CN113004378A

CN113004378A - Novel coronavirus-like particles, preparation method and application thereof

Info

Publication number: CN113004378A
Application number: CN202110333400.4A
Authority: CN
Inventors: 翟继先; 龙艳萍; 贾津布; 温舰宇; 鹿东东
Original assignee: Southwest University of Science and Technology
Current assignee: Southwest University of Science and Technology; Southern University of Science and Technology
Priority date: 2021-03-29
Filing date: 2021-03-29
Publication date: 2021-06-22

Abstract

The invention relates to a novel coronavirus-like particle, a preparation method and application thereof. The preparation method of the novel coronavirus-like particle comprises the following steps: constructing an expression vector for expressing S protein, E protein, M protein and N protein of the novel coronavirus, wherein the amino acid sequence of the S protein is shown as SEQ ID No.1, the amino acid sequence of the E protein is shown as SEQ ID No.2, the amino acid sequence of the M protein is shown as SEQ ID No.3, and the amino acid sequence of the N protein is shown as SEQ ID No. 4; after the expression vector is transfected into the host, culturing the host transfected with the expression vector; and extracting the novel coronavirus-like particles from the host transfected with the expression vector. The novel coronavirus-like particles prepared by the preparation method of the novel coronavirus-like particles have good safety, stability and effectiveness when used for vaccines.

Description

Novel coronavirus-like particles, preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a novel coronavirus-like particle and a preparation method and application thereof.

Background

A novel coronavirus (SARS-CoV-2) is an enveloped RNA virus whose positive-sense single-stranded RNA genome is approximately 30kb in length. Sequencing finds that the new coronavirus and atypical new coronavirus (SARS-CoV) and middle east respiratory syndrome coronavirus (MERS) belong to the same genus and the genus beta coronavirus respectively have the similarity of 80% and 50%. Research reports have shown that a complete viral transcript contains two large open reading frames: ORF1a and ORF1 b. ORF1a is translated into a polypeptide of about 450kD, which is cleaved into 11 non-structural proteins. During translation, frame shift mutations occurred rapidly upstream of the ORF1a stop codon. Thus, translation was smoothly transitioned to ORF1b, allowing for a larger polypeptide, perhaps around 800kD, to be obtained which would be hydrolyzed by viral proteases to 15 non-structural proteins. The function of these non-structural proteins is not yet fully determined.

Structural proteins constituting the novel coronavirus include: spike protein (S protein), membrane protein (M protein), envelope protein (E protein), nucleocapsid protein (N protein). The N protein is the only protein present in the nucleocapsid among the four structural proteins, and contains two independent domains at the N-terminal and C-terminal, and can be combined with RNA. In addition, it can bind to the M protein and other enzymes, helping to bind the viral genome to the replicase-transcriptase complex. The S protein is located at the outermost side of the virus and its main function is to recognize and bind to the host cell, mediating fusion of the viral envelope with the cell membrane. Notably, the RBD functional region on the S protein has two conformations. RBDs are active in binding to the host cell only when in the "up" conformation. In addition, S protein has been shown to interact with ACE2 protein expressed in various organs of the human body to infect host cells.

Clinically, the medicine for treating SARS-CoV-2 infected patient includes RNA polymerase inhibitor, proteinase inhibitor, blood plasma of convalescent patient and Chinese medicine. However, even though some drugs have already demonstrated good therapeutic effects, no specific drug effective against the virus has been determined so far, and no standardized therapeutic method exists. The concept of vaccination is in force, since infectious diseases such as hepatitis B and smallpox have been devastating worldwide and are effectively controlled by vaccination to achieve mass immunity.

At present, vaccines against SARS-CoV-2 are mainly classified into attenuated live vaccines, inactivated vaccines, attenuated influenza virus vector vaccines, adenovirus vector vaccines and nucleic acid vaccines.

The attenuated live vaccine is prepared by performing attenuation treatment (in vitro passage or gene recombination) on SARS-CoV-2 to obtain vaccine with reduced pathogenicity and good immunogenicity and comprising complete virus. The attenuated live vaccine has strong immunogenicity, can cause a wider range of immune response and has a more durable protective effect. However, live attenuated vaccines contain live pathogens, hidden infection hazards exist, and due to different pathogenic capacities of the pathogens in different live vaccines, the live attenuated vaccines have certain risk of virulence reversion, and the overall safety is not high.

The inactivated vaccine is prepared by inactivating SARS-CoV-2 by physicochemical method, and multiple tests and screens are needed to find SARS-CoV-2 which has lost pathogenic ability and does not completely lose the ability to activate human immune response. The inactivated vaccine retains the original surface protein of SARS-CoV-2, the antigenicity is similar to that of the live virus, but the inactivated vaccine can not be replicated in the host body and can not cause infection, and the problem of virulence reversion does not exist. Therefore, compared with attenuated live vaccines, inactivated vaccines have higher safety and do not cause human infection. However, inactivated vaccines do not mimic the natural infection of SARS-CoV-2 and are not sufficiently immunogenic and therefore less effective than live attenuated vaccines.

The attenuated influenza virus vector vaccine uses the attenuated influenza virus in the existing influenza virus vaccine as a vector, and transfers the gene of S protein of SARS-CoV-2 into the gene of the influenza virus vector, so that the S protein of SARS-CoV-2 is expressed on the surface of the influenza virus. The novel coronavirus vaccine with the influenza virus as the vector can prevent new coronary pneumonia and influenza at the same time, and the influenza virus has no DNA and does not have the risk of integrating the influenza virus DNA into a host genome. However, the capacity of the influenza virus is small, the range of inserting exogenous genes is limited, and the targeting of the vector is not high.

