGGTGGTCCTAAATAGTCA Primer 1R SEQ ID NO. 2 ATGCTGACTATTTAGGACCACCGTNNNNNNGCTTTATTTCACC Primer 2F SEQ ID NO. 3 GGTGAAATAAAGCATCTCTACNNNNNNCCTAAATAGTCAGCATAGTA Primer 2R SEQ ID NO. 4 ATGCTGACTATTTAGGACNNNNNNAGAGATGCTTTATTTCACC Primer 3F SEQ ID NO. 5 AAATAAAGCATCTCTACGGTGGTNNNNNNTAGTCAGCATAGTA Primer 3R SEQ ID NO. 6 TACTATGCTGACTANNNNNNACCACCGTAGAGATGCTTTA Figure 3

[0062] 4. Screening promoters with different high strengths and good orthogonality

[0063] With a 24nt subpromoter core sequence 5'ACUAUUUAGGACCACCGUAGAGAU3' ( Mutant siteUsing PCR primers as the research object, degenerate PCR primers were redesigned, and a randomized subpromoter library based on PCR primers and sequences was constructed. Next-generation sequencing technology (Illumina MiSeq sequencing platform) was used to sequence the subpromoters with different fluorescence intensities. The sequencing data was processed using MiSeq Reporter software, and the mutated nucleic acid sites were identified by comparing with the original promoter sequences. Promoters that expressed fluorescent proteins for longer periods than those expressed by the original promoters were screened out, and finally 20 promoters with different high intensities and good orthogonality were obtained (Table 2).

[0064] Table 2. Promoters with different high strength and good orthogonality and their sequences.

[0065] Sequence number Gene sequence 1A→G SEQ ID NO. 7 gTCTCTACGGTGGTCCTAAATACT 1A→C SEQ ID NO. 8 cTCTCTACGGTGGTCCTAAATACT 1A→T SEQ ID NO. 9 tTCTCTACGGTGGTCCTAAATACT 2T→A SEQ ID NO. 10 AaCTCTACGGTGGTCCTAAATACT 2T→G SEQ ID NO. 11 AgCTCTACGGTGGTCCTAAATACT 2T→C SEQ ID NO. 12 AcCTCTACGGTGGTCCTAAATACT 3C→A SEQ ID NO. 13 ATaTCTACGGTGGTCCTAAATACT 3C→G SEQ ID NO. 14 ATgTCTACGGTGGTCCTAAATACT 3C→T SEQ ID NO. 15 ATtTCTACGGTGGTCCTAAATACT 6T→A SEQ ID NO. 16 ATCTCaACGGTGGTCCTAAATACT 6T→G SEQ ID NO. 17 ATCTCgACGGTGGTCCTAAATACT 6T→C SEQ ID NO. 18 ATCTCcACGGTGGTCCTAAATACT 9G→A SEQ ID NO. 19 ATCTCTACaGTGGTCCTAAATACT 9G→C SEQ ID NO. 20 ATCTCTACcGTGGTCCTAAATACT 9G→T SEQ ID NO. 21 ATCTCTACtGTGGTCCTAAATACT 12G→A SEQ ID NO. 22 ATCTCTACaTGGTCCTAAATACT 12G→C SEQ ID NO. 23 ATCTCTACcTGGTCCTAAATACT 12G→T SEQ ID NO. 24 ATCTCTACtTGGTCCTAAATACT 15G→A SEQ ID NO. 25 ATCTCTACaTGGTCTTAATACT 15G→C SEQ ID NO. 26 ATCTCTACcTGGTCTTAATACT 15G→T SEQ ID NO. 27 ATCTCTACtTGGTCTTAATACT 18G→A SEQ ID NO. 28 ATCTCTACaTGGTCCTAAATACT 18G→C SEQ ID NO. 29 ATCTCTACcTGGTCCTAAATACT 18G→T SEQ ID NO. 30 ATCTCTACtTGGTCCTAAATACT SEQ ID NO. 22 ATCTCTACGGT a GTCCTAAATACT 12 G→C SEQ ID NO. 23 ATCTCTACGGT c GTCCTAAATACT 12 G→T SEQ ID NO. 24 ATCTCTACGGT t GTCCTAAATACT 18 A→T SEQ ID NO. 25 ATCTCTACGGT G GTCCT t AATACT 21 T→G SEQ ID NO. 26 ATCTCTACGGT G GTCCT AAAG ACT

