Method for enhancing nucleic acid expression effect

Abstract

The invention discloses a method for enhancing a nucleic acid expression effect. The core of the method is that mRNA encoding NADH dehydrogenase (ND) is co-delivered to the same cell with a nucleic acid molecule of interest. The expression product of the ND mRNA provides sufficient energy for translation of target nucleic acid molecules by improving the ATP level in cells, so that the protein expression quantity of the target nucleic acid molecules is remarkably amplified. The method is high in universality, is suitable for various nucleic acid types such as mRNA, circular RNA, self-amplified RNA and DNA, can be widely applied to the fields of vaccines, tumor immunotherapy, gene therapy and the like, and realizes multiplication of the curative effect and saving of the dosage.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, and specifically relates to a method for enhancing nucleic acid expression. Background Technology

[0002] Nucleic acid drugs, especially messenger RNA (mRNA) drugs, have shown great potential in vaccine development, tumor immunotherapy, protein replacement therapy, and gene editing. However, the efficacy of nucleic acid drugs is largely limited by their expression efficiency after entering cells. The expression level of the target protein within cells is a key factor determining their ultimate therapeutic effect.

[0003] Currently, strategies for improving nucleic acid expression efficiency mainly focus on optimizing the nucleic acid molecule itself (such as codon optimization and sequence modification) and delivery vectors (such as formulation optimization of lipid nanoparticles (LNPs)). These strategies primarily address extracellular factors or the stability of nucleic acid molecules, with insufficient attention paid to how to actively regulate the intracellular environment to create conditions more conducive to the efficient translation of exogenous nucleic acids.

[0004] mRNA translation is one of the most energy-intensive processes in cells, and its efficiency is closely related to the cell's metabolic state. When cellular energy supply is insufficient, the translation of exogenous mRNA is significantly inhibited. Previous studies have shown that delivering exogenous ATP along with mRNA to cells can enhance mRNA expression levels (J. Am. Chem. Soc. 2023, 145, 19800−19811). However, exogenously delivered ATP is easily inactivated, has low cellular uptake efficiency, limited enhancement of cellular metabolism, and the enhancement of target protein expression is not durable. Therefore, there is an urgent need in this field for a novel method that efficiently and persistently enhances nucleic acid expression at the intracellular level, enabling the delivery and enhancement of therapeutic nucleic acid efficacy while activating cellular metabolism. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for enhancing nucleic acid expression. This method does not rely on complex modifications to the target nucleic acid sequence or delivery vector, but rather achieves its purpose by co-delivering a metabolically regulatory nucleic acid (mRNA encoding NADH dehydrogenase, abbreviated as ND mRNA) to the target nucleic acid. NADH dehydrogenase is a key component of the intracellular mitochondrial oxidative phosphorylation system. It catalyzes NADH oxidation, initiates the electron transport chain, and actively pumps protons to establish a transmembrane electrochemical gradient, which is the direct driving force for ATP synthesis. Therefore, NADH dehydrogenase activity is a core regulatory point in cellular energy metabolism. The ND mRNA designed in this invention can be expressed as NADH dehydrogenase in cells, thereby increasing intracellular ATP levels, enhancing cellular metabolism, and fundamentally upgrading the cell's own translational capacity, achieving a significant amplification of the target nucleic acid expression level.

[0006] To solve the problems of the prior art, the technical solution adopted by the present invention is as follows: A method to enhance nucleic acid expression involves co-delivering mRNA encoding NADH dehydrogenase (ND mRNA) with a target nucleic acid molecule to cells, and enhancing the expression of the target nucleic acid molecule by increasing intracellular ATP levels through the expression product of ND mRNA.

[0007] Preferably, the antigen expressed by the mRNA of the NADH dehydrogenase is a fusion antigen or a mixed antigen.

[0008] More preferably, the fusion antigen is not limited to at least two of antigen I, antigen II, antigen III, antigen IV, antigen V, and antigen VI; the mixed antigen is not limited to at least two of antigen I, antigen II, antigen III, antigen IV, antigen V, and antigen VI.

[0009] More preferably, the mixed antigen includes any combination of antigen I, antigen II, and antigen IV in any order, or any combination of antigen III, antigen V, and antigen VI in any order.

[0010] Preferably, the mRNA of NADH dehydrogenase is ND mRNA A or ND mRNA B, and the nucleic acid sequence of ND mRNA A or ND mRNA B is shown in SEQ ID NO. 11 and SEQ ID NO. 12.

