CN115975042B - Beta coronavirus heteromultimeric antigen, preparation method and application thereof - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention relates to a beta-coronavirus heteromultimeric antigen, a preparation method and application thereof, wherein the amino acid sequence of the beta-coronavirus heteromultimeric antigen comprises the following steps: and a plurality of interconnected monomers derived from the beta coronavirus, wherein each monomer derived from the beta coronavirus is a part of or all of the amino acid sequence of the receptor binding region of the spike protein of the beta coronavirus, the number of the monomers is an integer not less than 3, and the plurality of monomers of the beta coronavirus heteromultimeric antigen comprises monomers derived from 2 heterobetacoronaviruses or monomers derived from more than 3 heterobetacoronaviruses. The beta coronavirus RBD multimer can be stably expressed, can induce strong immune response to various beta coronaviruses after immunizing mice, and can realize the immune effect of multivalent vaccine by only one antigen protein.
Description
Cross reference
The present application claims the benefit of the inventive patent application filed at month 8 and 26 of 2021, under application number 202110987719.9, entitled "a beta-coronavirus heteromultimeric antigen, method for its preparation and use", the entire contents of which are incorporated herein by reference.
Technical Field
The invention relates to the field of biological medicine, in particular to a beta coronavirus heteromultimeric antigen, a preparation method and application thereof.
Background
The coronaviridae contains 4 coronaviridae, α, β, γ, δ, respectively. Severe acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and novel coronavirus (2019-nCoV, hereinafter referred to as SARS-CoV-2) all belong to the genus beta coronavirus. They are all positive strand RNA envelope viruses, which can widely infect humans and animals, and can cause serious diseases and even death after infection of humans. The new coronavirus enters cells through human angiotensin converting enzyme 2 (human angiotensin-converting enzyme 2, hACE 2), and the hACE2 receptor is distributed in arterial and venous endothelial cells, arterial smooth muscle cells, intestinal epithelial cells, pulmonary alveoli, bronchi and other respiratory organs, and the viruses can infect the cells containing the hACE2 receptor. In addition, there are also coronaviruses that can infect humans, including HCoV-OC43 and HKU1 of the genus beta, HCoV-NL63 and HCoV-229E of the genus alpha, which are relatively light in symptoms after infection.
MERS cases are mainly concentrated in the middle east and europe, with most middle east regional work or sojourn history before the onset of the diagnosed cases in the foreign countries of the middle east, with mortality rates up to about 40%. Besides the cases of people, MERS-CoV is mainly transmitted to people by a dromedary, a large number of dromedaries are fed in the middle east, and researches report that MERS-CoV forms local epidemics in the dromedaries of Saint, so that MERS-CoV has a large transmission risk, and no vaccine or medicine is used at present. Currently, new coronaviruses are pandemic worldwide, causing serious damage. New and recurrent infectious diseases are increasingly frequent, so that a coronavirus vaccine antigen design method is developed, one antigen can be realized, multiple viruses can be effectively prevented, and the method has great application value.
The envelope of the beta coronavirus has mainly 3 glycoproteins: s protein (Spike protein, S), E protein (Envelope protein, E) protein, and M protein (Membrane protein, M). The S protein is closely related to the process of invasion of coronavirus into cells, is an important antigen in vaccine development, and can generate neutralizing antibodies. Wherein the receptor binding domain of the S protein (receptor binding domain, RBD) is the most predominant antigen target region for the body to induce the production of neutralizing antibodies.
Disclosure of Invention
Object of the Invention
The invention aims to provide a beta coronavirus heteromultimeric antigen, a preparation method and application thereof. The invention is based on the conclusion that RBD protein of beta coronavirus can excite organism to produce neutralizing antibody, and dimer RBD protein can more effectively excite organism immune response than monomer RBD protein, by connecting more than 3 beta coronavirus heteromonomers in series, a beta coronavirus heteromultimeric antigen is obtained, and corresponding vaccine is obtained by using the antigen. The vaccine can stimulate mice to generate strong antibody response to various beta coronaviruses, and achieves the effect of preventing various viruses by one antigen with high efficiency.
A beta coronavirus heteromultimeric antigen whose amino acid sequence comprises: and a plurality of interconnected monomers derived from the beta coronavirus, wherein each monomer derived from the beta coronavirus is a part of or all of the amino acid sequence of the receptor binding region of the spike protein of the beta coronavirus, the number of the monomers is an integer not less than 3, and the plurality of monomers of the beta coronavirus heteromultimeric antigen comprises monomers derived from 2 heterobetacoronaviruses or monomers derived from more than 3 heterobetacoronaviruses.
