CN112940138A - Trimerization new coronavirus receptor binding domain, preparation method and application thereof - Google Patents
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Abstract
The invention relates to the field of biomedicine, in particular to a trimerization new coronavirus receptor binding domain, and a preparation method and application thereof. The invention constructs and expresses trimerization new coronavirus RBD. The target fragment is connected to a pFastBac1 vector, and the recombinant bacmid is obtained after the target fragment is transformed into a DH10Bac competent cell. And (3) transfecting the recombinant bacmid to sf9 cells, performing virus rescue to obtain recombinant baculovirus, inoculating the recombinant baculovirus into suspension sf9 cells for 96 hours, and harvesting a culture medium for purification. PCR and WesternBlot results show that the recombinant baculovirus is successfully constructed and can successfully express the foreign protein RBD-6 HB. SDS-PAGE and WesternBlot identification are carried out on the purified protein, and the result shows that the purification effect is good, thereby laying the foundation for later vaccine and basic research.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to a trimerization new coronavirus receptor binding domain, and a preparation method and application thereof.
Background
Currently, new coronaviruses are still spreading worldwide. The novel coronavirus (SARS-CoV-2) belongs to the genus beta coronavirus of the family Coronaviridae, and is divided into four structural proteins (S, M, E, N), wherein S protein is involved in virus invasion and induction of specific antibody and production of neutralizing antibody. The S protein is divided into two parts of S1 and S2, S1 is related to receptor binding, S2 is related to membrane fusion, a Receptor Binding Domain (RBD) of a receptor binding site is positioned at the C end of S1, and the S1 protein presents a trimeric structure in a natural state.
Therefore, the simulation of the natural structure of the Receptor Binding Domain (RBD) of the novel coronavirus (SARS-CoV-2) has important practical significance for later vaccine acquisition and basic research.
Disclosure of Invention
In view of the above, the present invention provides trimerization new coronavirus receptor binding domain, and a preparation method and application thereof. PCR and WesternBlot results show that the recombinant baculovirus is successfully constructed and can successfully express the foreign protein RBD-6 HB. SDS-PAGE and Western Blot identification are carried out on the purified protein, and the result shows that the purification effect is good, thereby laying a foundation for later vaccine and basic research.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a trimerization new coronavirus receptor binding domain, wherein Human Immunodeficiency Virus (HIV) gp41 protein is added at the C end of the novel coronavirus receptor binding domain to obtain a target fragment, the target fragment is connected to a vector, competent cells are transformed to obtain recombinant bacmid, the recombinant bacmid is transfected to cells to obtain recombinant baculovirus, the cells are inoculated and cultured, a culture medium is collected, and the trimerization new coronavirus receptor binding domain is obtained by purification.
In some embodiments of the invention, the vector is a pFastBac1 vector; the competent cell is DH10Bac competent cell; the cells were sf9 cells.
The invention also provides a preparation method of the trimerization new coronavirus receptor binding domain, wherein Human Immunodeficiency Virus (HIV) gp41 protein is added at the C end of the novel coronavirus receptor binding domain to obtain a target fragment, the target fragment is connected to a vector, a cell is transformed to obtain recombinant bacmid, the recombinant bacmid is transfected to the cell to obtain recombinant baculovirus, the cell is inoculated and cultured, a culture medium is collected and purified to obtain the trimerization new coronavirus receptor binding domain.
In some embodiments of the invention, the vector is a pFastBac1 vector; the competent cell is DH10Bac competent cell; the cells were sf9 cells.
Specifically, a segment of Human Immunodeficiency Virus (HIV) gp41 protein is added at the C-terminal of a novel coronavirus receptor binding domain RBD, so that the RBD forms a trimer structure to simulate a natural structure of the RBD. The baculovirus expression system belongs to a eukaryotic expression system, has the advantages of safety and high efficiency, and the expressed protein is closer to a natural structure. The invention connects a target fragment to a pFastBac1 vector, obtains recombinant bacmid through transforming to a DH10Bac competent cell, obtains recombinant baculovirus after transfecting to an sf9 cell, obtains a culture medium 96h after inoculating the recombinant baculovirus to an sf9 cell, and purifies to obtain a target protein, namely a trimerization new coronavirus receptor binding domain.
The invention also provides the application of the trimerization novel coronavirus receptor binding domain or the trimerization novel coronavirus receptor binding domain prepared by the preparation method in the preparation of vaccines or novel coronavirus research.
In addition, the invention also provides a recombinant baculovirus, which is transformed into a purposeful fragment and can express the trimerization novel coronavirus receptor binding domain or the trimerization novel coronavirus receptor binding domain prepared by the preparation method; the target fragment is formed by adding Human Immunodeficiency Virus (HIV) gp41 protein to the C terminal of a novel coronavirus receptor binding domain.
