CN113150084A - Nanocrystallized coronavirus antigen and application thereof - Google Patents

Abstract

The invention discloses a nano coronavirus antigen and application thereof. The nano coronavirus antigen is obtained by polysaccharide covalent coupling of 2019-nCoV RBD protein; the 2019-nCoV RBD protein comprises: amino acids 319 to 541 of an S protein with a GenBank accession number of MN908947.3 or a variant of the amino acid sequence; the variants have at least 70% sequence homology to the pre-mutation sequence and retain at least the antigenicity of the pre-mutation sequence. The nano coupled RBD antigen overcomes the defect of insufficient immunogenicity of RBD monomers, and greatly improves the titer of a neutralizing antibody of a mouse against 2019-nCoV.

Description

Nanocrystallized coronavirus antigen and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a nano coronavirus antigen and application thereof, in particular to a novel nano coronavirus vaccine based on a polymer-coupled receptor binding region and application thereof.

Background

The novel coronavirus belongs to the genus coronavirus of the family Coronaviridae, is a positive-strand RNA virus with an envelope, and the genome of the positive-strand RNA virus is largest among all RNA viruses, and animals and humans are hosts of the coronavirus. The incubation period of 2019-nCoV infection of a human is generally 1-14 days, and common signs after infection comprise respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. By 30 days 12 months in 2020, more than 8 million global cumulative corroboration cases and more than 170 million deaths can be confirmed. Therefore, the development of a safe and effective vaccine against the novel coronavirus (2019-nCoV) is very urgent and has important significance.

The surface spike protein (S protein) is the major neutralizing antigen of the novel coronavirus. 2019-nCoV invades cells by binding to the host cell's Receptor angiotensin converting enzyme 2(ACE2) through the Receptor Binding Domain (RBD) region. Thus, the Receptor Binding Domain (RBD) of the S protein of the novel coronavirus is considered to be the most prominent antigen-targeting domain that induces the body to produce neutralizing antibodies. The RBD protein as vaccine antigen can focus the neutralizing antibody generated by body stimulation on the receptor combination aiming at the virus, and can improve the immunogenicity and the immune efficiency of the vaccine.

The target antigens for novel coronavirus vaccines are generally in the form of full-length S protein and truncated S protein (S1 and RBD). The types under investigation for novel coronavirus vaccines include adenoviral vector vaccines, mRNA vaccines, DNA vaccines, recombinant protein vaccines and inactivated vaccines. The invention aims to provide a novel coronavirus vaccine which is in a novel vaccine form and has enhanced immunogenicity and is based on the nanocrystallization of a receptor binding region coupled by a polymer.

In addition, previous studies of coronavirus vaccines have used RBD monomers, which have some neutralizing immunogenicity. However, current vaccines developed based on novel coronavirus S protein RBD monomers have limited immunogenicity. The RBD-based fusion protein (such as Fc fragment of IgG) has the defect of insufficient safety evaluation. Therefore, the development of a novel coronavirus vaccine with high immunogenicity and better safety is a problem to be solved.

Disclosure of Invention

The invention aims to solve the technical problem of providing a nano coronavirus antigen and application thereof in order to overcome the defect of limited immunogenicity of a coronavirus vaccine in the prior art. Compared with the RBD protein antigen, the coronavirus antigen disclosed by the invention can obviously improve the level of a host neutralizing antibody, is simple in preparation method, easy to purify and quick and low-cost, and the protein has no purification label and no biological safety risk and can be applied to clinical application.

The invention mainly solves the technical problems through the following technical scheme.

One of the technical schemes of the invention is as follows: a nano-sized coronavirus antigen obtained by covalently coupling a polysaccharide to a 2019-nCoV RBD protein; the 2019-nCoV RBD protein comprises:

amino acids 319 to 541 of an S protein with a GenBank accession number of MN908947.3 or a variant of the amino acid sequence; the variants have at least 70% sequence homology to the pre-mutation sequence and retain at least the antigenicity of the pre-mutation sequence.

For example, the 2019-nCoV RBD protein comprises or is amino acids 319-587 of an S protein with GenBank accession number MN 908947.3.

The mutation sites comprised by the variants may be conventional in the art, for example: starr et al, 2020, cell 182, 1295-. Preferably, the point mutations are one or more of C538S, C538A, S477N, N439K, N501Y, N354D, D364Y, V367F, W436R, I358F, Y365F, Y365W, and F392W.

