WO2024051266A1 - 用于表达水痘-带状疱疹病毒抗原蛋白的mRNA及其用途 - Google Patents

用于表达水痘-带状疱疹病毒抗原蛋白的mRNA及其用途 Download PDF

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WO2024051266A1
WO2024051266A1 PCT/CN2023/101292 CN2023101292W WO2024051266A1 WO 2024051266 A1 WO2024051266 A1 WO 2024051266A1 CN 2023101292 W CN2023101292 W CN 2023101292W WO 2024051266 A1 WO2024051266 A1 WO 2024051266A1
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seq
nucleotide sequence
mrna
untranslated region
varicella
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French (fr)
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葛君
李建强
石应辉
李玲
刘敏
孙娇娇
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远大赛威信生命科学(南京)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A61K39/25—Varicella-zoster virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/03—Herpetoviridae, e.g. pseudorabies virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/03—Herpetoviridae, e.g. pseudorabies virus
    • C07K14/04—Varicella-zoster virus
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to an mRNA for expressing the varicella-zoster virus antigen protein, a vector particle containing it, a recombinant plasmid and an immune composition, as well as their use in the preparation of prevention and/or treatment of varicella-zoster virus. Use in medicines for diseases or conditions associated with herpes zoster virus infection.
  • Herpes zoster is a viral skin disease caused by varicella-zoster virus (VZV). During primary infection, VZV enters local lymph nodes through the respiratory mucosal epithelium and replicates. The virus-infected lymphocytes then enter the blood circulation through the lymphatic circulation and infect peripheral blood mononuclear leukocytes. The virus spreads to the skin along with the bloodstream, and the clinical manifestation is chickenpox. After recovery from chickenpox, the virus replicates latently in the cranial ganglia and migrates to the skin along the peripheral nerves, causing clinical manifestations of herpes zoster. The most important complication of herpes zoster is post-herpetic neuralgia (PHN). About 20% of people over the age of 50 will develop PHN, and as age increases, the likelihood of developing PHN increases, with pain lasting for months or even years.
  • PPN post-herpetic neuralgia
  • Vaccination against herpes zoster is an effective means to prevent herpes zoster and reduce complications of PHN.
  • the main shingles vaccines on the market are Zostavax from Merck & Co. and Shingrix from GlaxoSmithKline. Among them, Zostavax was launched in 2006 as a live attenuated vaccine; Shingrix was launched in October 2017 as a recombinant herpes zoster vaccine.
  • mRNA vaccines can induce the body to produce humoral immunity and cellular immunity at the same time, protecting the body through a variety of mechanisms, and due to its own characteristics, it can be quickly degraded in the cytoplasm of transfected cells after immunity. This reduces security risks.
  • traditional vaccines attenuated, inactivated or recombinant subunit vaccines
  • the production process of mRNA vaccines is simple, does not require cell culture or animal-derived substrates, has fast synthesis speed and low cost.
  • DNA vaccines which are also nucleic acid vaccines
  • mRNA vaccines do not need to enter the cell nucleus to work, and there is no risk of integration into the host genome. Moreover, they are transiently expressed, and the half-life can be adjusted through modification.
  • Non-replicating mRNA mainly consists of 5' untranslated region (UTR), open reading frame (ORF), 3' untranslated region, 5' cap structure (Cap) and 100-250 bp polyadenosine Composed of acid tail [Poly(A)], among which, 5′- Regulatory elements in UTR and 3′-UTR can stabilize mRNA and increase antigen protein translation (Huang Huiyuan, Miao Mingsan, Zhu Yanhui, et al. Research progress on liposome-based mRNA vaccine delivery system [J]. International Journal of Pharmaceutical Research, 2019, 46(5):8). Herpes zoster vaccines developed based on this principle can be found in patent applications such as CN108472309A and CN114272369A.
  • the inventor conducted in-depth research on each component of the herpes zoster mRNA vaccine, and discovered the advantageous sequence suitable for preparing the herpes zoster mRNA vaccine, and the herpes zoster mRNA vaccine prepared therefrom The antigen expression level is high and the drug efficacy advantage is obvious.
  • the present invention provides an mRNA for expressing the varicella-zoster virus antigen protein, which sequentially includes a 5' untranslated region (5'UTR) and an open reading frame (ORF) from the 5' to 3' direction. , 3′ untranslated region (3′UTR) and polyadenylate (polyA) tail, wherein the nucleotide sequence of the 5′UTR is such as any one of SEQ ID NO. 23, 24, 25, 26 or 27 It is shown in the item, and the open reading frame encodes the varicella-zoster virus antigenic protein.
  • 5'UTR 5' untranslated region
  • ORF open reading frame
  • polyA polyadenylate
  • nucleotide sequence of the 3'UTR is as shown in any one of SEQ ID NO. 28 or 29.
  • the 5' untranslated region refers to the region in the mature messenger ribonucleic acid (mRNA) that is located upstream of the coding region (CDS) and is not translated into protein. It starts from the transcription start site and ends at the start codon. Ends at the previous nucleotide.
  • the median length of eukaryotic 5′ UTRs is generally approximately 150 nucleotides (nt), but some can be as long as several thousand bases.
  • the 3' untranslated region refers to an untranslated sequence downstream of the mature mRNA coding region, which plays an important role in mRNA transport, stability and translation regulation.
  • polyadenylic acid refers to 150-200 adenylic acid residues located in mRNA, which can protect mRNA from exonuclease attack, terminate transcription, and remove mRNA. Export from the nucleus and translation are both important.
  • an open reading frame is a sequence in a DNA sequence that has the potential to encode a protein. It is a continuous base sequence starting from the start codon and ending with the stop codon.
  • the nucleotide sequence of the 5'UTR is such as SEQ ID NO.23 is shown, and the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.28; the nucleotide sequence of the 5'UTR is shown in SEQ ID NO.24, and the 3' The nucleotide sequence of the UTR is shown in SEQ ID NO. 28; the nucleotide sequence of the 5' UTR is shown in SEQ ID NO. 25, and the nucleotide sequence of the 3' UTR is shown in SEQ ID NO. 28; the 5′ UTR nucleotide sequence is shown in SEQ ID NO. 26, and the 3′ UTR nucleotide sequence is shown in SEQ ID NO. 28; or the 5′ UTR The nucleotide sequence is shown in SEQ ID NO. 27, and the nucleotide sequence of the 3'UTR is shown in SEQ ID NO. 29.