In one possible implementation, the above-described beta-coronavirus heteromultimeric antigen is a severe respiratory syndrome coronavirus, a middle east respiratory syndrome coronavirus, or a 2019 novel coronavirus.
In one possible implementation, the above-mentioned heteromultimeric antigen of a β -coronavirus includes among the plurality of monomers of the heteromultimeric antigen of a β -coronavirus, monomers from the middle east respiratory syndrome coronavirus and monomers from the 2019 novel coronavirus.
In one possible implementation of the above-described beta coronavirus heteromultimeric antigen, the number of monomers is 3 or 4.
In one possible implementation, when the plurality of monomers of the heteromultimeric antigen of the betacoronavirus includes 2 or more than 3 monomers from the homologous betacoronavirus, the monomers from the homologous betacoronavirus are linked to each other first and then to the monomers from the heterologous betacoronavirus.
In one possible implementation of the above-described beta-coronavirus heteromultimeric antigen, the monomers are either directly linked in series or are linked in series by linking the amino acid sequences; alternatively, the linked amino acid sequences at different positions are independently selected from the following sequences: (GGS) n-linked sequences, wherein n represents the number of GGS and n is an integer of 1 or more; further alternatively, n is an integer selected from 1 to 10; still further alternatively, n is an integer selected from 1-5. GGS three letters represent amino acids G, G, S, respectively. The linking amino acid sequences at different positions are independent of each other: the linker amino acid sequence linking the first and second monomers from the N-terminus may be different from the linker amino acid sequence linking the second and third monomers from the N-terminus, and so on.
In one possible implementation, when the plurality of monomers of the heteromultimeric antigen of the β -coronavirus includes 2 or more than 3 monomers derived from the homologous β -coronavirus, the amino acid sequences of the monomers derived from the homologous β -coronavirus that are linked to each other are identical. That is, the sequences in homologous tandem are identical and are multiple repeated sequences. Of course, the monomers from the homologous β -coronavirus are not necessarily identical, e.g., the monomers from the homologous β -coronavirus may be one longer and one shorter.
In one possible implementation of the above-described heteromultimeric antigen of a β -coronavirus, the partial amino acid sequence of the receptor binding region of the spike protein of a β -coronavirus is at least 50%, 60%, 70%, 80%, 90%, 95%, 99% of the total amino acid sequence of the receptor binding region of the spike protein of a β -coronavirus.
In one possible implementation of the above-described beta-coronavirus heteromultimeric antigen, the plurality of interconnected monomers from the heterologous beta-coronavirus are:
one monomer from the middle east respiratory syndrome coronavirus is interconnected with two monomers from the 2019 novel coronavirus in tandem with each other. Namely: monomers of middle east respiratory syndrome coronavirus-monomers of 2019 novel coronavirus;
or, two monomers from the middle east respiratory syndrome coronavirus in tandem with two monomers from the 2019 novel coronavirus in tandem with each other are connected with each other. Namely: monomers of middle east respiratory syndrome coronavirus-monomers of 2019 novel coronavirus.
In one possible implementation of the above-described heterologous multimeric antigen of a β -coronavirus, the amino acid sequence of the heterologous antigen of a β -coronavirus comprises any one selected from the group consisting of:
the sequence is shown as SEQ ID NO. 7, namely: amino acids RBD 367-602 of the middle east respiratory syndrome coronavirus are directly connected with amino acids RBD 319-537 of two 2019 novel coronaviruses in series;
the sequence is shown as SEQ ID NO. 8, namely: amino acids RBD 367-602 of two middle east respiratory syndrome coronaviruses are directly connected with amino acids RBD 319-537 of two 2019 novel coronaviruses in series.
Wherein: the 367-602 region of the middle east respiratory syndrome coronavirus RBD is derived from the E367-N602 region of the spike protein sequence of MERS-CoV (GenBank: AFS88936.1 on NCBI); the 319-537 region of the 2019 novel coronavirus RBD is derived from the R319-K537 region of the S spike protein sequence of the WH01 strain of 2019 novel coronavirus (GenBank: YP_009724390 on NCBI).
The invention also provides a method for preparing the beta coronavirus heteromultimeric antigen, which comprises the following steps: adding a sequence for encoding a signal peptide to the 5 'end of a nucleotide sequence for encoding the beta-coronavirus heteromultimeric antigen, adding a sequence for encoding a histidine tag and a stop codon to the 3' end, cloning and expressing, screening a correct recombinant, then transfecting cells of an expression system for expression, collecting cell supernatant after expression, and purifying to obtain the beta-coronavirus heteromultimeric antigen.
