CN117777309A - Fusion protein construct comprising XBBQ 11 antibody, preparation method and application of fusion protein construct in vaccine - Google Patents
Fusion protein construct comprising XBBQ 11 antibody, preparation method and application of fusion protein construct in vaccine Download PDFInfo
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- 239000009792 yinqiao Substances 0.000 description 1
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Abstract
The invention relates to a fusion protein construct comprising an XBBQ 11 antibody, a preparation method and application thereof in vaccines, wherein the fusion protein construct comprises an RBD protein sequence of BQ11, an RBD protein sequence of XBB and a protein sequence of an antibody constant region, the RBD protein sequence of BQ11 and the RBD protein sequence of XBB are connected with different antibody constant region protein sequences, so that the fusion protein construct similar to the antibodies is formed, the fusion protein construct can be further used for preparing vaccines, and the vaccines can stimulate organisms to generate neutralizing antibodies aiming at various SARS-CoV-2 subtypes, and have broad spectrum.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a fusion protein construct comprising XBBQ 11 antibody, a preparation method and application thereof in vaccines.
Background
Coronaviruses are a class of enveloped single-stranded positive-stranded RNA viruses that primarily infect vertebrates, and are widely known as corolla-like viruses, found in human, mammalian and avian hosts, due to the presence of peripherally extending protrusions on the viral envelope. The international committee for classification of viruses purportedly classifies coronaviruses into four genera: α, β, γ, and δ. The beta genus coronavirus is divided into four pedigrees A, B, C, D, and the B pedigree (also called Sarbecovires) comprises 2019 novel coronavirus (SARS-CoV-2) and variant thereof, severe acute respiratory syndrome coronavirus (SARS-CoV) and variant thereof, and SARS-related coronavirus (SARSr-CoV).
The global SARS-CoV-2 mat can lead to COVID-19, which has great influence on human work and life, and different variant strains appear along with the spread of SARS-CoV-2 in different areas, and epidemic situation is still continuously stressed. Up to the present, variants with super-strong transmission have variants such as alpha (original strain) and beta, gamma, delta, lammda, omicron, and other types of variants are likely to appear in the future. Although research shows that vaccination can effectively prevent morbidity and reduce the serious disease rate and death rate after infection, breakthrough infection cases after vaccination appear at home and abroad, and research shows that the variant strains have different degrees of immune escape capability on the vaccine. Development of a universal vaccine for the beta coronavirus B lineage with a high potency and broad spectrum is urgent. The vaccine can prevent infection of SARS-CoV-2 and its variant, and also prevent infection of SARS-CoV or new SARS-related coronavirus.
So far, SARS-CoV-2 has mainly concentrated on RBD region, and only 1-2 neutralizing epitopes in NTD and S2 regions, so that there is an opportunity to activate NTD neutralizing antibodies outside RBD region by using S protein as vaccine immunogen, but it is generally difficult to activate neutralizing antibodies against S2 because neutralizing epitopes of S2 are too weak. However, when S protein is used as immunogen, the NTD will shade RBD area, resulting in neutralizing antibody response concentrated mainly in 3-4 epitopes on RBD top, and other 4-5 neutralizing epitopes are lost, so it is generally uneconomical; furthermore, because of the large number of epitopes of binding antibodies carried by S-proteins, which have substantially no protective effect and are likely to increase the risk of immune pathogenesis, RBD-based vaccines induce higher levels of neutralizing antibodies and T-cell immune responses, and lower levels of non-neutralizing antibodies or deleterious immune responses in immunized animals than S1 subunit-based, full-length S-proteins, and inactivated and attenuated vaccines. RBDs from different subgeneric beta coronaviruses (e.g., XBB, BQ.1.1) are connected in series or in parallel to form multi-specific anti-beta coronaviruses, so that immune response is widened and attack targets are increased, and therefore, the anti-beta coronaviruses can be more effectively applied to mutation and escape of the viruses, and the anti-beta coronaviruses are possible to become universal vaccines for preventing the COVID-19.
Disclosure of Invention
In order to overcome the defects, the invention provides a fusion protein construct comprising XBBQ 11 antibody, a preparation method and a technical scheme of application thereof in vaccines, in particular,
in a first aspect of the invention, there is provided a fusion protein construct comprising an antibody of the XBBQ 11 class, said fusion protein construct comprising the RBD protein sequence of BQ11, the RBD protein sequence of XBB, and the antibody constant region protein sequence.
Preferably, the constant regions include a light chain constant region (LC) and a heavy chain constant region (HC).
Preferably, the RBD protein sequence of the BQ11 and the RBD protein sequence of the XBB are connected with different constant regions, more preferably, the RBD protein sequence of the BQ11 is connected with the light chain constant region protein sequence to form a BQ11-LC antibody, the RBD protein sequence of the XBB is connected with the heavy chain constant region protein to form an XBB-HC antibody, or the RBD protein sequence of the BQ11 is connected with the heavy chain constant region protein sequence to form a BQ11-HC antibody, and the RBD protein sequence of the XBB is connected with the light chain constant region protein sequence to form an XBB-LC antibody.
Preferably, the linkage comprises a direct linkage and/or an indirect linkage, more preferably, the indirect linkage comprises a linkage via a linking peptide.
The light chain constant region is directly linked to the heavy chain constant region via disulfide bonds.
Preferably, the fusion protein construct further comprises a signal peptide.
More preferably, the RBD protein sequence of BQ11 includes:
(A1) SEQ ID No: as shown in positions 26-244 of 1,
(A2) Any protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in (A1);
(A3) A protein having 80% or more identity to any one of (A1) to (A2) and having the same function.
More preferably, the RBD protein sequence of XBB includes:
(B1) SEQ ID No: as shown in positions 26-244 of 2,
(B2) A protein with the same function obtained by substituting and/or deleting and/or adding any one of the (B1) through one or more amino acid residues;
(B3) A protein having 80% or more identity to any one of (B1) to (B2) and having the same function.
More preferably, the protein sequence of the antibody light chain constant region comprises:
(C1) SEQ ID No: as shown at positions 254-358 of 1,
(C2) A protein with the same function obtained by substituting and/or deleting and/or adding any one of (C1) through one or more amino acid residues;
(C3) A protein having 80% or more identity to any one of (C1) to (C2) and having the same function.