  • nucleotide sequence of the 5′ UTR is shown in SEQ ID NO. 24, and the nucleotide sequence of the 3′ UTR is shown in SEQ ID NO. 28.
  • the varicella-zoster virus antigen protein is gE protein; preferably, the amino acid sequence of the gE protein is as shown in SEQ ID NO. 1.
  • gE protein refers to varicella-zoster virus glycoprotein E.
  • the length of the polyA tail is 80 to 120 nucleotides, preferably 120 nucleotides. More preferably, the nucleotide sequence of the polyA tail is as shown in SEQ ID NO.5.
  • the nucleotide sequence of the mRNA is as shown in any one of SEQ ID NO. 30, 31, 32, 33 or 34.
  • the nucleotide sequence of the mRNA is shown in SEQ ID NO. 31.
  • the 5' end of the mRNA also has a cap structure.
  • the mRNA with a cap structure at the 5' end is represented by formula (I) or formula (II):
  • the molecular formula of formula (I) is C 21 H 26 N 10 O 17 P 3 2- and the molecular formula of formula (II) is C 22 H 28 N 10 O 17 P 3 + .
  • the cap structure refers to the unique cap structure at the 5' end of eukaryotic mRNA.
  • There are usually three types of hat structures m7G5′ppp5′Np, m7G5′ppp5′NmpNp and m7G5′ppp5′NmpNp), which are called type O, type I and type II respectively.
  • Type O means that the ribose at the terminal nucleotide is unmethylated
  • type I means that the ribose at one terminal nucleotide is methylated
  • type II means that the ribose at both terminal nucleotides is methylated.
  • This structure resists degradation by 5′-exonucleases. During protein synthesis, it helps ribosomes recognize and bind to mRNA so that translation can be initiated correctly.
  • the present invention provides a vector particle for expressing varicella-zoster virus antigenic protein, which contains the mRNA according to the present invention and a carrier material encapsulating the mRNA.
  • mRNA carrier particles refer to composite structures used to deliver exogenous mRNA into cells. Effective in vivo delivery is an important element for mRNA vaccines (drugs) to achieve therapeutic effects. Exogenous mRNA must penetrate the barrier of the lipid membrane before it can enter the cytoplasm and be successfully translated into functional proteins.
  • the carrier material is selected from one or more of protamine, lipid nanoparticles (LNP), polymer materials and inorganic nanoparticles.
  • the support material is LNP.
  • the present invention provides a recombinant plasmid for expressing the varicella-zoster virus antigen protein, which sequentially includes a 5' untranslated region (5'UTR), an open reading frame ( ORF), 3' untranslated region (3'UTR) and polyadenylic acid (polyA) tail, wherein the nucleotide sequence of the 5'UTR is as in SEQ ID NO. 9, 11, 12, 13 or 14 Any one is shown, and the open reading frame encodes the varicella-zoster virus antigenic protein.
  • 5'UTR 5' untranslated region
  • ORF open reading frame
  • 3'UTR 3' untranslated region
  • polyA polyadenylic acid
  • the nucleotide sequence of the 3'UTR is such as SEQ ID Shown in either NO.16 or 22.
  • nucleotide sequence of the 5′ UTR is shown in SEQ ID NO. 11, and the nucleotide sequence of the 3′ UTR is shown in SEQ ID NO. 16.
  • the varicella-zoster virus antigen protein is gE protein; preferably, the nucleotide sequence of the gE protein is as shown in SEQ ID NO. 2.
  • the length of the polyA tail is 80 to 120 nucleotides, preferably 120 nucleotides.
  • the nucleotide sequence of the polyA tail is as shown in SEQ ID NO.5.
  • the plasmid is pUC57 plasmid; preferably, the recombinant plasmid also includes an initial sequence (Ori), a T7 promoter and a resistance gene.
  • the initiation sequence is the replication origin site of the prokaryotic gene plasmid and is four highly conserved 19 bp direct repeat sequences.
  • the T7 promoter is a powerful and highly specific promoter controlled exclusively by T7 RNA polymerase.
  • the resistance gene that is, the genetic factor of resistance, is a kind of selection gene and belongs to the marker gene.
  • nucleotide sequence of the starting sequence is shown in SEQ ID NO. 3.
  • the nucleotide sequence of the T7 promoter is shown in SEQ ID NO. 4.
  • the resistance gene is a kanamycin sulfate resistance gene and/or an ampicillin resistance gene.
  • the nucleotide sequence of the kanamycin sulfate resistance gene is as shown in SEQ ID NO.6;
  • the nucleotide sequence of the ampicillin resistance gene is as shown in SEQ ID NO.7 .
  • the invention provides a varicella-zoster virus antigenic protein expressed from an mRNA according to the invention.
  • the present invention also provides a varicella-zoster virus antigen protein expressed by the vector particle according to the present invention.
  • the present invention also provides a varicella-zoster virus antigen protein expressed by the recombinant plasmid according to the present invention.
  • the present invention provides an immune composition comprising an immunologically effective amount of the mRNA according to the present invention or the antigenic protein expressed therefrom.
  • the present invention also provides an immunological composition, which comprises an immunologically effective amount of The vector particles or the antigenic proteins they express.
  • the present invention also provides an immune composition, which contains an immunologically effective amount of the recombinant plasmid according to the present invention or the antigen protein expressed therefrom.
  • the immune composition is a vaccine.
  • the present invention provides the use of the mRNA or the antigenic protein expressed by the mRNA according to the present invention in the preparation of a medicament for preventing and/or treating diseases or conditions related to varicella-zoster virus infection.
  • the present invention also provides the use of the vector particle according to the present invention or the antigen protein expressed therein in the preparation of a medicament for preventing and/or treating diseases or conditions related to varicella-zoster virus infection.
