Nucleotide sequence for encoding novel coronavirus antigen and application of nucleotide sequence

A nucleotide sequence, coronavirus technology, applied in the field of vaccines

Active Publication Date: 2021-04-09
ADVACCINE SUZHOU BIOPHARMACEUTICALS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] These data show that the new coronavirus is still a serious threat to human health, and there is currently no effective prevention and treatment for such diseases. The development of effective preventive vaccines is an effective means to alleviate the epidemic in addition to physical isolation

Method used

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  • Nucleotide sequence for encoding novel coronavirus antigen and application of nucleotide sequence
  • Nucleotide sequence for encoding novel coronavirus antigen and application of nucleotide sequence
  • Nucleotide sequence for encoding novel coronavirus antigen and application of nucleotide sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Optimization of S protein-encoding nucleic acid and vector construction

[0044] 1. The principle of nucleic acid sequence optimization is: (1) optimize the degenerate codons according to the host cell’s preference for nucleic acid codons, so that the optimized sequence contains more nucleic acid codons that are conducive to host cell recognition; On the basis of codon preference optimization, further optimize the GC content in the nucleic acid sequence, so that the GC content-optimized sequence can express more target proteins; (3) optimize the nucleic acid sequence so that it can transcribe more stable mRNA, which is beneficial to the target protein. (4) Change the codon frequency of host preference and increase the CAI index (codon adaptation index).

[0045] Optimization purpose: to increase the protein expression of the target protein in host cells.

[0046] Optimization strategy: optimize the third base of the amino acid coding codon from A to C or G, ...

Embodiment 2

[0051] Embodiment 2: Construction of nucleic acid vaccine (sequence after each optimization)

[0052] 1. Nucleic acid vaccine candidate sequence transformation

[0053] (1) Take 100 μL of competent cell (such as DH5α) suspension from a -70°C refrigerator and thaw on ice.

[0054] (2) Add the plasmid DNA solutions prepared in Example 1 (volume no more than 10 μL): pVAX1-S (WT), pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV402 into competent cells, shake gently , placed on ice for 30 minutes.

[0055] (3) Heat shock in a water bath at 42°C for 90 seconds, then quickly place on ice to cool for 5 minutes.

[0056] (4) Add 1 mL of LB liquid culture medium (without antibiotics) to the tube, mix well and incubate with shaking at 37°C for 45 minutes to restore the bacteria to normal growth state.

[0057] (5) Shake the above-mentioned bacterial liquid and apply 100 μL on a screening plate containing appropriate antibiotics, place it face up, and invert the culture dish after the bacteri...

Embodiment 3

[0072] Example 3: Identification of Candidate Nucleic Acid Vaccine Mammalian Cell Transcription

[0073] 1. In vitro transfection of nucleic acid vaccine

[0074] (1) Take out the frozen HEK293 cell line (ATCC, CRL-3216) from liquid nitrogen, centrifuge at 1000rpm for 5min after 37℃ water bath to remove DMSO;

[0075] (2) Add serum-free DMEM culture solution to wash once, in 5 mL of DMEM culture solution containing 10% calf serum, 37°C, 5% CO 2 nourish. 24 hours before transfection, cells were digested with trypsin (0.25%) at 37°C for 5 minutes and terminated, with (1-3)×10 5 The density of cells / well was spread on 6-well plate or 35mm culture dish, add 2mL growth medium and 37℃, 5%CO 2 Cultivate in the incubator for 20-24 hours until the cell density is 50-80%;

[0076] (3) Simultaneously transform pVAX1-ADV400, pVAX1-ADV401 and pVAX1-ADV40, wild-type pVAX1-S (WT), and empty plasmid pVAX1 for parallel experiments. Specifically, take 2 μg of the aforementioned 5 plasmids, ...

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Abstract

The invention provides a nucleotide sequence for encoding a novel coronavirus antigen and application of the nucleotide sequence. According to the nucleotide sequence, a wild-type DNA sequence for encoding SARS-CoV-2 virus surface protein Spike is optimized, a wild-type gene signal peptide is optimized and then inserted into an eukaryotic expression vector, the eukaryotic expression vector is introduced into a host cell, a virus Spike antigen is efficiently expressed in the host cell, and an antiviral humoral immune response and a cellular immune response are systematically activated after antigen presentation. The antibody generated by the activated humoral immune response can prevent invasion of the SARS-CoV-2 virus, and the activated cellular immune response can further remove cells infected by the virus and adjust adverse reactions caused by potential side effects of ADE.

Description

technical field [0001] The present invention relates to the technical field of vaccines, in particular to a nucleotide sequence encoding a novel coronavirus antigen and its application, specifically in the preparation of vaccines. Background technique [0002] Coronavirus (Coronaviruses) is an enveloped non-segment positive RNA virus, belonging to Coronaviridae (Coronaviridae) and non-segment virus order (Nidovirales), and is currently the largest known positive-strand RNA virus. According to the serological and genomic characteristics of the virus, the coronavirus subfamily can be divided into four genera: α, β, γ, and δ, among which β-coronaviruses are further divided into four lines: A, B, C, and D. The new coronavirus SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) belongs to the β genus of coronavirus, mainly transmitted through respiratory droplets, and can also cause pneumonia (Novel Coronavirus-infected Pneumonia, NCP) through contact transmission, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/50A61K39/215A61P31/14
CPCC07K14/005A61K39/12A61P31/14C12N2800/22C12N2770/20022C12N2770/20034A61K2039/53
Inventor 俞庆龄赵干何悦张世杰侯佳望程鑫程渊江秉谕吴宗圣睢诚董爱华
Owner ADVACCINE SUZHOU BIOPHARMACEUTICALS CO LTD
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