Nucleic acid for coding chicken ovalbumin and expression vector

Abstract

The invention relates to the technical field of gene engineering, in particular to nucleic acid for coding chicken ovalbumin and an expression vector. Coding nucleic acid codons of chicken ovalbumin are optimized, and four versions of optimized sequences are obtained. According to the obtained nucleic acid sequence, the expression quantity of OVA in a host is remarkably improved compared with that of a wild type and other contrast, mRNA prepared from the nucleic acid can have good transfection efficiency, and higher expression quantity can be obtained by transferring the nucleic acid into cells, especially 293T cells, so that the immune response of a human immune system can be better simulated.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to nucleic acids encoding chicken ovalbumin and their expression vectors. Background Technology

[0002] Chicken ovalbumin (OVA): the main protein in egg white. OVA is a typical globulin with a molecular weight of approximately 44.5 kDa, containing phosphorus and sugar, and possessing four free radicals and 385 amino acid residues. These amino acid residues intertwine and fold to form a highly spherical structure with a high degree of secondary structure, mostly α-helices and β-sheets. Chicken ovalbumin has various uses in immunization and vaccine preparation.

[0003] As a typical globulin, chicken ovalbumin has wide applications in the biotechnology and pharmaceutical fields. In the production of various vaccines and biopharmaceuticals, it is often used as a protein additive to improve the stability of the biopharmaceuticals or vaccines. This enhanced stability helps vaccines maintain their activity and potency during storage and transportation, thereby ensuring that the vaccine can exert the expected immune effect when administered to individuals.

[0004] Chicken ovalbumin also plays a crucial role in antibody preparation. It can act as a carrier protein for hapten conjugation. By binding to haptens, ovalbumin enhances the immunogenicity of the hapten, making it easier for the immune system to recognize and generate a corresponding immune response. This is essential for preparing antibodies against specific pathogens or antigens, as antibodies are key tools used by the immune system to neutralize foreign substances such as bacteria and viruses.

[0005] Chicken ovalbumin can also serve as an immune adjuvant, enhancing the immunogenicity of vaccines. Immune adjuvants stimulate the immune system, increasing the strength and duration of the immune response induced by vaccines. By combining chicken ovalbumin with vaccines, the immunogenicity of vaccines can be enhanced, enabling the immune system to more effectively produce antibodies and memory cells, thereby providing longer-lasting and more comprehensive protection.

[0006] In addition to the uses mentioned above, chicken ovalbumin is also frequently used as a universal model antigen in experimental immunology and vaccination research. As a typical globulin, chicken ovalbumin possesses well-defined chemical and biological properties, making it an ideal model antigen. In experimental immunology, researchers use chicken ovalbumin to mimic the behavior of real antigens to explore the response mechanisms of the immune system and the mechanisms of vaccine action. By introducing chicken ovalbumin into experimental animals through injection or other routes, processes such as antibody production, immune memory formation, and the dynamics of the immune response can be observed and studied.

[0007] Therefore, ovalbumin has a wide range of applications. Ovalbumin (OVA) was originally produced from the oviduct of hens. Early methods of obtaining OVA typically involved collecting it from animal urine or milk. However, extracting the protein from the body or from urine or milk is a cumbersome process, requiring purification steps and resulting in low efficiency. Previously, there were also methods to introduce the OVA-encoding sequence into a host for heterologous expression. However, host expression is often performed on microorganisms, which cannot accurately simulate OVA expression in the human environment or the immune response of the human immune system. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a nucleic acid encoding chicken ovalbumin and an expression vector.

[0009] This invention optimizes the codon ratio, GC content, sequence repeatability, RNA secondary structure, and RNA free energy in DNA sequences to obtain DNA sequences capable of high expression of OVA versions. In particular, it improves the expression level in human or mammalian cells, enabling the production of large quantities of OVA protein in a short time, saving working time and improving work efficiency.