More preferably, the protein sequence of the antibody heavy chain constant region comprises:
(D1) SEQ ID No: as shown at positions 254-581 of 2,
(D2) A protein with the same function obtained by substituting and/or deleting and/or adding any one of the (D1) through one or more amino acid residues;
(D3) A protein having 80% or more identity to any one of (D1) to (D2) and having the same function.
More preferably, the sequence of the connecting peptide comprises:
(E1) SEQ ID No: positions 245-253 of 1, or SEQ ID No: as shown in bits 245-253 of 2,
(E2) A polypeptide having the same function obtained by substitution and/or deletion and/or addition of one or more amino acid residues of any one of the polypeptides (E1);
(E3) A polypeptide having 80% or more identity to any one of (E1) to (E2) and having the same function.
More preferably, the sequence of the signal peptide comprises:
(F1) SEQ ID No:1, or positions 1-25 of SEQ ID No: the 1 st to 25 th positions of 2,
(F2) A polypeptide having the same function obtained by substitution and/or deletion and/or addition of one or more amino acid residues of any one of the polypeptides (F1);
(F3) A polypeptide having 80% or more identity to any one of (F1) to (F2) and having the same function.
Herein, identity refers to amino acid sequence identity. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and search is performed to calculate the identity of amino acid sequences, and then the value (%) of identity can be obtained.
Herein, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.
Preferably, the fusion protein construct further comprises a tag protein.
The tag protein refers to a polypeptide or protein which is fused and expressed together with the target protein by utilizing a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In a specific embodiment, the fusion protein construct comprises a BQ11-LC class antibody and an XBB-HC class antibody, which are directly linked by disulfide bonds.
Further preferred, the BQ11-LC class antibody comprises the amino acid sequence of SEQ ID No:1 and 254-358 or mutants thereof corresponding to A2, A3, C2, C3.
Further preferred, the XBB-HC antibody comprises the sequence of SEQ ID No:2 and positions 254-581 or mutants thereof corresponding to positions B2, B3, D2, D3.
In a second aspect of the invention there is provided a nucleotide molecule encoding a fusion protein construct of any one of the above.
Preferably, the nucleotide molecules comprise DNA and/or RNA, such as recombinant DNA, or mRNA.
More preferably, the nucleotide molecule comprises:
(G1) SEQ ID No: positions 76-732 of 3, SEQ ID No:3, positions 760-1074 of SEQ ID No: positions 76-732 of 4 and SEQ ID No: bits 760-1743 of 4,
(G2) Complementary, degenerate or transcribed sequences of (G1),
(G3) A DNA molecule or mRNA having more than 75% identity to the DNA molecule or mRNA defined in (G1) or (G2) and encoding the corresponding protein in the fusion protein construct.
The nucleotide sequences encoding the fusion protein constructs of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding the above fusion protein construct are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the above fusion protein construct and have the same function.
By identity is meant sequence similarity to a compared nucleic acid sequence. "identity" includes reference to SEQ ID No:3-4 has a nucleotide sequence of 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identity. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.
More preferably, the nucleotide molecules include sequences encoding BQ11-LC class antibodies and XBB-HC class antibodies.
In a specific embodiment, the nucleotide molecule comprises SEQ ID No:3 and SEQ ID No:4, or variants corresponding to (G2) - (G3).
In a third aspect of the invention there is provided a vector comprising a nucleotide molecule of any one of the above.
Preferably, the vector comprises two open reading frames encoding BQ11-LC class antibodies and XBB-HC class antibodies, respectively, or BQ11-HC class antibodies and XBB-LC class antibodies, respectively.
Preferably, the vector further comprises a regulatory factor, such as a promoter that initiates transcription of the gene sequence encoding the polypeptide or fusion protein construct, and may further comprise a terminator that terminates transcription of the gene sequence encoding the polypeptide or fusion protein construct. Further, the vector may also include enhancer sequences.
The vectors described herein refer to vectors capable of carrying exogenous DNA, mRNA or gene of interest into a host cell for amplification and expression, and may be cloning vectors or expression vectors, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, viral vectors (e.g., retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of the invention, the vector is a pUC57 vector and/or a pKS001 vector.
In a fourth aspect of the invention, there is provided a host cell comprising a nucleotide molecule or vector of any one of the above.
The host cell (also referred to as a recipient cell) described herein may be a plant cell or an animal cell. The host cell is understood to mean not only the particular recipient cell, but also the progeny of such a cell, and such progeny may not necessarily correspond, in their entirety, to the original parent cell, but are included in the scope of the host cell, due to natural, accidental, or deliberate mutation and/or alteration. Suitable host cells are known in the art, wherein: the plant cell may be, but is not limited to, plant cells such as arabidopsis thaliana (Arabidopsis thaliana), tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc.; the animal cells may be mammalian cells (e.g., chinese hamster ovary cells (CHO cells), vero cells, baby hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK 293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., xenopus laevis cells or giant salamander cells (Andrias davidianus) cells), fish cells (e.g., grass carp, rainbow trout or catfish cells), insect cells (e.g., sf21 cells or Sf-9 cells), etc., but are not limited thereto. In one or more embodiments of the invention, the host cell is a CHO-K1Q cell.
Preferably, the host cell may also be a microorganism, which may be a bacterium, fungus, actinomycete, protozoan, algae or virus. Wherein the bacteria may be derived from Escherichia sp, erwinia sp, agrobacterium sp, flavobacterium sp, alcaligenes sp, pseudomonas sp, bacillus sp, etc., but are not limited thereto, and for example, the bacteria may be Escherichia coli, bacillus subtilis Bacillus subtilis, or Bacillus pumilus. In one or more embodiments of the invention, the microorganism is a TOP10 competent cell.
In a fifth aspect of the present invention, there is provided a method for producing the above-described fusion protein construct, comprising introducing the above-described nucleotide molecule or vector encoding the fusion protein construct into a host cell, and culturing the host cell.
Preferably, the preparation method comprises screening monoclonal cell lines with high and stable expression level.
More preferably, the screening method includes screening methods conventional in the art, such as addition of screening reagents, ELISA methods to compare clone pool biological activities, and the like.
More preferably, the method of preparation comprises purifying the fusion protein construct under non-reducing conditions to obtain a fusion protein construct linked by disulfide bonds of LC and HC.