In one possible implementation of the above method, the cells of the expression system comprise cells that are mammalian cells, insect cells, yeast cells, or bacterial cells, optionally; the mammalian cells include HEK 293T cells, HEK293F cells, or CHO cells, and the bacterial cells include e.
The invention also provides a polynucleotide for encoding the beta coronavirus heteromultimeric antigen, a recombinant vector comprising the polynucleotide and an expression system cell comprising the recombinant vector.
The invention also provides application of the beta-coronavirus heteromultimeric antigen, the polynucleotide for encoding the beta-coronavirus heteromultimeric antigen, a recombinant vector comprising the polynucleotide or an expression system cell comprising the recombinant vector in preparation of the beta-coronavirus vaccine.
The invention also provides a beta coronavirus vaccine which comprises the beta coronavirus multimeric antigen and an adjuvant.
In one possible implementation of the above-mentioned beta coronavirus vaccine, the adjuvant is selected from the group consisting of aluminum adjuvants, MF 59-like adjuvants.
The invention also provides a beta coronavirus DNA vaccine, which comprises: a recombinant vector comprising a DNA sequence encoding the above-described beta-coronavirus heteromultimeric antigen.
The invention also provides a beta coronavirus mRNA vaccine, which comprises: a recombinant vector comprising an mRNA sequence encoding the above-described beta-coronavirus heteromultimeric antigen.
The invention also provides a beta coronavirus virus vector vaccine, which comprises: a recombinant viral vector comprising a nucleotide sequence encoding the above-described beta-coronavirus heteromultimeric antigen; optionally, the viral vector is selected from one or more of the following: adenovirus vectors, poxvirus vectors, influenza virus vectors, adeno-associated virus vectors, vesicular stomatitis virus vectors (Vesicular Stomatitis Virus, VSV).
Description of the drawings:
FIG. 1 shows molecular sieve analysis and gel electrophoresis analysis of MERS-RBD and SARS-CoV-2-RBD tandem dimer (MC-RBD-tr 2) antigenic proteins in example 1 of the present invention;
FIG. 2 shows molecular sieve analysis and gel electrophoresis analysis of single chain heterotrimeric antigen protein of example 4 of the present invention;
FIG. 3 shows molecular sieve analysis and gel electrophoresis analysis of the single-chain hetero-tetrameric antigen protein of example 4 of the present invention;
FIG. 4 is a schematic illustration of the immunization strategy for each group of immunized mice in example 5 of the present invention;
FIG. 5 is a graph showing the results of the ELISA method of example 6 for the detection of specific IgG binding antibody levels to MERS-RBD in the serum of example 5 of the present invention at 19 days after immunization of mice;
FIG. 6 is a graph showing the results of the ELISA method of example 6 for detecting the level of specific IgG-binding antibodies against SARS-CoV-2-RBD in the serum of example 5 of the present invention 19 days after immunization of the mice;
FIG. 7 is a graph showing the results of the ELISA method of example 6 for the detection of specific IgG binding antibody levels to MERS-RBD in serum 35 days after immunization of mice in example 5 of the present invention;
FIG. 8 is a graph showing the results of the ELISA method of example 6 on the specific IgG-binding antibody levels against SARS2-RBD detected in the serum 35 days after immunization of the mouse of example 5 according to the present invention;
FIG. 9 is a graph showing the results of neutralizing antibodies against SARS-CoV-2 detected by the pseudo-virus neutralization assay (example 7) in the mouse serum obtained in example 7 at day 35 post-immunization according to the present invention, as detected in example 8;
FIG. 10 is a graph showing the result of the virus load of lung tissue after virus challenge by SARS-CoV-2 live virus in example 10 according to the present invention, wherein the result is obtained by the SARS-CoV-2 live virus challenge protection test and RT-qPCR test; wherein, the ordinate indicates the copy number of the novel coronavirus sgRNA per gram of lung tissue and the abscissa indicates the immune group class.
