Novel coronavirus rbd fusion protein

Abstract

The present application relates to a novel coronavirus RBD fusion protein, an RBD protein dimer and a preparation method and application thereof for preventing novel coronavirus SARS-CoV-2 infection.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the novel coronavirus RBD fusion protein, RBD protein dimer, and their preparation and application. Background Technology

[0002] The pneumonia caused by the novel coronavirus SARS-CoV-2 has led to large-scale outbreaks in many countries worldwide, with a crude case fatality rate of approximately 2.3%. It has had a significant adverse impact on human life, health, and livelihoods, causing widespread social panic and inflicting heavy economic losses. Currently, there are no specific drugs available worldwide for the clinical treatment of pneumonia caused by SARS-CoV-2. Therefore, there is an urgent need to develop effective and safe vaccines to protect threatened populations.

[0003] As of May 22, 2020, according to WHO statistics, 10 COVID-19 vaccines were undergoing clinical trials globally (5 of which were from China), and 114 were in preclinical research. The core principle of vaccines is to enable the immune system to recognize the target virus in advance, especially the unique and functional structures of the target virus, to generate a high-quality immune response and thus develop immunity. Current strategies mainly include nucleic acid vaccines (RNA and DNA vaccines), adenovirus vector vaccines, recombinant protein vaccines, inactivated vaccines, and live attenuated virus vaccines. Different types of vaccines have different characteristics and advantages and disadvantages. Nucleic acid vaccines and adenovirus vector vaccines rely on the expression of virus-related proteins in the human body to generate an immune response, while recombinant protein vaccines, inactivated virus vaccines, and live attenuated virus vaccines directly use viral proteins. Recombinant protein vaccines are prepared by expressing a large number of functional genes of the SARS-CoV-2 virus in cells or microorganisms and then purifying them. In principle, recombinant protein vaccines are highly safe, especially those designed based on the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Their protein expression is controllable, and theoretically there is no risk of DNA integration into the genome. However, the preparation process is complex and technically challenging, and they often have weak immunogenicity, requiring the addition of adjuvants to improve immunogenicity.

[0004] The RBD derived from SARS-CoV-2 can induce highly effective antibody responses in immunized animals, including mice and horses (Xiaoyan Pan, Pengfei Zhou, et al. Immunoglobulin fragment F(ab')2 against RBD potently neutralizes SARS-CoV-2 in vitro. Antiviral Research 2020). These induced antibodies neutralize SARS-CoV-2 and inhibit viral infection of Vero-E6 cells. This indicates that the RBD sequence in the spike protein of SARS-CoV-2 can induce highly effective neutralizing antibody responses and could be developed into an effective and safe subunit vaccine for the prevention of COVID-19. Summary of the Invention

[0005] The spike protein (S protein) of the novel coronavirus SARS-CoV-2 contains two subunits, S1 and S2. S1 mainly contains a receptor-binding domain (RBD, sequence source: Genbank: QHR63260.2), which can specifically bind to the receptor of target cells, angiotensin-converting enzyme ACE2.

[0006] The inventors have discovered that by using the cleavage site of the SARS-CoV-2 RBD fusion protease and a tag that facilitates dimer formation, the expression level and purity of the RBD fusion protein of the present invention are significantly improved. Furthermore, the inventors have found that regardless of whether it is wild-type or mutant RBD, fusing it with, for example, an Fc fragment without a cysteine ​​residue in the hinge region or an Fc fragment without a hinge region (such as the sequence shown in SEQ ID NO: 13) significantly improves the expression level and purity of the RBD fusion protein, enhances the protease cleavage efficiency (e.g., increases the recovery rate of the target protein and reduces the generation of impurity proteins), reduces the proportion of high-polymers, and the obtained fusion protein, after protease cleavage, yields a higher concentration of RBD dimers. These RBD protein dimers exhibit better ACE2 binding activity. The fusion protein and the further formed RBD dimers can be used for vaccines against SARS-CoV-2 infection or for obtaining neutralizing antibodies in immunized animals, as well as for protection in individuals newly exposed to SARS-CoV-2.

[0007] Specifically, the present invention relates to the following aspects:

[0008] 1. The SARS-CoV-2 RBD fusion protein, comprising, in sequence, the SARS-CoV-2 RBD sequence (preferably the RBD sequence contains a cysteine ​​residue located at position 220 according to the sequence number of SEQ ID NO: 1 and has the activity of binding to the human ACE2 receptor; more preferably the RBD sequence contains an amino acid sequence as shown in any one of SEQ ID NO: 1-8), a protease cleavage site, and a tag that facilitates dimer formation.