  • the present invention also provides the use of the recombinant plasmid according to the present invention or the antigen protein expressed therein in preparing a medicament for preventing and/or treating diseases or conditions related to varicella-zoster virus infection.
  • the disease or condition is selected from one or more of chickenpox, herpes zoster, or post-herpetic neuralgia.
  • varicella is an acute infectious disease caused by primary infection with varicella-zoster virus.
  • herpes zoster is an acute infectious skin disease caused by the varicella-zoster virus. Because the virus is neurotropic, it can lurk in the neurons of the dorsal root ganglia of the spinal cord for a long time after infection. When the resistance is low, fatigue, infection, or cold, the virus can grow and reproduce again, and move to the skin along the nerve fibers. Causes intense inflammation of the affected nerves and skin.
  • the disease or condition is herpes zoster.
  • the experimental results of the present invention show that the average protein expression level of the mRNA screened out by the present invention is relatively high, reaching 250ng/ml, and the highest protein expression level can reach 400ng/ml.
  • the medicinal efficacy of the mRNA was verified, and the results proved that the mRNA provided by the invention all had immunostimulatory effects.
  • the immune effect is comparable to that of recombinant protein antigens.
  • Figure 1 shows the structure of the pUC57-kan-XgE-A plasmid.
  • Each nucleic acid fragment in Figure 1 is the transcription enhancer (Transcript enhancer), T7 promoter (T7 promoter), leader, and stop.
  • Improved gE546 ORF gE546 ORF Improved and initial sequence (Ori);
  • Figure 2 shows the gel electrophoresis results of mRNA transcribed from different combinations of 5′UTR and 3′UTR;
  • Figure 3 shows the expression levels of gE protein obtained from the expression of 14 mRNAs respectively. The results showed that the protein expression levels obtained from mRNA No. 01, 05, 07, 09, and 12 were all higher;
  • Figure 4 shows the effect of No. 05 mRNA in inducing immunity under different transfection reagents
  • Figure 5 shows the effect of mRNA-carrier particles in inducing immunity.
  • the DNA coding sequence was designed based on the amino acid sequence of human varicella-zoster virus gE protein (shown in SEQ ID NO.1). After codon optimization, the designed coding sequence was designed as shown in SEQ ID NO.2.
  • the recombinant plasmid contains an initial sequence (Ori) (SEQ ID NO.3), a T7 promoter sequence (SEQ ID NO.4), and a 5′ UTR sequence (shown in SEQ ID NO.8-15 respectively), 3 'UTR sequence (shown as SEQ ID NO.16-22 respectively), 3' terminal polyadenylic acid (polyA) sequence (SEQ ID NO.5), and kanamycin sulfate resistance gene (SEQ ID NO. 6) (can be replaced by other resistance genes, such as ampicillin resistance gene (SEQ ID NO. 7)).
  • recombinant plasmid 00 contains the 5′UTR sequence shown in SEQ ID NO.8 and the 3′UTR sequence shown in SEQ ID NO.16;
  • Recombinant plasmid 01 contains the 5′UTR sequence shown in SEQ ID NO.9 and the 3′UTR sequence shown in SEQ ID NO.16;
  • Recombinant plasmid 02 contains the 5′UTR sequence shown in SEQ ID NO.9 and the 3′UTR sequence shown in SEQ ID NO.17;
  • Recombinant plasmid 03 contains the 5′UTR sequence shown in SEQ ID NO.10 and SEQ ID NO. The 3′ UTR sequence shown in 16;
  • Recombinant plasmid 04 contains the 5′UTR sequence shown in SEQ ID NO.10 and the 3′UTR sequence shown in SEQ ID NO.18;
  • Recombinant plasmid 05 contains the 5′UTR sequence shown in SEQ ID NO.11 and the 3′UTR sequence shown in SEQ ID NO.16;
  • Recombinant plasmid 06 contains the 5′UTR sequence shown in SEQ ID NO.11 and the 3′UTR sequence shown in SEQ ID NO.19;
  • Recombinant plasmid 07 contains the 5′UTR sequence shown in SEQ ID NO.12 and the 3′UTR sequence shown in SEQ ID NO.16;
  • Recombinant plasmid 08 contains the 5′UTR sequence shown in SEQ ID NO.12 and the 3′UTR sequence shown in SEQ ID NO.20;
  • Recombinant plasmid 09 contains the 5′UTR sequence shown in SEQ ID NO.13 and the 3′UTR sequence shown in SEQ ID NO.16;
  • Recombinant plasmid 10 contains the 5′UTR sequence shown in SEQ ID NO.13 and the 3′UTR sequence shown in SEQ ID NO.21;
  • Recombinant plasmid 11 contains the 5′UTR sequence shown in SEQ ID NO.14 and the 3′UTR sequence shown in SEQ ID NO.16;
  • Recombinant plasmid 12 contains the 5′UTR sequence shown in SEQ ID NO.14 and the 3′UTR sequence shown in SEQ ID NO.22;
  • Recombinant plasmid 13 contains the 5′UTR sequence shown in SEQ ID NO.15 and the 3′UTR sequence shown in SEQ ID NO.16.
  • the coding region and polyA region of the pUC57-kan-XgE-A plasmid were sequenced.
  • the inserted target gene sequence was completely consistent with the reference sequence, and the recombination identification was correct.
  • the entire plasmid structure successfully constructed is shown in Figure 1.
  • Transcription uses the Novozant T7 transcription kit, uses ARCA (anti-reverse cap analog) to directly cap the 5' end during the transcription process, and reacts at 37°C for 3 hours.
  • the product is immediately stored in a -80°C refrigerator or in transfected cells.
  • the mRNA sequences obtained after in vitro transcription of the gE protein DNA sequence are shown in SEQ ID NO.30-34.
  • Figure 2 shows that each lane has only one band, indicating that the transcribed mRNA band is single and not degraded.
