CN113583137B - Novel recombinant subunit vaccine of coronavirus south Africa mutant strain and application thereof - Google Patents

Abstract

The invention discloses a fusion protein containing an epitope of a novel coronavirus south Africa mutant strain COVID-19 vaccine, which consists of an RBD fragment in an RBD epitope fragment S1 subunit, an SD1 fragment and an immunoglobulin Fc fragment of the novel coronavirus south Africa mutant strain COVID-19 vaccine. According to the invention, through research, the RBD fusion SD1 of the novel coronavirus south Africa mutant strain COVID-19 has excellent effects, especially has outstanding effects on neutralizing data, and therefore, the vaccine can be popularized as a vaccine aiming at the novel coronavirus south Africa mutant strain COVID-19.

Description

Novel recombinant subunit vaccine of coronavirus south Africa mutant strain and application thereof

Technical Field

The invention belongs to the technical field of biology and medicine, in particular to a novel recombinant subunit vaccine of coronavirus and application thereof, and more particularly relates to a method for expressing recombinant subunit protein with immunocompetence in eukaryotic cells by using virus genes artificially synthesized by a genetic engineering means and developing the vaccine by using the expressed recombinant protein.

Background

The novel coronavirus belongs to a novel coronavirus of beta genus, and has envelope, round or elliptical particle, usually polymorphism, and diameter of 60-140 nm. The structural protein of new coronavirus SARS-CoV-2 includes S protein, N protein, E protein and M protein. In SARS-CoV-2, only the neutralizing antibody directed against S protein can neutralize the virulence of virus and prevent the infection of virus to organism, the Spike protein (S protein) on the mantle of coronavirus is a key protein for recognizing host cell Receptor in the virus infection process, and the S protein is composed of two subunits S1 and S2, wherein the S1 subunit contains Receptor Binding Domain (RBD) to mediate adsorption, and the S2 subunit mainly shows fusion activity.

The mutant strain in south Africa is a popular call, and refers to a mutant lineage (linkage) of a novel coronavirus detected in south Africa at the earliest day 12/18 in 2020. This mutation is then propagated throughout the world at its very rapid rate, originally also referred to as 501y. v2, to distinguish it from the N501Y mutation in the british mutant. They all belong to the B lineage mutations. The south African mutant lineage was named according to the New crown lineage nomenclature (B.1.351). This variant has become the local leader since then. B.1.351 contained multiple spike mutations in addition to D614G, including a cluster of mutations in NTD (e.g., 242-244del and R246I), 3 mutations in RBD (K417N, E484K and N501Y) and one mutation near the furin cleavage site (A701V). Wangceng et al published a research paper in Nature journal (Antibody Resistance of SARS-Cov-2 Variants B.1.351 and B.1.1.7, Nature,2021) and revealed the protective efficiency of partially neutralizing antibodies and vaccine immune sera against mutants in south Africa. By comparison of the neutralizing activities, it was found that the neutralizing activity against the British mutant B.1.1.7 was substantially unchanged, but the neutralizing activity against the south African mutant B.1.351 was significantly reduced (12.4-fold, Moderna; 10.3-fold, feverer)

For the new coronavirus south Africa mutant strain COVID-19, some researches have been carried out to obtain related vaccines, and the vaccines which are on the market at present are developed for HuB strain new coronavirus, for example, the dominant antigen epitope fragment which can be used for preparing the new coronavirus south Africa mutant strain COVID-19 vaccine is screened and optimized in the patent application of CN 111333704A, and the protein fragment is fused with the human Fc fragment to prepare the new virus vaccine. And The first clinical test result of The CoVID-19 vaccine phase I of The institute of biological engineering of military medical research institute of military science in China published in The reissue of 22 th.e.5.2020 of China shows that The current test result of The novel coronavirus vaccine entering The human body test phase in China is good in feedback, and The specific antibody and T cell generated by a single dose of The novel adenovirus 5 vector COVID-19(Ad5-nCoV) vaccine in 14 days are patented by related vaccines. However, the existing vaccines have poor protection to the south African mutant strains, so that the development of effective vaccines for the south African strain novel coronavirus is urgently needed.

