Translation regulatory element, expression vector, pharmaceutical composition, vaccine composition, construction method, method for expressing target protein, and use thereof

Abstract

Provided are a translation regulatory element, an expression vector, a pharmaceutical composition, a vaccine composition, a construction method, a method for expressing a target protein, and the use thereof. The translation regulatory element is selected from recombinant IRES sequences having sequences as shown in SEQ ID NOs: 8-11, and / or one or more segments of recombinant IRES sequences complementary to the sequences as shown in SEQ ID NOs: 8-11; and can show high translation efficiency when being used for regulating gene expression, thereby realizing high-level expression of a target gene.

Description

A translation regulatory element, an expression vector, a pharmaceutical composition, a vaccine composition, a construction method, a method for expressing a target protein and application TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a translation regulatory element, an expression vector, a pharmaceutical composition, a vaccine composition, a construction method, a method for expressing a target protein and application. BACKGROUND

[0002] Messenger ribonucleic acid (mRNA) refers to a type of single-stranded ribonucleic acid transcribed from a strand of DNA as a template, and is a medium for transmitting genetic information from DNA to ribosomes in eukaryotic cells. The genetic information carried by mRNA serves as a direct template for protein biosynthesis and determines the amino acid sequence of the gene expression protein product. mRNA therapy uses in vitro synthesized mRNA as an information carrier to guide somatic cell protein synthesis, regulate gene expression, affect cell function and disease progression, and achieve therapeutic effects.

[0003] The translation process in eukaryotic cells requires the binding of the 5' end cap structure of mRNA to ribosomes to initiate translation; therefore, the 5' end cap structure needs to be introduced during the process of synthesizing RNA in vitro. The introduction of the cap structure increases the complexity and cost of RNA preparation; and transcription factors, RNA binding proteins and other regulatory factors in cells may affect the function of the cap structure, thereby affecting the regulation of gene expression; at the same time, the cap structure may be affected by certain regulatory mechanisms in cells, making the translation efficiency unstable in some cases; in addition, the cap structure usually limits the starting position of translation, making translation start only from the 5' end of RNA, which may have a significant adverse effect on the accuracy of translation, thereby affecting the therapeutic effect. Traditional mRNA gene expression therapy still faces great challenges in stability, specificity and effectiveness. Therefore, finding alternative translation regulatory elements for the 5' end cap structure, such as IRES sequences, can help overcome the above problems and improve the efficiency and stability of gene expression, bringing new opportunities for research and application in the field of gene therapy and protein expression.

[0004] The IRES sequence refers to an untranslated region (UTR) nucleotide sequence having a cap structure-independent translation initiation ability, capable of recruiting ribosomes to the translation initiation site of the viral mRNA by means of a trans-acting factor, and by integrating the IRES sequence into an exogenous ribonucleic acid vector, independent translation initiation can be achieved. In addition, the IRES sequence also has more flexible applicability, and researchers can select appropriate sources of IRES sequences according to needs. Therefore, the IRES sequence can be well applied to the construction of various gene vectors such as gene therapy and protein expression design. However, compared with the traditional cap structure-dependent translation initiation mechanism, the IRES sequence usually shows low translation efficiency, low protein expression amount, poor gene expression effect, which greatly limits its wide application in gene expression regulation, and has great limitations. SUMMARY

[0005] The first object of the present application is to solve the problems existing in the application of the existing IRES sequence as a translation regulatory element in gene expression regulation, and to provide a translation regulatory element comprising a recombinant IRES sequence having high translation efficiency, which can well achieve high-level expression of the target gene.

[0006] The second object of the present application is to provide an expression vector.

[0007] The third object of the present application is to provide a construction method of the above expression vector.

[0008] The fourth object of the present application is to provide a pharmaceutical composition.

[0009] The fifth object of the present application is to provide a vaccine composition.

[0010] The sixth object of the present application is to provide the application of the above-mentioned translation regulatory element, expression vector, pharmaceutical composition and / or vaccine composition in regulating the expression of the target gene of the cell.

