CN114057848B - Polypeptide and immunogenic conjugate for preventing novel coronavirus pneumonia COVID-19 and application thereof - Google Patents

Abstract

The invention relates to the fields of immunology technology, biotechnology and biological medicine, in particular to a polypeptide and an immunogenic conjugate for preventing novel coronavirus pneumonia COVID-19 and application thereof. An immunogenic conjugate comprising a polypeptide having the amino acid sequence shown in SEQ ID No.1 for use in the preparation of a vaccine for the prevention of covd-19. The immunogenic conjugate synthesizes target polypeptide based on a certain section of known or predicted antigen epitope amino acid sequence in pathogen antigen gene, the synthesized target polypeptide is combined with carrier protein to prepare polypeptide conjugate, and then an adjuvant is added to prepare the synthetic peptide vaccine. Compared with the traditional attenuated vaccine, inactivated vaccine and other novel vaccines, the vaccine can rapidly cope with virus variation; the field requirement for vaccine production is reduced, and the large-scale mass production is easy; the research and development cost and the research and development period are reduced; safety, innocuity and stability; because of its small and simple molecular structure, serious complications and iatrogenic infections are rarely caused.

Description

Polypeptide and immunogenic conjugate for preventing novel coronavirus pneumonia COVID-19 and application thereof

Technical Field

The invention relates to the fields of immunology technology, biotechnology and biological medicine, in particular to a polypeptide and an immunogenic conjugate for preventing novel coronavirus pneumonia COVID-19 and application thereof.

Background

Coronaviruses are forward enveloped viruses with RNA, whose genome is approximately 26-32 kb in size, and are the largest known RNA viruses. The genomic RNA and phosphorylated nucleocapsid (N) proteins are buried in phospholipid bilayers and covered with a spike glycoprotein trimer (S), with membrane (M) protein (type III transmembrane glycoprotein) and envelope (E) protein located between the S proteins of the viral envelope. Coronaviruses have a variety of hosts, including avian and mammalian species, particularly bats. Coronaviruses are a type of virus that is widely found in nature and can cause multiple system diseases including the respiratory tract, digestive tract and nervous system, and highly pathogenic coronavirus infection has become a public health problem of great concern for nearly 10 years. In 11 months 2002, the first Severe Acute Respiratory Syndrome (SARS) occurs in bergamot, china. In 2012, the middle east respiratory syndrome coronavirus (MERS-CoV) was the second highly pathogenic coronavirus found in the 21 st century. MERS-CoV has a high mortality rate, with up to 40% of patients dying. 2019 novel coronavirus (SARS-CoV-2) was first discovered in month 12 of 2019, and its infection caused novel coronavirus pneumonia (COVID-19) is affecting millions of patients worldwide.

The patient with covd-19 may develop symptoms of varying degrees, ranging from fever or mild cough to pneumonia, more severe and even death. At present, the mortality rate of the COVID-19 is about 2 to 4 percent, although the mortality rate is lower than that of SARS and MERS, the novel coronavirus (SARS-CoV-2) has the characteristics of long latency, strong infectivity and higher serious disease rate, and different from SARS viruses causing atypical pneumonia, part of cases have infectivity in latency, and other virus carriers do not show any obvious symptoms, so that the prevention and control difficulty of epidemic situation is increased. Thus, rapid development of prophylactic vaccines that can boost the immune level of the population and block viral transmission has become the most urgent current global need.

Since the first use of biologic vaccinia vaccines by humans at the end of the 18 th century, the vaccine has played an irreplaceable role in the elimination of a variety of infectious diseases. After the identification of the COVID-19, a plurality of institutions at home and abroad develop researches on the COVID-19 vaccine, at least 5 technical routes including nucleic acid vaccine, viral vector vaccine, inactivated vaccine, recombinant protein vaccine, attenuated influenza virus vector vaccine and the like are synchronously developed at present, but in view of the characteristics of the vaccine, the vaccine is strictly carried out according to the research and development production flow specified by the country, after animal experiments pass, pilot study, clinical declaration and clinical test study of I-III phase are also required to be completed, and the formal production stage is possible after approval is obtained. The SARS vaccine has been put aside until now, because the epidemic situation is completely controlled after phase II clinical trials, and phase III trials cannot be performed. The current design of the post clinical practice test of the COVID-19 vaccine is not clear, and a multi-country participation scheme is required to be formulated so as to determine the safety and effectiveness of the candidate vaccine. Thus, all of the vaccines under investigation have not been licensed for marketing.

