CN112023035A - Nano vaccine taking S protein RBD region of SARS-CoV-2 virus as antigen and preparation thereof - Google Patents

Abstract

The present invention discloses a nano vaccine using SARS-CoV-2 virus S protein RBD zone as antigen and its preparation method. Transfecting an expression vector with a gene sequence of an S protein RBD region of SARS-CoV-2 virus into 293T, 293i and CHO cell lines for antibody expression, and then carrying out protein extraction, ultrafiltration enrichment, antibody affinity chromatography purification, protein collection after purification, PBS dialysis and aluminum hydroxide adjuvant mixing incubation to obtain the required vaccine. The vaccine is a protein for expressing the amino acid sequence of the S protein RBD region of SARS-CoV-2 virus, 24 monomers in cells can be self-assembled into a sphere structure, and the fusion expressed SARS-CoV-2 virus S protein RBD peptide segment is displayed on the surface of the sphere. The vaccine has good neutralizing activity to SARS-CoV-2 virus, and can effectively inhibit the infection of vero cells by virus. The challenge experiment shows that the vaccine can obviously and effectively protect infection and damage of SARS-CoV-2 to rhesus monkeys, and provides an excellent candidate vaccine for the prevention and control of the current new crown epidemic situation.

Description

Nano vaccine taking S protein RBD region of SARS-CoV-2 virus as antigen and preparation thereof

Technical Field

The invention belongs to the technical field of preparation of coronavirus vaccines, and particularly relates to a nano self-assembled particle protein vaccine with good neutralization activity particularly for SARS-CoV-2 virus. Meanwhile, the invention also relates to a preparation method of the nano self-assembly particle protein vaccine.

Background

In recent years, vaccine preparation technology is continuously developed, and the newly appeared nano self-assembled particle vaccine has the following advantages: the preparation is rapid, the purification process is simple and convenient, the amplification is easy, multiple antigen monomers are polymerized and displayed, the antigenicity is higher, and the safety of the production is higher through the fermentation of human cells. For example, the technology of displaying the influenza virus conserved antigenic site on the surface of the nano self-assembled particle to enhance the antigenicity and safety of the vaccine has been widely applied to the development of general influenza vaccines.

SARS-CoV-2 virus (Severe acid Respiratory Syndrome Coronavir 2) belongs to the family of Coronaviridae, a Sarbecovirus subtype of the genus beta Coronavirus. This virus causes a new type of coronavirus infection pneumonia (COVID-19). The latent period of SARS-CoV-2 virus is longer than that of SARS-CoV virus, the infectivity is stronger than that of SARS-CoV virus, and although the fatality rate is weaker than that of SARS-CoV virus, it can also cause severe death of patient, especially chronic patient. At present, the special therapeutic drugs are still in the clinical experimental stage, the preventive vaccines are still in the clinical development stage, and the conventional SARS-CoV-2 virus inactivated vaccine preparation technology requires that the virus operation must be carried out in a laboratory with the biological safety level reaching more than three levels.

Although the nano self-assembled particle vaccine technology is expected to provide a new breakthrough for the prevention and treatment of SARS-CoV-2 virus, no relevant report is found in the prior art.

Disclosure of Invention

The invention aims to provide a nano self-assembly particle protein vaccine which can be rapidly prepared, takes an S protein RBD region of SARS-CoV-2 virus as an antigen and has good neutralizing activity to SARS-CoV-2 virus, aiming at the defects of the prior art, in particular to deal with the severe situation of prevention and control of COVID-19 epidemic situation.

The invention also aims to provide a method for preparing the nano self-assembled particle protein vaccine.

The purpose of the invention is realized by the following technical scheme.

A nano-vaccine taking S protein RBD zone of SARS-CoV-2 virus as antigen is characterized in that: the vaccine is a protein for expressing an amino acid sequence of an S protein RBD region of SARS-CoV-2 virus, the amino acid sequence of a monomer is shown as a sequence table SEQ ID NO.1, 24 monomers can be self-assembled into a sphere structure in a cell, and a fusion expressed SARS-CoV-2 virus S protein RBD peptide segment is displayed on the surface of the sphere.