  • RNA purification magnetic beads Remove from 2 to 8°C and equilibrate to room temperature, then mix the magnetic beads thoroughly by inverting or vortexing; add the mRNA solution and mix thoroughly; incubate at room temperature for 5 minutes to allow the mRNA to bind to the magnetic beads. Place the sample on the magnetic stand. After the solution is clarified, carefully remove the supernatant. Keep the sample in the magnetic stand and add 200 ⁇ l of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature for 30 s and carefully remove the supernatant. Repeat.
  • One step a total of 2 rinses; keep the sample in the magnetic rack, open the lid, and air dry the magnetic beads for 5 minutes. Take the sample out of the magnetic stand, add an appropriate volume of nuclease-free H 2 O, mix thoroughly, and let stand at room temperature for 5 minutes; place the sample on the magnetic stand for 5 minutes, and transfer it after the solution is clear. Transfer the supernatant to a new centrifuge tube (without nucleotides). Store in -80°C refrigerator.
  • the 14 mRNAs obtained in Example 1 were respectively translated to obtain gE protein.
  • the preparation steps are as follows: 293T cells were used for in vitro expression detection, and spread into a 6-well plate. After 6 hours of culture, the cell volume reached 80-90%, and the culture medium was removed. (The medium is composed of 90% DMEM + 10% FBS + 1% PS), change to serum-free medium for 2 hours, change to normal 2% FBS-DMEM medium during transfection, and add the prepared transfection mixture. The cells were then cultured in a cell culture incubator for 24 hours, and the cell supernatant was collected, and the gE protein expression was measured using the Elisa method.
  • Example 3 The expression results are shown in Figure 3.
  • the protein expression levels obtained by the mRNAs with X being 01, 05, 07, 09 and 12 were all relatively high, up to 250ng/ml.
  • the protein expression levels obtained by the mRNA with X being 05 were the highest. , can reach 400ng/ml, and subsequent efficacy experiments verified the mRNA with X as 05.
  • mice 24 female C57BL/6 mice were used and divided into 4 groups A to D (6 mice/group).
  • group A was also given transfection reagent (HG-TransGeneTM transfection reagent), 20 ⁇ l/animal
  • group B was also given transfection reagent (In vivo-jetRNA Transfection Reagent), 20 ⁇ l/animal.
  • Group C was given gE LNP-mRNA (LNP lipid nanoparticles encapsulating gE-mRNA in LNP, encapsulation rate 97.1%, yield 81.5%), 20 ⁇ g/animal;
  • Group D was given 2 ⁇ g gE antigen, QS21 lipid Body 1 ⁇ g, CpG 20 ⁇ g/animal.
  • Each group was immunized once, and the level of T lymphocyte spot number secreting gE-specific IFN- ⁇ in splenocytes was measured at the end of one week after the first immunization.