[0011] SEQ ID NO. 11: ND mRNA I-II-IV (3953nt) AGcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG AUGCCCAUGGCUAACCUGCUGCUCCUUAUUGUGCCUAUACUCAUU GCCAUGGCGUUUCUCAUGCUUACAGAGCGAAAGAUCCUCGGCUAUAUGCAGUUGAGAAAAGGUCCCAACGUGGUAG GCCCAUACGGUCUCUUGCAACCCUUCGCAGACGCCAUGAAGUUGUUUACAAAAGAGCCCCUGAAGCCGGCCACCUC AACUAUAACCCUGUACAUUACUGCACCUACGCUGGCCCUGACUAUCGCCCUGCUGCUGUGGACUCCCCUUCCUAUG CCUAAUCCACUGGUGAAUCUGAACCUCGGACUCCUUUUUAUCCUCGCAACGUCUAGUCUUGCCGUGUAUUCAAUAC UGUGGUCCGGCUGGGCUUCUAAUUCCAAUUAUGCACUGAUCGGCGCCUUGAGAGCCGUCGCACAGACUAUUUCCUA CGAGGUCACACUCGCUAUUAUUCUGCUGAGCACCCUCCUGAUGAGCGGCUCUUUCAACCUGAGCACUCUUAUUACA ACUCAGGAGCACUUGUGGUUGCUUCUGCCAAGUUGGCCCCUGGCAAUGAUGUGGUUUAUUUCUACCCUUGCCGAGA CAAAUCGAACUCCAUUUGACCUCGCGGAAGGAGAAUCAGAAUUGGUCUCCGGCUUCAACAUAGAGUAUGCCGCUGG ACCAUUUGCACUGUUCUUUAUGGCCGAAUAUACCAACAUCAUAAUGAUGAACACACUCACCACUACCAUUUUCCUG GGGACCACAUACGAUGCUCUGAGUCCUGAACUGUAUACCACGUACUUUGUUACCAAAACCCUGCUCCUGACGUCAC UCUUUCUCUGGAUAAGGACGGCCUAUCCACGCUUUAGAUACGAUCAGCUCAUGCAUCUGCUGUGGAAAAAUUUCCU GCCGCUCACGCUCGCAUUGCUGAUGUGGUACGUUAGUAUGCCAAUAACCAUCUCUUCUAUUCCUCCUCAGACA GGAGGAGGCGGUUCUgccacgaacuucucucuguuaaagcaagcaggagauguugaagaaaaccccgggccuAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG AUGAACCCCCUUGCCCAGCCCGUGAUUUACUCAAC CAUAUUUGCCGGAACCCUGAUCACGGCCCUGUCCAGCCAUUGGUUUUUUUACUUGGGUAGGCUUGAGAUGAAUAG UUGGCUUUAUAUCCCUGUUCUGACCAAGAAGAUGAACCCUCCGAUCUACCGAAGCUGCCAUCAAGUACUUUCUACCC AAGCUACAGCAUCUAUGAUUCUGCUCAUGGCCAUCCUGUUUAUAAUAUGCUCAGCGGGCAGUGGACAAUGACCAA UACCACCAACCAGUAUUCUAGUCUGAUGAUAAUGAUGCCAUGGCGAUGAAGCUGGGGAUGGCACCAUUCCACUUU UGGGUUCCUGAGGUCACUCAGGGAACACCUCUGACCUCCCGGUCUGCUUCUGCUUACAUGGCAAAAACUGGCUCCGA UUUCAAUCAUGUAUCAGACGCCAAGCUUGAAUGUGUCCCUGCUGUUGACCCUGCCAUCCUUUCUAUCAUGGC AGGUUCAUGGGGGGAUUGAUCAGACCCAGUUGCGGAAGAUACUGGCCUAUAUGUAGCAUUCACAUAUGGGUUGG AUGAUGGCAGUGCUUCCUUACAACCCAAACAUGACAAUCCUGAAUUUGACUAUUUACAUAUCCUCACCACGACUG CCUUCCUGCUGCUCAAUCUGAACAGCUCCACGACGACCCUCCUUCUGUCUAGGACUUGGAAACUGACUUGGCU GACUCCCCUGAUUCCCUAGUACGCUGCUUUCCCUGGGAGGACUUCCACCACUGACAGGGGUUCUUGCCAAAAUGGGCU AUCAUCGAAGAGUUCACAAAGAAUAACAGCCUCAUCUUCCAACUAUUAUGGCUACAAUCACGCUGCUGAAUCUGU ACUUUUAUUCCCGGCUGAUCUACUCUACCAGCAUAACAUUGCUCCUAUGUCUAACGUGAAGAAAUGGCA GUUUGAGCAUACUAAGCCUACCCCCGUUUCUGCCGACGCUGAUGCAUUGACCACUUUGCUUCUUCCAAUUUCCCCU UUCAUGCUGAUGAUCCUG GAGGAGGCGGUUCUGgccacgaacuucucucuguuaaagcaagcaggagauguugaagaaaaccccgggccuAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG AUGCUCAAACUUAU UGUCCCUACAAUCAUGCUCCUCCCCCUUACCUGGUUGAGCAAAAAGCACAUGAUCUGGAUCAACACGACUACCCAUU CUCUCAUAUAUCAGUAUCAUCCCUCUGCUGUUUUUAACCAGAUAAACAACAACCUUUUUUCCUGCAGCCCCACCUUC AGCAGCGAUCCACUUACAACCCCGCUCUUGAUGCUGACUACUUGGCUGUUGCCACUGACAAUUAUGGCGUCUCAACG CCAUUUGAGUAGCGAGCCGCUGUCAAGAAAGAAGCUGUAUCUGUCUAUGCUGAUUAGCCUGCAGAUAUAGCCUGAUUA UGACCUUCACAGCCACAGAGCUCAUUAUGUUCUAACAUUUUUUCGAAACCACACUCAUUCCGACUUUGGCCAUUAUA ACACGAUGGGGCAACCUAGCCGCCUGAAUGCCGGAACUUAUUUCCUCUUUUAUACCCUCGUCGGAUCACUCCC CCUCCUGAUCCGCUCUGAUUUACACGCAUAACACUCUCGGCUCCCUGAACAUUUUGCUUCUGACUCUGACUGCACAGG AGCUGAGUAACUCCUGGGCCAAUAACCUGAUGUGGUUGGCCUACACCAUGGCGUUCAUGGUGAAGAUGCCGUUGUAU GGAUUGCACUUGUGGCUCCCGAAAGCCAUGUGGAAGCACCUAUCGCUGGAUCUAUGGUCCUGGCCGCCGUACUGCU UAAACUUGGCGGAUACGGCAUGAUGCGACUGACACUGAUCCUCAAUCCGCUUACGAAACACAUGGCCUACCCUUUC UGGUGCUUUCUCUUUGGGGCAUGAUCAUGACUUCCAGCAUCUGCCUUCGCCAAACAGACCUUAAAAGCCUGAUUGCC UAUAGUAGUAUAUUCCCACAUGGCACUUGUCGUUACCGCCAAUAUUGAUCCAGACCCCCUGGUCUUUUACCGGGCAGU GAUCUUGAUGAUCGCCCACGGACUGACCUCAAGCGUGUUUCUGCCUCGCCAAUUAUCUACGAGAGGACACAUA GUAGGAUAUGAUCUCUCUCAGGCCUGCAGACCUUGCUCCACUGAUGGCUUUUUGGUGGCUUCUGGCGUCCCUU GCCAAUCUGGCCUUGCCCCCCCACAAUCAAUUUGUUGGGAGAACUGUCCGUGUUGGUGACAACAUUUUCAUGGAGUA CAUCACUCUGCUGCUGACAGGCCCUGAACAUGCUGGUGACAGCUCUUUACUCCCCUUUACAUGUUCACAACUACACAGU GGGGAUCCCUCACCCACCAAUUAACAUGAAGCCCUCUUUCACCCGCGAAAAUACCCUCAUGUUUGCACCUC UCUCCCAUUCUCCCUGCUGAGUCUUAACCCUGACAUCAUCACGGAUCAGUUCCUGAUGAAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAAAAAAAAAAAAAAAAAAAAAGCAUAUCACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Note: The underlined portion represents the antigen (subunit) RNA sequence; 5' Cap: AG (nucleotides 1-2); 5' UTR: cuguucuuuuugcagaagcucagaauaaacgcucaacuuugg (nucleotides 3-45); Kozak sequence : GCCACC (nucleotides 46-51); Flexible Linker: GGAGGAGGCGGUUCU and GAGGAGGCGGUUCUG; P2A: gccacgaacuucucucuguuaaagcaagcaggagauguugaagaaaaccccgggccu; Signal Peptide: AUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG; Stop Codon: UGAUGA (nucleotides 3712-3717); 3'UTR: AACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCAC (nucleotides 3718-3843); Poly A: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATCACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (nucleotides 3844-3953, where GCAUAUCACU is the internal linker of Poly A).