The adenovirus vector vaccine takes the modified harmless adenovirus as a vector, and transfers the gene of the S protein into the gene of the adenovirus (different from the influenza virus vector, the S protein is not expressed on the surface of the adenovirus). After the vaccine is inoculated, the adenovirus vector can enter cells, the adenovirus can not replicate due to gene defect, but S protein genes in the genes can transcribe and translate to express S protein, and the S protein can be transferred to the outside of the cells from the inside of the cells, so that the immune response of a human body is triggered, and the effect is good. However, for individuals infected with adenovirus (adenovirus antibody positive individuals), when adenovirus re-enters the body, the adenovirus antibody in the body will attack the vector and not the S protein it expresses, rendering the vaccine ineffective.

Nucleic acid vaccines include DNA vaccines and mRNA vaccines. The DNA vaccine directly injects the DNA sequence (common plasmid) of the coding virus antigen gene segment into a host, so that the antigen protein of the virus is expressed in the host, and then the humoral immunity and the cellular immunity are activated. The vaccine can imitate the infection mode of live virus to synthesize endogenous antigen, and the endogenous antigen is presented to MHC class I molecules to induce CD8+ T cells to generate killing effect. However, the DNA vaccine has a disadvantage in that such foreign DNA may be integrated with DNA of the cell itself, and thus the DNA vaccine particularly requires attention to safety. The technical route of RNA vaccine is similar to that of DNA vaccine, and the mRNA vaccine is based on the principle that mRNA of virus antigen is prepared into vaccine, and the mRNA injected into host can express antigen protein of virus, so as to induce organism to produce immune response. mRNA is directly used as vaccine, and does not need to enter the cell nucleus to complete the transcription process and then carry out the translation expression of protein in the cytoplasm like DNA vaccine, and mRNA vaccine can directly synthesize protein in the cytoplasm, which saves one step compared with DNA vaccine. The steps of the mRNA vaccine are simplified and there is no fear of integration of viral DNA into host DNA. However, mRNA is not as stable as DNA or protein, and the greatest drawback is its susceptibility to degradation.

Disclosure of Invention

Therefore, there is a need for a method for preparing novel coronavirus-like particles, and a novel coronavirus vaccine prepared from the novel coronavirus-like particles prepared by the method can improve the safety, stability and effectiveness of the novel coronavirus vaccine.

A method for preparing a novel coronavirus-like particle, comprising the following steps:

constructing an expression vector for expressing an S protein, an E protein, an M protein and an N protein of the novel coronavirus, wherein the amino acid sequence of the S protein is shown as SEQ ID No.1, the amino acid sequence of the E protein is shown as SEQ ID No.2, the amino acid sequence of the M protein is shown as SEQ ID No.3, and the amino acid sequence of the N protein is shown as SEQ ID No. 4;

transfecting the expression vector into a host, and culturing the host transfected with the expression vector, wherein the host is a plant; and

extracting novel coronavirus-like particles from a host transfected with the expression vector.

The novel coronavirus-like particle prepared by the method is a large particle assembled from structural proteins (S protein, E protein, M protein and N protein) of the novel coronavirus, and has no genetic material compared with the novel active coronavirus. Therefore, compared with the traditional attenuated live vaccine, inactivated vaccine, attenuated influenza virus vector vaccine, adenovirus vector vaccine and nucleic acid vaccine, the vaccine prepared by the novel coronavirus-like particles has high safety, good stability and high effectiveness.

In one embodiment, the nucleotide sequence of the nucleic acid segment encoding the S protein is shown as SEQ ID No.5, the nucleotide sequence of the nucleic acid segment encoding the E protein is shown as SEQ ID No.6, the nucleotide sequence of the nucleic acid segment encoding the M protein is shown as SEQ ID No.7, and the nucleotide sequence of the nucleic acid segment encoding the N protein is shown as SEQ ID No. 8.

In one embodiment, in the expression vector, the S protein, the E protein, the M protein, and the N protein are each independently expressed.

In one embodiment, the expression vector for expressing S protein, E protein, M protein and N protein of the novel coronavirus comprises a first recombinant expression vector for expressing the S protein and the E protein and a second recombinant expression vector for expressing the M protein and the N protein, the first recombinant expression vector and the second recombinant expression vector entering the host by co-transfection.

In one embodiment, the first recombinant expression vector is obtained by inserting a nucleic acid fragment encoding the S protein and a nucleic acid fragment encoding the E protein into a pHREAC vector, and the second recombinant expression vector is obtained by inserting a nucleic acid fragment encoding the M protein and a nucleic acid fragment encoding the N protein into a pHREAC vector.

In one embodiment, the host is tobacco.

In one embodiment, the expression vector is prepared by inserting the nucleic acid segment encoding the S protein, the nucleic acid segment encoding the E protein, the nucleic acid segment encoding the M protein and the nucleic acid segment encoding the N protein into the same empty vector.

A novel coronavirus-like particle is prepared by the preparation method of the novel coronavirus-like particle.

An expression vector is used for expressing S protein, E protein, M protein and N protein of a novel coronavirus, wherein the amino acid sequence of the S protein is shown as SEQ ID No.1, the amino acid sequence of the E protein is shown as SEQ ID No.2, the amino acid sequence of the M protein is shown as SEQ ID No.3, and the amino acid sequence of the N protein is shown as SEQ ID No. 4.

Use of the novel coronavirus-like particle as described above or the expression vector as described above for the preparation of a medicament for the prevention or treatment of a novel coronavirus infection.

A novel coronavirus vaccine comprising the novel coronavirus-like particle as described above.

