CN116726160A - Preparation and application of novel coronavirus mutant universal vaccine cRBD - Google Patents

Abstract

The application belongs to the technical field of biology, and particularly discloses preparation and application of a novel coronavirus mutant universal vaccine cRBD, which is characterized in that the novel coronavirus mutant universal vaccine cRBD is designed as shown in SEQ ID NO:2 or a nucleotide sequence as set forth in SEQ ID NO:1, the vaccine designed by the application has a certain immunogenicity and the produced antibody has broad spectrum, can generate neutralization effect on new variant strains such as Omicron, and the like, and suggests that the vaccine may have protective efficacy on various variant strains, thus indicating that the vaccine has potential as SARS-CoV-2 general vaccine.

Description

Preparation and application of novel coronavirus mutant universal vaccine cRBD

Technical Field

The application belongs to the technical field of biology, and particularly relates to preparation and application of a novel coronavirus mutant universal vaccine cRBD.

Background

The new coronavirus (SARS-CoV-2) is continuously transmitted in the population, so that new mutant strains are continuously appeared, and the large number of mutations of the mutant strains in the S protein region, especially RBD (Receptor binding domain) region, can not identify the neutralizing antibodies induced by the existing vaccine, so that the protection efficacy is lost, therefore, the development of a general vaccine capable of generating broad-spectrum protection efficacy against various mutant strains is urgently needed. The prior technical proposal mainly comprises: the inactivated vaccine is prepared by inactivating SARS-CoV-2 virus, and the preparation of the live virus requires repeated inactivation verification, has slow update times, does not have broad spectrum, and is difficult to cope with viruses with higher mutation frequency; mRNA vaccine is prepared into RNA preparation by utilizing RNA sequence design of virus protein, has large side effect, lower safety coefficient than other vaccines, needs a complex delivery system and is difficult to aim at the newly-developed strain; the recombinant protein vaccine directly expresses virus proteins by using an expression system, can be even designed into multivalent or multimeric vaccine, can simultaneously prevent a plurality of strains, but the multimeric protein can not completely display the whole appearance of the antigen after being folded, and is still difficult to cope with the newly-appearing strains; the different mutant strains are used for chimeric recombination of S protein structural domains and mutant reconstruction of recombinant protein vaccines, repeated screening and verification are needed, the protectiveness is difficult to determine, the working procedure is complex, and the cost is high.

There are many studies showing that multivalent or multimeric vaccines combining RBD or S proteins from different mutants have significantly improved protective efficacy against omacron and other variants. However, in previous studies we found that vaccines developed based on the omacron RBD protein containing the most variant mutation sites were very low in immunogenicity. The university of Xiamen team Xia Ningshao adopts a strategy of pedigree chimeric-mutation patch, namely, different mutant strains are selected for chimeric recombination and mutation transformation of the structural domain of the S protein, and the optimal combination is selected to realize wider antigen coverage. Based on the research and the discovery, we hypothesize whether the characteristic mutation sites of different strains can realize broad-spectrum antigen coverage by directly adopting a mutation transformation method on an original RBD sequence, based on the method, we design a plurality of brand-new transformation RBD sequences, and the RBD vaccine is expressed and purified by adopting a prokaryotic mode to immunize Balb/c mice and is detected by adopting a real and false virus neutralization experiment to immunize the mice so as to generate a neutralization antibody level, thereby solving the problem of insufficient broad-spectrum property of the existing vaccine, improving the broad-spectrum property of the vaccine and enabling the RBD vaccine to cope with the immune escape problem of the newly-appearing variant strain in the future.

Disclosure of Invention

The application mainly aims to provide a novel coronavirus mutant universal vaccine cRBD for solving the problems of insufficient broad spectrum of the existing vaccine, improving the broad spectrum of the vaccine and enabling the vaccine to cope with the immune escape problem of the mutant strain which is newly appeared in the future.

