CN112225814A - Novel coronavirus RBD fusion protein subunit vaccine and preparation method and application thereof - Google Patents
Novel coronavirus RBD fusion protein subunit vaccine and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological vaccines, and particularly relates to a novel coronavirus RBD fusion protein subunit vaccine, a preparation method and application thereof, wherein the RBD fusion protein is formed by fusing an RBD structural domain comprising a novel coronavirus S protein, HBM secretion signal peptide, flag immune enhancing peptide and hFc immune enhancing peptide, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 1; the subunit vaccine is obtained by expressing RBD fusion protein through a baculovirus insect cell expression system, and then purifying; the novel coronavirus RBD fusion protein is beneficial to forming a vaccine medicament for preventing the novel coronavirus, has important significance for preventing the novel coronavirus infection, obtains the pure novel coronavirus RBD fusion protein subunit vaccine with high purity and high recovery rate by extracting and purifying the vaccine, and can realize industrial production.
Description
Technical Field
The invention belongs to the technical field of biological vaccines, and particularly relates to a novel coronavirus RBD fusion protein subunit vaccine as well as a preparation method and application thereof.
Background
Coronaviruses (Coronavirus) are a class of single-stranded positive-strand RNA viruses that infect a variety of mammals, including humans. Coronaviruses can be divided into 4 families of α, β, γ and δ, and 7 kinds of coronaviruses known to infect humans are derived from both α and β families, including low-pathogenic HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-HKU1 and highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, wherein HCoV-229E, HCoV-NL63 belongs to the family α of coronaviruses, SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, and HCoV-HKU1 belong to the family β of coronaviruses. SARS CoV-2 is generally spherical, mostly has polymorphism, has a diameter of 60-140 nm, and has a unique spike of coronavirus on the surface of virus particle, and a length of about 9-12 nm. The SARS-CoV-2 gene is about 29.8kb in size, and the genome is annotated to contain 14 Open Reading Frames (ORFs), which encode 27-28 proteins in total. The 3' end of the genome encodes 4 structural proteins, which are conserved among coronaviruses, namely surface Spike glycoprotein (Spike, S), small Envelope protein (Envelope, E), outer Membrane protein (Membrane, M) and nucleocapsid protein (N).
A novel coronavirus pneumonia (hereinafter referred to as a novel coronavirus pneumonia) caused by a novel coronavirus SARS-CoV-2 in the end of 2019 is a serious public health problem in society, because the high infectivity of the novel coronavirus pneumonia seriously affects the health of people in all countries in the world and the social stability, and 2019-nCoV threatens the life health of human beings greatly. Immunoprophylaxis and disinfection are effective means of preventing the spread of coronavirus infection. Therefore, the research of the 2019-nCoV vaccine is very important scientific research work and has great significance for the diagnosis and prevention of the novel coronavirus pneumonia. The development direction of the novel coronavirus vaccine comprises a recombinant protein vaccine, an inactivated vaccine, a DNA vaccine, an mRNA vaccine and an adenovirus vector vaccine, and the recombinant novel coronavirus (2019-COV) vaccine (adenovirus vector) has not been reported in other achievements until the clinical stage.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a novel coronavirus RBD fusion protein subunit vaccine, which plays an important role in preventing novel coronavirus infection, and a preparation method and application thereof.
The technical content of the invention is as follows:
the invention provides a novel coronavirus RBD fusion protein, which is formed by fusing an RBD structural domain comprising a novel coronavirus S protein, HBM secretion signal peptide, flag immune enhancing peptide and hFc immune enhancing peptide;
the amino acid sequence of the novel coronavirus RBD fusion protein is shown as SEQ ID NO. 1;
the amino acid sequence of the RBD structure domain is shown as SEQ ID NO. 2;
the amino acid sequence of the HBM secretion signal peptide is shown as SEQ ID NO. 3;
the amino acid sequence of the flag immune enhancing peptide is shown as SEQ ID NO. 4;
the amino acid sequence of the hFc immune-enhancing peptide (Fc receptor) is shown in SEQ ID NO. 5.
