WO2023123517A1 - Vaccin à arncirc contre le virus de la nécrose infectieuse de la rate et des reins, son procédé de fabrication et son utilisation - Google Patents

Vaccin à arncirc contre le virus de la nécrose infectieuse de la rate et des reins, son procédé de fabrication et son utilisation Download PDF

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WO2023123517A1
WO2023123517A1 PCT/CN2021/144066 CN2021144066W WO2023123517A1 WO 2023123517 A1 WO2023123517 A1 WO 2023123517A1 CN 2021144066 W CN2021144066 W CN 2021144066W WO 2023123517 A1 WO2023123517 A1 WO 2023123517A1
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mcp
circrna
ires
necrosis virus
kidney necrosis
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PCT/CN2021/144066
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English (en)
Chinese (zh)
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贡成良
薛仁宇
胡小龙
朱敏
顾宇超
冯永杰
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苏州大学
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Priority to PCT/CN2021/144066 priority Critical patent/WO2023123517A1/fr
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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
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to the construction of a circRNA vaccine against infectious spleen and kidney necrosis virus.
  • common vaccines mainly include: inactivated vaccines, attenuated vaccines, subunit vaccines, synthetic peptide vaccines, recombinant live vector vaccines, DNA vaccines, and mRNA vaccines.
  • Different types of vaccines have their own advantages and disadvantages.
  • Traditional attenuated vaccines can activate humoral immunity and cellular immunity, but this type of vaccine has the potential risk of returning to pathogenicity and is less safe; compared with attenuated vaccines, subunit vaccines are safer, but induce cellular immunity Less effective in excluding intracellular pathogens; newer vaccine technologies based on viral vectors and nucleic acids (plasmid DNA and mRNA) induce humoral and cytotoxic T cell immune responses due to in situ expression of vaccine antigens.
  • mRNA Vaccines belong to nucleic acid vaccines and are the third generation of vaccines developed after live attenuated vaccines, inactivated vaccines and subunit vaccines. With the development of mRNA synthesis, modification and delivery technology, it has been confirmed that mRNA can play the role of vaccine after injection into animals. Because mRNA vaccines have no risk of integrating the host genome, no microbial contamination, can induce cellular and humoral immune responses, and have a short development time, low cost, and easy standardized production, they have become a hot spot in vaccine research and development in recent years. However, mRNA vaccines have poor stability and are easily degraded. They usually need cryopreservation and transportation, which greatly limits their application.
  • circRNA is a closed circular single-stranded RNA molecule without a 5'-end cap structure and a 3'-poly(A) tail. It is not easily degraded by RNase R and exonucleases. It has the advantages of mRNA vaccines and can complement mRNA vaccines. disadvantage.
  • Mandarin fish Siniperca chuatsi
  • largemouth bass Micropterus salmoides
  • Immunization technology has increasingly become a safe, effective and environmentally friendly new prevention and control technology in the prevention and control of fish diseases.
  • the inactivated vaccine for infectious spleen and kidney necrosis has obtained a new veterinary drug registration certificate (Class I).
  • the vaccine is obtained by infecting sensitive cell lines with viruses in vitro and then inactivating ISKNV. Due to the poor immune persistence of inactivated vaccines, the large dose of immunity will affect its use effect and coverage. Therefore, it is hoped that there will be a new type of vaccine for selective use in breeding production.
  • the inactivated vaccine against infectious spleen-kidney necrosis virus has poor immune persistence, and the immunization dose is large when used, which affects its use effect and coverage. Since the mRNA vaccine has no risk of integrating the host genome, it can induce cellular and humoral immune responses at the same time, and the research and development Short time, low cost, and easy standardized production have become a hot spot in vaccine research and development in recent years. However, the stability of mRNA vaccine is poor, and it is easy to degrade and affect the effect of use.
  • the purpose of the present invention is to provide a construction of a circRNA vaccine against infectious spleen and kidney necrosis virus, which can make up for the disadvantages of mRNA vaccines while giving full play to the advantages of mRNA vaccines, and provide a basis for the immune prevention and treatment of mandarin fish and largemouth bass infectious spleen and kidney necrosis. new options.
