阅读说明：本技术 基于嵌合型病毒样颗粒的新型冠状病毒疫苗 (Novel coronavirus vaccine based on chimeric virus-like particles ) 是由 孙祥明 曹文龙 孔迪 滕小锘 张大鹤 易小萍 于 2020-06-17 设计创作，主要内容包括：本发明公开了一种基于嵌合型病毒样颗粒的新型冠状病毒疫苗,其包含作为有效成分的嵌合型病毒样颗粒。所述嵌合型病毒样颗粒主要由一类重组的嵌合蛋白聚集形成,所述重组嵌合蛋白是通过将SARS-CoV-2病毒的S蛋白受体结合区域嵌合到HBcAg的主要免疫决定区而获得。本发明的疫苗能够在人类体内产生较强免疫响应,免疫后人类能够抵御新型冠状病毒感染。(The invention discloses a novel coronavirus vaccine based on chimeric virus-like particles, which comprises the chimeric virus-like particles as an effective component. The chimeric virus-like particles are mainly formed by aggregation of a recombinant chimeric protein obtained by chimerizing the S-protein receptor-binding region of SARS-CoV-2 virus to the major immunodominant region of HBcAg. The vaccine of the invention can generate stronger immune response in human bodies, and the human bodies can resist the infection of novel coronavirus after immunization.)

1. A chimeric protein obtained by chimerizing the S protein receptor binding region of SARS-CoV-2 virus to the major immunodominant region of HBcAg; preferably, the chimeric protein comprises SEQ ID NO: 2 or an amino acid sequence corresponding to seq id NO: 2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

2. A gene encoding the chimeric protein of claim 1; preferably, the coding gene comprises a sequence shown in SEQ ID NO: 1 or a nucleic acid molecule substantially identical to SEQ ID NO: 1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

3. A recombinant vector comprising the encoding gene of claim 2; preferably, the recombinant vector comprises a pET-22b (+) vector.

4. A recombinant bacterium comprising the coding gene of claim 2 or the recombinant vector of claim 3; preferably, the recombinant bacterium comprises escherichia coli.

5. A chimeric virus-like particle expressed by the recombinant bacterium according to claim 4.

6. Use of the chimeric protein of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, the recombinant bacterium of claim 4, or the chimeric virus-like particle of claim 5 in the preparation of novel coronavirus detection reagents.

7. Use of the chimeric protein of claim 1, the encoding gene of claim 2, the recombinant vector of claim 3, the recombinant bacterium of claim 4, or the chimeric virus-like particle of claim 5 in the preparation of a medicament for the prevention and/or treatment of a novel coronavirus infection.

8. A novel coronavirus vaccine, characterized by: the active component of the vaccine comprises the chimeric virus-like particle of claim 5.

9. The vaccine of claim 8, wherein: the vaccine further comprises a pharmaceutically acceptable carrier.

10. The process for producing a chimeric virus-like particle according to claim 5, which comprises: transforming Escherichia coli with the recombinant vector containing the coding gene of claim 2, culturing, and post-treating to obtain the chimeric virus-like particle.

Technical Field

The invention relates to a genetic engineering vaccine, in particular to a novel coronavirus vaccine based on chimeric virus-like particles, a preparation method and application thereof, belonging to the field of human vaccines and human biological products.

Background

Coronavir pneumonia (COVID-19) is an Acute Respiratory infectious Disease caused by a novel Coronavirus (SARS-CoV-2, hereinafter also referred to as "new Coronavirus"). SARS-CoV-2 is mainly transmitted by droplets and contact, and has the possibility of feces transmission and aerosol transmission, which is common and susceptible to the population and serious illness after the infection of the old and the people with basic diseases. SARS-CoV-2 mainly invades alveolar epithelial cells, the incubation period after infection is about 1-14 days, the maximum length can reach 24 days, clinical symptoms mainly comprise fever, dry cough and dyspnea, and patients with severe disease can rapidly progress to acute respiratory distress syndrome, septic shock, metabolic acidosis and blood coagulation dysfunction which are difficult to correct, and the like. The lung CT abnormality exists in 76.4 percent of patients, the CT is mainly shown as bilateral frosty glass-like lesion, and severe patients can be shown as bilateral multiple lobules and lung segment consolidation. The World Health Organization (WHO) classified the epidemic as an emergent Public health event of International interest (PHEIC) on 31/1/2020. On day 11/3/2020, the current epidemic is considered by the WHO under evaluation to be known as a global pandemic. China brings COVID-19 into class B infectious diseases specified in infectious disease prevention and control Law of the people's republic of China, takes prevention and control measures of class A infectious diseases and brings the COVID-19 into quarantine infectious disease management.