[0012] SEQ ID NO. 12: ND mRNA III-V-VI (3257nt) AGcuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggGCCACCAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG AUGAACUUGCCCUCAUCCUCAUGAUCAAUACCCUUCUUGCUCUUG CUUUUGAUGAUUAUAACUUUUUGGCUCCCUCAGCCUGAAUGGCUACAUGGGAGAAGUCCACCCUUAUGAAUGUGGGU UUGAUCCCUAUGAGCCCCGCAAGGGUUCCAUUCUCCAUGAGUUCUUUCUGGUGGCAAUCACAUUCCUCUUGUUCGA CCUUGAGAUGCAUUGCUUCCCCACUGCCUUGGGCCCUUCAGACGACCAAUCUUCCUCUGAUGGUGAUGAGCUCU CUGCUCCUGAUCAUCAUCUUGGCUCUGUCACUGGCAUAUGAAUGGCUGCAGAAGGGCCUUGAUUGGACAGAG GGAGGAGGCGGUUCUgccacgaacuucucucuguuaaagcaagcaggagauguugaagaaaaccccgggccuAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG AUGACUAUGCACACGACUAUGACACUUUGACCCCUU ACUAGCCUUAUCCCCCCUAUUUUGACCACCCUGGUGUACCCAAAUAAGAAGAAUAGCUACCCACACAUUGUGAAAA GCAUAGUGGCAAGUACCUUCAUCAUCUCACUGUUCCUACUACCAUGUUAUUGGCCUGGACCAGGAGGUGAUCAU CUCAAAUUGGCACUGGGCUACGACCCAGACUACACAGCUGUCUCUGAGCUUAAUAGUGGACUACUCUCCAUGAG UUCAUUCCCGUGGCCCUGUUUGUGACAUGGUCUAUCAUGGGAGUUUUCACUGUGGUACAUGAAUAGCGACCCUAACA UCAACCAGUUUUUUUAAAUAUCUCCUCAUAUUCUUGAUCACAAUGCUGAUACUGGUGACAGCCAACAAUCUGUUUCA GUUGUUCAUCGGGUGGGGAAGGUAGGCAUCAUGUCUUUCCUUCUGAUUUCCUGGUGUAUGCCAGGGCUGAUGCC AACACAGCCGCGAUCCAGGCCAUACUGUACAACCGAAUUGGUGUAUACGGUUUUAUACCUCGCCCUGGCAUGGUUCA UUCUGCAUUCAAAUAGCUGGGAUCCUCAGCAGCAGAUGGCCUUGUUGAAUGCCAACCCCUCUCUUACACCCCGUGUUGGG CUUGCUGCUUGCUGCAGCCGGCAAGAGUGCACAACUUGGACUGCAUCCGUGGCUGCCGAGCGCGAUGGAGGGCCCG ACACCUGUCAGUGCCCUGCUUCAUUCUCACCAUGGUGGUGGCUGGGAUUUUUUUGCUGAUCAGGUUCCAUCCAC UGGCAGAAAAUUCCCCCCUGAUCCAGACUCUCACACUCUCUCGGAGCUAUCACUACUCUGUUCGCGGCUGUGUUG UGCACUGACUCAGAAUGACAUCAAAAAGAUCGUCGCCUUCCAACCUCCAGUCAGCUCGGGUUGAUGAUGGUGACC AUAGGCAUCAACCAGCCUCACCUGGCCUUUCUUCACAUUUGCACACACGCCUUCUUAAGGCAAUGCUGUUUAUGU GCAGUGGAAGUAUCAUUCAUAACCUGAACAACGAGCAAGAUAUUCAGAAAAAUGGGGGACUUUUGAGAAGACCAUGCC CCUGACAUCUACCAGCCUCACCAUUGGAAGUCUCCGCUCUCGCUGGAAUGCCAUUUCUCACAGGAUCUCUAAG GACCAUAUCAUUGAAACGCCAACAUGAGUUACACCAACGCAUGGGCCCUCAGCAUACUCUGAUUGCAACUAGCC UCACCCUCCGCUUACAGUACUCGGAUGUAUACUCCUGACCCUCACAGGACAGCCCCGGUUCCCUACGCUGACUAAUAU UAUGAGAACAAUCCAACUCUUCUCAACCCAAUCAAGCGCCUGGCUGCGGAUCACUGUUCGCCGGUUUCCUCAUU ACCAAAUAUAUCACCCGCAUCCUCCAUUCCAGACUACUAUCCCACUUUACCUUAAGCUUAACAGCUGGCUGUGA CCUUUUUGGGCUUGCUGACCGCCCUCGAACCUGAACAUAUCUGAACGAACCUGAAAAUGAAGUCCUCUGUGUAC AUUCUACUUCUCCAAAUUGUUGGGGUUUUACCCAUCCAUCACACAUCGCACGAUUCCCAUAUCUGGGGCUCCUGACU UCACAAAACCUUCCCUCCUGCUCCUGGAUCUGACAUGGCUCGAAAAUUGCUCCCAAAACAAUUUCUCAACACC AGAUAUCCACUAGCAUUAUCACCAGCACUCAGGAAGGGGAUGAUCAAGCUGUAUUUUCUCAGUUUUUUUCUCCGCU UAUACUGACAUUGUUGCUGAUAACA GAGGAGGCGGUUCUGgccacgaacuucucucuguuaaagcaagcaggagauguugaagaaaaccccgggccuAUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG AUGAUGU ACGCGCUUUCUUGUGUGCAGUGGGACUUGUCAUGGGAUUUGUGUGCUUCUAGCAAACCUUCUCCCAUCUACGG GGGCUGGUGCUUAUAGUGAGUGGCGUGGUGGAUGCGUUAUAAUUCUCAACUUCGGCGGCGGUUACAUGGGGCUCAU GGUGUUCCUUAUAUACCUCGGUGGCAUGGUGUGUUCGGUUAACACACUGCCAUGGCAAUAGAAGAAGUACCCUG AGGCGUGGGGAUCUGGUGUGGGAAAGUCUUGGUGUCUGUGCUUGUGCUUGGGGCUGGCUAUGGAAGUUGGCCUCGUGUGUGUGGG GUCAAGGAAUACGACGGGGUGGUUGUGGUGGUGAACUUCAACAGUGUCGGCUCCUGGAUGAUUUACGAGGGCGAAGG CAGCGGGCUCAUUAGGGAAGAUCCUAUUGGCGCAGGAGCCCUUUAUGACUACGGUAGAUGGCUGGUUGUCGUGACUG GAUGGACUCUCUUUGUGGGCGUCUAUAUUGUGAUUGAAAUCGCCAGGGGGAAU UGAUGAAAACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCACAAAAAAAAAAAAAAAAAAAAAGCAUAUCACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Note: The underlined portion represents the antigen (subunit) RNA sequence; 5' Cap: AG (nucleotides 1-2); 5' UTR: cuguucuuuuugcagaagcucagaauaaacgcucaacuuugg (nucleotides 3-45); Kozak sequence : GCCACC (nucleotides 46-51); Flexible Linker: GGAGGAGGCGGUUCU and GAGGAGGCGGUUCUG; P2A: gccacgaacuucucucuguuaaagcaagcaggagauguugaagaaaaccccgggccu; Signal Peptide: AUGUUCGUGUUCCUGGUGCUGCUGCCUCUGGUGUCCAGCCAGUGUGUG; Stop Codon: UGAUGA (nucleotides 3016-3021); 3'UTR: AACCAGCCUCAAGAACACCCGAAUGGAGUCUCUAAGCUACAUAAUACCAACUUACACUUUACAAAAUGUUGUCCCCCAAAAUGUAGCCAUUCGUAUCUGCUCCUAAUAAAAAGAAAGUUUCUUCAC (nucleotides 3022-3147); Poly A: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATCACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (nucleotides 3148-3257, where GCAUAUCACU is the internal linker of Poly A) Preferably, the antigen combination expressed by the ND mRNA A is a combination of antigen I-antigen II-antigen IV, and the amino acid sequence of the antigen combination is shown in SEQ ID NO. 7, and the nucleotide sequence is shown in SEQ ID NO. 9.

[0013] SEQ ID NO. 7: ND mRNA antigen combination I-II-IV amino acid sequence (1204aa) MPMANLLLLIVPILIAMAFLMLTERKILGYMQLRKGPNVVGPYGLLQPFADAMKLFTKEPLKPATSTIT LYITAPTLALTIALLLWTPLPMPNPLVNLNLGLLFILATSSLAVYSILWSGWASNSNYALIGALRAVAQTISYEVTL AIILLSTLLMSGSFNLSTLITTQEHLWLLLPSWPLAMMWFISTLAETNRTPFDLAEGESELVSGFNIEYAAGPFALF FMAEYTNIIMMNTLTTTIFLGTTYDALSPELYTTYFVTKTLLLTSLFLWIRTAYPRFRYDQLMHLLWKNFLPLTLAL LMWYVSMPITISSIPPQT GGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCV MNPLAQPVIYSTIFAGTLI TALSSHWFFTWVGLEMNMLAFIPVLTKKMNPRSTEAAIKYFLTQATASMILLMAILFNNMLSGQWTMTNTTNQYSSL MIMMAMAMKLGMAPFHFWVPEVTQGTPLTSGLLLLTWQKLAPISIMYQISPSLNVSLLLTLSILSIMAGSWGGLNQT QLRKILAYSSITHMGWMMAVLPYNPNMTILNLTIYIILTTTAFLLLNLNSSTTTLLLSRTWNKLTWLTPLIPSTLLS LGGLPPLTGFLPKWAIIEEFTKNNSLIIPTIMATITLLNLYFYLRLIYSTSITLLPMSNNVKMKWQFEHTKPTPFLP TLIALTTLLLPISPFMLMIL GGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCV MLKLIVPTIMLLPLTWL SKKHMIWINTTTHSLIISIIPLLFFNQINNNLFSCSPTFSSDPLTTPLLMLTTWLLPLTIMASQRHLSSEPLSRKKL YLSMLISLQISLIMTFTATELIMFYIFFETTLIPTLAIITRWGNQPERLNAGTYFLFYTLVGSLPLLIALIYTHNTL GSLNILLLTLTAQELSNSWANNLMWLAYTMAFMVKMPLYGLHLWLPKAHVEAPIAGSMVLAAVLLKLGGYGMMRLTL ILNPLTKHMAYPFLVLSLWGMIMTSSICLRQTDLKSLIAYSSISHMALVVTAILIQTPWSFTGAVILMIAHGLTSSL LFCLANSNYERTHSRIMILSQGLQTLLPLMAFWWLLASLANLALPPTINLLGELSVLVTTFSWSNITLLLTGLNMLV TALYSLYMFTTTQWGSLTHHINNMKPSFTRENTLMFMHLSPILLLSLNPDIITGFSS Note: The underlined parts are antigens (subunits); GGGGS is a flexible linker; ATNFSLLKQAGDVEENPGP is a P2A dipeptide; MFVFLVLLPLVSSQCV is a signal peptide.