Drawings

FIG. 1 is a schematic diagram of the structure of pHREAC-S-E plasmid;

FIG. 2 is a schematic diagram of the structure of pHREAC-N-M plasmid;

FIG. 3 shows the expression of N protein in tobacco injected with an expression vector;

FIG. 4 shows the expression of the S protein in tobacco injected with an expression vector;

FIG. 5 shows the expression of N protein in wild-type tobacco after 4 days of culture in tobacco injected with the expression vector;

FIG. 6 is S protein expression in wild type tobacco after 4 days of culture in tobacco injected with expression vector;

FIG. 7 shows the results of 25% and 70% sucrose bedding crude extraction;

FIG. 8 is a SDS-PAGE pattern of cesium chloride after continuous density purification;

FIG. 9 shows the distribution of new coronavirus-like particles in samples numbered 40-64 in FIG. 8 as N-protein antibody;

FIG. 10 shows the distribution of new coronavirus-like particles in samples numbered 40-64 in FIG. 8 as S protein antibody;

FIG. 11 is an electron micrograph of a novel coronavirus-like particle.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It should be noted that the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance. 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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Interpretation of terms: virus-like particles (VLPs) are large particles assembled from viral structural proteins and have no genetic material present compared to active viruses.

One embodiment of the present invention provides a method for preparing a novel coronavirus-like particle, which comprises steps a to c:

step a: constructing expression vector for expressing S protein, E protein, M protein and N protein of novel coronavirus.

Specifically, the amino acid sequence of the S protein is shown as SEQ ID No.1, the amino acid sequence of the E protein is shown as SEQ ID No.2, the amino acid sequence of the M protein is shown as SEQ ID No.3, and the amino acid sequence of the N protein is shown as SEQ ID No. 4.

In this embodiment, the host is a plant. Compared with other hosts, the plant is used as the host of the expression vector for expressing the novel coronavirus-like particles, and the cost is lower and the scale production is easier. After the stable genetic material is obtained, the large-scale planting can be carried out, and the operation threshold is low without investment of fermentation plants and the like. In addition, the transportation and storage environment of the seeds is easy to reach, and the harsh external environment requirements such as temperature, humidity and the like are almost absent. More importantly, the plant is used as a host, so that the oral vaccine can be prepared, the production cost is greatly reduced, and the popularization of the globalized Xinguan vaccine is easier. Further, the host was nicotiana benthamiana (n. It is understood that in other embodiments, the host is not limited to tobacco, but may be other plants. Of course, the host is not limited to plants, and other hosts capable of producing recombinant proteins, such as engineered bacteria, are also possible.

Alternatively, the nucleotide sequence of the nucleic acid fragment for coding the S protein is shown as SEQ ID No.5, the nucleotide sequence of the nucleic acid fragment for coding the E protein is shown as SEQ ID No.6, the nucleotide sequence of the nucleic acid fragment for coding the M protein is shown as SEQ ID No.7, and the nucleotide sequence of the nucleic acid fragment for coding the N protein is shown as SEQ ID No. 8. The nucleic acid fragments corresponding to the nucleotide sequences shown in SEQ ID No. 5-SEQ ID No.8 are the nucleic acid fragments subjected to codon optimization according to the amino acid sequences of the S protein, the E protein, the M protein and the N protein, and can enable the nucleic acid fragments for coding the S protein, the E protein, the M protein and the N protein to be efficiently expressed in plants. It is understood that in other embodiments, the nucleic acid fragments encoding the S, E, M and N proteins are not limited to those described above, but may be other nucleic acid fragments encoding the amino acid sequences described above.

Specifically, the S protein, the E protein, the M protein and the N protein are independently expressed in an expression vector. The nucleic acid segment encoding the S protein, the nucleic acid segment encoding the E protein, the nucleic acid segment encoding the M protein, and the nucleic acid segment encoding the N protein are each independently present in an entire gene expression cassette (comprising a promoter, a terminator, a 5 'UTR, and a 3' UTR) in an expression vector.

In this embodiment, a complete gene expression cassette includes a 35S promoter, an NOS terminator, a 35S enhancer, a desired expression fragment (i.e., a nucleic acid fragment corresponding to each protein), a 5 'UTR, and a 3' UTR. The target expression fragment is efficiently expressed by using 35S promoter, NOS terminator and 35S enhancer. It is to be understood that, in other embodiments, the types of promoters, terminators, and enhancers are not limited to those described above, and other promoters, terminators, and/or enhancers may be used.

In this embodiment, the expression vector for expressing the S, E, M and N proteins of the novel coronavirus includes a first recombinant expression vector for expressing the S and E proteins and a second recombinant expression vector for expressing the M and N proteins. Specifically, the first recombinant expression vector is obtained by inserting a nucleic acid fragment encoding the S protein and a nucleic acid fragment encoding the E protein into a pHREAC vector, and the second recombinant expression vector is obtained by inserting a nucleic acid fragment encoding the M protein and a nucleic acid fragment encoding the N protein into a pHREAC vector. The pHREAC vector can realize high transient expression of recombinant protein in plants by adjusting the lengths of 5 'UTR and 3' UTR.

Of course, in other embodiments, the nucleic acid segment encoding the S protein, the nucleic acid segment encoding the E protein, the nucleic acid segment encoding the M protein, and the nucleic acid segment encoding the N protein may be placed on the same empty vector for expression, or the nucleic acid segment encoding the S protein, the nucleic acid segment encoding the E protein, the nucleic acid segment encoding the M protein, and the nucleic acid segment encoding the N protein may be inserted into different empty vectors to form expression vectors, and then the expression vectors may be co-transfected into the host, as long as the host can express the S protein, the E protein, the M protein, and the N protein.

Step b: after the expression vector is transfected into a host, the host transfected with the expression vector is cultured.

In this embodiment, the host is a plant. Thus, agrobacterium is used to transfect the expression vector into the host. Alternatively, the agrobacterium is agrobacterium tumefaciens GV 3101. In an alternative specific example, the first recombinant expression vector, the second recombinant expression vector, and the P19 vector are co-transfected into agrobacterium competent cells, and then agrobacterium containing the first recombinant expression vector, the second recombinant expression vector, and the P19 vector is injected into tobacco leaf, such that the agrobacterium infects the plant. The P19 vector can prevent the silencing of exogenous gene in tobacco. It will be appreciated that in other embodiments, an appropriate transfection method may be selected depending on the host.