In a preferred embodiment of the invention, the amino acid sequence of the 2019-nCoV RBD protein comprises a sequence shown as any one of SEQ ID NO 1-3; preferably, the nucleotide sequence encoding the 2019-nCoV RBD protein comprises a nucleotide sequence shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

As for the polysaccharide in the present invention, it may be a microbial natural polysaccharide, a plant natural polysaccharide or an animal natural polysaccharide.

Wherein the microbial natural polysaccharide preferably comprises a bacterial capsular polysaccharide, an O-specific polysaccharide of a bacterial lipopolysaccharide, a bacterial exopolysaccharide and a fungal polysaccharide; the bacterial capsular polysaccharide is preferably a streptococcus pneumoniae polysaccharide, more preferably a streptococcus pneumoniae polysaccharide type 9V;

the plant natural polysaccharide preferably comprises amylose, inulin, cellulose and pectin;

the animal natural polysaccharides preferably include chitin, glycogen, hyaluronic acid and heparin.

The covalent coupling in the present invention can be achieved by conventional means in the art, and preferably the chemical principle of the covalent coupling is reductive amination, carbodiimide-mediated condensation, thioalkylation, active ester or cyanation.

Among them, the coupling reagent used for carrying out the cyanation reaction is preferably 1-cyano-4- (dimethylamino) pyridyltetrafluoroborate. The coupling reagent used to perform the cyanation reaction preferably activates the hydroxyl groups on the polysaccharide, while coupling lysine residues on the 2019-nCoV RBD protein to form a coupled product.

In a preferred embodiment of the present invention, the method for preparing coronavirus antigen comprises: and mixing the polysaccharide activated by the coupling reagent with 2019-nCoV RBD protein, and reacting. The mass ratio of the polysaccharide activated by the coupling reagent to the 2019-nCoV RBD protein is preferably (0.2-2): 1, and more preferably 1: 1.

Preferably, after the reaction is finished, the reaction product is purified by dialysis or ultrafiltration through 0.9% sodium chloride solution.

The second technical scheme of the invention is as follows: a medicament comprising a coronavirus antigen according to any one of the preceding claims. The medicament preferably further comprises an aluminum adjuvant.

The third technical scheme of the invention is as follows: the application of the coronavirus antigen in the technical scheme in the preparation of vaccine medicines for preventing novel coronavirus. The vaccine medicament preferably further comprises an aluminium adjuvant.

The fourth technical scheme of the invention is as follows: a method for improving the immunogenicity of coronavirus antigens, which comprises the step of forming nano vaccine antigens by covalently coupling 2019-nCoV RBD protein and polysaccharide.

The technical scheme is one of the four further definitions of 2019-nCoV RBD protein, polysaccharide, covalent coupling and the like.

The fifth technical scheme of the invention is as follows: a method of preparing a nano-sized coronavirus antigen, the method comprising: mixing and reacting the polysaccharide activated by the coupling reagent with 2019-nCoV RBD protein;

the 2019-nCoV RBD protein comprises:

amino acids 319 to 541 of an S protein with a GenBank accession number of MN908947.3 or a variant of the amino acid sequence;

the variants have at least 70% sequence homology with the pre-mutation sequence and retain at least the antigenicity of the pre-mutation sequence.

The method preferably further comprises the step of purifying the reaction product by dialysis or ultrafiltration with a 0.9% sodium chloride solution.

The 2019-nCoV RBD protein can be directly synthesized by a biological company or obtained by expression and purification of engineering bacteria. In one embodiment of the invention, the 2019-nCoV RBD protein purification method comprises the steps of filtering cell supernatant expressing the antigen to remove cell debris, or renaturing bacterial inclusion bodies, removing impurities and polymers in a flow-through mode through a cation exchange column to roughly purify target protein, then passing a component containing the target protein through a pH-adjusted loading cation exchange column to carry out salt gradient elution, combining obtained elution peaks containing the target protein, carrying out retention concentration through a 10kDa MWCO membrane, transferring the sample into a buffer solution for preserving the sample, or further purifying and concentrating the sample through a hydrophobic chromatography column.

1) The bacterial inclusion bodies were washed, dissolved in a high concentration denaturant (8M urea or 6M guanidine hydrochloride) at 20mM DTT, pH7.0-10.0 at room temperature, and added to the renaturation solution at a concentration of 0.1-1 mg/ml. The denaturating solution contains 2-5M urea, oxidation-reduction agent (glutathione, cysteine/cystine, cysteamine/cystamine), oxidant concentration is 0.05-0.5mM, reducing agent concentration is 0.05-2mM, and renaturation is carried out for 16-60 hours at 4-10 ℃.