  • the gE-specific peptide library sequence refers to Chinese patent application CN2020114564359.
  • the mRNA advantageous sequence provided by the present invention which is suitable for preparing herpes zoster vaccine, has high protein expression, obvious pharmaceutical efficacy advantages, and good application prospects.

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Abstract

本发明公开了用于表达水痘-带状疱疹病毒抗原蛋白的mRNA及其用途，该mRNA从5'到3'方向依次包含5'非翻译区、开放阅读框、3'非翻译区和多聚腺苷酸尾，其中该5'非翻译区的核苷酸序列如SEQ ID NO.23、24、25、26或27中任一项所示，该3'非翻译区的核苷酸序列如SEQ ID NO.28或29中任一项所示，并且该开放阅读框编码水痘-带状疱疹病毒抗原蛋白。本发明获得的经序列优化的mRNA的平均蛋白表达量较高。经药效验证实验证实，该mRNA具有免疫刺激作用，当包裹LNP构成mRNA-载体颗粒时，免疫效果可与重组蛋白抗原相当，可用于制备mRNA疫苗。

Description

用于表达水痘-带状疱疹病毒抗原蛋白的mRNA及其用途 技术领域
本发明属于生物医药领域，具体涉及一种用于表达水痘-带状疱疹病毒抗原蛋白的mRNA、包含它的载体颗粒、重组质粒和免疫组合物，以及它们在制备预防和/或治疗水痘-带状疱疹病毒感染相关的疾病或病症的药物中的用途。
背景技术
带状疱疹是由水痘-带状疱疹病毒(varicella-zoster virus，VZV)引起的病毒性皮肤病。VZV原发感染时通过呼吸道黏膜上皮进入局部淋巴结复制，感染病毒的淋巴细胞随后经淋巴循环进入血液循环感染外周血单核白细胞，病毒随血流播散至皮肤，临床表现为水痘。水痘痊愈后，病毒潜伏在颅脑神经节复制，并沿外周神经移行至皮肤，临床表现为带状疱疹。带状疱疹的最主要并发症是疱疹感染后神经痛(post-herpetic neuralgia，PHN)。50岁以上的人群中约20％会发生PHN，并且随着年龄增大，出现PHN的可能性增高，疼痛持续数月甚至数年。
接种带状疱疹疫苗是预防带状疱疹，减轻PHN并发症等的有效手段。目前，已上市的带状疱疹疫苗主要有默沙东的Zostavax与葛兰素史克的Shingrix。其中，Zostavax于2006年上市，为减毒活疫苗；Shingrix于2017年10月上市，为重组带状疱疹疫苗。
mRNA疫苗作为第三代疫苗，可以诱导机体同时产生体液免疫和细胞免疫，通过多种机制来保护机体，并且由于其自身的特性，免疫后在转染的细胞的细胞质中能够很快被降解，从而降低了安全性风险。相比传统疫苗(减毒、灭活或重组亚单位等疫苗)，mRNA疫苗的生产工艺简单、无需细胞培养或动物源基质、合成速度快、成本低。相比同属核酸疫苗的DNA疫苗，mRNA疫苗发挥作用无需进入细胞核，没有整合至宿主基因组的风险，而且为一过性表达，半衰期可以通过修饰进行调整。
非复制型mRNA主要由5′非翻译区(untranslated region，UTR)、开放阅读框(open reading frame，ORF)、3′非翻译区、5′帽子结构(Cap)和100～250bp多聚腺苷酸尾巴〔Poly(A)〕组成，其中，5′- UTR和3′-UTR中的调节元件可稳定mRNA并增加抗原蛋白质翻译(黄慧媛，苗明三，朱艳慧等。基于脂质体的mRNA疫苗递送系统研究进展[J]。国际药学研究杂志，2019，46(5)：8)。基于此原理的开发的带状疱疹疫苗可见于如专利申请CN108472309A和CN114272369A等。
但是，如何筛选可以高效表达的核酸序列仍是本领域的技术难点。发明人据此进行了大量实验，以期开发药效好、安全性风险低、生产工艺简单、合成速度快、成本低的带状疱疹疫苗。
发明内容
基于上述现有技术的研究现状，本发明人对带状疱疹mRNA疫苗的各元件进行了深入研究，发现了适合用于制备带状疱疹mRNA疫苗的优势序列，由其制备的带状疱疹mRNA疫苗的抗原表达量高，且药效优势明显。
一方面，本发明提供了一种用于表达水痘-带状疱疹病毒抗原蛋白的mRNA，其从5′到3′方向依次包含5′非翻译区(5′UTR)、开放阅读框(ORF)、3′非翻译区(3′UTR)和多聚腺苷酸(polyA)尾，其中所述5′UTR的核苷酸序列如SEQ ID NO.23、24、25、26或27中任一项所示，并且所述开放阅读框编码水痘-带状疱疹病毒抗原蛋白。
作为本发明的一些实施方式，所述3′UTR的核苷酸序列如SEQ ID NO.28或29中任一项所示。
根据本发明，5′非翻译区是指成熟信使核糖核酸(mRNA)中位于编码区(CDS)上游的不被翻译为蛋白质的区域，其从转录起始位点开始，在起始密码子的前一个核苷酸处结束。真核生物的5′UTR的中位数长度一般约为150核苷酸(nt)，但有些可长达数千碱基。
根据本发明，3′非翻译区是指成熟mRNA编码区下游的一段不被翻译的序列，其在mRNA转运、稳定性和翻译调节中起重要作用。
根据本发明，多聚腺苷酸(polyA)是指位于mRNA中的150-200个腺苷酸残基，其能够保护mRNA，使其免受核酸外切酶攻击，并且对转录终结、将mRNA从细胞核输出以及进行翻译都十分重要。
根据本发明，开放阅读框(Open Reading Frame，ORF)是DNA序列中具有编码蛋白质潜能的序列，其为从起始密码子开始，结束于终止密码子连续的碱基序列。
作为本发明的几种实施方式，所述5′UTR的核苷酸序列如SEQ ID  NO.23所示，并且所述3′UTR的核苷酸序列如SEQ ID NO.28所示；所述5′UTR的核苷酸序列如SEQ ID NO.24所示，并且所述3′UTR的核苷酸序列如SEQ ID NO.28所示；所述5′UTR的核苷酸序列如SEQ ID NO.25所示，并且所述3′UTR的核苷酸序列如SEQ ID NO.28所示；所述5′UTR的核苷酸序列如SEQ ID NO.26所示，并且所述3′UTR的核苷酸序列如SEQ ID NO.28所示；或者所述5′UTR的核苷酸序列如SEQ ID NO.27所示，并且所述3′UTR的核苷酸序列如SEQ ID NO.29所示。
