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Novel coronavirus vaccine and application thereof

A coronavirus, a new type of technology, applied in the fields of genetic engineering and molecular biology, can solve problems such as high production costs, high drug costs for patients, and increased clinical risks

Active Publication Date: 2021-01-26
BEIJING NORTHLAND BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result, higher doses are required in clinical practice. On the one hand, it leads to high production costs, high drug costs and heavy burdens for patients; clinical risk

Method used

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  • Novel coronavirus vaccine and application thereof
  • Novel coronavirus vaccine and application thereof
  • Novel coronavirus vaccine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0090] Example 1. Construction of recombinant plasmids

[0091] For the sequence of the Spike protein used in this embodiment, refer to GenBank accession number NC_045512.2, wherein the 1-12th amino acid is the sequence of the signal peptide of the natural Spike protein (SEQ ID NO: 10), and the remaining amino acid sequences are the natural Spike protein The amino acid sequence (as shown in SEQ ID NO: 1, its encoding nucleotide sequence is shown in SEQ ID NO: 2). The 67 amino acids at the end of the natural Spike protein were removed to obtain a truncated Spike protein (the amino acid sequence of which is shown in SEQ ID NO: 3).

[0092] According to the codon preference of human cells, the nucleotide sequences encoding the native Spike protein and the truncated Spike protein were optimized to obtain the nucleotide sequences shown in SEQ ID NO:4 and 5.

[0093] Then, the nucleotide sequence (96 nucleotides) of coding HGF signal peptide (SEQID NO: 11) was connected respectiv...

Embodiment 2

[0100] Example 2. Production and detection of recombinant plasmids

[0101] In this example, construction and screening, fermentation and plasmid purification of conventional engineering bacteria were carried out. In short, the recombinant plasmids containing the sequences of SEQ ID NO: 6-9 were respectively transformed into host bacteria DH5α to obtain stable strains with high plasmid yields, which were preserved as engineering bacteria. The engineering bacteria were fermented, and the obtained bacterial liquid was centrifuged to collect the bacterial sludge, and purified according to the conventional plasmid purification method. After electrophoresis detection, the purity of the obtained four plasmids was greater than 95%, the supercoiled content was greater than 90%, and the ratio of OD260 to OD280 was greater than 1.8, indicating that the quality of the plasmids met the requirements. The specific test results are shown in the table below.

[0102] Table 4. Detection re...

Embodiment 3

[0105] Example 3. Detection of Spike protein expression

[0106] 1. HEK293T cells transfected with recombinant plasmids

[0107] (1) Cell preparation: take the HEK293T cells to be transfected, inoculate them in 24-well cell culture plates, 500 μl / well, and put them in 37°C, 5% CO 2 Cultivate overnight in an incubator so that the cell confluency is 90-95% on the day of transfection.

[0108] (2) Plasmid transfection: For each well of cells, use 50 μL DMEM serum-free medium to dilute 2 μL Lipofectamine 2000; for each well of cells, use 50 μl DMEM serum-free medium to dilute the plasmid, add 0.8 μg of each of the above four recombinant plasmids; room temperature After incubation for 5 minutes, the diluted DNA and diluted Lipofectamine2000 were mixed gently. And keep warm at room temperature for 20min. The experimental group was: medium + plasmid + transfection reagent; the negative control group was: medium + transfection reagent; the above experimental group and control gro...

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Abstract

The invention relates to a novel coronavirus vaccine and application thereof, in particular to a truncated Spike protein, a fusion protein containing the truncated Spike protein, a nucleic acid molecule containing a nucleotide sequence encoding the truncated Spike protein or the fusion protein, and a vector and a host cell containing the nucleic acid molecule. The invention further relates to a pharmaceutical composition containing the truncated Spike protein, fusion protein, nucleic acid molecule or vector.

Description

technical field [0001] This application relates to the fields of genetic engineering and molecular biology, specifically to the truncated 2019 novel coronavirus Spike protein, the fusion protein comprising the truncated Spike protein, and the nucleoside encoding the truncated Spike protein or fusion protein Nucleic acid molecules of acid sequences, and vectors and host cells comprising said nucleic acid molecules. In addition, the present application also relates to a pharmaceutical composition comprising the truncated Spike protein, fusion protein, nucleic acid molecule or carrier. Background technique [0002] Novel coronavirus pneumonia (COVID-19) is pneumonia caused by 2019 new coronavirus infection. With the spread of the epidemic, a large number of cases have appeared in many places around the world, posing a greater threat to people's lives and health. cause serious impact. [0003] For COVID-19, there is no specific therapeutic drug yet, so vaccine development has ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/165C07K19/00C12N15/50C12N15/62C12N15/63A61K39/215A61P31/14A61P11/00
CPCC07K14/005C12N15/63A61K39/12A61P31/14A61P11/00C12N2770/20022C12N2770/20023C12N2770/20034C12N2800/22C07K2319/02C07K2319/60C07K2319/61A61K2039/53
Inventor 梁明征马杉姗汤晓闯聂李亚马素永韩成权许松山
Owner BEIJING NORTHLAND BIOTECH
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