阅读说明：本技术 一种新型冠状病毒疫苗及其应用 (Novel coronavirus vaccine and application thereof ) 是由 董春升 熊思东 梁明龙 于 2020-05-14 设计创作，主要内容包括：本发明公开了一种新型冠状病毒疫苗及其应用。本发明在Balb/c小鼠模型中,给予小鼠肌肉注射或者滴鼻新型冠状病毒刺突蛋白S表达质粒pCov-S,能够诱导小鼠产生抗新型冠状病毒刺突蛋白S的抗体,体外实验证明此抗体具有中和作用。本发明在开发预防新型冠状病毒疫苗方面有重要意义。本发明在预防新型冠状病毒疫苗研发及生产中,具有重要的应用和推广价值。(The invention discloses a novel coronavirus vaccine and application thereof. In a Balb/c mouse model, the expression plasmid pCov-S of the novel coronavirus spike protein S is given to a mouse for intramuscular injection or nasal drip, and the antibody for resisting the novel coronavirus spike protein S can be induced to generate by the mouse. The invention has important significance in developing a novel coronavirus prevention vaccine. The invention has important application and popularization value in the research, development and production of novel coronavirus prevention vaccines.)

1. A novel coronavirus spike protein S expression plasmid pCov-S is formed by carrying a novel coronavirus spike protein S gene sequence shown as SEQ ID NO:1 through a eukaryotic expression plasmid vector.

2. The vaccine of claim 1, wherein the eukaryotic expression plasmid is pcDNA3.1^+、pCAGGS or p3 xFlag.

3. The application of the novel coronavirus vaccine in preparing the medicine for preventing the novel coronavirus is characterized in that the vaccine comprises a novel coronavirus spike protein S expression plasmid pCov-S, and the novel coronavirus spike protein S expression plasmid pCov-S is formed by loading a novel coronavirus spike protein S gene sequence shown as SEQ ID NO:1 through a eukaryotic expression plasmid vector.

4. The use according to claim 3, wherein the eukaryotic expression plasmid is pcDNA3.1^+、pCAGGS or p3 xFlag.

5. The use according to claim 3 or 4, wherein the novel coronavirus spike protein S gene sequence encodes the novel coronavirus spike protein S full-length protein sequence shown in SEQ ID NO. 2.

6. The use according to claim 5, wherein the vaccine is administered by intramuscular injection or nasal drop administration after chitosan encapsulation.

Technical Field

The invention belongs to the field of immunology and preparation of vaccines for viral infectious diseases, and particularly relates to a novel coronavirus vaccine and application thereof.

Background

The novel coronavirus (SARS-CoV-2) is mainly transmitted by respiratory droplets or close contact, and the virus is never popular in people, and has no immune barrier of people, so people in all ages are generally susceptible. At present, there is no specific antiviral medicine for SARS-CoV-2 and no clinical vaccine for preventing it, so that the research on SARS-CoV-2 vaccine is not slow.

Coronaviruses (Coronaviride) are a single-stranded positive-strand RNA virus, and the genome size is 27-32 kb. Among all known RNA viruses (e.g. influenza virus, poliovirus, HIV, etc.), the genome of coronaviruses is the largest. The virus is named because the envelope protein S in the shape of spike (spike) is contained on the capsule membrane of the virus, and the virus is similar to the big bulge of the pearl inlaid on the imperial crown in the structure of an electron microscope. Coronaviruses can be divided into a/β/γ/four genera, each with different strains, according to phylogenetic differences, with a/β genus coronavirus infecting mammals and a γ genus coronavirus infecting birds.