Detailed Description
New coronaviruses are pandemic worldwide, causing serious harm. New and recurrent infectious diseases are increasingly frequent, so that a coronavirus vaccine antigen design method is developed, one antigen can be realized, multiple viruses can be effectively prevented, and the method has great application value. In this study, we first selected new coronaviruses and MERS-CoV. MERS has high mortality rate, can be transmitted by people, can be transmitted by camels to people, and is fed with a large number of dromedaries in the middle east. We developed a novel vaccine design approach using the novel coronavirus and MERS-CoV design multivalent antigens as models. In previous studies, we found that subunit vaccines prepared based on single chain dimeric RBD proteins of the novel coronavirus were more immunogenic than monomeric RBD protein vaccines and induced better antibody levels (PMID: 32645327). In order to further improve the vaccine effect and realize the simultaneous prevention of various coronaviruses, we designed heterologous RBD multimeric antigens derived from different viruses, and detect whether the obtained protein can induce stronger immune response than the dimeric RBD protein after immunizing animals, and has better effect on various viruses at the same time, and is used for vaccine research and development.
Example 1: expression and purification of MERS-CoV RBD and SARS-CoV-2RBD chimeric dimer (MC-RBD-tr 2) proteins
MERS-RBD (367-602) amino acid sequence is connected with SARS-CoV-2-RBD (319-537) amino acid in series, named MC-RBD-tr2 (its sequence is shown as SEQ ID NO: 1), 6 histidines are added at the C end of the sequence, and MERS-S protein self signal peptide (MIHSVFLLMFLLTPTES, SEQ ID NO: 2) is connected at the N end. The nucleotide sequence encoding MC-RBD-tr2 with MERS-S protein self-signal peptide and histidine was inserted into EcoRI and XhoI cleavage sites of pCAGGS vector (the above nucleotide sequence is shown in SEQ ID NO:3, which includes sequences encoding histidine and signal peptide). The promoter contained a Kozak sequence GCCGCCACC upstream. The plasmid pCAGGS-MC-RBD-tr2 expressing the heterodimer was obtained by molecular cloning. The plasmid was transfected into HEK293F cells, and after 5 days, the supernatant was collected, centrifuged to remove the precipitate, and then filtered through a 0.22 μm filter membrane to further remove impurities. The resulting cell supernatant was adsorbed by a nickel affinity column (His Trap, GE Healthcare) at 4℃and washed with buffer A (20mM Tris,150mM NaCl,pH 8.0) to remove non-specific binding proteins. Then eluting the target protein from His Trap with buffer B (20mM Tris,150mM NaCl,pH 8.0,300mM imidazole), concentrating the eluate with 30kD concentration tube for more than 30 times to molecular sieve chromatography buffer PBS (8 mM Na) 2 HPO4,136mM NaCl,2mM KH 2 PO4,2.6mM KCl, pH 7.2) final volume was less than 1ml. Then pass through Superdex TM 200 The target protein was further purified by molecular sieve chromatography on an Increase 10/300GL column (GE Healthcare). The molecular sieve chromatography buffer is PBS buffer. A typical molecular sieve chromatographic chart is shown in figure 1, after molecular sieve chromatography: SDS-PAGE analysis was performed on the eluted peak around 14ml of the eluted volume, and the size of the target protein was about 60Kd under non-reducing conditions (without DTT) and under reducing conditions (with DTT), which confirmed that the peak was a dimer (monomer size: 30 Kd). SDS-PAGE analysis was performed on the eluted peak around 16ml of elution volume, and the size of the target protein was about 30kD under non-reducing conditions (without DTT) and under reducing conditions (with DTT), demonstrating that the peak was mainly RBD monomer.
Example 2: expression purification of tandem repeat RBD dimer proteins
In order to compare with the tandem repeat RBD dimer antigen, plasmids expressing single-chain repeat dimer MERS-RBD-tr2 (the amino acid sequences of two repeated MERS-RBD 367-602 are directly connected in series, the amino acid sequences are shown as SEQ ID NO: 4), SARS2-RBD-tr2 (the amino acid sequences of two repeated SARS-CoV-2-RBD 319-537 are directly connected in series, the amino acid sequences are shown as SEQ ID NO: 5) are respectively constructed. 6 histidines are added to the C end of the sequence, and the N end is connected with MERS-S protein self signal peptide (MIHSVFLLMFLLTPTES, SEQ ID NO: 2). The nucleotide sequence encoding MERS-RBD-tr2 or SARS-CoV-2-RBD-tr2 with MERS-S protein self signal peptide and histidine was inserted into the EcoRI and XhoI cleavage sites of the pCAGGS vector. Upstream of the promoter there is a Kozak sequence GCCACC. Plasmids pCAGGS-MERS-RBD-tr2, pCAGGS-SARS-CoV-2-RBD-tr2 expressing the repeat dimers were obtained by molecular cloning (see Dai, L., et al, A Universal Design of Betacoronavirus Vaccines against COVID-19,MERS,and SARS.Cell,2020.182 (3): p.722-733e11 for details).
