CN115925992A

CN115925992A - SARS-CoV-2 tetramer RBD fusion protein and its application

Info

Publication number: CN115925992A
Application number: CN202211317921.1A
Authority: CN
Inventors: 王彦明; 刘征; 王玉记; 杨铖璐
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2022-10-26
Filing date: 2022-10-26
Publication date: 2023-04-07

Abstract

The invention provides a SARS-CoV-2 tetramer RBD fusion protein and application thereof, belonging to the technical field of genetic engineering. The invention provides a SARS-CoV-2 tetramer RBD fusion protein, comprising two 2XS ^RBD Fc recombinant proteins, two 2XS ^RBD Fc recombinant proteins form tetrameric RBD fusion proteins through Fc-mediated disulfide bonding. The SARS-CoV-2 tetramer RBD fusion protein has higher ACE2 binding affinity than 2XS ^RBD -Fc recombinant protein pair ACThe E2 binding affinity is improved by 7 times, and the competitive binding on the ACE2 receptor is realized, so that the infection path of the novel coronavirus is blocked. Meanwhile, the SARS-CoV-2 tetramer RBD fusion protein can also be used as immunogen to stimulate the organism and generate neutralizing antibody, thereby improving the immunity of the organism to the new coronavirus and controlling the virus pandemics.

Description

SARS-CoV-2 tetramer RBD fusion protein and its application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a SARS-CoV-2 tetramer RBD fusion protein and application thereof.

Background

Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to a series of viruses with corolla lipid membrane and protein shell, and also includes MERS-CoV and SARS-CoV-1 ^[1] . The coronavirus coat is typically composed of four structural proteins, including spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N) ^[2] . The S protein of SARS-CoV-2 virus is a glycoprotein that mediates membrane fusion and viral entry into host cells. The S protein forms itself a trimer of approximately 180kDa each, the monomer consisting of two subunits, S1 and S2 ^[3] . As with most coronaviruses, activation of SARS-CoV-2 requires proteolytic cleavage of S into two subunits, S1 and S2 ^[3] . The S1 subunit mediates attachment of the S protein to host receptors, while the S2 subunit is involved in fusion of viruses and cells ^[4，5] . The C-terminus of the S1 subunit comprises the Receptor Binding Domain (RBD), and the RBD of SARS-CoV-2 has 73% amino acid homology with the RBD of SARS-CoV-1 and only 22% homology with the RBD of MERS-CoV ^[6，7] . The difference of the amino acid sequences determines that SARS and MERS-CoV bind to different cell receptors ^[8] . Similar to SARS-CoV-1, the S protein RBD of SARS-CoV-2 binds to angiotensin converting enzyme 2 (ACE-2), a metallopeptidase, but with higher affinity and faster binding kinetics ^[9，10] . SARS-CoV-2S protein enters cells by binding to ACE2, and S protein RBD of SARS-CoV-2 and SARS-CoV has similar affinity to human ACE2 ^[11] . Structural analysis of the S1 trimer showed that only one of the three RBD domains was in the "up" conformation prior to binding to the ACE-2 receptor. This is an unstable transient state, transitioning between trimeric subunits, and this exposed state can serve as a target for neutralizing antibodies ^[12] . The interaction between the powerful antibody and RBD is generally in an 'upward' state, and SRBD in SARS-CoV-2 cannot be combined with ACE2 after being occupied by the antibody, so that the effect of neutralizing virus infection by the antibody is achieved ^[13] 。

Polyclonal antibodies against the SARS-CoV-2S protein RBD have been shown to inhibit its interaction with the ACE-2 receptor, and thus RBD is an attractive target for vaccines and antiviral therapy ^[14] . The single dose administration of the monoclonal antibody therapeutic drug AZD7442 is effective in preventing Covid-19 without obvious safety problem ^[15] . Studies have shown that S-RBD antigen can interact with antibodies in the blood of new coronary patients, consistent with the development of S-RBD antibody immunity after exposure to SARS-CoV-2 ^[16] . A number of new SARS-CoV-2 subtype genomes have been identified to date, including Omicron-B.1.1.529. The first Omicron variant was first discovered in south Africa at 11 months 2021 and soon became the dominant SARS-CoV-2 alarming epidemic Variant (VOC). The Omicron variant contains 15 mutations in the RBD domain, which may affect the adaptability and transmission of the virus. Most mutations involved in the binding of the S protein to ACE-2, resulting in higher binding affinity of the S protein to ACE-2, which may result in greatly increased infectivity of viral mutants (e.g., omicron) ^[17，18] . Some of these mutations also promote immune escape and reduce the neutralizing activity of several monoclonal antibodies ^[17] . At present, mRNA, recombinant virus and inactivated virus vaccines against new coronavirus have been developed and showed great effects ^[19，20] . RBD-mRNA vaccine can completely protect mice from the virus challenge experiment of SARS-CoV-2 mutant virus ^[21] . It was found that self-amplifying RNA encoding SARS-CoV-2S protein encapsulated in Lipid Nanoparticles (LNP) as a vaccine showed dose-dependent high titers of SARS-CoV-2 specific antibodies in the serum of immunized mice and strong neutralizing effects against both pseudoviruses and wild-type viruses ^[22] . However, mRNA vaccines need to be encapsulated otherwise they are not stable under physiological conditions, have short half-lives and may have problems or side effects beyond those expected ^[23] . The multivalent SARS-CoV-2 spike receptor binding domain nanoparticle (RBD-NP) vaccine in clinical stage can reduce the infection of SARS-CoV-2 virus to mice after single immunization, and can be used as another potential strategy ^[24] . Due to the timely vaccination of various types of vaccines, the current onesThe pandemic results show that the new crown mortality and infection rates are much lower than those initially predicted ^[25] 。