In a sixth aspect of the invention there is provided the use of a fusion protein construct, nucleotide molecule, vector or host cell of any one of the above, said use comprising any one of the following:
(1) Use in the manufacture of a product for the prevention and/or treatment of a disease caused by infection with the SARS-CoV-2 virus;
(2) Use in the preparation of a product for inducing an immune response to a SARS-CoV-2 virus antigen;
(3) Use in the prevention and/or treatment of diseases caused by SARS-CoV-2 virus infection;
(4) Use in inducing an immune response to a SARS-CoV-2 virus antigen.
The products described herein may be reagents, medicaments, or vaccines.
(1) And (2) the product may be a vaccine or antibody against SARS-CoV-2 virus, including, but not limited to, full length antibodies or antigen binding fragments (e.g., fab fragments, fv fragments, fab 'fragments, F (ab') 2 fragments, single chain antibodies (ScFv), nanobodies (single domain antibodies), bispecific antibodies, or Minimal Recognition Units (MRU), etc.
Further, the SARS-CoV-2 antibody can be a neutralizing antibody that specifically binds to the SARS-CoV-2 spike protein (S protein) receptor binding region (receptor binding domain, RBD). The neutralizing antibodies can be high titer neutralizing antibodies against a variety of epidemic strains of novel coronaviruses.
In the above application, the disease caused by SARS-CoV-2 virus infection may include respiratory system infection, digestive system infection, cardiovascular system infection, and/or nervous system infection, etc.
Preferably, the respiratory infection may comprise a respiratory tract infection and/or a lung infection.
Preferably, the digestive system infection may include intestinal disease, anorexia, nausea, vomiting, abdominal pain and/or diarrhea.
More preferably, the respiratory tract infection may comprise severe acute respiratory syndrome, hypoxic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngolaryngitis, tracheitis, and/or bronchitis.
More preferably, the pulmonary infection may include pneumonia and/or lung injury.
More preferably, the pneumonia may include pneumonia caused by a novel coronavirus infection (covd-19).
Preferably, the product for preventing and/or treating diseases caused by SARS-CoV-2 virus infection includes vaccine.
Preferably, the novel coronavirus comprises a mutant or a branch of original strain (alpha), armuronate XBB, BA.1, BA.5, BA.2.75, CH.1.1, BQ 1.1.1, etc.
In a seventh aspect of the invention there is provided a vaccine comprising a fusion protein construct, nucleotide molecule, vector or host cell of any of the above.
Preferably, the vaccine further comprises an adjuvant.
More preferably, the adjuvant may be a substance that stimulates the body to mount a more intense humoral and/or cellular immune response against the antigen with which it is vaccinated. Adjuvants described herein may be well known to those skilled in the art, including, but not limited to: plant adjuvants (e.g., alkylamines, phenolic components, quinines, sapocins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, and the like), bacterial adjuvants (e.g., cholera toxin, escherichia coli heat labile toxin, bacterial lipopolysaccharide, and the like), aluminum adjuvants and other inorganic component adjuvants (e.g., calcium adjuvants), cytokines and nucleic acid adjuvants (e.g., monocyte clone stimulating factors, leukocyte factors IL-1, IL-2, IL-4, IL-5, IL-6, IFN-gamma, cpG motifs, nucleic acid vectors, and the like), emulsion adjuvants (e.g., freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.
It is well known to those skilled in the art that in order to enhance the immunogenicity of antigen proteins, in addition to the addition of a compound having an immunopotentiating effect as an adjuvant, the gene combination can be adjusted to be expressed as a particulate structure; or agglomerating in vitro, and encapsulating in liposome or capsule microsphere.
Preferably, the vaccine further comprises a vaccine delivery system.
The vaccine delivery system may be a class of substances that are capable of carrying antigenic substances to the immune system of the body and storing and exerting their antigenic effects therein for a longer period of time. The vaccine delivery system described herein may be a Lv Yan gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nanoadjuvant vaccine delivery system.
Further, the vaccine may further comprise one or more pharmaceutically acceptable carriers.
The pharmaceutically acceptable carrier may be a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, an adsorption carrier, a surfactant, or a lubricant, but is not limited thereto.
The vaccine for preventing infection can be intramuscular liquid injection, intravenous liquid injection, intranasal liquid injection, intradermal liquid injection or subcutaneous liquid injection.
In an eighth aspect of the invention there is also provided a method of generating an immune response, the method comprising administering to a subject a vaccine as described above.
In the above method, after administration of the vaccine to a subject, an immune response against SARS-CoV-2 virus can be elicited in the subject. The immune response may be a cellular immune response, or a humoral immune response, or both.
The cellular immune response may include a B cell immune response and a T cell immune response.
The subject described herein can be a human or a non-human animal.
Further, the non-human animal may be a non-human mammal.
The non-human mammal may be any one of, but not limited to, a mouse, a rat, a guinea pig, a hamster, a pig, a dog, a sheep, a monkey, a rabbit, a cat, a cow, and a horse.
The subject described herein includes, but is not limited to, healthy subjects, symptomatically infected subjects, asymptomatic infected subjects, or recovered subjects (subjects recovered after infection).
Administration as described herein includes, but is not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral nasal spray, or aerosol inhalation.
In summary, compared with the prior art, the invention has the following beneficial effects:
1. the fusion protein construct of the invention connects the RBD region of the virus with the antibody constant region to increase the molecular weight thereof, thereby improving the stability of the antigen, and simultaneously, the RBD sequence of BQ11 and the RBD of XBB are connected with different antibody constant regions to form a dimer fusion protein construct similar to the antibody, and the connection of the light chain constant region and the heavy chain constant region ensures that the antigen has a multivalent function and increases the effect of inducing the organism to produce the antibody.
2. The selection of the RBD regions of the XBB and BQ11 subtypes as antigens in combination can produce neutralizing antibodies against multiple SARS-CoV-2 subtypes, including the original strain, multiple branches of the armonk, the vaccine of the present invention can be used as a broad-spectrum vaccine for the coronavirus B lineage.
3. The antibody-like construct of the invention can exert the function similar to the Fc fragment after entering the organism because of comprising the antibody constant region, and can be combined with the receptor of the Fc fragment on the surface of antigen presenting cells, namely FcR, so that the antibody-like construct is easier to be phagocytized by DC cells, thereby having higher antigen presenting efficiency and improving the immune response level.
