Circular RNA vaccine for influenza viruses

A circular and vaccine technology, applied in the field of circular RNA vaccines, can solve the problems of the advent of mRNA vaccines and poor stability of mRNA

Active Publication Date: 2020-12-25
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to reasons such as poor mRNA stability, no successful mRNA vaccine has yet been developed.

Method used

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  • Circular RNA vaccine for influenza viruses
  • Circular RNA vaccine for influenza viruses
  • Circular RNA vaccine for influenza viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, construction recombinant plasmid

[0042] Through the analysis of a large number of strain sequences, the H1N1-WSN-HA antigen was truncated and modified, and multiple point mutations were introduced based on virus variation to integrate H1N1-M2e antigen, H3N2-M2e antigen, H5-M2e (H5N1, H5N6) antigen, H7N9- For the M2e antigen and the H9N2-M2e antigen, the segments are connected by GGGGS, and the protein shown in sequence 1 of the sequence table is designed. The interleukin-2 signal peptide is added to the N-terminal of the protein shown in sequence 1 in the sequence listing, and the Flag tag is added to the C-terminal to design the protein shown in sequence 2 in the sequence listing.

[0043] Through codon optimization, the DNA molecule shown in sequence 3 of the sequence listing is obtained, which encodes the protein shown in sequence 2 of the sequence listing. The IRES sequence is added downstream of the DNA molecule shown in sequence 3 in the sequence ...

Embodiment 2

[0046] Embodiment 2, preparation circularized RNA

[0047] 1. Take the recombinant plasmid IAV-circmRNA-1.0-pBluescript II KS(+), digest it with restriction endonuclease XhoI, and recover the linearized plasmid.

[0048] 2. Transcribing RNA

[0049] Reaction system (20 μl): 1 μg linearized plasmid, 2 μl NTP mixture, 2 μl 10× reaction buffer and T7 transcriptase, the balance is DEPC water. The active ingredients provided by the NTP mix are ATP, CTP, GTP and UTP. In the reaction system, the concentrations of ATP, CTP, GTP and UTP are all 100mM, and the content of T7 transcriptase is 100U. NTP mixture: Beijing Shengke Boyuan Biotechnology Co., Ltd., catalog number 70906. 10× reaction buffer: Shanghai Yuanmu Biotechnology Co., Ltd., catalog number YM-MY162J. T7 transcriptase: Beijing Biolab Biotechnology Co., Ltd., catalog number JN0010.

[0050] Reaction conditions: 37°C water bath, 16h.

[0051] 3. After completing step 2, use QIAGEN's RNA purification kit for purification ...

Embodiment 3

[0062] Example 3. Preparation of HA2-M2e protein using HA2-M2e circ mRNA

[0063] 1. Take the recombinant plasmid IAV-circmRNA-1.0-pBluescript II KS(+), digest it with restriction endonuclease XhoI, and recover the linearized plasmid.

[0064] 2. Transcribed and purified RNA

[0065] Take step 1 to get the linearized plasmid, use HiScribe TM T7 High Yield RNA Synthesis Kit (New England Biolabs (UK) Ltd) was used for in vitro transcription, and the specific operation steps were carried out according to the instructions.

[0066] 3. Take the product of step 2, and use RNeasy Mini Kit (QIAGEN) to purify to obtain purified RNA.

[0067] 4. Cyclization identification

[0068] Method is the same as step 6 of embodiment 2.

[0069] Electropherogram see figure 1 Lane 2 of , shows a target band of about 250bp, indicating that circularized RNA was obtained.

[0070] Therefore, the purified RNA obtained in step 3 was named HA2-M2e circ mRNA.

[0071] 5. In vitro expression identif...

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Abstract

The invention discloses a circular RNA vaccine for influenza viruses. The invention provides a circular RNA molecule (HA2-M2e circ mRNA). The circular RNA molecule is shown as a sequence 8 in a sequence table. The invention also provides a protein (HA2-M2e circ mRNA translated protein), which is shown as a sequence 2 in the sequence table. The invention also protects a coding gene of the protein.The invention also protects a DNA molecule containing the coding gene. The circular RNA molecule, the protein and the gene can be used for preparing vaccines with broad spectrum on various influenza viruses. The invention has important application and popularization values for prevention and control of influenza viruses.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a circular RNA vaccine of influenza virus. Background technique [0002] Messenger RNA, that is, messenger RNA (mRNA), is transcribed from the DNA template strand, and its sequence is the same as that of the coding strand, and is complementary to the template strand. Unlike prokaryotes, the mRNA carrying genetic information in eukaryotes is composed of protein-coding exons and introns without coding functions. Only mature mRNA that has been correctly modified and spliced ​​can be transported into the cytoplasm as an information template for further translation to produce proteins. [0003] Circular RNA, or circular RNA (circRNA), is a type of endogenous non-coding RNA that exists widely in prokaryotic and eukaryotic cells. Circular RNAs in eukaryotic cells originate from back-splicing of pre-mRNA. The circRNAs discovered so far can be divided into the following three c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/44C07K14/11A61K39/145A61P31/16
CPCC12N15/113C07K14/005A61K39/12A61P31/16C12N2310/532C12N2760/16122C12N2760/16134A61K2039/53
Inventor 刘文军李晶闵洁白志华
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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