Example 3: design and preparation of MERS-RBD and SARS-CoV-2RBD single chain heteromultimer
The N-terminal and the C-terminal of the MERS-RBD and the SARS-CoV-2-RBD respectively have a flexible sequence, and an attempt is made to connect three or four heteroRBD subunits in series to obtain RBD single-chain heterotrimers or RBD single-chain heterotrimers so as to induce the generation of antibody responses against both MERS-CoV and SARS-CoV-2 coronaviruses.
We designed the construction of single stranded heterotrimers and single stranded heterotrimers as follows:
(1) Amino acids 367-602 of a MERS-RBD partial sequence are connected with amino acids 319-537 of two SARS-CoV-2-RBD partial sequences in series, and the amino acid sequence is named MCC-RBD-tr3 (the amino acid sequence is shown as SEQ ID NO: 6);
(2) The amino acid sequence 367-602 of the two MERS-RBD partial sequences are connected with the amino acid sequence 319-537 of the two SARS-CoV-2-RBD partial sequences in series and named MMCC-RBD-tr4 (the amino acid sequence is shown as SEQ ID NO: 7).
The DNA sequence encoding MCC-RBD-tr3 or MMCC-RBD-tr4 with N-terminal connecting signal peptide (MERS-S protein self signal peptide MIHSVFLLMFLLTPTES, SEQ ID NO: 2) and C-terminal 6 histidines was inserted between EcoRI and XhoI cleavage sites of pCAGGS vector (the nucleotide sequence encoding MCC-RBD-tr3 with N-terminal connecting signal peptide and histidine is shown as SEQ ID NO:8, and the nucleotide sequence encoding MMCC-RBD-tr4 with N-terminal connecting signal peptide and histidine is shown as SEQ ID NO: 9). Upstream of the promoter there is a Kozak sequence GCCACC. Plasmids pCAGGS-MCC-RBD-tr3 and pCAGGS-MMCC-RBD-tr4 expressing the heterotrimer and the heterotrimer are obtained by molecular cloning.
Example 4: expression purification of MERS-RBD and SARS-CoV-2RBD single chain heterotrimers and tetrameric proteins
MCC-RBD-tr3 single-chain heterotrimer and MMCC-RBD-tr4 single-chain heterotrimer were expressed using HEK293F cells. HEK293F cells were transfected with plasmids pCAGGS-MCC-RBD-tr3 and pCAGGS-MMCC-RBD-tr4, respectively, and after 5 days, the supernatant was collected, and the precipitate was removed by centrifugation and filtered through a 0.22 μm filter membrane to further remove impurities. The resulting cell supernatant was adsorbed by a nickel affinity column (His Trap, GE Healthcare) at 4℃and washed with buffer A (20mM Tris,150mM NaCl,pH 8.0) to remove non-specific binding proteins. Then eluting the target protein from His Trap with buffer B (20mM Tris,150mM NaCl,pH 8.0,300mM imidazole), concentrating the eluate with 30kD concentration tube for more than 30 times to molecular sieve chromatography buffer PBS (8 mM Na) 2 HPO4,136mM NaCl,2mM KH 2 PO4,2.6mM KCl, pH 7.2) final volume was less than 1ml. Then pass through Superdex TM 200 The target protein is further purified by molecular sieve chromatography on an Increase 10/300GL column (GE Healthcare), and the molecular sieve chromatography buffer solution is PBS buffer solution. Through molecular sieve chromatography, MCC-RBD-tr3 (figure 2) and MMCC-RBD-tr4 (figure 3) respectively have an elution peak at about 14ml and 13ml, and SDS-PAGE analysis is carried out, so that MCC-RBD-tr3 and MMCC-RBD-tr4 proteins with sizes of about 90KD and 120KD under the conditions of non-reduction (without adding DTT) and reduction (with adding DTT) are respectively formed into trimers and tetramers.
As can be seen from FIGS. 2 and 3, MCC-RBD-tr3 and MMCC-RBD-tr4 can be properly folded and secreted, and antigen proteins with high purity can be obtained by purification.
Example 5: MCC-RBD-tr3 and MMCC-RBD-tr4 protein immunized mice experiments
We immunized mice after mixing the different antigen components with MF 59-like adjuvant, SWE adjuvant (SEPPIC). The grouping of mice is shown in Table 1. The immunization groups of the mice immunization experiments were set up using MCC-RBD-tr3 and MMCC-RBD-tr4 as immunogens; PBS as a negative control; the positive control groups were MERS of MERS-CoV and SARS-CoV-2 as immunogens (MERS-RBD-tr 2 and SARS2-RBD-tr 2), respectively.