The nucleocapsid of SARS-CoV-2 is formed by the encapsulation of a single-stranded positive strand RNA by a nucleocapsid protein (Nucleocapsid protein, N); the outer envelope is embedded with three glycoproteins: spike protein (S), envelope protein (E), and membrane protein (M). The S protein is positioned at the outermost layer of the virus, is regularly arranged into a coronal structure on the membrane, participates in the process of combining the virus with a virus receptor on the surface of a host cell and mediating the virus to enter the cell through membrane fusion, and plays an important role in inducing the host to generate neutralizing antibodies. At present, the research and development of the COVID-19 vaccine at home and abroad all take S protein as a primary target antigen, but the S protein of SARS-CoV of the same genus coronavirus is reported to be possible to induce antibody dependent immune enhancement reaction (ADE), so specific fragments (namely polypeptides) in the S protein are screened as antigens, and the occurrence of potential ADE can be effectively prevented while the COVID-19 is avoided.

Protein epitope profiling (the "cross-over" polypeptide compounds) was studied using combinatorial chemistry techniques, and minimal epitopes were rapidly discovered and a determinant profile was constructed. The method is characterized in that a cross overlapping fragment is synthesized by a certain number of amino acid residue peptides (such as 1 to 50 amino acid residues), protein sequences are synthesized in a way of one-by-one dislocation (or interval dislocation comprises total amino acid positions from 2 to the synthesized peptide fragments), then antigen-antibody reaction (or screening reaction for other biological purposes, such as screening to find T-cell immune epitopes, receptor ligands of SARS-CoV, and the like) is carried out, and all shortest polypeptide epitopes can be obtained at one time, so that an epitope spectrum is drawn. Then, these active short peptides are subjected to appropriate extension or ordered linear connection and then subjected to anti-SARS-CoV human positive serum screening reaction, so that the B-cell polypeptide compounds and their maps which can be used for preparing SARS-CoV diagnostic reagent, medicine and vaccine can be confirmed. SARS-CoV-2 has very high homology with SARS-CoV. The SARS-CoV-2 sequence with its antigenic determinant obtained by screening by constructing SARS-CoV's chemical library and its substitution to its position is an effective way to quickly find out new coronavirus antigen.

Traditional vaccines are prepared by inactivating or attenuating pathogens, and have the problems of biological hazard, loss of potency of original vaccines caused by genetic variation and the like. The epitope vaccine is prepared by using antigen epitopes, and is the current direction for developing infectious disease vaccines. Compared with the traditional attenuated vaccine, inactivated vaccine and other novel vaccines, the synthetic peptide vaccine is safe, nontoxic and stable, and has small and simple molecular structure, so that serious complications and iatrogenic infection are rarely caused. The synthetic peptide vaccine is prepared by synthesizing short peptide with antigenicity through chemical technology according to a certain section of antigen epitope amino acid sequence known or predicted in pathogen antigen gene, combining the short peptide with a carrier and adding an adjuvant, is the most ideal and safe novel vaccine, and is also the novel vaccine for preventing and controlling infectious diseases and treating malignant tumors at present.

Disclosure of Invention

In view of the problems of the prior art, the present invention aims to provide a polypeptide for preventing novel coronavirus pneumonia covd-19, an immunogenic conjugate and uses thereof. The invention synthesizes target polypeptide based on a certain section of known or predicted antigen epitope amino acid sequence in pathogen antigen gene, prepares the synthesized target polypeptide into polypeptide conjugate by combining with carrier protein, and then prepares the synthetic peptide vaccine by adding an adjuvant, and is used for preventing the COVID-19 virus, and compared with the traditional attenuated vaccine, inactivated vaccine and other novel vaccines, the synthetic peptide vaccine provided by the invention can rapidly cope with virus variation; the field requirement for vaccine production is reduced, and the large-scale mass production is easy; the research and development cost and the research and development period are reduced; safety, innocuity and stability; because of its small and simple molecular structure, serious complications and iatrogenic infections are rarely caused.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

A polypeptide for preventing novel coronavirus pneumonia covd-19, the amino acid sequence of said polypeptide is selected from any one of the following.