The method for preparing the nano vaccine specifically comprises the following steps:

(1) transfecting an expression vector with a SARS-CoV-2 virus S protein RBD region gene sequence into 293T, 293i and CHO cell lines for antibody expression, wherein the SARS-CoV-2 virus S protein RBD region gene sequence is shown as a sequence table SEQ ID NO. 2; wherein, the 293T cell expression system uses a serum-free culture medium, and the Transfection reagent is FuGENE HD Transfection (Promega); 293i and CHO cell expression system is suspension expression system, serum-free culture medium is used, and transfection reagent is PEI 25000; the rest operations are carried out according to conventional operation methods of 293T, 293i and CHO cell expression systems;

(2) extracting proteins from cells using non-denaturing extraction methods including grinding, freeze-thawing, and sonication; directly ultrafiltering and enriching the culture medium;

(3) carrying out affinity chromatography purification of the antibody by using a Ni-NTA column, and collecting the purified protein; wherein, a gradient elution method is used during elution, and the basic buffer solution consists of 50mM sodium dihydrogen phosphate, 300mM sodium chloride, 0.05% Tween 20 and 5% glycerol, and has the pH value of 7.5; the elution buffer solution is prepared by adding imidazole with different concentrations into a basic buffer solution; the gradient elution method is that elution is carried out by using elution buffers of 10mM, 20mM and 40mM of imidazole until no protein flows out, then elution is carried out by using elution buffers of 200mM and 300mM of imidazole in sequence, the elution buffers of 200mM and 300mM of imidazole are collected, and the collected elution buffers of 200mM and 300mM of imidazole are enriched;

(4) dialyzing the purified protein with 2mM PBS (pH 7.5) overnight, and detecting the concentration of the protein for later use;

(5) and mixing the total amount of the target protein with an aluminum hydroxide adjuvant with the final concentration of 1mg/ml, and incubating for more than 2h to form a stable mixture, namely the required nano self-assembled particle protein vaccine.

In order to identify the biological function of the nano self-assembly particle protein vaccine, the invention immunizes mice and rhesus monkeys with the prepared nano self-assembly particle protein vaccine, collects immune serum to carry out a SARS-CoV-2 virus neutralization experiment, and carries out a rhesus monkey challenge protective experiment. The immunization dose of the mice is 2ug of each nose drop, 6ug of each subcutaneous injection is injected subcutaneously, and the immunization dose of the rhesus monkey is 20ug of each nose drop or intramuscular injection. Experiments show that the neutralizing antibody generated by the nano self-assembled particle protein vaccine can effectively inhibit the infection of SARS-CoV-2 virus to vero cells, and rhesus monkey virus challenge protective experiments show that the nano self-assembled particle protein vaccine can obviously and effectively protect the infection and damage of SARS-CoV-2 to rhesus monkeys.

Compared with the prior art, the invention has the following advantages:

1. the invention uses the nano self-assembly particle technology to prepare the vaccine for resisting SARS-CoV-2 virus for the first time, the obtained vaccine has good neutralization activity to SARS-CoV-2 virus, the generated neutralizing antibody can effectively inhibit the infection of SARS-CoV-2 virus to vero cells, and the challenge protective experiment of rhesus monkey shows that the nano self-assembly particle protein vaccine can obviously and effectively protect the infection and damage of SARS-CoV-2 to rhesus monkey, and provides an excellent candidate vaccine for the prevention and control of the current new crown epidemic situation.

2. The nano self-assembly particle technology can be used for quickly preparing emergency vaccines, has a plurality of antigenic sites and is displayed on the surface of nano particles, and is favorable for generating good antigenicity.

3. The preparation process of the vaccine can be completed only in a common laboratory, has high safety and is beneficial to process expansion.