Reference documents

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Disclosure of Invention

In view of the above, the present invention provides a SARS-CoV-2 tetrameric RBD fusion protein and its application, the SARS-CoV-2 tetrameric RBD fusion protein is 2XS ^RBD The S-RBD tetramer formed by Fc mediated disulfide bond can greatly reduce the infection probability of the new coronavirus and can be used as immunogen to generate neutralizing antibody, thus providing a new means for preparing the medicine for preventing and treating the new coronavirus.

The invention provides a SARS-CoV-2 tetramer RBD fusion protein, comprising two 2XS ^RBD Fc recombinant proteins, two 2XS ^RBD -Fc recombinant proteins form tetrameric RBD fusion proteins by Fc mediated disulfide bonding;

said 2XS ^RBD the-Fc recombinant protein is IgG kappa signal peptide-2 XS ^RBD -an Fc-tag.

Preferably, said 2XS ^RBD The amino acid sequence of the-Fc recombinant protein is shown as SEQ ID NO. 1.

The invention provides a preparation method of SARS-CoV-2 tetramer RBD fusion protein, which comprises the following steps:

will code2×S ^RBD Inserting a gene sequence of the mFc recombinant protein into a vector to obtain a recombinant vector;

and (3) carrying out recombinant expression after transfecting the recombinant vector to cells, collecting cell supernatant, and purifying to obtain the recombinant protein.

Preferably, the encoding is 2 × S ^RBD The gene sequence of the mFc recombinant protein is shown as SEQ ID NO. 2;

the carrier is pcDNA3.4 plasmid;

the cells are expihho cells;

the purification method is affinity chromatography purification, and the protein separation and identification method is Bis-Tris SDS-PAGE.

The invention provides a medicine for blocking new coronavirus infection, which comprises SARS-CoV-2 tetramer RBD fusion protein and medically acceptable auxiliary materials.

Preferably, the dosage form of the medicament comprises a nasal spray.

The invention provides a vaccine for blocking new coronavirus infection, which comprises SARS-CoV-2 tetramer RBD fusion protein and adjuvant.

Preferably, the final concentration of the SARS-CoV-2 tetrameric RBD fusion protein is 1mg/ml.

The invention provides application of the SARS-CoV-2 tetramer RBD fusion protein or the SARS-CoV-2 tetramer RBD fusion protein obtained by the preparation method in preparing a medicament for preventing and/or treating new coronavirus infection.

The invention provides application of the SARS-CoV-2 tetramer RBD fusion protein or the SARS-CoV-2 tetramer RBD fusion protein obtained by the preparation method in preparing vaccines for preventing and controlling new coronavirus infection.

The invention provides a SARS-CoV-2 tetramer RBD fusion protein, which comprises two 2xS ^RBD Fc recombinant proteins, two 2XS ^RBD -Fc recombinant proteins form tetrameric RBD fusion proteins by Fc mediated disulfide bonding; said 2XS ^RBD the-Fc recombinant protein is IgGkappa signal peptide-2 XS ^RBD -an Fc-tag. The SARS-CoV-2 tetramer RBD fusion protein has higher ACE2 binding affinity. Experiments show that SARS-CoV-2 tetrameric RBD fusion protein efficiently binds to hACE2 in a dose-dependent manner with a dissociation constant of 0.13nM and 2XS ^RBD The dissociation constant for efficient binding of the Fc recombinant protein to hACE2 was 0.9nM, and it can be seen that the SARS-CoV-2 tetrameric RBD fusion protein is 2XS ^RBD The Fc recombinant protein has 7-fold improved binding affinity to ACE2, and can be bound to ACE2 receptor competitively, so as to block infection path of new coronavirus. Meanwhile, the SARS-CoV-2 tetramer RBD fusion protein can also be used as immunogen to stimulate the organism and generate neutralizing antibody, thereby improving the immunity of the organism to the new coronavirus and controlling the virus pandemics.