The invention also provides a preparation method of the novel coronavirus RBD fusion protein, which comprises the following steps of fusing the RBD structural domain of the novel coronavirus S protein, HBM secretion signal peptide, flag immune enhancing peptide and hFc immune enhancing peptide fragment, and synthesizing on a plasmid vector to obtain the fusion protein;
the fusion mode comprises tandem connection;
the plasmid vector comprises a pfastBac1 vector.
The invention provides a novel coronavirus RBD fusion protein subunit vaccine, which is obtained by expressing RBD fusion protein through a baculovirus insect cell expression system.
The invention also provides a preparation method of the novel coronavirus RBD fusion protein subunit vaccine, wherein the subunit vaccine is obtained by expressing the RBD fusion protein through a baculovirus insect cell expression system, and then the preparation method further comprises an extraction and purification process, wherein the extraction and purification process comprises the steps of pretreating culture supernatant obtained after centrifuging the baculovirus insect system, performing EDTA and alkali resistant Ni filler affinity chromatography, and performing ultrafiltration concentration dialysis treatment;
the pretreatment comprises adding imidazole into culture supernatant after a baculovirus insect system is centrifuged to a final concentration of 5mM-50mM, adjusting the conductance to 15 ms/cm-50 ms/cm by using NaCl, adjusting the pH to 6.2-8.0, filtering by using a membrane below 0.45um, and using filtrate for chromatographic purification;
the operation of affinity chromatography comprises the following steps:
1) the upstream construction is provided with a histidine tag at the C terminal, so that high-purity target protein can be conveniently and quickly obtained, and the affinity chromatography filler is an EDTA and alkali resistant Ni filler; the concentration, the conductivity and the pH value of the filler balance liquid imidazole are the same as those of the pretreatment liquid: 10 mM-0.1M PB (Na)2HPO4，NaH2PO4) (ii) a The NaCl concentration is 50 mM-0.6M; the concentration of imidazole is 5-50 mM; pH6.2-8.0; the column height is 5-25 cm, and the flow speed is 60-300 cm/h; the equilibrium volume is 2 CV-20 CV;
2) after filtration, the sample is loaded, the flow rate of the sample loading is 60 cm/h-300 cm/h, and the loading amount is 2 mg-40 mg per ml of filler;
3) after the sample is loaded, the column is washed by balance liquid until the base line is flat, and the flow speed is 60 cm/h-300 cm/h;
4) washing impurities, namely, the target protein with the histidine tag is bound on the column under the conditions, and part of the impurity protein is non-specifically adsorbed on the column, so that the proper conditions are selected, the non-specifically bound impurity protein can be washed, the target protein cannot be washed under the conditions, the purity is improved, and the recovery rate is not reduced;
the impurity washing buffer mainly comprises 10 mM-0.1M PB (Na)2HPO4，NaH2PO4) (ii) a The NaCl concentration is 50 mM-0.6M; the concentration of imidazole is 10-80 mM; the pH value is 6.2-8.0, and the flow speed is 60-300 cm/h;
eluting the target protein by using an elution buffer solution after impurity washing, wherein the elution buffer solution comprises 10 mM-0.1M PB (Na)2HPO4，NaH2PO4) (ii) a NaCl concentration is 0 mM-0.6M; the concentration of imidazole is 100-1M; the pH value is 6.2-8.0, and the flow speed is 60-300 cm/h;
the ultrafiltration concentration dialysis treatment comprises the following steps: because the affinity chromatography collection fluid has high-concentration imidazole and the protein is not easy to store after no stable component is purified, buffer solution is required to be replaced, an ultrafiltration method is selected for buffer solution replacement, the size of an ultrafiltration membrane package is selected from 3.5 KD-30 KD, and the ultrafiltration buffer solution comprises but is not limited to: 10 mM-0.1M PB (Na)2HPO4，NaH2PO4) 1 to 3 portions of mannitolPercent, 1 to 4 percent of arginine hydrochloride and pH6.0 to 8.0;
the process has the main characteristics of simple process route, high purity after purification and high recovery rate, wherein the purity after purification can reach more than 90 percent, and the recovery rate is more than 85 percent;
the sample after ultrafiltration is vaccine stock solution, and the vaccine stock solution and the adjuvant are prepared into a vaccine finished product together to form a final product.