Drawings

FIG. 1: and (3) detecting the RBD fusion expression ferritin in 293T system cells, 293i system culture supernatant and CHO system culture supernatant by using his and SARS Spike protein antibody western blot.

FIG. 2: laser confocal detection of RBD fusion expressed ferritin using his and SARS spike protein antibodies in 293T.

FIG. 3: after protein purification, RBD fusion expression ferritin in 293T system cells, 293i system culture supernatant and CHO system culture supernatant is detected by using western blot under the condition of SARS Spike S1 protein antibody denaturation.

FIG. 4: after protein purification, using a western blot under the non-denaturing condition of SARS Spike S1 protein antibody to detect RBD fusion expression ferritin in 293T system cells, 293i system culture supernatant and CHO system culture supernatant.

FIG. 5: and (3) detecting RBD fusion expression ferritin in 293T system cells, 293i system culture supernatant and CHO system culture supernatant by PAGE-Coomassie brilliant blue staining under a non-denaturing condition after protein purification.

FIG. 6: and (3) observing 293T system cells, 293i system culture supernatant and CHO system culture supernatant by using a projection electron microscope after protein purification, and performing RBD fusion expression on ferritin nanoparticles.

FIG. 7: antibody detection of RBD fusion expression ferritin in serum of BALB/C mice, C57 mice and rhesus monkeys after immunization.

FIG. 8: mouse subcutaneous injection and nose drop immunization, and serum neutralization SARS-CoV-2 virus experiment.

FIG. 9: experiments on neutralizing SARS-CoV-2 virus in serum after intramuscular injection and nasal drop immunization of rhesus monkeys.

FIG. 10: and (3) detecting the nasal and pharyngeal toxin expelling virus load after SARS-CoV-2 infects rhesus monkeys.

FIG. 11: SARS-CoV-2 infects rhesus monkey lung HE staining to observe pathological injury.

Detailed Description

The invention is described in further detail with reference to the following drawings and examples, which are not intended to limit the technical scope of the invention, and all changes and equivalents that can be made based on the teachings of the invention shall fall within the protective scope of the invention.

Example 1: preparing the nano self-assembled particle protein vaccine.

Cell transfection plasmid preparation

1) A gene sequence (shown as a sequence table SEQ ID NO. 2) of an S protein RBD region of SARS-CoV-2 virus is optimized according to a codon of a human-derived expression system, and is subjected to whole gene synthesis to construct a pc3.1+ vector framework.

2) The expression frame is verified to be correct by constructing plasmids through sequencing, and a target plasmid escherichia coli seed bank is established.

3) Extracting expression plasmid of S protein RBD region of SARS-CoV-2 virus by using endotoxin removing large quality-improving particle kit.

Different cell line protein expression conditions

1. Protein expression in 293T cell System

Transient transfection of the expression plasmid for the S protein RBD region of the SARS-CoV-2 virus into 293T cells:

1) performing transfection experiment by using a 10cm culture plate, and starting transfection when the density of 293T cells reaches 70-90%;

2) the original culture medium is discarded before transfection, and 8ml of new culture medium of 2% fetal calf serum is added;

3) preparing transfection reagent suspension, adding 20ug of target plasmid into each plate, and adding the plasmid into serum-free culture medium according to the proportion of 50ul per 0.8ug of plasmid to prepare solution A; adding 60 ul of transfection reagent into each plate, and adding the transfection reagent into a serum-free culture medium according to the proportion of 50ul of transfection reagent into every 2ul of plasmids to prepare a solution B;

4) placing the prepared solution A and solution B for 5 minutes, adding the solution A and the solution B together, uniformly mixing, placing the mixture at room temperature for more than 20 minutes, and slowly adding the mixture into a cell culture plate;

5) mixing the culture plates by cross-mixing method, and adding 5% CO at 37 deg.C₂Culturing, and harvesting cells after 48 hours. The cell culture was carried out for 48h, and the protein expression was observed by confocal laser microscopy (FIG. 2).