1) The amino acid sequence is the polypeptide shown in SEQ ID NO. 1.

2) A polypeptide having an amino acid sequence more than 85% identical to the amino acid sequence shown in SEQ ID NO. 1.

3) The polypeptide with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 1.

An immunogenic composition comprising a polypeptide as described above and at least one pharmaceutical carrier or excipient.

An immunogenic conjugate comprising the polypeptide described above, the immunogenic conjugate being prepared by cross-linking the polypeptide to a carrier protein by a polypeptide conjugation method.

Further, the carrier protein is any one of diphtheria nontoxic mutant CRM197, tetanus toxoid TT, diphtheria toxoid DT or meningococcal outer membrane protein M-OMP.

Further, the molar ratio of the polypeptide to the carrier protein is: 1:1-1:30.

further, the binding rate of the polypeptide to the carrier protein is 40% or more.

A vaccine for preventing novel coronavirus pneumonitis covd-19, the active ingredient of which comprises the polypeptide, the immunogenic composition, or the immunogenic conjugate of any one of the above.

Further, the vaccine further comprises a pharmaceutically acceptable adjuvant.

Further, the adjuvant is one or more combinations of aluminum hydroxide (Al (OH) 3), aluminum phosphate (AlPO 4), monophosphoryl lipid a (MPL), or an oligonucleotide (CpG).

Further, for administration to humans, the adjuvant content is 1 μg to 1000 μg per dose, preferably 10 μg to 500 μg per dose, more preferably 10 μg to 200 μg per dose, more preferably 10 to 100 μg per dose, most preferably 10 to 50 μg per dose.

Further, the vaccine is in any pharmaceutically acceptable dosage form, including intramuscular injection, subcutaneous injection, intradermal injection, and microneedle injection.

Further, the vaccine is in any pharmaceutically acceptable dose.

The preparation method of the vaccine specifically comprises the following steps.

Step 1, using a full-automatic microwave polypeptide synthesizer, adopting Fmoc solid-phase synthesis method, adding solid-phase carriers, different amino acids, removing agents, polypeptide condensing reagents and the like, and carrying out full-automatic synthesis of target polypeptides.

And 2, respectively carrying out conjugation and combination on the polypeptide synthesized in the step 1 and pharmaceutically acceptable carrier proteins (including any one of diphtheria nontoxic mutant CRM197, tetanus toxoid TT, diphtheria toxoid DT or meningococcal outer membrane proteins) through a Linker to form an immunogenic conjugate.

And 3, mixing the immunogenic conjugate prepared in the step 2 with a pharmaceutically acceptable adjuvant to prepare the vaccine.

Further, the specific method in the step 3 is that the immunogenic conjugate is mixed with an adjuvant and PBS solution, and the mixture is subjected to shaking table at room temperature for 1 hour and 30RPM, and the final concentration contains 100 mug/ml of polypeptide antigen and 0.5mg/ml of adjuvant, namely the final sample; or firstly adsorbing the immunogenic conjugate on an aluminum adjuvant, shaking at room temperature for 1 hour at 30RPM, then adding MPL or CpG adjuvant, shaking at room temperature for 1 hour at 30RPM, and obtaining final concentration containing 100 mug/ml polypeptide antigen, 0.5mg/ml aluminum adjuvant and 0.5mg/ml MPL or CpG, namely the final sample.

Use of a polypeptide as defined above, an immunogenic composition as defined above, or an immunogenic conjugate as defined in any one of the above, in the manufacture of a medicament for the prophylaxis or treatment of novel coronavirus pneumonitis covd-19.

The medicament is in any pharmaceutically acceptable dosage form.