2) Cation columns used in the course of rough purification include SP Sepharose Big beads of Cytiva, SP Sepharose Fast Flow, Toyopearl SP-550C of Tokyo, Toyopearl SP-650M, SP Bestarose FF of Booglon, and the like.

3) Cation columns used in the fine purification include SP Sepharose HP, Source30S, from Cytiva, Eshmuon CPS, from Merck, Toyopearl Sulfate-650F, from Tokyo, SP Bestarose HP, from Booglon, and the like.

4) The filler used for hydrophobic purification includes Phenyl Sepharose Fast Flow (high Sub) of Cytiva, Phenyl Sepharose Fast Flow (Low Sub), Butyl Sepharose Fast Flow, Phenyl Bestarose FF (HS) of Bordeten, Butyl Bestarose 4FF and the like.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the invention forms the nano vaccine antigen by recombining RBD protein antigen and polymers such as microbial polysaccharide through chemical coupling. And respectively immunizing Balb/c mice by using purified RBD protein monomers and nano-coupled RBD antigens. The nano-coupled RBD antigen overcomes the defect of insufficient immunogenicity of RBD monomers, greatly improves the titer of neutralizing antibodies of mice to 2019-nCoV, and has strong positive inhibition rate results under 20-fold dilution of serum.

Drawings

FIG. 1 is a SDS-PAGE pattern of E.coli expressed RBD antigen; m is protein marker, S is cell lysis supernatant, P is cell lysate sediment, R is Reduced SDS-PAGE, and NR is Non-Reduced SDS-PAGE.

FIG. 2 is a SDS-PAGE picture of RBD antigen expression in HEK293 cells.

FIG. 3 is a graph of the mass-spectrometric accurate molecular weight of an E.coli expressed RBD antigen.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods in the following examples, in which specific conditions are not specified, were selected according to conventional methods and conditions, or according to the commercial instructions.

The vector used for the plasmid of the target gene is pETK, which is purchased from Suzhou Jinzhi Biotechnology GmbH.

BL21(DE3) competent cells were purchased from Beijing Quanjin Biotechnology, Inc.

The formula of the liquid LB culture medium is 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and pH7.0.

Example 1: preparation of escherichia coli engineering bacteria for expressing 2019-nCoV recombinant RBD antigen

And (3) trusting Suzhou Jinzhi to synthesize a plasmid for expressing the target protein, and sequencing to verify the correctness.

The correct plasmid containing the gene of interest (sequences shown in SEQ ID NOS: 4, 5 and 6, respectively) was transformed into BL21(DE3) competent cells, and the cells were monoclonally inoculated into 3mL of liquid LB medium containing 100ng/mL of kanamycin, and cultured overnight at 220rpm at 30 ℃ with shaking, to serve as seeds.

The seeds were inoculated at 1:500(v/v) into TB medium containing 100ng/mL kanamycin, cultured with shaking at 220rpm and 37 ℃ until OD600 became 2, added with 0.5mM IPTG at the final concentration, cultured for 4 hours, and centrifuged to collect the cells for purification.

The 10OD bacteria were resuspended in 1ml lysis buffer (10mM Tris-HCl, pH8.0), disrupted by sonication, total protein determination by SOP, SDS-PAGE electrophoretic analysis of supernatant and pellet fractions, and the results are shown in FIG. 1.

Example 2: preparation of Expi293 recombinant cells expressing 2019-nCoV recombinant RBD antigen

And extracting the plasmid of the target gene with correct sequencing by using a QIAGEN plasmid extraction kit for later use.

Expi293 cell suspensions freshly passaged to 2.0-2.5 × 10E 6/mL were prepared according to the volume required for transfection.

Transfection reagent-plasmid complexes were prepared, namely, solution A (plasmid/Opti-MEM (1. mu.g/50. mu.L)) and solution B (PEI/Opti-MEM (5. mu.L/50. mu.L)), which were incubated for 10 minutes independently.

Adding the solution B dropwise into the solution A, incubating at room temperature for 10min, adding the cell suspension dropwise, and culturing under shaking (115rpm, 36.8 deg.C, 5% CO)₂) After 24 hours, 0.4mL of KTfeed can be added, the shaking culture is continued for 5 to 7 days under the same conditions, and the cell supernatant is collected by centrifugation at 8500rpm for 15min and then is subjected to downstream purification.

SDS-PAGE analysis of the supernatant according to SOP was carried out, and the analysis results are shown in FIG. 2.