SARS-CoV-2 is a positive-strand single-stranded RNA virus, genome length is about 30kb, and belongs to Togaviridae, Coronaviridae, β genus coronavirus, gene function research finds that SARS-CoV-25' segment is about 2/3 gene used for coding non-structural proteins, these non-structural proteins form multimers and perform replicase and translation functions, and the remaining about 1/3 gene encodes four structural proteins, including S Protein (Spike Protein), M Protein (membrane Protein), E Protein (Envelope Protein) and N Protein (nucleomapped Protein) (Chen Y, Liu Q, Guo D.Emerging coronaviruses: genome structure, reproduction, and pathogenesis [ J ] Protein]J MedVirol, 2020, 92 (4): 418-423.). The S protein is glycoprotein formed by 1273 amino acid residues, and mainly has the functions of mediating viruses to recognize host cell receptors, promoting membrane fusion and inducing immune response to generate neutralizing antibodies. The S protein consists of two subunits, S1 and S2, wherein the S1 subunit comprises 1 signal peptide, an N-terminal Domain (NTD) and a Receptor-binding Domain (RBD). Receptor binding region of the S protein (S)_RBD) Is a part directly combined with a host cell receptor and plays an important role in the processes of virus adsorption and entering host cells. It has been recently reported that SARS-CoV-2 can infect human, bat and pig through ACE2 receptor (Zhom P, Yang X L, Wang X G, et al. A pnemmomonia outbreaak associatedwith a new coronavirus of probable bat origin [ J]Nature, 2020, doi: 10.1038/S41S 86-020-. S_RBDThe sequence has two deletions, the non-deleted part of which can also bind ACE2 to invade cells (Chan JF, Kok K H, ZhmZ, et al genomic characterization of the 2019novel hmman-pathogenic coronavirus from a tissue with an active plasmid pnuemonia aft visitingWuhan [ J]Emerg Microbes infestations, 2020, 9 (1): 221-_RBDA certain specific sequence plays an important role in the binding process of the virus and ACE2 and the adsorption process of host cells. Thus, S_RBDMay be used as important action target point for developing SARS-CoV-2 resisting medicine and vaccine.

However, COVID-19 has no specific pharmaceutical application at present, and clinical treatment mainly aims at symptom and support. Vaccines against covi-19 are still in the development stage, and up to now, mainly rely on controlling the infectious agent and cutting off the transmission path to control the epidemic situation.

Disclosure of Invention

The invention mainly aims to provide a novel coronavirus vaccine, a preparation method and application thereof, so as to overcome the defects in the prior art.

In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:

the embodiment of the invention provides a chimeric protein, which is obtained by chimeric of the S protein receptor binding region of SARS-CoV-2 virus to the main immune determining region of HBcAg. Further, the chimeric protein comprises SEQ ID NO: 2 or an amino acid sequence corresponding to SEQ ID NO: 2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

The embodiment of the invention also provides a coding gene of the chimeric protein. Further, the coding gene comprises a sequence shown as SEQ ID NO: 1 or a nucleic acid molecule substantially identical to SEQ ID NO: 1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

The embodiment of the invention also provides a recombinant vector containing the coding gene.

The embodiment of the invention also provides a recombinant bacterium containing the coding gene or the recombinant vector.

The embodiment of the invention also provides the chimeric virus-like particle expressed by the recombinant bacterium.

The embodiment of the invention also provides application of the chimeric protein, the coding gene, the recombinant vector, the recombinant bacterium or the chimeric virus-like particle in preparing a novel coronavirus detection reagent or in preparing a medicament for preventing and/or treating novel coronavirus infection.

The embodiment of the invention also provides a novel coronavirus vaccine, and the active ingredient of the novel coronavirus vaccine comprises the chimeric virus-like particle. Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

The embodiment of the invention also provides a method for preparing the chimeric virus-like particle, which comprises the following steps: and transforming the recombinant vector containing the coding gene into escherichia coli, and culturing and post-treating to obtain the chimeric virus-like particle.