In order to achieve the above object, the present application provides the following technical solutions:

as one embodiment, the sequence set forth in SEQ ID NO:2 in the preparation of novel coronavirus mutant universal vaccine.

Further, the present application provides a polypeptide as set forth in SEQ ID NO:1, which consists of an amino acid sequence as set forth in SEQ ID NO:2, and performing transcription and translation on the nucleotide sequence shown in the formula 2.

As set forth in SEQ ID NO:1 in the preparation of novel coronavirus mutant universal vaccine.

Further, the application provides a recombinant vector comprising a sequence as set forth in SEQ ID NO:2, and a nucleotide sequence shown in the following formula. Preferably, the vector backbone is pET-28a (+).

Furthermore, the recombinant vector provided by the application can be applied to the preparation of novel coronavirus mutant universal vaccines.

Preferably, the novel coronavirus mutant is: alpha or Beta or Delta or omicon.

Further, the present application provides a specific application, comprising the steps of:

s1: the sequence set forth in SEQ ID NO:2 is connected to pET-28a (+), constructs a pET-28a (+) -cRBD1 recombinant vector, and then is transferred into escherichia coli BL21 (DE 3) for expression;

s2: after induced expression and culture of escherichia coli BL21 (DE 3) containing the recombinant vector, centrifugally collecting thalli, re-suspending thalli by using a lysate to crack the thalli, and collecting sediment;

s3: the expressed cRBD proteins are all in inclusion body precipitation, and then the cRBD proteins are extracted and purified from inclusion bodies;

s4: mixing the purified cRBD protein with AddaS03 oil-in-water type nano emulsion adjuvant according to the volume ratio of 1:1 to obtain the novel coronavirus mutant universal vaccine.

In step S2, the lysate is composed of the active protein extraction reagent of the Biyun bacteria, 1% protease inhibitor, 10mM EDTA, 0.1mg/ml lysozyme and 10U/ml super nuclease.

Further, in step S3, the steps of extracting and purifying the cRBD protein from inclusion bodies are as follows:

s3-1: washing inclusion bodies with a washing buffer consisting of 20mM Tris-HCl,0.5M NaCl,2M urea, 1% Triton-X100, pH=8.0, resuspending the pellet in an inclusion body wash, sonicating, collecting the pellet for 20min, and repeating the step once;

s3-2: then dissolving inclusion bodies, dissolving the washed inclusion bodies in a denaturation buffer solution, wherein the denaturation buffer solution consists of 20mM Tris-HCl,0.5M NaCl,8M urea, 20mM beta-mercaptoethanol and pH=8.0, completely resuspending the inclusion bodies by ultrasound, and stirring and dissolving at 4 ℃ for overnight;

s3-3: collecting the protein solution dissolved and denatured, centrifuging the denatured protein solution at 8000rpm for 30min at 4deg.C, retaining supernatant, filtering with 0.22 μm filter membrane, and measuring protein concentration (well known as century) by BCA method; recovering inclusion body protein, slowly dripping the supernatant of the dissolved inclusion body into inclusion body renaturation buffer solution to ensure that the concentration after dilution is about 0.1mg/ml, wherein the renaturation buffer solution consists of 20mM Tris-HCl,0.5M NaCl,2M urea, 20% glycerol and 2mMGSH,0.2mM GSSG,pH =8.0;

s3-4: standing at 4 ℃ for renaturation for 24 hours; purifying cRBD protein after renaturation, adding imidazole with the final concentration of about 20mM into the obtained renaturation solution, and filtering with a 0.22um filter membrane;

s3-5: protein purification was performed using Akta protein purifier; after loading, the column is equilibrated with 20mM imidazole, 20mM Tris-HCl,0.5M NaCl,pH =8.0 buffer until the ultraviolet absorption at 280nm is leveled with the baseline, 1.5ml/min is loaded, 500mM imidazole, 20mM Tris-HCl and 0.5M NaCl buffer are used for eluting protein, and outflow near the peak of the ultraviolet absorption at 280nm is collected to obtain purified RBD protein;

s3-5: protein solutions were desalted using a Hi Prep 26/10 desalting column and replaced into PBS buffer.

