CN117070535A - Novel coronavirus mutant broad-spectrum vaccine with humoral immunity and cellular immunity functions - Google Patents

Abstract

The application belongs to the technical field of biomedicine, and particularly discloses a novel coronavirus mutant broad-spectrum vaccine with humoral immunity and cellular immunity functions, which comprises the following components in percentage by weight: 1 or SEQ ID NO:3 in the preparation of a novel coronavirus mutant universal vaccine, wherein the novel coronavirus mutant comprises the following components: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16, immunizing Balb/c mice after expression purification by adopting a prokaryotic mode, and detecting the level of neutralizing antibodies generated after the RBD vaccine is immunized by adopting a real and pseudovirus neutralization experiment, thereby solving the problem of insufficient broad-spectrum property of the existing vaccine and improving the broad-spectrum property of the vaccine.

Description

Novel coronavirus mutant broad-spectrum vaccine with humoral immunity and cellular immunity functions

Technical Field

The application belongs to the technical field of biomedicine, and particularly relates to a novel coronavirus mutant broad-spectrum vaccine with humoral immunity and cellular immunity functions.

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.

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 above research and 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 the original RBD sequence, based on the method, we design a plurality of brand-new modified RBD sequences, such as cRBD1 disclosed in patent 202310999162.X, the RBD vaccine is subjected to expression purification in a prokaryotic mode, and then the RBD vaccine is subjected to immunity of Balb/c mice and is detected by adopting a real and pseudovirus neutralization experiment to generate a neutralization antibody level, so that the defect of the broad-spectrum property of the existing vaccine is solved, the broad-spectrum property of the vaccine is improved, the mutation sites of different strains are different, and the screening of the vaccine with stronger resistance and higher broad-spectrum property is needed for coping with the problem of immune escape of more variant strains.

Disclosure of Invention

The application mainly aims to provide a novel coronavirus mutant broad-spectrum vaccine with humoral immunity and cellular immunity functions, so as to solve the problem of insufficient broad-spectrum property of the existing vaccine and improve the broad-spectrum property of the vaccine.

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

the present application provides a polypeptide as set forth in SEQ ID NO:1 or SEQ ID NO:3 in the preparation of a novel coronavirus mutant universal vaccine, wherein the novel coronavirus mutant is: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16.

Further, provided are the amino acid sequences set forth in SEQ ID NOs: 2 or SEQ ID NO:4, which consists of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and performing transcription and translation on the nucleotide sequence shown in the formula 3.

Further, provided are the amino acid sequences set forth in SEQ ID NOs: 2 or SEQ ID NO:4 in the preparation of a novel coronavirus mutant universal vaccine, wherein the novel coronavirus mutant is: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16.

Further, the application also provides a recombinant vector, which comprises a sequence shown as SEQ ID NO:1 or SEQ ID NO:3, and a nucleotide sequence shown in 3.

Preferably, the recombinant vector backbone is pET-28a.

Further, the recombinant vector is applied to preparing a novel coronavirus mutant universal vaccine, and the novel coronavirus mutant is: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16.

Further, the preparation of the novel coronavirus mutant universal vaccine comprises the following steps:

s1: the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, connecting the nucleotide sequence shown in the 3 to pET-28a, constructing a pET-28a-cRBD recombinant vector, and transferring into escherichia coli BL21 for expression;

s2: after the escherichia coli BL21 containing the recombinant vector is subjected to induced expression and culture, the thalli are collected by centrifugation, the thalli are resuspended by adopting a lysate, and the thalli are lysed and the precipitate is collected.

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.

The application achieves the following effects:

according to the application, 2 brand-new modified RBD sequences are designed, balb/c mice are immunized by adopting a prokaryotic mode after expression and purification, and neutralizing antibody levels are generated after the RBD vaccine is used for immunizing the mice by adopting 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, xbb.1.5, eg.5.1 mutants was 10 ² -10 ³ The synthesis of cRBD2 and cEBD3 sequences was designed before the occurrence of the BA.5, XBB.1.5, EG.5.1 mutants. 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 appeared variants such as Omicron, and suggests that it may have protective efficacy on a variety of variants, demonstrating that the vaccine has a broad spectrum as SARS-CoV-2 vaccineIs not limited by the potential of (a).