The medicament is in any pharmaceutically acceptable dosage.

Compared with the prior art, the invention has the following beneficial effects.

The synthetic polypeptide provided by the invention is a sequence with specific antigenicity screened out based on the sequence of the S protein of the COVID-19 virus, and is synthesized into target polypeptide, so that the site requirement required by vaccine production is reduced, and the large-scale mass production is easy; the synthetic target polypeptide is combined with carrier protein to prepare polypeptide conjugate, and an adjuvant is added to prepare the synthetic peptide vaccine which is used for preventing the COVID-19 virus, and compared with the traditional attenuated vaccine, the inactivated vaccine and other novel vaccines, the synthetic peptide vaccine is safe, nontoxic and stable, and has small and simple molecular structure, so serious complications and iatrogenic infection are rarely caused.

The polypeptide provided by the invention screens out sequences with specific antigenicity based on the sequence of the S protein of the COVID-19 virus, synthesizes short peptides in vitro, combines various short peptides, and can reduce the occurrence risk of side effects compared with the whole sequence; the prior report shows that the COVID-19 virus has mutation, compared with the polypeptide sequence provided by the invention, the phenomenon that all sequences are mutated simultaneously does not occur, so that the antigenicity loss caused by mutation is effectively reduced, meanwhile, the sequence mutation part can be rapidly screened out, new short peptides are added, and new vaccines are prepared by synthesis, so that the virus mutation can be rapidly handled, and the research and development cost and the research and development period are reduced.

The vaccine for preventing the COVID-19 provided by the invention adopts a polypeptide and protein carrier combination technology, enhances the immune response of a human body, amplifies the antigenicity of the polypeptide, increases the generation of a neutralizing antibody, enhances the effectiveness of the vaccine, adopts different adjuvants and different administration modes, has universal applicability, and is suitable for people of multiple ages.

Drawings

FIG. 1 is a comparison of antibody levels of polypeptides after combination with different carriers and different adjuvants.

Detailed Description

The present invention is further illustrated by the following examples, which are given solely by way of illustration and not limitation, and various modifications and alterations of the invention will become apparent to those skilled in the art and are deemed to be within the spirit and principles of the invention as defined herein.

Example 1 Synthesis of a polypeptide of interest (the amino acid sequence of which is shown in SEQ ID NO. 1).

1. The polypeptide with the amino acid sequence shown as SEQ ID NO.1 is prepared by adopting a conventional solid phase synthesis method, and the specific steps are as follows.

(1) Fmoc protection was removed.

Commercial Rink Amide AM resin was placed in a reaction tube with a filter and resistant to organic solvents and closed with a lid resistant to organic solvents. After washing with DMF (dimethylformamide) for 1 min, an excess of 20% piperidine/DMF (volume ratio) solution was added, the reaction tube was capped and gently shaken to mix and keep Fmoc protection removed for 15 min. After draining, the mixture was washed three times with DMF.

(2) Peptide bond condensation.

Fmoc-protected amino acid in 3 times the molar amount of resin amino after preactivation (2-3 minutes after preactivation), HBTU as an activator in 3 times the molar amount of resin amino, and DIPEA (N, N-diisopropylethylamine) in 6 times the molar amount of resin amino were added to the reaction tube, DMF was used as a solvent, and complete dissolution of the above reagents was ensured, allowing complete resin coverage. The resin is mixed uniformly by shaking every 2 to 3 minutes and reacted for 20 to 30 minutes.

Then, a 50-fold molar excess of acetic anhydride DMF solution was added at room temperature and then the mixture was drained for 10 minutes, and an excess of 20% piperidine/DMF (volume ratio) solution was added at room temperature to react for 30 minutes. The solution was filtered off and the resin was washed 5 times with DMF.

The above steps are repeated in cycles until all Fmoc protected amino acids fitted into the polypeptide are attached to the resin in a linear fashion.

(3) And (5) polypeptide cleavage.