[0009] 2. The RBD fusion protein of the novel coronavirus SARS-CoV-2 of claim 1, wherein the tag that facilitates dimer formation is selected from the group consisting of a leucine zipper (preferably a sequence that does not contain HHHHHH in the sequence shown in SEQ ID NO: 30 or 31) and an Fc fragment, wherein the Fc fragment does not contain cysteine ​​in the hinge region (preferably the Fc fragment is an Fc fragment derived from human IgG, mouse IgG or horse IgG, more preferably, the Fc fragment does not contain a hinge region, and even more preferably the Fc fragment contains an amino acid sequence as shown in any one of SEQ ID NO: 13-16, 29), preferably the leucine zipper is derived from c-JUN or c-FOS protein, and more preferably, the C-terminus of the leucine zipper is further connected to a His, Flag, C-myc or HA tag.

[0010] 3. The RBD fusion protein of the novel coronavirus SARS-CoV-2 according to claim 1 or 2, wherein the protease cleavage site and the tag are linked by a linker peptide, preferably, the linker peptide is a flexible peptide, more preferably, the linker peptide is selected from SEQ ID NO: 26, 27 or 28.

[0011] 4. The RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-3, wherein the protease is selected from thrombin, enterokinase, TEV protease or HRC-3C protease; preferably, the amino acid sequence of the cleavage site of the thrombin, enterokinase, TEV protease or HRC-3C protease is as shown in any one of SEQ ID NO: 17-20; more preferably, the amino acid sequence of the protease cleavage site does not repeat with the amino acid sequence in the RBD sequence or Fc fragment.

[0012] 5. The RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-4, wherein the N-terminus of the fusion protein further comprises a signal peptide, preferably, the amino acid sequence of the signal peptide comprises the amino acid sequence shown in any one of SEQ ID NO: 21-23.

[0013] 6. The RBD dimer of the novel coronavirus SARS-CoV-2, characterized in that, according to the position numbering of the RBD monomer sequence shown in SEQ ID NO: 1, cysteine ​​residues at positions 18 and 43, 61 and 114, 73 and 207, and 162 and 170 of the monomer RBD form intrachain disulfide bonds, and an interchain disulfide bond is formed between cysteine ​​residues at position 220 of the two monomer RBDs. Preferably, the monomer RBD sequence contains the amino acid sequence shown in any one of SEQ ID NO: 1 and 5-8. More preferably, the two monomer RBD sequences of the dimer are identical. Preferably, the RBD dimer is prepared by the method described in 8 below.

[0014] 7. A method for preparing the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-5, comprising sequentially linking the RBD sequence of the novel coronavirus SARS-CoV-2, a protease cleavage site, and a tag that facilitates dimer formation, preferably, the protease cleavage site and the tag are linked by a linker peptide.

[0015] 8. A method for preparing the RBD dimer of the novel coronavirus SARS-CoV-2 according to claim 6, comprising cleaving the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-5 with a protease, preferably, the method further comprising purifying the RBD fusion protease cleavage product, preferably, the purification comprising chromatography (e.g., affinity chromatography, ion exchange chromatography).

[0016] 9. A polynucleotide, characterized in that it encodes the RBD fusion protein of the novel coronavirus SARS-CoV-2 as described in any one of claims 1-5 or the RBD dimer of the novel coronavirus SARS-CoV-2 as described in claim 6.

[0017] 10. A carrier comprising the polynucleotide of claim 9.

[0018] 11. A host cell comprising the polynucleotide of claim 9 or the vector of claim 10.

[0019] 12. A vaccine, preferably for the prevention of infection with the novel coronavirus, characterized in that it comprises the RBD fusion protein of the novel coronavirus SARS-CoV-2 as described in any one of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 as described in claim 6, or the polynucleotide as described in claim 9.

[0020] 13. The RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 according to claim 6, or the polynucleotide according to claim 9, for use in preventing human infection with the novel coronavirus SARS-CoV-2, or for immunizing animals to obtain antiviral serum, wherein the animals are preferably mammals, more preferably horses, and preferably the antiviral serum can be used for the prevention or treatment of human infection with the novel coronavirus SARS-CoV-2.

[0021] 14. A method for preventing or treating infection with the novel coronavirus SARS-CoV-2 or for obtaining neutralizing antibodies against the novel coronavirus SARS-CoV-2, comprising immunizing an animal with the RBD fusion protein of the novel coronavirus SARS-CoV-2 as described in any one of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 as described in claim 6, or the polynucleotide as described in claim 9 to obtain antiviral serum, wherein the animal is preferably a human or a non-human mammal, more preferably a horse.

[0022] In some embodiments of the present invention, the Fc segment is an Fc fragment derived from human IgG, preferably the sequence shown in SEQ ID NO: 13.

[0023] In some embodiments of the present invention, the Fc segment is an Fc fragment derived from equine IgG, preferably the sequence shown in SEQ ID NO: 15.