[0011] Specifically, the translation regulatory element provided by the present application comprises one or more segments of the recombinant IRES sequence as shown in the nucleotide sequence of SEQ ID NO: 8-11; and / or the translation regulatory element comprises one or more segments of the recombinant IRES sequence complementary to the sequence as shown in SEQ ID NO: 8-11.

[0012] In some specific embodiments, the recombinant IRES sequence is obtained by DNA shuffling of the CVB3 IRES sequence; the CVB3 IRES sequence comprises a nucleotide fragment as shown in SEQ ID NO: 1 or a nucleotide fragment complementary to the sequence as shown in SEQ ID NO: 1.

[0013] In some embodiments, the DNA shuffling comprises: subjecting the CVB3 IRES sequence to random fragmentation by a deoxyribonuclease to obtain a fragmented product; subjecting the fragmented product to primer-free PCR to obtain a primer-free PCR product; and subjecting the primer-free PCR product to primer-dependent PCR to obtain the primer-dependent PCR product.

[0014] In some embodiments, the primer system of the primer-dependent PCR comprises a primer 750-IRES-F having a nucleotide sequence as set forth in SEQ ID NO: 2 and / or a primer 750-IRES-R having a nucleotide sequence as set forth in SEQ ID NO: 3.

[0015] The expression vector provided by the present application comprises the translation regulatory element described above.

[0016] In some embodiments, the expression vector comprises a coding region of a protein of interest.

[0017] In some embodiments, the expression vector is linear or circular.

[0018] In some embodiments, the expression vector is DNA or RNA.

[0019] In some embodiments, the expression vector is circRNA.

[0020] In some embodiments, the expression vector is linear mRNA.

[0021] The construction method of the above expression vector provided by the present application comprises: the construction method comprises: Gibson assembly of the translation regulatory element and a plasmid to obtain the expression vector.

[0022] The pharmaceutical composition provided by the present application comprises the expression vector described above.

[0023] The vaccine composition provided by the present application comprises the expression vector described above.

[0024] The method for expressing a protein of interest provided by the present application comprises: introducing the expression vector described above into a host cell to express the protein of interest.

[0025] The translation regulatory element, the expression vector, the pharmaceutical composition and / or the vaccine composition described above provided by the present application are applied to regulate expression of a gene of interest in a cell. Advantages:

[0026] The translation regulatory element provided by the present application comprises a specific recombinant IRES sequence, which shows high translation efficiency when applied in gene expression regulation, and can realize high-level expression of the target gene. The reason for its high translation efficiency is that the recombinant IRES sequence comprising a nucleotide fragment as shown in SEQ ID NO: 8-11 or a nucleotide fragment complementary to the sequence shown in SEQ ID NO: 8-11 has high structuralization and tightness, has low dependence on IRES trans-acting factors and translation initiation factors compared with the original CVB3 IRES sequence, can better bind to ribosomes, realize the initiation of translation, and thus show higher translation efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0027] Fig. 1 is an agarose gel electrophoresis diagram of the fragmented DNA product prepared in Example 1 of the present application;

[0028] Fig. 2 is an agarose gel electrophoresis diagram of the primer-free PCR product prepared in Example 1 of the present application;

[0029] Fig. 3 is an agarose gel electrophoresis diagram of the primer-containing PCR product prepared in Example 1 of the present application;

[0030] Fig. 4 is a structural schematic diagram of the linear plasmid prepared in Example 1 of the present application;

[0031] Fig. 5 is an agarose gel electrophoresis diagram of circRNAs 1, 2, 3 and 4 prepared in Example 2 of the present application. DETAILED DESCRIPTION

[0032] The translation regulatory element provided by the present application comprises one or more segments of the recombinant IRES sequence as shown in SEQ ID NO: 8-11; and / or, the translation regulatory element comprises one or more segments of the recombinant IRES sequence complementary to the sequence as shown in SEQ ID NO: 8-11.