The application adopts a mutation transformation method to introduce characteristic mutation sites and immune escape key sites of different strains on an original RBD sequence to realize broad-spectrum antigen coverage, and specifically designs 1 brand-new modified RBD sequence cRBD-1, such as SEQ ID NO:2, the coded amino acid is shown as SEQ ID NO:1, expressing and purifying Balb/c mice by adopting a prokaryotic mode, and detecting the neutralizing antibody level of the RBD vaccine after the mice are immunized by adopting a real and fake virus neutralization experiment.

In the present application, it was found that the mice developed neutralizing antibodies against various mutants (Alpha, beta, delta, omicron) after three immunizations, wherein the average neutralizing antibody titer against Omicron ba.1, ba.2 mutant was 10 ³ -10 ⁴ Average neutralizing antibody titre against Omicron ba.5 mutant was 10 ² -10 ³ The first synthetic RBD sequence is designed before the BA.5 mutant strain appears, which shows that the designed vaccine has certain immunogenicity and the generated antibody has broad spectrum, can generate neutralization effect on the newly appearing variant strains such as Omicron and the like, and shows that the vaccine possibly has protective efficacy on various variant strains, thus showing that the vaccine has potential as SARS-CoV-2 general vaccine.

Drawings

FIG. 1 is a schematic diagram of a novel RBD sequence key mutation site designed based on an original strain S protein RBD 1;

FIG. 2 shows the effect of successfully expressing and purifying cRBD1 proteins; (M: protein Maker cRBD1: expressed purified cRBD1 protein NC: negative cell control PC: RBD standard control);

FIG. 3 graphs of serum BA.2 binding antibody effects after cRBD1 immunization of BALB/c mice; (Low: 5 μg Low dose group high:25 μg high dose group)

FIG. 4 shows a graph of the effect of neutralizing antibodies against serum of different strains after cRBD1 immunization; (Low: 5 μg Low dose group high:25 μg high dose group)

FIG. 5 shows a graph of pseudovirus effects on newly emerging different strains after cRBD1 immunization; (Low: 5 μg Low dose group high:25 μg high dose group)

FIG. 6 graphs of the results of changes in body temperature, body weight, and nasopharyngeal swab of Omacron BA.5 variant after immunization of BALB/c mice with cRBD1 vaccine; (Low: 5 μg Low dose group high:25 μg high dose group)

FIG. 7 shows graphs of results of lung, turbinate and tracheal viral load detection of Omacron BA.5 variant after immunization of BALB/c mice with cRBD1 vaccine; (Low: 5 μg Low dose group high:25 μg high dose group)

FIG. 8 lung pathology of 5dpi challenge to Omacron BA.5 variant after immunization of BALB/c mice with cRBD1 vaccine;

FIG. 9 shows the results of lung pathology detection analysis of 5dpi of Omacron BA.5 variant after immunization of BALB/c mice with cRBD1 vaccine; (Low: 5 μg Low dose group high:25 μg high dose group);

Detailed Description

The conception and the technical effects produced by the present application will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, features and effects of the present application. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have meanings commonly understood by those of ordinary skill in the art, and the purchase of goods in the test methods, such as those not explicitly stated herein, is performed under conventional conditions or conditions suggested by the manufacturer, and reagents or equipment used, such as those not explicitly stated by the manufacturer, may be obtained by commercially available purchase of conventional products.

Examples

1. Construction of antigen sequences

The antigen analysis and comparison of the B cell antigen epitope prediction tool Bepipred Linear Epitope Prediction 2.0.0 of an on-line platform of IEDB (www.iedb.org) are used for carrying out antigen analysis and comparison on the S protein RBD regions of different mutant strains of SARS-CoV-2, the sites without obvious change of antigenicity in each mutant strain are reserved, the mutation sites which appear in each mutant strain are selected, and simultaneously, the sites which are reported by literature and are critical to immune escape in each mutant strain are combined, so that a brand-new cRBD1 antigen sequence (figure 1) is designed on the basis of the S protein RBD of the original strain, and a nucleic acid sequence is generated by carrying out E.coli codon optimization on the sequence, wherein the sequence is shown as SEQ ID NO:2, the amino acid sequence obtained by the nucleotide coding is shown as SEQ ID NO: 1.