[0024] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0025] The terms used in this invention have their conventional meanings as understood by those skilled in the art. Where a term has two or more definitions as used and / or where acceptable in this art, the definitions used herein are intended to encompass all meanings.

[0026] When determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called “conserved” amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which has little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art, for example, from WO 04 / 037999, GB-A-3357 768, WO 98 / 49185, WO 00 / 46383, and WO 01 / 09300; and the (preferred) type and / or combination of such substitutions can be selected based on the relevant teachings of WO 04 / 037999 and WO 98 / 49185 and other references cited therein.

[0027] "Conservative amino acid substitution" refers to the substitution of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, non-essential amino acid residues of immunoglobulin polypeptides are preferably substituted with other amino acid residues from the same side chain family. In other embodiments, a string of amino acids may be substituted with a structurally similar string of amino acids that differ in sequence and / or composition of the side chain family.

[0028] The table below provides non-limiting examples of conserved amino acid substitutions, where a similarity score of 0 or higher indicates a conserved substitution between the two amino acids.

[0029] C G P S A T D E N Q H K R V M I L F Y W W -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 -0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5

[0030] M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12

[0031] In some embodiments, the conservative substitution is preferably a substitution in which one amino acid in the following groups (a)-(e) is replaced by another amino acid residue in the same group: (a) small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0032] The preferred conservative substitutions are as follows: Ala is replaced with Gly or Ser; Arg is replaced with Lys; Asn is replaced with Gln or His; Asp is replaced with Glu; Cys is replaced with Ser; Gln is replaced with Asn; Glu is replaced with Asp; Gly is replaced with Ala or Pro; His is replaced with Asn or Gln; Ile is replaced with Leu or Val; Leu is replaced with Ile or Val; Lys is replaced with Arg, or Gln or Glu; Met is replaced with Leu, or Tyr or Ile; Phe is replaced with Met, or Leu or Tyr; Ser is replaced with Thr; Thr is replaced with Ser; Trp is replaced with Tyr; Tyr is replaced with Trp; and / or Phe is replaced with Val, or Ile or Leu. Attached Figure Description

[0033] Figure 1 A schematic diagram of the recombinant fusion protein RBD-Fc.

[0034] Figure 2 .RBD-His SDS-PAGE non-reduction electrophoresis detection image. Figure 2 A, Ni affinity chromatography samples, lanes 1-3 are for the same elution peak collected in separate tubes; Figure 2 B, cation exchange chromatography sample, lane 1 is the protein marker, lanes 2-10 are different components eluted from low salt to high salt.

[0035] Figure 3 .RBD-Fc SDS-PAGE electrophoresis detection image. Figure 3A, Lane 1, RBD-TFc non-reduced; Lane 2, RBD-YFc non-reduced; Lane 3, RBD-TFc reduced; Lane 4, RBD-YFc reduced; Lane 5, RBD-eqIgG1Fc non-reduced; Lane 6, RBD-eqIgG1Fc reduced; Lane 7, RBD-eqIgG4Fc non-reduced; Lane 8, RBD-eqIgG4Fc reduced; MK, Protein Marker. Figure 3 B, Lane 1, RBD L455I F456V-YFc non-reducing; Lane 2, RBD G476S-YFc non-reducing; Lane 3, RBD V483A-YFc non-reducing; Lane 4, RBD D494P-YFc non-reducing; Lane 5, RBD L455I F456V-YFc reducing; Lane 6, RBD G476S-YFc reducing; Lane 7, RBD V483A-YFc reducing; Lane 8, RBD S494P-YFc reducing; MK, Protein Marker.

[0036] Figure 4 .RBD-Fc HPLC-SEC detection chromatogram.

[0037] Figure 5 Enzyme digestion of RBD-TFc and RBD-YFc was detected by SDS-PAGE non-reducing electrophoresis. Lane 1: RBD-TFc before digestion; Lane 2: RBD-TFc after digestion; Lane 3: RBD-YFc before digestion; Lane 4: RBD-YFc after digestion.

[0038] Figure 6 Unlabeled products (RBD) TFc and RBD YFc The identification. Figure 6 A, SDS-PAGE electrophoresis detection, lane 1, RBD TFc Non-reduced; lane 2, RBD YFc Non-reduced; lane 3, RBD TFc Restoration; Lane 4, RBD YFc reduction; Figure 6 B, HPLC-SEC detection chromatogram; Figure 6 C, Mass spectrometry molecular weight detection.

[0039] Figure 7 Enzyme digestion of horse Fc-tagged RBD was detected by SDS-PAGE non-reducing electrophoresis. Lane 1: RBD-eqIgG1Fc digestion before digestion; Lane 2: RBD-eqIgG1Fc digestion after digestion; Lane 3: RBD-eqIgG4Fc digestion before digestion; Lane 4: RBD-eqIgG4Fc digestion after digestion.