Drawings

FIG. 1 is a schematic diagram of key mutation sites of novel cRBD2 and cRBD3 sequences designed based on the RBD of an original strain S protein;

FIG. 2 shows a graph of the results of electrophoresis staining of cRBD2 and cRBD3 proteins successfully expressed and purified; ( A. Protein electrophoresis staining results of the expressed and purified cRBD2 and cRBD 3; B. identifying the results of the cRBD2 and cRBD3 recombinant proteins by WB detection using the RBD antibody; m: protein Maker cRBD2/cRBD3: expression of purified cRBD2/cRBD3 protein NC: negative cell control PC: RBD standard control )

FIG. 3 is a graph showing the results of detection of IgG-specific binding antibodies at various time points after mice are immunized with recombinant protein vaccines; ( Crbd2; crbd3, low: low dose group high: high dose group )

FIG. 4 shows graphs of experimental results of cross-neutralization of serum of a three-way mice with a recombinant protein vaccine cRBD2 against true strains of different novel coronavirus variants; (Low: high dose group)

FIG. 5 graph of the effect of pseudoviruses on different strains, which are newly developed or not, on the formation of a major epidemic after immunization of mice (A. Measurement of the titer of neutralizing antibodies to pseudoviruses of serum portions of the novel coronavirus variants at different time points after immunization of mice with cRBD2; B. Measurement of the titer of neutralizing antibodies to pseudoviruses of serum portions of the novel coronavirus variants at different time points after immunization of mice with cRBD 3; low: low: high: dose group)

FIG. 6 is a representative graph of results of Elispot method on positive memory cells of IFN-gamma and IL-2 after stimulation of spleen PBMC with S protein of XBB strain after immunization with different vaccines;

FIG. 7 shows the results of detection of body temperature, body weight and throat swab viral load after Omicron XBB.1.16 challenge after immunization of cRBD2 vaccine, (A. XBB.1.16 challenge body weight change monitoring results after immunization of cRBD2 vaccine, (B. XBB.1.16 challenge body temperature change monitoring results after immunization of cRBD2 vaccine, (C. XBB.1.16 challenge throat swab viral load change monitoring results after immunization of cRBD2 vaccine), (D. XBB.1.16 challenge throat swab change monitoring results after immunization of cRBD2 vaccine; low: high: low: high: dose group

FIG. 8 shows the results of detection of body temperature, body weight and throat swab viral load after Omicron XBB.1.16 challenge after immunization of cRBD3 vaccine, (A. XBB.1.16 challenge body weight change monitoring results after immunization of cRBD3 vaccine, (B. XBB.1.16 challenge body temperature change monitoring results after immunization of cRBD3 vaccine, (C. XBB.1.16 challenge throat swab viral load change monitoring results after immunization of cRBD3 vaccine), (D. XBB.1.16 challenge throat swab change monitoring results after immunization of cRBD3 vaccine; low: high: low: high: dose group

FIG. 9 shows graphs of results of lung and rhinovirus load detection after Omicron XBB.1.16 challenge with cRBD2 vaccine, (A. CRBD2 vaccine, omicron XBB.1.16 challenge with lung viral genome load (gRNA) detection after B. CRBD2 vaccine, omicron XBB.1.16 challenge with rhinovirus genome load (gRNA) detection after C. CRBD2 vaccine, omicron XBB.1.16 challenge with lung viral subgenomic load (sgRNA) detection after D. CRBD2 vaccine, omicron XBB.1.16 challenge with rhinovirus subgenomic load (sgRNA) detection after Low-dose group high-dose group