Disclosure of Invention

The invention finally determines the effective vaccine aiming at the novel coronavirus south Africa mutant strain based on the analysis research of the novel coronavirus south Africa mutant strain and the analysis of the novel coronavirus vaccine. Firstly, based on the vaccine constructed by fusing the RBDSD1 sequence and the human Fc sequence, the research and analysis on the applicability of the vaccine to the novel coronavirus south African mutant strain unexpectedly find that the vaccine constructed by fusing the epitope fragment of the novel coronavirus south African mutant strain, namely the RBD fragment in the S1 subunit and the SD1 fragment, with the human immunoglobulin Fc sequence has unexpected technical effects.

The invention firstly provides a fusion protein containing an antigen epitope of a novel coronavirus south African mutant strain COVID-19 south African mutant vaccine, which is characterized in that the fusion protein is formed by fusing an RBD fragment in an S1 subunit, an SD1 fragment and a human immunoglobulin Fc sequence of the antigen epitope fragment of the novel coronavirus south African mutant strain COVID-19 south African mutant vaccine, and the amino acid sequence of the RBD fragment is as shown in SEQ ID NO: 2 is shown in the specification; the amino acid sequence of the SD1 fragment is shown as SEQ ID NO: 3 and the immunoglobulin Fc fragment is selected from human, mouse, rabbit, cow, goat, pig, mouse, rabbit, hamster, rat, or guinea pig immunoglobulin Fc fragment; further, the immunoglobulin Fc fragment is selected from an Fc fragment of IgG, IgA, IgD, IgE, or IgM; wherein preferably the immunoglobulin Fc fragment is selected from an IgG1 Fc fragment, an IgG2 Fc fragment, an IgG3 Fc fragment, or an IgG4 Fc fragment; particularly preferably, the immunoglobulin Fc fragment is a human IgG Fc fragment, most preferably it is the amino acid sequence shown as SEQ ID No. 5.

Most preferably, the fusion protein comprises the amino acid sequence shown in SEQ ID No. 7.

The invention correspondingly provides a nucleic acid for coding the fusion protein, preferably, the nucleotide sequence for coding the RBD fragment is shown as SEQ ID NO: 1 or a degenerate sequence thereof; the encoding nucleotide sequence of the SD1 fragment is shown as SEQ ID NO: 4 or a degenerate sequence thereof; the nucleotide sequence of the human immunoglobulin Fc fragment is shown as SEQ ID NO: 6 or a degenerate sequence thereof. Most preferably, the nucleotide sequence of the nucleic acid encoding the fusion protein is as set forth in SEQ ID NO: 8 or a degenerate sequence thereof.

The invention further provides a recombinant vector and a recombinant cell containing the encoding nucleic acid.

The invention also provides a novel coronavirus south Africa mutant strain COVID-19 south Africa mutant strain vaccine which is characterized by comprising the fusion protein. Further included are adjuvants such as aluminum hydroxide. Wherein, the fusion protein is obtained by chemical synthesis or gene recombination method.

Through research and exploration, the invention discovers that for a novel coronavirus south African mutant strain COVID-19 south African mutant strain, SD1 is fused on the basis of RBD of the novel coronavirus south African mutant strain COVID-19 south African mutant strain, and the effect of the novel coronavirus south African mutant strain is obviously superior to that of only fusing RBD in the aspects of immunogenicity and stability.

Drawings

FIG. 1ProteinA affinity chromatography each buffer system elution peak reduction electrophoresis chart.

FIG. 2HAc-NaAc buffer system SP elution non-reducing electrophoretogram.

FIG. 3PB buffer SP elution non-reducing electropherogram.

FIG. 4 is a photograph of an SDS-PAGE electrophoretic test of example four.

FIG. 5 detection profiles and data from size exclusion chromatography in example five.

Detailed Description

The following examples and figures of the present invention are merely illustrative of specific embodiments for practicing the invention and are not to be construed as limiting the invention. Meanwhile, the experimental techniques and experimental methods used in this example are all conventional techniques unless otherwise specified. The materials, reagents and the like used in the present examples are all available from normal commercial sources unless otherwise specified.