2. Vector construction and expression

This sequence was ligated into pET-28a (+) prokaryotic expression vector and then transferred into E.coli BL21 (DE 3) (Prinsepia TSC-E01) for expression, first, 4ml of pET-28a-cRBD 1-transformed overnight broth was inoculated into 400ml of medium (800 ml Erlenmeyer flask), shake-cultured at 37℃at 220rpm until OD600 = 0.4-0.8 (2-4 h), and then 0.5mM IPTG (s Soxhlatin: I1020) was added for induction of expression for 6h.

The cells were collected by centrifugation, 30ml of the lysate was resuspended, lysed at room temperature 15Min, and the pellet was collected by centrifugation at 8000rpm for 20 Min. The lysate contained Biyun bacterial active protein extraction reagent, 1% protease inhibitor (b Biyun (P1026)), 10mM EDTA (P1026)), 0.1mg/ml lysozyme (Biyun (ST 206)), and 10U/ml supernuclease (Biyun (D7121)). The RBD proteins expressed at this time are all in inclusion body pellet, and then the proteins are extracted and purified from inclusion bodies.

3. Mycoprotein acquisition and purification

First, washing buffer (20 mM Tris-HCl (Bio (B548140-0500)), 0.5M NaCl (bio (B548140-0500)), 2M urea (bio (A600148-0002)), 1% Triton-X100 (Soliebao (IT 9100)), washing inclusion bodies, and re-suspending the pellet in inclusion body washing liquid by ultrasonic, centrifuging at 8000rpm at 4 ℃ for 20min, repeating for 2 times; the inclusion bodies were then dissolved, the washed inclusion bodies were dissolved in denaturation buffer (20 mM Tris-HCl,0.5M NaCl,8M urea, 20mM beta-mercaptoethanol (Sigma (M3148)), sonicated to completely resuspend the inclusion bodies, stirred at 4℃overnight (14-16 h), denatured protein solution was centrifuged at 8000rpm at 4℃for 30min, the supernatant was retained, filtered through a 0.22 μm filter, the protein concentration was measured by BCA method, inclusion body proteins were recovered, the supernatant of the dissolved inclusion bodies was slowly dropped into inclusion body renaturation buffer (20 mM Tris-HCl,0.5M NaCl,2M urea, 20% glycerol (BioFrox, 1280ML 500)), 2mM GSH (1.5 mM GSSG (2 mM GSSG) (524-0005)), and allowed to stand at 4℃for about 24h after dilution, and purified by adding imidazole D to a final solution (20 mM 100-6000.277)), thereby obtaining a final solution of imidazole (20 mM biological filter, which was filtered.

Protein purification was performed using an Akat Go protein purifier, and after column loading according to the packing procedure, the Ni column (bio (C600792)) was equilibrated with baseline for uv absorption at 280nm using 20mM imidazole, 20mM Tris-HCl,0.5M NaCl buffer; then the renaturated protein solution is slowly injected into a sample inlet at a flow rate of 1.5 ml/min; the column was then equilibrated with 50mM imidazole, 20mM Tris-HCl,0.5M NaCl buffer to 280nm UV absorbance level with baseline; finally, 500mM imidazole, 20mM Tris-HCl and 0.5M NaCl buffer solution are used for eluting protein, and the eluent is collected when a 280nm ultraviolet absorption curve peaks, so that the purified RBD protein is obtained.