Step c: novel coronavirus-like particles are extracted from a host transfected with an expression vector.

In this embodiment, tobacco leaves are collected after 2 to 7 days of culture, and the novel coronavirus-like particles in the tobacco leaves are extracted. Preferably, the tobacco cultured for 2 to 4 days is selected to extract the novel coronavirus-like particles. Specifically, the step of extracting the novel coronavirus-like particles in the tobacco lamina comprises the steps of c 1-c 4:

step c 1: grinding the tobacco leaves, and mixing the ground tobacco leaves with the protein extracting solution to prepare a mixed solution.

Specifically, in order to prevent the formed new coronavirus-like particles from being damaged during grinding, liquid nitrogen is not added during grinding, but the protein extracting solution, a mortar and a pestle are precooled in advance or a precooled stirrer is used during extraction, and chemical substances capable of denaturing the protein are avoided in the protein extracting solution. Optionally, the protein extract is 0.1M sodium phosphate buffer pH 5-8 (protease inhibitor is added). The mass ratio of the protein extracting solution to the tobacco leaves is (2-4): 1.

step c 2: removing the plant tissue fragments.

Specifically, the mixture was filtered through a 0.22 μm filter and centrifuged to remove plant tissue debris. The diameter of the novel coronavirus-like particles is about 80nm, and therefore, the novel coronavirus-like particles can be separated from most tissues of plants using a 0.22 μm filter. Alternatively, the mixture was centrifuged at 4 ℃ at 10000g for 30min, and the supernatant was retained.

Step c 3: the filtrate obtained in step c2 was subjected to a preliminary purification.

Specifically, the novel coronavirus-like particles in the filtrate obtained in step c2 were primarily purified by bottoming with 25% (w/v) sucrose and 70% (w/v) sucrose to obtain a primarily purified product.

Optionally, adding the filtrate obtained in step c2 into an ultracentrifuge tube, then sequentially injecting 500 μ L of 70% sucrose solution and 1mL of 25% sucrose into the bottom of the ultracentrifuge tube (firstly adding 25% sucrose and then adding 70% sucrose) by using a 1mL syringe with an elongated needle, and then centrifuging at 4 ℃ for 30000g for 3 hours; and after the centrifugation is finished, a green band appears between sucrose gradients, the solution at the position is sucked out by using a shearing gun head, the obtained solution is concentrated by using a concentration tube, a sodium phosphate buffer solution is continuously added while the centrifugation is carried out, so that the sucrose solution is replaced by the sodium phosphate buffer solution, and finally the primary purified product is obtained by filtering through a 0.22 mu m filter membrane.

Step c 4: and (3) refining and purifying the primarily purified product by adopting density gradient centrifugation to prepare the novel high-purity coronavirus-like particles.

Specifically, the density gradient centrifugation includes continuous density gradient centrifugation and discontinuous density gradient centrifugation, and the centrifugation medium can be selected from sucrose, cesium chloride, glycerol, etc. In one embodiment, the primary purified product is purified by continuous density centrifugation with cesium chloride.

The preparation method of the novel coronavirus-like particle comprises the steps of constructing an expression vector for expressing S protein, E protein, M protein and N protein of the novel coronavirus, and transfecting the expression vector into a host, so that the host produces the novel coronavirus-like particle, and the novel coronavirus-like particle can be extracted from the host. The novel coronavirus-like particles prepared by the method for preparing the novel coronavirus-like particles do not contain viral nucleic acid and thus have high safety, and the novel coronavirus-like particles prepared by the method for preparing the novel coronavirus-like particles have the same shell structure as natural novel coronavirus and the same immunogenicity as natural novel coronavirus, so that the novel coronavirus-like particles are good materials for preparing a material for preventing or treating the novel coronavirus.

Based on the above, an embodiment of the present invention further provides a novel coronavirus-like particle, which is prepared by the above preparation method of the novel coronavirus-like particle, and the novel coronavirus-like particle does not contain a nucleic acid of a novel coronavirus, and is formed by a three-dimensional structure of S protein, E protein, M protein and N protein, wherein an amino acid sequence of the S protein is shown as SEQ ID No.1, an amino acid sequence of the E protein is shown as SEQ ID No.2, an amino acid sequence of the M protein is shown as SEQ ID No.3, and an amino acid sequence of the N protein is shown as SEQ ID No. 4.

In addition, the embodiment of the invention also provides an expression vector, which is used for expressing the S protein, the E protein, the M protein and the N protein of the novel coronavirus, wherein the amino acid sequence of the S protein is shown as SEQ ID No.1, the amino acid sequence of the E protein is shown as SEQ ID No.2, the amino acid sequence of the M protein is shown as SEQ ID No.3, and the amino acid sequence of the N protein is shown as SEQ ID No. 4.

In addition, an embodiment of the present invention further provides an application of the above novel coronavirus-like particle or the above expression vector in the preparation of a medicament for preventing or treating a novel coronavirus infection. For example, the above-mentioned novel coronavirus-like particles mimic a novel coronavirus, and a drug for treating infection with the novel coronavirus is developed. The novel coronavirus-like virus is used as an active ingredient of a novel coronavirus vaccine to prepare the novel coronavirus vaccine.

In view of the above, an embodiment of the present invention also provides a novel coronavirus vaccine, wherein the effective component of the novel coronavirus vaccine comprises the novel coronavirus-like particle.

The novel coronavirus vaccine contains the novel coronavirus-like particles as an active ingredient, and has high safety, high stability and good effectiveness.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

Example 1

(1) Artificially synthesizing nucleic acid segments for coding S protein, E protein, M protein and N protein of the novel coronavirus, wherein the nucleotide sequence of the nucleic acid segment for coding the S protein is shown as SEQ ID No.5, the nucleotide sequence of the nucleic acid segment for coding the E protein is shown as SEQ ID No.6, the nucleotide sequence of the nucleic acid segment for coding the M protein is shown as SEQ ID No.7, and the nucleotide sequence of the nucleic acid segment for coding the N protein is shown as SEQ ID No. 8.