6) Harvested cells were washed with PBS, centrifuged at 3000rpm for 5min, and washed twice. PBS was discarded, RIPA was added, ice-cooled for 30min, centrifuged at 12000rpm for 30min, and the supernatant was harvested. The harvested supernatant is subjected to Western-Blot detection (shown in figure 1), and the result shows that the 293T cell expression system can express the target protein.

2. Protein mass expression in 293i cell System

1) Shaking glycerol bacillus of escherichia coli with target genes, carrying out large-scale extraction by using an endotoxin-free plasmid large-scale extraction kit, and filtering the large-scale extracted plasmids by using a 0.22 mu m filter; the plasmid concentration is preferably 1mg/ml, and concentration is required when the plasmid concentration is low.

2) The day before transfection, cells to be transfected were taken out from the incubator and counted, and the density and the survival rate of the cells were roughly calculated.

3) The cells were harvested and replaced with fresh medium, transferred to a 1L flask with a final volume of 360mL, placed at 37 ℃ in 8% CO₂And culturing for 16h in a 125r/min incubator.

4) Before transfection, cell counting was carried out, and the density of transfected cells was 5X 10 per 250ml of the system⁸And (4) cells. Will be 5X 10⁸The cells were gently resuspended in pre-warmed 30mL RPMI 1640 medium and the resuspended cells were transferred to 250mL flasks.

5) Add 500. mu.g of plasmid to 28mL of RPMI 1640 medium, mix well with shaking, add 1.5mL of PEI to each tube, vortex immediately for 20s, incubate transfection mixture at room temperature for 20 min. The transfection mixture was slowly added to the cells and placed at 37 ℃ with 8% CO₂And culturing in a 125r/min incubator.

6) After 4-6 hours, the transfected cells were transferred to a 1L flask and medium was added to 250 mL; the cells were then incubated at 37 ℃ with 8% CO₂And culturing in a 125r/min incubator for 5 days.

7) The supernatant was harvested and protein concentrated using a 10kD ultrafiltration tube. The harvested supernatant is subjected to Western-Blot detection (shown in figure 1), and the result shows that the 293i cell expression system can express the target protein.

3. Mass expression of proteins using CHO cells

1) Shaking glycerol bacillus of escherichia coli with target genes, carrying out large-scale extraction by using an endotoxin-free plasmid large-scale extraction kit, and filtering the large-scale extracted plasmids by using a 0.22 mu m filter; the concentration of plasmid is preferably 1mg/ml, and when the concentration of plasmid is low, concentration is required.

2) Mixing the plasmid, Opti-MEM solution, FreeStyle^TMThe CHO cell culture medium was removed from the refrigerator and returned to room temperature. In FreeStyle^TMAdding glutamine (the final concentration is 8mM) into a CHO cell culture medium according to a volume ratio of 1/100, and supplementing double antibodies;

3) before transfection, the density of CHO suspension cells is ensured to be 1.2X 10⁶-1.5×10⁶Per mL, the proportion of viable cells should be greater than 95% in order to ensure high transfection efficiency.

4) In using FreeStyle^TMMixing the solution slightly and uniformly for several times before MAX transfection reagent without shaking and mixing;

5) add 37.5. mu.g of plasmid DNA to the Opti-MEM solutionMaking the total volume to be 0.6ml, and mixing uniformly; mix 37.5. mu.l FreeStyle^TMMAX Transfection Reagent was added to Opti-MEM solution to make 0.6ml total;

6) immediately diluting the diluted FreeStyle^TMAdding MAX transfection reagent diluent into the diluted DNA solution to make the total volume be 1.2ml, and gently mixing uniformly;

7) DNA-FreeStyle^TMThe MAX mixed solution is placed for 10 minutes at room temperature, so that a DNA-FreeStyleTM MAX complex is formed; 1.2ml of DNA-FreeStyle^TMAdding the MAX compound into a 125-ml suspension cell culture bottle; culturing the transfected cells in a CO2 incubator at 37 ℃, and culturing in a shaking table for 6-7 days;

8) the supernatant was harvested and protein concentrated using a 10kD ultrafiltration tube. The harvested supernatant is subjected to Western-Blot detection (shown in figure 1), and the result shows that the CHO cell expression system can express the target protein.