[0014] SEQ ID NO. 9: ND DNA sequence antigen combination I-II-IV (5823bp) CCAATGATGAATTCTAATACGACTCACTATAGcttgttctttttgcagaagctcagaataaacgctcaactttggGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGCCCATGGCTAAC CTGCTGCTCCTTATTGTGCCTATACTCATTGCCATGGCGTTTCTCATGCTTACAGAGCGAAAGATCCTCGGCTATA TGCAGTTGAGAAAAGGTCCCAACGTGGTAGGCCCATACGGTCTCTTGCAACCCTTCGCAGACGCCATGAAGTTGTT TACAAAAGAGCCCCTGAAGCCGGCCACCTCAACTATAACCCTGTACATTACTGCACCTACGCTGGCCCTGACTATC GCCCTGCTGCTGTGGACTCCCCTTCCTATGCCTAATCCACTGGTGAATCTGAACCTCGGACTCCTTTTTATCCTCG CAACGTCTAGTCTTGCCGTGTATTCAATACTGTGGTCCGGCTGGGCTTCTAATTCCAATTATGCACTGATCGGCGC CTTGAGAGCCGTCGCACAGACTATTTCCTACGAGGTCACACTCGCTATTATTCTGCTGAGCACCCTCCTGATGAGC GGCTCTTTCAACCTGAGCACTCTTATTACAACTCAGGAGCACTTGTGGTTGCTTCTGCCAAGTTGGCCCCTGGCAA TGATGTGGTTTATTTCTACCCTTGCCGAGACAAATCGAACTCCATTTGACCTCGCGGAAGGAGAATCAGAATTGGT CTCCGGCTTCAACATAGAGTATGCCGCTGGACCATTTGCACTGTTCTTTATGGCCGAATATACCAACATCATAATG ATGAACACACTCACCACTACCATTTTCCTGGGGACCACATACGATGCTCTGAGTCCTGAACTGTATACCACGTACT TTGTTACCAAAACCCTGCTCCTGACGTCACTCTTTCTCTGGATAAGGACGGCCTATCCACGCTTTAGATACGATCA GCTCATGCATCTGCTGTGGAAAAATTTCCTGCCGCTCACGCTCGCATTGCTGATGTGGTACGTTAGTATGCCAATA ACCATCTCTTCTATTCCTCCTCAGACA GGAGGAGGCGGTTCTgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGAA CCCCCTTGCCCAGCCCGTGATTTACTCAACCATATTTGCCGGAACCCTGATCACGGCCCTGTCCAGCCATTGGTTT TTTACTTGGGTAGGGCTTGAGATGAATATGTTGGCTTTTATCCCTGTTCTGACCAAGAAGATGAACCCTCGATCTA CCGAAGCTGCCATCAAGTACTTTCTTACCCAAGCTACAGCATCTATGATTCTGCTCATGGCCATCCTGTTTAATAA TATGCTCAGCGGGCAGTGGACAATGACCAATACCACCAACCAGTATTCTAGTCTGATGATAATGATGGCCATGGCG ATGAAGCTGGGGATGGCACCATTCCACTTTTGGGTTCCTGAGGTCACTCAGGGAACACCTCTGACCTCCGGTCTGC TTCTGCTTACATGGCAAAAACTGGCTCCGATTTCAATCATGTATCAGATCAGCCCAAGCTTGAATGTGTCCCTGCT GTTGACCCTGTCCATCCTTTCTATCATGGCAGGTTCATGGGGGGGATTGAATCAGACCCAGTTGCGGAAGATACTG GCCTATAGTAGCATTACACATATGGGTTGGATGATGGCAGTGCTTCCTTACAACCCAAACATGACAATCCTGAATT TGACTATTTACATTATCCTCACCACGACTGCCTTCCTGCTGCTCAATCTGAACAGCTCCACGACGACCCTCCTTCT GTCTAGGACTTGGAACAAACTGACTTGGCTGACTCCCCTGATTCCTAGTACGCTGCTTTCCCTGGGAGGACTTCCA CCACTGACAGGGTTCTTGCCAAAATGGGCTATCATCGAAGAGTTCACAAAGAATAACAGCCTCATCATTCCAACTA TTATGGCTACAATCACGCTGCTGAATCTGTACTTTTATCTCCGGCTGATCTACTCTACCAGCATAACATTGCTCCC TATGTCTAACAACGTGAAGATGAAATGGCAGTTTGAGCATACTAAGCCTACCCCGTTTCTGCCGACGCTGATTGCA TTGACCACTTTGCTTCTTCCAATTTCCCCTTTCATGCTGATGATCCTG GAGGAGGCGGTTCTGgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG ATGCTCAAACTTATTGTCCCTACAATCATGCTCCTCCCCCTTACCTGGTTGAGCAAAAAGC ACATGATCTGGATCAACACGACTACCCATTCTCTCATTATCAGTATCATCCCTCTGCTGTTCTTTAACCAGATAAAC AACAACCTTTTTTCCTGCAGCCCCACCTTCAGCAGCGATCCACTTACAACCCCGCTCTTGATGCTGACAACTTGGCT GTTGCCACTGACAATTATGGCGTCTCAACGCCATTTGAGTAGCGAGCCGCTGTCAAGAAAGAAGCTGTATCTGTCTA TGCTGATTAGCCTGCAGATTAGCCTGATTATGACCTTCACAGCCACAGAGCTCATTATGTTCTACATCTTTTTCGAA ACCACACTCATTCCGACTTTGGCCATTATAACACGATGGGGCAACCAACCTGAGCGCCTGAATGCCGGAACTTATTT CCTCTTTTATACCCTCGTCGGATCACTCCCCCTCCTGATCGCTCTGATTTACACGCATAACACTCTCGGCTCCCTGA ACATTTTGCTTCTGACTCTGACTGCACAGGAGCTGAGTAACTCCTGGGCCAATAACCTGATGTGGTTGGCCTACACC ATGGCGTTCATGGTGAAGATGCCGTTGTATGGATTGCACTTGTGGCTCCCGAAAGCCCATGTGGAAGCACCTATCGC TGGATCTATGGTCCTGGCCGCCGTACTGCTTAAACTTGGCGGATACGGCATGATGCGACTGACACTGATCCTCAATC CGCTTACGAAACACATGGCCTACCCTTTTCTGGTGCTTTCTCTTTGGGGCATGATCATGACTTCCAGCATCTGCCTT CGCCAAACAGACCTTAAAAGCCTGATTGCCTATAGTAGTATATCCCACATGGCACTTGTCGTTACCGCCATATTGAT CCAGACCCCCTGGTCTTTTACCGGGGCAGTGATCTTGATGATCGCCCACGGACTGACCTCAAGCCTGTTGTTCTGCC TCGCCAATTCTAACTACGAGAGGACACATAGTAGGATTATGATCCTGTCTCAGGGCCTGCAGACCTTGCTCCCACTG ATGGCTTTTTGGTGGCTTCTGGCGTCCCTTGCCAATCTGGCCTTGCCCCCCACAATCAATTTGTTGGGAGAACTGTC CGTGTTGGTGACAACATTTTCATGGAGTAACATCACTCTGCTGCTGACAGGCCTGAACATGCTGGTGACAGCTCTTT ACTCCCTTTACATGTTCACAACTACACAGTGGGGATCCCTCACCCACCACATTAACAACATGAAGCCCTCTTTCACC CGCGAAAATACCCTCATGTTTATGCACCTCTCTCCCATTCTCCTGCTGAGTCTTAACCCTGACATCATCACCGGATT CAGTTCC Note: The underlined portion represents the antigen (subunit) DNA sequence; EcoRI restriction site: GAATTC (nucleotides 9-14); T7 promoter: TAATACGACTCACTAT (nucleotides 15-30); 5' Cap: AG (nucleotides 31-32); 5' UTR: cttgttctttttgcagaagctcagaataaacgctcaactttgg (nucleotides 33-75); Kozak sequence : GCCACC (nucleotides 76-81); Flexible Linker: GGAGGAGGCGGTTCT and GAGGAGGCGGTTCTG; P2A: gccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcct; Signal Peptide: ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG; Stop Codon: TGATGA (nucleotides 3742-3747); 3'UTR: AACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCAC (nucleotides 3748-3873); Poly A: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATCACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (nucleotides 3874-3983, where GCATATCACT is the internal linker of Poly A).