(2) The four synthesized structural protein coding sequences are connected into pHREAC vector through Bsa I enzyme cutting site to obtain recombinant plasmids pHREAC-N, pHREAC-M, pHREAC-S and pHREAC-E. In the recombinant plasmid, each coding sequence contains a complete gene expression cassette including the 35S promoter, 35S enhancer, NOS terminator and 5 'UTR and 3' UTR.

(3) Primers (shown as SEQ ID No.9 and SEQ ID No. 10) are designed on the plasmid pHREAC-M and the plasmid pHREAC-E, the complete gene expression frames of the M protein and the E protein (including a 35S promoter, a 35S enhancer, a 5 'UTR, a coding sequence of the M protein and the E protein, a 3' UTR and an NOS terminator) are amplified, the complete gene expression frames of the M protein and the E protein are purified and recovered, and are connected through KpnI enzyme cutting sites on the pHREAC-N plasmid and the pHREAC-S plasmid, and finally the pHREAC-S-E plasmid and the pHREAC-N-M plasmid are obtained. The pHREAC-S-E plasmid is shown in FIG. 1, and the pHREAC-N-M plasmid is shown in FIG. 2.

(4) pHREAC-S-E plasmid, pHREAC-N-M plasmid and P19 vector are respectively transferred into Agrobacterium tumefaciens GV3101 competence to prepare Agrobacterium tumefaciens containing pHREAC-S-E plasmid, Agrobacterium tumefaciens containing pHREAC-N-M plasmid and Agrobacterium tumefaciens containing P19 vector, and then the three kinds of Agrobacterium tumefaciens and Nicotiana benthamiana are co-cultured to obtain the coronavirus-like particles. Specifically, the method comprises the following steps:

a. pHREAC-S-E plasmid, pHREAC-N-M plasmid and P19 vector were transferred into Agrobacterium tumefaciens GV3101 competent cells, respectively, and Agrobacterium tumefaciens containing pHREAC-S-E plasmid, Agrobacterium tumefaciens containing pHREAC-N-M plasmid and Agrobacterium tumefaciens containing P19 vector were cultured in LB medium supplemented with 50ng/mL kanamycin and 50ng/mL rifampicin, respectively, and cultured at 28 ℃ with shaking at 220 rpm.

b. After 24 hours of culture, OD600 of the culture was 0.6 at 3000rpm for 10min, and three kinds of Agrobacterium (Agrobacterium tumefaciens with pHREAC-S-E plasmid, Agrobacterium tumefaciens with pHREAC-N-M plasmid, and Agrobacterium tumefaciens with P19 vector) were collected, respectively. Then 4mL of MgCl were added separately₂Solution (10mM MES,10mM MgCl)₂) Resuspending, and centrifuging at 3000rpm for 10min again at 4 deg.C to obtain Agrobacterium pellet. Subsequently, 4mL of MES solution and 6. mu.L of acetosyringone were added thereto, and the mixture was incubated at room temperature for 1 hour with shaking. Then, the cells were centrifuged and MgCl was used for the cells obtained by the centrifugation₂Resuspending the solution and mixing MgCl in a 1:1 ratio₂And (5) filling. The mixture was manually injected into tobacco lamina using a 1mL syringe and the tobacco was cultured.

c. After culturing the tobacco of step b for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days, tobacco leaves transformed with pHREAC-S-E plasmid, pHREAC-N-M plasmid, and P19 vector were collected, and total proteins in the tobacco leaves were extracted, and the expression of structural proteins of the novel coronavirus in tobacco after different culturing times was detected by Western blot using S protein antibody and N protein antibody (S protein antibody: GTX632604, N protein antibody: GTX635712, the same applies below), and the results are shown in FIGS. 3 to 6. FIGS. 3 to 4 show the expression of the N protein and S protein in tobacco transformed with the expression vector after culturing for 1 to 7 days, wherein the lane number "M" corresponds to marker and the lane numbers "1 to 7" correspond to culturing time for 1 to 7 days in FIGS. 3 and 4; FIGS. 5 and 6 show the expression of N protein and S protein in tobacco transformed with the expression vector and wild-type tobacco after 4 days of culture.

As is clear from FIGS. 3 to 6, both the N protein and the S protein can be expressed in tobacco leaves.

d. The tobacco lamina collected in step c was weighed and added to a pre-chilled mortar and pestle to grind at 4 ℃, and then 0.1M sodium phosphate buffer pH 7.0 (protease inhibitor added) was mixed with tobacco lamina in a mass ratio of 3: 1, and standing for 15min in an ice bath after uniformly mixing. Then, the mixed solution after standing was passed through a 0.22 μm filter to preliminarily remove plant tissue fragments. Then, the filtrate was centrifuged at 4 ℃ at 10000g for 30min, and the supernatant was retained.

e. The supernatant from step d was subjected to a first purification step by bedding with 25% (w/v) and 70% (w/v) sucrose (sucrose solution prepared in 0.1M sodium phosphate buffer pH 7.0): adding all the supernatant obtained in the step d into an ultracentrifuge tube, then using a 1mL syringe with an elongated needle to sequentially inject 500. mu.L of 70% sucrose solution and 1mL of 25% sucrose into the bottom of the ultracentrifuge tube (firstly adding 25% sucrose and then adding 70% sucrose), and then centrifuging at 30000g for 3 hours at 4 ℃. After centrifugation, green bands appeared between the sucrose gradients in the tubes (as shown in FIG. 7). And then sucking out the solution at the position by using a shearing gun head, concentrating the obtained solution by using a concentration pipe, continuously adding a sodium phosphate buffer solution while centrifuging to replace the sucrose solution with the sodium phosphate buffer solution, and finally filtering by using a 0.22-micron filter membrane to obtain the primarily purified new coronavirus-like particles.