TABLE 1 grouping and dosing of coronavirus RBD single chain heterotrimer and tetramer vaccine immunized mice
BALB/c mice used in the present invention were purchased from Vetong Lihua, inc., female, 6-8 weeks old. The immunization strategy is shown in FIG. 4. Diluting antigen protein to 200 mug/ml by PBS, mixing and emulsifying the MF 59-like adjuvant and immunogen according to the volume ratio of 1:1 to prepare the vaccine. The mixed vaccine was used to immunize BALB/c mice, and all mice were vaccinated with 2 vaccine doses on day 0 and day 21, respectively, with a vaccination volume of 100 μl each (where 50 μl antigen was mixed with 50 μl adjuvant, 200 μg/ml 50 μl = 10 μg). Mice were pooled by centrifugation on day 19 and day 35 and serum was stored in a-80 ℃ freezer and then used to titrate antigen specific antibody titers.
Example 6: ELISA (enzyme-linked immunosorbent assay) for detecting antigen-specific antibody titer generated by vaccine
(1) RBD monomeric proteins of MERS-CoV or SARS-CoV-2 were diluted to 3. Mu.g/ml with ELISA coating (Soxhlet, C1050), and 96-well ELISA plates (Coring, 3590) were added at 100. Mu.l per well and left at 4℃for 12 hours.
(2) Pouring the coating liquid, adding PBS, and washing once. 5% skim milk prepared with PBS was used as a blocking solution, and 100. Mu.l of each well was added to a 96-well plate, blocked, and left at room temperature for 1 hour. After blocking, the cells were washed once with PBS.
(3) Mice serum was diluted during blocking. Serum samples were also diluted with blocking solution. Serum samples were serially diluted from 20-fold starting at 4-fold gradient. The first well was mixed with 152. Mu.l of blocking solution and 8. Mu.l of serum, and the second dilution was mixed with 120. Mu.l of blocking solution and 40. Mu.l of solution from the first well, and diluted sequentially. After dilution 100 μl was added to each well of ELISA plate, negative control was blocking solution added, incubated at 37℃for 1.5 hours, and then washed 4 times with PBST.
(4) Goat anti-mouse secondary antibody (Abcam, ab 6789) conjugated with HRP diluted 1:2000 in blocking solution was added and incubated for 1.5 hours at 37 ℃ before 5-6 washes with PBST. After the reaction was completed by adding 60. Mu.l of TMB color-developing solution and reacting for a proper time, the reaction was terminated by adding 60. Mu.l of 2M hydrochloric acid, and the OD450 reading was measured on an ELISA reader. Antibody titer values were defined as the highest dilution of serum with a response value greater than 2.5 times the negative control value. When the reaction value of the lowest dilution (limit of detection) is still less than 2.5 times background value, the titer of the sample is defined as half the lowest dilution, i.e. 1:10.
Analysis of results:
ELISA results of serum after primary immunization against MERS-RBD are shown in FIG. 5, and MCC-RBD-tr3 and MMCC-RBD-tr4 can induce specific IgG against MERS-RBD with a titer as high as 10 3 The MMCC-RBD-tr4 group was significantly different from MERS-RBD-tr2 group (p)<0.01 The MCC-RBD-tr3 group was significantly different from MERS-RBD-tr2 group (×p)<0.05 A) is provided; but the MCC-RBD-tr3 group was not significantly different from the MMCC-RBD-tr4 group.
ELISA results of serum after primary immunization against SARS-CoV-2-RBD are shown in FIG. 6, and the MCC-RBD-tr3 and MMCC-RBD-tr4 can induce specific IgG against SARS-CoV-2-RBD with a titer as high as 10 3 The above is significantly different from the SARS2-RBD-tr2 group (.p)<0.05)。
ELISA results of serum after two immunizations against MERS-RBD as shown in FIG. 7, MCC-RBD-tr3 and MMCC-RBD-tr4 induced 1:10 production 5 Antigen-specific IgG titers above. The MMCC-RBD-tr4 group was significantly different from MERS-RBD-tr2 group (×p)<0.05)。
ELISA results of serum after two immunizations against SARS-CoV-2-RBD are shown in FIG. 8, MCC-RBD-tr3 induction produced 1:10 5 The antigen-specific IgG titer, MMCC-RBD-tr4 induction produced 1:10 6 Antigen-specific IgG titers above; the MCC-RBD-tr3 group and MMCC-RBD-tr4 group both have significant differences (P) compared to the PBS group<0.0001 However, there was no statistical difference compared to the SARS2-RBD-tr2 group.