[0010] The nucleic acid encoding the chicken ovalbumin provided by this invention is any one of the following I) to III):

[0011] I) Nucleic acids having a sequence as shown in SEQ ID NO: 1, 2, 3 or 4;

[0012] II) A nucleic acid that, in the sequence of the nucleic acid described in I), has been substituted, deleted, added and / or replaced one or more bases and encodes chicken ovalbumin;

[0013] III) Nucleic acids that have at least 80% identity with the nucleic acid sequences described in I) or II).

[0014] In this embodiment of the invention, four nucleic acids encoding OVA, as shown in SEQ ID NO:1, 2, 3, or 4, were optimized. Compared with the wild type, the expression levels of the nucleic acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3 were significantly increased. Compared with the optimized scheme in the prior art, the expression level of the nucleic acid sequence shown in SEQ ID NO:1 was significantly increased.

[0015] In this invention, the at least 80% identity includes: at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, at least 99% identity, at least 99.5% identity, at least 99.6% identity, at least 99.7% identity, at least 99.8% identity, or at least 99.9% identity.

[0016] The present invention also provides an expression unit comprising a promoter and a nucleic acid as described above.

[0017] In some embodiments, the expression unit includes, from the 5' end to the 3' end, a promoter, a 5'UTR, a Kozak, a nucleic acid as described above, a 3'UTR, a polyA tail, and a restriction enzyme site.

[0018] In this invention, the promoter is selected based on the host, and this invention does not limit it. It can be a eukaryotic promoter or a prokaryotic promoter. As a credible example, the promoter is the T7 promoter, SFFV promoter, MSCV promoter, EF1a promoter, EFS promoter, CMV promoter, CAG promoter, PGK promoter, U6 and H1 promoters, CBh promoter, SV40 promoter, TRE promoter, or UBC promoter.

[0019] In this invention, the sequence of the 5'UTR, 3'UTR, or Kozak is not limited.

[0020] The sequence of the restriction enzyme site is repeated once in the plasmid vector of the present invention. In the embodiments of the invention, SapI is used as the restriction enzyme site for plasmid linearization.

[0021] In this invention, the length of the polyA tail is 110 bp.

[0022] In addition to the elements mentioned above, the expression unit may also include other elements, such as enhancers, terminators, and / or nuclear localization signals.

[0023] In some embodiments, the nuclear localization signal is located between the promoter and the nucleic acid as described above. The nuclear localization signal is SV40 NLS, and its amino acid sequence is PKKKRKV.

[0024] In some embodiments, the enhancer is selected from SV40 enhancer, CMV enhancer, SV-1 enhancer, EF1A enhancer, PGK enhancer, UBC enhancer, ROSA26 enhancer, EF1α enhancer, HARE5 enhancer, COPIA enhancer, CAGG enhancer or ACT5C enhancer.

[0025] In some embodiments, the terminator is selected from T0 phage terminator, hGH terminator, T7 phage terminator, λ phage terminator, SV40 terminator, rrnB terminator, bGH terminator, CMV terminator, or rbGlob terminator.

[0026] Furthermore, the present invention provides a plasmid vector containing at least one of the following i) to ii):

[0027] i) Nucleic acids as described above;

[0028] ii) Expression units as described above.

[0029] The plasmid vectors described in this invention are used for nucleic acid amplification, preservation, transformation, and / or transfection, and this invention does not limit their application. This invention does not limit the insertion site of the nucleic acid and / or expression units in the plasmid vector; preferably, they can be inserted at the multiple cloning site of the plasmid vector, or they can be inserted into other regions. The plasmid vectors described in this invention can be circular or linear, and this invention does not limit their application either. In some embodiments, they are cloning vectors, expression vectors, herpes simplex virus vectors, retroviral vectors, lentiviral vectors, adenovirus vectors, or adeno-associated virus vectors. For example, the cloning vectors are pUC series plasmid vectors, pBR322 plasmid vectors, pGEM series plasmid vectors, pET series plasmid vectors, Yeast series plasmid vectors, or Gateway plasmid vectors, etc. For example, the adeno-associated virus vector is an rAAV vector, whose serotypes include AAV1, AAV2, AAV5, AAV6, AAV8, or AAV9. In some embodiments, the pmRVac vector is used as a backbone for protein expression verification.