An application of a novel coronavirus RBD fusion protein in preparing a novel coronavirus resistant medicament.
An application of a novel coronavirus RBD fusion protein in a novel coronavirus subunit vaccine.
The invention has the following beneficial effects:
the RBD fusion protein capable of expressing the novel coronavirus S protein provided by the invention is beneficial to forming a vaccine medicament for preventing the novel coronavirus, has good immunogenicity on a mouse, and has important significance for preventing the novel coronavirus from being infected.
Drawings
FIG. 1 is a map of the HBM-flag-RBD-hFc _ pfastbA 1 plasmid of example 1;
FIG. 2 shows the results of transformation of the plasmid of example 1 into blue and white spots;
FIG. 3 shows the results of PCR identification of the plasmid transformation of example 1;
FIG. 4 shows the result of PCR electrophoresis in bacmid extraction of example 1;
FIG. 5 shows WB assay results of protein expression of example 1;
FIG. 6 shows SDS-PAGE of the purified sample of example 1;
FIG. 7 shows WB assay results of the sample after protein purification of example 1.
Detailed Description
The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Example 1
Preparation of a novel coronavirus RBD fusion protein subunit vaccine:
1. construction of recombinant plasmid
Selecting C-terminal domain RBD, HBM (secretion signal peptide), flag (immune enhancement peptide) and hFc (immune enhancement peptide region) fragments in an S protein sequence of a novel coronavirus (COVID-19), fusing in a series connection mode, designing and synthesizing to a pfastBac1 carrier by adopting a codon optimization system, wherein the structural schematic diagram of the constructed recombinant plasmid HBM-flag-RBD-hFc _ pfastBac1 is shown in figure 1;
2. construction of recombinant bacmids
The recombinant plasmid HBM-flag-RBD-hFc _ pfastBac1 is transformed into escherichia coli DH10Bac competent cells, a blue-white spot screening recon (shown in figure 2) is carried out on an LB plate containing X-Gal/IPTG, 5 white colonies are selected for PCR identification, the size of a target fragment is 1650bp, the identification result is shown in figure 3, and the 5 white single colonies all carry target genes.
2 positive recombinants identified by PCR are picked for amplification culture, bacmid DNA is separated by an alkaline lysis method, and the result of bacmid electrophoresis is shown in figure 4, wherein the concentration of bacmid is higher and the band is correct.
3. Expression of baculovirus
0.5ml of ESF 921 was cultured at a density of 1.0X 106SF9 cells per ml were added to a 24deep well block and the following reagents were mixed in an EP tube:
a: 2 μ g of recombinant vector was added to 100 μ l of serum-free transfection medium;
b: adding 6 μ l transfection agent into 100 μ l serum-free culture medium, and standing the solution for at least 5 min;
c: the A, B solution was gently mixed and incubated at room temperature for 30 min.
After incubation, adding 800 mul of serum-free culture medium into an EP tube of a transfection agent-DNA compound incubated for 30min, gently mixing, adding the DNA-transfection agent compound with the total volume of 1ml into a 24well deep block added with cells, incubating for 4-5 hours, adding 3ml of ESF 921 culture medium into the 24well deep block hole (the final volume is 4ml), if the cell growth state is not good, adding antibiotics (using gentamicin with the concentration of 10 mg/ml), keeping the DNA transfection agent compound in the culture medium without influencing the cell transfection efficiency (if the cell growth state is adherent, sucking out the DNA transfection agent compound), covering a cover on the culture medium, culturing for 4-5 days in a shaking table, sampling, observing the pathological change condition, and harvesting the virus P0.
Passage: passage of virus P0 (virus P1); passage of virus P1 (virus P2); the 6-well plate was infected with the P2 virus generation (prepared with P3 virus), one well of cells and the supernatant were harvested on days 3, 4, 5, and 6, and protein expression was detected by WB, and the results of protein continuous expression were shown in FIG. 5(PS: cell culture supernatant, LS: cell lysis supernatant, RC: thawing pellet), where protein expression started in the cell culture supernatant on day 3 and increased in the culture supernatant on days 4, 5, and 6.