[0033] In the present application, the translation regulatory element refers to a type of nucleotide sequence fragment having a regulatory effect on the translation process of gene expression, which can be DNA or RNA. When the translation regulatory element is DNA, the nucleotide sequence thereon is transferred from DNA to RNA during transcription to play a role in regulating translation. When the translation regulatory element is RNA, the nucleotide sequence thereon recruits and binds ribosomes to realize the initiation of translation.

[0034] In the present application, the recombinant IRES sequence is obtained by using genetic engineering technology to perform directed mutation or non-directed mutation based on the CVB3 IRES sequence comprising the nucleotide fragment as shown in SEQ ID NO: 1 or the nucleotide fragment complementary to the sequence as shown in SEQ ID NO: 1. The genetic engineering technology is a method commonly used in the existing biological field, and the modification from the CVB3 IRES sequence to the recombinant IRES sequence is limited, and specific examples include but are not limited to PCR site-directed mutagenesis technology and / or DNA shuffling technology.

[0035] In the present application, the recombinant IRES sequence is preferably obtained by PCR site-directed mutagenesis of the CVB3 IRES sequence. The PCR site-directed mutagenesis specifically refers to designing corresponding mutant primers based on the CVB3 IRES sequence and according to the nucleotide fragments as shown in SEQ ID NO: 8-11, so as to introduce corresponding mutations on the CVB3 IRES sequence during PCR amplification to obtain the recombinant IRES sequence. The design of the mutant primer can be obtained by primer design software, or the primer obtained by primer design software is further artificially designed, and the present application does not particularly limit the way of obtaining it, as long as the directed modification from the CVB3 IRES sequence to the recombinant IRES sequence is limited.

[0036] In the present application, the recombinant IRES sequence is preferably obtained by DNA shuffling of the CVB3 IRES sequence. The DNA shuffling specifically refers to randomly fragmenting the CVB3 IRES sequence by taking a deoxyribonuclease to obtain a fragmentation product, taking the fragmentation product to perform primerless PCR to obtain a primerless PCR product, and taking the primerless PCR product to perform primer PCR to obtain the primer PCR.

[0037] In some specific embodiments, the deoxyribonuclease specifically refers to a kind of nuclease capable of specifically cutting the phosphodiester bond in DNA, which has randomness in cutting the phosphodiester bond, so that the fragmentation product has high abundance, and specific examples include but are not limited to DNase I enzyme.

[0038] In some preferred embodiments, based on the input mass of the CVB3 IRES sequence, the addition amount of the deoxyribonuclease is preferably 0.1-0.3 U / 1 μg, and specifically can be 0.1 U / 1 μg, 0.13 U / 1 μg, 0.15 U / 1 μg, 0.2 U / 1 μg, 0.24 U / 1 μg, 0.28 U / 1 μg, 0.3 U / 1 μg or any value therebetween.

[0039] In some preferred embodiments, the conditions for the random fragmentation include a temperature of preferably 10-15°C, specifically 10°C, 10.2°C, 10.8°C, 11°C, 13°C, 14°C, 15°C, or any value therebetween; and a time of preferably 5-20 min, specifically 5 min, 6 min, 8 min, 10 min, 15 min, 20 min, or any value therebetween.

[0040] In some specific embodiments, the length of the fragmented product is preferably 50-100 bp, specifically 50 bp, 55 bp, 58 bp, 60 bp, 68 bp, 75 bp, 80 bp, 99 bp, 100 bp, or any value therebetween. In this case, the length of the fragmented product is suitable, which is conducive to the subsequent primer-free PCR and primer-based PCR, and realizes a higher success rate of DNA shuffling.

[0041] In the present application, the primer-free PCR specifically refers to the use of the homology between the fragmented products to replace primers to realize the amplification of nucleotide fragments, which is a method commonly used in the prior art. Those skilled in the art can make adaptive adjustments to the reaction system and reaction conditions of the primer-free PCR according to the nucleotide sequence of the fragmented product, and the present application does not particularly limit it.

