CN112851825A - Recombinant ferritin nanoparticle for expressing novel coronavirus RBD and construction method thereof - Google Patents
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Abstract
The invention relates to the field of biomedicine, in particular to a recombinant ferritin nanoparticle for expressing a novel coronavirus RBD and a construction method thereof. The invention relates to a recombinant ferritin nanoparticle expressing a novel coronavirus Receptor Binding Domain (RBD) by connecting a novel coronavirus (SARS-CoV-2) and ferritin according to the self-assembly characteristic of the ferritin. RT-PCR and Western Blot results show that the recombinant baculovirus is successfully constructed and can express the foreign protein RBD-HF. The observation of a transmission electron microscope shows that 20-40nm nanoparticles can be seen under the electron microscope, which proves that the recombinant ferritin nanoparticles presenting SARS-CoV-2RBD are successfully constructed by the invention, and a foundation is laid for the subsequent vaccine research.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a recombinant ferritin nanoparticle for expressing a novel coronavirus RBD and a construction method thereof.
Background
SARS-CoV-2 belongs to the genus of beta coronavirus, and it has been found that its specific receptor is Angiotensin converting enzyme 2 (ACE 2), and the region of the viral protein binding to it is the Receptor Binding Domain (RBD).
Vaccines will play a crucial role in the control of this virus. According to data of World Health Organization (WHO), 52 candidate vaccines are in clinical trials as long as 12-8 days of 2020, and 162 vaccines are in preclinical research, so that the development of a safe and effective candidate vaccine is still a current urgent task.
Particle vaccines can bring the protein of interest closer to its native conformation, and the self-assembling protein nanoparticles most studied to date include Virus like particles (Virus like particles), Serum albumin (Serum albumin), silk protein (silk protein), and Ferritin (Ferritin).
Ferritin has stable structure and can resist high temperature and various denaturants, and the outer surface of ferritin can be modified by gene fusion method to express and present exogenous protein. Viruses that have been reported to date using ferritin as a nanoparticle carrier are: EB virus, Hepatitis C Virus (HCV), Human Immunodeficiency Virus (HIV), Influenza virus (Influenza), and the like.
Therefore, the recombinant ferritin nanoparticle for expressing the novel coronavirus RBD and the construction method thereof have important practical significance.
Disclosure of Invention
In view of the above, the invention constructs the recombinant ferritin nanoparticles presenting SARS-CoV-2RBD by using ferritin as a nanoparticle vector and using a baculovirus expression system, and lays a foundation for the next research.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a recombinant ferritin nanoparticle for expressing a novel coronavirus RBD, wherein a novel coronavirus receptor binding domain and a recombinant fragment of ferritin are connected to a vector, a competent cell is transformed to obtain a recombinant bacmid, and the recombinant bacmid is transfected to the cell to obtain a recombinant baculovirus.
In some embodiments of the invention, the vector is a pFastBac Dual vector; the competent cell is DH10Bac competent cell; the cells were sf9 cells.
The invention also provides a preparation method of the recombinant ferritin nanoparticle, which comprises the steps of connecting the novel coronavirus receptor binding domain and the recombinant fragment of ferritin to a vector, transforming cells to obtain recombinant bacmids, and transfecting the recombinant bacmids to the cells to obtain the recombinant baculovirus. In some embodiments of the invention, the vector is a pFastBac Dual vector; the competent cell is DH10Bac competent cell; the cells were sf9 cells.
The invention also provides the application of the recombinant ferritin nano-particle or the recombinant ferritin nano-particle prepared by the preparation method in the preparation of a novel coronavirus receptor binding domain and ferritin recombinant protein, vaccine or novel coronavirus research.
In addition, the invention also provides recombinant protein, and the recombinant ferritin nanoparticle or the recombinant ferritin nanoparticle prepared by the preparation method is expressed.
Based on the research, the invention also provides the application of the recombinant protein in preparing a medicament for treating diseases or symptoms; the condition or disease includes conditions or diseases caused by the novel coronavirus.
More importantly, the invention also provides a vaccine prepared from the recombinant protein.
In some embodiments of the invention, the vaccine further comprises a pharmaceutically acceptable protective agent, stabilizer, adjuvant, diluent, carrier or adjuvant.
The invention also provides the use of the vaccine in the manufacture of a medicament for the treatment of a condition or disease; the condition or disease includes conditions or diseases caused by the novel coronavirus.