The above results demonstrate that MCC-RBD-tr3 and MMCC-RBD-tr4 are able to induce mice to produce high levels of RBD-specific IgG against MERS-CoV and SARS-CoV-2 simultaneously, comparable to the same dose of homodimer-induced antibody levels (SARS-CoV-2), even better (MERS-CoV).
Example 7: MC-RBD-tr2 and MMCC-RBD-tr4 protein immunization mice experiments
To further compare the immunogenicity of MMCC-RBD-tr4 heteromultimers with MC-RBD-tr2 heterodimers, we immunized mice after mixing different antigen components with SWE adjuvant. The grouping of mice is shown in Table 2. The immunization groups set up in the mouse immunization experiments used MC-RBD-tr2 and MMCC-RBD-tr4 as immunogens; PBS as a negative control; the positive control group was a homodimer of SARS-CoV-2 as an immunogen (SARS 2-RBD-tr 2).
TABLE 2 grouping and dosing of coronavirus RBD single chain heterotrimer and tetramer vaccine immunized mice
BALB/c mice used in the present invention were purchased from Vetong Lihua, inc., female, 6-8 weeks old. Immunization strategies were the same as in example 5. Mice were subjected to blood collection by centrifugation on day 35, stored in a-80 ℃ refrigerator, and then used to titrate neutralizing antibody titers.
Example 8: pseudo-virus neutralization assay of immune serum
We diluted the serum ratio obtained in example 7 at day 35 post immunization (i.e. 14 days post two immunizations) (20-fold initiation, dilution with 2-fold gradient) and the serial dilutions were each mixed with 100TCID 50 SARS-CoV-2 pseudovirus (according to Jianhui Nie et al Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2,Emerging Microbes)&Prepared by the method described in the informations 2020, vol.9) were mixed and incubated at 37℃for 1 hour. The mixture was added to 96-well plates that had been confluent with Huh7 cells. Discard the culture 24 hoursAnd (3) adding a cell lysate after the nutrient solution, and detecting the luciferase activity value.
The level of neutralizing antibodies against SARS-CoV-2 in serum after two immunizations is shown in FIG. 9, and the results of FIG. 9 show: SARS-CoV-2RBD-tr2, MC-RBD-tr2 and MMCC-RBD-tr4 all induce 1:10 production in mice 3 Above (90% neutralization potency, pVNT 90 ) Is directed against SARS-CoV-2, and MMCC-RBD-tr4 induces significantly increased levels of neutralizing antibodies compared to MC-RBD-tr2 group (<0.05). In addition, MC-RBD-tr2 induced weak neutralizing antibody levels (.p) compared to SARS2-RBD-tr2<0.05 But the multimeric MMCC-RBD-tr4 produced neutralizing antibodies at levels comparable to homodimers (ns), this result indicated that MMCC-RBD-tr4 had better immunogenicity than MC-RBD-tr2, stimulating the production of higher levels of neutralizing antibodies.
Example 9: neutralization of immune serum by SARS-CoV-2 original strain true virus
In this example, neutralization experiments of the SARS-CoV-2 original strain of real virus were performed using the serum obtained after the two immunizations in example 7.
Specifically, we performed a double dilution (20-fold initial, dilution with 2-fold gradient) of the serum obtained in example 7 at day 35 post immunization (i.e., 14 days post two immunizations), and the resulting serial dilutions were each diluted with 100TCID 50 SARS-CoV-2 real virus (hCoV-19/China/CAS-B001/2020,GISAID No.EPI_ISL_514256-7) at 37℃for 1 hour; adding the mixed solution into a 96-well plate which is fully paved with Vero-E6 cells; after 72 hours, cytopathy (cytopathic effect, CPE) was observed under a microscope. The experiment was performed in the institute of biosafety class 3 laboratory (BSL 3).
The results show that: MC-RBD-tr2 induces mice to produce neutralizing antibodies with a titer of 1:1413 or higher, compared with MMCC-RBD-tr4 which induces neutralizing antibodies with a titer of 1:2100 or higher, which is significantly higher than that of MC-RBD-tr2 group; these results demonstrate that MMCC-RBD-tr4 is able to induce mice to produce a higher neutralizing antibody against SARS-CoV-2 than MC-RBD-tr2, as demonstrated by SARS-CoV-2 euvirus neutralization experiments.