FIG. 10 shows graphs of results of lung and rhinovirus load detection after Omicron XBB.1.16 challenge with cRBD3 vaccine, (A. CRBD3 vaccine, omicron XBB.1.16 challenge with lung viral genome load (gRNA) detection after B. CRBD3 vaccine, omicron XBB.1.16 challenge with rhinovirus genome load (gRNA) detection after C. CRBD3 vaccine, omicron XBB.1.16 challenge with lung viral subgenomic load (sgRNA) detection after D. CRBD3 vaccine, omicron XBB.1.16 challenge with rhinovirus subgenomic load (sgRNA) detection after Low-dose group high-dose group

FIG. 11 shows the results of lung pathology tests at 5dpi by Omicron XBB.1.16 challenge after immunization with cRBD2 and cRBD3 vaccines (Low: low: high: low

FIG. 12 shows the results of the Omicron XBB.1.16 challenge 5dpi lung histopathological scoring analysis after immunization with cRBD2 and cRBD3 vaccines;

FIG. 13 shows graphs of experimental results of cross-neutralization of serum of a triple-immunity mouse with a cRBD3 recombinant protein vaccine against different strains of novel coronavirus; (Low: high dose group)

FIG. 14 shows the results of the cell immunity test (A. Analysis of IFN-gamma after cRBD1 immunization, B. Analysis of IL-2 after cRBD1 immunization, C. Analysis of IFN-gamma after cRBD2 immunization, D. Analysis of IL-2 after cRBD2 immunization, E. Analysis of IFN-gamma after cRBD3 immunization, F. Analysis of IL-2 after cRBD3 immunization).

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.

As some embodiments, the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 can be applied to preparing novel coronavirus mutant universal vaccines, wherein the novel coronavirus mutant comprises the following components: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16, etc.

As further embodiments, the amino acid sequence represented by SEQ ID NO:1 or SEQ ID NO:3, and the nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4, can be applied to preparing novel coronavirus mutant universal vaccines, wherein the novel coronavirus mutant comprises the following components: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16, etc.

As a more specific embodiment, the present application also provides a recombinant vector comprising a sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3, and a nucleotide sequence shown in 3. The recombinant vector can be applied to the preparation of novel coronavirus mutant universal vaccines, and the novel coronavirus mutants are as follows: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16, etc.

Example 1

1. Construction of antigen sequences

The antigen analysis and comparison of the S protein RBD regions of different mutant strains of SARS-CoV-2 are carried out by using a B cell antigen epitope prediction tool Bepipred Linear Epitope Prediction 2.0.0 of an IEDB (www.iedb.org) online platform, the sites without obvious change of antigenicity in each mutant strain are reserved, the mutation sites appearing in each mutant strain are selected, and simultaneously, the sites which are reported by the combined literature and are critical to immune escape in each mutant strain are combined, so that brand-new cRBD2 and cRBD3 antigen sequences (figure 1) are designed on the basis of the S protein RBD of the original strain, and the nucleotide sequence of the cRBD2 is shown as SEQ ID NO:1, the amino acid sequence is shown as SEQ ID NO:2, the nucleotide sequence of the cRBD3 is shown as SEQ ID NO:3, the amino acid sequence is shown as SEQ ID NO: 4.

2. Vector construction and expression

E.coli codon optimization is carried out on antigen sequences of cRBD2 and cRBD3, a nucleic acid sequence is respectively generated, the sequences are respectively connected to pET-28a prokaryotic expression vectors, pET-28a-cRBD2 and pET-28a-cRBD3 recombinant vectors are respectively constructed, the recombinant vectors are transferred into E.coli BL21 for expression, 4ml of overnight culture bacterial liquid transformed with pET-28a-cRBD is inoculated into 400ml of culture medium (800 ml bottle), shaking culture is carried out at 37 ℃ and 220rpm until OD600 = 0.4-0.8 (4 h), and then 0.5mM IPTG is added for induction 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 contains Biyun bacteria active protein extraction reagent, 1% protease inhibitor, 10mM EDTA, 0.1mg/ml lysozyme and 0.5% supernuclease. 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. Protein acquisition and purification