Example one vector construction

Through a chemical synthesis method, a fusion protein related gene sequence of the antigen epitope of the novel coronavirus south Africa mutant strain COVID-19 south Africa mutant strain vaccine is synthesized by Beijing Liu-He Hua Dagenescience and technology limited company. Wherein the content of the first and second substances is controlled,

the nucleotide sequence of the novel coronavirus south African mutant COVID-19 south African mutant RBD is as follows (SEQ ID NO: 1):

the encoded RBD amino acid sequence is as follows (SEQ ID NO: 2):

the encoded SD1 amino acid sequence is as follows (SEQ ID NO: 3):

the SD1 nucleotide sequence is as follows (SEQ ID NO: 4):

the encoded hFc amino acid sequence is as follows (SEQ ID NO: 5):

the hFc nucleotide sequence is as follows (SEQ ID NO: 6):

RBDSD1-hFc amino acid sequence (SEQ ID NO: 7)

The nucleotide sequence of RBDSD1-hFc is as follows (SEQ ID NO: 8):

the target genes (i.e., RBD-Fc and RBDSD1-Fc) were constructed into pcDNA3.1 expression vector by direct synthesis from gene company to obtain DNA plasmids pcDNA3.1-RBD, pcDNA3.1-RBD-Fc and pcDNA3.1-RBDSD1-Fc, respectively.

Example II transient cell transfer

One day before transfection, according to 2.0X 10 ⁶ cells are inoculated in the density of 4.0 multiplied by 10 in the next day of culture ⁶ cells/mL or so;

② after the cells are counted the next day of culture, the cell survival rate is more than 95%, the viable cell density is more than or equal to 4.0

10 ⁶ cells/mL, can be used directly; if the cell density is less than 4.0X 10 ⁶ cells/mL, collected by centrifugation (800rpm, 5min)Collecting cells, and mixing the cells at 4.0 × 10 ⁶ cells/mL density was resuspended in Transpro CD01 medium;

thirdly, preparing a DNA and PEI mixed solution according to the optimized transient conversion process;

adding the mixed solution into the culture solution for culture;

fifthly, after 18h of culture, 2mM valproic acid (VPA) and 5% + 0.5% DN feed 2+ DN feed B2 of initial culture volume are recommended to be added once, the living cell density and the protein expression can be further improved, and 0.05-0.10g/L dextran sulfate can be added if the cell agglomeration phenomenon occurs in the process of culturing after transient transformation.

Sixthly, culturing for 7 days or the activity is lower than 60 percent, finishing the culture, and collecting cell supernatant for protein purification.

EXAMPLE III protein purification

The protein obtained in example two was purified by affinity chromatography followed by ion exchange as described below.

First, the following Protein A affinity chromatography method was adopted for purification of RBDSD1-Fc

One) the overall procedure of the Protein A affinity chromatography method is as follows

A chromatographic column: AT protein a Diamond affinity chromatography medium, column volume CV: 10ml, flow rate: 4-6 ml/min, pressure limiting: less than or equal to 0.3 MPa.

Pretreatment: at least 3 CV's were washed first with 0.1M NaOH and then with purified water for at least 10 CV's.

Balancing: washing 2-3 CVs with a washing buffer (0.1M HAc-NaAc, pH3.0), and then fully balancing at least 10 CVs with a binding buffer (0.02M PB,0.5M NaCl, pH7.0) for later use.

Loading: and adjusting the conductance of the filtered CHO suspension cell culture to be consistent with that of the binding buffer solution by using 5M NaCl, then loading the CHO suspension cell culture to a well-balanced chromatographic column, collecting the loaded sample, flowing through the chromatographic column, and sampling 100 mu l of the CHO suspension cell culture from the sample for electrophoretic detection.

Leaching: after loading was completed, at least 4 CVs were rinsed with binding buffer to UV baseline plateau, followed by rinsing with rinsing buffer (0.1M HAc-NaAc, pH5.0) at least 4 CVs to UV baseline plateau.

And (3) elution: eluting with elution buffer (0.1M HAc-NaAc, pH3.8), collecting each eluted fraction in separate tubes, adding appropriate amount of neutralization buffer (0.02M PB, pH8.0) according to the volume of the eluate in each tube, and mixing.

II) ProteinA affinity chromatography process optimization

1. Optimization of buffer system for protein affinity chromatography elution

Experiments are carried out according to the whole steps of the Protein A affinity chromatography method, glycine-hydrochloric acid, acetic acid-sodium acetate and citric acid buffer systems are selected for purification, and purified samples are collected for SDS-PAGE electrophoresis detection.