作为本发明的一种优选实施方式，所述5′UTR的核苷酸序列如SEQ ID NO.24所示，并且所述3′UTR的核苷酸序列如SEQ ID NO.28所示。
作为本发明的一种实施方式，所述水痘-带状疱疹病毒抗原蛋白为gE蛋白；优选地，所述gE蛋白的氨基酸序列如SEQ ID NO.1所示。根据本发明，gE蛋白是指水痘-带状疱疹病毒糖蛋白E。
作为本发明的一种实施方式，所述polyA尾的长度为80～120个核苷酸，优选为120个核苷酸。更优选地，所述polyA尾的核苷酸序列如SEQ ID NO.5所示。
作为本发明的一种实施方式，所述mRNA的核苷酸序列如SEQ ID NO.30、31、32、33或34中任一项所示。优选地，所述mRNA的核苷酸序列如SEQ ID NO.31所示。
作为本发明的一种实施方式，所述mRNA的5′端还具有帽结构。
优选地，所述5′端具有帽结构的mRNA如式(I)或式(II)所示：

其中，式(I)的分子式为C21H26N10O17P3 2-，式(II)的分子式为C22H28N10O17P3 +。
根据本发明，帽结构是指真核生物mRNA的5′端特有的帽子(cap)结构。帽子结构通常有三种类型(m7G5′ppp5′Np、m7G5′ppp5′NmpNp和m7G5′ppp5′NmpNmpNp)，分别称为O型、I型和II型。O型是指末端核苷酸的核糖未甲基化，I型是指末端一个核苷酸的核糖被甲基化，II型是指末端两个核苷酸的核糖均被甲基化。这种结构能够对抗5′-核酸外切酶的降解作用。在蛋白质合成过程中，它有助于核糖体对mRNA的识别和结合，使翻译得以正确起始。
另一方面，本发明提供了一种用于表达水痘-带状疱疹病毒抗原蛋白的载体颗粒，其包含根据本发明的mRNA和包封所述mRNA的载体材料。
根据本发明，mRNA载体颗粒是指用于将外源mRNA递送到细胞内的复合结构。有效的体内递送是mRNA疫苗(药物)实现治疗效果的重要要素。外源mRNA必须穿透脂质膜的屏障，才能进入细胞质，进而被成功翻译成功能性蛋白质。
作为本发明的一种实施方式，所述载体材料选自鱼精蛋白、脂质纳米颗粒(LNP)、高分子材料和无机纳米颗粒中的一种或多种。在优选的实施方式中，所述载体材料是LNP。
再一方面，本发明提供了一种用于表达水痘-带状疱疹病毒抗原蛋白的重组质粒，其从5′到3′方向依次包含5′非翻译区(5′UTR)、开放阅读框(ORF)、3′非翻译区(3′UTR)和多聚腺苷酸(polyA)尾，其中所述5′UTR的核苷酸序列如SEQ ID NO.9、11、12、13或14中任一项所示，并且所述开放阅读框编码水痘-带状疱疹病毒抗原蛋白。
作为本发明的一种实施方式，所述3′UTR的核苷酸序列如SEQ ID  NO.16或22中任一项所示。
作为本发明的一种优选实施方式，所述5′UTR的核苷酸序列如SEQ ID NO.11所示，并且所述3′UTR的核苷酸序列如SEQ ID NO.16所示。
作为本发明的一种实施方式，所述水痘-带状疱疹病毒抗原蛋白为gE蛋白；优选地，所述gE蛋白的核苷酸序列如SEQ ID NO.2所示。
作为本发明的一种实施方式，所述polyA尾的长度为80～120个核苷酸，优选为120个核苷酸。优选地，所述polyA尾的核苷酸序列如SEQ ID NO.5所示。
作为本发明的一种实施方式，所述质粒为pUC57质粒；优选地，所述重组质粒还包含起始序列(Ori)、T7启动子和抗性基因。
根据本发明，起始序列(Ori)是原核生物基因质粒的复制起始位点，是四个高度保守的19bp的正向重复序列。
根据本发明，T7启动子是专一受控于T7RNA聚合酶的功能强大且专一性高的启动子。
根据本发明，抗性基因即抗性的遗传因子，是选择基因的一种，属于标记基因。
作为本发明的一种优选实施方式，所述起始序列的核苷酸序列如SEQ ID NO.3所示。
作为本发明的一种优选实施方式，所述T7启动子的核苷酸序列如SEQ ID NO.4所示。
作为本发明的一种优选实施方式，所述抗性基因为硫酸卡那霉素抗性基因和/或氨苄青霉素抗性基因。优选地，所述硫酸卡那霉素抗性基因的核苷酸序列如SEQ ID NO.6所示；优选地，所述氨苄青霉素抗性基因的核苷酸序列如SEQ ID NO.7所示。
又一方面，本发明提供了一种水痘-带状疱疹病毒抗原蛋白，其由根据本发明的mRNA表达。
本发明还提供了一种水痘-带状疱疹病毒抗原蛋白，其由根据本发明的载体颗粒表达。
本发明还提供了一种水痘-带状疱疹病毒抗原蛋白，其由根据本发明的重组质粒表达。
还一方面，本发明提供了一种免疫组合物，其包含免疫有效量的根据本发明的mRNA或其表达的抗原蛋白。
本发明还提供了一种免疫组合物，其包含免疫有效量的根据本发明 的载体颗粒或其表达的抗原蛋白。
本发明还提供一种免疫组合物，其包含免疫有效量的根据本发明的重组质粒或其表达的抗原蛋白。
作为本发明的一种优选实施方式，所述免疫组合物为疫苗。
另一方面，本发明提供了根据本发明的mRNA或其表达的抗原蛋白在制备预防和/或治疗水痘-带状疱疹病毒感染相关疾病或病症的药物中的用途。
本发明还提供了根据本发明的载体颗粒或其表达的抗原蛋白在制备预防和/或治疗水痘-带状疱疹病毒感染相关疾病或病症的药物中的用途。
本发明还提供了根据本发明的重组质粒或其表达的抗原蛋白在制备预防和/或治疗水痘-带状疱疹病毒感染相关疾病或病症的药物中的用途。
作为本发明的一种实施方式，所述疾病或病症选自水痘、带状疱疹或疱疹感染后神经痛中的一种或多种。
根据本发明，水痘(varicella，chickenpox)是由水痘-带状疱疹病毒初次感染引起的急性传染病。
根据本发明，带状疱疹是由水痘-带状疱疹病毒引起的急性感染性皮肤病。由于病毒具有亲神经性，感染后可长期潜伏于脊髓神经后根神经节的神经元内，当抵抗力低下或劳累、感染、感冒时，病毒可再次生长繁殖，并沿神经纤维移至皮肤，使受侵犯的神经和皮肤产生强烈的炎症。
作为本发明的一种优选的实施方式，所述疾病或病症为带状疱疹。