[0042] In some specific embodiments, the length of the primer-free PCR product is preferably 50-500 pb, specifically 50 pb, 58 pb, 60 pb, 75 pb, 80 pb, 100 pb, 120 pb, 180 pb, 200 pb, 300 pb, 400 pb, 500 pb, or any value therebetween. In this case, the primer-free PCR product has a more suitable length increase compared to the fragmented product, wherein a certain number of mutations are introduced, which is conducive to realizing a higher success rate of DNA shuffling.

[0043] In the present application, the primer-based PCR specifically refers to the use of the CVB3 IRES sequence reagent to obtain a corresponding primer system to amplify the primer-free PCR product, which is a method commonly used in the prior art. Those skilled in the art can make adaptive adjustments to the reaction system and reaction conditions of the primer-free PCR according to the nucleotide sequence of the primer-free PCR product, and the present application does not particularly limit it.

[0044] In some specific embodiments, the primer system in the primer-based PCR preferably includes the primer 750-IRES-F with the nucleotide sequence shown in SEQ ID NO: 2 and / or the primer 750-IRES-R with the nucleotide sequence shown in SEQ ID NO: 3.

[0045] The expression vector provided by the present application comprises the translation regulatory element described above, and can realize ribosome-dependent recognition and binding of the expression vector or the expression vector transcription product, and mediate the start of translation.

[0046] In the present application, the expression vector further comprises a protein coding region of interest, and the protein coded by the protein coding region of interest has a therapeutic effect and / or a protective effect. Specific examples of the protein coded by the protein coding region of interest can be, but are not limited to, one or more of an immunogenic compound, a lymphokine and a cytokine. The immunogenic compound is a kind of substance with immunogenicity and / or reactivity. The lymphokine is a kind of protein molecule that plays a regulatory and modulatory role in the lymphatic system. The cytokine is a kind of molecule with biological activity that mainly acts on cell growth, proliferation, differentiation and function.

[0047] In the present application, the number of the protein coding region of interest on the expression vector is specifically an integer not less than 1, and can be 1, 2, 3, 4 or any integer greater than them.

[0048] In the present application, the expression vector can be linear or circular. According to the structure of the expression vector, the recombinant IRES sequence and the protein coding region of interest can have different relative positions. When the expression vector is linear, the recombinant IRES sequence is located upstream of the protein coding region of interest. When the expression vector is circular, the recombinant IRES sequence is located upstream or downstream of the protein coding region of interest. It should be noted that “... is located upstream / downstream of...” is defined in the direction of 5'-3' end of the nucleotide fragment.

[0049] In the present application, the expression vector can be DNA or RNA. When the expression vector is preferably DNA, it can be used as a template for transcription to synthesize the protein of interest in cells, and can also be replicated in cells to realize stable and efficient expression of the target gene.

[0050] In the present application, when the expression vector is preferably RNA, it can be directly used as a template for translation to synthesize the protein of interest, and realize efficient expression of the target gene. In some specific embodiments, the expression vector can be more specifically circRNA and / or linear mRNA, and the corresponding structure can be selected according to the actual application requirements.

[0051] In the present application, when the expression vector is RNA, the expression vector preferably further comprises a 5' untranslated region and / or a 3' untranslated region. Among them, the 5' untranslated region is a nucleotide sequence between the 5' end and the start codon of the coding region of the target protein, which has high sensitivity for translation initiation. The 3' untranslated region is a nucleotide sequence from the stop codon of the coding region of the target protein to the 3' end.

[0052] The method for constructing the above expression vector provided by the present application specifically comprises Gibson assembly of the translation regulatory element and a plasmid, and obtaining the expression vector through amplification and / or transcription.

[0053] In the present application, the Gibson assembly specifically comprises homologous treatment of the recombinant IRES sequence and a plasmid to obtain an insertion fragment with homologous arms and a linear plasmid; Gibson assembly of the insertion fragment and the linear plasmid to obtain the expression vector.