f. And (3) purifying the primarily purified new coronavirus-like particles by adopting a cesium chloride continuous density centrifugation method: 11% (w/w) and 33% (w/w) CsCl solutions were prepared using 0.1M sodium phosphate buffer pH 7.0. Equal volumes of 11% CsCl and 33% CsCl were added to an ultracentrifuge tube and a continuous density gradient solution from 11% CsCl to 33% CsCl was obtained by a continuous density prep. Carefully spreading the primarily purified new coronavirus-like particles on CsCl solution, centrifuging at 4 ℃ for 16 hours at 30000g, taking 150 mu L as a unit from top to bottom after centrifugation, sampling by a shearing gun head, and detecting SDS-PAGE. The SDS-PAGE detection steps include: preparing 10% separating gel and 5% concentrating gel, adding 5 μ L5 × loading buffer solution into 20 μ L sample per 150 μ L CsCl solution, mixing, rapidly placing in ice bath at 98 deg.C for 10min to prepare electrophoresis sample, performing polyacrylamide gel electrophoresis, and silver staining after the electrophoresis is finished, wherein the electrophoresis is shown in FIG. 8.

g. SDS-PAGE gel, 20V 60min transfer to PVDF membrane, block the membrane into 5% skim milk in PBS for one hour, 1: commercial S protein antibody and N protein antibody were added at 1000 deg.C and incubated at 37 deg.C for 1h followed by three PBST washes, ten minutes each. The film was transferred to 1: in 5000 dilution of secondary antibody (sheep anti mouse), after 1h incubation at 37 ℃ PBST washed three times, each time for ten minutes, developed, the results are shown in FIG. 9 and FIG. 10.

h. The sample in lane 58 was negatively stained and observed by transmission electron microscopy (transmission electron microscopy is shown in FIG. 11).

As is clear from FIG. 9, the novel coronavirus-like particles prepared by the above method have a steric structure, have a diameter of about 100nm, and contain S protein.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val

1 5 10 15

Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu

35 40 45

His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp

50 55 60

Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile

115 120 125

Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr

195 200 205

Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala

275 280 285

Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val

305 310 315 320

Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala

340 345 350

Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe

385 390 395 400

Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly

405 410 415

Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly

485 490 495

Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val

565 570 575

Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe

580 585 590

Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val

595 600 605

Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile

610 615 620

His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser

625 630 635 640

Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val

645 650 655

Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala

660 665 670

Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala

675 680 685

Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile

705 710 715 720

Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val

725 730 735

Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr

755 760 765

Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln

770 775 780

Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly

820 825 830

Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp

835 840 845

Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile

885 890 895

Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr

900 905 910

Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn

915 920 925

Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn

945 950 955 960

Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val

965 970 975

Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln

980 985 990

Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val

995 1000 1005

Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu

1010 1015 1020

Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val

1025 1030 1035 1040

Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala

1045 1050 1055

Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu

1060 1065 1070

Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His

1075 1080 1085

Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val

1090 1095 1100

Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr

1105 1110 1115 1120

Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr

1125 1130 1135

Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu

1140 1145 1150

Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1155 1160 1165

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1170 1175 1180

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu

1185 1190 1195 1200

Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile

1205 1210 1215

Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile

1220 1225 1230

Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val

1250 1255 1260

Leu Lys Gly Val Lys Leu His Tyr Thr

1265 1270

<210> 2

<211> 75

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

1 5 10 15

Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

20 25 30

Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

35 40 45

Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

50 55 60

Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val

65 70 75

<210> 3

<211> 222

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu

1 5 10 15

Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile

20 25 30

Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile

35 40 45

Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys

50 55 60

Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile

65 70 75 80

Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe

85 90 95

Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe

100 105 110

Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile

115 120 125

Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile

130 135 140

Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp

145 150 155 160

Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu

165 170 175

Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly

180 185 190

Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr

195 200 205

Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln

210 215 220

<210> 4

<211> 419

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

1 5 10 15

Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

20 25 30

Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

65 70 75 80

Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

85 90 95

Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

100 105 110

Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

130 135 140

His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

145 150 155 160

Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

165 170 175

Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

180 185 190

Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

210 215 220

Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

225 230 235 240

Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

245 250 255

Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

275 280 285

Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

305 310 315 320

Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala

325 330 335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

385 390 395 400

Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

405 410 415

Thr Gln Ala

<210> 5

<211> 3822

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgtttgtgt tcctggtact tcttccactt gtttcatcac aatgcgttaa ccttacaaca 60