Example 3: purification and identification of 2019-nCoV recombinant RBD antigen escherichia coli or cell expression

The E.coli-expressing cells of example 1 were resuspended at 1:20(W/V) in Tris buffer pH8.0, disrupted by high pressure homogenizer at 900bar 3 times, and centrifuged to obtain inclusion bodies. Inclusion bodies were denatured by dissolution in 20mM ethylene glycol, 8M Urea, 50mM DTT pH8.0 and renatured with 20mM Tris, pH8.0, 3M Urea, 0.05mM cystamine, 1mM cysteamine renaturation solution for 20 hours. Adjusting the pH value of the renaturation supernatant fluid to 7, centrifuging for 1 hour at 10000 Xg, taking the supernatant fluid to purify and remove HCP (host cell protein) and polymer to carry out coarse purification on the target protein by virtue of Cytiva SP Sepharose Fast Flow in a Flow-through mode; adjusting the pH value of the flow-through solution to 5, performing further fine purification by using Cytiva SP sepharose HP in a binding mode, performing gradient elution on target protein by using 1M NaCl, combining elution peaks containing the target protein, performing dense concentration by using 10kDa MWCO, and changing the solution into 2 XPBS buffer solution for further polysaccharide coupling. The molecular weight identification map is shown in FIG. 3, and the complete molecular weight is determined according to liquid phase mass spectrometry: 30285.7Da corresponds to the theoretical molecular weight (30284.79 Da).

The cell supernatants expressing the antigens of example 2 were filtered to remove cell debris. Adjusting the pH of cell supernatant to 7.0, removing HCP and polymers by a Cytiva SP Fast Flow cation exchange column in a Flow-through mode to carry out coarse purification on target protein, adjusting the pH of Flow-through liquid to 4.0, then further carrying out fine purification on the SP sepharose HP in the cation exchange column, carrying out gradient elution by using 1M NaCl, combining elution peaks containing the target protein, carrying out dense concentration by using a 10kDa MWCO dense tube, and exchanging the liquid into 2 XPBS buffer solution for further polysaccharide coupling.

Example 4: fermentation, purification and identification of bacterial hyaluronic acid polysaccharide

Culturing the streptococcus zooepidemicus bacterial strain expressing hyaluronic acid polysaccharide at 37 ℃ for 10 hours by a tryptone culture medium to obtain a supernatant or thallus containing the corresponding bacterial polysaccharide. The bacterial culture solution is inactivated by 0.5 percent of formaldehyde, and then centrifuged at 8000g for 30 minutes to collect supernatant. Ultrafiltering and concentrating the centrifugal supernatant by using a 100kDa membrane package, performing fractional precipitation by using 25-80% ethanol, and collecting the precipitate to obtain crude polysaccharide; dissolving polysaccharide in sterilized water for injection, ultrafiltering, dialyzing, refining with ion exchange filler by series chromatography or fractional chromatography, collecting flow-through peak, desalting by ultrafiltration dialysis, lyophilizing to obtain refined hyaluronic acid polysaccharide, and storing at-20 deg.C.

Measuring the content of residual protein (required to be less than or equal to 1%) in the refined hyaluronic acid polysaccharide by adopting a Lowry method; measuring the content of residual nucleic acid (required to be less than or equal to 1%) by using an ultraviolet spectrophotometry; measuring the content of uronic acid (required to be more than or equal to 44%) by a sulfuric acid-carbazole method; the weight average molecular weight is determined by HPLC-SEC-MALLS method (requirement is more than or equal to 10)⁵Da)。

Table 1: quality attributes of refined hyaluronic acid polysaccharides

Example 5: fermentation, purification and identification of pneumococcal capsular polysaccharide

The bacterial strain of streptococcus pneumoniae expressing 9V type capsular polysaccharide is cultured for 10 hours at 37 ℃ by a tryptone culture medium to obtain thalli containing the corresponding bacterial polysaccharide. The bacterial culture solution is inactivated by 0.5% sodium deoxycholate, centrifuged at 8000g for 30 minutes, and the supernatant is collected. Ultrafiltering and concentrating the centrifugal supernatant by using a 100kDa membrane package, performing fractional precipitation by using 25-80% ethanol, and collecting the precipitate to obtain crude polysaccharide; dissolving polysaccharide in sterilized water for injection, ultrafiltering, dialyzing, refining with ion exchange filler by series chromatography or fractional chromatography, collecting flow-through peak, desalting by ultrafiltration dialysis, lyophilizing to obtain refined 9V pneumococcal polysaccharide, and storing at-20 deg.C.