4. The antibody-like construct of the present invention includes an antibody constant region, and can increase the expression level of an antigen molecule: because the Fc part structure of the antibody molecule has very high expression quantity in cells, the high-efficiency expression of the fusion gene in CHO can be promoted, the yield of antigen molecules can be improved, and the cost can be reduced.
5. The antibody-like construct of the present invention is easy to purify: after the antigen molecule is added with the Fc structure, a protein A affinity purification process can be adopted in the production and purification process, so that the purification steps are simplified, the yield is improved, the production loss is reduced, and the cost is reduced.
Drawings
Fig. 1: schematic of plasmid structure.
Fig. 2: schematic representation of fusion protein construct structure.
Fig. 3: protein purification results of fusion protein constructs.
Fig. 4: neutralizing antibody titers after mouse immunization, each group corresponds to XBBQ 11-class antibodies, XBBB-class antibodies and BQ.1.1-class antibodies from left to right.
Fig. 5: the neutralization experimental results of the vaccine on the pseudoviruses are that each group corresponds to XBBQ 11-type antibody, XBBB-type antibody and BQ.1.1-type antibody from left to right.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples are listed below, and others are commercially available unless otherwise specified.
1. Reagent consumables, as shown in table 1:
table 1: reagent consumable
| Sequence number | Name of the name | Goods number | Suppliers (suppliers) |
| 1 | HEK293-ACE2 | DD1401 | Vazyme |
| 2 | SARS-CoV-2-FlucWT | DD1702 | Vazyme |
| 3 | Pseudo virus neutralizing antibody standard | DD1602 | Vazyme |
| 4 | Bio-LiteLuciferaseAssaySystem | DD1201 | Vazyme |
| 5 | DMEM | C11995500BT | Gibco |
| 6 | OPM-293CD05Medium | 81075-001 | Shanghai ao Pu Mai |
| 7 | FBS | FSP500 | ExCell |
| 8 | Double antibody | 450-201-CL | Weisente (Chinese character) |
| 9 | PBS | 311-010-CL | Weisente (Chinese character) |
2. Device information, as in table 2:
table 2: device information
Example 1 preparation of fusion protein constructs
The amino acid sequence of XBBQ 11 antibody is added with signal peptide sequence, and then optimized synthesis is carried out by Nanjing Jinsri biotechnology Co., ltd, and the synthesized gene comprises enzyme cutting site, kozak sequence, signal peptide, target gene and stop codon, and the total length is 3287bp. The recombinant gene was synthesized with codon optimization to facilitate expression in chinese hamster ovary cells Cricetulus griseus (CHO cells).
1. Fusion protein construct (XBBQ 11 class antibody, XBBQ 11-class antibody) expression vector construction:
1. XBBQ 11 antibody expression plasmid construction materials are shown in Table 3:
table 3: XBBQ 11 antibody expression plasmid construction material
| Name of the name | Suppliers (suppliers) | Goods number |
| pKS001 plasmid | QuaCell | A13201 |
| pUC57 plasmid | Gold srey synthesis | —— |
| NotI | NEB | R3189L |
| HindIII | NEB | R3104V |
| Q5Start high-fidelity enzyme | Biolabs | M0492S |
| 5×cutsmartbuffer | Biolabs | B6004S |
| Ligase | NEB | M2200L |
| 2×LigaseBuffer | Biolabs | B2200S |
| top10 competent cells | All-type gold | CD101-02 |
| LB medium | —— | Self-priming |
| 2×PCRTaqMasterMix | Trans | AS111 |
Amino acid sequence of XBBQ 11 antibody:
the amino acid sequence of the BQ11-LC antibody is shown as SEQ ID No:1 is shown as follows:
MLRGPGPGLLLAVLCLGTAVRCTEA
RVQPTESIVRFPNITNLCPFDEVFNATTFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSTVGGNYNYRYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGVNCYFPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSGSGGGSGGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID No：1)
wherein, the 1 st to 25 th positions are signal peptide, the 26 th to 244 th positions are RBD of BQ11, the 245 th to 253 th positions are connecting peptide, and the 254 th to 358 th positions are antibody light chain constant region (LC).
The amino acid sequence of the XBB-HC antibody is shown as SEQ ID No:2 is shown as follows:
MLRGPGPGLLLAVLCLGTAVRCTEA
RVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLFRKSKLKPFERDISTEIYQAGNKPCNGVAGSNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKSGSGGGSGASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID No：2)
wherein, the 1 st to 25 th positions are signal peptide, the 26 th to 244 th positions are RBD of XBB, the 245 th to 253 th positions are connecting peptide, and the 254 th to 581 th positions are antibody heavy chain constant region (HC).
Sequence of nucleotide molecules:
the nucleotide sequence of the coded BQ11-LC is shown as SEQ ID No:3, shown in the following:
ATGCTGAGAGGACCAGGACCAGGACTGCTGCTGGCTGTGCTGTGCCTGGGAACCGCTGTGAGGTGTACAGAGGCCCGGGTGCAGCCAACCGAGAGCATCGTGCGCTTCCCAAACATCACAAATCTGTGCCCCTTCGACGAGGTGTTTAACGCCACCACATTTGCTTCTGTGTACGCCTGGAACAGGAAGCGGATCTCTAATTGTGTGGCTGATTATTCCGTGCTGTACAATTTCGCTCCCTTCTTTGCCTTTAAGTGCTACGGCGTGTCTCCTACCAAGCTGAACGACCTGTGTTTCACAAATGTGTATGCCGATTCCTTTGTGATCAGGGGCAACGAGGTGAGCCAGATCGCTCCAGGCCAGACCGGCAACATCGCCGACTACAATTATAAGCTGCCCGACGATTTCACCGGCTGCGTGATCGCCTGGAACTCCAATAAGCTGGATAGCACAGTGGGCGGCAACTACAATTATAGATACCGCCTGTTCAGAAAGAGCAAGCTGAAGCCTTTTGAGCGCGACATCTCTACCGAGATCTACCAGGCTGGCAACAAGCCATGCAATGGCGTGGCCGGCGTGAACTGTTATTTCCCTCTGCAGTCCTACGGCTTTAGGCCAACATATGGAGTGGGACACCAGCCATATAGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCATGCTCCAGCTACCGTGTGCGGACCTAAGAAGTCCACAAACCTGGTGAAGAATAAGTCCGGAAGCGGAGGAGGATCTGGAGGACAGCCTAAGGCTGCTCCATCCGTGACCCTGTTCCCACCTTCCAGCGAGGAGCTGCAGGCCAATAAGGCTACCCTGGTGTGTCTGATCAGCGACTTTTACCCAGGAGCTGTGACAGTGGCTTGGAAGGCTGATTCTTCCCCTGTGAAGGCCGGCGTGGAGACCACAACCCCAAGCAAGCAGTCTAACAATAAGTATGCCGCTAGCTCTTACCTGAGCCTGACCCCCGAGCAGTGGAAGTCTCACCGGTCTTATTCCTGCCAGGTGACACATGAGGGCTCCACAGTGGAGAAGACCGTGGCCCCTACAGAGTGTAGC(SEQ ID No：3)。
wherein, the 76 th to 732 th positions code RBD protein sequence of BQ11, 760 th to 1074 th positions code LC protein sequence.