Example 10: live virus challenge protection experiment and RT-qPCR experiment for detecting lung tissue viral load
For the mice immunized twice in example 7, the receptor hACE2 of SARS-CoV-2 was transiently expressed in the lung by nasal drop infection with adenovirus expressing hACE2, thereby rendering the mice susceptible to SARS-CoV-2; after 5 days, use 5x10 5 TCID 50 Is attenuated by SARS-CoV-2 original strain live virus (hCoV-19/China/CAS-B001/2020,GISAID No.EPI_ISL_514256-7); 3 days after virus challenge, the mice were dissected, lung tissue was taken, and the supernatant was homogenized after grinding to extract viral RNA. The quantitative PCR experiment is adopted to detect the viral load (sgRNA), specifically, 5 μl of nucleic acid is taken to prepare a PCR reaction system, and the real-time fluorescent RT-PCR reaction is carried out on a Bio-Rad fluorescent quantitative PCR instrument. The primer sequences were as follows:
the forward primer sequences were: CGATCTCTTGTAGATCTGTTCTC (SEQ ID NO: 10);
the reverse primer sequences were: ATATTGCAGCAGTACGCACACA (SEQ ID NO: 11);
the fluorescent probe sequence is: FAM-ACACTAGCCATCCTTACTGCGCTTCG (SEQ ID NO: 12) -TAMRA;
qRT-PCR experiments were performed using FastKing one-step reverse transcription-fluorescence quantitative kit (Probe method, cat. Number FP 314) from Tiangen Biochemical science and technology company, according to the kit instruction method; the reaction parameters are as follows: one cycle at 50 ℃ for 30min and 95 ℃ for 3 min; and (3) cycling for 40 times at 95 ℃ for 15s and 60 ℃ for 30s, and collecting fluorescent signals after extension.
The experimental results are shown in fig. 10, and fig. 10 shows: after the live virus of the new coronavirus attacks the virus for 3 days, the lung tissue virus load of 6 mice in 7 mice in the MMCC-RBD-tr4 group is cleared, namely, the virus zero clearing rate is about 86%; the lung tissue viral loads of only 2 mice and 1 mouse in SARS2-RBD-tr2 group and MC-RBD-tr2 group are cleared, and the viral clear rates are about 29% and 14% respectively. The result shows that MMCC-RBD-tr4 has very remarkable protective effect on the living toxin of SARS-CoV-2 as vaccine, and the protective effect is obviously better than MC-RBD-tr2 and SARS2-RBD-tr2.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A beta coronavirus heteromultimeric antigen characterized by: the amino acid sequence of the beta-coronavirus heteromultimeric antigen comprises any one of the amino acid sequences selected from the group consisting of:
the sequence is shown as SEQ ID NO. 6;
the sequence is shown as SEQ ID NO. 7.
2. A method of preparing the βcoronavirus heteromultimeric antigen of claim 1, wherein: the method comprises the following steps: adding a sequence for coding a signal peptide to the 5 'end of a nucleotide sequence for coding the beta coronavirus heteromultimeric antigen, adding a sequence for coding a histidine tag and a stop codon to the 3' end, cloning and expressing, screening a correct recombinant, then transfecting cells of an expression system for expression, collecting cell supernatant after expression, and purifying to obtain the beta coronavirus heteromultimeric antigen.
3. The method according to claim 2, characterized in that: cells of the expression system include mammalian cells, insect cells, yeast cells, or bacterial cells.
4. A method according to claim 3, characterized in that: the mammalian cells include HEK 293T cells, HEK293F cells, or CHO cells;
and/or, the bacterial cells include E.coli cells.
5. A polynucleotide encoding the beta coronavirus heteromultimeric antigen of claim 1.
6. A recombinant vector comprising the polynucleotide of claim 5.
7. An expression system cell comprising the recombinant vector of claim 6.
8. Use of a βcoronavirus heteromultimeric antigen according to claim 1, a polynucleotide according to claim 5, a recombinant vector according to claim 6 or an expression system cell according to claim 7 in the preparation of a βcoronavirus vaccine.
9. A beta coronavirus vaccine characterized by: comprising the beta-coronavirus heteromultimeric antigen of claim 1 and an adjuvant.
10. The beta coronavirus vaccine of claim 9, wherein: the adjuvant is selected from aluminum adjuvant, MF59 adjuvant or MF 59-like adjuvant; wherein the MF 59-like adjuvant is a SWE adjuvant.
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