[0030] Furthermore, the present invention also provides an mRNA comprising at least one of the following A) to B):

[0031] A) 5' cap-like structure and nucleic acid as described above;

[0032] B) 5' cap-like structure and the expression unit as described above.

[0033] Furthermore, the method for preparing the mRNA includes linearizing the plasmid vector as described above, followed by capping and purification.

[0034] Specifically, the preparation method of mRNA includes linearizing the plasmid vector with restriction endonuclease, adding transcriptase and other materials to carry out transcription reaction, adding capping enzyme and other materials to carry out capping, and then purifying with magnetic beads to obtain mRNA sample.

[0035] Furthermore, the present invention also provides a host cell that is transformed or transfected with the plasmid vector as described above, or whose genome integrates the nucleic acid as described above, or whose genome integrates the expression unit as described above.

[0036] In this invention, the host is a human cell or a mammalian cell. Alternatively, the host can be a prokaryotic or eukaryotic microorganism. The eukaryotic host includes, but is not limited to, yeast and insect cells, while the prokaryotic host includes, but is not limited to, *Escherichia coli*. For example, the host may be *E. coli* BL21(DE3), BL21(DE3)pLysS, DH5α, JM109, JM110, TOP10, HB101, or Xl1-Blue. The human cell is a 293T cell.

[0037] Furthermore, the present invention also provides a method for constructing host cells, which includes treating cells with mRNA and lipids as described above. In embodiments of the present invention, the treatment includes transfection and / or transformation.

[0038] The present invention also provides products obtained by culturing host cells as described above.

[0039] The present invention also provides the use of the host cells and / or the products described above in the preparation of formulations for the preparation of antigen mimics.

[0040] The present invention also provides formulations of mimic antigens, which include the use of host cells as described above and / or the products described above in the preparation of formulations of mimic antigens.

[0041] Methods for screening drugs and / or vaccines using mimic antigens, including validation using the formulations described above as test subjects.

[0042] This invention optimizes the coding codons of chicken ovalbumin, obtaining four optimized sequences. The resulting nucleic acid sequences show significantly higher OVA expression levels in the host compared to wild-type and other comparative sequences. The mRNA prepared from these nucleic acids exhibits good transfection efficiency, achieving higher expression levels, particularly in 293T cells, thus better mimicking the immune response of the human immune system. Attached Figure Description

[0043] Figure 1 Western blot images showing the expression of cells in each group;

[0044] Figure 2 The images show SDS-PAGE electrophoresis results of each group of cells after expression.

[0045] Figure 3 The grayscale analysis of the gel images after Western blot detection of cell expression in each group is shown (****P<0.0001);

[0046] Figure 4 The pmRVac vector spectrum is shown;

[0047] Figure 5 Example 1 shows the GC content analysis chart;

[0048] Figure 6 This is a schematic diagram illustrating the sequence repetition analysis in Example 1.

[0049] Figure 7 Schematic diagram of RNA secondary structure shown in Example 1;

[0050] Figure 8 Indicates the meaning of the standard codon;

[0051] Figure 9 This demonstrates the intended use of the species codon for "Homo sapiens".

[0052] Figure 10 Example 2 shows the GC content analysis chart;

[0053] Figure 11 This is a schematic diagram illustrating the sequence repetition analysis in Example 2.

[0054] Figure 12 Example 2 shows a schematic diagram of the RNA secondary structure;

[0055] Figure 13 Example 3 shows the GC content analysis chart;

[0056] Figure 14 This is a schematic diagram illustrating the sequence repetition analysis in Example 3.

[0057] Figure 15 Example 3 shows a schematic diagram of the RNA secondary structure;

[0058] Figure 16 Example 4 shows the GC content analysis chart;

[0059] Figure 17 This is a schematic diagram illustrating the sequence repetition analysis in Example 4.

[0060] Figure 18 Example 4 shows a schematic diagram of the RNA secondary structure;

[0061] Figure 19 This shows a graph illustrating the GC content analysis of the first version of the DNA sequence.