[0054] In the present application, the homologous treatment preferably comprises: (1) enzyme digestion of the plasmid to obtain a linear plasmid with 5' and 3' ends, and optional PCR amplification of the linear plasmid to obtain a linear plasmid with non-homologous 5' and 3' ends; (2) designing corresponding primers based on the 5' and 3' ends of the linear plasmid and the nucleotide sequence of the translation regulatory element, and using PCR amplification to obtain an insertion fragment with homologous 5' and 3' ends to the linear plasmid, so that the ends of the linear plasmid can be base-paired complementary to the insertion fragment.

[0055] In some specific embodiments, the primer system used in step (1) of the homologous treatment preferably comprises FKplasmid-R with a nucleotide sequence as shown in SEQ ID NO: 4 and / or FKplasmid-F with a nucleotide sequence as shown in SEQ ID NO: 5.

[0056] In some specific embodiments, the primer system used in step (2) of the homologous treatment preferably comprises primer GR-F with a nucleotide sequence as shown in SEQ ID NO: 6 and / or primer GR-R with a nucleotide sequence as shown in SEQ ID NO: 7.

[0057] In the present application, the plasmid is a kind of substance commonly used in the biological field, which can be a recombinant plasmid pre-inserted with a target gene, or a blank plasmid not inserted with a target gene, and specific examples thereof include but are not limited to pUC19 and / or pUC18.

[0058] In the present application, the assembly preferably comprises transforming the competent cells after co-incubation of the insert and the linear plasmid in the presence of T5 exonuclease, husion DNA polymerase, Taq DNA ligase, etc.

[0059] In the present application, the transformed competent cells are subjected to corresponding expression, extraction and purification according to the structure (linear or circular) and chemical nature (DNA or RNA) of the expression vector to be obtained, and the methods used are the technical means commonly used in the art, which are not particularly limited in the present application.

[0060] The pharmaceutical composition provided in the present application specifically comprises the above-mentioned expression vector, and the expression vector comprises a coding region of a target protein.

[0061] In the present application, the pharmaceutical composition preferably further comprises an adjuvant for improving the delivery effect of the expression vector, and specific examples of the adjuvant include but are not limited to one or more of polyethyleneimine, polyethylene glycol and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride.

[0062] The vaccine composition provided in the present application specifically comprises the above-mentioned expression vector, and the expression vector comprises a coding region of a target protein.

[0063] In the present application, the vaccine composition preferably further comprises an adjuvant for improving the immunostimulatory properties of the expression vector, and specific examples of the adjuvant include but are not limited to one or more of interleukin, interferon, immune-activating protein, cytidine, double-stranded polyinosinic acid, aluminum hydroxide, alum, polyethyleneimine, polyethylene glycol and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride.

[0064] The method for expressing a target protein provided in the present application specifically comprises introducing the above-mentioned expression vector comprising a coding region of a target protein into a host cell for expression of the target protein.

[0065] In the present application, specific examples of the method for introducing the expression vector into a host cell include but are not limited to one or more of electroporation, microinjection, biolistics, calcium phosphate co-precipitation, liposome carrier and virus.

[0066] The present application also provides the use of the above-mentioned translation regulatory element, expression vector, pharmaceutical composition and / or vaccine composition in regulating the expression of a target gene in a cell.

[0067] Hereinafter, examples of the present application are described in detail. Examples are intended to explain the present application, and cannot be construed as limiting the present application. Unless otherwise specified, technical or conditions in examples are performed according to the techniques or conditions described in the literature in the art or according to the product manual. Unless otherwise specified, reagents or instruments used are all conventional products that can be commercially available.