  • the technical scheme adopted in the present invention is: a method for constructing a circRNA molecule against infectious spleen and kidney necrosis virus, comprising the following steps: (1) Synthesizing the 5' end in order of in vitro transcription promoter, internal ribosome The fusion sequence of entry site IRES, N6-methyladenine m6A site, open reading frame of infectious spleen and kidney necrosis virus (P-IRES-m6A-MCP); (2) P-IRES-m6A-MCP sequence Cloned into the vector to obtain the recombinant plasmid pP-IRES-m6A-MCP; (3) Recover the P-IRES-m6A-MCP fragment after digesting pP-IRES-m6A-MCP or amplify it by PCR with pP-IRES-m6A-MCP template P-IRES-m6A-MCP fragment; (4) Use the P-IRES-m6
  • the specific construction method of the above-mentioned circRNA molecule against infectious spleen and kidney necrosis virus is as follows: (1) The 5' end of the synthesis is in vitro transcription promoter, internal ribosome entry site IRES, N6-methyladenine m6A position Point, the fusion sequence of the open reading frame of the main capsid protein MCP of infectious spleen and kidney necrosis virus ISKNV (P-IRES-m6A-MCP); (2) Cloning the sequence of P-IRES-m6A-MCP into the vector to obtain the recombinant plasmid pP-IRES-m6A-MCP; (3) Recover the P-IRES-m6A-MCP fragment after digesting pP-IRES-m6A-MCP, or obtain P-IRES-m6A by PCR amplification with pP-IRES-m6A-MCP template -MCP fragment; (4) Use the P-IRES-m6A-MCP fragment as
  • the in vitro transcription promoter is a T7 or SP6 promoter, and the sequences are SEQ ID NO:1, SEQ ID NO:2, described internal ribosome entry site IRES selects the internal ribosome entry site IRES of encephalomyocarditis virus, and its sequence is SEQ ID NO: 3; the N6-methyladenine m6A site is SEQ ID NO: 4; the open reading frame sequence of the main capsid protein MCP of infectious spleen and kidney necrosis virus ISKNV is Sequence in GenBank ID: sequence shown in AF370008.1.
  • the fusion sequence P-IRES-m6A-MCP can be chemically synthesized, or the IRES can be amplified from the IRES-containing plasmid by PCR, and the MCP coding sequence can be amplified from the ISKNV by PCR, and then passed through with primers containing the m6A sequence.
  • the P-IRES-m6A-MCP sequence is obtained by PCR bridging, and further, enzyme cutting sites can be added on both sides of the P-IRES-m6A-MCP sequence.
  • step (2) clones the P-IRES-m6A-MCP sequence into a vector, which is a conventional cloning vector, for example: pFasTBac TM Dual, pBlueScript II SK vector. If there are restriction sites on both sides of the P-IRES-m6A-MCP sequence, it can be cloned into the vector after digestion; if there are no restriction sites on both sides of the P-IRES-m6A-MCP sequence, it can be seamlessly cloned cloned into the vector.
  • a vector which is a conventional cloning vector, for example: pFasTBac TM Dual, pBlueScript II SK vector.
  • step (3) can use enzymes to cut out the P-IRES-m6A-MCP fragment from the pP-IRES-m6A-MCP plasmid in step (2), or use PCR to cut out the pP-IRES-m6A-MCP fragment from pP-IRES-m6A-
  • the P-IRES-m6A-MCP fragment amplified in MCP; step (4) using the P-IRES-m6A-MCP fragment as a template, using RNA polymerase to transcribe in vitro, and using DNase Digest and remove DNA to obtain in vitro transcribed RNA; when P-IRES-m6A-MCP fragments are transcribed in vitro, select the corresponding RNA polymer according to the type of promoter (T7 or SP6) contained in the P-IRES-m6A-MCP sequence enzymes for in vitro transcription; use DNase After digestion, it must be further extracted with phenol and chloroform, and then precipitated with ethanol to obtain
  • step (5) the RNA transcribed in vitro is ligated with RNA ligase, and further treated with RNase R to remove linear RNA molecules.
  • RNA ligase In order to obtain circular RNA with higher purity, it is preferable to extract it with phenol and chloroform, and then obtain circular RNA by ethanol precipitation.
  • the circular RNA is circRNA-MCP, which is a circRNA vaccine against infectious spleen and kidney necrosis virus.
  • the present invention discloses a circRNA vaccine against infectious spleen and kidney necrosis virus constructed according to the construction method of the above-mentioned circRNA vaccine against infectious spleen and kidney necrosis virus.