Measuring the residual protein content (required to be less than or equal to 1%) of the refined 9V pneumococcal polysaccharide by adopting a Lowry method; measuring the content of residual nucleic acid (required to be less than or equal to 1%) by using an ultraviolet spectrophotometry; measuring the content of uronic acid (required to be more than or equal to 15%) by a sulfuric acid-carbazole method; hexosamine content (required to be more than or equal to 13%); the weight average molecular weight is determined by HPLC-SEC-MALLS method (requirement is more than or equal to 10)⁵g.mol^-1)。

Table 2: quality attribute of refined 9V type pneumococcal polysaccharide

Example 6: covalent coupling of microbial polysaccharides of 2019-nCoV recombinant RBD antigens

The recombinant RBD antigen obtained in example 3 is subjected to polysaccharide product hyaluronic acid polysaccharide (HA) fermentation through coupling reagent 1-cyano-4- (dimethylamino) pyridine tetrafluoroborate (CDAP) activated microorganisms, then RBD protein and activated polysaccharide are mixed and reacted according to the feeding mass ratio of 1:1, amino groups of lysine on a sequence are directly subjected to covalent coupling and are subjected to continuous crosslinking polymerization, and after the reaction is finished, the nano antigen is obtained after dialysis or ultrafiltration purification for 3 times through 0.9% sodium chloride solution. And (4) determining the RBD protein content in the purified antigen by adopting a Lowry method.

Example 7: immunization experiment of mice

The conjugated antigens obtained in examples 4 and 5 were diluted in physiological saline and formulated with an aluminum adjuvant according to the method shown in Table 2. Balb/C female mice, 4-6 weeks old, were then immunised in groups. The immunization procedure is as in table 3, i.e. by means of inguinal subcutaneous injection, each mouse received 2 vaccine immunizations each at day 0, day 14, in a vaccination volume of 100 μ l each time. On day 28 (i.e., day 14 after the 2-pin immunization), the mice were subjected to orbital bleeds. The mouse serum is obtained by centrifugation at 3000rpm for 10 minutes after the serum is separated out after standing for a period of time, and is stored at-20 ℃ and below for detection of neutralizing antibody titer and pseudovirus neutralizing titer.

Table 3: animal immunization group status

Example 8: serum IgG Total antibody Titer assay

The test adopts an indirect ELISA method to detect the IgG total antibody titer aiming at the new coronavirus RBD in the serum of mice of each test group.

i) Antigen coating

S1 protein and RBD protein are respectively used as coating antigens, an ELISA plate is coated in each hole with the protein concentration of 10 mug/ml and 100 mul, and the ELISA plate is coated overnight at 4 ℃.

ii) serum dilution and incubation

The test group sera (10-fold initial) were diluted 2-fold, 100. mu.l per well, added to the microplate, and incubated for 120 min at 37 ℃.

iii) plate washer alkaline phosphatase IgG Secondary antibody incubation

Add 200 mul of washing liquid into each hole to wash the enzyme label plate for 5 times. Then, a 4000-fold dilution of goat anti-mouse IgG alkaline phosphatase secondary antibody was added and incubated at 37 ℃ for 120 minutes.

iv) plate washing and color development

Adding 200 mul of cleaning solution into each hole to clean the enzyme label plate, and cleaning for 5 times; after the plate is washed, 100 mu L of color development liquid is added into each hole. After development at 20-25 ℃ for 60 minutes under dark conditions, 50. mu.L of stop solution was added.

v) standards for reading and result determination

And (4) placing the enzyme label plate into an enzyme label instrument, and reading at 405 nm. When analyzing the result, the positive and negative Cutoff values are calculated, and the calculation formula is as follows. Based on the Cutoff value, the IgG antibody titer of the sample was calculated.

Cutoff value-mean OD405 +2SD of sample well in negative group

vi) analysis of test results

In the test groups (test groups 5 and 6) of prokaryotic and eukaryotic expression RBD monomeric proteins, after 2-needle immunization at a dose of 10 mu g, the generation of IgG antibody level with high titer aiming at the 2019-nCoVRBD region can be induced in a mouse body; RBD protein test groups (test groups 1, 2) covalently coupled with bacterial polysaccharides induced high titer IgG antibody levels against the 2019-ncorvrbd region in mice after 2-needle immunizations at a dose of 10 μ g. And the RBD protein test groups (test groups 3 and 4) after covalent coupling of bacterial polysaccharides can also produce higher neutralizing antibody levels after 2-needle immunization at a low dose of 1 mu g and are comparable to test group 1. The results are detailed in Table 4.