The invention also provides the use of the recombinant baculovirus in the preparation of a medicament for treating a disorder or disease; the condition or disease includes conditions or diseases caused by the novel coronavirus.
The invention also provides a vaccine prepared by the recombinant baculovirus.
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 RBD can form a trimer structure by adding a 6HB sequence of Human Immunodeficiency Virus (HIV) transmembrane protein gp41 at the C terminal of the RBD so as to simulate the natural structure of the RBD. The baculovirus expression system belongs to a eukaryotic expression system, has the advantages of safety and high efficiency, and the expressed protein is closer to a natural structure. The target fragment is connected to a pFastBac1 vector, is transformed into a DH10Bac competent cell to obtain recombinant bacmid, is transfected into an sf9 cell to obtain recombinant baculovirus, is inoculated into an sf9 cell, is cultured for 96 hours to obtain a culture medium, and is purified to obtain the target protein, so that the foundation is laid for the subsequent vaccine and basic research.
Drawings
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 diagram of rB-SARS-CoV2-RBD-6 HB;
FIG. 2 shows recombinant bacmid RT-PCR identification; wherein, M: DL 5000; 1-2, rB-RBD-6 HB; 3: water; 4: pFastBac 1;
FIG. 3 shows visualization of recombinant baculovirus infected sf9 cytopathic effect (200 ×); a: normal sf9 cells; b: 48 hours after sf9 cells are infected with rBV-SARS-CoV2-RBD-6 HB; 72h after sf9 cells are infected with rBV-SARS-CoV2-RBD-6 HB;
FIG. 4 shows recombinant baculovirus RT-PCR identification; wherein, M: DL 2000; 1: rBV-SARS-CoV2-RBD-6 HB; 2: pFastBac 1; 3: MOCK (DH10 Bac); 4: water;
FIG. 5 shows Western Blot to identify recombinant baculovirus foreign gene expression; wherein, M: the protein MARKER; 2: protein RBD-6 HB; 3: MOCK;
FIG. 6 shows purification of a protein of interest; wherein, M: MARKER; 1: stock solution; 2-5: 1-4 parts of eluent.
Detailed Description
The invention discloses a trimerization new coronavirus receptor binding domain, a preparation method and application thereof, and can be realized 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.
In the trimerization new coronavirus receptor binding domain, the preparation method and the application thereof, the used raw materials and reagents can be purchased from the market.
The invention is further illustrated by the following examples:
plasmids, cells, strains
SARS-CoV-2RBD gene sequence (shown as SEQ ID No. 1) was synthesized by Nanjing Kingsrey Biotech, 6HB (shown as SEQ ID No. 2) was synthesized by Biotechnology engineering (Shanghai) Inc., pFastBac1 vector was purchased from Invitrogen; sf9 cells were stored in the laboratory; DH10BacTMCompetent cells were purchased from Saimer Feishale science (China) Co.
SARS-CoV-2RBD gene sequence:
AGAGTGCAGCCTACCGAGAGCATCGTGCGTTTCCCTAACATCACCAACCTCTGCCCTTTCGGCGAGGTGTTCAACGCCACCCGTTTCGCATCCGTCTACGCCTGGAACCGTAAGCGTATCAGCAACTGCGTGGCTGACTACTCCGTGCTGTACAACTCCGCATCGTTCTCCACCTTCAAGTGCTACGGCGTCAGCCCTACCAAGCTGAACGACTTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTCATCCGCGGTGACGAGGTCAGGCAGATCGCCCCCGGTCAGACCGGAAAGATCGCTGACTACAACTACAAGTTGCCCGACGACTTCACCGGATGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAGGTCGGCGGAAACTACAACTACTTGTACCGTTTGTTCCGTAAGAGCAACTTGAAGCCATTCGAGCGCGACATCAGCACCGAGATATACCAGGCCGGTAGCACCCCATGCAACGGTGTGGAGGGTTTCAACTGCTACTTCCCATTGCAGTCCTACGGATTCCAGCCTACCAACGGAGTGGGTTACCAGCCATACAGGGTCGTGGTGCTGTCCTTCGAGTTGTTGCACGCCCCTGCTACCGTCTGCGGTCCTAAGAAGTCCACCAACCTCGTGAAGAACAAGTGCGTGAACTTC
6HB sequence:
GGCGGTGGTGGTAGCGGCGGCGGTGGTAGCTCCGGCATCGTGCAGCAGCAGAACAACCTGCTGCGTGCCATCGAAGCTCAGCAGCACCTGCTGCAGCTGACTGTGTGGGGTATCAAGCAGCTGCAGGCTCGTATCCTGGCTGGTGGTAGCTCCCTGCTGACCGAAGTGGAGACCCCTATCCGTAACGAGTGGGGCTGTCGCTGTAACGGTAGCAGCGATTCCGGAGGCCACACCACCTGGATGAACTGGACCCGTGAAATCAACAACTACACCTCCCTGATCCACAACCTGACCGAGGAATCCCAGAACCAGACTGAAAAGAACGAAAACGAAACTCTGGAA
primary reagent
sf-900TMⅡSFM、Grace’s Insect Medium、
II Reagent and restriction endonuclease purchased from Saimer Feishale (China) Co., Ltd.; IPTG, X-Gal, kanamycin sulfate, tetracycline, gentamicin were purchased from Beijing Solebao technologies, Inc.;
HD Cloning Kit was purchased from Takara, Inc. (Beijing) of Baozi's medical technology, Inc. Fetal bovine serum, penicillin/streptomycin was purchased from Hyclone; Anti-RBD mouse polyclonal antibody is presented by the laboratory of Dr. Harzewski; horse radish peroxidase-labeled goat anti-mouse IgG (H + L) was purchased from shanghai bi yunnan biotechnology limited,
XT
the line of products was purchased from IBA corporation.