[0040] Figure 8 .RBD eqG1FcIdentification. Figure 8 A, SDS-PAGE electrophoresis detection, lane 1, RBD eqG1Fc Non-reduced; lane 2, RBD eqG1Fc reduction; Figure 8 B, HPLC-SEC detection chromatogram; Figure 8 C, Mass spectrometry molecular weight detection graph.

[0041] Figure 9 Biacore assay of the binding of unlabeled products of different RBD-Fc after enzyme digestion and affinity chromatography to hACE2-Fc.

[0042] Figure 10 .RBD YFc Non-reducing SDS-PAGE and Coomassie brilliant blue staining images of different components eluted by cation exchange chromatography.

[0043] Figure 11 .RBD YFc Biacore detection images of the dimer (A) and monomer (B) binding to hACE2-Fc, respectively.

[0044] Figure 12 .RBD YFc Schematic diagram of the dimer structure.

[0045] Figure 13 Label-free RBD products obtained by enzyme digestion and affinity chromatography of different RBD-Fc groups. YFc9 RBD YFc10 and RBD YFc11 Non-reducing and reduced SDS-PAGE Coomassie Brilliant Blue staining images. Detailed Implementation

[0046] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings. The present invention can be implemented in many ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the scope of protection of the present invention is defined by the claims and is not limited to the specific embodiments disclosed below.

[0047] Example 1: Fusion Protein Structure Design

[0048] The RBD protein sequence was obtained from GenBank: QHR63260.2. The RBD gene fragment was synthesized from the whole genome and constructed into a eukaryotic expression vector (e.g., pcDNA3.1 vector, Invitrogen) between multiple cloning restriction enzyme sites. A signal peptide (e.g., CD33 signal peptide, IL2 signal peptide, human albumin HSA signal peptide, etc.) was added to its N-terminus, and a protein tag (e.g., His, Flag, HA, myc, or Fc tag, etc.) was added to its C-terminus to facilitate purification. Furthermore, a protease restriction enzyme coding sequence was added downstream of the RBD gene and upstream of the tag gene. The protease includes, but is not limited to, thrombin (Sigma), enterokinase (New England Biolabs), TEV protease (Invitrogen), or HRV3C protease (Novagen). Optionally, the protease restriction enzyme site and, for example, the Fc tag are linked by a linker peptide. The specific structure of the fusion protein is as follows: Figure 1 As shown in Table 1, the specific sequence information of the constructs is detailed in Table 1.

[0049] RBD is a domain in the S protein of the SARS-CoV-2 novel coronavirus, located in the region of amino acid residues 319-541 of the entire S protein (SEQ ID NO: 1). Studies have reported the selection of different regions of the RBD for individual or fusion expression with Fc to obtain recombinant RBD proteins. For example, exogenous expression of residues 319-545 of the S protein (SEQ ID NO: 2) is performed (Jingyun Yang, Wei Wang, Zimin Chen, et al. A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity. Nature 2020.), or exogenous expression of residues 331-524 of the S protein (SEQ ID NO: 3) fused with Fc is performed (Hongjing Gu, Qi Chen, Guan Yang, et al. Adaptation of SARS-CoV-2 in BALB / c mice for testing vaccine efficacy. Science 2020.), or exogenous expression of residues 319-537 of the S protein (SEQ ID NO: 4) is performed (Lianpan Dai, Tianyi Zheng, Kun Xu, et al. A universal design of betacoronavirus vaccines against COVID-19, MERS and...). (SARS.Cell 2020.). For the RBD sequences of the above different regions, fusion Fcs were constructed, as shown in Table 1: RBD-YFc6, RBD-YFc7, and RBD-YFc8. The RBD regions correspond to residues 319-545, 331-524, and 319-537 of the S protein, respectively.

[0050]

[0051]

[0052] Example 2: Expression and purification of RBD proteins with different tags

[0053] Plasmids were extracted using standard plasmid extraction methods and used for chemical transfection of 293 (ATCC) or CHO-S (Gibco) cells. Transfected cells were cultured in suspension with shaking at 37°C and 5% CO2 for 7–10 days. The supernatant was harvested by centrifugation at 3000×g and filtered through a 0.22 μm filter membrane.

[0054] RBD-His feed solution was harvested and subjected to Ni column affinity chromatography. The expression level was less than 1 mg / L, and the sample was then analyzed by SDS-PAGE. Figure 2 As shown in Figure A, the sample after Ni column affinity chromatography was impure and of low purity. Therefore, cation exchange chromatography was used for purification. The sample was linearly eluted with low salt (5 mM NaCl) and high salt (500 mM NaCl). Different components eluted from low salt to high salt were collected separately and analyzed by SDS-PAGE. Figure 2 As shown in Figure B, lane 2 contains the highest purity fraction with a band size of 50 kDa, consistent with the theoretical molecular weight of RBD-His. However, the sample purification yield is extremely low, not exceeding 20%. Similarly, the RBD-His1 expression solution underwent the same Ni column and cation exchange chromatography purification process as described above, yielding the same protein purity and band size, with no significant difference in purification yield. This indicates that using different signal peptides for RBD-fused His-tagged protein expression does not result in significant differences in expression level or purification yield, and both protein expression and yield remain low.