本发明的实验结果表明，本发明筛选出的mRNA平均蛋白表达量较高，可达250ng/ml，其中最高蛋白表达量可以达到400ng/ml。对所述mRNA进行药效验证，结果证明本发明所提供的mRNA均具有免疫刺激作用。当其以脂质纳米颗粒(LNP)包裹构成mRNA-载体颗粒后，免疫效果可与重组蛋白抗原相当。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示了pUC57-kan-XgE-A质粒的结构，图1中各核酸片段分别是转录增强子(Transcript enhancer)，T7 promoter(T7启动子)，引导区(leader)，终止(stop)，改进gE546 ORF(gE546 ORF Improved)和起始序列(Ori)；
图2显示了由不同5′UTR和3′UTR的组合转录得到的mRNA的凝胶电泳结果；
图3显示了分别由14个mRNA表达得到gE蛋白的表达量。结果表明，01、05、07、09、12号mRNA得到的蛋白表达量均较高；
图4显示了05号mRNA在不同转染试剂下诱导免疫的效果；
图5显示了mRNA-载体颗粒诱导免疫的效果。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
本发明实施例中所用的化学药品购自常规商业途径。除具体说明的之外，所用生物材料均从商业途径获得。
实施例1 mRNA的制备
以人水痘-带状疱疹病毒gE蛋白的氨基酸序列(如SEQ ID NO.1所示)为基础设计其DNA编码序列，经密码子优化，设计得到的编码序列如SEQ ID NO.2所示。
在上述DNA编码序列的基础上增加5′UTR、3′UTR和polyA序列，通过同源重组插入至pUC57质粒中进行构建获得重组质粒pUC57-kan-XgE-A(XgE代表不同5′UTR和3′UTR组合的5′UTR-gE-3′UTR序列，A为polyA结构)。所述重组质粒包含起始序列(Ori)(SEQ ID NO.3)、T7启动子序列(SEQ ID NO.4)、5′UTR序列(分别如SEQ ID NO.8-15所示)，3′UTR序列(分别如SEQ ID NO.16-22所示)、3′端聚腺苷酸(polyA)序列(SEQ ID NO.5)，以及硫酸卡那霉素抗性基因(SEQ ID NO.6)(可替换为其他抗性基因，如氨苄青霉素抗性基因(SEQ ID NO.7))。
其中，重组质粒00包含SEQ ID NO.8所示的5′UTR序列和SEQ ID NO.16所示的3′UTR序列；
重组质粒01包含SEQ ID NO.9所示的5′UTR序列和SEQ ID NO.16所示的3′UTR序列；
重组质粒02包含SEQ ID NO.9所示的5′UTR序列和SEQ ID NO.17所示的3′UTR序列；
重组质粒03包含SEQ ID NO.10所示的5′UTR序列和SEQ ID NO. 16所示的3′UTR序列；
重组质粒04包含SEQ ID NO.10所示的5′UTR序列和SEQ ID NO.18所示的3′UTR序列；
重组质粒05包含SEQ ID NO.11所示的5′UTR序列和SEQ ID NO.16所示的3′UTR序列；
重组质粒06包含SEQ ID NO.11所示的5′UTR序列和SEQ ID NO.19所示的3′UTR序列；
重组质粒07包含SEQ ID NO.12所示的5′UTR序列和SEQ ID NO.16所示的3′UTR序列；
重组质粒08包含SEQ ID NO.12所示的5′UTR序列和SEQ ID NO.20所示的3′UTR序列；
重组质粒09包含SEQ ID NO.13所示的5′UTR序列和SEQ ID NO.16所示的3′UTR序列；
重组质粒10包含SEQ ID NO.13所示的5′UTR序列和SEQ ID NO.21所示的3′UTR序列；
重组质粒11包含SEQ ID NO.14所示的5′UTR序列和SEQ ID NO.16所示的3′UTR序列；
重组质粒12包含SEQ ID NO.14所示的5′UTR序列和SEQ ID NO.22所示的3′UTR序列；
重组质粒13包含SEQ ID NO.15所示的5′UTR序列和SEQ ID NO.16所示的3′UTR序列。
对pUC57-kan-XgE-A质粒的编码区及polyA区域进行测序，插入的目的基因测序与参考序列完全一致，重组鉴定为正确。构建成功的整个质粒结构如图1所示。
1.2转录和加帽
转录使用诺唯赞T7转录试剂盒，使用ARCA(抗反向帽类似物)直接在转录过程中在5′端加帽，于37℃条件下反应3h，产物立即保存于-80℃冰箱或用于转染细胞。gE蛋白DNA序列经体外转录后得到的mRNA序列分别如SEQ ID NO.30-34所示。使用凝胶电泳系统对转录得到的mRNA大小及完整性进行分析。结果如图2所示，图2显示各泳道均只有一条条带，说明转录的mRNA条带单一，无降解。
1.3 mRNA纯化
对1.2转录得到的mRNA进行纯化：将RNA纯化磁珠(诺唯赞)提 前从2～8℃取出，平衡至室温，然后通过颠倒或旋涡振荡使磁珠充分混匀；加入mRNA溶液充分混匀；室温下孵育5分钟，使mRNA结合到磁珠上。将样品置于磁力架，待溶液澄清后，小心移除上清；保持样品始终处于磁力架中，加200μl新鲜配制的80％乙醇漂洗磁珠；室温孵育30s，小心移除上清；重复上一步骤，总计漂洗2次；保持样品始终处于磁力架中，开盖，空气干燥磁珠5分钟。将样品从磁力架上取出，加入适量体积的无核苷酸酶水(Nuclease-free H2O)充分混匀，室温静置5分钟；将样品置于磁力架5分钟，待溶液澄清后转移上清至一个新的离心管(无核苷酸)。存于-80℃冰箱。
实施例2 mRNA的表达验证
将实施例1得到的14个mRNA分别翻译得到gE蛋白，制备步骤如下：体外表达检测使用293T细胞，将其铺到6孔板中，培养6h后，细胞量达到80～90％，去掉培养基(培养基由90％DMEM+10％FBS+1％PS组成)，换成无血清培养基2小时，转染时换成正常2％FBS-DMEM培养基，加入配制好的转染混合液，再于细胞培养箱培养24小时收细胞上清，使用Elisa方法测定其gE蛋白表达量。
表达结果见图3，实施例1中X为01、05、07、09和12的mRNA得到的蛋白表达量均较高，可达250ng/ml，其中X为05的mRNA得到的蛋白表达量最高，可以达到400ng/ml，后续药效实验验证了X为05的mRNA。
实施例3 mRNA疫苗的药效验证
使用24只雌性C57BL/6小鼠，共分为A-D 4组(6只/组)。A组和B组分别给予实施例1制备的X＝05的mRNA，20μl/只(1.5μg/μl)。此外，A组还给予转染试剂(HG-TransGeneTM transfection reagent)，20μl/只，B组还给予转染试剂(In vivo-jetRNA Transfection Reagent)，20μl/只。C组给予gE LNP-mRNA(将gE-mRNA包封在LNP中的LNP脂质纳米颗粒，包封率97.1％，收率81.5％)，20μg/只；D组给予gE抗原2μg、QS21脂质体1μg、CpG 20μg/只。各组均免疫1次，一免后一周终点时进行脾细胞分泌gE特异性IFN-γ的T淋巴细胞斑点数水平检测。其中，gE特异性肽库序列参考中国专利申请CN2020114564359。