  • circRNA-MCP and appropriate adjuvants for example: liposome
  • the immunized fish can prevent the occurrence and prevalence of infectious spleen and kidney necrosis.
  • the invention discloses the application of the above-mentioned circRNA vaccine against infectious spleen and kidney necrosis virus in the preparation of vaccines against infectious spleen and kidney necrosis virus; or the application of the above-mentioned circRNA vaccine against infectious spleen and kidney necrosis virus in the preparation of anti-infectious spleen and kidney necrosis virus Application of viruses in medicine.
  • the present invention discloses the construction of a circRNA vaccine against infectious spleen and kidney necrosis virus, including claim 2 and an anti-infectious spleen and kidney according to claim 1
  • the construction of the circRNA vaccine of necrotic virus, the in vitro transcription promoter is T7 or SP6 Promoter; T7 promoter sequence is SEQ ID NO:1, T7 promoter sequence is SEQ ID NO: 2; including claim 3
  • the internal ribosome entry site IRES is the internal ribosome entry site IRES of encephalomyocarditis virus , whose sequence is SEQ ID NO: 3; including claim 4 according to the construction of a circRNA vaccine against infectious spleen and kidney necrosis virus according to claim 1
  • the N6-methyladenine m6A site is SEQ ID NO: 4;
  • the present invention has the following advantages compared with the prior art: 1.
  • the construction of a circRNA vaccine against infectious spleen and kidney necrosis virus disclosed by the present invention has not been reported; the circRNA vaccine disclosed by the present invention is a A nucleic acid vaccine.
  • circRNA vaccines Compared with DNA vaccines, circRNA vaccines have no security risks of being integrated into the genome, have high biological safety, and can directly translate antigenic proteins to cause immune responses without going through the transcription process; compared with mRNA vaccines, circRNA vaccines have a stable structure, Anti-RNase The degradation caused by R enzymes and exonucleases, while having the advantages of mRNA vaccines, can also make up for the disadvantages of mRNA vaccines.
  • the existing anti-infectious spleen and kidney necrosis virus vaccine is an inactivated vaccine, which can only stimulate the body's humoral immunity, and the duration of immunity is short, so there are problems such as unsatisfactory immune effect and poor immunity persistence.
  • CircRNA vaccines can induce cellular and humoral immune responses, and the development time is short, the cost is low, and it is easy to standardize production.
  • Fig. 1 is the enzyme digestion identification of the recombinant plasmid pFAST TM Dual-IRES-m6A-MCP in Example 1.
  • Lane M standard DNA molecular weight, lane 1, pFAST TM Dual-IRES-m6A-MCP double-digested product with Bam HI and Xba I.
  • FIG. 2 is a Western blot detection of circRNA-MCP expressing MCP in EPC cells in Example 1. After 2 ⁇ 10 5 fish EPC cells were transfected with circRNA-MCP for 48 h, the cells were collected, the total protein was extracted, separated by SDS-PAGE, and then detected by Western blot. The primary antibody was mouse anti-MCP, and the secondary antibody was HRP-labeled goat anti-mouse IgG. The internal reference was ⁇ -Tubulin. Lane 1, normal fish EPC cells; lane 2, EPC cells transfected with 4 ⁇ g circRNA-MCP; lane 3, EPC cells transfected with 8 ⁇ g circRNA-MCP.
  • Fig. 3 is the tissue immunohistochemical detection of MCP gene expression in Example 1.
  • 50 ⁇ g circRNA-MCP immunized perch (length 8-12 cm, body weight 35-55 g)
  • the control group was injected with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • kidney tissue was collected for immunohistochemical experiments.
  • the primary antibody was MCP antibody
  • the secondary antibody was FITC-labeled goat anti-mouse IgG. Nuclei were stained with DAPI.
  • Figure 4 is the titer detection of the immunization injection of circRNA-MCP induced perch to produce antibodies in Example 1.
  • 50 ⁇ g circRNA-MCP was injected into immunized sea bass (length 8-12 cm, body weight 35-55 cm) g)
  • immunized sea bass length 8-12 cm, body weight 35-55 cm
  • 3 perch were randomly selected, blood was collected from the tail vein, and the antibody titer was detected by ELISA.
  • Figure 5 is the circRNA-MCP PCR identification in Example 2.
  • Lane M standard molecular weight DNA, lane 1, linear molecule of circRNA-MCP; lane 2, circRNA-MCP.