The results show that the RBD monomeric protein has certain immunogenicity and can generate higher total IgG antibody level by coupling with bacterial polysaccharide. This suggests the immunological feasibility of bacterial polysaccharide-conjugated RBD antigens as vaccines, and further confirmation of whether the produced IgG antibodies have neutralizing protective activity is required.

TABLE 4 detection of neutralizing antibody levels in antigen-challenged mice (20-fold dilution)

Example 9: serum neutralizing antibody titer determination assay

The test was carried out using a commercially available cPass from Kinry corporation^TMThe sVNT Kit detects the titer of the neutralizing antibody of the new coronavirus in the serum of mice of each test group. cPass^TMThis technique allows rapid detection of total neutralizing antibodies (NAbs) in a sample by mimicking the process of interaction between the virus and host cells. In order for a virus to infect a host cell, the viral receptor binding protein (RBD) first needs to interact with the membrane receptor protein (ACE2) of the host cell. The interaction of the virus with the host cell and subsequent infection with the virus results in the activation of an immune response in the individual, thereby generating a population of antibodies against the virus. Some of these antibodies bind to the virus, but do not necessarily prevent viral infection. Other antibodies may bind to RBD in a manner that blocks interaction with the ACE2 receptor. This result helps to distinguish whether the sample contains NAbs that may specifically block the interaction and thus the entry of the virus into the host cell.

i) Serum dilution and incubation

The positive quality control serum, the negative quality control serum and the test group serum (20 times of the original serum) are diluted according to a 2-fold ratio, mixed with the RBD protein marked by the HRP according to a volume of 1:1, and the mixed solution is incubated for 30 minutes at 37 ℃.

ii) incubation of Mixed-solution ELISA plates

And (3) sucking 100 mu L of mixed liquid of the positive quality control serum, the negative quality control serum and the test group serum, adding the mixed liquid into an ELISA plate hole, and incubating for 15 minutes at 37 ℃.

iii) washing the plate and developing the color

Adding 260 mu L of cleaning solution into each hole, and washing the plate for 4 times; after the plate is washed, 100 mu L of TMB color developing solution is added into each hole. After development at 20-25 ℃ for 15 minutes under dark conditions, 50. mu.L of stop solution was added.

iiii) reading and result criteria

The microplate was placed in the microplate reader and read at 450 nm. In the analysis of the results, it is necessary to determine whether the system negative and positive serum results are satisfied, and the determination criteria are shown in Table 5.

TABLE 5 systematic negative and positive determination criteria

Quality control project	OD 450nm	Quality control qualification requirement
			Negative quality control	＞1.0	Negative quality control establishment
Positive quality control	＜0.3	Positive quality control establishment

The serum neutralizing antibody titer of the test group is calculated by inhibiting the ACE2 binding rate, and the calculation formula is as follows:

the inhibition rate is (1-test group sample OD value/negative quality control group OD value) × 100%

The Cutoff value criteria for the negative and positive inhibition of neutralizing antibody titers in the sample groups are shown in Table 6:

TABLE 62019-criteria for positive and negative inhibition Cutoff values of nCoV neutralizing antibody titers

iiii) analysis of test results

In prokaryotic and eukaryotic expression RBD monomeric protein test groups (test groups 5 and 6), after 2-needle immunization at a dose of 10 mu g, neutralizing antibodies aiming at 2019-nCoV are generated at inducible parts in a mouse body, but the neutralizing antibodies are lower; the RBD protein test groups (test groups 1 and 2) after covalent coupling of bacterial polysaccharide can induce the generation of neutralizing antibody level against 2019-nCoV in mice after 2 times of immunization at the dose of 10 mu g, and the antibody level is higher. And the RBD protein test groups (test groups 3 and 4) after covalent coupling of bacterial polysaccharides also produced higher levels of neutralizing antibodies after 2-needle immunization at a low dose of 1 μ g and were comparable to test groups 1 and 2. The results are detailed in Table 7.

TABLE 7 detection of neutralizing antibody levels in test antigen-primed mice

The results show that the RBD monomeric protein has certain immunogenicity, and weak neutralizing antibody levels are generated after immunization. After coupling of various bacterial polysaccharides, the immunogenicity of the RBD protein is obviously enhanced, and a higher level of neutralizing antibodies can be generated at a lower dose. This suggests that the immunogenicity of the 2019-nCoV vaccine can be significantly improved when the bacterial polysaccharide-coupled RBD antigen is used as a vaccine, and the protective neutralizing antibody level has obvious advantages.