[0066] 5. Cellular experimental verification

[0067] Using the 9G→T mutation site as an example, a subpromoter cell system containing the 9G→T mutation site was obtained according to the above-described method for constructing a fluorescence detection system. The fluorescence intensity of the replicon transcript plasmid pSINrep5(9G→T) in BHK-21 cells was observed, indicating that the fluorescence intensity of the subpromoter in BHK-21 cells is preserved. Figure 4 ).

[0068] Depend on Figure 4 As shown in section A, cells began to grow and exhibited weak fluorescence intensity 6 hours after transfection with the replicon transcription plasmid pSINrep5(9G→T), indicating that the fluorescent protein had begun to be expressed at this time; Figure 4 As shown in section B, cells transfected with the replicon transcription plasmid pSINrep5(9G→T) 24 hours later exhibited greater growth and stronger fluorescence intensity, indicating that the fluorescent protein was already highly expressed at this time; Figure 4 As shown in Figure C, cells transfected with the replicon transcription plasmid pSINrep5(9G→T) 72 h showed no growth compared to 24 h, but the fluorescence intensity was stronger, indicating that the fluorescent protein was already expressed in large quantities and had not degraded at this time; simultaneously, from Figure 4 and Figure 2 In comparison, the expression time of the fluorescent protein was earlier and the fluorescence intensity was increased, indicating that the 9G→T mutation at the site accelerated the expression time and increased the expression level of the protein.

[0069] Other promoters screened in Table 2 were also found to accelerate protein expression time and increase protein expression levels through experiments.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A promoter with high strength and good orthogonality, characterized in that, The promoter includes any of the nucleotide sequences shown in SEQ ID NO.7-26.

2. A recombinant vector, characterized in that, It includes the promoter as described in claim 1.

3. A recombinant engineered bacterium, characterized in that, It includes the recombinant vector as described in claim 2.

4. The use of the promoter as described in claim 1, the recombinant vector as described in claim 2, or the recombinant engineered bacteria as described in claim 3 in the preparation of products that accelerate the expression of the target protein or increase the expression level of the target protein.

5. The application as described in claim 4, characterized in that, The target protein may include a fluorescent protein or an antigen.

6. The use of the promoter as described in claim 1, the recombinant vector as described in claim 2, or the recombinant engineered bacteria as described in claim 3 in the construction of saRNA vaccines.

7. A method for screening promoters with high strength and good orthogonality, characterized in that, Includes the following steps: Genes encoding alphavirus replicase protein and fluorescent protein were transferred into replicon transcription plasmids to construct recombinant replicon transcription plasmids; Using the core sequence of the alphavirus replicase protein subpromoter and its upstream and downstream sequences as the research object, multiple sets of PCR primers were designed, and then PCR amplification was performed using the recombinant replicon transcription plasmid as a template. The amplification product was then transferred into eukaryotic cells to obtain an alphavirus replicase protein subpromoter library. Using sequencing technology, subpromoters with different fluorescence intensities were sequenced and compared with the original promoter sequences to obtain promoters with high intensity and good orthogonality.

8. The method as described in claim 7, characterized in that, The gene sequence encoding the alphavirus replicase protein is shown in SEQ ID NO.28; the core sequence of the alphavirus replicase protein subpromoter is shown in SEQ ID NO.

27.

9. The method as described in claim 7, characterized in that, The nucleotide sequences of the multiple sets of PCR primers are shown in SEQ ID NO. 1-6.

10. A saRNA vaccine, characterized in that, Includes the promoter described in claim 1.

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