The invention relates to a recombinant ferritin nanoparticle expressing a novel coronavirus Receptor Binding Domain (RBD) by connecting a novel coronavirus (SARS-CoV-2) and ferritin according to the self-assembly characteristic of the ferritin. The invention connects SARS-CoV-2RBD and ferritin recombinant fragment to the position under pFastBac Dual vector pH promoter, and transforms them to DH10Bac competent cell to obtain recombinant bacmid, and obtains recombinant baculovirus after transfecting adherence sf9 cell. RT-PCR and Western Blot results show that the recombinant baculovirus is successfully constructed and can express the foreign protein RBD-HF. The observation of a transmission electron microscope shows that 20-40nm nanoparticles can be seen under the electron microscope, which proves that the recombinant ferritin nanoparticles presenting SARS-CoV-2RBD are successfully constructed by the invention, and a foundation is laid for the subsequent vaccine research.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a schematic representation of rBD-SARS-CoV 2-RBD-HF;
FIG. 2 shows recombinant bacmid RT-PCR identification; wherein, M: DL 5000 MARKER; 1-2: rBD-SARS-CoV 2-RBD-HF; 3: water; 4: empty vector recombinant bacmid;
FIG. 3 shows visualization of recombinant baculovirus infected sf9 cytopathic effect (200 ×); wherein, A: normal sf9 cells; b: 48 hours after sf9 cells are infected with rBDV-SARS-CoV 2-RBD-HF; c: 72h after sf9 cells are infected with rBDV-SARS-CoV 2-RBD-HF;
FIG. 4 shows recombinant baculovirus RT-PCR identification; wherein, M: DL2000 MARKER; 1-2: rBDV-SARS-CoV 2-RBD-HF; 3: pFastBac Dual; 4: MOCK (DH10 Bac); 5: water;
FIG. 5 shows Western Blot to identify recombinant baculovirus foreign gene expression; wherein, M: the protein MARKER; 1: protein RBD-HF; 2: MOCK;
figure 6 shows recombinant ferritin nanoparticle observations under an electron microscope.
Detailed Description
The invention discloses a recombinant ferritin nanoparticle expressing a novel coronavirus RBD and a construction method thereof, and a person skilled in the art can realize the expression by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Ferritin is present in most organisms and may have the property of self-assembly. The invention fuses SARS-CoV-2RBD and ferritin, and constructs the recombinant ferritin nano-particle presenting SARS-CoV-2RBD through a baculovirus expression system according to the characteristics of ferritin. The successful expression and self-assembly of SARS-CoV-2 into nano-particles are proved by RT-PCR, WesternBlot and transmission electron microscope identification, and then we can proceed immunogenicity research on the recombinant ferritin nano-particles.
In the recombinant ferritin nanoparticles for expressing the novel coronavirus RBD and the construction method thereof, used raw materials and reagents can be purchased from the market.
The invention is further illustrated by the following examples:
plasmid, cell, strain:
SARS-CoV-2RBD-HF gene sequence (shown as SEQ ID No. 1) entrusted Nanjing King Musry Biotechnology Ltd to synthesize and connect to pFastBac Dual vector, named pFBD-SARS-CoV 2-RBD-HF; sf9 cells were stored in the laboratory; DH10BacTMPurchased from Saimer Feishale science and technology (China) Co.
SARS-CoV-2RBD-HF gene sequence:
GGATCCAACTTAAAAAAAAAAATCAAAATGATCACCAACCTGTGCCCATTCGGAGAGGTGTTCAACGCTACTAGGTTCGCCTCCGTCTACGCTTGGAACCGCAAGCGTATCTCTAACTGCGTCGCCGACTACTCAGTGCTGTACAACTCCGCTTCCTTCTCTACCTTCAAGTGCTACGGAGTGTCCCCTACCAAGCTGAACGACCTGTGCTTCACTAACGTCTACGCCGACAGCTTCGTGATCAGGGGTGACGAAGTCAGACAGATCGCCCCAGGCCAGACCGGAAAGATCGCTGACTACAACTACAAGCTGCCTGACGACTTCACTGGCTGCGTGATCGCTTGGAACAGCAACAACCTGGACTCTAAGGTCGGTGGCAACTACAACTACCTGTACAGGCTGTTCAGAAAGTCTAACCTGAAGCCCTTCGAGCGCGACATCTCCACCGAAATCTACCAGGCCGGTAGCACTCCATGCAACGGTGTGGAGGGCTTCAACTGCTACTTCCCACTGCAGTCATACGGCTTCCAGCCTACCAACGGAGTCGGTTACCAGCCCTACCGTGTGGTCGTGCTGTCTTTCGAACTGCTCCACGCTCCTGCTACTGTGTGCGGACCCAAGAAGTCAACTAACCTGGTCAAGAACAAGTGCGTGAACTTCAACTTCAACGGACTGACCGGTACTGGAGGTGGCGGAGGTTCAGGCGGAGGTGGCTCCGGAGGTGGCGGAAGCATGACCACTGCCTCAACTTCCCAGGTCCGCCAGAACTACCACCAGGACTCTGAGGCTGCCATCAACCGTCAGATCAACCTGGAACTGTACGCTTCATACGTGTACCTGAGCATGTCTTACTACTTCGACAGGGACGACGTCGCCCTGAAGAACTTCGCTAAGTACTTCCTGCACCAGTCCCACGAGGAAAGAGAGCACGCTGAAAAGCTGATGAAGCTGCAGAACCAGCGCGGTGGCCGTATCTTCCTGCAGGACATCAAGAAGCCCGACTGCGACGACTGGGAGTCCGGTCTGAACGCCATGGAGTGCGCTCTGCACCTGGAAAAGAACGTGAACCAGAGCCTGCTGGAACTGCACAAGCTGGCCACCGACAAGAACGACCCTCACCTGTGCGACTTCATCGAGACTCACTACCTGAACGAACAGGTGAAGGCTATCAAGGAGCTGGGCGACCACGTCACCAACCTCAGGAAGATGGGTGCTCCTGAGTCTGGACTGGCTGAATACCTGTTCGACAAGCACACTCTGGGAGACTCAGACAACGAATCCTAAAAGCTT