[0062] Figure 20 This diagram illustrates the analysis of DNA sequence duplication in the first version.

[0063] Figure 21 This is a schematic diagram of the secondary structure of the first version of the DNA sequence RNA. Detailed Implementation

[0064] This invention provides a nucleic acid encoding chicken ovalbumin and its expression vector. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0065] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0066] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0067] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0068] In this application, "and / or" describes the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone.

[0069] In this application, "identity" refers to the proportion of two sequences that are identical in nucleotides or amino acids at the same site. To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0070] In this application, the terms "comprising," "including," and "having" are used interchangeably to indicate the inclusiveness of a solution, meaning that the solution may contain elements other than those listed. It should also be understood that the use of "comprising," "including," and "having" herein also provides for solutions that are "composed of" or "as shown."

[0071] In this application, "nucleic acid" includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'.

[0072] In this application, "plasmid vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors integrated into the genome of a host cell into which the vector has been introduced.

[0073] In this application, the nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers containing mixtures of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derived sugar or phosphate backbones bonded or chemically modified residues.

[0074] In this application, "host cell" refers to a cell in which a foreign nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their progeny, regardless of the number of passages. Progeny may not be completely identical to parental cells in terms of nucleic acid contents and may contain mutations. This document includes mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells.

[0075] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0076] The test materials used in this invention are all commercially available products. The sequences involved in the text are as follows:

[0077] Comparative Example 2

[0078] ATGGGCTCCATCGGTGCAGCAAGCATGGAATTTTGTTTTGATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATTGCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAAATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAGAGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCAGCTCAGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCT(SEQ ID NO:5)

[0079] Comparative Example 1

[0080] ATGGGCTCCATCGGCGCAGCAAGCATGGAATTTTGTTTTGATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATTGCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAGATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAGAGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCTCTAGCGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCT(SEQ ID NO:6)

[0081] Example 1

[0082] ATGGGCTCCATCGGTGCAGCAAGCATGGAATTTTGTTTTGATGTATTCAAGGAGCTCAAAGTCCACCATGCCAATGAGAACATCTTCTACTGCCCCATTGCCATCATGTCAGCTCTAGCCATGGTATACCTGGGTGCAAAAGACAGCACCAGGACACAAATAAATAAGGTTGTTCGCTTTGATAAACTTCCAGGATTCGGAGACAGTATTGAAGCTCAGTGTGGCACATCTGTAAACGTTCACTCTTCACTTAGAGACATCCTCAACCAAATCACCAAACCAAATGATGTTTATTCGTTCAGCCTTGCCAGTAGACTTTATGCTGAAGAGAGATACCCAATCCTGCCAGAATACTTGCAGTGTGTGAAGGAACTGTATAGAGGAGGCTTGGAACCTATCAACTTTCAAACAGCTGCAGATCAAGCCAGAGAGCTCATCAATTCCTGGGTAGAAAGTCAGACAAATGGAATTATCAGAAATGTCCTTCAGCCAAGCTCCGTGGATTCTCAAACTGCAATGGTTCTGGTTAATGCCATTGTCTTCAAAGGACTGTGGGAGAAAGCATTTAAGGATGAAGACACACAAGCAATGCCTTTCAGAGTGACTGAGCAAGAAAGCAAACCTGTGCAGATGATGTACCAGATTGGTTTATTTAGAGTGGCATCAATGGCTTCTGAGAAAATGAAGATCCTGGAGCTTCCATTTGCCAGTGGGACAATGAGCATGTTGGTGCTGTTGCCTGATGAAGTCTCAGGCCTTGAGCAGCTTGAGAGTATAATCAACTTTGAAAAACTGACTGAATGGACCAGTTCTAATGTTATGGAAGAGAGGAAGATCAAAGTGTACTTACCTCGCATGAAGATGGAGGAAAAATACAACCTCACATCTGTCTTAATGGCTATGGGCATTACTGACGTGTTTAGCAGCTCAGCCAATCTGTCTGGCATCTCCTCAGCAGAGAGCCTGAAGATATCTCAAGCTGTCCATGCAGCACATGCAGAAATCAATGAAGCAGGCAGAGAGGTGGTAGGGTCAGCAGAGGCTGGAGTGGATGCTGCAAGCGTCTCTGAAGAATTTAGGGCTGACCATCCATTCCTCTTCTGTATCAAGCACATCGCAACCAACGCCGTTCTCTTCTTTGGCAGATGTGTTTCCCCT(SEQ IDNO:1)