[0068] The nucleotide sequences involved in the present application are specifically shown in Table 1:

[0069] Table 1. Nucleotide sequence table

[0070] Example 1

[0071] This example is used to illustrate the construction of an expression vector comprising a recombinant IRES sequence as a translation regulatory element, which is based on the construction of a recombinant IRES sequence and an expression vector using a CVB3 IRES sequence (nucleotide sequence shown as SEQ ID NO: 1) derived from an original enterovirus:

[0072] 1. Construction of a recombinant IRES sequence

[0073] The recombinant IRES sequence is constructed based on DNA shuffling technology, through DNA fragmentation, primerless PCR and primer PCR steps, specifically including:

[0074] (1) Random fragmentation of CVB3: S11, take 1 μL of DNase I enzyme with a concentration of 10 U / μL, 25 μL of Tris-HCl (pH 7.4), 50 μL of MgCl2 solution with a concentration of 20 mM and 424 μL of sterile deionized water, mix and stir at 15°C for 10 min, to obtain an enzyme digestion reaction solution with a final concentration of DNase I enzyme of 0.15 U / μL.

[0075] S12, take 10 μg of CVB3 and mix with 500 μL of enzyme digestion reaction solution, digest at 15°C for 10 min, then terminate the reaction at 90°C for 10 min; use a DNA recovery kit (Nanjing Novogene Bio-tech Co., Ltd., product number DC201-01) and recover the fragmented DNA product by agarose gel electrophoresis. Figure 1 is an agarose gel electrophoresis map of the obtained fragmented DNA product, which shows that the length of the fragmented DNA product is 30-50 bp.

[0076] (2) Primer-free PCR: 10 μg of the fragmented DNA product was subjected to primer-free PCR, and the primer-free PCR product was recovered by using a DNA recovery kit and agarose gel electrophoresis. Fig. 2 is an agarose gel electrophoresis graph of the obtained primer-free PCR product. As can be seen from Fig. 2, the band is diffuse and not clear, and the length of the primer-free PCR product is 50-900 bp.

[0077] The reaction system of the primer-free PCR comprises: 10 μL of the fragmented DNA product, 10 μL of 5x fastpfu buffer, 4 μL of dNTPs (2.5 mM), 1.5 μL of Fastpfu, and ddH2O is added to a total volume of 50 μL; the reaction conditions comprise: 94°C pre-denaturation for 60 s, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 30 s, 40 cycles.

[0078] (3) Primer PCR: primer 750-IRES-F (nucleotide sequence as shown in SEQ ID NO: 2) and primer 750-IRES-R (nucleotide sequence as shown in SEQ ID NO: 3) were used to amplify the primer-free PCR product by primer PCR, and the primer PCR product was recovered by using a DNA recovery kit and agarose gel electrophoresis. Fig. 3 is an agarose gel electrophoresis graph of the obtained primer PCR product. As can be seen from Fig. 3, the length of the primer PCR product is about 750 bp.

[0079] The reaction system of the primer PCR comprises: 10 μg of the fragmented DNA product, 10 μL of 5x fastpfu buffer, 4 μL of dNTPs (2.5 mM), 1.5 μL of Fastpfu, 1 μL of primer 750-IRES-F, 1 μL of primer 750-IRES-R, and ddH2O is added to a total volume of 50 μL; the reaction conditions comprise: 94°C pre-denaturation for 60 s, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 5 min, 30 cycles.

[0080] 2. Construction of expression vector

[0081] The expression vector is obtained based on Gibson assembly technology, specifically comprising:

[0082] (1) Plasmid reverse amplification: S11, the custom pUC19-Circ-Nanoluc plasmid was reverse amplified by using primers FKplasmid-R (nucleotide sequence as shown in SEQ ID NO: 4) and FK plasmid-F (nucleotide sequence as shown in SEQ ID NO: 5), and the linear plasmid was recovered by using a DNA recovery kit and agarose gel electrophoresis method. The structure of the linear plasmid is shown in FIG. 4. It should be noted that the structure shown in FIG. 4 only shows the fragments related to the construction of the expression vector, and other fragments are omitted.

[0083] The reaction system for reverse amplification includes: 10 μg of pUC19-Circ-Nanoluc plasmid, 10 μL of 5×fastpfu buffer, 4 μL of dNTPs (2.5 mM), 1.5 μL of Fast pfu, 1 μL of primer FK plasmid-R, 1 μL of primer FK plasmid-F, and ddH2O added to a total volume of 50 μL; the reaction conditions include: 94°C pre-denaturation for 60 s, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 5 min, 30 cycles.