the main reagents are as follows:
sf-900TMII SFM medium, Grace's InsectMedium,
II Reagent transfection Reagent and restriction endonuclease from Saimer Feishel (China) company; X-Gal, IPTG, tetracycline, kanamycin and gentamicin were purchased from Beijing Solebao technologies, Inc.; fetal bovine serum, azure, streptomycin were all purchased from Hyclone; anti-SARS-CoV 2RBD mice were multi-resistant given by Dr. Harzepine of this group of subjects; horseradish peroxidase-labeled goat anti-mouse IgG (H + L) was purchased from shanghai bi yunnan biotechnology limited.
Example 1 recombinant bacmid preparation and characterization
The plasmid pFBD-SARS-CoV2-RBD-HF was transformed into DH10Bac, and then applied to a selection plate containing tetracycline (10. mu.g/ml), kanamycin sulfate (50. mu.g/ml), gentamicin (7. mu.g/ml), X-gal (100. mu.g/ml) and IPTG (40. mu.g/ml), and cultured in a 37 ℃ incubator for 48 hours. Picking white single colony to SOC culture medium, shake culturing for 3h at 37 ℃, and carrying out culture by using a universal primer PH: TTCATACCGTCCCACCAT (shown as SEQ ID No. 2) and pUC/M13R: AGCGGATAACAATTTCACACAGG (shown in SEQ ID No. 3), PCR identification is carried out on the bacterial liquid, and bacmid is extracted from the correctly identified strain and named rBD-SARS-CoV 2-RBD-HF.
The results of colony PCR using the primer set rBD-SARS-CoV2-RBD-HF and empty vector recombinant bacmid (FIG. 1) with PH and pUC/M13R are shown in FIG. 2, wherein the amplification of the rBD-SARS-CoV2-RBD-HF fragment 1956bp long and the amplification of the empty vector recombinant bacmid fragment 678bp long are consistent with the expectation.
EXAMPLE 2 rescue of recombinant baculovirus
Sf9 cells were seeded in six well plates when cell density was 2X 106At individual cells/ml, the medium was changed to 1ml Grace medium per well. Mu.l of Cellffectin. RTII and 3. mu.g of rBD-SARS-CoV2-RBD-HF were diluted in 100. mu.l of Grace medium, and mixed well. Mixing the rod particles diluted with Grace culture medium with
II, mixing and standing for 20 minutes at room temperature. Transfectants were added to six-well plates, incubated at 27 ℃ for 5 hours in a constant temperature incubator, the transfection medium was discarded, and replaced with complete medium. Culturing at 27 deg.C in incubator until cell lesion. The supernatant was harvested, centrifuged at 3000rpm for 5 minutes, filtered through a 0.22 μm filter to give the first generation (P1) of recombinant baculovirus, designated rBDV-SARS-CoV2-RBD-HF, inoculated into sf9 cells at 1% volume of the recombinant baculovirus of P1 generation, harvested after 72 hours, and treated in the same manner as P1 generation to give the second generation (P2) of rBDV-SARS-CoV 2-RBD-HF.
As shown in fig. 3: after the rBDV-SARS-CoV2-RBD-HF is inoculated to the adherent sf9 cells, observation is carried out under the 48h and 72h mirrors respectively, and after the rBDV-SARS-CoV2-RBD-HF is infected, a large number of sf9 cells fall off and become round.
Example 3 identification of recombinant baculovirus by RT-PCR
rBDV-SARS-CoV2-RBD-HF was inoculated into sf9 cells, cultured in a incubator at 27 ℃ for 72 hours, and the cells were collected, and a certain volume (e.g., 500. mu.l) of Trizol reagent was added thereto and left at room temperature for 10 minutes to extract total RNA. After reverse transcription of the extracted total RNA, the RNA was purified by reverse transcription using the primer RBD-HF-F: ATCTCGAGCCATGGTGCTAGCATGATCACCAACCTGTGCCC (shown in SEQ ID No. 4) and RBD-HF-R:
CCATCTCCCGGTACCGCATGCTTAGGATTCGTTGTCTGAGTCTCCC (shown in SEQ ID No. 5) were subjected to PCR identification.