[0083] Example 2

[0084]

[0085] Example 3

[0086]

[0087] Example 4

[0088] ATGGGCAGCATCGGCGCCGCCAGCATGGAGTTCTGCTTCGACGTGTTCAAGGAGCTGAAGGTCCACCACGCCAACGAGAACATCTTCTACTGTCCCATCGCAATCATGAGCGCCCTGGCCATGGTGTACCTGGGCGCCAAAGACAGCACCCGGACACAGATCAACAAGGTGGTGAGATTCGACAAGCTGCCCGGCTTCGGCGACAGCATCGAGGCCCAGTGCGGCACCAGCGTGAACGTGCACAGCAGCCTGCGGGACATCCTGAACCAGATCACAAAGCCCAACGACGTGTACAGCTTCAGCCTGGCAAGCCGGCTGTACGCCGAGGAGCGGTACCCCATCCTGCCCGAGTACCTGCAGTGTGTGAAGGAGCTGTACAGAGGAGGCCTGGAGCCCATCAACTTCCAGACCGCCGCCGACCAGGCCCGGGAGCTGATCAACAGCTGGGTGGAGAGCCAGACCAACGGCATCATCCGGAACGTGCTGCAGCCCAGCAGCGTGGACAGCCAGACAGCCATGGTGCTGGTGAACGCCATCGTGTTCAAAGGCCTGTGGGAGAAGGCCTTCAAGGACGAAGACACACAGGCAATGCCCTTCCGGGTGACCGAGCAAGAGAGCAAGCCCGTGCAGATGATGTACCAGATCGGGCTGTTCAGAGTGGCCAGCATGGCCAGCGAGAAGATGAAGATCCTGGAGCTGCCCTTCGCCAGCGGCACAATGAGCATGCTGGTGCTGCTCCCCGACGAAGTGAGCGGCCTGGAGCAGCTGGAGAGCATCAACTTCGAAAAGCTGACCGAGTGGACCAGCAGCAACGTCATGGAGGAACGGAAGATCAAGGTCTACCTGCCCAGGATGAAAATGGAGGAAAAGTACAACCTGACCAGCGTGCTGATGGCAATGGGCATCACCGACGTGTTCAGCAGCAGCGCCAACCTGAGCGGCA TCAGCAGCGCCGAGAGCCTGAAGATCAGCCAGGCCGTGCACGCCGCCCACGCCGAGATCAACGAGGCCGGCCGGGAAGTGGTGGGAAGCGCCGAAGCCGGCGTGGACGCCGCCAGCGTGAGCGAGGAGTTCAGAGCCGACCACCCCTTCCTGTTCTGCATCAAGCACATCGCCACCAACGCCGTGCTGTTCTTCGGCAGATGCGTGAGCCCC (SEQ ID NO:4)

[0089] The invention will be further described below:

[0090] Example

[0091] 1. Codon optimization

[0092] The optimized sequence was derived from the wild-type CDS region sequence of the species *Gallus gallus*.

[0093] Related information

[0094] Sequence source: https: / / www.ncbi.nlm.nih.gov / nuccore / 2099367237

[0095] The DNA sequence optimization method in this invention is as follows.

[0096] (1) Amino acid sequence analysis

[0097] This invention relates to a wild-type version of the amino acid sequence of chicken ovalbumin, as shown in Table 1.