Protein purification

The gradient elution method is used during purification and elution, and the basic buffer solution consists of 50mM sodium dihydrogen phosphate, 300mM sodium chloride, 0.05% Tween 20 and 5% glycerol, and has the pH value of 7.5; the elution buffer was prepared by adding imidazole at different concentrations to the base buffer.

1) The Ni-NTA Agarose beads and all solutions were equilibrated to room temperature;

2) digesting the non-denatured extract protein with DNAse I for 30 minutes, then adding 1M imidazole to a final concentration of 10mM, filtering the protein solution into beads using a 0.22um filter, and mixing by inversion at room temperature for 2 hours;

3) the protein-beads slurry was added to the column, and the column was washed with 10mM, 20mM, and 40mM imidazole in the order of elution buffer.

4) And detecting the protein concentration of the effluent eluent and the 40mM imidazole elution buffer solution by using a spectrophotometer, adding 200mM and 300mM imidazole elution buffer solution to carry out gradient elution on the target protein when the protein concentration of the effluent eluent of the column is less than or equal to the protein concentration of the 40mM imidazole elution buffer solution, and collecting the effluent 200mM and 300mM imidazole elution buffer solution.

5) The eluted target protein is enriched by using a 10kD ultrafiltration tube, dialyzed overnight by using 2mM PBS (pH 7.5), and collected and subjected to native-PAGE Coomassie brilliant blue staining (shown in figure 5), so that the target protein with high purity of a single band is obtained, and Western-Blot detection (shown in figure 3 and figure 4) is carried out, and the result shows that the target band with correct size is obtained. The purified enriched protein was observed using transmission electron microscopy to form nanoparticles (shown in fig. 6).

6) Protein quantification was performed on the purified protein using BCA method.

In conclusion, the 293T, 293i and CHO expression systems can produce target proteins and assemble into nanoparticles, wherein the 293T expression system can be used for preparing small and medium-sized vaccines, and the 293i and CHO expression systems can be used for preparing medium and large-sized vaccines.

The application example is as follows: verification of antiviral efficacy of vaccines

Purified protein immunized animals

1) Animal experiments all accord with the 3R principle, namely substitution, reduction and optimization; the Animal experiments were approved by the institute of medical biology, IACUC (institutional Animal Care and Use Committee) organization of the Chinese academy of medical sciences. Six healthy rhesus monkeys, 8 months old, weighed 1.5kg ± 0.5 kg. The BALB/C mice and C57BL/6 mice used were all 4-week-old mice.

2) Preparation of intramuscular or subcutaneous injection vaccine: the purified protein was mixed with aluminium hydroxide adjuvant at the desired concentration, the adjuvant final concentration being 1 mg/ml. Preparing a nasal drop vaccine: 1ml of 100ug/ml purified protein was added to 10ug of recombinant human interferon alpha 1b dry powder.

3) The nanometer self-assembly particle protein vaccine is injected into rhesus monkey via upper respiratory tract nasal drop at 20ug/200ul or intramuscular injection at 20 ug/0.5 ml. BALB/C mice and C57BL/6 mice were injected nasally via the upper respiratory tract with 2ug/20ul or subcutaneously with 6ug/50ul of the nano self-assembling particle protein vaccine. Equal amounts of blanks were run in a simultaneous experiment using PBS instead of protein vaccine. Second and third boosts were performed three weeks apart.

4) Three weeks after booster immunization animal sera were taken and used as primary antibodies according to 1: the protein was diluted at a ratio of 200 and subjected to western blot detection of purified protein (shown in FIG. 7), which revealed that the immunized animal could produce antibodies against the RBD peptide fragment of the S protein of SARS-CoV-2 virus.