  • Figure 6 shows the expression of MCP in fish EPC cells by the cellular immunofluorescence circRNA-MCP in Example 2.
  • 1 ⁇ 10 4 EPC cells were transfected with 4 ⁇ g of circRNA-MCP, and immunofluorescence experiments were performed 48 hours later.
  • the primary antibody was a self-made MCP mouse polyclonal antibody, and the secondary antibody was FITC-labeled goat anti-mouse IgG. Nuclei were stained with DAPI.
  • Fig. 7 is the qRT-PCR detection of circRNA-MCP in spleen and kidney tissues of immunized perch in Example 2.
  • the left picture is the spleen tissue, and the right picture is the kidney tissue; control is the control group, injected with phosphate buffer; Circ-MCP is the experimental group, injected with circRNA-MCP.
  • the 5' end of the synthesis is the internal ribosome entry site IRES of encephalomyocarditis virus (SEQ ID NO:3), N6-methyladenine m6A site (SEQ ID NO:4), infectious spleen and kidney necrosis virus
  • IRES internal ribosome entry site
  • SEQ ID NO:3 encephalomyocarditis virus
  • SEQ ID NO:4 N6-methyladenine m6A site
  • infectious spleen and kidney necrosis virus The fusion sequence of the open reading frame of the main capsid protein MCP of ISKNV (Sequence ID:AF370008.1), and the T7 promoter sequence (SEQ ID NO:1) was added at the 5' end to obtain the fusion sequence T7-IRES-m6A-MCP , cloned into the Bam HI and Xba I of the pFastBac TM Dual vector (Novagen Company) and then sequenced for verification, and the
  • T7-IRES-m6A-MCP fragment as a template, use T7 RNA polymerase for in vitro transcription, then digest the DNA template with DNaseI, and further extract with phenol/chloroform to remove T7 RNA polymerase, followed by ethanol precipitation to obtain pure in vitro transcribed RNA.
  • RNA-MCP circRNA-MCP was obtained by ethanol precipitation , the sequence is SEQ ID NO:5, Xho ⁇ Eco R Sites can be replaced with other restriction sites or removed from the sequence.
  • circRNA-MCP and liposome mixture (5 ⁇ g: 1 ⁇ L) were injected into perch (length 8-12 cm, body weight 35-55 g), each tail was injected with 50 ⁇ g circRNA-MCP.
  • 3 perch were randomly selected at the 1st, 2nd, 3rd, and 4th week respectively, and blood was collected.
  • the antibody titer produced by the serum was determined by ELISA ( Figure 4). The antibody titer in the serum was 1:1600, and the antibody titer in the perch serum in the fourth week after injection was 1:800.
  • step (8) On the 28th day of perch immunization with circRNA-MCP, the infectious spleen and kidney necrosis virus solution in step (8) was artificially inoculated, and a blank control group was also set up (200 ⁇ l of phosphate buffer solution per tail). The injected fish were raised at 27-29°C and fed 3 times a day to observe the activity of perch and check and record the disease.
  • the incidence rates of the blank control group and the immune group were 70% and 50%, respectively, and the incidence rate of the immune group was 20% lower than that of the control group.
  • Example 2 A circRNA vaccine against infectious spleen-kidney necrosis virus was constructed based on in vitro transcription of SP6 RNA polymerase.
  • the 5' end synthesized according to SEQ ID NO:5 is the internal ribosome entry site IRES of encephalomyocarditis virus (SEQ ID NO:3), the N6-methyladenine m6A site (SEQ ID NO:4), The fusion sequence of the open reading frame of the main capsid protein MCP of infectious spleen and kidney necrosis virus ISKNV (Sequence ID:AF370008.1), and the SP6 promoter sequence (SEQ ID NO:2) was added at the 5' end to obtain the fusion sequence SP6 -IRES-m6A-MCP, cloned into the Bam HI and Xba I of the pBlueScript II SK(+) vector (Youbao Biological Co., Ltd.) for sequencing verification, and the clone with the correct sequence was named pBlue-IRES-m6A-MCP.
  • RNA-MCP-F divergent primers circMCP-F (SEQ ID NO: 7) respectively Perform PCR amplification with circMCP-R (SEQ ID NO:8), and the amplified product is subjected to agarose gel electrophoresis.