[0098] Table 1. Amino acid count of chicken egg albumin

[0099]

[0100] Amino acid sequence 1: MGSIGAASMEFCFDVFKELKVHHANENIFYCPIAIMSALAMVYLGA KDSTRTQINKVVRFDKLPGFGDSIEAQCGTSVNVHSSLRDILNQITKPNDVYSFSLASRLYAEERYPILPEYLQCVKELYRGGLEPINFQTAADQARELINSWVESQTNGIIRNVLQPSSVDSQTAMVLVNAIVFKGLWEKAFKDEDTQAMPFRVTEQESKPVQMMYQIGL FRVASMASEKMKILELPFASGTMSMLVLLPDEVSGLEQLESIINFEKLTEWTSSNVMEERKIKVYLPRMKMEEKYNLTSVLMAMGITDVFSSSANLSGISSAESLKISQAVHAAHAEINEAGREVVGSAEAGVDAASVSEEFRADHPFLFCIKHIATNAVLFFGRCVSP*.

[0101] (2) Converting amino acid sequences into DNA sequences

[0102] To convert the entire amino acid sequence to the most commonly used codon sequence for the desired species, for example, for the species "Homosapiens," replace all instances of "alanine" with "GCC." The commonly used codon sequence for a species can be found at the following website (link: https: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=9606).

[0103] After following the above procedure, the first version of the DNA sequence (SEQ ID NO:7) was obtained:

[0104]

[0105] (3) DNA sequence analysis

[0106] GC content analysis was performed on the first version of the DNA sequence obtained in step 2. For example... Figure 19 As shown.

[0107] GC content analysis tool website: https: / / www.vectorbuilder.cn / tool / gc-content- calculator.html

[0108] Sequence repetition analysis was performed on the first version of the DNA sequence obtained in step 2. For example... Figure 20 As shown.

[0109] Website for sequence repetition analysis tool: https: / / www.vectorbuilder.cn / tool / sequence- dot-plot.html

[0110] The DNA sequence of the first version was analyzed for RNA secondary structure and free energy. The minimum free energy (MFE) of this RNA sequence was -475.50 kcal / mol. The RNA secondary structure is as follows: Figure 21 As shown.

[0111] The website for RNA secondary structure and free energy analysis tools is: http: / / rna.tbi.univie.ac.at / / cgi-bin / RNAWebSuite / RNAfold.cgi

[0112] (4) DNA sequence adjustment

[0113] By combining parameters such as codon bias, GC content, sequence repetition, RNA secondary structure, and RNA free energy, an optimized version of the DNA sequence is obtained.

[0114] The optimization of each parameter is described below.

[0115] Codon bias: After obtaining the first version of the DNA sequence based on the most frequently used codons, the first-most frequently used codons are replaced sequentially with the second, third, and fourth most frequently used codons. The principle is that the percentages of the first, second, third, and fourth most frequently used codons in the total sequence of this protein should be controlled within the following ranges: 40–100%, 0–50%, 0–25%, and 0–15%, respectively. Figure 8 (As shown in https: / / zh.wikipedia.org / wiki / DNA%E5%AF%86%E7%A0%81%E5%AD%90%E8%A1%A8), the codons corresponding to amino acids are identified based on the standard codon table, and then combined with... Figure 9The codon tables of Homo sapiens were used to obtain codon preferences. In the Homo sapiens species, the proportion of glycine GGC was 40-100%, GGA was 0-50%, GGG was 0-25%, and GGT was 0-15%.

[0116] 1) GC content percentage: Higher than 0%–15% of the total GC content of the species. For example, the total GC content of *Homo sapiens* is 52.27%, so the optimized DNA sequence should have a total GC content within the range of 52.27%–67.27%. GC content may vary between different species. For instance, the total GC content of *Macaca fascicularis* is 49.64%, so the optimized DNA sequence should have a total GC content within the range of 49.64%–64.64%. The local GC content of the DNA sequence should be controlled between 30% and 95%.

[0117] 2) Sequence repetition: Optimize the number and length of repetitive sequences as much as possible. For example, replace a codon in a repetitive sequence segment longer than 20 bases with a synonymous codon to reduce the number of bases in the repetitive sequence.

