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Expression vector, recombinant adeno-associated virus and application of recombinant adeno-associated virus in preparation of novel 2019 coronavirus vaccine

A coronavirus and expression vector technology, used in the preparation of 2019 new coronavirus vaccine applications, can solve the lack of long-acting research on AAV new crown vaccine and other problems, and achieve reduced adverse reactions, good stability, and antibody titers. high effect

Active Publication Date: 2022-01-11
BRAINVTA WUHAN CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] Compared with other types of vaccines, there are less studies on recombinant AAV-based COVID-19 vaccines at present, and there is a lack of long-acting research on AAV-based COVID-19 vaccines. There is an urgent need to develop a long-acting and stable vaccine against the new coronavirus SARS-CoV-2. AAV vector vaccine

Method used

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  • Expression vector, recombinant adeno-associated virus and application of recombinant adeno-associated virus in preparation of novel 2019 coronavirus vaccine
  • Expression vector, recombinant adeno-associated virus and application of recombinant adeno-associated virus in preparation of novel 2019 coronavirus vaccine
  • Expression vector, recombinant adeno-associated virus and application of recombinant adeno-associated virus in preparation of novel 2019 coronavirus vaccine

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preparation example Construction

[0063]The present invention also provides a preparation method of recombinant adeno-associated virus, comprising the following steps: co-incubating the above-mentioned expression vector, helper plasmid pHelper and serotype plasmid pRepCap, and transfecting host cells in the presence of transfection reagent polyethyleneimine After the cells are cultivated, the cells are collected by centrifugation, lysed and purified to obtain a purified solution containing recombinant adeno-associated virus.

[0064] In some embodiments, the serotype plasmid pRepCap is pRep2Cap6, wherein the nucleotide sequence of the cap gene is shown in SEQ ID No.7.

[0065] The preparation of the recombinant adeno-associated virus can adopt the methods known to those skilled in the art. In the embodiment of the present invention, HEK293T-based AAV helper-free system (AAVHelper-FreeSystem) is used for production, that is, the three-plasmid co-transfection method. The AAV helper-free system contains expressio...

Embodiment 1

[0075] Single-stranded AAV is used in the prior art to express the new crown vaccine molecule. This example verifies the difference in the expression levels of single- and double-stranded viruses in cells.

[0076] 1. Plasmid construction

[0077] Two kinds of rAAV plasmids, rAAV-CMV-mCHERRY-BGHpA (ID: PB2-0948) and pFD-scAAV-CMV-mCHERRY-BGHpA (ID: GT-0118), were constructed for the preparation of single-chain AAV and double-chain AAV, respectively.

[0078] 2. Virus packaging and titer detection

[0079] HEK293T cells were treated with 1.5×10 per dish 24 hours before transfection 6 The density of cells was inoculated in a 100mm culture dish, and the core plasmid pAAV-GOI (PB2-0948 or GT-0118): serotype plasmid pRepCap: helper plasmid pHelper=5μg: 10μg: 7.5μg and 45μL transfection reagent polyethylene glycol were added. Amine solution (PEI, 1 mg / mL) was used to incubate the transfection. After 72 hours of transfection, the cells were collected by centrifugation, lysed and pu...

Embodiment 2

[0086] Embodiment 2 Preparation of different antigen molecule carriers

[0087] The SARS-CoV-2 (2019-nCoV-WIV04) full-length S protein coding sequence used in this example is the nucleotide sequence shown in SEQ ID No. 1 commissioned by GenScript Company. The sequence was optimized with human codons and named 2019-HnCOV-S. Select the main antigen recognition epitope RBD region of S protein, as shown in SEQ ID No.2, and name it RBD; the amino acid sequence corresponding to this sequence is shown in SEQ ID No.3. On the basis of this sequence, the following signal peptide TPA and three targeting peptide sequences were designed, as shown in Table 1.

[0088] Table 1 Signal peptide and targeting peptide sequence information

[0089]

[0090] Wherein TPA is a secretion signal peptide, added to the N-terminal of the RBD sequence when the vector is constructed, and targeting peptides are respectively added to the C-terminal of the RBD sequence, and the targeting peptides used in ...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses an expression vector, a recombinant adeno-associated virus and application of the recombinant adeno-associated virus in preparation of a novel 2019 coronavirus vaccine. The expression vector comprises a target gene expression cassette and adeno-associated virus reverse terminal repetitive sequences located at the two ends of the target gene expression cassette, wherein the target gene expression cassette comprises promoters which are from 5' to 3' and can be connected in an operable mode, a nucleotide sequence for encoding TPA secretion signal peptide and a nucleotide sequence for encoding a coronavirus spike protein Receptor Binding Domain (RBD); the expression vector is used for preparing the recombinant double-stranded adeno-associated virus. The recombinant adeno-associated virus prepared by means of the expression vector can efficiently, stably and chronically secrete and express coronavirus RBD protein in vivo, induces generation of a serum neutralizing antibody, has a neutralizing effect on the novel 2019 coronavirus including a variant, achieves continuous expression and has good application prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to an expression vector, a recombinant adeno-associated virus and their application in preparing a 2019 novel coronavirus vaccine. Background technique [0002] Coronavirus (Coronavirus) is a class of enveloped single-stranded positive-strand RNA viruses, and it is the virus with the largest genome among known RNA viruses. At present, a total of 7 kinds of coronaviruses that can infect humans have been found, namely human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), Hong Kong type I human coronavirus (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and 2019 novel coronavirus (SARS-CoV-2). Among them, SARS-CoV, MERS-CoV and SARS-CoV-2 are highly pathogenic human coronaviruses discovered so far, which have brought great harm to ...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/50C12N7/01A61K39/215A61P31/14
CPCC12N15/86C12N7/00C07K14/005A61K39/12A61K9/0019A61K9/0043A61P31/14C12N2750/14143C12N2750/14121C12N2800/107C12N2800/22C12N2770/20022C12N2770/20034A61K2039/523A61K2039/543
Inventor 不公告发明人
Owner BRAINVTA WUHAN CO LTD
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