Drawings

FIG. 1 construction and identification of HEK293T-hACE2 cells; wherein (A) the immunoblot shows that hACE 2is expressed in HEK293T-hACE2 cells, but not in HEK293T mother cells; (B) Flow cytometry assays showed expression of hACE2 in HEK293T-hACE2 cells compared to HEK293T progenitor cells stained with anti-hACE 2 antibody; (C) Microscopic images showed expression of human hACE2 in HEK293T-hACE2 cells (DNADAPI stained blue, hACE2 antibody stained green, 10 μm scale);

FIG. 2.S ^RBD -mFc and 2XS ^RBD Expression and analysis of mFc fusion proteins, in which (A) S after purification ^RBD -mFc and 2XS ^RBD SDS-PAGE analysis of the mFc fusion protein under reducing and non-reducing conditions, respectively; (B) Confocal microscope image display S ^RBD -mFc and 2XS ^RBD Binding of mFc to human hACE2 expressed on the cell surface of HEK293T-hACE2 (DNADAPI staining in blue, S) ^RBD -mFc or 2XS ^RBD -mFc staining red, scale bar 10 μm);

FIG. 3. HACCE 2 and S ^RBD -mFc and 2XS ^RBD Affinity assay between mFc, where (A-B) flow cytometric Table shows S serially diluted in a 2-fold gradient ^RBD -mFc (A) and 2xS ^RBD -the mFc (B) fusion protein binds to hACE2 expressed on the surface of HEK293T cells; (C) Based on the flow cytometry detection result, S is established ^RBD -mFc or 2XS ^RBD -binding profile of an mFc fusion protein;

FIG. 4.S ^RBD -mFc and 2XS ^RBD -the mFc fusion protein blocks the infection of HEK293T-hACE2 cells by SARS-CoV-2S protein pseudovirus; wherein (A) the microscope image shows the expression of GFP protein in HEK293T-hACE2 cells 48 hours after pseudovirus transfection; pseudoviruses and S ^RBD -mFc and 2XS ^RBD Reduction of GFP positive cells after mFc co-incubation; (B) Analyzing the number of GFP expression cells by flow cytometry, and taking HEK293T-hACE2 cells without virus infection as a negative control; the other cell groups were treated in a manner similar to that described in (A); (C) percentage and mean of GFP positive cells (n = 3);

FIG. 5.2 XS ^RBD -mFc as a vaccine produces neutralizing antibody assay results against infection with SARS-CoV-2S protein pseudovirus; wherein (A) the RBD antibody activity is measured by ELISA method before the immunization and on the 35 th day after the inoculation; (B) S ^RBD The antibody activity in the antisera immunized with-mFc is significantly lower than 2XS ^RBD -antibody activity in the antisera immunized with mFc (×, P ≦ 0.01, n = 6); (C) Flow cytometry analysis shows the inhibition effect of No. 8 mouse antiserum on pseudovirus transfected HEK293T-hACE2 cells under 2-fold gradient dilution condition; (D) Establishing a competitive inhibition curve of No. 8 antiserum according to the flow cytometry result;

FIG. 6.2 XS ^RBD -mFc as vaccine results in neutralizing antibody experiments against infection of S protein pseudovirus of SARS-CoV-2 ormer variant strain; wherein (A) the inhibition of Oncuronan strain S protein pseudovirus transfected HEK293T-hACE2 cells by mouse antiserum No. 8 was analyzed by flow cytometry; (B) A competitive inhibition curve for antiserum No. 8 was established based on flow cytometry results.

Detailed Description

The invention provides a SARS-CoV-2 tetramer RBD fusion protein, comprising two 2XS ^RBD Fc recombinant proteins, two 2XS ^RBD -Fc recombinant proteins form tetrameric RBD fusion proteins by Fc mediated disulfide bonding; said 2XS ^RBD the-Fc recombinant protein is IgGkappa signal peptide-2 XS ^RBD -an Fc-tag.