[0055] Replacing the C-terminal protein tag of RBD with other tags, including but not limited to Flag, HA, and c-Myc tags, yields RBD-Flag, RBD-HA, and RBD-myc fusion tag proteins, respectively. After transient transfection of the constructed expression plasmids into 293 or CHO-S cells and subsequent suspension-shaking culture for 7-10 days, the protein expression levels in the harvested plasmids were similar to those of RBD-His, all less than 1 mg / L. Following affinity chromatography for the tags, the protein yield was also less than 20%. Therefore, in mammalian expression systems, fusing short peptide protein tags to the C-terminus of RBD results in low expression levels and purification yields.

[0056] The protein expression solution fused with the Fc tag by RBD was harvested and subjected to protein A affinity chromatography. The purified protein was analyzed by SDS-PAGE, such as... Figure 3 As shown, the band size is approximately 110 kDa, consistent with the theoretical molecular weight of the RBD fused with the Fc tag. Further analysis of the polymer content using high-performance size exclusion chromatography (HPLC-SEC) is shown in Table 2. Figure 4As can be seen, changing the RBD tag from a short peptide tag to an Fc tag significantly improved expression levels and purity. Furthermore, the expression level using the horse IgG1 Fc tag was significantly higher than that using the human Fc tag. Optimization of the RBD-Fc fusion protein showed that the HPLC-SEC purity of the unoptimized RBD-TFc was 89.43%, while the purity of the optimized RBD-YFc increased to 95.82%, with a reduced proportion of polymers. In addition, for the optimized RBD L455I F456V-YFc, RBD G476S-YFc, and RBD V483A-YFc, the proportion of polymers was significantly reduced, and the purity increased by 5-10%. Compared to the unoptimized RBD S494P-TFc, the optimized RBD S494P-YFc also showed a significantly reduced proportion of polymers and a 5% increase in purity. The expression levels and purity of RBD-YFc9, RBD-YFc10, and RBD-YFc11 were not significantly different from those of RBD-YFc. The expression levels and purity of RBD-epIG1Fc1 were not significantly different from those of RBD-epIG1Fc. The expression levels and purity of RBD-epIG4Fc1 were not significantly different from those of RBD-epIG4Fc.

[0057] Table 2. Expression levels and purity of different RBD-Fc cells

[0058] Buildings Expression level HPLC-SEC RBD-TFc 80mg / L 89.43％ RBD-YFc 100mg / L 95.82％ RBD-YFc1 98mg / L 94.56％ RBD-YFc2 95mg / L 95.11％ RBD-YFc3 99mg / L 95.63％ RBD-YFc4 87mg / L 95.02％ RBD-YFc5 88mg / L 94.75％ RBD-YFc6 90mg / L 90.91％ RBD-YFc7 92mg / L 77.78％ RBD-YFc8 89mg / L 94.59％ RBD-YFc9 95mg / L 92.77％ RBD-YFc10 99mg / L 94.64％ RBD-YFc11 99mg / L 93.18％ RBD L455I F456V-YFc 108mg / L 60.70％ RBD G476S-YFc 94mg / L 94.41％ RBD V483A-YFc 97mg / L 95.28％ RBD S494P-TFc 43mg / L 55.00％ RBD S494P-YFc 54mg / L 60.66％ RBD-eqIgG1Fc 180mg / L 89.84％ RBD-eqIgG4Fc 92mg / L 78.13％

[0059] Example 3: Preparation and Identification of Label-Free RBDs

[0060] To eliminate non-specific antibodies induced by the Fc tag in vivo and their impact on RBD, the Fc tag was cleaved using a protease to obtain tag-free RBD protein. The specific method is as follows: The corresponding protease was added to the RBD fusion Fc protein purified by affinity chromatography, and incubation was performed under specific conditions according to the manufacturer's instructions. The RBD protein was then obtained through flow-through chromatography using protein A or protein G affinity chromatography, resulting in the tag-free product. The enzyme digestion efficiency of the RBD fusion Fc protein before and after digestion was detected by SDS-PAGE, and the recovery rate of the RBD protein was calculated. The final tag-free RBD protein was then analyzed.

[0061] Preparation of ACE2 protein: A eukaryotic expression plasmid (vector pcDNA3.1) of hACE2-Fc (SEQ ID NO: 24), a protein fused with the extracellular domain of human ACE2 and Fc, was constructed. The plasmid was transiently transfected into 293 or CHO-S cells, cultured for 7-10 days, and the supernatant was harvested. The hACE2-Fc protein was obtained by affinity chromatography with Protein A.