Washing the inclusion body with a washing buffer (20 mM Tris-HCl,0.5M NaCl,2M urea, 1% Triton-X100), and re-suspending the precipitate in the inclusion body washing solution by ultrasonic, centrifuging at 8000rpm at 4deg.C for 20min, repeating for 2 times; then dissolving inclusion bodies, dissolving the washed inclusion bodies in a denaturation buffer solution (20 mM Tris-HCl,0.5M NaCl,8M urea, 20mM beta-mercaptoethanol), completely suspending the inclusion bodies by ultrasound, stirring and dissolving overnight (16 h) at 4 ℃, centrifuging the denatured protein solution at 8000rpm for 30min at 4 ℃, retaining the supernatant, filtering with a 0.22 mu m filter membrane, and measuring the protein concentration by a BCA method; inclusion body protein recovery followed by renaturation of inclusion body protein the supernatant of the solubilized inclusion body was slowly added dropwise to inclusion body renaturation buffer (20 mM Tris-HCl,0.5M NaCl,2M urea, 20% glycerol, 2mMGSH,0.2mM GSSG) to give a concentration of about 0.1mg/ml after dilution. Standing at 4 ℃ for renaturation for about 24 hours; purifying RBD protein after renaturation, adding imidazole with the final concentration of about 20mM into the obtained renaturation solution, filtering with a 0.22um filter membrane, and purifying by using a protein purifier.

The purifier first equilibrates the column to 280nm UV absorbance level with baseline 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 starts to peak, so that the purified RBD protein is finally obtained.

4. Vaccine preparation

The purified RBD protein is mixed with AddaS03 ™ oil-in-water type nanoemulsion adjuvant in a ratio of 1:1 to prepare the vaccine, and the effectiveness and broad spectrum of the vaccine are proved by serum binding antibodies, serum neutralizing antibodies, pseudovirus neutralizing antibodies, virus attack protection experiments and the like after the BALB/c mice are immunized.

cRBD-2 nucleic acid (SEQ ID NO: 1)

CGTGTTCAACCTACCGAATCTATAGTGCGTTTTCCTAATATCACCAATTTATGTCCTTTCGGTGAAGTTTTTAACGCAACCCGTTTTGCTTCTGTTTATGCTTGGAATCGTAAAAGAATTAGTAACTGTGTTGCAGATTACAGTGTTCTGTACAATTTTGCCAGCTTTTCTACCTTTAAGTGTTATGGTGTTAGTCCCACCAAGTTAAATGATCTGTGTTTTACCAATGTCTACGCCGATAGCTTTGTTATTCGTGGTGATGAGGTTCGCCAGATTGCACCGGGTCAGACCGGTAATATTGCCGATTATAATTATAAGCTGCCGGATGATTTTACCGGTTGTGTTATTGCATGGAATTCAAATAAACTGGATAGTAAAGTGGGTGGTAATTATAATTACCGTTATCGGCTGTTTCGTAAGAGCAATCTGAAGCCGTTTGAGAGAGATATTTCTACAGAAATTTACCAAGCAGGCAGCAAACCATGTAATGGTGTTGCAGGTTTTAACTGTTACTTTCCTCTGCGTAGCTATGGTTTCCGGCCCACCTACGGTGTTGGTCACCAGCCGTATCGCGTTGTTGTTTTAAGTTTTGAGCTGCTGCATGCACCGGCTACCGTTTGTGGTCCTAAAAAGTCAACCAATTTAGTTAAGAACAAGTGTGTTAACTTC

cRBD-2 amino acid (SEQ ID NO: 2)

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNFASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

cRBD-3 nucleic acid (SEQ ID NO: 3)