[0015] More preferably, the amino acid sequences encoding antigen I (subunit 1), antigen II (subunit 2), and antigen IV (subunit 4) are as shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 4, respectively.

[0016] SEQ ID NO. 1: NADH dehydrogenase subunit 1 (318aa) MPMANLLLLIVPILIAMAFLMLTERKILGYMQLRKGPNVVGPYGLLQPFADAMKLFTKEPLKPATSTITLYITAPTLALTIALLLWTPLPMPNPLVNLNLGLLFILATSSLAVYSILWSGWASNSNYALIGALRAVAQTISYEVTLAIILLSTLLMSGSFNLSTLITTQEHLWLLLPSWPLAMMWFISTLAETNRTPFDLAEGESELVSGFNIEYAAGPFALFFMAEYTNIIMMNTLTTTIFLGTTYDALSPELYTTYFVTKTLLLTSLFLWIRTAYPRFRYDQLMHLLWKNFLPLTLALLMWYVSMPITISSIPPQT SEQ ID NO. 2: NADH dehydrogenase subunit 2 (subunit 2) (347aa) MNPLAQPVIYSTIFAGTLITALSSHWFFTWVGLEMNMLAFIPVLTKKMNPRSTEAAIKYFLTQATASMILLMAILFNNMLSGQWTMTNTTNQYSSLMIMMAMAMKLGMAPFHFWVPEVTQGTPLTSGLLLLTWQKLAPISIMYQISPSLNVSLLLTLSILSIMAGSWGGLNQTQLRKILAYSSITHMGWMMAVLPYNPNMTILNLTIYIILTTTAFLLLNLNSSTTTLLLSRTWNKLTWLTPLIPSTLLSLGGLPPLTGFLPKWAIIEEFTKNNSLIIPTIMATITLLNLYFYLRLIYSTSITLLPMSNNVKMKWQFEHTKPTPFLPTLIALTTLLLPISPFMLMIL SEQ ID NO. 4: NADH dehydrogenase subunit 4 (subunit 4) (459aa) MLKLIVPTIMLLPLTWLSKKHMIWINTTTHSLIISIIPLLFFNQINNNLFSCSPTSSSDPLTTPLLMLTTWLLPLTIMASQRHLSSEPLSRKKLYLSMLISLQISLIMTFTATE LIMFYIFFETTLIPTLAIITRWGNQPERLNAGTYFLFYTLVGSLPLLIALIYTHNTLGSLNILLLTLTAQELSNSWANNLMWLAYTMAFMVKMPLYGLHLWLPKAHVEAPIAGSM VLAAVLLKLGGYGMMRLTLILNPLTKHMAYPFLVLSLWGMIMTSSICLRQTDLKSLIAYSSISHMALVVTAILIQTPWSFTGAVILMIAHGLTSSLLFCLANSNYERTHSRIMIL SQGLQTLLPLMAFWWLLASLANLALPPTINLLGELSVLVTTFSWSNITLLLTGLNMLVTALYSLYMFTTTQWGSLTHHINNMKPSFTRENTLMFMHLSPILLLSLNPDIITGFSS Preferably, the antigen combination expressed by the ND mRNA B is a combination of antigen III-antigen V-antigen VI, the amino acid sequence of the antigen combination is shown in SEQ ID NO. 8, and the DNA nucleic acid sequence is shown in SEQ ID NO. 10.

[0017] SEQ ID NO. 8: ND mRNA antigen combination III-V-VI amino acid sequence (972aa) MNFALILMINTLLALLLMIITFWLPQLNGYMEKSTPYECGFDPMSPARVPFSMKFFLVAITFLLFDLE IALLLPLPWALQTTNLPLMVMSSLLLIIILALSLAYEWLQKGLDWTE GGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCV MTMHTTMTTLTLTSLIPPILTTLVNPNKKNSYPHYVKSIVASTFIISLFPTTMFMCLDQEVIISN WHWATTQTTQLSLSFKLDYFSMMFIPVALFVTWSIMEFSLWYMNSDPNINQFFKYLLIFLITMLILVTANNLFQLF IGWEGVGIMSFLLISWWYARADANTAAIQAILYNRIGDIGFILALAWFILHSNSWDPQQMALLNANPSLTPLLGLL LAAAGKSAQLGLHPWLPSAMEGPTPVSALLHSSTMVVAGIFLLIRFHPLAENSPLIQTLTLCLGAITTLFAAVCAL TQNDIKKIVAFSTSSQLGLMMVTIGINQPHLAFLHICTHAFFKAMLFMCSGSIIHNLNNEQDIRKMGGLLKTMPLT STSLTIGSLALAGMPFLTGFYSKDHIIETANMSYTNAWALSITLIATSLTSAYSTRMILLTLTGQPRFPTLTNINE NNPTLLNPIKRLAAGSLFAGFLITNNISPASPFQTTIPLYLKLTALAVTFLGLLTALDLNYLTNKLKMKSPLCTFY FSNMLGFYPSITHRTIPYLGLLTSQNLPLLLLDLTWLEKLLPKTISQHQISTSIITSTQKGMIKLYFLSFFFPLIL TLLLIT GGGGSATNFSLLKQAGDVEENPGPMFVFLVLLPLVSSQCV MMYALFLLSVGLVMGFVGFSSKPSPIYGGL VLIVSGVVGCVIILNFGGGYMGLMVFLIYLGGMMVVFGYTTAMAIEEYPEAWGSGVEVLVSVLVGLAMEVGLVLWV KEYDGVVVVVNFNSVGSWMIYEGEGSGLIREDPIGAGALYDYGRWLVVVTGWTLFVGVYIVIEIARGN Note: The underlined parts are antigens (subunits); GGGGS is a flexible linker; ATNFSLLKQAGDVEENPGP is a P2A dipeptide; MFVFLVLLPLVSSQCV is a signal peptide.