The nucleotide sequence for encoding XBB-HC is shown as SEQ ID No:4, as follows:
ATGCTGAGGGGACCAGGACCAGGACTGCTGCTGGCTGTGCTGTGCCTGGGAACCGCTGTGAGGTGTACAGAGGCCCGGGTGCAGCCAACCGAGTCTATCGTGCGGTTCCCTAACATCACAAATCTGTGCCCATTCCACGAGGTGTTTAACGCCACCACATTTGCTTCCGTGTACGCCTGGAACAGGAAGCGGATCAGCAATTGTGTGGCTGACTATTCTGTGATCTACAATTTCGCTCCATTCTTTGCCTTTAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGTTTCACAAATGTGTATGCCGATAGCTTTGTGATCAGGGGCAACGAGGTGTCTCAGATCGCTCCAGGCCAGACCGGCAACATCGCCGACTACAATTATAAGCTGCCCGACGATTTCACAGGCTGCGTGATCGCCTGGAACTCTAATAAGCTGGATTCCAAGCCTAGCGGCAACTACAATTATCTGTACAGACTGTTCCGCAAGTCCAAGCTGAAGCCCTTTGAGAGAGATATCAGCACCGAGATCTACCAGGCTGGCAACAAGCCTTGCAATGGCGTGGCCGGCTCTAACTGTTATTCCCCCCTGCAGAGCTACGGCTTCAGGCCTACATATGGCGTGGGCCACCAGCCATATCGGGTGGTGGTGCTGAGCTTTGAGCTGCTGCATGCTCCAGCTACCGTGTGCGGACCCAAGAAGTCTACAAACCTGGTGAAGAATAAGTCCGGAAGCGGAGGAGGATCTGGAGCTTCCACCAAGGGCCCATCCGTGTTTCCTCTGGCCCCATCCAGCAAGTCTACCTCCGGAGGAACAGCTGCTCTGGGATGTCTGGTGAAGGACTACTTCCCTGAGCCAGTGACCGTGAGCTGGAACTCTGGCGCTCTGACCTCCGGCGTGCATACATTTCCAGCCGTGCTGCAGTCTTCCGGCCTGTACAGCCTGAGCTCTGTGGTGACCGTGCCCTCCAGCTCTCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCTAGCAATACAAAGGTGGACAAGAGAGTGGAGCCAAAGTCTTGTGATAAGACCCATACATGCCCCCCTTGTCCAGCTCCTGAGCTGCTGGGAGGACCATCCGTGTTCCTGTTTCCACCCAAGCCTAAGGACACCCTGATGATCTCCCGCACCCCTGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAGGATCCAGAGGTGAAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACCAAGCCCAGAGAGGAGCAGTATAACTCTACCTACCGCGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGTCTAATAAGGCCCTGCCAGCTCCCATCGAGAAGACCATCTCCAAGGCTAAGGGCCAGCCCAGAGAGCCTCAGGTGTACACACTGCCTCCATCTCGCGAGGAGATGACCAAGAACCAGGTGTCCCTGACATGTCTGGTGAAGGGCTTCTATCCCTCTGACATCGCCGTGGAGTGGGAGTCCAATGGCCAGCCTGAGAACAATTACAAGACCACACCCCCTGTGCTGGACTCCGATGGCAGCTTCTTTCTGTATTCCAAGCTGACCGTGGATAAGAGCAGGTGGCAGCAGGGCAACGTGTTTAGCTGTTCTGTGATGCATGAGGCCCTGCACAATCATTACACACAGAAGTCCCTGAGCCTGTCTCCTGGC(SEQ ID No：4)。
wherein, the 76 th to 732 th bit codes RBD protein sequence of XBB, 760 th to 1743 th bit codes HC protein sequence.
2. The construction method comprises the following steps:
template synthesis: the nucleotide sequences for encoding the XBB antibody and the BQ11 antibody are subjected to CHO cell codon optimization, subjected to total gene synthesis by Nanjing Jinsri biotechnology limited company, inserted into pUC57 plasmid to obtain PUC57-XBB-HC and PUC57-BQ11-LC, and the optimized XBB-HC and BQ11-LC nucleic acid sequences are cut off from a PUC57 carrier and sequentially connected to the same carrier to express the XBBQ 11 antibody, wherein the plasmid structure schematic diagram is shown in figure 1.
2.1 double cleavage of the Gene of interest and the vector plasmid
The PUC57-XBB-HC was digested with restriction enzymes and the system is shown in Table 4:
table 4: XBB-HC restriction endonuclease double-enzyme cutting system
| Reactants | Dosage of |
| DNA | 2-6μg |
| ECORI | 1.5 |
| HindIII | 1.5 |
| 10×cutsmartbuffer | 5 |
| Nuclease-FreeWater | Fill up to 50. Mu.l |
The PUC57-BQ11-LC was double digested with restriction endonucleases and the system is shown in Table 5:
table 5: BQ11-LC restriction endonuclease double-enzyme cutting system
| Reactants | Dosage of |
| DNA | 2-6μg |
| XbaI | 1.5 |
| NotI | 1.5 |
| 10×cutsmartbuffer | 5 |
| Nuclease-FreeWater | Fill up to 50. Mu.l |
After mixing, enzyme digestion is carried out at 37 ℃ for 1.5hr, nucleic acid electrophoresis identification is carried out, gel recovery is carried out after the correct strip is displayed, the DNA concentration is measured, and the next test is carried out.