In the present invention, the 2XS ^RBD The amino acid sequence of the Fc recombinant protein is preferably as shown in SEQ ID NO 1 (RVQPTESIVRFFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYS)<xnotran> VLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGGGGSGGGGSGGGGSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFLECICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK) . </xnotran> Said 2XS ^RBD <xnotran> -Fc SEQ ID NO:2 (AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCGGTGGCGGTGGCTCGGGTGGCGGCGGATCTGGTGGCGGTGGCTCGAGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAA </xnotran><xnotran> AGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCCTCGAGTGCATCTGTACCGTGCCTGAGGTGTCCAGCGTGTTTATCTTCCCCCCTAAGCCCAAGGACGTGCTGACCATCACCCTGACCCCTAAGGTGACCTGTGTGGTGGTGGACATCAGCAAGGATGACCCCGAGGTGCAGTTTAGCTGGTTCGTGGATGACGTGGAGGTGCACACCGCCCAGACCCAGCCCAGAGAGGAGCAGTTTAACAGCACCTTCAGGAGCGTGTCCGAGCTGCCCATCATGCACCAGGACTGGCTGAACGGCAAGGAGTTTAAGTGTCGGGTGAACTCCGCTGCTTTTCCCGCCCCTATCGAGAAGACCATCTCCAAGACCAAGGGCAGGCCTAAGGCCCCCCAGGTGTACACCATCCCTCCCCCCAAGGAGCAGATGGCTAAGGACAAGGTGAGCCTGACCTGCATGATCACCGACTTTTTCCCCGAGGACATCACCGTGGAGTGGCAGTGGAACGGCCAGCCCGCCGAGAATTACAAGAATACCCAGCCTATCATGGACACCGACGGCTCCTACTTCGTGTACAGCAAGCTGAATGTGCAGAAGTCCAATTGGGAGGCTGGCAATACCTTCACCTGCTCCGTGCTGCACGAGGGCCTGCACAACCACCACACCGAGAAGAGCCTGTCCCACAGCCCTGGCAAG) . </xnotran> The sequence is residues 319-541 of SARS-CoV-2 (GenBank accession number: NC-045512) spike protein. <xnotran> Fc SEQ ID NO:3 (LECICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK) . </xnotran> S is ^RBD <xnotran> SEQ ID NO:4 (RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN) , SEQ ID NO:5 (AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAA </xnotran>ACAATGTGTGTCAATTTC).

will encode 2XS ^RBD Inserting a gene sequence of the mFc recombinant protein into a vector to obtain a recombinant vector;

In the present invention, the vector is preferably pcDNA3.4 plasmid; the preferred multiple cloning site for insertion into the vector is HindIII/XhoI. The cells are preferably expihho cells; the purification method is preferably affinity chromatography purification for separation. The method of the present invention for the insertion and transfection is not particularly limited,

in the present invention, a single band having a molecular weight of about 200kDa was observed for the isolated recombinant protein under non-reducing conditions, while a single band having a molecular weight of about 100kDa was observed under reducing conditions, indicating that the fusion protein forms a homodimer through Fc fragment-mediated disulfide bonding.

In the invention, the SARS-CoV-2 tetrameric RBD fusion protein blocks the infection path of the new coronavirus by competing with the new coronavirus to bind to hACE2 receptor. The dosage form of the medicament preferably comprises a nasal spray. The preparation method of the nasal spray medicament is not particularly limited, and the preparation method of the nasal spray medicament known in the field can be adopted. In the present example, 2 × S ^RBD The transfection capability of the pseudovirus on cells after the-mFc protein co-incubation is obviously reduced, which indicates that the fusion protein obviously blocks the infection of the pseudovirus on HEK293T-hACE2 cells.

In the present invention, the final concentration of the SARS-CoV-2 tetrameric RBD fusion protein is preferably 1mg/ml. The method for preparing the vaccine is not particularly limited, and the method for preparing the recombinant protein vaccine well known in the field can be adopted. In the examples of the present invention, 2XS ^RBD After mice were immunized with the-mFc protein, the antibody activity in mouse antiserum was analyzed by ELISA using His6-S-RBD fusion protein, and the results showed that 2XS ^RBD The mFc protein can be used as an effective antigen to generate antibodies against SRBD. 2xS ^RBD Mice immunized with mFc protein vs S ^RBD The higher antibody activity exhibited by the mFc immunized mice, indicating that the tetrameric SRBD protein can increase neutralizing antibody titers.