[0118] 3) RNA secondary structure and free energy: Reduce the number of consecutive base-pairing regions to lower the minimum free energy. If both cannot be achieved simultaneously, prioritize reducing the number of consecutive base-pairing regions.

[0119] Based on the above optimization method, the sequences of Examples 1 to 4 of this invention were obtained. The free energies of Examples 1 to 4 are -446.09 kcal / mol, -444.51 kcal / mol, -450.96 kcal / mol, and -445.90 kcal / mol, respectively.

[0120] The percentages of the first, second, third, and fourth codons in the total sequence of the protein in Example 1 (SEQ ID NO:1) ranged from 44% to 100%, 0% to 50%, 0% to 14%, and 0% to 3%, respectively.

[0121] The percentages of the first, second, third, and fourth codons in the total sequence of the protein in Example 2 (SEQ ID NO:2) ranged as follows: 44–100%, 0–50%, 0–14%, and 0–3%, respectively.

[0122] The percentages of the first, second, third, and fourth codons in the total sequence of the protein in Example 3 (SEQ ID NO:3) ranged from 44% to 100%, 0% to 50%, 0% to 14%, and 0% to 3%, respectively.

[0123] The percentages of the first, second, third, and fourth codons in the total sequence of the protein in Example 4 (SEQ ID NO:4) ranged from 44% to 100%, 0% to 50%, 0% to 14%, and 0% to 3%, respectively.

[0124] Table 2. Sequence Comparison Information of This Experiment

[0125]

[0126] 2. Carrier Construction

[0127] The DNA sequence of chicken ovalbumin was used as the antigen coding region and inserted into the pmRVac backbone using the Gibson method (the DNA sequences of all elements except the target sequence in the comparative and example samples were consistent; the pmRVac vector was modified from the pUC19k vector; the map is shown below). Figure 4 ), to obtain pmRVac-OVA ( Figure 4 Gibson reaction buffer for the vector. The reaction buffer was then transferred to VB UltraStable competent cells by electroporation, SOC culture medium was added, and the cells were incubated on a shaker for 1 hour. A portion of the culture medium was then taken out and spread and streaked on LB plates containing Kana antibiotics, and incubated overnight at 37°C.

[0128] 3. Cloning identification

[0129] Multiple single clones were picked from the Kana plates cultured overnight in step 2, dissolved in sterile water to obtain bacterial culture, and the bacterial culture was used for PCR amplification. The correct clones were identified by gel electrophoresis. The bacterial culture of the correct clones was added to LB medium containing Kana antibiotic and incubated overnight at 37°C on a shaker.

[0130] 4. Plasmid extraction

[0131] Take the bacterial culture from step 3 that has been cultured overnight and use a plasmid extraction kit to extract plasmids. Then, perform enzyme digestion, sequencing, and other identifications on the obtained plasmids to obtain the correct plasmids.

[0132] 5. Plasmid linearization

[0133] The plasmid from step 4 was digested with SapI and purified using a PCR product purification kit to obtain the purified linearized plasmid.

[0134] 6. In vitro transcription of mRNA

[0135] The linearized plasmid from step 5 was added to the transcription system and reacted at 37°C to obtain crude mRNA. After purification and denaturation by heating, it was added to the capping system and capped at 37°C. After purification with magnetic beads, the mRNA sample was obtained.

[0136] 7. Cell transfection

[0137] mRNA transfection dose: 1ug / well.

[0138] Take the mRNA from step 6 and add it to 293T cells in a 12-well plate (cell density at transfection was 80%). After culturing at 37°C for 24 hours, collect the supernatant and place it on ice for labeling.

[0139] 8. BCA method for determining protein concentration

[0140] Dilute the BCA protein standard with a specific gradient concentration and add appropriate amounts to 96-well microplates. Mix BCA working solution reagents A and B at a ratio of 50:1, prepare fresh, and add BCA working solution to each well of the microplate. Shake the microplate on a shaker and incubate at 37°C. Measure the absorbance at 562 nm using a microplate spectrophotometer. Plot a standard curve with protein concentration (µg) on ​​the x-axis and absorbance on the y-axis and fit a linear equation.