2.2 ligation with pKS001vector and transformation
(1) The fragment of interest was ligated to the pKS001vector according to the ligation system as shown in table 6 (20 μl system):
table 6: target fragment and pKS001vector connecting system
| pKS001Vector (double enzyme cutting) | 1μl |
| DNA template (double enzyme cutting) | 8μl |
| Ligase | 1μl |
| 2×LigaseBuffer | 10μl |
The mixed reaction solution is placed at room temperature (25 ℃) to react for 5 to 10 minutes.
(2) Conversion of ligation products
The ligation product was added to 50-100. Mu.l of TOP10 competent cells thawed on ice bath and allowed to stand on ice for 30min to allow the competent cells to mix well with the ligation product. The above product was placed in a 42 ℃ heat shock for 90s and then rapidly transferred to an ice bath on ice for 2min. 400. Mu.l of sterilized LB medium (without antibiotics) was added to the above product, and after mixing uniformly, it was placed in a shaking table at 37℃and 220rpm for 1hr. After all bacterial solutions are blown evenly, the bacterial solutions are evenly coated on two LB agar media with ampicillin resistance. The plates were placed in an incubator at 37℃overnight.
(3) Identification of recombinant plasmids
Individual clones on the plates were randomly picked, inoculated into 500. Mu.l of LB liquid medium containing ampicillin resistance, shake-cultured at 37℃for 2hr, and 0.5. Mu.l of the bacterial liquid was aspirated for colony PCR verification.
Colony PCR verification system (10. Mu.l system) is as in Table 7:
table 7: PCR verification system
| PCR system component | Addition amount of |
| 2×PCRTaqMasterMix | 5μl |
| Upstream primer | 0.5μl |
| Downstream primer | 0.5μl |
| Bacterial liquid | 1μl |
| Nuclease-FreeWater | 3μl |
The PCR reaction parameters are as in table 8:
table 8: PCR reaction parameters
And (5) detecting the PCR amplification product by agarose gel electrophoresis.
Positive clones amplified out of the band of interest were selected for sequencing.
(5) The plasmid strain sequenced correctly is amplified in a large quantity in LB culture medium containing ampicillin resistance, and expression plasmids are extracted in a large quantity by using a deiotoxin plasmid extraction kit.
2. Cell expression:
the aim of the experiment is to screen out monoclonal cell strains with high and stable expression. Transferring target DNA into CHO-K1Q cells by electrotransformation, inoculating the cells into 96-well plates in proportion, adding screening reagent, culturing under pressure, and comparing the biological activity of the clone pool by ELISA method to select high biological activity and continuously performing amplification culture; and finally, selecting a clone pool with higher activity, and finally selecting a monoclonal strain with high expression by using a limiting dilution method.
Experimental method
Screening of high-expression clone strains
1. Transfection
The materials are shown in table 9:
table 9: transfection materials
| Material | Suppliers (suppliers) | Goods number |
| CHO-K1Q cells | QuaCell | A13101 |
| CHOCD04 medium | QuaCell | A11004 |
| Glutamin | gibco | 25030-081 |
| DPBS | gibco | 14190-136 |
| Electric turn-over instrument | Yida living things | —— |
| EBELBuffer | Yida living things | H10305 |
| Electric rotating cup | Yida living things | ETTA2mm |
And taking out the Buffer, the cell culture solution and the D-PBS in advance 30min before electrotransformation, and returning to room temperature.
1.1 passage of cells: the day before transfection, cells were counted, centrifuged at 1200rpm,4min at 1.5x10 per bottle 6 Passaging at individual cell concentrations.
1.2 cell collection, count: cells were uniformly suspended and placed in an EP tube, and trypan blue staining was performed for counting.
1.3 centrifugation: placing the cells of the culture solution into a new centrifuge tube, centrifuging at 1000rpm for 4min;
1.5D-PBS wash: removing the supernatant culture solution to obtain the required cells, adding 1mL of D-PBS to resuspend the cells, centrifuging at 1000rpm for 4min;
1.5 mixing DNA, cells, buffer: discarding the D-PBS, adding the required amount of buffer and plasmid, lightly blowing and uniformly mixing;
1.6 electric conversion: the plasmid-mixed cell suspension was added to the H1 electrocuvette at 200. Mu.l+DNA volume/cup, and the electrocuvette was inserted into the base, and the correct electrotransformation conditions were selected for electrotransformation.
The experimental parameters are shown in table 10:
table 10: electric conversion experimental parameters
1.7 culturing: to a previously prepared T75 flask, 20ml of CD04 medium (containing 4mM glutamine) was added, and after electrotransfer, the solution was transferred to the flask, gently mixed and incubated in a 5% CO2 incubator at 37 ℃.
2. Screening of monoclonal high-expression strains.
The materials are shown in table 11:
table 11: monoclonal high-expression strain screening material
| QuaMonoPlusCHOCloningMediumA | QuaCell | A11602A |
| QuaMonoPlusCHOCloningMediumB | QuaCell | A11602B |
| QuaMonoPlusCHOCloningMediumC | QuaCell | A11602C |
| CHOCD04 medium | QuaCell | A11004 |
| 96-well plate | NEST | 701001 |
| 24-well plate | NEST | 702001 |
| 6-hole plate | NEST | 703001 |
| T25 square bottle | NEST | 707003 |
| 125 shake flask | NEST | 781011 |
2.1 preparation of Medium: preparing a mixed solution of Medium A and Medium B according to a ratio of 100:1 (1 mlB plus 100ml A)
2.2 cell plating: cell count, centrifugation 1200rpm,4min, number of plates calculated at 0.5 cells per well, 200 μl/well AB mix, 96 well plates plated, diagonally sealed with tape, and incubated in a 5% CO2 incubator at 37℃for 7 days.
2.3 cell replacement fluid: after the incubation days, medium C was added, 100. Mu.l/well, and incubated in a 5% CO2 incubator at 37℃for 7 days.