CGTGTTCAACCTACCGAATCTATAGTGCGTTTTCCTAATATCACCAATTTATGTCCTTTCGGTGAAGTTTTTAACGCAACCCGTTTTGCTTCTGTTTATGCTTGGAATCGTAAAAGAATTAGTAACTGTGTTGCAGATTACAGTGTTCTGTACAATTTTGCCAGCTTTTCTACCTTTAAGTGTTATGGTGTTAGTCCCACCAAGTTAAATGATCTGTGTTTTACCAATGTCTACGCCGATAGCTTTGTTATTCGTGGTAATGAGGTTTCTCAGATTGCACCGGGTCAGACCGGTAATATTGCCGATTATAATTACAAACTGCCCGACGATTTTACCGGTTGTGTTATTGCATGGAATTCAAATAATCTGGATAGTAAAGTGGGTGGTAATTACAATTATCGTTATCGCCTGTTTCGTAAAAGCAACCTGAAGCCCTTCGAACGGGATATTAGCACGGAAATTTATCAAGCGGGAAGCAAACCGTGTAATGGTGTTGCAGGTGTTAACTGTTACTTTCCACTGCGTAGCTATGGTTTCCGGCCGACCTATGGTGTTGGACATCAGCCGTATAGAGTGGTTGTTTTAAGTTTTGAGCTGCTGCATGCCCCGGCGACCGTTTGTGGTCCGAAGAAGTCTACTAATTTAGTTAAGAATAAGTGCGTGAACTTC

cRBD-3 amino acid (SEQ ID NO: 4)

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNFASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVAGVNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

Vaccine effectiveness and broad-spectrum experiments prove

1. Successful expression and purification of the cRBD2 and cRBD3 proteins

As shown in FIG. 2, coomassie brilliant blue staining indicates that the sizes of expressed and purified cRBD2 and cRBD3 proteins are correct and single, and WB experiments indicate that the RBD proteins can be identified by RBD specific antibodies and the sizes are consistent with RBD standards, indicating that we successfully express and purify the cRBD2 and cRBD3 proteins.

2. Serum has obviously raised S protein antigen binding antibody titer to BA.2 after BALB/c mice are immunized by the cRBD2 and cRBD3 recombinant protein vaccine: immunization was performed three times at 0d,21d and 42d, respectively. As can be seen in FIG. 3, BA.2 serum-binding antibodies can be produced by post-one-shot 14d, while post-two-shot and three-shot 14d (d 35 and d 56) significantly increased the serum-binding antibody levels.

3. After three times of immunization, the cRBD2 and the cRBD3 generate certain serum neutralizing antibodies to different strains

As shown in fig. 4 and 13, it can be seen that 21d (63 d) after three immunizations all produced higher neutralizing antibodies against WT, alpha, beta, delta, BA.1, BA.2, BA.5, and also produced certain neutralizing antibodies against xbb.1.5 and eg.5.1, but the 2 sequences were designed so as not to appear ba.5 and XBB and eg.5 strains, indicating that the vaccine has cross-protection potential against the variants that have not appeared in the past except for the protection against the existing strains.

4. certain serum neutralizing antibodies are also generated to pseudoviruses of different strains which newly appear or do not form the main epidemic strains after the cRBD2 and the cRBD3 are immunized

As shown in fig. 5, both post-secondary immunization 14d (35 d) and post-tertiary immunization 14d (56 d) produced higher neutralizing antibodies against bf.7, bq.1.1 and ch.1.1 pseudoviruses, as well as some neutralizing antibodies against xbb.1.16, demonstrating the protective potential of cRBD2 and cRBD3 vaccines against variants that have not yet emerged.

5. The spleen PBMC immunocytes of mice after three immunizations of the cRBD1 disclosed in patent CN202310999162.X and the cRBD2, cRBD3 vaccines of the application were subjected to detection of the S protein antigen-stimulated cellular immune effect against XBB variant by the Elispot method, and the cellular immune levels of the two vaccines were assessed by IFN-gamma and IL-2 positive cell ratios, respectively.