Then protein desalting is carried out, a protein dissolving system is replaced by PBS buffer solution, and after a desalting column is washed by PBS with double column volume, a protein eluent is put on the column with the flow rate of 2ml/min; eluting the protein by using PBS, and collecting the eluent when the ultraviolet absorption of 280nm starts for the first time to obtain desalted cRBD protein.

4. Vaccine preparation

Protein is diluted to 0.5mg/ml and 0.1mg/ml respectively after being quantified by BCA, and is mixed with AddaS03 ™ oil-in-water nano emulsion adjuvant (InvivoGen (vac-as 03-10)) according to the ratio of 1:1 respectively to prepare high-dose group vaccine and low-dose group vaccine, and 100 ul/BALB/c mice are immunized and then the effectiveness and broad spectrum of the vaccine are proved through experiments such as serum binding antibody, serum neutralizing antibody, pseudovirus infection and the like.

In the application, the following components are added:

the cRBD1 amino acid sequence SEQ ID NO:1:

RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN

the nucleic acid sequence SEQ ID NO after cRBD1 optimization: 2: CGTGTTCAACCTACCGAATCTATAGTGCGTTTTCCTAATATCACCAATTTATGTCCTTTCGATGAAGTTTTTAACGCAACTCGTTTTGCTTCAGTTTATGCTTGGAATCGTAAAAGAATTAGTAACTGTGTTGCAGATTACAGTGTTCTGTATAATAGCGCCTCATTTAGTACCTTTAAGTGTTATGGTGTTAGTCCGACCAAGCTGAATGATCTGTGTTTTACCAATGTGTATGCGGATTCCTTTGTTATACGTGGTGATGAGGTTCGTCAGATTGCACCGGGTCAGACCGGTAATATTGCAGATTATAATTATAAGCTGCCCGATGATTTTACCGGTTGTGTTATTGCCTGGAATAGTAATAAACTGGATAGTAAAGTGGGTGGTAATTATAATTACCGTTATCGGCTGTTCCGTAAAAGCAACCTGAAACCGTTTGAACGGGATATTAGCACAGAAATTTACCAAGCCGGCAGCAAACCGTGTAATGGAGTGGCAGGATTTAACTGTTACTTCCCTCTGAGATCTTATGGTTTCCGGCCGACCTATGGTGTTGGACATCAGCCGTATCGCGTTGTGGTTTTATCATTTGAGCTGTTACATGCACCGGCAACAGTTTGTGGTCCGAAGAAATCCACCAATCTTGTTAAGAATAAGTGTGTTAACTTC

The effect achieved by the embodiment 1 of the application is verified:

1. the pER-28a prokaryotic expression vector is used for successfully expressing cRBD1 protein in BL21 (DE 3) competent cells, recovering the protein after washing, ultrasonic crushing, dissolving and filtering of inclusion bodies, diluting the protein by renaturation buffer solution for renaturation, purifying the protein by a protein purifier, and finally, detecting and identifying by RBD specific antibody.

As can be seen from FIG. 2, coomassie brilliant blue (Solebao (C8430)) staining was performed according to conventional procedures, and the results showed that the expressed and purified cRBD1 protein was of correct size and relatively single, whereas WB experiments showed that the RBD protein could be recognized by RBD specific antibodies and the size was consistent with the RBD standard, indicating that we successfully expressed and purified the cRBD1 protein.

2. The RBD binding antibody titer of serum BA.2 was significantly increased after the cRBD-1 immunization of BALB/c mice: immunization was performed three times at 0d,21d and 42d, respectively.

As can be seen in fig. 3, RBD-specific binding antibodies to ba.2 were generated with post-one-shot 14d serum, whereas post-two-shot and three-shot 14d (d 35 and d 56) significantly increased their serum binding antibody levels.

After cRBD-1 immunization, certain serum neutralizing antibodies are generated for different strains

As can be seen from fig. 4, the post-two-exemption 14d (35 d) and the post-three-exemption 14d (56 d) both generate higher neutralizing antibodies against strains such as Prototype, alpha, beta, ba.1, ba.2 and the like, and also generate certain neutralizing antibodies against newly-appeared strains such as ba.5 and XBB, but the strains such as ba.5 and XBB do not appear yet in the sequence design, which indicates that the vaccine has protection potential against variant strains which do not appear yet besides the protection effect on the strains appearing in the past.