[0062] like Figure 5As shown in lanes 2 and 4, the optimized construct RBD-YFc exhibits significantly better enzymatic digestion performance than the unoptimized RBD-TFc. Table 3 shows that the optimized RBD-YFc, RBD-YFc1-RBD-YFc11, and RBD-eqIgG1Fc show significantly improved enzyme digestion and purification recovery rates compared to the unoptimized RBD-TFc. Furthermore, regardless of whether the RBD is wild-type or mutant, the optimized RBD-YFc demonstrates superior enzymatic digestion performance compared to RBD-TFc, increasing the target protein recovery rate by 20-30%. For unlabeled RBD products… TFc and RBD YFc To conduct identification, from purity ( Figure 6 A and 6B), molecular weight (A and 6B) Figure 6 Regarding C), both are consistent, and the mass spectrometry molecular weight analysis shows that the RBD protein contains both monomeric (31KD) and dimeric (62KD) forms, a result consistent with SDS-PAGE and SEC results. The binding activity with hACE2-Fc ( Figure 9 For unlabeled products, RBD YFc Its binding activity with hACE2-Fc is higher than that with RBD. TFc .

[0063] Table 3. RBD Recovery Rate Statistics

[0064] Buildings RBD after removing Fc Target protein recovery rate RBD-TFc <![CDATA[RBD TFc ]]> 58.6％ RBD-YFc <![CDATA[RBD YFc ]]> 82.3％ RBD-YFc1 <![CDATA[RBD YFc1 ]]> 78.8％ RBD-YFc2 <![CDATA[RBD YFc2 ]]> 79.3％ RBD-YFc3 <![CDATA[RBD YFc3 ]]> 77.5％ RBD-YFc4 <![CDATA[RBD YFc4 ]]> 77.4％ RBD-YFc5 <![CDATA[RBD YFc5 ]]> 78.9％ RBD-YFc6 <![CDATA[RBD YFc6 ]]> 79.2％ RBD-YFc7 <![CDATA[RBD YFc7 ]]> 78.9％ RBD-YFc8 <![CDATA[RBD YFc8 ]]> 77.36％ RBD-YFc9 <![CDATA[RBD YFc9 ]]> 76.9％ RBD-YFc10 <![CDATA[RBD YFc10 ]]> 80.1％ RBD-YFc11 <![CDATA[RBD YFc11 ]]> 79.3％ RBD-eqIgG1Fc <![CDATA[RBD eqG1Fc ]]> 70.7％

[0065] Note: Since each milligram of RBD-Fc protein consists of 0.5 milligrams of RBD and 0.5 milligrams of Fc tag, the RBD target protein recovery rate = the final amount of RBD protein obtained / (RBD-Fc protein amount × 50%).

[0066] RBD fusion Fc proteins constructed using different protease cleavage sites, such as RBD-YFc (thrombin), RBD-YFc1 (enterokinase), RBD-YFc2 (TEV protease), and RBD-YFc3 (HRV-3C protease), showed superior enzymatic digestion compared to RBD-TFc containing the corresponding cleavage sites before optimization (i.e., the thrombin cleavage site in RBD-TFc was replaced with an enterokinase cleavage site, a TEV protease cleavage site, and an HRV-3C protease cleavage site). The recovery rate of the target RBD protein was significantly improved, and after enzymatic digestion, affinity chromatography, and ion exchange chromatography, the obtained unlabeled RBD product... YFc RBD YFc1 RBD YFc2 and RBD YFc3 There were no significant differences in purity, molecular weight, and hACE2-Fc binding activity.

[0067] RBD fusion Fc proteins, such as RBD-YFc6, RBD-YFc7, and RBD-YFc8, were constructed from different regions of the S protein. After enzyme digestion and affinity chromatography, the tag-free product RBD was obtained. YFc6 RBD YFc7 and RBD YFc8 Purity was determined by non-reducing SDS-PAGE, as shown in Table 4. Among them, RBD... YFc6 Dimer content exceeds 80%, RBD YFc7 The proportion of dimers is less than 30%, and the proportions of polymers (molecular weight exceeding 90kD) and monomers both exceed 30%, RBD YFc8 Only monomers were used. RBD-YFc9-11, which are different from the linker peptides of RBD-YFc, yielded the tagless product RBD after enzyme digestion and affinity chromatography. YFc9 RBD YFc10 and RBD YFc11 Both contain nearly 90% dimer components. Figure 13 ).

[0068] Table 4. Purity of RBD unlabeled products as determined by non-reducing SDS-PAGE.