[0018] SEQ ID NO. 10:ND DNA sequence III-V-VI (5128bp) CCAATGATGAATTCTAATACGACTCACTATAGcttgttctttttgcagaagctcagaataaacgctcaactttggGCCACCATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGGTGTGTG ATGAACTTTGCCCTC ATCCTCATGATCAATACCCTTCTTGCTCTGCTTTTGATGATTATAACTTTTTGGCTCCCTCAGCTGAATGGCTACA TGGAGAAGTCCACCCCTTATGAATGTGGGTTTGATCCTATGAGCCCCGCAAGGGTTCCATTCTCCATGAAGTTCTT TCTGGTGGCAATCACATTCCTCTTGTTCGACCTTGAGATCGCATTGCTTCTCCCACTGCCTTGGGCCCTTCAGACG ACCAATCTTCCTCTGATGGTGATGAGCTCTCTGCTCCTGATCATCATCTTGGCTCTGTCACTGGCATATGAATGGC TGCAGAAGGGCCTTGATTGGACAGAG GGAGGAGGCGGTTCTgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggccttATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGGTGTGTG ATGAC TATGCACACGACTATGACCACTTTGACCCTTACTAGCCTTATCCCCCCTATTTTGACCACCCTGGTGAACCCAAAT AAGAAGAATAGCTACCCACACTATGTGAAAAGCATAGTGGCAAGTACCTTCATCATCTCACTGTTTCCTACTACCA TGTTTATGTGCCTGGACCAGGAGGTGATCATCTCAAATTGGCACTGGGCTACGACCCAGACTACACAGCTGTCTCT GAGCTTTAAGTTGGACTACTTCTCCATGATGTTCATTCCCGTGGCCCTGTTTGTGACATGGTCTATCATGGAGTTT TCACTGTGGTACATGAATAGCGACCCTAACATCAACCAGTTTTTTAAATATCTCCTCATATTCTTGATCACAATGC TGATACTGGTGACAGCCAACAATCTGTTTCAGTTGTTCATCGGGTGGGAAGGTGTAGGCATCATGTCTTTCCTTCT GATTTCCTGGTGGTATGCCAGGGCTGATGCCAACACAGCCGCGATCCAGGCCATACTGTACAACCGAATTGGTGAT ATCGGTTTTATCCTCGCCCTGGCATGGTTCATTCTGCATTCAAATAGCTGGGATCCTCAGCAGATGGCCTTGTTGA ATGCCAACCCCTCTCTTACACCCCTGTTGGGCTTGCTGCTTGCTGCAGCCGGCAAGAGTGCACAACTTGGACTGCA TCCGTGGCTGCCGAGCGCGATGGAGGGCCCGACACCTGTCAGTGCCCTGCTTCATTCTTCCACCATGGTGGTGGCT GGGATTTTTTTGCTGATCAGGTTCCATCCACTGGCAGAAAATTCTCCCCTGATCCAGACTCTCACACTCTGTCTCG GAGCTATCACTACTCTGTTCGCGGCTGTGTGTGCACTGACTCAGAATGACATCAAAAAGATCGTCGCCTTCTCAAC CTCCAGTCAGCTCGGGTTGATGATGGTGACCATAGGCATCAACCAGCCTCACCTGGCCTTTCTTCACATTTGCACA CACGCCTTCTTTAAGGCAATGCTGTTTATGTGCAGTGGAAGTATCATTCATAACCTGAACAACGAGCAAGATATCA GAAAAATGGGGGGACTTTTGAAGACCATGCCCCTGACATCTACCAGCCTCACCATTGGAAGTCTCGCTCTCGCTGG AATGCCATTTCTCACAGGATTCTACTCTAAGGACCATATCATTGAAACCGCCAACATGAGTTACACCAACGCATGG GCCCTCAGCATAACTCTGATTGCAACTAGCCTCACCTCCGCTTACAGTACTCGGATGATACTCCTGACCCTCACAG GACAGCCCCGGTTCCCTACGCTGACTAATATTAATGAGAACAATCCAACTCTTCTCAACCCAATCAAGCGCCTGGC TGCGGGATCACTGTTCGCCGGTTTCCTCATTACCAACAATATATCACCCGCATCTCCATTCCAGACTACTATCCCA CTTTACCTTAAGCTTACAGCTCTGGCTGTGACCTTTTTGGGCTTGCTGACCGCCCTCGACCTGAACTATCTGACGA ACAAGCTGAAAATGAAGAGTCCTCTGTGTACATTCTACTTCTCCAATATGTTGGGGTTTTACCCATCCATCACACA TCGCACGATTCCATATCTGGGGCTCCTGACTTCACAAAACCTTCCCCTCCTGCTCCTGGATCTGACATGGCTCGAA AAATTGCTCCCCAAAACAATTTCTCAACACCAGATATCCACTAGCATTATCACCAGCACTCAGAAGGGGATGATCA AGCTGTATTTTCTCAGTTTTTTCTTCCCGCTTATACTGACATTGTTGCTGATAACA GAGGAGGCGGTTCTGgccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcctATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGGTGTGTG ATGATGTACGCGCTTTTCTTGTTGTCAGTGGGACTTGTCATGGGATTTGTTGG CTTCTCTAGCAAACCTTCTCCCATCTACGGGGGGCTGGTGCTTATAGTGAGTGGCGTGGTGGGATGCGTTATAATTC TCAACTTCGGCGGCGGTTACATGGGGCTCATGGTGTTCCTTATATACCTCGGTGGCATGATGGTAGTGTTCGGTTAC ACAACTGCCATGGCAATAGAAGAGTACCCTGAGGCGTGGGGATCTGGTGTGGAAGTCTTGGTGTCTGTGCTTGTGGG GCTGGCTATGGAAGTTGGCCTCGTGTTGTGGGTCAAGGAATACGACGGGGTGGTTGTGGTGGTGAACTTCAACAGTG TCGGCTCCTGGATGATTTACGAGGGCGAAGGCAGCGGGCTCATTAGGGAAGATCCTATTGGCGCAGGAGCCCTTTAT GACTACGGTAGATGGCTGGTTGTCGTGACTGGATGGACTCTCTTTGTGGGCGTCTATATTGTGATTGAAATCGCCAG GGGGAAT Note: The underlined portion represents the antigen (subunit) DNA sequence; EcoRI restriction site: GAATTC (nucleotides 9-14); T7 promoter: TAATACGACTCACTAT (nucleotides 15-30); 5' Cap: AG (nucleotides 31-32); 5' UTR: cttgttctttttgcagaagctcagaataaacgctcaactttgg (nucleotides 33-75); Kozak sequence : GCCACC (nucleotides 76-81); Flexible Linker: GGAGGAGGCGGTTCT and GAGGAGGCGGTTCTG; P2A: gccacgaacttctctctgttaaagcaagcaggagatgttgaagaaaaccccgggcct; Signal Peptide: ATGTTCGTGTTCCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGTGTGTG; Stop Codon: TGATGA (nucleotides 3742-3747); 3'UTR: AACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTTACAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCAC (nucleotides 3748-3873); Poly A: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATCACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (nucleotides 3874-3983, where GCATATCACT is the internal linker of Poly A).

[0019] More preferably, the amino acid sequences encoding antigen III (subunit 3), antigen V (subunit 5) and antigen VI (subunit 6) are as shown in SEQ ID NO.3, antigen II (subunit 2) in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0020] SEQ ID NO. 3: NADH dehydrogenase subunit 3 (115aa) MNFALILMINTLLALLLMIITFWLPQLNGYMEKSTPYECGFDPMSPARVPFSMKFFLVAITFLLFDLEIALLLPLPWALQTTNLPLMVMSSLLLIIILALSLAYEWLQKGLDWTE SEQ ID NO. 5: NADH dehydrogenase subunit 5 (subunit 5) (603aa) MTMHTTMTTLTLTSLIPPILTTLVNPNKKNSYPHYVKSIVASTFIISLFPTTMFMCLDQEVIISNWHWATTQTTQLSLSFKLDYFSMMFIPVALFVTWSIMEFSLWYMNSDPNINQFFKYLLIFLITMLILVTANNLFQLFIGWEGVGIMSFLLISWWYARADANTAAIQAILYNRIGDIGFILALAWFILHSNSWDPQQMALLNANPSLTPLLGLLLAAAGKSAQLGLHPWLPSAMEGPTPVSALLHSSTMVVAGIFLLIRFHPLAENSPLIQTLTLCLGAITTLFAAVCALTQNDIKKIVAFSTSSQLGLMMVTIGINQPHLAFLHICTHAFFKAMLFMCSGSIIHNLNNEQDIRKMGGLLKTMPLTSTSLTIGSLALAGMPFLTGFYSKDHIIETANMSYTNAWALSITLIATSLTSAYSTRMILLTLTGQPRFPTLTNINENNPTLLNPIKRLAAGSLFAGFLITNNISPASPFQTTIPLYLKLTALAVTFLGLLTALDLNYLTNKLKMKSPLCTFYFSNMLGFYPSITHRTIPYLGLLTSQNLPLLLLDLTWLEKLLPKTISQHQISTSIITSTQKGMIKLYFLSFFFPLILTLLLIT SEQ ID NO. 6: NADH dehydrogenase subunit 6 (subunit 6) (174aa) MMYALFLLSVGLVMGFVGFSSKPPSPIYGGLVLIVSGVVGCVIILNFGGGYMGLMVFLIYLGGMMVVFGYTTAMAIEEYPEAWGSGVEVLVSVLVGLAMEVGLVLWVKEYDGVVVVVNFNSVGSWMIYEGEGSGLIREDPIGAGALYDYGRWLVVVTGWTLFVGVYIVIEIARGN Preferably, the co-delivery is co-transfection or co-encapsulation.