The SARS-CoV-2 tetramer RBD fusion protein and its application provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Preparation method of SARS-CoV-2 tetramer RBD fusion protein

1. Construction of recombinant vectors

2XS is added ^RBD (RBD- (GGGGS-GGGGS-GGGGS, SEQ ID NO: 6) -RBD) addition of IgGkappa signal peptide at the N-terminus and mouse Fc tag at the C-terminus to give 2XS ^RBD -mFc (amino acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO: 2), by means of artificial synthesis the artificial sequence is inserted into the HindIII/XhoI multiple cloning site of pcDNA3.4, obtaining pcDNA3.4-2 XS ^RBD -mFc。

2. Protein expression and purification

Mixing pcDNA3.4-2 XS ^RBD -the mFc recombinant expression plasmid was transfected into expichho cells, and the cells were cultured for 7 days. Thereafter, the cell culture supernatant was collected and purified using affinity chromatographyAnd (4) transforming. Proteins were separated using Bis-TrisSDS-PAGE at 4% to 20% and then proteins in the gel were stained using coomassie brilliant blue to visualize protein bands.

2XS was analyzed using SDS-PAGE under reducing and non-reducing conditions ^RBD -an mFc protein. As shown in FIG. 2, A, the 2 XSRB-mFc fusion protein had a molecular weight of about 100kDa under reducing conditions and about 200kDa under non-reducing conditions, indicating that the fusion protein forms homodimers through Fc fragment-mediated disulfide bonds.

Comparative example 1

Preparation method of SARS-CoV-2 dimer RBD fusion protein

1. Construction of recombinant vectors

Adding IgGkappa signal peptide to the N-terminal of RBD and adding mouse Fc label to the C-terminal of RBD to obtain S ^RBD -mFc (amino acid sequence SEQ ID NO:7, nucleotide sequence SEQ ID NO: 8), inserting the artificial sequence into HindIII/XhoI multiple cloning site of pcDNA3.4 by artificial synthesis method to obtain pcDNA3.4-S ^RBD -mFc。

2. Protein expression and purification

Mixing pcDNA3.4-S ^RBD -the mFc recombinant expression plasmid was transfected into expihcho cells, and the cells were cultured for 7 days. Thereafter, the cell culture supernatant was collected and purified using affinity chromatography. Proteins were separated using Bis-TrisSDS-PAGE at 4% to 20% and then proteins in the gel were stained using coomassie brilliant blue to visualize protein bands.

S was analyzed using SDS-PAGE under reducing and non-reducing conditions ^RBD -an mFc protein. As shown in FIG. 2A, S ^RBD The molecular weight of the mFc fusion protein was about 57kDa under reducing conditions and about 115kDa under non-reducing conditions, indicating that the fusion protein forms homodimers through Fc fragment mediated disulfide bonds.

Example 2

Construction method of HEK293T cell with hACE2 expressed on surface

To create a host cell line that can be efficiently infected with the SARS-CoV-2S protein pseudovirus, HEK293T cells were transduced with a lentiviral vector expressing human ACE2 under the EF1a promoter by the following specific method:

1. the construction method of the lentivirus vector comprises the following steps:

1) PCR amplification of the hACE2 sequence (2470 bp)

Primer F TGTCGTGAGGATCTATTTCCGGTGCCACCATGTCAAGCTCTTCCTG (SEQ ID NO: 9);

primer R CGGTAGAATTATCTAGAGTCCGTCAAAAGGAGTCTGAACATCAG (SEQ ID NO: 10).

The PCR amplification reaction system is as follows:

the PCR amplification reaction program is as follows:

and (3) cutting the PCR product to recover the gel, and recovering the fragment to perform double enzyme digestion.

1.2% agarose detection recovery

2) Double-restriction enzyme pCDH-EF1 alpha-MCS-PGK-Puro

1.2% agalose detection recovery

3) Homologous recombination hACE2 and double enzyme digestion pCDH-EF1 alpha-MCS-PGK-Puro are as follows:

4) Transformation of ligation products into competent cells (chemical transformation)

Taking one competent cell from-80 deg.C, thawing in ice bath

Adding 10 μ l of the ligation product into competent cells, flicking with index finger to mix the two, placing on ice for 30min, thermally shocking at 42 deg.C for 45s, cooling on ice for 3min, adding 900 μ l of LB liquid culture medium, reversing up and down, mixing, resuscitating at 37 deg.C and 150r/min for 45min,4000r/min, centrifuging for 2-3min, discarding 800 supernatant, resuspending thallus precipitate, taking 120 μ l of coated Amp plate (the remaining bacteria solution is preserved at 4 deg.C for use), placing at 37 deg.C for 30min, and culturing overnight.

5) Colony PCR identification of positive clones

The sequence (2692 bp) was amplified by PCR with the following primers:

primer F CAGCTTGGCACTTGATTAATT (SEQ ID NO: 11);

primer R is GTGGATGTGGAATGTGTGCGAG (SEQ ID NO: 12).