[0069] RBD unlabeled products Polymer ratio Dimer ratio Monomer ratio <![CDATA[RBD TFc ]]> 0 65％ 35％ <![CDATA[RBD YFc ]]> 0 90％ 10％ <![CDATA[RBD YFc6 ]]> 0 81％ 19％ <![CDATA[RBD YFc7 ]]> 31％ 29％ 40％ <![CDATA[RBD YFc8 ]]> 0 0 100％ <![CDATA[RBD YFc9 ]]> O 87％ 13％ <![CDATA[RBD YFc10 ]]> 0 88％ 12％ <![CDATA[RBD YFc11 ]]> 0 89％ 11％

[0070] Note: RBD YFc7 and RBD YFc8 Neither of them contains cysteine ​​located at position 220 according to the position number of SEQ ID NO; 1.

[0071] like Figure 7 As shown in lanes 2 and 4, the restriction enzyme cleavage efficiency of the construct RBD-eqIgG1Fc is significantly better than that of RBD-eqIgG4Fc, and as... Figure 8 and Figure 9 As shown, RBD obtained after RBD-eqIgG1Fc enzyme digestion. eqG1Fc In terms of purity, molecular weight, and hACE2-Fc binding activity, it is comparable to RBD. YFc Highly consistent. RBD obtained after RBD-eqIgG4Fc enzyme digestion. eqG4Fc It also contains a dimeric protein with a molecular weight of 62kD and a monomer with a molecular weight of 31kD. Biacore detection shows it is similar to RBD. eqG1Fc They exhibit consistent activity.

[0072] Example 4: Detection of the binding activity of RBD protein to human ACE2

[0073] Numerous studies have reported that the SARS-CoV-2 SARS-CoV-2 novel coronavirus RBD has high binding activity with the human ACE2 receptor.

[0074] Unlabeled products RBDYFc (That is, the tag was removed by protease) Further elution was performed using cation exchange chromatography (e.g., using GE's Capto SP ImpRes packing material) with a high-salt (500 mM NaCl) linear gradient. The eluted components were then analyzed by SDS-PAGE. Figure 10 As shown, monomers and dimers can be effectively separated. The collected and combined lanes 2-4 are RBDs. YFc Monomeric components were collected and combined in lanes 7-12 as RBD. YFc The purity of the dimer component was determined by HPLC-SEC, and the purity of both the monomer and dimer components exceeded 97%.

[0075] RBD respectively YFc The binding activity of the monomeric and dimeric components to hACE2-Fc protein was analyzed using Biacore assays, such as... Figure 11 As shown, RBD YFc The affinity of the dimer for hACE2-Fc is K. D =0.192nM, while RBD YFc The affinity of the monomer for hACE2-Fc is K. D =15.60 nM. RBD YFc The ability of the dimer to bind to hACE2-Fc is significantly stronger than that of the RBD. YFc The ability of monomers to bind hACE2-Fc.

[0076] RBD YFc The amino acid residue codes of the monomers are shown in Table 5. RBD YFc Mass spectrometry analysis of the dimer after enzymatic digestion with thymotrypsin (Sigma) revealed that a pair of interchain disulfide bonds formed at cysteine ​​residue 220 of the two monomers. This indicates that RBD... YFc The dimer consists of two RBDs. YFc The monomers are covalently linked by an interchain disulfide bond formed by a pair of cysteine ​​residues at position 220.

[0077] Table 5 RBD YFc The amino acid residues of the monomer encode (SEQ ID NO: 1)

[0078]

[0079] In summary, when selecting the RBD region of the SARS-CoV-2 virus S protein (e.g., residues 319-541), a tag conducive to dimer formation (e.g., an Fc tag derived from IgG) is fused to the C-terminus. Preferably, a protease cleavage site and a linker peptide and hinge region optimized between the protease cleavage site and the tag are added between the RBD and the tag to construct an RBD-cleavage site-tag fusion protein. This fusion protein exhibits good expression levels and purity in mammalian cells. Furthermore, after protease digestion, affinity chromatography, and ion exchange chromatography, a high-recovery RBD dimer component can be obtained, and the dimer component shows higher binding activity with hACE2-Fc. The molecular weight of the RBD dimer is 62 kDa, and it is formed by two RBD monomers covalently linked by a pair of interchain disulfide bonds (specifically, a disulfide bond formed between cysteine ​​residues at position 220 as shown in Table 5). Each RBD monomer contains four pairs of intrachain disulfide bonds (see Table 5). Figure 12 The disulfide bonds are: (1) the disulfide bond between cysteine ​​at position 18 and position 43, (2) the disulfide bond between cysteine ​​at position 61 and position 114, (3) the disulfide bond between cysteine ​​at position 73 and position 207, and (4) the disulfide bond between cysteine ​​at position 162 and position 170. The sequence codes are shown in Table 5.