Compared with the prior art, the embodiment of the invention is realized by adding S of SARS-CoV-2_RBDChimeric to MIR Region (Major immune determinant) of HBcAg (hepatitis B virus core antigen), S can be displayed on the surface of HBcAg-forming virus-like particles_RBDThereby enhancing the immune system of the human body to S by virtue of the virus-like particle structure_RBDThe immune response of the vaccine can obtain good immune effect, so that immune people can obtain good immune protection, and the infection of novel coronavirus can be avoided.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is an SDS-PAGE pattern of a sample obtained in example 2 of the present invention;

FIG. 2 is an electron micrograph of a virus-like particle obtained in example 3 of the present invention;

FIG. 3 is a standard curve based on the test results of the kit standards in example 4 of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

One aspect of the embodiments of the present invention provides a chimeric protein obtained by chimeric binding of the S protein receptor binding region of SARS-CoV-2 virus to the major immunodominant region of HBcAg, and thus may also be referred to as S_RBDHBcAg chimeric antigen.

Preferably, in said S_RBDIn the/HBcAg chimeric antigen, S is added_RBDThe two sides of the antigen are respectively added with connecting peptide, and the C terminal is added with connecting peptide and 6 histidines, which greatly enhances the stability of the antigen particles.

Further, the chimeric protein comprises SEQ ID NO: 2 or an amino acid sequence corresponding to SEQ ID NO: 2, and an amino acid sequence which is 95% or more identical to the full-length amino acid sequence of the polypeptide.

In another aspect of the embodiments of the present invention, the encoding gene of the chimeric protein includes a sequence shown in SEQ id no: 1 or a nucleic acid molecule substantially identical to SEQ ID NO: 1, or a nucleic acid molecule having a nucleotide sequence that is 95% or more identical to the nucleotide sequence of 1.

In another aspect of the embodiments of the present invention, there is also provided a recombinant vector comprising the encoding gene.

Further, the recombinant vector includes, but is not limited to, pET-22b (+) vector and the like, and for example, other E.coli expression vectors (e.g., pET series vectors), animal cell expression vectors (e.g., pCDNA3.1), yeast cell expression vectors (e.g., pPIC9.0), baculovirus expression vectors (e.g., pFASTBA AC), and the like may also be used.

In another aspect of the embodiments of the present invention, there is also provided a recombinant bacterium comprising the coding gene or the recombinant vector.

Further, the recombinant bacteria can be selected from, but not limited to, escherichia coli and the like. For example, yeast, insect cell/baculovirus expression systems, mammalian cells such as CHO cells, plant cells, transgenic animals (e.g., mammary gland bioreactor), and the like can also be used.

In another aspect of the embodiments of the present invention, chimeric virus-like particles expressed by the recombinant bacteria are also provided.

Furthermore, the chimeric virus-like particles can be formed by aggregating the recombinant chimeric proteins, can rapidly induce the immune response of a human body, and can provide complete protection for the human body with only small dose when being used as a novel coronavirus vaccine.

In particular, in use, a safe and effective amount of the chimeric virus-like particles of the invention is administered to a mammal (e.g., a human), wherein the safe and effective amount is generally at least about 1 microgram per kilogram of body weight, and in most cases does not exceed about 10 milligrams per kilogram of body weight, preferably the dose is from about 1 microgram per kilogram of body weight to about 1 milligram per kilogram of body weight. Of course, the particular dosage will also take into account factors such as the route of administration, the health of the user, and the like, which are within the skill of the skilled practitioner.

The embodiment of the invention also provides application of the chimeric protein, the coding gene, the recombinant vector, the recombinant bacterium or the chimeric virus-like particle in preparing a novel coronavirus detection reagent.

The embodiment of the invention also provides application of the chimeric protein, the coding gene, the recombinant vector, the recombinant bacterium or the chimeric virus-like particle in preparing a medicament for preventing and/or treating novel coronavirus infection.

In another aspect of the embodiments of the present invention, there is provided a novel coronavirus vaccine, the active ingredient of which comprises the chimeric virus-like particle.

Further, the vaccine may further comprise a pharmaceutically acceptable carrier.

The pharmaceutically acceptable carriers refer to such pharmaceutical carriers as: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. For example, a full description of pharmaceutically acceptable carriers can be found in Remington's pharmaceutical Sciences (Mack pub. co., n.j.1991) and the like.