4. Purification of novel coronavirus RBD fusion protein
Sample pretreatment:
centrifuging a novel coronavirus RBD fusion protein subunit vaccine insect rod system, adding imidazole into 500ml of culture supernatant to a final concentration of 10mM, and fully stirring until the mixture is completely dissolved;
the conductivity of the above treated solution is adjusted to 30ms/cm by NaCl, the pH is adjusted to 7.2, the treated solution is filtered by a 0.45um membrane, and the filtrate is used for chromatographic purification.
(II) affinity chromatography
(1) The affinity chromatography filler is an EDTA and alkali resistant Ni filler; the concentration, the conductivity and the pH value of the filler balance liquid imidazole are the same as those of the pretreatment liquid: 20mM PB, NaCl concentration 0.3M, imidazole concentration 10mM, pH7.2 balance; the column height is 5cm, and the flow rate is 150 cm/h; equilibrium volume 10 CV;
(2) after filtration, loading a sample, wherein the loading flow rate is 150cm/h, and the loading amount is 2-40 mg per ml of filler;
(3) after the sample is loaded, the column is washed by the equilibrium liquid until the base line is flat, and the flow rate is 150 cm/h;
(4) impurity washing: washing mixed buffer solution 20mM PB, NaCl concentration 0.3M, imidazole concentration 30mM, pH7.2, flow rate 150 cm/h;
(5) and (3) elution: elution buffer 20mM PB, NaCl concentration 0.3M, imidazole concentration 300mM, pH7.2, flow rate in 150 cm/h;
(III) Ultrafiltration dialysis
The affinity chromatography collection liquid contains high-concentration imidazole, an ultrafiltration method is selected for buffer solution replacement, the size of an ultrafiltration membrane package is 10KD, and the ultrafiltration buffer solution: 20mMPB (Na)2HPO4，NaH2PO4) Mannitol 1.5%, arginine hydrochloride 2%, pH7.4, dialysis multiple 6 times volume. After dialysis, the mixture is concentrated to the concentration of 2 mg/ml;
purity was 92% and recovery 90% by coomassie brilliant blue staining.
Identification of the purified protein: taking 15 mu L of each purified protein, mixing the protein with an equal amount of 2X electrophoresis sample-adding buffer solution, performing SDS-PAGE electrophoresis, wherein the electrophoresis result is shown in figure 6, one part of the protein is stained by Coomassie brilliant blue, the purification condition is observed, the molecular weight of a sample lane of figure 6 is the target molecular weight, and the staining result after digestion almost has no other impurity band; and performing Western blot identification on the other part of the sample, wherein the WB detection result is shown in figure 7, and the molecular weight corresponding to the sample band is the target molecular weight.
5. Preparation of the vaccine:
diluting the prepared antigen with PBS at a dilution ratio of 1:10000, adding AS01B adjuvant into the diluted antigen at a ratio of 1:2, mixing completely, and packaging to obtain vaccine sample.
The application of the vaccine is as follows: 20 mice, divided into 2 groups, male and female halves. HBM-flag-RBD-hFc-pfastba 1 fusion protein subunit vaccine was injected intramuscularly in experimental group, and sterile PBS was injected intramuscularly in blank control group mice. Observing all animals participating in the experiment every day, observing crawling, eating, body temperature, respiratory tract symptoms and other conditions, wherein the immune group and the control group have no difference;
sera were obtained by tail vein blood collection on day 14 and 28 post immunization, respectively.
Immunological evaluation of the vaccine: the antibody titers detected in the sera obtained on day 14 and day 28, as shown by the results in Table 1, indicate that the immunized group had high titers of antibody production.