As shown in fig. 6 and 14, the analysis results show that the cRBD1 vaccine has a lower level of cellular immunity effect after immunization, the cRBD2 can see a significant increase in cellular immunity effect, and the cRBD3 has a significant increase in cellular immunity effect; the enhancement of the cellular immunity effect shows that the two vaccines of the cRBD2 and the cRBD3 have a certain long-term immunity effect, the broad-spectrum effect of the vaccine on variant strains is better, and more effects refer to the table 1, and as can be seen from the table 1, the effects of the cRBD2 and the cRBD3 are better than those of the cRBD1.

TABLE 1 comparison of the effects of cRBD1, cRBD2, cRBD3 vaccines

6. Protective effect against Omicron XBB.1.16 variant after immunization of BALB/c mice with cRBD2 and cRBD3 vaccines

(1) As shown in fig. 7, the body temperature, the body weight and the virus load of the nasal/pharyngeal swab after the toxicity attack by Omicron xbb.1.16 after the immunization of the cRBD2 vaccine, the body weight and the body temperature fluctuation range of the high-dose and low-dose groups of the cRBD2 vaccine after the toxicity attack by the xbb.1.16 variant are smaller; 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.

(2) As shown in fig. 8, the body temperature, the body weight and the virus load of the nasal/pharyngeal swab after the toxicity of the Omicron xbb.1.16 after the immunization of the cRBD3 vaccine, the body weight and the body temperature fluctuation range of the high-dose and low-dose groups of the cRBD3 vaccine after the toxicity of the xbb.1.16 variant are smaller; 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.

(3) Results of lung and turbinate viral load detection after ompcron xbb.1.16 challenge following cRBD2 vaccine immunization as shown in figure 9, the lung and turbinate viral loads were significantly lower in the high and low dose groups following xbb.1.16 variant challenge at 5dpi than in the model control group. It is demonstrated that the replication of the virus in the upper and lower respiratory tract of the respiratory system can be significantly reduced after immunization with the vaccine.

(4) Results of lung and turbinate viral load detection after ompcron xbb.1.16 challenge following cRBD3 vaccine immunization as shown in figure 10, the lung and turbinate viral loads were significantly lower in the high and low dose groups following xbb.1.16 variant challenge at 5dpi than in the model control group. It is demonstrated that the replication of the virus in the upper and lower respiratory tract of the respiratory system can be significantly reduced after immunization with the vaccine.

(5) As shown in FIG. 11, the lung pathological damage scores of the high-dose and low-dose groups immunized by the cRBD2 and cRBD3 vaccines after the vaccine is immunized by the cRBD2 and cRBD3 are obviously lower than those of the model control group after the vaccine is immunized by the mutant strain XBB.1.16 by 5dpi, and the lung pathological damage protection has a certain dose dependence on the lung pathological damage protection. 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.

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 (7)

1. As set forth in SEQ ID NO:1 or SEQ ID NO:3 in the preparation of a novel coronavirus mutant universal vaccine, wherein the novel coronavirus mutant is: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16.

2. As set forth in SEQ ID NO:2 or SEQ ID NO:4, characterized in that the amino acid sequence consists of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3, and performing transcription and translation on the nucleotide sequence shown in the formula 3.

3. As set forth in SEQ ID NO:2 or SEQ ID NO:4 in the preparation of a novel coronavirus mutant universal vaccine, wherein the novel coronavirus mutant is: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16.

4. A recombinant vector comprising a sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3, and a nucleotide sequence shown in 3.

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

6. Use of the recombinant vector according to claim 4 or 5 for the preparation of a novel coronavirus mutant universal vaccine, said novel coronavirus mutant being: WT, alpha, beta, delta, BA.1, BA.2, BA.5, XBB.1.5, EG.5.1, BF.7, BQ.1.1, CH.1.1 or XBB.1.16.

7. The use according to claim 1, characterized by the steps of:

s1: the sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, connecting the nucleotide sequence shown in the 3 to pET-28a, constructing a pET-28a-cRBD recombinant vector, and transferring into escherichia coli BL21 for expression;

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.