The polypeptide lysate is treated with a strong acid, such as TFA (trifluoroacetic acid). The resin was treated with TFA\water\EDT dimercaptoethanol\phenol (volume ratio: 92.5:2.5:2.5:2.5) for 2 hours at room temperature. The lysate was then carefully collected in a glass collector and diethyl ether pre-chilled with ice was added, the precipitated polypeptide was collected and washed with cold diethyl ether for 5-6 further times to give the crude peptide.

(4) And (5) purifying the polypeptide.

The crude peptide was purified by HPLC (high performance liquid chromatography), collected, lyophilized, and finally checked for purity (214 nm wavelength) of greater than 85% by HPLC and for correct molecular weight by mass spectrometry.

2. And (5) identifying results.

The synthetic peptide shown in SEQ ID NO.1 has a purity of 95% and a molecular weight of 3276.63. The polypeptide has strong antigenicity through specific antigenicity experiment measurement, and can induce neutralizing antibody in human body.

Example 2 preparation of synthetic peptide immunogenic conjugate shown in SEQ ID No. 1.

The synthetic peptides prepared in example 1 were combined with CRM197, TT, DT, bacterial outer membrane proteins by Linker to form consensus polypeptides, respectively.

The TT tetanus toxin protein and tetanus toxin carrier protein TT can be obtained through fermentation, pyrolysis, centrifugation and chromatographic purification of tetanus bacillus.

DT diphtheria toxin protein and diphtheria toxin carrier protein DT can be obtained by fermentation, pyrolysis, centrifugation and chromatography purification of diphtheria bacillus.

CRM197 recombinant diphtheria toxin protein, wherein CRM197 is diphtheria bacillus with reconstructed gene sequence, and is obtained through fermentation, cracking, centrifugation and chromatographic purification.

Meningococcal outer membrane proteins: the meningococcal strain is obtained by fermenting, cracking, centrifuging and purifying by chromatography.

1. Synthetic peptides were conjugated to CRM197 via SMCC Linker or DSAP Linker to form a consensus polypeptide.

The CRM197 protein was formulated in PBS containing 2mM EDTA at a concentration of 1 mg/ml. The required amount of CRM197 solution was taken, sulfo-SMCC (10 mg/ml) was added thereto in a molar ratio of 1:2 to 1:40, and the reaction was carried out at room temperature for 1 hour, and the reaction solution was concentrated by ultrafiltration with an ultrafiltration concentration tube at 4℃and centrifugation at 3500 rpm. After the volume concentration was 1/10 of the reaction solution, PBS was added to the original reaction solution volume, and the centrifugation concentration was continued, followed by 5 times of continuous centrifugation concentration to remove free sulfoSMCC. The final stock concentration of CRM197-SMCC was 10mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or PBS solution containing 10% DMSO, and CRM197-SMCC stock solution was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume, reacted for 1h at room temperature, SDS-PAGE was used to detect the conjugation, protein loading was 2. Mu.g, and SDS-PAGE was used to detect the electropherogram.

The desired amount of synthetic peptide (example 1) was weighed, dissolved in DMSO, DSAP (10 mg/ml DMSO solution) was added at a molar ratio of 1:2-1:30, mixed, triethylamine (30-fold molar ratio) was added, reacted overnight at room temperature, 500. Mu.l NaPi (pH 7.4, 0.1M) was added, extracted three times with 1ml chloroform, centrifuged, and the aqueous phase was added to a solution of CRM197 (2 mg/ml NaPi (pH 7.4, 0.1M)) at a molar ratio of synthetic peptide to CRM197 protein of 10:1-1:1, reacted at room temperature for 24 hours, and SDS-PAGE was used to detect the conjugation.

2. Synthetic peptides bind to TT proteins via SMCC Linker or DSAP Linker to form consensus polypeptides.

TT protein was formulated in PBS containing 2mM EDTA at a concentration of 1 mg/ml. The required amount of TT solution is taken, sulfo-SMCC (10 mg/ml) is added according to the mol ratio of 1:2-1:40, the reaction is carried out for 1 hour at room temperature, and the reaction solution is concentrated by ultrafiltration with an ultrafiltration concentration tube at 4 ℃ and is concentrated by centrifugation at 3500 rpm. After the volume concentration was 1/10 of the reaction solution, PBS was added to the original reaction solution volume, and the centrifugation concentration was continued, followed by 5 times of continuous centrifugation concentration to remove free sulfoSMCC. The final TT-SMCC stock concentration obtained was 10mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or PBS solution containing 10% DMSO, and TT-SMCC stock solution was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume, reacted for 1h at room temperature, and SDS-PAGE was used to detect the conjugation.