The reaction system of PCR amplification is as follows:

the reaction procedure for PCR amplification was:

the PCR amplification products were subjected to 1.2% agarose electrophoresis detection. Positive clones were picked and sequenced.

2. Lentiviral packaging method

Day1: cell plating: every 100mm dish 300 ten thousand HEK293T cells, DMEM medium (containing 10% FBS, no double antibody) in the constant temperature incubator overnight;

day2 transfection packaging plasmid

(1) The normal culture medium is discarded 2h before transfection, and a DMEM culture medium (without serum or double antibody) is used;

(2) Preparation of transfection reagent and plasmid

Solution A: opti MEM 1500. Mu.l/dish, lipofectamine ^TM 2000 36 mul/dish, mixing evenly, standing for 5min at room temperature, and not being suitable for mixing evenly by violent vortex.

And B, liquid B: opti MEM 1500. Mu.l/dish, pMD2. G1.5. Mu.g, psPAX 2.5. Mu.g, target plasmid 6.0. Mu.g, vortex, mix well, then stand at room temperature for 5min.

Mixing the solution A and the solution B in equal volume, incubating at room temperature for 5-10 min, then carefully dripping the AB mixed solution (3 mL/dish) into a culture dish, slightly mixing, and placing in a constant-temperature incubator.

(3) The supernatant was discarded 12h after transfection and the medium was replaced (DMEM medium containing 10% fbs, no double antibody);

day4 viral supernatants were collected 24h,48h post transfection, centrifuged at 2000g for 10min and filtered through a 0.45um filter, which may be stored at 4 ℃ if used recently or at-80 ℃.

3. Infecting the cells of interest

Day1, plating of target cells, plating of HEK293T cells in good growth state, and making the density of the cells in infection 30% -50%.

Preparing a Day2 virus and Polybrene, mixing the Polybrene with the virus at a working concentration of 8 mug/mL, adding part of fresh culture medium, adding the mixed solution into a culture dish, and then placing the culture dish in a constant temperature incubator;

day3, second infection, observing the growth state of cells one Day later, discarding the supernatant, and adding new virus supernatant in the same way to continue infection;

day4: specific selection, cells after transfection were selected using puromycin 3. Mu.g/mL, and fresh medium was replaced every 48 h.

4. Monoclonal culture:

(1) Using the gradient dilution method, the selected cells were subjected to gradient dilution, and then plated in a 96-well plate for culture. Culture conditions were DMEM medium, 10% FBS,1% double antibody.

(2) After 4 to 5 days, the growth of the single clone was observed, and if one cell clone had grown in one well of a 96-well plate, the cell clone was trypsinized, transferred to a 24-well plate, and expanded in culture under DMEM medium, 10% FBS,1% double antibody.

(3) After the 24-well plate is full of cells, pancreatin is used for digestion, half of the cells are used for WB detection, and the rest cells are continuously cultured.

5. And (3) monoclonal identification:

(1) For each clone sample, immunoblot hybridization analysis was performed using antibodies against β actin and hACE2, and the expression level of hACE2 was analyzed using β actin as an internal reference, and the clone with the highest expression level of hACE2 was selected.

Next, the position of hACE2 in HEK293T-hACE2 cells was analyzed.

The HEK293T-hACE2 cell is detected by immunofluorescence according to the following specific method:

HEK293T-hACE2 cells on cell culture coverslips at 1X 10 ⁵ Cell/well density was cultured in 24-well plates. After 24 hours, the cells were washed with PBS, fixed in 4% formalin in PBS for 30 minutes, washed with PBS, and then washed with anti-human ACE2 antibody or S ^RBD -mFc and 2XS ^RBD -incubation with an mFc protein. And then. ACE2 antibodies were labeled with either Alexa Fluor488 goat anti-rat IgG (Abcam ab 175473) secondary antibody (1. After staining, photographs were observed using a laser scanning confocal microscope (Zeiss).

Immunofluorescence staining and confocal microscopy using anti-ACE 2 antibody revealed that hACE2 was localized on the cell membrane surface (C in fig. 1).

Furthermore, flow cytometry was used to detect expression of hACE2 and S ^RBD Binding of the fusion protein to HEK293T-hACE2 cells. HEK293T-hACE2 cells were incubated with anti-human ACE2 antibody for 2h, using Alexa Fluor488 goat anti-rat IgG (Abcam ab 175473) against HEK293T cells as control.

The flow cytometric analysis showed that the location of hACE2 was detected on the surface of HEK293T-hACE2 cells (B in FIG. 1). Microscopic and flow analysis showed that hACE2 was localized to the cell membrane with the correct orientation of the extracellular domain.