药效结果见图4和图5。图4和5中A、B、C和D分别对应各给药 组，各组中gE代表的柱子为检测值，培养基代表的柱子为空白校正。图4和图5中结果显示：在转染试剂的辅助下，本发明提供的mRNA具有免疫刺激作用(图4)。为了提高核苷酸单体在体内的药效，以LNP包裹的mRNA的IFN-γ产生水平相较单体有了显著提高，效果可与重组蛋白抗原相当(图5)。
综上所述，本发明提供的适用于制备带状疱疹疫苗的mRNA优势序列的蛋白表达量高，药效优势明显，具有较好的应用前景。
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，所述修改和改变应归属于权利要求书的范围。

Claims (16)

  1. 一种用于表达水痘-带状疱疹病毒抗原蛋白的mRNA，其从5′到3′方向依次包含5′非翻译区、开放阅读框、3′非翻译区和多聚腺苷酸尾，其中所述5′非翻译区的核苷酸序列如SEQ ID NO.23、24、25、26或27中任一项所示，并且所述开放阅读框编码水痘-带状疱疹病毒抗原蛋白。
  2. 根据权利要求1所述的mRNA，其中，所述3′非翻译区的核苷酸序列如SEQ ID NO.28或29中任一项所示。
  3. 根据权利要求1或2所述的mRNA，其中，所述5′非翻译区的核苷酸序列如SEQ ID NO.23所示，并且所述3′非翻译区的核苷酸序列如SEQ ID NO.28所示；
    所述5′非翻译区的核苷酸序列如SEQ ID NO.24所示，并且所述3′非翻译区的核苷酸序列如SEQ ID NO.28所示；
    所述5′非翻译区的核苷酸序列如SEQ ID NO.25所示，并且所述3′非翻译区的核苷酸序列如SEQ ID NO.28所示；
    所述5′非翻译区的核苷酸序列如SEQ ID NO.26所示，并且所述3′非翻译区的核苷酸序列如SEQ ID NO.28所示；或者
    所述5′非翻译区的核苷酸序列如SEQ ID NO.27所示，并且所述3′非翻译区的核苷酸序列如SEQ ID NO.29所示；
    优选地，所述5′非翻译区的核苷酸序列如SEQ ID NO.24所示，并且所述3′非翻译区的核苷酸序列如SEQ ID NO.28所示。
  4. 根据权利要求1至3中任一项所述的mRNA，其中，所述水痘-带状疱疹病毒抗原蛋白为gE蛋白；优选地，所述gE蛋白的氨基酸序列如SEQ ID NO.1所示；
    优选地，所述多聚腺苷酸尾的长度为80～120个核苷酸，优选为120个核苷酸；更优选地，所述多聚腺苷酸尾的核苷酸序列如SEQ ID NO.5所示。
  5. 根据权利要求1至4中任一项所述的mRNA，其中，所述mRNA的核苷酸序列如SEQ ID NO.30、31、32、33或34中任一项所示；更优选地，所述mRNA的核苷酸序列如SEQ ID NO.31所示。
  6. 根据权利要求1至5中任一项所述的mRNA，其5’端还具有帽结构；
    优选地，所述5’端具有帽结构的mRNA如式(I)或式(II)所示：
  7. 一种用于表达水痘-带状疱疹病毒抗原蛋白的载体颗粒，其包含根据权利要求1至6中任一项所述的mRNA和包封所述mRNA的载体材料；
    优选地，所述载体材料选自鱼精蛋白、脂质纳米颗粒、高分子材料和无机纳米颗粒中的一种或多种；更优选地，所述载体材料为脂质纳米颗粒。
  8. 一种用于表达水痘-带状疱疹病毒抗原蛋白的重组质粒，其从5’到3’方向依次包含5’非翻译区、开放阅读框、3’非翻译区和多聚腺苷酸尾，其中所述5’非翻译区的核苷酸序列如SEQ ID NO.9、11、12、13或14中任一项所示，并且所述开放阅读框编码水痘-带状疱疹病毒抗原蛋白；
    优选地，所述3′非翻译区的核苷酸序列如SEQ ID NO.16或22中任一项所示。
  9. 根据权利要求8所述的重组质粒，其中，所述5′非翻译区的核苷酸序列如SEQ ID NO.11所示，并且所述3′非翻译区的核苷酸序列如SEQ ID NO.16所示。
  10. 根据权利要求8或9所述的重组质粒，其中，所述水痘-带状疱疹病毒抗原蛋白为gE蛋白；优选地，所述gE蛋白的核苷酸序列如SEQ  ID NO.2所示；
    优选地，所述多聚核苷酸尾的长度为80～120个核苷酸，优选为120个核苷酸；更优选地，所述多聚核苷酸尾的核苷酸序列如SEQ ID NO.5所示。
  11. 根据权利要求8至10中任一项所述的重组质粒，其中所述质粒为pUC57质粒；优选地，所述重组质粒还包含起始序列、T7启动子和抗性基因；
    优选地，所述起始序列的核苷酸序列如SEQ ID NO.3所示；
    优选地，所述T7启动子的核苷酸序列如SEQ ID NO.4所示；
    优选地，所述抗性基因为硫酸卡那霉素抗性基因和/或氨苄青霉素抗性基因；更优选地，所述硫酸卡那霉素抗性基因的核苷酸序列如SEQ ID NO.6所示；更优选地，所述氨苄青霉素抗性基因的核苷酸序列如SEQ ID NO.7所示。
  12. 一种水痘-带状疱疹病毒抗原蛋白，其由根据权利要求1至6中任一项所述的mRNA、根据权利要求7所述的载体颗粒和/或根据权利要求8至11中任一项所述的重组质粒表达。
  13. 一种免疫组合物，其包含免疫有效量的根据权利要求1至6中任一项所述的mRNA或其表达的抗原蛋白、根据权利要求7所述的载体颗粒或其表达的抗原蛋白和/或根据权利要求8至11中任一项所述的重组质粒或其表达的抗原蛋白。
  14. 根据权利要求13所述的免疫组合物，其为疫苗。
  15. 根据权利要求1至6中任一项所述的mRNA或其表达的抗原蛋白、根据权利要求7所述的载体颗粒或其表达的抗原蛋白和/或根据权利要求8至11中任一项所述的重组质粒或其表达的抗原蛋白在制备预防和/或治疗水痘-带状疱疹病毒感染相关疾病或病症的药物中的用途。
  16. 根据权利要求15所述的用途，其中所述疾病或病症选自水痘、带状疱疹或疱疹感染后神经痛中的一种或多种；
    优选地，所述疾病或病症为带状疱疹。
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