The desired amount of synthetic peptide (example 1) was weighed, dissolved in DMSO, DSAP (10 mg/ml DMSO solution) was added at a molar ratio of 1:2-1:30, mixed, triethylamine (30-fold molar ratio) was added, reacted overnight at room temperature, 500. Mu.l NaPi (pH 7.4, 0.1M) was added, extracted three times with 1ml chloroform, centrifuged, and the aqueous phase was added to TT (2 mg/ml NaPi (pH 7.4, 0.1M)) solution, the molar ratio of synthetic peptide to TT protein was 10:1-1:1, reacted at room temperature for 24 hours, and SDS-PAGE was used to detect the conjugation.

3. Synthetic peptides are conjugated to DT via SMCC Linker or DSAP Linker to form a consensus polypeptide.

DT protein was prepared in 1mg/ml solution with PBS containing 2mM EDTA. The required amount of DT solution is taken, sulfo-SMCC (10 mg/ml) is added according to the mol ratio of 1:2-1:40, the reaction is carried out for 1 hour at room temperature, and the reaction solution is concentrated by ultrafiltration with an ultrafiltration concentration tube at 4 ℃ and 3500 rpm. After the volume concentration was 1/10 of the reaction solution, PBS was added to the original reaction solution volume, and the centrifugation concentration was continued, followed by 5 times of continuous centrifugation concentration to remove free sulfoSMCC. The final DT-SMCC stock concentration obtained was 10mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or PBS solution containing 10% DMSO, and DT-SMCC stock solution was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume, reacted for 1h at room temperature, and SDS-PAGE was used to detect the conjugation.

The desired amount of synthetic peptide (example 1) was weighed, dissolved in DMSO, DSAP (10 mg/ml DMSO solution) was added at a molar ratio of 1:2-1:30, mixed, triethylamine (30-fold molar ratio) was added, reacted overnight at room temperature, 500. Mu.l NaPi (pH 7.4, 0.1M) was added, extracted three times with 1ml chloroform, centrifuged, and the aqueous phase was added to DT (2 mg/ml NaPi (pH 7.4, 0.1M)) solution, the molar ratio of synthetic peptide to DT protein was 10:1-1:1, reacted at room temperature for 24 hours, and SDS-PAGE was used to detect the conjugation.

4. Synthetic peptides are conjugated to M-OMP via SMCC Linker or DSAP Linker to form a consensus polypeptide.

M-OMP protein was formulated in PBS containing 2mM EDTA at a concentration of 1 mg/ml. The required amount of M-OMP solution was taken, sulfoSMCC (10 mg/ml) was added thereto in a molar ratio of 1:2-1:40, the reaction was carried out at room temperature for 1 hour, and the reaction solution was concentrated by ultrafiltration at 4℃and centrifugation at 3500 rpm. After the volume concentration was 1/10 of the reaction solution, PBS was added to the original reaction solution volume, and the centrifugation concentration was continued, followed by 5 times of continuous centrifugation concentration to remove free sulfoSMCC. The final stock solution concentration of M-OMP-SMCC was 10mg/ml. The desired amount of synthetic peptide (example 1) was weighed, dissolved in PBS or PBS solution containing 10% DMSO, stock solution of M-OMP-SMCC was added at a molar ratio of 15:1-1:1, PBS was added to the appropriate reaction volume, reacted for 1h at room temperature, and SDS-PAGE was used to detect the conjugation.