TABLE 1 results of antibody titer produced after animal immunization
Example 2
Extracting and purifying a novel coronavirus RBD fusion protein subunit vaccine insect rod system:
sample pretreatment:
centrifuging a novel coronavirus RBD fusion protein subunit vaccine insect rod system, adding imidazole into 500ml of culture supernatant to a final concentration of 30mM, and fully stirring until the mixture is completely dissolved;
adjusting the conductivity of the above treated solution to 50ms/cm with NaCl, adjusting pH to 6.2, filtering with 0.45um membrane, and purifying the filtrate by chromatography;
(II) affinity chromatography
(1) The affinity chromatography filler is an EDTA and alkali resistant Ni filler; the concentration, the conductivity and the pH value of the filler balance liquid imidazole are the same as those of the pretreatment liquid: 40mM PB, NaCl concentration 0.6M, imidazole concentration 30mM, pH6.2 balance; the column height is 15cm, and the flow rate is 300 cm/h; equilibrium volume 20 CV;
(2) after filtration, loading a sample, wherein the loading flow rate is 300cm/h, and the loading amount is 2-40 mg of target protein per ml of filler;
(3) after the sample is loaded, the column is washed by the equilibrium liquid until the base line is flat, and the flow rate is 300 cm/h;
(4) impurity washing: washing impurity buffer solution 40mM PB, NaCl concentration 0.6M, imidazole concentration 60mM, pH6.2, flow rate 300 cm/h;
(5) and (3) elution: elution buffer 40mM PB, NaCl concentration 0.3M, imidazole concentration 500mM, pH6.2, flow rate in 300 cm/h;
(III) Ultrafiltration dialysis
The affinity chromatography collection fluid contains high-concentration imidazole, the ultrafiltration method is selected for buffer solution replacement, the size of an ultrafiltration membrane package is 3.5KD, and the ultrafiltration buffer solution: 40mMPB (Na)2HPO4，NaH2PO4) 1 percent of mannitol, 3 percent of arginine hydrochloride and pH6.2, wherein the dialysis multiple is 6 times of the volume, and the concentration is concentrated to 2mg/ml after the dialysis is finished;
purity 93% and recovery 86.5% were analyzed by coomassie brilliant blue staining.
Example 3
Extracting and purifying a novel coronavirus RBD fusion protein subunit vaccine insect rod system:
sample pretreatment:
centrifuging a novel coronavirus RBD fusion protein subunit vaccine insect rod system, adding imidazole into 500ml of culture supernatant to a final concentration of 5mM, and fully stirring until the mixture is completely dissolved;
the conductivity of the above treated solution is adjusted to 20ms/cm by NaCl, the pH is adjusted to 8.0, the treated solution is filtered by a 0.45um membrane, and the filtrate is used for chromatographic purification.
(II) affinity chromatography
(1) The affinity chromatography filler is an EDTA and alkali resistant Ni filler; the concentration, the conductivity and the pH value of the filler balance liquid imidazole are the same as those of the pretreatment liquid: 0.1M PB, NaCl concentration 0.2M, imidazole concentration 5mM, pH8.0 balance; the column height is 10cm, and the flow rate is 100 cm/h; equilibrium volume 10 CV;
(2) after filtration, the sample is loaded, the loading flow rate is 60cm/h, and the loading amount is 2-40 mg per ml of filler;
(3) after the sample is loaded, the column is washed by balance liquid until the base line is flat, and the flow speed is 100 cm/h;
(4) impurity washing: washing buffer solution 0.1M PB, NaCl concentration 0.2M, imidazole concentration 20mM, pH7.2, flow rate 100 cm/h;
(5) and (3) elution: elution buffer 0.1M PB, NaCl concentration 0.2M, imidazole concentration 0.1M, pH7.2, flow rate in 100 cm/h;
(III) Ultrafiltration dialysis
The affinity chromatography has high yieldAnd (3) replacing the buffer solution by adopting an ultrafiltration method, wherein the size of an ultrafiltration membrane package is 30KD, and the ultrafiltration buffer solution: 0.1MPB (Na)2HPO4，NaH2PO4) 1 percent of mannitol, 4 percent of arginine hydrochloride and pH8.0, wherein the dialysis multiple is 6 times of the volume, and the concentration is concentrated to 2mg/ml after the dialysis is finished;
purity was 91.5% and recovery 87% by coomassie brilliant blue staining.