Preparing samples to be electrophoresed (boiling water bath for 5min) according to the proportion of 100 mu l of sample and 25 mu l of Loading dye (non-reduction type) of each sample collected in the purification process, and then taking a proper amount of sample to carry out electrophoresis under the electrophoresis conditions: the gel concentration is 10%, the gel is firstly run for 15min at constant voltage of 100V, then run to the front edge at constant voltage of 200V to just run out, and then Coomassie brilliant blue staining is carried out, boiling water is used for decoloring, and the electrophoresis result is recorded by photographing (the electrophoresis image is shown in figure 1). And (4) carrying out electrophoretic purity analysis on the corresponding lane of the Protein A affinity chromatography collected sample by using Labworks software, and recording an analysis result. As shown in Table 1, the protein obtained by purifying the acetic acid-sodium acetate system has relatively highest purity, the average particle size is also smallest, and the small particle size indicates that the protein aggregation degree is low, so that the acetic acid-sodium acetate system is optimal.

TABLE 1 analysis table of particle size and purity of main peak eluted by each buffer system of ProteinA affinity chromatography

Secondly, the purification of RBDSD1-Fc was performed by the following SP cation exchange chromatography

One) the overall steps of the SP cation exchange chromatography method are as follows:

a chromatographic column: monomix HC60-SP cation exchange chromatography column, column volume CV: 10ml, flow rate: 4-6 ml/min, pressure limiting: less than or equal to 0.3 MPa.

Pretreatment: at least 3 CV's were washed with 1M NaOH followed by at least 10 CV's with purified water.

Balancing: eluting 2-3 CVs with an elution buffer (0.02M PB,1M NaCl, pH6.0), and then fully balancing at least 10 CVs with a binding buffer (0.02M PB, pH6.0) for later use.

Loading: and (3) eluting and collecting neutralized samples by ProteinA affinity chromatography, loading the samples to a well-balanced chromatographic column, collecting the loaded samples to flow through, and respectively sampling 100 mu l of the samples for electrophoretic detection.

Leaching: after the loading is finished, the sample is firstly eluted to 6-8 CV to UV baseline flatness by using a binding buffer solution, and then eluted to 6-8 CV to UV baseline flatness by using 3% B.

And (3) elution: elution was performed with a 10% B gradient followed by a 100% B gradient and fractions were collected in separate tubes.

Di) SP cation exchange chromatography process optimization

1. SP cation exchange chromatography buffer system optimization

Protein purification is carried out by using the SP cation exchange chromatography method, acetic acid-sodium acetate and PB (phosphate buffer) systems are respectively selected as buffer systems, purified samples are collected after purification, and the purity of the samples is analyzed by electrophoresis, so that the two systems are used for purifying the collected proteins, and two obvious impurity bands (shown in figure 2) exist between about 50-70 Kd, but the two impurity bands are obviously reduced (shown in figure 3) when the number of collection tubes is increased and the number of samples obtained by purifying the PB system is reduced. Therefore, the PB system can collect protein samples with better purity. Therefore, for the purification of SP by cation exchange chromatography, a buffer system of PB is preferably used.

EXAMPLE four detection of SDS-PAGE

Taking a sample collected after the proteinA A and cation exchange chromatography purification to prepare an electrophoresis sample (boiling water bath for 5min) according to the proportion of 100 mu l sample and 25 mu l Loading dye, and then taking a proper amount of sample to carry out electrophoresis, wherein the electrophoresis conditions are as follows: the gel concentration is 10%, the gel is firstly run for 15min at constant voltage of 100V, then run to the front edge at constant voltage of 200V to just run out, and then Coomassie brilliant blue staining is carried out, boiling water is used for decoloring, and the electrophoresis result is recorded by photographing (an electrophoretogram is shown in the figure). The result shows that the molecular weight of the denatured protein is between 60 and 70Kd, and the purity of the protein obtained after two-step purification is more than 95 percent and meets the requirement (figure 4).

EXAMPLE V size exclusion chromatography detection

Soaking the chromatographic column for 48 hours before filling the column to fully swell the column, stirring to remove air bubbles, slowly pouring the column into glass or other suitable materials, finishing one-time filling to avoid layering, flattening the column surface of the chromatographic column, and continuously washing the newly filled chromatographic column with water for 4-6 hours to discharge the bubbles in the column.