  • the former has no specific PCR product, and the latter can amplify a band consistent with the theoretical size ( Figure 5) , indicating that circRNA-MCP has been constructed as designed.
  • the actin gene was used as an internal reference, and the quantitative primer for the expression level of the internal reference gene was ⁇ -actin-1 (SEQ ID NO:11) and ⁇ -actin-2 (SEQ ID NO:12), the results are shown in Figure 7, indicating that after immunization, circRNA-MCP enters spleen and kidney tissues.
  • the incidence rates of the blank control group and the immune group were 75% and 45%, respectively, and the incidence rate of the immune group was 30% lower than that of the control group.
  • the DNA sequence is: SEQ ID NO: 1: TAATACGACTCACTATAGG.
  • SEQ ID NO: 2 ATTTAGGTGACACTATAGAA.
  • SEQ ID NO: 4 TCTGGACTAAAGCCGGACTTGT.
  • SEQ ID NO: 6 TTACAGGATAGGGAAGCCTGC.
  • SEQ ID NO: 7 TACACGGTGGCCCAAAAGTT.
  • SEQ ID NO: 8 ACACCGGCCTTATTCCAAGC.
  • SEQ ID NO: 9 CAGGCGTTCCAGAAGTCAAGG.
  • SEQ ID NO: 10 CGTGAGACCGTGCGTAGTTC.
  • SEQ ID NO: 11 CCCAGAGCAAGAGAGGTATC.
  • SEQ ID NO: 12 GCTGTGGTGGTGAAGGAGTAG.

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Abstract

La présente invention concerne un vaccin à ARNcirc contre un virus de la nécrose infectieuse de la rate et des reins, ainsi qu'un procédé de construction et son utilisation. Le procédé consiste à : synthétiser une séquence de fusion P-IRES-m6A-MCP avec un promoteur de transcription T7 (ou S6) in vitro, un site d'entrée interne du ribosome IRES d'un virus de l'encéphalomyocardite, un site N6-méthyladénine (m6A) et un cadre de lecture ouvert d'une protéine de capside majeure (MCP) d'un virus de la nécrose infectieuse de la rate et du rein (ISKNV) successivement à l'extrémité 5', soumettre l'ARN à une transcription in vitro avec une transcriptase T7 (ou S6) en utilisant P-IRES-m6A-MCP comme matrice, en éliminant l'ADN matrice avec de la DNaseI, puis en effectuant une liaison et une cyclisation avec une ARN ligase T4, en éliminant l'ARN linéaire avec une RNase R, et enfin en obtenant un ARN circulaire, c'est-à-dire un ARNcirc-MCP. L'immunisation avec l'ARNcirc-MCP permet de réduire l'incidence de la nécrose infectieuse de la rate et des reins.
PCT/CN2021/144066 2021-12-31 2021-12-31 Vaccin à arncirc contre le virus de la nécrose infectieuse de la rate et des reins, son procédé de fabrication et son utilisation WO2023123517A1 (fr)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
US20190345503A1 (en) * 2016-06-20 2019-11-14 The Board Of The Leland Stanford Junior University Circular rnas and their use in immunomodulation
WO2021041541A1 (fr) * 2019-08-28 2021-03-04 The Board Of Trustees Of The Leland Stanford Junior University Arn circulaires modifiés et leurs procédés d'utilisation
CN113476597A (zh) * 2021-07-26 2021-10-08 苏州培恩特生物科技有限公司 一种抗传染性脾肾坏死病毒的载体疫苗的构建方法及应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190345503A1 (en) * 2016-06-20 2019-11-14 The Board Of The Leland Stanford Junior University Circular rnas and their use in immunomodulation
WO2021041541A1 (fr) * 2019-08-28 2021-03-04 The Board Of Trustees Of The Leland Stanford Junior University Arn circulaires modifiés et leurs procédés d'utilisation
CN113476597A (zh) * 2021-07-26 2021-10-08 苏州培恩特生物科技有限公司 一种抗传染性脾肾坏死病毒的载体疫苗的构建方法及应用

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OBI PRISCA; CHEN Y. GRACE: "The design and synthesis of circular RNAs", METHODS, ACADEMIC PRESS, NL, vol. 196, 2 March 2021 (2021-03-02), NL , pages 85 - 103, XP086875211, ISSN: 1046-2023, DOI: 10.1016/j.ymeth.2021.02.020 *

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