SEQUENCE LISTING

<110> Jiangsu Kunli biopharmaceutical Limited liability company;

BEIJING ZHIDAO BIOLOGICAL TECHNOLOGY Co.,Ltd.

<120> nano coronavirus antigen and application thereof

<130> P21011424C

<160> 6

<170> PatentIn version 3.5

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Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser

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

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

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

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260 265 270

<210> 4

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<223> 2019-nCoV RBD protein nucleotide sequence

<400> 4

atgcgcgttc agcctacaga atcaattgtt cgctttccta acattacaaa cctttgtcct 60

ttcggagagg tcttcaatgc aacacgcttt gcttcagttt atgcttggaa ccgcaaacgc 120

atttcaaact gtgttgctga ttattcagtt ctttataact cagcttcatt ctccacgttt 180

aaatgttatg gcgtttcacc tacaaagctg aatgatcttt gtttcactaa tgtttatgct 240

gattcatttg ttattcgcgg cgatgaagtt cgccagattg ctcctggcca gacaggcaag 300

atagcggatt ataactataa acttcctgat gatttcaccg gatgtgttat tgcttggaac 360

tcaaacaacc tcgattcaaa ggtgggtggc aactataact atctttatcg cctattcaga 420

aagtcaaacc ttaaaccttt cgagcgggat atttcaacag aaatttatca ggctggctca 480

acaccttgta acggcgttga aggctttaac tgttatttcc cgttacagtc ttatggcttt 540

cagcctacaa acggcgttgg ctatcagcct tatcgcgttg ttgttctttc atttgaactt 600

cttcatgctc ctgctacagt ttgtggccct aagaagagca ctaaccttgt taagaataag 660

tgtgttaact ttaactttaa cggccttaca ggcacaggcg ttcttacaga atcaaacaag 720

aagtttctgc catttcagca gtttggccgc gatattgctg atacaaccga cgccgtcaga 780

gaccctcaga cacttgaaat tcttgatatt taa 813

<210> 5

<211> 813

<212> DNA

<213> Artificial Sequence

<220>

<223> 2019-nCoV RBD protein nucleotide sequence

<400> 5

atgcgcgttc agcctacaga atcaattgtt cgctttccta acattacaaa cctttgtcct 60

ttcggagagg tcttcaatgc aacacgcttt gcttcagttt atgcttggaa ccgcaaacgc 120

atttcaaact gtgttgctga ttattcagtt ctttataact cagcttcatt ctccacgttt 180

aaatgttatg gcgtttcacc tacaaagctg aatgatcttt gtttcactaa tgtttatgct 240

gattcatttg ttattcgcgg cgatgaagtt cgccagattg ctcctggcca gacaggcaag 300

atagcggatt ataactataa acttcctgat gatttcaccg gatgtgttat tgcttggaac 360

tcaaacaacc tcgattcaaa ggtgggtggc aactataact atctttatcg cctattcaga 420

aagtcaaacc ttaaaccttt cgagcgggat atttcaacag aaatttatca ggctggctca 480

acaccttgta acggcgttga aggctttaac tgttatttcc cgttacagtc ttatggcttt 540

cagcctacaa acggcgttgg ctatcagcct tatcgcgttg ttgttctttc atttgaactt 600

cttcatgctc ctgctacagt ttgtggccct aagaagagca ctaaccttgt taagaataag 660

tctgttaact ttaactttaa cggccttaca ggcacaggcg ttcttacaga atcaaacaag 720

aagtttctgc catttcagca gtttggccgc gatattgctg atacaaccga cgccgtcaga 780

gaccctcaga cacttgaaat tcttgatatt taa 813

<210> 6

<211> 672

<212> DNA

<213> Artificial Sequence

<220>

<223> 2019-nCoV RBD protein nucleotide sequence

<400> 6

agagtgcaac ctacagaatc aatcgtgaga tttcctaaca tcacaaacct ttgccctttc 60

ggagaagtgt tcaatgctac aagatttgca tcagtgtacg catggaacag aaagaggata 120

tcaaactgcg tggcagatta ctcagtgctt tacaactcag catcattctc tacctttaaa 180

tgctacggag tgtcacctac aaagttaaat gatctttgct ttacaaacgt gtacgcagat 240

tcatttgtga tcagaggaga tgaagtgaga caaatcgcac ctggacaaac aggaaagata 300

gctgattaca actacaaact tcctgatgat ttcaccgggt gcgtgatcgc atggaactca 360

aacaacttgg attcaaaggt gggaggcaat tataattatt tatatcgttt atttaggaag 420

tccaacctca aacctttcga gcgagatatc tcaacagaaa tctaccaagc aggatcaaca 480

ccttgcaacg gagtggaagg atttaactgc tactttcctc ttcaatcata cggatttcaa 540

cctacaaacg gagtgggata ccaaccttac agagtggtgg tgctttcatt tgaacttctt 600

cacgcacctg caacagtgtg cggacctaag aagagtacga accttgtgaa gaataagtgc 660

gtgaactttt ag 672

Claims (10)