In the vaccine provided in the embodiments of the present invention, the pharmaceutically acceptable carrier may contain a liquid, such as water, saline, glycerin and sorbitol. In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin and the like, may also be present in these carriers.

In some embodiments, the pharmaceutically acceptable carrier may also contain other types of adjuvants such as immunostimulants, cell transfection reagents, and the like. Preferably, part of the adjuvant is produced by Suzhou Shino biotechnology, Inc. to improve the effect of the vaccine.

In embodiments of the invention, the vaccine may be formulated in a variety of dosage forms suitable for mammalian administration, including, but not limited to: injection, capsule, tablet, emulsion, and suppository; preferably an injection.

In another aspect of the embodiments of the present invention, there is provided a method for producing the chimeric virus-like particle, comprising: and transforming the recombinant vector containing the coding gene into escherichia coli, and culturing and post-treating to obtain the chimeric virus-like particle. Such post-treatments include cell lysis, protein purification, and the like, all of which are well known to those skilled in the art.

In the above examples of the present invention, the novel coronavirus S protein receptor binding domain (S)_RBDAnd at least part of the sequence is SEQ ID NO: 4) to the MIR region of HBcAg (at least a portion of the sequence is SEQ ID NO: 3, wherein the assembly domain is located at positions 1-143 and the C-terminal domain CTD is located at positions 150-183, can be formed on the virus-like particle (hereinafter also referred to as S)_RBDHBcAg chimeric virus-like particle) surface displays the receptor binding region of the S protein, and enhances the immune response of the human immune system to the receptor binding region of the S protein by means of the virus-like particle structure to obtain good immune effect, thereby ensuring immune people to obtain good immune protectionProtecting and avoiding the infection of the novel coronavirus.

The hepatitis B virus core antigen (HBcAg) mentioned in the present specification is composed of core protein subunits and has a regular icosahedral symmetric particle structure. Single core protein subunits (comprising polypeptide chains of 183-185 amino acids) are first assembled into homodimers (Homodimer), which are then further multimerized to form HBcAg particles. The 150-183 part of the HBcAg C-terminal is a nucleic acid binding region, exists in the interior of the virus capsid, has the capacity of binding nucleic acid RNA, but is not necessary for the shape and size maintenance and self-assembly matching of the virus core particle, and the HBcAg with the fragment cut off has better stability. The 1-149 part of the N-terminus is the particle assembly region, responsible for the formation of virus-like particles. A number of studies have reported that the N-terminus of HBcAg (1-149) can form homodimers through C61 and assemble into virus-like particles, in which the L76-D83 form the top of the protuberance, which becomes the Major Immunodominant Region (MIR), and the two sides form the base and handle of the dimer. The MIR region present in VLPs formed by HBcAg is well suited to carry foreign peptides without affecting the formation and immunogenicity of their VLPs. HBcAg can be expressed in different systems (e.g.E.coli, yeast, vaccinia Virus, insect cells, etc.) and assembles into a 21kD polypeptide that can spontaneously assemble to form HBcAg, which self-assembly in heterologous systems provides advantages for HBcAg as an immune vector (Ulrich, R.M.Nassal, H.Meisel, et al.core particulate matters B viruses as carrier for infection epitopes.adv Virus Res, 1998.50: p.141-82.).

In the above embodiments of the present invention, it is preferable to use a coliform expression system, which has a high expression level, strong expansibility, and good protein immunogenicity.

The above embodiment of the present invention expresses S using recombinant Escherichia coli_RBDThe HBcAg chimeric virus-like particle has antigenicity, immunogenicity and function similar to those of natural protein, high expression level, strong immunogenicity and no pathogenicity to human when being applied as an active component of the vaccine, and the vaccine can be cultured in a bioreactor without serum in a large scaleThe quality control is easy, the safety is high, the immunogenicity is good, the batch is stable, the production cost of the vaccine is greatly reduced, and the requirement of large-scale industrial production can be well met.

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The reagents and starting materials used in the following examples are commercially available, and the test methods in which specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the respective manufacturers. Further, unless otherwise indicated, the assays, detection methods, and preparations disclosed herein may be performed using any of the techniques well known in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and the like. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORYMANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and Methodsin Molecular BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.