Recent sequence analysis showed that SARS-CoV-2 and bat-derived coronavirus RaTG13 isolated from clinical patients had 96% identity and SARS coronavirus was also 79.3% identity. The coronan genome structure is relatively conservative, and comprises 11 functional open reading frames, namely ORF1a, ORF1b, S, ORF 3, ORF 4a, ORF 4b, ORF 5, E, M, ORF 8b and N in sequence. Wherein ORF1a and ORF1b encode the two replicase enzymes PL2pro and 3CLpro, respectively, which are conserved in all coronaviruses. ORFs 3, 4a, 4b, 5 and 8b encode 5 accessory proteins of the virus, respectively. ORFs S, E, M and N encode 4 major structural proteins, Spike Protein (S), envelope Protein (E), Membrane Protein (M) and Nucleocapsid Protein (N). Wherein, the E protein is a transmembrane protein which forms an ion channel on the surface of the virus and is related to the virulence of the virus. The N protein is a major component of the viral genomic RNA replication complex and binds to the C-terminal domain of the M protein. The M protein is a protein that forms the shape of a coronavirus particle and is involved in the integration of viral components into the virion. The S protein protrudes out of the surface of the virion, is I-type transmembrane glycoprotein, is related to virion adsorption and membrane fusion, is reported to be a main immunogen for inducing an organism to generate a neutralizing antibody, and becomes an important target for development of MERS vaccines and genetic engineering drugs of middle east respiratory syndrome virus.

The most effective method for preventing SARS-CoV-2 from spreading in large area is vaccination. At present, all related vaccines are in a laboratory research stage, and no preventive vaccine can be used as a clinical drug and is approved to be used in the market.

Disclosure of Invention

The invention provides a novel coronavirus vaccine and application thereof, which play a certain role in preventing novel coronavirus infection and have important significance in the aspect of developing and preventing the novel coronavirus vaccine.

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, a novel coronavirus vaccine is provided, the vaccine comprises a novel coronavirus spike protein S expression plasmid pCov-S, and the novel coronavirus spike protein S expression plasmid pCov-S is formed by a eukaryotic expression plasmid vector carrying a novel coronavirus spike protein S gene sequence shown as SEQ ID NO: 1.

In some preferred embodiments, the eukaryotic expression plasmid is pcDNA3.1^+、pCAGGS or p3 xFlag.

In a second aspect, there is provided the use of a novel coronavirus vaccine comprising a novel coronavirus spike protein S expression plasmid pCov-S, in the preparation of a medicament for the prevention of a novel coronavirus, said novel coronavirus spike protein S expression plasmid pCov-S being formed by a eukaryotic expression plasmid vector carrying a novel coronavirus spike protein S gene sequence as set forth in SEQ ID NO: 1.

In some preferred embodiments, the eukaryotic expression plasmid is pcDNA3.1^+、pCAGGS or p3 xFlag.

In some preferred embodiments, the novel coronavirus spike protein S gene sequence encodes the novel coronavirus spike protein S full-length protein sequence shown in SEQ ID NO. 2.

In some preferred embodiments, the vaccine is administered by intramuscular injection or nasal drop administration after chitosan encapsulation.

The technical scheme provided by the embodiment of the invention has the following beneficial effects: the invention provides a novel coronavirus spike protein S expression plasmid pCov-S and application thereof, wherein the novel coronavirus spike protein S expression plasmid pCov-S comprises a plasmid DNA immune mouse, can induce a specific antibody of the novel coronavirus spike protein S and neutralize novel coronavirus spike protein S pseudovirus particles in vitro, prevents viruses from infecting cells, and plays a role in protection.

Drawings

FIG. 1 is a diagram of the construction of pCov-S plasmid in the first example;

FIG. 2 is a Western blot of S protein of 293T cells transfected with pCov-S plasmid in the first example;

FIG. 3 is a flow chart of 293T cells transfected with pCov-S plasmid in example one;

FIG. 4 is an ELISA test chart of serum S antibody of pCov-S immunized mouse in example two;

FIG. 5 is a schematic diagram of the preparation of SARS-Cov-2 pseudovirions in example three;

FIG. 6 is a diagram showing the verification of SARS-Cov-2 pseudovirions in example three;

FIG. 7 is the experimental picture of neutralizing SARS-Cov-2 pseudovirion in mouse immune serum of example four.

SEQ ID NO. 1 is the gene sequence of the novel coronavirus spike protein S;

2 is a novel coronavirus spike protein S full-length protein sequence;

SEQ ID NO 3 is the key amino acid sequence of the novel coronavirus spike protein S.

Detailed Description