Example 3

2×S ^RBD -mFc and S ^RBD Method for detecting binding affinity of (E) -mFc to hACE2

1. Culturing the cells, discarding the old culture medium when the cell growth reaches 60% -70%, and washing the cells for 3 times by using PBS.

2. Digesting the cells with 0.25% pancreatin until the cells become round, adding a cell culture medium to stop digestion, blowing off all adherent cells, gently blowing off the cells, and collecting the cells in a centrifugal tube.

The cells were pelleted by centrifugation at 3.200 g for 5min.

4. The supernatant was carefully aspirated, leaving 50. Mu.l of medium.

5. Approximately 1ml of pre-cooled PBS was added, the cells were resuspended, and transferred to a 1.5ml centrifuge tube. 20 μ l of the cells were counted, the cells were again pelleted by centrifugation, the supernatant carefully aspirated, and the cells were diluted to 10 with PBS ⁶ One per ml.

6. Gently flick the bottom of the centrifuge tube to properly disperse the cells and avoid cell clumping.

7. In a 1.5ml centrifuge tube, S was added ^RBD -mFc and 2XS ^RBD -mFc was diluted to 6.6nM separately and then diluted in 2-fold gradients; add 200. Mu.l PBS to blank tube; 200 mul of S after gradient dilution is added into the other tubes respectively ^RBD -mFc and 2XS ^RBD -mFc。

8. 100. Mu.l of cell suspension (about 10) was added to each 1.5ml centrifuge tube ⁵ Cells) and mixed gently.

9. Incubate in a refrigerator at 4 ℃ for 60min in the dark.

Centrifuging at 10.200g4 deg.C for 5min, discarding supernatant, and repeating the washing process 3 times. 100 ul PBS resuspended cells and then detected by the flow-up meter.

TABLE 1 detection of fusion protein binding Positive cells by flow cytometry

	S ^RBD -S ^RBD -mFc	S ^RBD -mFc
			nM	Percentage of	Percentage of
0.0515625	3.99	0.43
			0.103125	35.26	0.52
0.20625	80.27	0.76
			0.4125	96.89	2.99
0.825	99.21	40.32
			1.65	99.39	94.05
3.3	99.08	99.1
			6.6	99.13	98.77

Using flow cytometry, we investigated hACE2 and S expression on the surface of HEK293T cells ^RBD -mFc and 2XS ^RBD Affinity of binding of the mFc fusion protein (A in FIG. 3, B in FIG. 3). Discovery S ^RBD -mFc and 2XS ^RBD the-mFc bound efficiently to hACE2 in a dose-dependent manner with dissociation constants of 0.9 and 0.13nM (C in fig. 3), respectively, differing by a factor of 7.

Example 4

S ^RBD -mFc and 2XS ^RBD Experiment of infection with-mFc blocking SARS-CoV-2S protein pseudovirus

Analysis of S Using SARS-CoV-2S protein pseudovirus expressing GFP ^RBD -mFc and 2XS ^RBD Whether or not mFc blocks virus binding and entry into HEK293T-hACE2 cells. The interaction of ACE2 with S Protein drives pseudovirus into and infects target cells (see Crawford, K.H.D., et al, protocols and Reagents for pseudoviral Particles with SARS-CoV-2 Spike Protein for neural differentiation assays. Viruses,2020.12 (5)). In the assay, the S protein pseudovirus was set 10-fold higher than that of HEK293T-hACE2 cells. First, pseudoviruses are mixed with S ^RBD -mFc and 2XS ^RBD -mFc protein mixture, after 30 minutes of co-incubation the mixture was added to HEK293T-hACE2 cells. As shown in FIG. 4A, 48 hours after infection, with S ^RBD -mFc and 2XS ^RBD After co-incubation with the mFc fusion protein, there were many fewer pseudovirus infected cells with green fluorescent signal than the control.

To quantize S ^RBD -mFc and 2XS ^RBD Efficiency of competition of the mFc protein with the pseudovirus, we first put the pseudovirus with S ^RBD -mFc and 2XS ^RBD -mFc protein mixed incubation, then transfected HEK293T-hACE2 cells. 48 hours after transfection, HEK293T-hACE2 cells were trypsinized and flow cytometric assayed to analyze green fluorescence signal and viral infection efficiency (B in FIG. 4).

The results showed that cells infected with pseudovirus alone had a stronger GFP signal, accounting for 38% of infected cells (B and C in FIG. 4). In contrast, S ^RBD -mFc and 2XS ^RBD The transfection capability of the pseudovirus on cells after the-mFc protein co-incubation is obviously reduced, which indicates that the fusion protein obviously blocks the infection of the pseudovirus on HEK293T-hACE2 cells.