[0141] Dilute the sample to a suitable concentration, add BCA working solution, mix thoroughly, incubate at 37°C, and then measure the absorbance. Calculate the protein concentration from the absorbance using the standard curve, and multiply by the sample dilution factor to obtain the sample concentration.

[0142] 9. Western blot (WB) test and SDS-PAGE staining and destaining

[0143] Mix the supernatant obtained in step 7 thoroughly, add 4x SDS loading solution, mix well, heat to denature, and then place on ice. Assemble two precast gels and place them in the electrophoresis tank. Add buffer and spot the samples in the same order and by the same volume. Electrophoresis at 150mV for 1 hour. Cut an appropriate 0.45µm PVDF membrane and immediately transfer one of the precast gels after electrophoresis. Transfer at 250mA for 45 minutes. After electrophoresis, remove the membrane with tweezers and place it in a container. Rinse with TBST and add blocking buffer. Block at room temperature for 1 hour. Rinse twice with TBST and incubate overnight with primary antibody. Rinse three times with TBST and incubate with secondary antibody at room temperature for 1.5 hours. After rinsing three times with TBST, drop ECL Western blot substrate onto the membrane and perform luminescence imaging using a gel electrophoresis apparatus to observe the protein expression results.

[0144] For the other pre-made gel, after removing the gel, immerse it in a glass dish containing an appropriate amount of Coomassie brilliant blue staining solution. Place the glass dish on a circumferential decolorizing shaker and incubate at room temperature for 1 hour. Discard the staining solution, add an appropriate amount of decolorizing solution, and decolorize at room temperature for 4-24 hours, changing the decolorizing solution 2-4 times during this period.

[0145] 100 μg / well sample was used for WB and SDS-PAGE experiments. The experimental data are as follows: Figures 1-2 As shown:

[0146] 10. Conclusion

[0147] At the same dosage, OVA protein expression was strongest in Example 1, weakest in Comparative Example 2, and worst in Example 3. No significant protein expression was observed in other samples.

[0148] The band strength of each sample in the SDS-PAGE gel was basically consistent, indicating that the amount of sample loaded into the WB was consistent.

[0149] Example 1 showed significant differences compared to Comparative Example 1 and Comparative Example 2.

[0150] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The nucleic acid encoding chicken ovalbumin, characterized in that, It is any one of the following: I) to III): I) Nucleic acids having a sequence as shown in SEQ ID NO: 1, 2, 3 or 4; II) A nucleic acid that, in the sequence of the nucleic acid described in I), has been substituted, deleted, added and / or replaced one or more bases and encodes chicken ovalbumin; III) Nucleic acids that have at least 80% identity with the nucleic acid sequences described in I) or II).

2. An expression unit comprising a promoter and the nucleic acid of claim 1.

3. The expression unit according to claim 2, characterized in that, The sequence from the 5' end to the 3' end includes a promoter, a 5' UTR, a Kozak, the nucleic acid as described in claim 1, a 3' UTR, a polyA tail, and a restriction enzyme site.

4. A plasmid vector containing at least one of the following: i) to ii) i) The nucleic acid as described in claim 1; ii) The expression unit as described in claim 2 or 3.

5. mRNA, which includes at least one of the following: A) to B) A) A 5' cap-like structure and the nucleic acid as described in claim 1; B) 5' cap-like structure and the expression unit as described in claim 2 or 3.

6. The method for preparing the mRNA according to claim 5, comprising linearizing the plasmid vector according to claim 4 or 5, followed by capping and purification.

7. A host cell having the nucleic acid of claim 1 integrated into its genome, or having the expression unit of claim 2 or 3 integrated into its genome, or being transformed or transfected with the plasmid vector of claim 4.

8. The method for constructing host cells according to claim 7, comprising transfecting cells after mixing the reagent and lipids according to claim 5.

9. The use of the host cell of claim 7 and / or the culture product of the host cell of claim 7 in the preparation of formulations for mimicking antigens.

10. An antigen formulation comprising the host cell of claim 7 and / or a culture product of the host cell of claim 7.

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