Example 1 construction of pFB-SARS-CoV-RBD-6HB recombinant shuttle plasmid
SARS-CoV-2RBD and 6HB are connected to pFastBac1 vector by means of homologous recombination, named as pFB-SARS-CoV2-RBD-6HB, and the sequencing by Jilin province American biotechnology limited company shows that the sequence of the target fragment is correct.
Example 2 recombinant bacmid preparation and characterization
The pFB-SARS-CoV2-RBD-6HB plasmid was transformed into DH10Bac competent cells and plated on a selection plate containing 10. mu.g/ml tetracycline, 50. mu.g/ml kanamycin sulfate, 7. mu.g/ml gentamicin, 100. mu.g/ml X-gal and 40. mu.g/ml IPTG, and incubated at 37 ℃ for 48 hours. White colonies were picked to SOC medium, shake-cultured at 37 ℃ for 3h, and the mixture was cultured using the universal primer pUC/M13F: CCCAGTCACGACGTTGTAAAACG (shown as SEQ ID No. 3) and pUC/M13R: AGCGGATAACAATTTCACACAGG (shown in SEQ ID No. 4), and performing PCR identification of bacterial liquid, and extracting bacmid after the bacterial strain is correctly identified, and the strain is named as rB-SARS-CoV2-RBD-6 HB.
In addition, the empty vector pFastBac1 was transformed, screened, cultured as described above and used as an empty vector recombinant bacmid control.
RT-PCR was performed using pUC/M13F and pUC/M13R primers for rB-SARS-CoV2-RBD-6HB and empty vector recombinant bacmid (FIG. 1). As a result, as shown in FIG. 2, rB-SARS-CoV2-RBD-6HB amplified a fragment of about 3518bp in length, and an empty fragment amplified a fragment of about 2300bp in length, in accordance with the expectation.
EXAMPLE 3 rescue of recombinant baculovirus
Seeding sf9 cells into six well plates at a cell density of 2X 106At individual cells/ml, the replacement medium was 1ml Grace medium per well. Taking 8 μ l
II and 3. mu.g rB-SARS-CoV2-RBD-6HB were diluted in 100. mu.l Grace medium, respectively, and mixed well. Mixing the diluted particles with
II, mixing and standing for 20 minutes at room temperature. The transfection mixture was added to a six well plate, incubated at 27 ℃ for 5 hours, the transfection medium was discarded and replaced with complete medium. Incubation at 27 ℃ until cytopathic effects are observed. Recovering the supernatant, centrifuging at 3000rpm for 5min, filtering with 0.22 μm filter membrane to obtain the first generation (P1) recombinant baculovirus named rBV-SARS-CoV2-RBD-6HB, P1 generation recombinant baculovirus is inoculated to sf9 cell by 1% volume, supernatant is harvested after 72 hours, and after centrifugation and filtration by the same method of P1 generation, the second generation (P2) rBV-SARS-CoV2-RBD-6HB is obtained.
As shown in fig. 3: after rBV-SARS-CoV2-RBD-6HB to sf9 cells are inoculated, observation is carried out under the 48h and 72h mirrors respectively, and after the recombinant baculovirus is infected, a large amount of sf9 cells drop off, and adherent cells are sparse and swell.