cggacccagt tgccgcctgc ttatacaaac tcatttacaa gaggtgtata ctacccggac 120

aaagtattcc gctcttcagt tcttcatagc acgcaggacc tattcctccc tttcttcagc 180

aatgttacat ggtttcatgc aatccatgtt tcaggaacaa acggaacaaa gcggttcgat 240

aacccagttc ttccatttaa cgatggagtt tatttcgctt ctacagagaa gtcgaatatc 300

atcagaggat ggatctttgg aacaacactt gattcaaaga cccagtcact tcttatcgtt 360

aacaacgcaa caaacgttgt tatcaaagtt tgcgagttcc agttctgtaa tgaccctttc 420

ttaggtgtat attatcataa gaacaacaag tcctggatgg aatcagaatt tagagtttat 480

tcatcagcaa acaactgcac atttgaatat gtttcacaac catttcttat ggacctcgag 540

ggtaaacaag gcaatttcaa gaatttgcgg gagttcgtct ttaagaatat cgacggttac 600

tttaagatat attcaaagca cactccaatc aaccttgtta gagatcttcc acaaggattc 660

tccgctcttg aaccacttgt tgatcttcca atcggaatca acatcacaag atttcaaaca 720

cttcttgcac ttcatagatc atatcttaca ccaggagatt catcatcagg atggacagca 780

ggagcagcag catattatgt tggatatctt caaccaagaa catttcttct taaatataac 840

gagaatggta caatcacaga tgcagttgat tgcgcacttg atccactttc cgagactaaa 900

tgcactttga agtctttcac cgtggagaag ggcatctatc aaacatcaaa ctttagagtt 960

caaccaacag aatcaatcgt tagatttcca aacatcacaa acctttgccc atttggagaa 1020

gtattcaatg ctacaagatt tgcatcagtt tatgcatgga acagaaagcg tatttcaaac 1080

tgcgttgcag attattcagt tctttataac tcagcatcat tctcgacgtt taaatgctat 1140

ggagtttcac caacaaagtt aaatgatctt tgctttacaa acgtttatgc agattcattt 1200

gttatcagag gagatgaagt tagacaaatc gcaccaggac aaacaggaaa gatagctgat 1260

tataactata aacttccaga tgatttcact ggctgcgtta tcgcatggaa ctcaaacaat 1320

ctggactcga aagttggcgg taattacaat tacctatacc gtctattcag gaaatccaac 1380

ctcaagccgt ttgaaagaga tatctcaaca gaaatctatc aagcaggatc aacaccatgc 1440

aacggagttg aaggatttaa ctgctatttc ccgttgcagt catatggatt tcaaccaaca 1500

aacggagttg gatatcaacc atatagagtt gttgttcttt catttgaact tcttcatgca 1560

ccagcaacag tttgcggacc aaagaagtct actaaccttg ttaagaataa gtgcgttaac 1620

tttaacttta acggacttac aggaacagga gttcttacag aatcaaacaa gaagttcttg 1680

ccatttcaac aatttggaag agatatcgca gatacaacag atgcagttag agatccacaa 1740

acacttgaaa tccttgatat cacaccatgc tcatttggag gagtttcagt tatcacacca 1800

ggaacaaaca catcaaacca agttgcagtt ctttatcaag atgttaactg cacagaagtt 1860

ccagttgcaa tccatgcaga tcaacttaca ccaacatgga gagtttattc aacaggatca 1920

aacgtgttcc agaccagagc aggatgcctt atcggagcag aacatgttaa caactcatat 1980

gaatgcgata tcccaatcgg agcaggaatc tgcgcatcat atcaaacaca aacaaactca 2040

ccaagaagag caagatcagt tgcatcacaa tcaatcatcg catatacaat gtcacttgga 2100

gcagagaata gcgttgcata ttcaaacaac tcaatcgcaa tcccaacaaa ctttacaatc 2160

tcagttacaa cagagatact cccagtcagt atgacaaaga cgagtgttga ttgcacaatg 2220

tatatctgcg gagattcaac agaatgctca aaccttcttc ttcaatatgg atcattctgt 2280

actcaactta acagagcact tacaggaatc gcagttgaac aagataagaa tacccaagaa 2340

gtattcgccc aggttaaaca aatctataag acaccgccta ttaaagattt cggcggtttc 2400

aatttctcac agatattgcc cgacccttca aagccctcca agcgtagctt tatcgaagat 2460

ctgttgttca acaaagttac acttgcagat gcaggattta tcaaacaata tggagattgc 2520

ctgggtgaca ttgccgccag agatcttatc tgcgcacaga agttcaatgg acttacagtt 2580

cttccaccac ttcttacaga tgaaatgatc gcacaatata catcagcact tcttgcagga 2640

acaatcacat caggatggac atttggagca ggagcagcac ttcaaatccc atttgcaatg 2700

caaatggcat atagatttaa cggaatcgga gttacacaga atgtacttta tgagaatcag 2760

aaactgatag caaaccaatt taactcagca atcggaaaga ttcaggattc actttcatca 2820

acagcatcag cacttggaaa gttacaggat gttgttaacc agaatgccca agcacttaac 2880

acacttgtta aacaactttc atcaaacttt ggagcaatct catcagttct taacgatatc 2940

ctttcaagac ttgataaagt tgaagcagaa gttcagattg accgtctcat aactggaaga 3000

cttcaatcac ttcaaacata tgttacacaa caacttatca gagcagcaga aatcagagca 3060

tcagcaaacc ttgcagcaac aaagatgtcc gaatgcgttc ttggacaatc aaagcgtgtc 3120

gatttctgtg gtaagggtta tcaccttatg tcatttccac aatcagcacc acatggagtt 3180

gtcttcctcc acgttacata tgttccagca caagagaaga atttcacaac agcaccagca 3240

atctgccatg atggaaaggc gcacttccct cgggaaggag tgttcgtaag caacggaaca 3300

cattggtttg ttacacaaag aaacttctac gagccacaaa tcatcacaac agataacaca 3360

tttgtttcag gaaactgcga tgttgttatc ggaatcgtta acaacacagt ttatgatcca 3420

ctgcagccgg agctcgactc cttcaaggag gagctcgaca agtactttaa gaatcataca 3480

tcaccagatg ttgatctggg tgacataagc ggcatcaacg catcagttgt taacatccag 3540

aaggaaattg accgcctgaa tgaggttgca aagaatttga acgaatcact tatcgatctt 3600

caagaacttg gaaagtacga gcaatatatc aaatggccat ggtatatctg gcttggattt 3660

atcgcaggac ttatcgcaat cgttatggtt acaatcatgc tttgctgcat gacatcatgc 3720

tgctcgtgct tgaaaggatg ctgctcatgc ggttcctgct gcaaatttga tgaagatgat 3780

tcagaaccag ttcttaaagg agttaaactt cattatacat ga 3822

<210> 6

<211> 228

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgtattcat ttgtttcaga agaaacagga acacttatcg ttaactcagt gttactcttc 60