Neutralizing experiment detection of SARS-CoV-2 virus by immune animal serum

1) Sample serum inactivated monkey inactivated serum sample: 56 degrees, 30 minutes. Mouse sera were treated with RDE II for 16h, followed by 56 degrees for 30min to stop the reaction.

2) The virus was neutralized with different dilutions of animal serum and allowed to react for 2 hours at room temperature.

3) Using vero cells in logarithmic growth phase, after trypsinization, using complete cell culture medium to blow, mix uniformly, dilute to 3X 10⁴100ul of the virus was added to the neutralized virus-serum mixture at 100. mu.l/well. The cell culture plate was incubated at 37 ℃ for an additional 72-96 hours with 5% CO2 to observe cytopathic effects. Experiments show that the neutralizing antibody generated by the nano self-assembly particle protein vaccine can effectively inhibit the infection of the SARS-CoV-2 virus to vero cells (shown in figure 8 and figure 9).

SARS-CoV-2 attacking and immunizing rhesus monkey protective experiment

Immunization of rhesus monkeys and control rhesus monkey rhinorrhea infection 1 x 10⁵TCID50 SARS-CoV-2. Rhesus nasal swabs and pharyngeal swabs were collected daily after infection for virus detoxification detection (shown in fig. 10). Rhesus monkeys were sacrificed 7 days after infection, and tissues of rhesus monkeys were collected for pathological examination (shown in fig. 11). The results show that the nano-particle protein vaccine effectively protects the infection and the damage of SARS-CoV-2 to rhesus monkeys after three times of immunization.

Sequence listing

<110> institute of medical science and biology of China academy of medical sciences

<120> a nano vaccine using SARS-CoV-2 virus S protein RBD region as antigen and its preparation

<141> 2020-08-06

<150> 2020102642338

<151> 2020-04-07

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 460

<212> PRT

<213> Artificial sequence

<223> based on the RBD sequence of SARS-Cov2 virus S protein and the ferritinin sequence of nano self-assembly particle framework protein

<400> 1

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp His His His His His His Asn Ile Thr Asn Leu

20 25 30

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

35 40 45

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

100 105 110

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

115 120 125

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

130 135 140

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

145 150 155 160

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro

165 170 175

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

180 185 190

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

195 200 205

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

210 215 220

Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe

225 230 235 240

Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe

245 250 255

Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala

260 265 270

Val Arg Asp Pro Gln Thr Leu Glu Ser Gly Gly Met Leu Ser Lys Asp

275 280 285

Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys Glu Met Asn Ser Ser

290 295 300

Thr Val Tyr Met Ser Met Ser Ser Trp Cys Tyr Thr His Ser Leu Asp

305 310 315 320

Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His

325 330 335

Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln

340 345 350

Leu Thr Ser Ile Ser Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln

355 360 365

Ile Phe Gln Lys Ala Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile

370 375 380

Asn Asn Ile Val Asp His Ala Ile Lys Ser Lys Asp His Ala Thr Phe

385 390 395 400

Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu

405 410 415

Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His

420 425 430

Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg

435 440 445

Lys Ser His His His His His His His His His His

450 455 460

<210> 2

<211> 1383

<212> DNA

<213> Artificial sequence

<223> based on the RBD sequence of SARS-Cov2 virus S protein and the ferritinin sequence of nano self-assembly particle framework protein

<400> 2

atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60

gaccaccacc atcatcacca caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 120