Example 4 identification of recombinant baculovirus by RT-PCR
rBV-SARS-CoV2-RBD-6HB was inoculated into sf9 cells, the cells were collected after 72 hours of culture, a certain volume of Trizol was added, and total RNA was extracted after standing at room temperature for 10 minutes. After reverse transcription, primer RBD-6HB-F (shown as SEQ ID No. 5) is used:
CCCACCATCGGGCGCGGATCCAACTTAAAAAAAAAAATCAAAATGAAGTT and RBD-6HB-R (shown as SEQ ID No. 6):
CTAGTACTTCTCGACAAGCTTTTATTCCAGAGTTTCGTTTTCGTTC PCR identification.
As shown in fig. 4: after the sf9 cells infected with rBV-SARS-CoV2-RBD-6HB for 72 hours are harvested, and the total RNA extraction, reverse transcription and PCR identification are carried out, the result is shown in FIG. 4, a 1260bp long strip can be seen, which is consistent with the expectation.
Example 5 Western Blot identification of recombinant baculovirus-expressed foreign proteins
rBV-SARS-CoV2-RBD-6HB was inoculated into sf9 cells, cultured for 72 hours, the cells were collected, and then disrupted by ultrasonication after addition of an IP lysate, centrifuged at 12000rpm at 4 ℃ for 3 minutes to collect the supernatant. After 5 XSDS was added to the supernatant, SDS-PAGE was performed after 10 minutes in a boiling water bath. 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, washing with TBST, and dripping ECL onto NC membrane for exposure.
The sf9 cells infected with rBV-SARS-CoV2-RBD-6HB for 72 hours were harvested, sonicated, subjected to SDS-PAGE, protein was transferred to NC membrane, and developed after 5% skim milk blocking, primary antibody incubation, goat anti-mouse secondary antibody incubation, as shown in FIG. 5, a band of about 41kDa was seen, consistent with the expectation.
EXAMPLE 6 purification of the protein of interest
rBV-SARS-CoV2-RBD-6HB was inoculated into suspension cells, cultured for 96 hours, the cells and the medium were harvested, centrifuged at 3000rpm at 4 ℃ for 5min, and the supernatant was collected. Using products of IBA corporation
XT
Protein purification is carried out. The purified protein was verified by SDS-PAGE and Western Blot.
And (3) harvesting a culture medium of sf9 suspension cells infected with rBV-SARS-CoV2-RBD-6HB for 96 hours, and eluting after column balance, protein adsorption and washing. Eluting with 0.6 times column volume of eluent to obtain eluent, adding 0.8 times column volume of eluent three times by the above method after eluent 1 completely flows out, recovering eluents 2, 3, and 4 respectively. The eluates were named eluates 1 to 4, respectively. As shown in FIG. 6, the eluates 2 to 4 were purified very well except for the eluent 1.
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 trimerization new coronavirus receptor binding domain is characterized in that human immunodeficiency virus gp41 protein is added at the C end of the novel coronavirus receptor binding domain to obtain a target fragment, the target fragment is connected to a vector, competent cells are transformed to obtain recombinant bacmids, the recombinant baculoviruses are transfected to the cells to obtain recombinant baculoviruses, then the cells are inoculated and cultured, and a culture medium is collected and purified to obtain the trimerization new coronavirus receptor binding domain.
2. The trimerized neo-coronavirus receptor binding domain of claim 1, wherein said vector is the pFastBac1 vector;
the competent cell is DH10Bac competent cell;
the cells were sf9 cells.
3. The method for preparing the trimerization new coronavirus receptor binding domain according to claim 1 or 2, wherein the trimerization new coronavirus receptor binding domain is obtained by adding human immunodeficiency virus gp41 protein to the C-terminal of the new coronavirus receptor binding domain to obtain a target fragment, connecting the target fragment to a vector, transforming cells to obtain recombinant bacmids, transfecting the recombinant baculoviruses to the cells to obtain recombinant baculoviruses, inoculating the cells, culturing, collecting a culture medium, and purifying.
4. The method according to claim 3, wherein the vector is a pFastBac1 vector;
the competent cell is DH10Bac competent cell;
the cells were sf9 cells.
5. Use of the trimerized novel coronavirus receptor-binding domain of claim 1 or 2 or the trimerized novel coronavirus receptor-binding domain prepared by the preparation method of claim 3 or 4 for the preparation of a vaccine or novel coronavirus research.
6. Recombinant baculovirus transformed with a fragment of interest capable of expressing the trimerized novel coronavirus receptor-binding domain of claim 1 or 2 or the trimerized novel coronavirus receptor-binding domain prepared by the preparation process of claim 3 or 4;
the target fragment is that human immunodeficiency virus gp41 protein is added at the C end of a novel coronavirus receptor binding domain.
7. Use of a recombinant baculovirus as defined in claim 6 for 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.
8. A vaccine made from the recombinant baculovirus 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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