ELISA method for detecting biological activity and picking up the amplified culture with high activity.
The final selection of monoclonal strains showed 50-fold higher than the results of the monoclonal pool protein expression ELISA. The obtained protein has higher purity, and is purified by using ECA filler, and the yield is about 60mg/L.
3. The indirect ELISA method detects the activity of XBBQ 11 antibody protein.
The detection system is shown in Table 12:
table 12: indirect ELISA PBS method detection system for detecting XBBQ 11 antibody protein activity
3.1. Reagent preparation
3.1.1ELISA coating liquid (1X) is prepared: ELISA coating (10X) was diluted to 1X with sterile distilled water.
Pbs formulation: the PBS powder was removed and each bag was dissolved in 2L of sterile distilled water.
3.1.3. Washing liquid: PBST (PBS containing 0.05% Tween-20) is measured 1L of filtered PBS into a blue cap bottle, 500 mu LTwen-20 is added, and the mixture is fully mixed and stored at 2-8 ℃ for standby. According to the actual situation, the PBST with the required volume is prepared according to the method.
3.1.4. Blocking solution and sample dilution (PBS containing 5% skimmed milk powder. 5% skimmed milk powder by volume of PBS (milk powder mass/g=PBS volume/mLx 5%) were weighed, PBS taken out from 2-8deg.C was equilibrated to room temperature, PBS solution of the required volume was measured into centrifuge tubes to which milk powder had been added, and then dissolved well for use.
3.1.5. Diluting goat anti-mouse secondary antibody: and (3) balancing the secondary antibody with the specification of 1mL to room temperature, sub-packaging, and storing at-20+/-5 ℃. The sample was removed prior to each use and diluted 1:4000, where the diluent was PBS.
3.2 coating: cell supernatants were diluted 10-fold, 10. Mu.l were added with 90. Mu.l of coating solution, coated with enzyme-labeled plate, 100. Mu.l/well, positive and negative controls were set, and left overnight at 4 ℃.
3.3 closing: taking out the coated plate from the temperature of 2-8 ℃, washing the plate for 3 times, wherein the volume of each washing liquid is 300 mu l/hole, and if the washing liquid remains in the hole after washing, beating the coated plate on absorbent paper; then adding a pre-prepared sealing liquid into the coated holes, sealing the plate film at the temperature of 37 ℃ for 60-90min at 300 mu l/hole.
3.4 washing the plate: the sealed coated plate was washed 3 times with a washing solution volume of 300. Mu.l/well each time, and if washing solution remained in the well after washing, the plate was then dried on absorbent paper.
3.5 primary anti-dilution: primary antibodies were diluted to appropriate concentrations with PBS in centrifuge tubes. Adding the diluted primary antibody to the sample well at 100 μl/well; cover the sealing plate membrane and incubate at 37℃for 60min.
3.6 adding secondary antibody: discarding the sample, washing the plate for 3 times, wherein the volume of each washing liquid is 300 mu l/hole, and if the washing liquid remains in the hole after washing, beating the plate on absorbent paper; the diluted secondary antibody was added, 100. Mu.l/well, the membrane was covered and incubated at 37℃for 60min.
3.7 developing: washing the 96-well plate 3 times, wherein the volume of each washing solution is 300 μl/well, if washing solution remains in the well after washing, beating the plate on absorbent paper, adding single-component TMB developing solution 1 (from the beginning in advance)Taken out, equilibrated to room temperature), 100 μl/well, developed at 25deg.C in the dark for 15min.
3.8 termination: immediately after the color development, a stop solution was added to terminate the reaction, 50. Mu.l/well, and the mixture was gently shaken and mixed.
3.9 detection: the ELISA plate was placed in an ELISA reader and absorbance was measured at wavelengths of 450nm and 650 nm.
3.10, judging: and the positive value is judged as being 2.1 times greater than the negative OD value.
4 antigen purification
The materials are shown in table 13 below,
table 13: antigen purification material
| Material | Suppliers (suppliers) | Goods number |
| UniMab50 | Nawei | 17010-050100 |
| Polyacrylamide gel | solarbio | A1010 |
| Protein electrophoresis Marker | solarbio | PR1910 |
4.1 centrifuging: the cell suspension was centrifuged at 8000 rpm for 20 minutes and the supernatant was taken.
4.2, the specific steps are as follows:
1. washing the column: washing the column with PBS (phosphate buffer solution) to more than 40ml, removing bubbles before eluting, and if bubbles exist in the column, refilling
2. Loading: flow-through and liquid-penetrating are performed, and the air bubbles enter after staring.
3. Balance: PBS was equilibrated between 40-50ml.
4. Eluting: 0.1M glycine pH3 (effluent from peak recovery, concentrated HCl (3M) pH adjusted), and the collected eluate was pH adjusted to 7 with 2M Tris-Base.
5. Washing the column: about 30ml of the solution was washed with 1M NaoH.
The column was washed with PBS until pH 7.
And (3) preserving: 20ml-30ml of 20% ethanol, and preserving at-4deg.C.
4.9SDS-PAGE is performed to verify that the size of the XBB-HC target band is 64KD, the size of the BQ11-LC target band is 35KD, and the yield is 120mg/L.
The structural schematic diagram of the fusion protein construct is shown in fig. 2, wherein the light chain and the heavy chain are connected through disulfide bonds, and the heavy chain are also connected through disulfide bonds, so that a structure similar to an antibody is formed.
The purification results are shown in FIG. 3, and under reducing conditions, two target bands were obtained, the XBB-HC target band and the BQ11-LC target band (see column corresponding to "elution-reduction" in FIG. 3), and under eluting non-reducing conditions, LC and HC were combined by disulfide bonds to form a single target band of antibody-like structure (see first upper band corresponding to "elution-non-addition" in FIG. 3).