The desired amount of synthetic peptide (example 1) was weighed, dissolved in DMSO, DSAP (10 mg/ml DMSO solution) was added at a molar ratio of 1:2-1:30, mixed, triethylamine (30-fold molar ratio) was added, reacted overnight at room temperature, 500. Mu.l NaPi (pH 7.4, 0.1M) was added, extracted three times with 1ml chloroform, centrifuged, and the aqueous phase was added to M-OMP (2 mg/ml NaPi (pH 7.4, 0.1M)) solution, the molar ratio of synthetic peptide to M-OMP protein was 10:1-1:1, reacted at room temperature for 24 hours, and SDS-PAGE was used to detect the conjugation.

Example 3 preparation of vaccine and immunization efficacy.

1. Vaccine preparation.

1. A material.

Polypeptide protein: the amino acid sequence is the polypeptide shown in SEQ ID NO. 1.

Carrier protein: diphtheria toxin null mutant (CRM 197), tetanus Toxoid (TT), diphtheria Toxoid (DT), meningococcal outer membrane protein (Meningococcus OMP).

Adjuvants: aluminum hydroxide (Al (OH) 3), aluminum phosphate (AlPO 4), MPL, cpG.

2. The preparation method.

Mixing the immunogenic conjugate with an adjuvant and PBS (phosphate buffer solution), and shaking at room temperature for 1 hour and 30RPM to obtain final concentration of polypeptide antigen 100 mug/ml and adjuvant 0.5mg/ml, namely the final sample; or firstly adsorbing the immunogenic conjugate on an aluminum adjuvant, shaking at room temperature for 1 hour at 30RPM, then adding MPL or CpG adjuvant, shaking at room temperature for 1 hour at 30RPM, and obtaining final concentration containing 100 mug/ml polypeptide antigen, 0.5mg/ml aluminum adjuvant and 0.5mg/ml MPL or CpG, namely the final sample.

Table 1. Cross-combination scheme of polypeptide conjugates with adjuvants.

Note that: combination sequence number naming convention: "vector initials" + "adjuvant number".

2. And (5) performing immune effect experiments.

Vaccine immunized mice in Table 1 were subjected to immunogenicity studies, and healthy BALB/c mice weighing 16-18g were randomly grouped, with 8-10 mice per group; the samples to be tested in Table 1 were subjected to intramuscular injection on the hind limbs of mice 1 time on days 0, 14 and 21, and each time the injection volume was 100 uL/dose, blood was collected 1 time before the injection on days 0, 21 and 28, serum was separated, and 20-fold dilution was performed on the serum, and serum antibody levels on days 0, 21 and 28 were measured by enzyme-linked immunosorbent assay (ELISA), specifically.

(1) The coating liquid (0.05M carbonate-bicarbonate buffer solution) dilutes the polypeptide stock solution into working solution with the final concentration of 5 mug/ml (DMSO content is not higher than 0.05%), and after fully mixing, the enzyme label plate is added according to 100 mug/hole, the sealing paper is attached, and the coating is carried out at 2-8 ℃ overnight.

(2) The supernatant was aspirated, washed 5 times with PBST, and blocked with 100ul of 1% BSA in PBS for 1h at room temperature.

(3) PBST was washed 5 times.

(4) Sample adding: 100ul of diluted serum sample was added, incubated at room temperature for 2h, and washed 5 times with PBST.

(5) Adding enzyme-labeled antibody: 100ul of goat anti-mouse IgG HRP enzyme-labeled secondary antibody was added and incubated for 1h at room temperature.

(6) PBST was washed 5 times.

(7) Adding a substrate solution for color development: 100ul TMB was added for color development.

(8) Terminating the reaction: the reaction was quenched by the addition of 50ul of 1N sulfuric acid.

(9) And (3) result judgment: OD measurement ₄₅₀ Values, results are shown in Table 2.

Table 2. Antibody levels were compared after polypeptides were combined with different carriers and different adjuvants.

As shown in table 2, the carrier protein can significantly increase the antibody titer of the polypeptide; after the polypeptide is combined with carrier protein, the antibody titer can reach more than 0.8. The antibody titer of the carrier-free group is not more than 0.2 after the adjuvant is added, which is obviously lower than that of the carrier-free group, thus indicating that the combination of the polypeptide and the adjuvant cannot generate expected immunogenicity; the same dosage, the same carrier protein and the addition of the adjuvant can obviously increase the antibody titer, and no obvious difference exists between different adjuvants.