Example 5

S ^RBD Anti-serum neutralization of SARS-CoV-Determination of the Activity of 2S protein pseudovirus

With S ^RBD -mFc and 2XS ^RBD After immunization of mice with the mFc protein, his6-S ^RBD <xnotran> (SEQ ID NO:13,RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFHHHHHH) ELISA , : </xnotran> ELISA plates were prepared with carbonate buffer containing 0.02. Mu.g/mL His6-RBD protein at 4 ℃. After washing with 5% milk PBS solution and blocking, antiserum diluted in 5% milk PBS solution was added to each well and incubated at 37 ℃ for 4 hours. Plates were washed with PBS and incubated with 1. After washing, TMB solution was added to the plate and incubated at 37 ℃ for 20 minutes, followed by 1.0MH with the same volume ₂ SO ₄ To terminate the reaction. Absorbance at 450nm was measured by a microplate reader (Synergy neo 2).

The antisera potency after the last immunization was significantly higher than before immunization (A in FIG. 5), indicating S ^RBD -mFc and 2XS ^RBD The mFc protein can be used as an effective antigen to generate the antigen against S ^RBD The antibody of (1). 2XS ^RBD Mice immunized with mFc protein vs S ^RBD Higher antibody activity was shown in mice immunized with-mFc (B in FIG. 5), indicating tetrameric S ^RBD The protein can increase neutralizing antibody titer.

Example 6

To evaluate the activity of antisera neutralizing antibodies, virus infection experiments were performed after incubation of SARS-CoV-2 and Omicron BA.4S protein pseudoviruses in admixture with antisera (see Li, W., et al., angiotensin-converting enzyme 2is a functional receptor for the SARS coronavirus. Nature,2003.426 (6965): p.450-4). Antiserum from mouse # 8 neutralized infection with the SARS-CoV-2S protein pseudovirus in a concentration-dependent manner, indicating that the antiserum had neutralizing antibody activity (C in FIG. 5 and D in FIG. 5).

Meanwhile, antiserum from mouse # 8 neutralized the infection with the Omicron BA.4S protein pseudovirus in a concentration-dependent manner, indicating that the antiserum had neutralizing antibody activity against the S protein of the Omicron variant virus (A in FIG. 6 and B in FIG. 6).

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.

Claims

1. A SARS-CoV-2 tetramer RBD fusion protein is characterized in that it comprises two 2XS ^RBD Fc recombinant proteins, two 2XS ^RBD -Fc recombinant proteins form tetrameric RBD fusion proteins by Fc mediated disulfide bonding;

said 2XS ^RBD the-Fc recombinant protein is IgGkappa signal peptide-2 XS ^RBD -an Fc-tag.

2. The SARS-CoV-2 tetrameric RBD fusion protein of claim 1, wherein the 2xs is ^RBD The amino acid sequence of the-Fc recombinant protein is shown as SEQ ID NO. 1.

3. A method for preparing SARS-CoV-2 tetrameric RBD fusion protein of claim 1 or 2, comprising the steps of:

and transfecting the recombinant vector to a cell, performing recombinant expression, collecting cell supernatant, and purifying to obtain the recombinant protein.

4. The method for preparing SARS-CoV-2 tetrameric RBD fusion protein as claimed in claim 3, wherein the code is 2XS ^RBD The gene sequence of the mFc recombinant protein is shown as SEQ ID NO. 2;

the carrier is pcDNA3.4 plasmid;

the cells are expihho cells;

5. A medicament for blocking a new coronavirus infection comprising the SARS-CoV-2 tetrameric RBD fusion protein of claim 1 or 2 and a pharmaceutically acceptable excipient.

6. The medicament of claim 5, wherein the dosage form of the medicament comprises a nasal spray.

7. A vaccine for blocking a new coronavirus infection, comprising the SARS-CoV-2 tetrameric RBD fusion protein of claim 1 or 2 and an adjuvant.

8. The vaccine of claim 7, wherein the final concentration of SARS-CoV-2 tetrameric RBD fusion protein is 1mg/ml.

9. Use of the SARS-CoV-2 tetrameric RBD fusion protein of claim 1 or 2 or the SARS-CoV-2 tetrameric RBD fusion protein obtained by the preparation method of claim 3 or4 in the preparation of a medicament for preventing and/or treating a neocoronavirus infection.

10. Use of the SARS-CoV-2 tetrameric RBD fusion protein of claim 1 or 2 or the SARS-CoV-2 tetrameric RBD fusion protein obtained by the preparation method of claim 3 or4 in the preparation of a vaccine for preventing and controlling a new coronavirus infection.