[0084] S12, the obtained primer PCR product was PCR amplified by using primers GR-F (nucleotide sequence as shown in SEQ ID NO: 6) and primer GR-R (nucleotide sequence as shown in SEQ ID NO: 7), and was recovered by using a DNA recovery kit and agarose gel electrophoresis method to obtain an insertion fragment with homologous arms to the linear plasmid.

[0085] The reaction system for PCR amplification includes: 10 μg of C18, C35, C36 or C38, 10 μL of 5×fastpfu buffer, 4 μL of dNTPs (2.5 mM), 1.5 μL of Fast pfu, 1 μL of primer F, 1 μL of primer R, and ddH2O added to a total volume of 50 μL; the reaction conditions include: 94°C pre-denaturation for 60 s, 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 5 min, 30 cycles.

[0086] (2) Assembly of linear plasmid and insert: Gibson Assembly Cloning Kit (Nanjing Novozyme Bio-tech Co., Ltd., Catalog No. C1115-02-AA) was used to connect the linear plasmid and the insert according to the instructions to obtain a reaction solution containing the expression vector. 5 μL of the reaction solution was used to transfect 50 uL of E. coli DH5a competent cells by fast transformation, which were then plated on LB solid medium (Biosharp, Catalog No. BL1057A) containing 5 g / L Amp antibiotic. 200 single colonies with good growth were selected for sequencing. After comparison, 38 different sequences were obtained. Four recombinant IRES sequences C18, C35, C36 and C38 were obtained by screening and identifying the above sequences, i.e. the expression vector library was successfully constructed.

[0087] The amino acid sequences of the recombinant IRES sequences C18, C35, C36 and C38 constructed in this example are shown in Table 1.

[0088] Example 2

[0089] This example is used to illustrate the construction of circRNA, which specifically includes:

[0090] 1. In vitro transcription: the expression vector library constructed in Example 1 was used to obtain competent cells, and the expression vector was obtained. The expression vector was subjected to in vitro transcription to obtain RNA linear precursor.

[0091] The reaction system for in vitro transcription includes: 1.5 μL of ATP (100 mM), 1.5 μL of UTP (100 mM), 1.5 μL of CTP (100 mM), 1.5 μL of GTP (100 mM), 1 μg of RNA linear precursor, 2 μL of T7 RNA polymerase, 2 μL of 10× DNA reaction buffer, 1 μL of pyrophosphatase, 0.3 μL of RNase inhibitor, and RNase-free water added to a total volume of 20 μL. The reaction conditions include an incubation temperature of 37°C and a time of 3 h.

[0092] 2. Cyclization reaction: the RNA linear precursor was subjected to in vitro cyclization, and then 5 U of RNase R was used to digest the cyclization product to degrade the unreacted RNA linear precursor to obtain circRNAs 1, 2, 3 and 4. Agarose gel electrophoresis was used to verify the obtained circRNAs 1, 2, 3 and 4, and the results are shown in Figure 5. As shown in Figure 5, the bands of circRNAs 1, 2, 3 and 4 are clear, and the length is 1524 nt.

[0093] The in vitro circularization reaction system consisted of: 125 μg of linear RNA precursor, 5 μL of T4 RNase reaction buffer, 1 μL of GTP (100 mM), 0.3 μL of RNase inhibitor, and RNase-free water added to a total volume of 250 μL. The prepared reaction system was mixed 10 times by pipetting or vortexed, and then incubated at 55°C in a metal bath for 10 min.

[0094] Example 3

[0095] This embodiment utilizes luciferin reporter gene technology to illustrate the translation efficiency of the circRNA provided in Example 2. Specifically, it includes: HFB cells being seeded into DMEM complete medium (Gibco, catalog number 11965092) containing 10% fetal bovine serum (Gibco, catalog number 10099-141) and 1% penicillin / streptomycin (Gibco, catalog number 15140122) and cultured at 37°C until the cell density is approximately 60%; THP-1 and T cells being seeded into RPMI complete 1640 medium (Gibco, catalog number 11875119) containing 10% fetal bovine serum and 1% penicillin / streptomycin and cultured at 37°C until the cell density is approximately 60%.