Sequence listing

<110> university of Qinghua, liaoning university biological stock Co., ltd

<120> a polypeptide for preventing novel coronavirus pneumonia COVID-19, immunogenic conjugate and use thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 29

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15 20

Pro Thr Trp Arg Val Tyr Ser Thr Gly

25

Claims (15)

1. A polypeptide for preventing novel coronavirus pneumonia COVID-19, characterized in that the polypeptide

The amino acid sequence is a polypeptide shown as SEQ ID NO. 1.

2. An immunogenic conjugate comprising the polypeptide of claim 1, wherein the immunogenic conjugate is prepared by cross-linking the polypeptide of claim 1 to a carrier protein by polypeptide conjugation; the carrier protein is any one of diphtheria nontoxic mutant CRM197, tetanus toxoid TT, diphtheria toxoid DT or meningococcal outer membrane protein.

3. The immunogenic conjugate of claim 2, wherein the molar ratio of the polypeptide to the carrier protein is: 1:1-1:30.

4. the immunogenic conjugate of claim 2, wherein the polypeptide binds to the carrier protein at a rate of greater than 40%.

5. A vaccine for the prophylaxis of novel coronavirus pneumonitis covd-19, characterized in that its active ingredient comprises a polypeptide according to claim 1, or an immunogenic conjugate according to any one of claims 2-4.

6. The vaccine of claim 5, further comprising a pharmaceutically acceptable adjuvant.

7. The vaccine of claim 6, wherein the adjuvant is one or more of aluminum hydroxide Al (OH) 3, aluminum phosphate AlPO4, monophosphoryl lipid a MPL, or an oligonucleotide CpG.

8. The vaccine of claim 5, wherein the adjuvant is administered to a human in an amount of 1 μg to 1000 μg per dose.

9. The vaccine of claim 5, in any pharmaceutically acceptable dosage form, including intramuscular injection, subcutaneous injection, intradermal injection, microneedle injection.

10. The vaccine of claim 5, wherein the vaccine is in any pharmaceutically acceptable dose.

11. A method of preparing a vaccine according to any one of claims 5 to 10, comprising in particular the steps of:

step 1, using a full-automatic microwave polypeptide synthesizer, adopting Fmoc solid phase synthesis method, adding solid phase carrier, different amino acids, remover and polypeptide condensation reagent to perform full-automatic synthesis of target polypeptide,

step 2, respectively carrying out conjugation and combination on the polypeptide synthesized in the step 1 and pharmaceutically acceptable carrier proteins through a Linker to form an immunogenic conjugate;

and 3, mixing the immunogenic conjugate prepared in the step 2 with a pharmaceutically acceptable adjuvant to prepare the vaccine.

12. The method for preparing the vaccine according to claim 11, wherein the specific method in the step 3 is that the immunogenic conjugate is mixed with the adjuvant and the PBS solution, and the mixture is subjected to shaking table at room temperature for 1 hour and 30RPM, and the final concentration of the polypeptide antigen is 100 mug/mL and the adjuvant is 0.5mg/mL, thus obtaining a final sample; or firstly adsorbing the immunogenic conjugate on an aluminum adjuvant, shaking at room temperature for 1 hour at 30RPM, then adding MPL or CpG adjuvant, shaking at room temperature for 1 hour at 30RPM, and obtaining final concentration containing 100 mug/mL of polypeptide antigen, 0.5mg/mL of aluminum adjuvant and 0.5mg/mL of MPL or CpG, namely the final sample.

13. Use of a polypeptide according to claim 1, or an immunogenic conjugate according to any one of claims 2 to 4, in the manufacture of a medicament for the prophylaxis or treatment of novel coronavirus pneumonitis covd-19.

14. The medicament of claim 13, wherein the medicament is in any pharmaceutically acceptable dosage form.

15. The medicament of claim 13, wherein the medicament is in any pharmaceutically acceptable dosage.