[0021] Preferably, the molar ratio of the ND mRNA to the target nucleic acid molecule is (0.1-10):1.

[0022] More preferably, the co-transfection involves encapsulating ND mRNA and the target nucleic acid molecule in different delivery vectors, and then co-delivering them into the cell.

[0023] More preferably, the co-encapsulation involves encapsulating the ND mRNA and the target nucleic acid molecule together in the same delivery vector and then co-delivering them to the cell.

[0024] Preferably, the target nucleic acid molecule is selected from small interfering nucleic acid (siRNA), messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), or plasmid DNA.

[0025] Preferably, the target nucleic acid molecule encodes a therapeutic protein, a preventative antigen, or a gene-editing tool.

[0026] Beneficial effects: Compared with existing technologies, this invention specifically relates to a method for significantly enhancing the intracellular expression of target nucleic acid molecules by regulating cellular metabolic states. More specifically, this invention relates to a strategy of co-delivering mRNA encoding NADH dehydrogenase (ND mRNA) and the target nucleic acid molecule to the same cell, and increasing cellular energy levels (ATP) through ND mRNA, thereby amplifying the expression of the target nucleic acid molecule. This novel approach to enhancing nucleic acid expression through metabolic regulation has the following significant advantages and positive effects: 1. Innovative principles, remarkable results.

[0027] This invention proposes a novel strategy for the first time to actively enhance cellular metabolic levels through co-delivery of ND mRNA, thereby amplifying the expression of target nucleic acids. This strategy addresses the fundamental problem of intracellular resource bottlenecks and has demonstrated a strong amplification effect on target protein expression in various cell and mouse models, with enhancements ranging from tens to hundreds of times.

[0028] 2. Highly applicable.

[0029] The method of this invention is not dependent on a specific target nucleic acid type and exhibits significant expression enhancement effects on linear mRNA, circular RNA, self-amplifying RNA, and even plasmid DNA. This indicates that the method is a universal platform technology that can be widely applied in various fields of nucleic acid drug development.

[0030] 3. High security.

[0031] This method utilizes the cell's own endogenous metabolic pathways for regulation, rather than introducing exogenous chemicals, thus avoiding potential toxic side effects. Animal experiments have shown that this method effectively increases cellular protein levels while exhibiting good safety.

[0032] 4. Synergistic effect, achieving dose saving.

[0033] Due to the significantly improved expression efficiency, the dosage of the target nucleic acid can be significantly reduced while achieving the same or even better therapeutic effect (e.g., 1 / 10 of the dosage in the example is still better than the traditional full dose). This helps to reduce drug costs and reduce the toxic side effects that may be caused by high doses, which has important clinical significance.

[0034] 5. The method of this invention can be seamlessly integrated with existing technologies. This method can be combined with various existing nucleic acid optimization technologies (such as sequence optimization and vector optimization) to produce a synergistic effect and further push the therapeutic boundaries of nucleic acid drugs. Attached Figure Description

[0035] Figure 1 This is a schematic diagram illustrating the working mechanism of the method of the present invention.

[0036] Figure 2 The enhancement effect of ND mRNA on Spike protein expression in cells (a) and mice (b) was demonstrated by co-encapsulation of LNP in Example 1.

[0037] Figure 3 Example 2 compares the enhancement effect of ND mRNA on Spike protein expression under two methods: co-transfection and co-encapsulation.

[0038] Figure 4 This is an example of how the co-encapsulation of LNP in Example 3 enhances the anti-tumor effect of ND mRNA on HPV therapeutic vaccines.

[0039] Figure 5 In Example 4, the SARS-CoV-2 linear mRNA vaccine optimized using the method of the present invention significantly enhanced the serum IgG antibody titers induced in mice at different doses.

[0040] Figure 6This is a comparison chart showing the effects of the optimized messenger RNA (mRNA), self-amplified RNA (saRNA), circular RNA (circRNA), and plasmid DNA, obtained using the method of the present invention in Example 5, on cell expression compared with traditional methods. Specific Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the implementation of the present invention is not limited thereto.

[0042] Example 1: ND mRNA enhanced the expression of SARS-CoV-2 Spike mRNA in (a) cells and (b) mice.

[0043] Using SARS-CoV-2 Spike mRNA (refer to published literature) Antiviral Research 212(2023): 105556.) was used as the target nucleic acid to be paired with ND mRNA. A traditional ethanol injection-microfluidic mixing method was employed (refer to published literature). Antiviral Research 212 (2023): 105556), LNPs co-encapsulated with Spike mRNA and ND mRNA (Spike mRNA:ND mRNA molar ratio = 1:1) were prepared. LNPs encapsulated with only Spike mRNA served as a control. HEK 293T cells (commercially available products) were transfected with the above LNPs. 24 hours after transfection, cellular ATP levels and intracellular Spike protein expression were measured. ND mRNA was ND mRNA A.

[0044] The simplified steps using a commercially available ATP assay kit are as follows: Cells transfected with different LNPs were washed with PBS and lysed in ATP assay buffer. 50 μL of cell lysate was mixed with an equal volume of reconstituted assay reagent in an opaque 96-well plate. After incubation at 25°C for 10 minutes, the luminescence signal was detected using a microplate reader. Total protein concentration was determined using the BCA method to standardize and calibrate the ATP concentration.

[0045] The method for quantitative detection of Spike protein is as follows: First, commercially available 293T cells were incubated overnight in 24-well plates, and the medium was replaced with fresh medium before transfection. LNP was then added to the cells for transfection, and the cells were cultured for 24 hours. After transfection, the cells were collected, digested with trypsin, and stained with commercially available anti-SARS-CoV-2 Spike antibody and APC-labeled anti-mouse IgG1 secondary antibody. Finally, the cells were resuspended in PBS and analyzed by flow cytometry.

[0046] The results are as follows Figure 2As shown in (a), compared with the control group, the experimental group co-encapsulated with ND mRNA showed an approximately 2.1-fold increase in cellular ATP levels and an approximately 8.5-fold increase in Spike protein expression. This demonstrates that ND mRNA can effectively exert a metabolic amplification effect.

[0047] Following this, mouse immunization experiments were conducted. The LNPs prepared above (co-encapsulated group and control group) were administered intramuscularly to BALB / c mice (n=6 / group) at a dose of 10 μg Spike mRNA per mouse. On day 7 post-primary immunization, mouse serum was collected, and the titer of specific IgG antibodies against SARS-CoV-2 Spike protein in the serum was detected by ELISA. Figure 2 As shown in (b), the experimental group co-encapsulated with ND mRNA induced a geometric mean titer (GMT) of IgG antibodies that was 12.3 times that of the control group. This indicates that ND mRNA can also significantly enhance the expression and immunogenicity of the target antigen in vivo.

[0048] Example 2: Comparison of the effects of co-transfection and co-encapsulation methods SARS-CoV-2 Spike mRNA (encoding Omicron BA.1 Spike protein) was used as the target nucleic acid in combination with ND mRNA. Co-encapsulated LNPs (ND mRNA:Spike mRNA molar ratio = 1:1), co-transfected LNPs (Spike mRNA-LNP and ND mRNA-LNP were prepared independently and mixed at a Spike mRNA:ND mRNA molar ratio of 1:1 during transfection), and a control group (Spike mRNA-LNP only) were prepared. All LNP groups were transfected into HEK 293T cells (commercially available products), ensuring a consistent final Spike mRNA dosage across groups. Spike protein expression levels were measured 24 hours later. Detection methods included ELISA to detect S protein in the supernatant or flow cytometry using an S protein antibody to detect intracellular and extracellular S protein levels. ND mRNA was ND mRNA B.