[0080] RBD dimer components, namely RBD-mIgG2aFc, RBD-JUN, and RBD-FOS (Table 2), obtained after purification, enzymatic digestion, affinity chromatography, and ion exchange chromatography, possess [specific properties related to...]. Figure 12 The RBD dimer shown has the same sequence, structure, and binding activity with hACE2-Fc.

[0081]

[0082]

[0083]

Claims

1. The RBD fusion protein of the novel coronavirus SARS-CoV-2 is composed of the following sequence: The sequence is the RBD sequence of coronavirus SARS-CoV-2 shown in SEQ ID NO:1 or 2, the protease cleavage site shown in SEQ ID NO:17, and the tag shown in SEQ ID NO:13, 14 or 15.

2. The RBD fusion protein of the novel coronavirus SARS-CoV-2, which is selected from the group consisting of the following: (1) A fusion protein consisting of the following sequence: the signal peptide shown in SEQ ID NO:21, the RBD sequence of coronavirus SARS-CoV-2 shown in SEQ ID NO:1, the protease cleavage site shown in SEQ ID NO:17, the linker peptide sequence shown in SEQ ID NO:26, and the tag sequence shown in SEQ ID NO:

13. (2) A fusion protein consisting of the following sequence: the signal peptide shown in SEQ ID NO:21, the RBD sequence of coronavirus SARS-CoV-2 shown in SEQ ID NO:2, the protease cleavage site shown in SEQ ID NO:17, the linker peptide sequence shown in SEQ ID NO:26, and the tag sequence shown in SEQ ID NO:

13. (3) A fusion protein consisting of the following sequence: the signal peptide shown in SEQ ID NO:21, the RBD sequence of coronavirus SARS-CoV-2 shown in SEQ ID NO:1, the protease cleavage site shown in SEQ ID NO:17, and the tag sequence shown in SEQ ID NO:

13. (4) A fusion protein consisting of the following sequence: the signal peptide shown in SEQ ID NO:21, the RBD sequence of coronavirus SARS-CoV-2 shown in SEQ ID NO:1, the protease cleavage site shown in SEQ ID NO:17, the linker peptide sequence shown in SEQ ID NO:27, and the tag sequence shown in SEQ ID NO:14, and (5) A fusion protein consisting of the following sequence sequence: the signal peptide shown in SEQ ID NO:21, the RBD sequence of coronavirus SARS-CoV-2 shown in SEQ ID NO:1, the protease cleavage site shown in SEQ ID NO:17, the linker peptide sequence shown in SEQ ID NO:28, and the tag sequence shown in SEQ ID NO:

14.

3. A method for preparing the RBD fusion protein of the novel coronavirus SARS-CoV-2 as described in claim 1, comprising sequentially linking the RBD sequence of the novel coronavirus SARS-CoV-2, a protease cleavage site, and a tag that facilitates dimer formation.

4. A method for preparing the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to claim 2, wherein when the RBD fusion protein of the novel coronavirus SARS-CoV-2 is the RBD fusion protein of the novel coronavirus SARS-CoV-2 as described in (1), (2), (4) or (5), the method comprises sequentially linking the signal peptide, the RBD sequence of the coronavirus SARS-CoV-2, the protease cleavage site, the linker peptide sequence and the tag sequence; or When the novel coronavirus SARS-CoV-2 RBD fusion protein is the novel coronavirus SARS-CoV-2 RBD fusion protein described in (3), the method includes sequentially linking the signal peptide, the coronavirus SARS-CoV-2 RBD sequence, the protease cleavage site, and the tag sequence.

5. A method for preparing the RBD dimer of the novel coronavirus SARS-CoV-2 according to claim 1 or 2, comprising cleaving the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-2 with thrombin.

6. The method according to claim 5, wherein the method further comprises purifying the RBD fusion protease digestion product.

7. The method of claim 6, wherein the purification comprises chromatography.

8. The method according to claim 7, wherein the chromatography is affinity chromatography or ion exchange chromatography.

9. A polynucleotide, characterized in that... Encoding the RBD fusion protein of the novel coronavirus SARS-CoV-2 as described in any one of claims 1-2.

10. A carrier comprising the polynucleotide of claim 9.

11. A host cell comprising the polynucleotide of claim 9 or the vector of claim 10.

12. A vaccine, preferably for the prevention of infection with the novel coronavirus, characterized in that... It comprises the RBD fusion protein of the novel coronavirus SARS-CoV-2 as described in any one of claims 1-2 or the polynucleotide as described in claim 9.

13. The use of the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any one of claims 1-2 or the polynucleotide according to claim 9 in the preparation of a reagent for preventing human infection with the novel coronavirus SARS-CoV-2 or for immunizing animals to obtain antiviral serum.

14. The application according to claim 13, wherein the animal is a mammal.

15. The application according to claim 13, wherein the animal is a horse.

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