A certain serum neutralizing antibody is also generated on the newly developed pseudoviruses with different strains after the cRBD1 is immunized

As can be seen from the graph, the post-di-exemption 14d (35 d) and the post-tri-exemption 14d (56 d) both generate high neutralizing antibodies against BF.7 and BQ.1.1 pseudoviruses, and also generate certain neutralizing antibodies against XBB.1.16 and CH.1.1, so that the vaccine has the protection potential against variant strains which do not appear.

Protective effect of cRBD1 vaccine on Omacron BA.5 variant after immunization of BALB/c mice

Results of changes in body temperature, body weight, and nasal/pharyngeal swab following Omicron BA.5 challenge

As shown in fig. 6, both the body weight and body temperature fluctuation range of the cRBD1 vaccine immunized high dose and low dose groups after challenge with the ba.5 variant were smaller than those of the control group; both nasal and pharyngeal swabs had lower viral loads than the control group. It is demonstrated that the vaccine can improve and alleviate clinical symptoms after virus infection.

Lung, turbinate and trachea viral load detection result after virus attack

As shown in fig. 7, the high and low dose groups immunized with crbd1 vaccine had significantly lower viral genome viral loads (gRNA) in lung, tracheal and turbinate tissues after 5dpi challenge with the ba.5 variant than the model control group; subgenomic viral load (sgRNA), representing the level of new coronavirus replication, was undetectable in lung tissue and organ tissue, with lower levels detected in turbinate tissue, indicating that new coronavirus ba.5 had stopped replication in the first two respiratory tissues above, and lower levels in turbinate tissue. It is demonstrated that the vaccine can significantly reduce and inhibit the replication of viruses in the upper and lower respiratory tracts of the respiratory system after immunization.

Lung pathology detection result of 5dpi

As shown in fig. 8 and 9, the high-dose and low-dose groups immunized with the crbd1 vaccine had significantly lower lung tissue pathological injury scores than the model control group after 5dpi challenge with the ba.5 variant, and had a certain dose-dependence on the pathological injury protection of lung tissue. The vaccine can obviously reduce the pathological damage degree of virus infection to lung tissues after immunization, and the effect of high dose is obviously better than that of low dose.

In combination, the application designs 1 brand newThe modified RBD sequence is expressed and purified in a prokaryotic mode, and then Balb/c mice are immunized and the level of neutralizing antibodies is generated after the RBD vaccine is used for detecting the mice immunized by using a real and pseudovirus neutralization experiment. It was found that mice developed neutralizing antibodies to different mutants (Alpha, beta, delta, omacron) after three immunizations, wherein the average neutralizing antibody titer against omacron ba.1, ba.2 mutants was 10 ³ -10 ⁴ Average neutralizing antibody titre against Omicron ba.5 mutant was 10 ² -10 ³ While the first synthetic RBD sequence was designed to be completed before the ba.5 mutant appeared. This demonstrates that the designed vaccine has a certain immunogenicity and the produced antibodies have a broad spectrum, can produce a neutralizing effect on newly emerging variants such as Omicron, and suggests that the vaccine may have protective efficacy on various variants, demonstrating the potential of the vaccine as a general type SARS-CoV-2 vaccine.

The foregoing is only a preferred embodiment of the present application. It should be noted that the above examples are only for illustrating the present application and are not intended to limit the scope of the present application. It will be apparent to those skilled in the art that modifications may be made without departing from the principles of the application, and such modifications are intended to be within the scope of the application.

Claims (10)

1. As set forth in SEQ ID NO:2 in the preparation of novel coronavirus mutant universal vaccine.

2. As set forth in SEQ ID NO:1, characterized in that the amino acid sequence consists of the amino acid sequence shown in SEQ ID NO:2, and performing transcription and translation on the nucleotide sequence shown in the formula 2.