[0096] circRNA1, 2, 3, and 4 were used. Transfection kit ( HFB, THP-1, and T cells were transfected with a luciferase reporter assay kit (Promega, catalog number N1110) for 48 hours according to the instructions. Nanolucase expression was detected using the kit, following the instructions. circRNA 0 (containing only the CVB3 IRES sequence) obtained from linear plasmids via in vitro transcription and circularization was used as a control. Relative fluorescence intensity was calculated using the following formula, and the results are shown in Table 2. Relative fluorescence intensity = Fo x / F0

[0097] In the formula, F x F0 represents the fluorescence intensity measured in the experimental group (circRNA 1, 2, 3, or 4), while F0 represents the fluorescence intensity measured in the control group (CVB3).

[0098] Table 2. Translation efficiency of circRNA

[0099] From the test results, compared with the original CVB3 IRES sequence, the recombinant IRES sequences C18, C35, C36 and C38 provided by the embodiment of the application can well improve the expression amount of Nanoluc enzyme in HFB, THP-1 and T cells, that is, it is explained that the translation efficiency is high when the IRES sequence is applied as a translation regulatory element in gene expression regulation, and has a good application prospect.

[0100] Although the embodiments of the present application have been shown and described above, it should be understood by those skilled in the art that the above embodiments are exemplary and cannot be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and purposes of the present application within the scope of the present application.

Claims

1. A translation regulatory element, comprising, The translation regulatory element comprises one or more segments of a recombinant IRES sequence having a nucleotide sequence as shown in SEQ ID NO: 8-11. And / or, the translation regulatory element comprises one or more segments of a recombinant IRES sequence complementary to a sequence as shown in SEQ ID NO: 8-11.

2. The translational regulatory element according to claim 1, wherein The recombinant IRES sequence is obtained by DNA shuffling from a CVB3 IRES sequence; the CVB3 IRES sequence comprises a nucleotide fragment as shown in SEQ ID NO: 1 or a nucleotide fragment complementary to a sequence as shown in SEQ ID NO:

1.

3. The translational regulatory element of claim 2, wherein The DNA shuffling specifically comprises: subjecting the CVB3 IRES sequence to random fragmentation by a deoxyribonuclease to obtain a fragmented product; subjecting the fragmented product to a primer-free PCR to obtain a primer-free PCR product; and subjecting the primer-free PCR product to a primer-containing PCR to obtain the primer-containing PCR product.

4. The translation regulatory element of claim 3, wherein The primer system of the primer-containing PCR comprises a primer 750-IRES-F having a nucleotide sequence as shown in SEQ ID NO: 2 and / or a primer 750-IRES-R having a nucleotide sequence as shown in SEQ ID NO:

3.

5. An expression vector, characterized by, The translation regulatory element of claim 1.

6. The expression vector of claim 5, wherein, The expression vector comprises a coding region of a protein of interest.

7. The expression vector of claim 5, wherein, The expression vector is linear or circular.

8. The expression vector of claim 5, wherein, The expression vector is DNA or RNA.

9. The expression vector of claim 5, wherein, The expression vector is a circRNA.

10. The expression vector of claim 5, wherein, The expression vector is a linear mRNA.

11. The method for constructing an expression vector according to claim 5, wherein The construction method comprises: Gibson assembly of the translation regulatory element and a plasmid to obtain the expression vector.

12. A pharmaceutical composition, characterized by, The expression vector of claim 5.

13. A vaccine composition, characterized in that, The expression vector of claim 5.

14. A method of expressing a protein of interest, comprising, The method comprises: introduction of the expression vector of claim 5 into a host cell for expression of a protein of interest.

15. Use of the translation regulatory element of claim 1 in regulating expression of a gene of interest in a cell.

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