ctcgctttcg tcgtttttct attagtgacg cttgcaatcc ttacagcact tagactttgc 120

gcatattgct gcaatattgt aaatgtaagt ctagtgaagc cgtctttcta cgtatacagc 180

agagttaaga atttaaactc atcaagagtt ccagatcttc ttgtttga 228

<210> 7

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggcagatt caaacggaac aatcacagtt gaagaactta agaagttgtt ggaacaatgg 60

aacttggtaa ttggcttcct gtttcttaca tggatctgcc ttcttcaatt tgcatatgca 120

aacagaaaca gatttcttta tatcatcaaa cttatctttc tttggcttct ttggccagtt 180

acacttgcat gctttgttct tgcagcagtt tatagaatca actggatcac aggaggaatc 240

gcaatcgcaa tggcatgcct tgttggactt atgtggcttt catatttcat agcgtcattt 300

agattattcg cgaggacaag atcaatgtgg tcatttaacc cagaaacaaa catccttctt 360

aacgttccac ttcatggaac aatccttaca agaccacttc ttgaatcaga actcgtcata 420

ggcgctgtga tccttagagg acatcttaga atcgcaggac atcatcttgg aagatgcgat 480

atcaaagatc ttccaaagga gattacagtt gcaacatcaa gaacactttc atattataaa 540

cttggagcat cacaaagagt tgcaggagat tcaggatttg cagcatattc aagatataga 600

atcggaaact ataaacttaa cacagatcat tcatcatcat cagataacat cgcacttctt 660

gttcaatga 669

<210> 8

<211> 1260

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgtcagata acggaccaca gaatcagaga aacgcaccaa gaatcacatt tggaggacca 60

tcagattcaa caggatcaaa ccagaatggc gaaagatcag gagcaagatc aaagcagcgc 120

agaccacaag gacttccaaa caacacagca tcatggttta cagcacttac acaacatgga 180

aaggaggacc ttaaatttcc aagaggacaa ggagttccaa tcaacacaaa ctcatcacca 240

gatgatcaaa tcggatatta tagaagagca acaagaagaa tcagaggagg agatggaaag 300

atgaaggatc tttcaccaag atggtatttc tactaccttg gaacaggacc agaagcagga 360

cttccatatg gagcaaacaa agatggaatc atctgggttg caacagaagg agcacttaac 420

acaccaaagg accacatcgg aacaagaaac ccagcaaaca acgcagcaat cgttcttcaa 480

cttccacaag gaacaacact tccaaagggt ttctatgccg aaggatcaag aggaggatca 540

caagcttctt cgcgttcgtc cagtcgctcc cgcaacagtt cgagaaattc gacgcctggc 600

tcatcaagag gaacatcacc agcaagaatg gcaggaaacg gaggagatgc agcacttgca 660

cttcttcttc ttgatagact taaccaactt gaatcaaaga tgtcgggaaa gggtcagcaa 720

caacaaggac agacggtcac gaagaagtca gcagcagaag catcaaagaa gccgcggcag 780

aaacggaccg caacaaaggc ttacaacgtt acacaagcat ttggaagaag aggaccagaa 840

caaacacaag gaaactttgg agatcaagaa cttatcagac aaggaacaga ttataaacat 900

tggccacaaa tcgcacaatt tgcaccatca gcatcagcat tcttcggtat gtcaagaatc 960

ggaatggaag ttacaccatc aggaacatgg cttacatata caggcgccat aaagctcgac 1020

gacaaggacc caaactttaa ggaccaggta attctgctga ataagcatat cgatgcatat 1080

aagactttcc caccaaccga gcccaagaag gacaagaaaa aaaaggcgga cgaaacacaa 1140

gcacttccac aaagacagaa gaagcagcaa acggtaaccc tattgccagc agcagatctt 1200

gatgatttct cgaaacagct acaacaatca atgtcatcag cagattcaac acaagcatga 1260

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

catcgttgaa gatgcctctg ccgac 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gatctagtaa catagatgac accgc 25

Claims

1. A method for preparing a novel coronavirus-like particle, comprising the steps of:

2. The method of claim 1, wherein the host is tobacco.

3. The method according to claim 1, wherein the nucleotide sequence of the nucleic acid fragment encoding the S protein is represented by SEQ ID No.5, the nucleotide sequence of the nucleic acid fragment encoding the E protein is represented by SEQ ID No.6, the nucleotide sequence of the nucleic acid fragment encoding the M protein is represented by SEQ ID No.7, and the nucleotide sequence of the nucleic acid fragment encoding the N protein is represented by SEQ ID No. 8.

4. The method according to any one of claims 1 to 3, wherein the S protein, the E protein, the M protein and the N protein are each independently expressed in the expression vector.

5. The method according to claim 4, wherein the expression vectors for expressing S, E, M and N proteins of the novel coronavirus include a first recombinant expression vector for expressing the S and E proteins and a second recombinant expression vector for expressing the M and N proteins, and the first and second recombinant expression vectors are introduced into the host by co-transfection.

6. The method according to claim 5, wherein the first recombinant expression vector is obtained by inserting a nucleic acid fragment encoding the S protein and a nucleic acid fragment encoding the E protein into a pHREAC vector, and the second recombinant expression vector is obtained by inserting a nucleic acid fragment encoding the M protein and a nucleic acid fragment encoding the N protein into a pHREAC vector.

7. The method according to claim 4, wherein the expression vector is prepared by inserting the nucleic acid fragment encoding the S protein, the nucleic acid fragment encoding the E protein, the nucleic acid fragment encoding the M protein and the nucleic acid fragment encoding the N protein into the same empty vector.

8. A novel coronavirus-like particle produced by the method for producing a novel coronavirus-like particle according to any one of claims 1 to 7.

9. Use of the novel coronavirus-like particle of claim 8 for the preparation of a medicament for the prevention or treatment of a novel coronavirus infection.

10. A novel coronavirus vaccine comprising the novel coronavirus-like particle of claim 8.