[0049] like Figure 3 As shown, compared with the control group, the expression levels of Spike protein in the co-transfection group and the co-encapsulation group increased by approximately 7.2-fold and 8.5-fold, respectively. This demonstrates the effectiveness of ND mRNA under different delivery methods.

[0050] Example 3: Application in therapeutic cancer vaccines The target mRNA (E6-E7 mRNA) encoding the HPV16 E6-E7 antigen was used in combination with the ND mRNA. The LNP vaccine was prepared using a co-encapsulation method (ND mRNA:E6-E7 mRNA molar ratio = 1:1). Subsequently, a TC1 tumor-bearing mouse model was established by subcutaneous inoculation of a TC1 tumor cell suspension. Specifically, 100 μL of a PBS suspension containing 5 × 10^5 TC1 cells was subcutaneously injected into the right back of C57BL / 6 mice. Tumor volume was monitored periodically, and modeling was considered successful when the tumor grew to approximately 50 mm³. Evaluation was then conducted via intramuscular injection of the LNP vaccine. The ND mRNA consisted of an equimolar mixture of ND mRNA A and ND mRNA B.

[0051] like Figure 4 As shown, compared with the traditional E6-E7 mRNA-LNP vaccine, the vaccine co-encapsulated with ND mRNA showed a stronger tumor growth inhibition effect and a trend of prolonging the survival of mice, demonstrating the application potential of the present invention in tumor immunotherapy.

[0052] The above examples fully demonstrate that using ND mRNA as a metabolic amplification tool and co-delivering it with the target nucleic acid is a universal, effective and easy-to-implement strategy to enhance nucleic acid expression.

[0053] Example 4: Dosage-saving effect in prophylactic vaccines A co-encapsulated LNP vaccine was prepared using SARS-CoV-2 Spike mRNA (referencing the published paper Antiviral Research 212 (2023):105556.) as the target nucleic acid, combined with ND mRNA (ND mRNA:Spike mRNA molar ratio = 1:1). The ND mRNA consisted of an equimolar mixture of ND mRNA A and ND mRNA B.

[0054] Mouse immunization experiments showed that ( Figure 5 (a) At the same dose (10 μg), the vaccine co-delivered with ND mRNA and Spike mRNA induced IgG antibody titers 62.3 times higher than those of the conventional vaccine. However, the co-delivered vaccine using only 1 / 10th the dose (1 μg) of the conventional vaccine still induced antibody titers 6.6 times higher than those of the conventional full-dose vaccine. Figure 5 (b)).

[0055] This indicates that the method of the present invention has a strong dose-saving effect, allowing for better results at lower doses, while potentially reducing the risk of immunogenicity-related side effects.

[0056] Example 5: Validation of universality across different target nucleic acid types like Figure 6 As shown in (a), mRNA, saRNA, circRNA, and plasmid DNA encoding luciferase were used as target nucleic acids paired with ND mRNA. Co-encapsulated LNPs (ND mRNA:target nucleic acid molar ratio = 1:1) and a control group (target nucleic acid - LNP only) were prepared. Each group of LNPs was transfected into HEK 293T cells, maintaining a consistent target nucleic acid dosage. Cellular fluorescence signal expression levels were detected after 24 hours. Detection methods included ELISA to detect S protein in the supernatant or flow cytometry to detect intracellular and extracellular S protein content using an S protein antibody. ND mRNA was a mixture of ND mRNAA and ND mRNA B in an equimolar ratio.

[0057] The method for detecting cellular fluorescence signal expression levels is as follows: HEK 293T cells were transfected with LNPs containing the target nucleic acid encoding luciferase and cultured at 37°C for 24 hours. The culture medium was discarded, and lysis buffer containing luciferin substrate was added. After lysis, the cell lysate was collected, and the relative light unit value was read using a chemiluminescence detector to quantify the protein expression level of the reporter gene.

[0058] like Figure 6 As shown in (b), compared with the control groups, the present invention increased the cellular fluorescence intensity of the target nucleic acid by more than ten to dozens of times, which is statistically significant, further proving that the method of the present invention has a universal enhancement effect on different types of target nucleic acids.

[0059] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention, such as replacing NDmRNA with other metabolic regulatory genes that can effectively increase cellular ATP levels (such as other mitochondrial-related genes), or using other delivery systems that can achieve sequential release of nucleic acids, should be covered within the scope of protection of the present invention.

Claims

1. A method for enhancing the expression effect of a nucleic acid, characterized by, The mRNA encoding NADH dehydrogenase is co-delivered with the target nucleic acid molecule to the cell, and the expression product of the ND mRNA enhances the expression effect of the target nucleic acid molecule by increasing the intracellular ATP level.

2. The method for enhancing the expression effect of a nucleic acid according to claim 1, wherein The antigen expressed by the mRNA of the NADH dehydrogenase is a fusion antigen or a mixed antigen.

3. The method for enhancing the expression effect of a nucleic acid according to claim 2, wherein The fusion antigen includes but is not limited to at least two of antigen I, antigen II, antigen III, antigen IV, antigen V, and antigen VI fused together; the mixed antigen includes but is not limited to at least two of antigen I, antigen II, antigen III, antigen IV, antigen V, and antigen VI mixed together.

4. The method for enhancing the expression effect of a nucleic acid according to claim 3, wherein The mixed antigen includes a combination of antigen I, antigen II, and antigen IV in any order, or a combination of antigen III, antigen V, and antigen VI in any order.

5. The method of enhancing the expression effect of a nucleic acid according to claim 3, wherein The mRNA of the NADH dehydrogenase is ND mRNA A or ND mRNA B, and the nucleic acid sequence of the ND mRNA A or ND mRNA B is shown in SEQ ID NO. 11 or SEQ ID NO.

12.

6. The method of enhancing the expression effect of a nucleic acid according to claim 5, wherein The antigen combination expressed by the ND mRNA A is a combination of antigen I-antigen II-antigen IV, the amino acid sequence of the antigen combination is shown in SEQ ID NO. 7, and the nucleotide sequence is shown in SEQ ID NO. 9; the amino acid sequences of the encoded antigen I (subunit 1), antigen II (subunit 2), and antigen IV (subunit 4) are shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 4, respectively.

7. The method of enhancing the expression effect of a nucleic acid according to claim 5, wherein The antigen combination expressed by the ND mRNA B is a combination of antigen III-antigen V-antigen VI, the amino acid sequence of the antigen combination is shown in SEQ ID NO. 8, and the DNA nucleic acid sequence is shown in SEQ ID NO. 10; the amino acid sequences of the encoded antigen III (subunit 3), antigen V (subunit 5), and antigen VI (subunit 6) are shown in SEQ ID NO. 3, SEQ ID NO. 5, and SEQ ID NO. 6, respectively.

8. The method of enhancing the expression of nucleic acid according to claim 1, wherein, The co-delivery is co-transfection or co-encapsulation, and the molar ratio of the ND mRNA to the target nucleic acid molecule is (0.1-10):

1.

9. The method of enhancing the expression of nucleic acid according to claim 1, wherein, The co-transfection is to separately encapsulate the ND mRNA and the target nucleic acid molecule in different delivery vectors, and then co-deliver them to the cell; the co-encapsulation is to co-encapsulate the ND mRNA and the target nucleic acid molecule in the same delivery vector, and then co-deliver them to the cell.

10. The method of enhancing the expression of nucleic acid according to claim 1, wherein, The target nucleic acid molecule is selected from small interfering nucleic acid (siRNA), messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), or plasmid DNA.

11. The method of enhancing the expression of nucleic acid according to claim 1, wherein, The target nucleic acid molecule encodes a therapeutic protein, a prophylactic antigen, or a gene editing tool.

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