3. As set forth in SEQ ID NO:1 in the preparation of novel coronavirus mutant universal vaccine.

4. A recombinant vector comprising a sequence as set forth in SEQ ID NO:2, and a nucleotide sequence shown in the following formula.

5. The recombinant vector according to claim 4, wherein the vector backbone is pET-28a (+).

6. The use of the recombinant vector according to claim 4 for the preparation of novel coronavirus mutant universal vaccines.

7. The use according to claim 1 or 3 or 6, wherein the novel coronavirus mutant is: alpha or Beta or Delta or omicon.

8. The use according to claim 7, characterized by the steps of:

s1: the sequence set forth in SEQ ID NO:2 is connected to pET-28a (+), and then transferred into escherichia coli BL21 for expression after constructing a pET-28a (+) -cRBD1 recombinant vector;

s2: after induced expression and culture of escherichia coli BL21 containing the recombinant vector, centrifugally collecting thalli, and re-suspending thalli by using a lysate to crack the thalli and collecting sediment;

s3: the expressed cRBD proteins are all in inclusion body precipitation, and then the cRBD proteins are extracted and purified from inclusion bodies;

s4: mixing the purified cRBD protein with AddaS03 oil-in-water type nano emulsion adjuvant according to the volume ratio of 1:1 to obtain the novel coronavirus mutant universal vaccine.

9. The use according to claim 8, wherein in step S2, the lysate consists of the active protein extraction reagent of the bacteria Biyun, 1% protease inhibitor, 10mM EDTA, 0.1mg/ml lysozyme and 10U/ml supernuclease.

10. The use according to claim 8, wherein in step S3, the steps of extracting and purifying the cRBD protein from inclusion bodies are:

s3-1: washing inclusion bodies with a washing buffer consisting of 20mM Tris-HCl,0.5M NaCl,2M urea, 1% Triton-X100, pH=8.0, resuspending the pellet in an inclusion body wash, sonicating, collecting the pellet for 20min, and repeating the step once;

s3-2: then dissolving inclusion bodies, dissolving the washed inclusion bodies in a denaturation buffer solution, wherein the denaturation buffer solution consists of 20mM Tris-HCl,0.5M NaCl,8M urea, 20mM beta-mercaptoethanol and pH=8.0, completely resuspending the inclusion bodies by ultrasound, and stirring and dissolving at 4 ℃ for overnight;

s3-3: collecting the protein solution dissolved and denatured, centrifuging the denatured protein solution at 8000rpm at 4deg.C for 30min, retaining supernatant, filtering with 0.22 μm filter membrane, and measuring protein concentration by BCA method; recovering inclusion body protein, slowly dripping the supernatant of the dissolved inclusion body into inclusion body renaturation buffer to ensure that the concentration is 0.1mg/ml after dilution, wherein the renaturation buffer consists of 20mM Tris-HCl,0.5M NaCl,2M urea, 20% glycerol and 2mMGSH,0.2mM GSSG,pH =8.0;

s3-4: standing at 4 ℃ for renaturation for 24 hours; purifying cRBD protein after renaturation, adding imidazole with the final concentration of 20mM into the obtained renaturation solution, and filtering by a 0.22um filter membrane;

s3-5: protein purification was performed using Akta protein purifier; after loading, the column is equilibrated with 20mM imidazole, 20mM Tris-HCl,0.5M NaCl,pH =8.0 buffer until the ultraviolet absorption at 280nm is leveled with the baseline, 1.5ml/min is loaded, 500mM imidazole, 20mM Tris-HCl and 0.5M NaCl buffer are used for eluting protein, and outflow near the peak of the ultraviolet absorption at 280nm is collected to obtain purified RBD protein;

s3-5: protein solutions were desalted using a Hi Prep 26/10 desalting column and replaced into PBS buffer.