The sample introduction can adopt an automatic sample introduction valve, or can directly add the sample to the surface of the bed, and adopts molecular exclusion chromatography and hydrophilic methacrylic resin size exclusion chromatography columns. The mobile phase is 20mmol/L Histidine solution (pH5.7-6.2), 0.5-1.35 mol/L NaCl, 0.02% polysorbate 80, and the flow rate is 0.5mL/min isocratic elution; the sample loading amount is 20 mu l; the detection wavelength is 280 nm. The purity was calculated by area normalization. The result showed a purity of 95.972 (FIG. 5).

EXAMPLE six mouse immunization and neutralizing antibody detection

Each recombinant protein was separately mixed with an aluminum hydroxide adjuvant and mice were immunized. Female BALB/c mice, 20, 6-8 weeks old, were purchased and randomly divided into 2 groups (experimental and negative control), 10 per group. Each mouse was muscle immunized with 10ug of protein. Boosts were performed every two weeks for two boosts. From the first immunization, mice were bled every two weeks, and supernatants were centrifuged for detection of neutralizing antibody titers.

The neutralizing antibody titer detection assay was as follows:

1. cell preparation: the day before the experiment, will be about 1x10 ⁴ Inoculum size of individual cells/well cells to be infected were seeded in 96-well cell culture plates.

2. Pseudovirus infection: taking out the frozen pseudovirus, thawing, sucking required pseudovirus, adding the pseudovirus into a cell culture system to infect target cells after complete thawing, and replacing a fresh culture medium for continuous culture after 6-8H after virus infection.

3. Infection detection: after the cells are infected with pseudovirus 48-72H, the infection efficiency is determined by observing the activity of the detected luciferase.

4. The maximum dilution of serum at which 50% of infection was inhibited was taken as the neutralizing antibody titer.

The experimental results are shown in the table below, and the same serum samples can be found, and the neutralizing antibody titer detected by using the south Africa strain pseudovirus is far higher than that detected by using the HuB strain pseudovirus, which indicates that the vaccine has better protective effect on the south Africa strain. RBDSD1-Fc has a higher neutralizing activity than RBD-Fc.

EXAMPLE seventhly, stability on standing

The purified proteins RBD-Fc and RBDSD1-Fc were placed in an incubator at 25 ℃ for 60 days, mice were immunized with the antibody of the sixth example and tested for neutralizing antibodies with a south African pseudovirus, and it was found that RBDSD1-Fc protein, which was placed in the incubator at 25 ℃ for 60 days, still induced the body to produce high-titer neutralizing antibodies, while RBD-Fc protein, which was placed in the incubator at 25 ℃ for 60 days, induced the body to have relatively low neutralizing antibody titer (results are shown in the following table).

The results show that SD1 has the function of stabilizing the structure of RBDSD1-Fc protein.

Sequence listing

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<120> a novel recombinant subunit vaccine of coronavirus south Africa mutant strain and application thereof

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aactgtgtgg ctgactactc tgtgctctac aactctgcct ccttcagcac cttcaagtgt 240

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acctatggag tgggctacca accatacagg gtggtggtgc tgtcctttga actgctccat 660

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ctgccattcc aacagtttgg cagggacatt gctgacacca cagatgctgt gagggaccca 840

cagaccttgg agattctgga catcacacca tgttccgaca aaactcacac atgcccaccg 900

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 960

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 1020

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 1080

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 1140

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 1200

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 1260

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 1320

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 1380

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 1440

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 1500

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 1560

ctcgag 1566

Claims (7)

1. A fusion protein containing the epitope of a novel coronavirus south African mutant COVID-19 vaccine is characterized in that the fusion protein is composed of the epitope fragment of the novel coronavirus south African mutant COVID-19 vaccine and an immunoglobulin Fc fragment, wherein the epitope fragment of the novel coronavirus south African mutant COVID-19 vaccine is an RBD fragment in an S1 subunit and an SD1 fragment;

the fusion protein consists of SEQ ID NO: 9, and (b) is encoded by the nucleotide sequence shown in the figure.

2. The fusion protein of claim 1.

3. The coding nucleic acid of claim 2, having the nucleotide sequence of SEQ ID NO: shown at 9.

4. Recombinant vector, recombinant cell containing a nucleic acid encoding according to claim 2 or 3.

5. A novel coronavirus south african mutant covi-19 vaccine, comprising the fusion protein of claim 1.

6. The vaccine of claim 5, further comprising an adjuvant.

7. The vaccine of claim 5, wherein the fusion protein is obtained by chemical synthesis or by genetic recombination.

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