gccacccgct tcgccagcgt gtacgcctgg aaccgcaagc gcatcagcaa ctgcgtggcc 180

gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta cggcgtgagc 240

cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatccgc 300

ggcgacgagg tgcgccagat cgcccccggc cagaccggca agatcgccga ctacaactac 360

aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggacagc 420

aaggtgggcg gcaactacaa ctacctgtac cgcctgttcc gcaagagcaa cctgaagccc 480

ttcgagcgcg acatcagcac cgagatctac caggccggca gcaccccctg caacggcgtg 540

gagggcttca actgctactt ccccctgcag agctacggct tccagcccac caacggcgtg 600

ggctaccagc cctaccgcgt ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc 660

gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttcaacttc 720

aacggcctga ccggcaccgg cgtgctgacc gagagcaaca agaagttcct gcccttccag 780

cagttcggcc gcgacatcgc cgacaccacc gacgccgtgc gcgaccccca gaccctggag 840

agcggcggca tgctgagcaa ggacatcatc aagctgctga acgagcaggt gaacaaggag 900

atgaacagca gcaccgtgta catgagcatg agcagctggt gctacaccca cagcctggac 960

ggcgccggcc tgttcctgtt cgaccacgcc gccgaggagt acgagcacgc caagaagctg 1020

atcatcttcc tgaacgagaa caacgtgccc gtgcagctga ccagcatcag cgcccccgag 1080

cacaagttcg agggcctgac ccagatcttc cagaaggcct acgagcacga gcagcacatc 1140

agcgagagca tcaacaacat cgtggaccac gccatcaaga gcaaggacca cgccaccttc 1200

aacttcctgc agtggtacgt ggccgagcag cacgaggagg aggtgctgtt caaggacatc 1260

ctggacaaga tcgagctgat cggcaacgag aaccacggcc tgtacctggc cgaccagtac 1320

gtgaagggca tcgccaagag ccgcaagagc caccatcacc accatcatca tcaccaccac 1380

tag 1383

Claims (2)

1. A nano-vaccine taking S protein RBD zone of SARS-CoV-2 virus as antigen is characterized in that: the vaccine is a protein for expressing an amino acid sequence of an S protein RBD region of SARS-CoV-2 virus, the amino acid sequence of a monomer is shown as a sequence table SEQ ID NO.1, 24 monomers can be self-assembled into a sphere structure in a cell, and a fusion expressed SARS-CoV-2 virus S protein RBD peptide segment is displayed on the surface of the sphere.

2. The method for preparing the nano vaccine of claim 1, which comprises the following steps:

(1) transfecting an expression vector with a SARS-CoV-2 virus S protein RBD region gene sequence into 293T, 293i and CHO cell lines for antibody expression, wherein the SARS-CoV-2 virus S protein RBD region gene sequence is shown as a sequence table SEQ ID NO. 2; wherein, the 293T cell expression system uses a serum-free culture medium, and the Transfection reagent is FuGENE HD Transfection (Promega); 293i and CHO cell expression system is suspension expression system, serum-free culture medium is used, and transfection reagent is PEI 25000; the rest operations are carried out according to conventional operation methods of 293T, 293i and CHO cell expression systems;

(2) extracting proteins from cells using non-denaturing extraction methods including grinding, freeze-thawing, and sonication; directly ultrafiltering and enriching the culture medium;

(3) carrying out affinity chromatography purification of the antibody by using a Ni-NTA column, and collecting the purified protein; wherein, a gradient elution method is used during elution, and the basic buffer solution consists of 50mM sodium dihydrogen phosphate, 300mM sodium chloride, 0.05% Tween 20 and 5% glycerol, and has the pH value of 7.5; the elution buffer solution is prepared by adding imidazole with different concentrations into a basic buffer solution; the gradient elution method is that elution is carried out by using elution buffers of 10mM, 20mM and 40mM of imidazole until no protein flows out, then elution is carried out by using elution buffers of 200mM and 300mM of imidazole in sequence, the elution buffers of 200mM and 300mM of imidazole are collected, and the collected elution buffers of 200mM and 300mM of imidazole are enriched;

(4) dialyzing the purified protein with 2mM PBS (pH 7.5) overnight, and detecting the concentration of the protein for later use;

(5) and mixing the total amount of the target protein with an aluminum hydroxide adjuvant with the final concentration of 1mg/ml, and incubating for more than 2h to form a stable mixture, namely the required nano self-assembled particle protein vaccine.

Images