1. A nano coronavirus antigen is characterized in that the antigen is obtained by polysaccharide covalent coupling of 2019-nCoV RBD protein; the 2019-nCoV RBD protein comprises:

amino acids 319 to 541 of an S protein with a GenBank accession number of MN908947.3 or a variant of the amino acid sequence;

the variants have at least 70% sequence homology to the pre-mutation sequence and retain at least the antigenicity of the pre-mutation sequence.

2. The coronavirus antigen of claim 1, wherein the 2019-nCoV RBD protein comprises amino acids 319-587 of the S protein of GenBank accession No. MN 908947.3;

and/or, the variant comprises one or more of the point mutations C538S, C538A, S477N, N439K, N501Y, N354D, D364Y, V367F, W436R, I358F, Y365F, Y365W and F392W.

3. The coronavirus antigen of claim 1, wherein the amino acid sequence of the 2019-nCoV RBD protein comprises a sequence as set forth in any one of SEQ ID NOs 1-3; preferably, the nucleotide sequence encoding the 2019-nCoV RBD protein comprises a nucleotide sequence shown as SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6.

4. The coronavirus antigen of claim 1, wherein the polysaccharide comprises: natural polysaccharides of microorganisms, natural polysaccharides of plants and natural polysaccharides of animals; wherein:

the microbial natural polysaccharides preferably include bacterial capsular polysaccharides, O-specific polysaccharides of bacterial lipopolysaccharides, bacterial exopolysaccharides and fungal polysaccharides; the bacterial capsular polysaccharide is preferably a streptococcus pneumoniae polysaccharide, more preferably a streptococcus pneumoniae polysaccharide type 9V;

the plant natural polysaccharide preferably comprises amylose, inulin, cellulose and pectin;

the animal natural polysaccharides preferably include chitin, glycogen, hyaluronic acid and heparin.

5. The coronavirus antigen of claim 1, wherein the covalent coupling is based on the chemical principles of reductive amination, carbodiimide-mediated condensation, thioalkylation, active ester or cyanation; wherein the content of the first and second substances,

the cyanation reaction is preferably carried out using a coupling reagent such as 1-cyano-4- (dimethylamino) pyridyltetrafluoroborate;

and/or, the coupling reagent used to perform the cyanation reaction preferably activates hydroxyl groups on the polysaccharide while coupling lysine residues on the 2019-nCoV RBD protein to form a coupled product.

6. The coronavirus antigen according to any one of claims 1 to 5, which is prepared by a method comprising: mixing and reacting the polysaccharide activated by the coupling reagent with 2019-nCoV RBD protein;

the mass ratio of the polysaccharide activated by the coupling reagent to the 2019-nCoV RBD protein is preferably (0.2-2): 1, and more preferably 1: 1.

7. The coronavirus antigen of claim 6, wherein after completion of the reaction, the reaction product is purified by dialysis or ultrafiltration with 0.9% sodium chloride solution.

8. Use of a coronavirus antigen as defined in any one of claims 1 to 7 in the preparation of a medicament for the prevention of a coronavirus; preferably, the medicament further comprises an aluminum adjuvant.

9. A medicament comprising a coronavirus antigen as defined in any one of claims 1 to 7;

preferably, the medicament further comprises an aluminum adjuvant.

10. A method of increasing the immunogenicity of a coronavirus antigen, the method comprising forming a nanovaccine antigen by covalent coupling of a 2019-nCoV RBD protein as defined in any one of claims 1 to 3 with a polysaccharide as defined in claim 4;

the covalent coupling is preferably effected by reductive amination, carbodiimide-mediated condensation, thioalkylation, reactive ester or cyanation reactions; wherein the content of the first and second substances,

the cyanation reaction is preferably carried out using a coupling reagent such as 1-cyano-4- (dimethylamino) pyridyltetrafluoroborate;

and/or, the coupling reagent used to perform the cyanation reaction preferably activates hydroxyl groups on the polysaccharide while coupling lysine residues on the 2019-nCoV RBD protein to form a coupled product.

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