Sf9 cells are infected with rBDV-SARS-CoV2-RBD-HF for 72h, the cells are harvested, and after total RNA extraction, reverse transcription and PCR identification, a band with the length of 1299bp can be seen, which is consistent with the expectation, as shown in FIG. 4.
Example 4 identification of recombinant baculovirus expressing foreign protein by Western Blot
rBDV-SARS-CoV2-RBD-HF is inoculated to sf9 cells, the cells are collected after being cultured in a constant temperature incubator at 27 ℃ for 72 hours, and are added with IP lysate and then are subjected to ultrasonic disruption, and the mixture is centrifuged at 12000rpm at 4 ℃ for 3 minutes to collect supernatant. After addition of 5 XSDS, SDS-PAGE was performed in a boiling water bath for 10 minutes. Transferring by semi-dry transfer method, sealing with 5% skimmed milk at room temperature for 2 hr, incubating with Anti-RBD mouse polyclonal antibody, washing with TBST, incubating with goat Anti-mouse secondary antibody, and dripping ECL onto NC membrane for exposure.
Sf9 cells were infected with rBDV-SARS-CoV2-RBD-HF for 72h, the cells were harvested, lysed with IP lysate and sonicated, electrophoresed by SDS-PAGE, proteins were transferred to NC membranes, blocked with 5% skim milk, incubated with Anti RBD polyclonal antibody, incubated with goat Anti-mouse secondary antibody and developed by exposure, as shown in FIG. 5, a band of approximately 48kDa was seen, consistent with the expectation.
EXAMPLE 5 Electron microscopy
rBDV-SARS-CoV2-RBD-HF was inoculated to sf9 cells, cultured in a incubator at 27 ℃ for 72 hours, followed by cell harvest and sonication, centrifuged at 12000rpm at 4 ℃ for 3 minutes, and the supernatant was collected. The supernatant was ultracentrifuged (20% sucrose cushion, 35000rpm, 4 ℃, 2h), and the supernatant was discarded and resuspended in an appropriate amount of PBS. The sample was dropped onto a transmission electron microscope copper mesh, incubated for 5min, negatively stained with 1% phosphotungstic acid for 3 min, and observed using a transmission electron microscope.
As shown in fig. 6: after Sf9 cells are infected with rBDV-SARS-CoV2-RBD-HF for 72h, the cells are harvested, after ultrasonic disruption and high-speed centrifugation, the heavy suspension precipitate is observed under a projection electron microscope, and the recombinant ferritin nanoparticles with the particle size of 20-40nm can be seen.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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Claims (10)
1. The recombinant ferritin nanoparticle for expressing the novel coronavirus RBD is characterized in that a novel coronavirus receptor binding domain and a recombinant fragment of ferritin are connected to a vector, competent cells are transformed to obtain recombinant bacmids, and the recombinant baculoviruses are transfected to the cells to obtain the recombinant baculovirus.
2. The recombinant ferritin nanoparticle of claim 1 wherein the vector is a pFastBacDual vector;
the competent cell is DH10Bac competent cell;
the cells were sf9 cells.
3. The method of claim 1 or 2, wherein the recombinant fragment of ferritin and the novel coronavirus receptor binding domain are ligated to a vector, the cells are transformed to obtain recombinant bacmid, and transfected into cells to harvest the recombinant baculovirus.
4. The method according to claim 3, wherein the vector is a pFastBacDual vector;
the competent cell is DH10Bac competent cell;
the cells were sf9 cells.
5. Use of the recombinant ferritin nanoparticles according to claim 1 or 2 or prepared by the preparation method according to claim 3 or 4 for the preparation of recombinant proteins, vaccines or novel coronavirus research of novel coronavirus receptor binding domain and ferritin.
6. Recombinant protein expressed from the recombinant ferritin nanoparticles according to claim 1 or 2 or the recombinant ferritin nanoparticles produced by the production method according to claim 3 or 4.
7. Use of a recombinant protein according to claim 6 for the manufacture of a medicament for the treatment of a disorder or disease; the condition or disease includes conditions or diseases caused by the novel coronavirus.
8. A vaccine made from the recombinant protein of claim 7.
9. The vaccine of claim 8, further comprising a pharmaceutically acceptable protective agent, stabilizer, adjuvant, diluent, carrier, or adjuvant.
10. Use of a vaccine according to claim 8 or 9 in the manufacture of a medicament for the treatment of a disorder or disease; the condition or disease includes conditions or diseases caused by the novel coronavirus.
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