EXAMPLE 2 antibody titre after mouse immunization
The specific method comprises the following steps: the Balb/c mice of 6-8 weeks of age were randomly grouped, 5 mice per group were intramuscular injected with the vaccine composition, and vaccine groups, control group (PBS), immunized at weeks 0, 3, and blood was collected at weeks 3, 5. Antigen starting strain RBD-his (original strain), omicron1 RBD-his (BA.1), omicron5 RBD-his (BA.5), XBB RBD-his (XBB), and BQ11 RBD-his (BQ 11) protein (all of which were purchased from Yinqiao) were tested by ELISA method for their antibody titer (i.e., total IgG) expressed as EC50 value (EC 50 refers to the dilution of mouse serum that causes 50% of the maximum effect), and the results are shown in FIG. 4 and Table 14:
table 14: antibody titre after immunization of mice of each experimental group
The results show that the XBBQ 11 antibody vaccine provided by the invention can stimulate organisms to generate a broad spectrum of antibodies, and can generate antibodies with higher titer in all subtypes (original strains and various branches of currently popular Omicron strains) including SARS-CoV-2, and compared with a control group, the XBBQ 11 vaccine has a remarkable difference, and the XBBQ 11 vaccine also has a synergistic effect, such as remarkable improvement of the antibody generated against XBB compared with the single XBB-antibody, remarkable improvement of the antibody generated against BQ11 compared with the single BQ 11-antibody, and remarkable improvement of the sum of the antibody generated against other virus subtypes.
EXAMPLE 3 pseudo-Virus experiment
The neutralization test of the vaccine-induced serum of example 2 on WT, XBB, BA.1, BA.5, BA.2.75, CH.1.1, BQ.1.1 strain was examined by conducting the neutralization test of Nanjinouzan pseudovirus, and the results were expressed as IC 50 Values (IC 50 is serum dilution that inhibits 50% of infection of cells) are shown in fig. 5 and table 15:
table 15 neutralization experiments of pseudoviruses by vaccine-induced serum
The results show that the antibody induced by the vaccine provided by the invention can neutralize each branch of an original strain and an Omicron strain which is popular at present, so that the vaccine has broad-spectrum antiviral effect, has obvious difference relative to a control group, and meanwhile, the combination of XBBQ 11 also has synergistic effect, for example, the neutralization effect is obviously improved relative to separate XBB antibody and separate BQ11 antibody, and the synergistic effect is also reflected on other virus subtypes.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (10)
1. A fusion protein construct comprising an XBBBQ 11-like antibody, wherein the fusion protein construct comprises the RBD protein sequence of BQ11, the RBD protein sequence of XBB, and the antibody constant region protein sequence.
2. The fusion protein construct of claim 1, wherein the constant region sequences comprise a light chain constant region (LC) and a heavy chain constant region (HC), preferably the RBD protein sequence of BQ11 and the RBD protein sequence of XBB are linked to different constant regions, more preferably the RBD protein sequence of BQ11 is linked to a light chain constant region protein sequence to form a BQ11-LC class antibody, the RBD protein sequence of XBB is linked to a heavy chain constant region protein to form an XBB-HC class antibody, or the RBD protein sequence of BQ11 is linked to a heavy chain constant region protein sequence to form a BQ11-HC class antibody, and the RBD protein sequence of XBB is linked to a light chain constant region protein sequence to form an XBB-LC class antibody.
3. A fusion protein construct according to claim 1 or 2, characterized in that,
the RBD protein sequence of BQ11 comprises:
(A1) SEQ ID No: as shown in positions 26-244 of 1,
(A2) Any protein with the same function obtained by substituting and/or deleting and/or adding one or more amino acid residues in (A1);
(A3) A protein having 80% or more identity to any one of (A1) to (A2) and having the same function;
preferably, the RBD protein sequence of XBB comprises:
(B1) SEQ ID No: as shown in positions 26-244 of 2,
(B2) A protein with the same function obtained by substituting and/or deleting and/or adding any one of the (B1) through one or more amino acid residues;
(B3) A protein having 80% or more identity to any one of (B1) to (B2) and having the same function;
preferably, the protein sequence of the antibody light chain constant region comprises:
(C1) SEQ ID No: as shown at positions 254-358 of 1,
(C2) A protein with the same function obtained by substituting and/or deleting and/or adding any one of (C1) through one or more amino acid residues;
(C3) A protein having 80% or more identity to any one of (C1) to (C2) and having the same function,
preferably, the protein sequence of the antibody heavy chain constant region comprises:
(D1) SEQ ID No: as shown at positions 254-581 of 2,
(D2) A protein with the same function obtained by substituting and/or deleting and/or adding any one of the (D1) through one or more amino acid residues;
(D3) A protein having 80% or more identity to any one of (D1) to (D2) and having the same function.
4. A nucleotide molecule encoding the fusion protein construct of any one of claims 1-3.
5. The nucleotide molecule according to claim 4, characterized in that it comprises DNA and/or RNA, preferably that it comprises:
(G1) SEQ ID No: positions 76-732 of 3, SEQ ID No:3, positions 760-1074 of SEQ ID No: positions 76-732 of 4 and SEQ ID No: bits 760-1743 of 4,
(G2) Complementary, degenerate or transcribed sequences of (G1),
(G3) A DNA molecule or mRNA having more than 75% identity to the DNA molecule or mRNA defined in (G1) or (G2) and encoding the corresponding protein in the fusion protein construct.
6. A vector comprising the nucleotide molecule of any one of claims 4-5.
7. A host cell comprising the nucleotide molecule of any one of claims 4-5 or the vector of claim 6.
8. A method of preparing a fusion protein construct according to any one of claims 1-3, comprising introducing a nucleotide molecule according to any one of claims 4-5 or a vector according to claim 6 into a host cell, culturing the host cell, preferably, purifying the fusion protein construct under non-reducing conditions to obtain a fusion protein construct linked by disulfide bonds of LC and HC.
9. Use of the fusion protein construct of any one of claims 1-3, the nucleotide molecule of any one of claims 4-5, the vector of claim 6 or the host cell of claim 7, comprising any one of the following:
(1) Use in the manufacture of a product for the prevention and/or treatment of a disease caused by infection with the SARS-CoV-2 virus;
(2) Use in the preparation of a product for inducing an immune response to a SARS-CoV-2 virus antigen;
(3) Use in the prevention and/or treatment of diseases caused by SARS-CoV-2 virus infection;
(4) Use in inducing an immune response to a SARS-CoV-2 virus antigen.
10. A vaccine comprising the fusion protein construct of any one of claims 1-3, the nucleotide molecule of any one of claims 4-5, the vector of claim 6 or the host cell of claim 7, preferably the vaccine further comprising an adjuvant.
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