Example 4
Extracting and purifying a novel coronavirus RBD fusion protein subunit vaccine insect rod system:
(I) pretreatment
Centrifuging a novel coronavirus RBD fusion protein subunit vaccine insect rod system, adding imidazole into 500ml of culture supernatant to a final concentration of 50mM, and fully stirring until the mixture is completely dissolved;
the conductivity of the above treated solution is adjusted to 40ms/cm by NaCl, the pH is adjusted to 7.6, the treated solution is filtered by a 0.45um membrane, and the filtrate is used for chromatographic purification.
(II) affinity chromatography
(1) The affinity chromatography filler is an EDTA and alkali resistant Ni filler; the concentration, the conductivity and the pH value of the filler balance liquid imidazole are the same as those of the pretreatment liquid: 30mM PB, NaCl concentration 0.5M, imidazole concentration 50mM, pH7.6 balance; the column height is 25cm, and the flow rate is 60 cm/h; equilibrium volume 15 CV;
(2) after filtration, the sample is loaded, the loading flow rate is 60cm/h, and the loading amount is 2-40 mg per ml of filler;
(3) after the sample is loaded, the column is washed by balance liquid until the base line is flat, and the flow speed is 60 cm/h;
(4) impurity washing: washing impurity buffer solution 30mM PB, NaCl concentration 0.5M, imidazole concentration 80mM, pH7.6, flow rate 60 cm/h;
(5) eluting with 30mM PB, NaCl concentration of 0.5M, imidazole concentration of 1M, pH7.6, and flow rate of 60 cm/h;
(III) Ultrafiltration dialysis
The affinity chromatography collection liquid contains high-concentration imidazole, an ultrafiltration method is selected for buffer solution replacement, the size of an ultrafiltration membrane package is 10KD, and the ultrafiltration buffer solution: 30mMPB (Na)2HPO4，NaH2PO4) 3 percent of mannitol, 1.5 percent of arginine hydrochloride, pH7.6, the dialysis multiple is 6 times of volume, and the mixture is concentrated to the concentration of 2mg/ml after the dialysis is finished;
purity was 95% and recovery 85.1% by coomassie brilliant blue staining.
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Claims (9)
1. The novel coronavirus RBD fusion protein is characterized by being formed by fusing an RBD structural domain comprising a novel coronavirus S protein, an HBM secretion signal peptide, a flag immune enhancing peptide and an hFc immune enhancing peptide.
2. The novel coronavirus RBD fusion protein of claim 1, wherein the amino acid sequence of the novel coronavirus RBD fusion protein is set forth in SEQ ID No. 1.
3. A method for preparing the novel coronavirus RBD fusion protein as defined in claim 1 or 2, which comprises the step of fusing the RBD domain of the novel coronavirus S protein, HBM secretion signal peptide, flag immunopotentiation peptide and hFc immunopotentiation peptide fragment, and synthesizing the fused protein on a plasmid vector.
4. The method of claim 3, wherein said fusion comprises tandem.
5. The method for preparing a novel coronavirus RBD fusion protein according to claim 3, wherein the plasmid vector comprises a pfastBac1 vector.
6. A novel coronavirus RBD fusion protein subunit vaccine is characterized in that the RBD fusion protein subunit vaccine is obtained by expressing RBD fusion protein through a baculovirus insect cell expression system.
7. The preparation method of the novel coronavirus RBD fusion protein subunit vaccine as claimed in claim 6, characterized in that the subunit vaccine RBD fusion protein is obtained by expressing a baculovirus insect cell expression system, and then the preparation method further comprises an extraction and purification process, wherein the extraction and purification process comprises the steps of pretreatment of culture supernatant obtained after centrifugation of the baculovirus insect cell expression system, EDTA and alkali resistant Ni filler affinity chromatography and ultrafiltration concentration dialysis treatment.
8. Use of a novel coronavirus RBD fusion protein according to claim 1 or 2 for the preparation of a medicament against a novel coronavirus.
9. Use of a novel coronavirus RBD fusion protein according to claim 1 or 2 in a novel coronavirus subunit vaccine.
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| WO2023220842A1 (en) * | 2022-05-19 | 2023-11-23 | Shenzhen Genius Biotech Service Co.,Ltd. | A fusion protein as a subunit vaccine immunogen against sars-cov-2 and the preparation thereof |
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